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EFFECTS OF THE NEUROTOXICANT METHYLMERCURY
ON NEURONAL Ca2+ CHANNELS:
EFFECTS ON Ca2+ CHANNELS INVOLVED IN TRANSMITTER RELEASE

Volume I
By

Timothy Joseph Shafer

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Pharmacology and Toxicology
and Institute for Environmental Toxicology

1991

ABSTRACT
EFFECTS OF THE NEUROTOXICANT METHYLMERCURY

ON NEURONAL Ca2+ CHANNELS:
EFFECIS ON Ca2+ CHANNELS INVOLVED IN TRANSMITTER RELEASE

By
Timothy Joseph Shafer

The ability of the environmental neurotoxicant methylmercury (MeHg) to disrupt
neuronal Ca2+ channel function was examined. In rat brain synaptosomes, MeHg (25-125
is M) blocked depolarization-induced 45Ca2+ inﬂux. Increasing the concentration of
extracellular Ca2+ ([Ca“L) only slightly antagonized block by MeHg of the Ca2+ channel-
mediated component of “Ca inﬂux, whereas block of the Na+ /Ca2* exchange-mediated
component of inﬂux was relieved. MeHg also disrupted Ca2+ channel ftmction by altering
ionic selectivity and inactivation kinetics of synaptosomal Ca2+ channels. Eﬂects of MeHg
did not depend on the state of synaptosomal Ca2+ channels but were exacerbated by
increasing membrane depolarization. MeHg also inhibited [’lﬂ-nitrendipine binding to
synaptosomal preparations. MeHg blocked depolarization induced “Ca2+ inﬂux into
differentiated and undiﬂerentiated rat pheochromocytoma (PC12) cells, and inhibited
binding of the Ca2+ channel ligand to -conotoxin (Cg’Ik) to its receptors in this cell line.
Studies of effects of MeHg on Ca channel function in PC12 cells using the whole-cell patch
voltage clamp technique demonstrated that 5-20 u M MeHg reduced rapidly Ba“ current
mediated by N- and btype Ca“ channels in PC12 cells. Increasing [Ba’m antagonized
block by MeHg between 10 and 20 mM Ba“ but not between 20 and 30 mM Ba“. MeHg

disrupted Ca” channel function in a manner which was voltage- but not state-dependent.
Ionic selectivity and inactivation of whole cell current were altered by MeHg. Effects of
MeHg on nerve terminal Ca“ channels in intact nerve muscle preparations were examined
by measuring effects of MeHg on Ba2*-dependcnt voltage changes in the perineurial sheath
of mouse aimgularis stemi preparations. 50-100 u M MeHg blocked the Bah-dependent
potential as well as the Na*-dependent component. These results suggest that MeHg: 1)
blocks Ca2+ channels in synaptosomal preparations and intact motor nerve terminals, 2)
disrupts function of N- and L-type Ca channels in PC12 cells, 3) disrupts Ca2+ channel ionic
selectivity and inactivation, and 4) disrupts Ca channel function in a manner which is
voltage-, but not state-dependent in synaptosomes and PC12 cells. These actions may
contribute to the ability of MeHg to disrupt synaptic transmission as well as other neurotoxic

effects of Mel-1g.

For Kathryn, Rosie and Loren

iv

ACKNOWLEDGEMENTS

I would like to thank my parents, family and friends for their love, support and
encouragement. Also, I would like to thank my wife Jenny for her love, patience and help.
These people have contributed to this dissertation in ways that cannot be measured.

I would like to thank my advisor, Dr. William Atchison, for his genuine interest in
my graduate training. He has provided signiﬁcant ideas, suggestions and criticisms which
shaped this dissertation and has been an excellent teacher of science, laboratory techniques
and writing skills. In addition, I would like to express my sincere thanks to my guidance
committee for their input, ideas and suggestions, as well as their willingness to share their
time to serve on the committee.

The time and effort expended by student workers, technicians and other graduate
students to help with experiments is appreciated. The excellent technical assistance from Ms.
Lynne Adgate and Aizhen Yao and Drs. Paul Levesque and John Spitsbergen is especially
appreciated. Finally, I would like to thank Ms. Diane Hummell and Mrs. Gretchen

Humphries for their excellent clerical assistance.

‘

PREFACE
Some of the data contained in this dissertation has been published previously.
Chapter Three appeared as Shafer and Atchison (1989), Chapter Four as Shafer er a1. (1990)
and Chapter Five as Shafer and Atchison (1991).

TABLE OF CONTENTS

LIST OF TABLES ................................................. xi
LIST OF FIGURES ........................................... ’ ..... xii
LIST OF ABBREVIATIONS .......................................... xv
CHAPTER ONE: INTRODUCTION ..................................... 1
A) GENERAL INTRODUCTION ................................. 2

B) BACKGROUND ........................................... 7

1) Methylmercury in humans and the environment. ............... 7

2) Effects of Mel-lg on Synaptic Transmission. .................. 10

3) Synaptic Transmission .................................. 16

4) Types of Ca2+ channel .................................. 18

5) Environmental contaminants, natural toxins and pharmaceutical

agents which affect Ca2+ channels ....................... 27

CHAPTER TWO: SYNAPTOSOMES AND PHEOCHROMOCYTOMA CELLS AS

MODEL SYSTEMS FOR STUDY on Ca" CHANNELS ............... 32
A) SYNAPTOSOMES AND SYNAPTOSOMAL Ca2+ CHANNELS ...... 33
B) Caz” CHANNEL FUNCTION IN PC12 CELLS ................... 38

CHAPTER THREE: BLOCK OF ”Ca“ INFLUX INTO SYNAPTOSOMES BY

METHYLMERCURY: Ca2+ AND Na‘ DEPENDENCE ................ 48
A) ABSTRACT .............................................. 49
B) INTRODUCIION ......................................... 51
C) MATERIALS AND METHODS ............................... 54
D) RESULTS ............................................... 58
E) DISCUSSION ............................................. 72

CHAPTER FOUR: _ CHARACTERIZATION OF THE INTERACTIONS OF
METHYLMERCURY WITH Ca2+ CHANNELS IN SYNAPTOSOMES AND

PHEOCHROMOCYTOMA CELLS: RADIOTRACER FLUX AND BINDING

STUDIES .................................................. 78
A) ABSTRACT .............................................. 79
B) INTRODUCTION ......................................... 82
C) MATERIALS AND METHODS ............................... 86
D) RESULTS ..... . .......................................... 92
E) DISCUSSION ............................................ 115

CHAPTER FIVE: METHYLMERCURY BLOCKS N- AND L-TYPE Cal2+
CHANNELS IN NERVE GROWTH FACTOR-DIFFEREN'I‘IATED
PI-IEOCI-IROMOCYTOMA (PC12) CELLS ........................ 127
A) ABSTRACT ............................................. 128

B) INTRODUCTION ........................................ 130

C) MATERIALS AND METHODS .............................. 132
D) RESULTS .............................................. 135
E) DISCUSSION ............................................ 153

CHAPTER SIX: EFFECTS OF METHYLMERCURY ON Na+ CHANNELS IN

NEUROBLASTOMA CELLS ................................... 161
A) ABSTRACT ............................................. 162
B) INTRODUCIION ........................................ 163
C) MATERIALS AND METHODS .............................. 165
D) RESULTS .............................................. 167
E) DISCUSSION ............................................ 170

CHAPTER SEVEN: EFFECTS OF METHYLMERCURY ON Na+ AND

Ca’*CURRENTs IN INTACT NEUROMUSCULAR JUNCTIONS ....... 174
A) ABSTRACT ............................................. 175
B) INTRODUCTION ........................................ 176
C) MATERIALS AND METHODS .............................. 179
D) RESULTS .............................................. 182
E) DISCUSSION ............................................ 190
CHAPTER EIGHT: SUMMARY AND CONCLUSIONS .................... 194

APPENDIX: TRANSMITTER, ION CHANNEL AND RECEPTOR PROPERTIES

OF PHEOCHROMOCYTOMA (PC12) CELLS: A MODEL FOR

NEUROTOXICOLOGICAL STUDIES ............................
A) INTRODUCTION ........................................

B) PC12 CELL DIFFERENTTATION. ...........................

C) TRANSMITTERS, ION CHANNELS AND RECEPTORS IN PC12

CELLS ...............................................
1) Neurotransmitter Release from PC12 cells. .................

2) Ion channels in PC12 cells. .............................

3) Receptors on PC12 cell membranes. ......................
D) CLONAL VARIABILITY AND SUBCLONES OF PC12 ...........
E) SUMMARY .............................................

204
204

205

207
214
218
222

223

LIST OF TABLES
Table 1.1 Biophysical and Pharmacological Characteristics of Ca2+ channel Types . . 23
Table 4.1 N on-Linear least squares regression values for apparent inactivation of
45C3“ inﬂux into synaptosomes ............................... 101
Table A1 Activity and Content of Noradrenergic and Cholinergic

Synthetic Enzymes and Neurotransmitters in PC12 Cells .............. 211

LIST OF FIGURES

Figure 1.1 Effects of stimulus intensity and Ca2+c on EPP amplitude in the presence

Figure 1.2.
Figure 2.1.
Figure 2.2.
Figure 2.3.
Figure 2.4.
Figure 3.1.
Figure 3.2.

Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 3.6.
Figure 3.7.

Figure 3.8.

Figure 4.1.

of MeHg. ............................................... 14
Sequence of events during synaptic transmission. .................. 19
Components of Ca2+ inﬂux into synaptosomes. ................... 35
N and L type Ca2+ channels in PC12 cells. ...................... 40
N orepinephrine release from PC 12 cells. ........................ 42
Effects of EtOH on Ca“ channel density in PC12 cells .............. 47
MeHg blocks resting ”Ca“ entry into synaptosomes ................ 59
Inﬂux of “Ca“ into synaptosomes vs duration of depolarization. ...... 60

Inﬂux of 45Ca“ into synaptosomes as a function of protein

concentration. ............................................ 61
MeHg blocks depolarization-induced “Ca2+ inﬂux into synaptosomes. . . 63
Block of Ca2+ channels by MeHg is not reversed by increasing [Ca2*]e. . 64
Fast and slow components of inﬂux have differential dependence on

N a,. ................................................... 67
In the absence of Na,, block of slow inﬂux by MeHg is much greater in
magnitude. .............................................. 68
In the absence of Na,, block of slow inﬂux by MeHg is not reversed by

increasing [Caz‘]e .......................................... 71

Block of ”Ca“ inﬂux into synaptosomes vs. time. ................. 93

Figure 4.2. Voltage-dependence of “Ca“ inﬂux into synaptosomes and effects of

MeHg. ................................................. 95
Figure 4.3. State-dependence of the actions of MeHg on “Ca2+ inﬂux. .......... 97
Figure 4.4. Effects of MeHg on apparent inactivation of synaptosomal 45Ca2"

inﬂux. ................................................. 100
Figure 4.5. Effects of MeHg on the inﬂux of 45Ca“, "SSrz+ and 133Ba2+ into

synaptosomes. ........................................... 103
Figure 4.6. Effects of MeHg on the relative inﬂux of 45Ca“, “Sr“ and 1”Ba2+ into

synaptosomes. ........................................... 104
Figure 4.7. Inﬂux of ”Ca“ into PC12 cells vs duration of depolarization. ....... 106
Figure 4.8. Inﬂux of “Ca“ into PC12 cells as a function of protein concentration. . 107
Figure 4.9. Effects of nifedipine on depolarization-induced inﬂux of 45Ca“ in PC12

cells ................................................... 108
Figure 4.10. Effects of MeHg on depolarization-induced ”Ca2+ inﬂux into PC12

cells. .................................................. 110
Figure 4.11. Effects of MeHg on nitrendipine binding to synaptosomes .......... .113

Figure 4.12. Effects of MeHg on binding of conotoxin to synaptosomes and PC12

Figure 5.2. Current responses to a series of test potentials. .................. 137

Figure 53. Effects of o-conotoxin and (+)-s202-791 on Ba2+ current in PC12

cells ................................................... 138
Figure 5.4. Effects of MeHg on currents in PC12 cells. ..................... 140
Figure 5.5. Use-dependent effects of MeHg on In. in PC12 cells ............... 142
Figure 5.6. Resting block of In. by MeHg in PC12 cells. .................... 144

Figure 5.7. Effects of MeHg at different holding and test potentials in PC12 cells. . 145

Figure 5.8. Voltage-dependent block by MeHg of In. in PC12 cells is not

irreversible. ............................................ 148
Figure 5.9. Ba” can antagonize block of Ca2+ channels by MeHg. ............ 149
Figure 5.10. MeHg alters ionic permeability of Ca2+ channels in PC12 cells. ..... 152
Figure 6.1. Effects of MeHg on Na+ channels in NIB-115 cells. .............. 168
Figure 6.2. Effects of MeHg on time to peak Na+ current. .................. 169
Figure 7.1. Local circuits in the perineurial sheath of motor neurons. .......... 178
Figure 7.2. Diagram of mouse triangulari: stemi muscle. .................... 181
Figure 7.3. Voltage responses recorded from perineurial sheaths. ............. 183

Figure 7.4. Ca2+ channel-mediated component of voltage potentials in the perineurial

sheath. ................................................ 186
Figure 7.5. Effects of MeHg on Na‘/Caz* potentials in the perineurial sheath. . . . 187
Figure 7.6. Effects of MeHg on repetitive ﬁring of motor nerves. ............. 189
Figure A]. Vesicular nature of ACh release from PC12 cells. ................ 213
Figure A.2. Voltage-dependent Na‘ channels in PC12 cells. ................. 217

xiv

LIST OF ABBREVIATIONS

ACh - acetylcholine

AN OVA - analysis of variance

ATP - adenosine 5’-triphosphate (disodium salt)

[Bar'],3 - extracellular Ba2+ concentration

Bum - density of binding sites for a ligand

CAT - choline acetyltransferase

CAMP - cyclic adenosine monophosphate

[Ca2+]: - sum of the extracellular concentration of all calcium isotopes
[Ca2+], - intracellular Ca2+ concentration

Cng - moconotoxin GVIA

DA - dopamine

DEX - dexamethasone

DI-IP - dihydropyridine

DRG - dorsal root ganglion

EGF - epidermal growth factor

EGTA - ethyleneglycol bis-(B—aminoethyl ether)-N,N,N’,N’,-tetraacetic acid
EPP - endplate potential

EtOH - ethanol

FGF - ﬁbroblast growth factor

FTX - synthethic funnel-web spider toxin

GTP - guanosine triphosphate

HEPES - N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid

IA - whole cell current through the inactivating K+ channel

IBa - whole cell current mediated by barium

ICa - whole cell current mediated by calcium

IK - whole cell current mediated by the delayed rectiﬁer K+ channel
INa - whole cell current mediated by sodium

IC$0 - concentration of chemical that causes 50% inhibition of the maximal response
of the system

KD - equilibrium dissociation constant for ligand binding
lsd - least signiﬁcant difference

MEPP - miniature endplate potential
MeHg - methylmercury

ms - milliseconds

[Na*],z - extracellular concentration of Na+
N GF - nerve growth factor

N MJ - neuromuscular junction

pA - picoamperes

PC12 cells - pheochromocytoma cells
PKA - protein kinase A

PKC - protein kinase C

PI - phosphoinositide

SEM - standard error of the mean

TEACl - tetraethylammonium chloride

TI'X - tetrodotoxin

xvii

CHAPTER ONE

INTRODUCTION

A) GENERAL INTRODUCTION

Methylmercury (MeHg) is a neurotoxic heavy metal found as a contaminant
throughout the environment, but especially in aquatic ecosystems. The organic
component of this molecule imparts to MeHg the ability to bioconcentrate in aquatic
organisms and biomagnify within the food chain. Consumption of contaminated
seafood has resulted in numerous cases of MeHg-intoxication in human populations.
In addition, agricultural use of MeHg has resulted in human intoxication from
accidental consumption of MeHg-treated grain. The neurotoxic potential of MeHg
was dramatically illustrated by chronic and acute episodes of human exposure in
Japan (Takeuchi et al., 1962; 1968) and Iraq (Bakir et aL, 1973), respectively. The
characteristic clinical symptoms of MeHg-intoxication observed included cerebellar
ataxia, speech and visual impairments, and, in the Iraqi episode, weakness of the
extremities (Chang, 1980). Central and peripheral lesions of the nervous system have
been observed in cases of MeHg intoxication (Chang and Hartmann, 1972a,b), but
the cellular mechanisms responsible for these lesions are unknown.

In individuals from the Iraqi episode, neostigmine alleviated clinically
observable symptoms of motor weakness produced by MeHg (Rustam et aL, 1975),
indicating that MeHg possibly disrupts cholinergic transmission at the motor end-
plate. This prompted further in vitro study of the effects of MeHg on neuromuscular
transmission. In isolated nerve-muscle preparations, MeHg exerts two prominent

presynaptic actions: 1) block of nerve-evoked neurotransmitter release, and 2)

3

increased frequency, followed by complete block of spontaneous neurotransmitter
release (Barrett et al., 1974; Juang, 1976; Atchison and Narahashi, 1982).

Release of neurotransmitter following invasion of the action potential into the
nerve terminal is dependent on inﬂux of Ca2+ through voltage-sensitive Ca2+
channels in the membrane (Katz and Miledi, 1967a,b). Several divalent heavy metal
cations which produce effects on evoked and spontaneous transmitter release similar
to those of MeHg block the movement of Ca2+ through these channels. Traxinger
and Atchison (1987b) demonstrated that, under certain conditions, effects of MeHg
on nerve-evoked transmitter release are relieved by increasing the extracellular Ca2+
concentration ([Ca2+]c). MeHg also blocks depolarization-induced 45Ca2“ inﬂux into
synaptosomes. Effects of MeHg on 45Ca2+ inﬂux are also partially antagonized by
increasing [Ca2+]e (Atchison et 611., 1986). These results indicate that the ability of
MeHg to disrupt synaptic transmission at the neuromuscular junction may, in part,
be due to a disruption of Ca2+-dependent events at the motor nerve terminal. This
dissertation reports results of experiments designed to examine the following
hypothesis: disruption of voltage-sensitive Ca2+ channel function by MeHg
contributes to block of synaptic transmission observed in neuromuscular preparations.
The emphasis of studies undertaken was to characterize effects of MeHg on Ca2+
channels, determine types of Ca2+ channels affected by MeHg, and examine the
ability of MeHg to affect Ca2+ channels in intact motor nerve terminals.

Entry of Ca2+ into nerve terminals at the neuromuscular junction is difﬁcult

to measure directly. Thus, effects of MeHg on channel-mediated 45Ca2+ inﬂux into

4

synaptosomes (nerve terminals isolated from the central nervous system) were
examined in initial experiments. In addition, non-channel mediated entry of 45Ca“
was also examined as a measure of the speciﬁcity of MeHg for Ca2+ channel
function. MeHg readily blocks the channel-mediated components of inﬂux in a
manner which is not completely antagonized by increasing [Ca2+]e. MeHg also affects
non-channel-mediated components of inﬂux, including Na+ /Ca2+ exchange and
depolarization-independent entry of Ca2+. For components of Ca2+ inﬂux not
mediated by Ca2+ channels, the ability of increasing [Ca2+]e to antagonize the actions
of MeHg and/ or the concentrations at which MeHg effectively blocks entry are
different than for effects on voltage-sensitive Ca2+ channels by MeHg (Shafer and
Atchison, 1989). Furthermore, the ability of Ca“., to antagonize the block of channel-
mediated 45Ca2+ inﬂux by MeHg is decreased when compared to block by divalent
cations.

When compared to the actions of inorganic Ca2+ channel blockers, among the
unique effects of MeHg on Ca2+ channel function in synaptosomes are alteration of
the apparent rate of inactivation of synaptosomal Ca2+ channels as well as the ionic
permeability of Ca2+, Sr2+ and Ba2+ through the Ca2+ channel. In addition, the
effects of MeHg do not depend directly on the state of the channel, but block of
45Ca“ inﬂux is increased in magnitude as a function of the magnitude of the
depolarizing stimulus (Shafer et aL, 1990).

Because effects of MeHg are expressed in the central as well as the peripheral

nervous system, the use of nerve terminal preparations derived from mammalian

5

central neurons provides meaningful and relevant data about effects of MeHg.
Continued studies in central nervous system preparations in the future will be
necessary to understand completely the mechanisms of MeHg’s neurotoxic effects.
Because synaptosomal preparations are heterogeneous with respect to the
neurotransmitter content of individual terminals within the preparation, the ability
of MeHg to block Ca2+ uptake into synaptosomes may represent a common action
of MeHg at all types of terminals, and because Ca2+ entry is required for
neurotransmitter release, the ability of MeHg to disrupt this process may be a
component underlying its neurotoxicity. However, while these experiments
demonstrate that MeHg is capable of producing unique alterations of Ca2+ channel
function in a population of isolated nerve terminals, they provide only preliminary
evidence to support the stated hypothesis. The purpose of studies performed in the
latter portion of this dissertation was to collect data which would provide stronger
evidence for or against the hypothesis.

Transmitter release from central and peripheral neurons is likely to be
mediated at least in part by N- and/ or L-type Ca2+ channels (Fox et aL, 1987a,b) or
as yet uncharacterized Ca2+ channel types which inactivate Slowly (Smith and
Augustine, 1988; Suszkiw et at, 1989). The hypothesis predicts that MeHg should be
capable of blocking one or both of these types of channels if block of Ca“ channels
by MeHg contributes to the disruption of synaptic transmission. Therefore, the ability
of MeHg to interact with N- and/ or L-type Ca2+ channels was determined using

whole-cell patch voltage-clamp techniques to examine effects of MeHg on Ca2+ (or

6
Ba“) current in rat pheochromocytoma (PC 12) cells. PC12 cells express both N - and

L-type Ca2+ channels when cultured in the presence of nerve growth factor. The use
of patch voltage-clamp techniques allows unambiguous identiﬁcation of Ca2+ channel
types as well as the ability to measure channel function with temporat resolution that
approaches physiological rates of channel function. However, this method of
examining Ca2+ channel function requires use of supraphysiological concentrations
of Ca2+ or other permeant ions such as Ba2+ or Sr“. If MeHg is able to block one
or both of the aforementioned types of Ca2+ channel, this would be strong evidence
in favor of the hypothesis.

The hypothesis was also examined by measuring directly Ca2+ channel
function at the terminal regions of motor neurons. Whole cell patch-voltage clamp
recording, while allowing for unambiguous determination of Ca2+ channel types,
nonetheless measures function of predominantly somatic rather than terminal
currents. By precisely placing electrodes in the perineurial sheath near the motor
nerve terminal, one can record voltage changes within the perineurial sheath in
response to movement of Ba“ through Ca2+ channels in the nerve terminal (Penner
and Dreyer, 1986). Measurement of effects of MeHg on Ca2+-dependent voltage
waveforms from the perineurial Sheath will provide the most direct assessment of the
ability of MeHg to alter Ca2+ channel function in intact motor nerve terminals.

MeHg is a highly reactive compound, especially with sulfhydryl groups. As
such, MeHg binds to a large variety of proteins and disrupts a large variety of

cellular processes, including channel function, mitochondrial function (Levesque and

7
Atchison, 1987; 1988; 1991), microtubule structure (Abe et at, 1975; Sager elf £11.,

1983) and protein synthesis (Yoshino et aL, 1966). Furthermore, exposures to MeHg
which produce signiﬁcant pathological changes are also likely to alter numerous
cellular functions. Thus, it is important to characterize all events which might
contribute to compromised cellular function in the presence of MeHg rather than
attempt to deﬁne a single critical event which results in altered cellular function
(Reuhl, 1987). The plasma membrane, along with its resident channels, receptors and
transporter proteins is the initial Site of interaction for all foreign and endogenous
compounds with the cell (See Narahashi, 1980) and plays a vital role in cellular
homeostasis. In the nervous system, the proper functioning of Na+ and K+ channels
is crucial for propagation of action potentials, and Ca2+ channel function is critical
to the process of neurotransmitter release. By characterizing the ability of MeHg to
disrupt Ca2+ channel function, the effects at this initial site of interaction with the
cell will be better understood. The signiﬁcance of this information may exceed
possible effects on neurotransmitter release, as Ca2+ channels are involved in cellular
Ca2+ homeostasis; alterations of Ca2+ homeostasis may therefore contribute to

disruption of cellular function by MeHg.

B) BACKGROUND
1) Methylmercury in humans and the environment.
Chronic and acute episodes of human intoxication with MeHg have occurred

previously. In the mid- to late- 1950’s, ingestion of MeHg-contaminated shellﬁsh from

8
Minamata Bay in Japan led to 1500 cases of intoxication and 46 deaths (Takeuchi

et aL, 1962, 1968). The cause of contamination was due to discharge of organic and
inorganic mercurial waste into the bay from a chemical plant producing acetaldehyde
from acetylene. Mercury metal used as a catalyst reacted with the acetylene,
ultimately forming MeHg as a contaminant. In Iraq, the use of MeHg-treated seed
grain for bread (despite government warnings of adverse health effects) resulted in
450 deaths due to acute MeHg poisoning in 1972 (Bakir, et al., 1973). Thus,
environmental contamination with MeHg, as well as any signiﬁcant exposure to
MeHg, represent legitimate human health concerns. MeHg still remains a
considerable human health hazard, and certian populations, particularly those which
consume large omounts of ﬁsh, remain at risk (Hansen, 1990).

There are two main sources of environmental contamination by MeHg: MeHg
itself, and elemental Hg which is converted to MeHg. Because of its antifungal
properties, MeHg has been employed by the agricultural, lumber, paper and leather
industries, primarily as a preservative. Consequently, it is found as a contaminant in
seed grain dressings, agricultural mnoff, and waste-water discharges from these
industries. Elemental Hg has been used by the electrical apparatus, instrumentation,
paint and chloralkali industries, and is released into the environment via atmospheric
venting or discharge along with waste-water. Mercury is also released into the
environment during the combustion of fossil fuels. In the aquatic environment,
microorganisms can methylate inorganic mercury (Wood et aL, 1968; Ridley et aL,

1977), thus increasing the pool of MeHg in the environment.

9

The conversion of Hg to MeHg in the environment has important toxicological
implications. Prior to knowledge of this conversion, inorganic Hg discharged into the
environment was considered to be relatively harmless to humans as it is poorly
absorbed by the gastrointestinal tract. By contrast, the more lipophilic MeHg tends
to bioaccumulate and biomagnify in the food chain, and up to 95% is absorbed by
the gastrointestinal tract.

MeHg is transported in the plasma and red blood cells and distributes evenly
to most tissues, but concentrates in the blood and central nervous system. In rats
chronically exposed to MeHg, the spinal dorsal root ganglia accumulated the highest
levels of MeHg, while the cerebral cortex and cerebellum also contained high levels
of MeHg (Somjen et al., 1973). The blood-brain barrier is easily crossed and also
damaged by MeHg (Steinwall and Klatzo, 1966; Steinwall and Olsson, 1969; Chang
and Hartmann 1972c). Pathological examination of victims of MeHg toxicity in
Minamata indicated that MeHg lesions the calcarine (visual) cortex, as well as
cerebellar granule cells and the dorsal root ganglia (Chang, 1977; 1979).
Histochemical analysis has shown that MeHg is closely associated with the
mitochondria, golgi, endoplasmic reticulum, and the nuclear envelope. In nerves,
MeHg is found primarily in myelin sheaths and mitochondria, and causes axonal
degeneration (Chang, 1977). The high afﬁnity of MeHg for membranous structures
in the neuron may contribute to the toxicity of this compound by disrupting important

cellular functions which take place at these sites.

10

MeHg is Slowly excreted; its half-life in humans is approximately 70 days.
MeHg slowly decomposes to inorganic Hg, is completely reabsorbed from the bile,
and has a low rate of urinary excretion compared to elemental Hg (Magos, 1975).
Thus, frequent consumption of MeHg-contaminated food or water can result in
signiﬁcant accumulation of MeHg in the body. Consumption of contaminated seafood
has resulted in the accumulation of toxic doses of MeHg in Japanese (Takeuchi et
at, 1962; 1968) and Swedish (Berglund et al., 1971) populations. Such episodes have
resulted in concern regarding the safety of ﬁsh from the Great Lakes. However,
mercury levels in ﬁsh in the Great Lakes have declined since 1976, and the average
Hg concentration in ﬁsh in each of the ﬁve Great Lakes is below the action level
established by the Food and Drug Administration (1.0 mg/kg) and the Michigan

Department of Public Health (0.5 mg/kg) (D’Itri, 1985).

2) Effects of MeHg on Synaptic Transmission.

The observation that neostigmine alleviated symptoms of motor weakness
spurred research into the mechanism underlying this action of MeHg. Neuromuscular
preparations such as the rat phrenic nerve-hemidiaphram preparation have served
as useful model systems for study of synaptic transmission between nerve and muscle.
The neuromuscular junction (NMJ) has been well characterized with respect to its
structure, function and pharmacological response to various drugs and toxins. In
addition, the synthesis, storage and Caz+-dependent release of acetylcholine (ACh)

at the NMJ are similar in many respects to the synthesis, storage and release of other

ll

neurotransmitters in central and peripheral neurons. As MeHg not only affects
neurons which release ACh, but also those neurons which release catecholamines,
serotonin, GABA, glutamate and glycine (Borowitz, 1974; Nakazato, et al., 1979;
Bondy, et al., 1979; Tuomisto and Komulainen, 1983), information obtained using this
preparation may also provide preliminary indications as to how MeHg affects other
neurons as well.

Following acute bath application to isolated nerve-muscle preparations, MeHg
causes a rapid block of nerve-evoked transmitter release and an increase in the
frequency, followed by a complete block, of spontaneous neurotransmitter release
(Barrett et al., 1974; Juang, 1976; Atchison and Narahashi, 1982; Miyamato,1983;
Atchison et al., 1984; Atchison, 1986; Levesque and Atchison 1987; 1988). At the
neuromuscular junction, these effects are observed electrophysiologically as
alterations of the amplitude of end plate potentials measured in the muscle cell in
response to nerve-evoked (EPP) or spontaneous (MEPP) neurotransmitter release.
Block of the EPP by 100 11M MeHg occurs rapidly and with no apparent gradual
reduction; after 8-20 min the EPP is completely abolished (Atchison and N arahashi,
1982; Atchison et al., 1984; Traxinger and Atchison, 1987b). By contrast, changes in
MEPP frequency occur after a latent period, which can be shortened without
increasing the peak MEPP frequency observed by increasing the concentration of
MeHg (Atchison and N arahashi, 1982) or by K+-induced depolarization (Atchison,
1986). The ultimate block of transmitter release produced by MeHg is not due to

depletion of ACh stores; treatment with La3+ after complete block of MEPPs by

12
MeHg results in their restoration (Atchison, 1986; Levesque and Atchison, 1988),

indicating that the nerve terminal has not been depleted of ACh-containing vesicles.

Although post-synaptic actions of MeHg have not yet been carefully examined,
several observations indicate that the block of EPP and increase in MEPP frequency
caused by MeHg are the result of actions of MeHg on the presynaptic terminal. The
experimental evidence supporting a presynaptic action of MeHg include the following
observations: 1) the response of the endplate to iontophoretically applied ACh is
unchanged even after 1 hr of exposure to 100 MM MeHg (Atchison and Narahashi,
1982). At this time, neither EPPS nor MEPPS are observed. 2) MEPP amplitude is
not affected at the time of complete EPP block by MeHg (Atchison and N arahashi,
1982). These results indicate that the postsynaptic cell still responds to ACh at a time
when synaptic transmission is impaired by MeHg. In addition to this experimental
evidence, changes in MEPP frequency are thought to be due to presynaptic and not
postsynaptic events.

Not only do the actions of MeHg appear to be presynaptic, but the differences
in the time course of MeHg’S effects on 'EPPs and MEPPS suggest different
mechanisms of action by MeHg on each of these forms of release. Indeed, the
mechanisms underlying the EPP and MEPP themselves are different. The MEPP is
a depolarization of the end-plate membrane resulting from release of one quantum
of neurotransmitter and subsequent interaction with receptors located on the
postsynaptic membrane (Fatt and Katz, 1952). Although MEPPS are affected by

extracellular Ca“, spontaneous release can occur in Ca2*-free solutions. The

l3
frequency of MEPP occurence reﬂects the intraterminal [Ca2+] (Hubbard et al.,

1968). Thus, it has been proposed that the actions of MeHg on MEPP frequency are
due to a disruption of intraterminal Ca2+ buffering organelles, such as the smooth
endoplasmic reticulum and mitochondria, leading to an increase in [Ca2+]i (Atchison
et al., 1984; Atchison 1986; Levesque and Atchison, 1987; 1988; 1991). The EPP,
however, is the postsynaptic response resulting from synchronous release of many
quanta of neurotransmitter following the depolarization of the nerve terminal by an
action potential and subsequent entry of extracellular Ca2+ (Katz and Miledi,
1967a,b; Llinas et al., 1981). Under physiological conditions, EPPS require Ca2+e. The
rapid and complete block of EPPS by MeHg indicates that MeHg may block
conduction of the action potential, electrotonic Spread of depolarization into the
terminal and/ or entry of Ca2+ into the nerve terminal.

Initially, MeHg-induced block of evoked release at the NMJ is not reversible
simply by methods designed to overcome a competitive block of divalent cation inﬂux
by MeHg. Block of the EPP by MeHg is not reversed by perfusing the preparation
with MeHg-free solutions containing normal or elevated [CaZI] or with MeHg-free
solutions containing agents (4-aminopyridine) which prolong Ca2+ entry into the
nerve terminal. However, increasing the intensity or duration of the stimulus in
MeHg-free solutions does partially and occasionally reverse MeHg-induced block of
evoked release (Traxinger and Atchison, 1987b) (Figure 1.1). This indicates that
MeHg may affect impulse conduction at either the axonal membrane or the

unmyelinated portion of the nerve terminal. Previous studies have indicated that

l4

 

 

 

9 e
E \u. 4 mM Ca
Lu 5 a)” 0..” o MeHg
O
D lT / MeHg
t A
._i 00
CL 3 .MeHg 'T‘V.. e—e—e
2 '\
< Ca
8.
LIJ 0 t i
0 6 12 32 40
TIME (min)

Figure 1.1 Effects of stimulus intensity and Ca2+e on EPP amplitude in the presence of MeHg. EPPS
were evoked in rat hemidiaphram by stimulation of the phrenic nerve at 0.25 Hz throughout the
experiment. MeHg was applied continuously for 9 min by which time the EPP was blocked. Immediately
following block, the preparation was perfused with a MeHg-free Ca2+ ([Ca“) = 4 mM) solution for
15 min. During this time, EPPS remained blocked. While maintaining the preparation in 4 mM Ca2+
solution, the stimulus intensity was increased (t V); this brought about an immediate return of EPPS.
Subsequent rc-exposure of the preparation to MeHg in normal (2 mM) Ca2+ solutions caused EPPS
to be abolished within 3 min. Wash of the preparation at this time with MeHg-free, 4mM Ca2+ solution
caused a prompt recovery of the EPP. All values are from a single representitive preparation.

Modiﬁed from: Traxinger and Atchison, T axicol. Appl. Pharmacol. 90:23-33 (1987b).

15
MeHg may interact with Na+ channels or sulfhydryl groups in the membrane which

are important in action potential propagation (Shrivistav, et al., 1976; Quandt et al.,
1982). An increase in the duration or intensity of stimulation would be expected to
overcome a MeHg-induced decrease in excitability in these regions (Konishi,
1985). Under conditions of increased stimulus intensity, re-exposure of the
preparation to MeHg resulted in a re-occurrence of block of EPPS. This subsequent
block of EPPS could be reversed by increasing [Ca2+], (Traxinger and Atchison,
1987b). Thus, block of EPPS by MeHg may in part be due to block by MeHg of Ca2+
entry associated with neurotransmitter release. This hypothesis is strengthened by the
observation that MeHg blocks depolarization-induced 45Ca2+ inﬂux into rat forebrain
synaptosomes, and that the block of inﬂux by MeHg over 10 s is antagonized,
although not completely overcome by increasing [Ca2"],3 (Atchison et al., 1986).

In addition to experimental evidence suggesting that MeHg may block Ca2+
entry associated with neurotransmitter release, one may suspect, based on its
similarities in physico-chemical properties (i.e., relatively similar ionic radius, positive
charge, ability to gain or lose electrons) (Miller, 1984) with other heavy and/ or
transition metals, that MeHg could exert this action. Other heavy metals such as N i2+
(Kita and Van der Kloot, 1973), Cd2+ (F orshaw, 1977; Cooper and Manalis 1983)
and Pb2+ (Kostial and Vouk, 1957; Manalis and Cooper, 1973; Silbergeld et al.,
1974a,b; Atchison and Narahashi, 1984; Cooper and Manalis, 1983; Biisselberg et
al. ,1990), block evoked transmitter release in a fashion Similar to MeHg. However,

in contrast to the effects of MeHg, block of synaptic transmission at the NMJ by Pb2+

16
(Kober and Cooper, 1976; Cooper and Manalis, 1983; Atchison and Narahashi, 1984;

Pickett and Bornstein, 1978; 1984), Cd2+ (Nilson and Volle, 1976; Cooper and
Manalis, 1984a,b), or Co2+ (Weakly, 1973) is reversed by simply increasing [Ca2+ 1,
or washing with normal, metal-free solutions. In synaptosomes, block of 45Ca2+ inﬂux
by divalent cations such as Pb“, Ni“, Cd2+ and Hg“ (N achshen, 1984; Atchison et
al., 1986), but not MeHg (Atchison et al., 1986; Shafer and Atchison, 1989), is
completely reversed by increasing [Ca2+]e concentration. As MeHg has enhanced
lipophilicity and a monovalent charge, differences in charge and lipophilicity may
underlie the differences in the action of MeHg and inorganic, divalent cations on

nerve terminal Ca2+ ﬂuxes.

3) Synaptic Transmission
Successful transmission of neuronal signals, whether between nerve and muscle,
post-synaptic nerve or other cell type, requires a complex sequence of events.
Disruption of any one event, such as block of Na” channels by tetrodotoxin
(N arahashi er al. , 1964) or inhibition of acetylcholinesterase by organophosphate
insecticides, can lead to deleterious consequences. Many neurotoxic compounds
(including heavy metals) disrupt processes at the synapse. Thus, it is conceivable that
MeHg may also act at this Site.
Transmission at peripheral and central chemical synapses involves the
conversion of an electrical signal into a chemical signal in the presynaptic cell, and

then, depending on the postsynaptic cell, into an electrical or biochemical (i.e., 2nd

l7

messenger) signal. Although the complete sequence of events resulting in
neurotransmitter release is not yet known, several processes which take place prior
to release have been identiﬁed. Action potentials propagated along the nerve axon
by movement of Na+ and K+ into and out of the axon through their respective
channels (Hodgkin and Huxley, 1952) cause depolarization of the non-myelinated
terminal membrane by electrotonic spread into the terminal region (Brigant and
Mallart, 1982; Mallart, 1985). This depolarization causes voltage-sensitive Ca2+
channels in the nerve terminal membrane to open, allowing extracellular Ca2+ to
move down its electrochemical gradient into the nerve terminal (Katz and Miledi,
1967a,b; Llinas er al., 1981). It is thought that these channels, whose activation is
crucial for evoked release, are clustered in areas in which vesicles containing
neurotransmitter are arranged in close apposition to the cell membrane (Pumplin et
al., 1981; Robitaille et al., 1990; Cohen et al., 1991). Inﬂux of Ca2+ in these areas, or
"active zones”, gives rise to locally high [Ca2+]i, resulting in increased interaction of
Ca2+ ions with CaZI-binding proteins. This interaction between Ca2+ and binding
proteins is thought to trigger directly or indirectly the fusion of synaptic vesicles with
the cell membrane, resulting in the release of chemical neurotransmitters into the
synaptic cleft (Augustine er al., 1987). The protein or proteins to which Ca2+ binds
have yet to be identiﬁed, although synapsin I, synexin or other members of the
calelectrin family and p38-synaptophysin have been suggested as the
macromolecule(s) responsible for coupling Ca2+ entry to neurotransmitter release

(Smith and Augustine, 1988). Injection of dephosphorylated synapsin I into the

18

preterminal region of squid giant synapse inhibited neurotransmitter release (Llinas
et al., 1985) and reduced spontaneous release (Lin et al., 1990a). Furthermore,
depolarization of synaptosomes caused translocation of synapsin I from the
particulate (presumably cytoskeletal) fraction to the soluble (cytoplasmic) fraction in
a Ca2+ and phosphorylation-dependent manner (Sihra et al., 1989). It has been
suggested that this protein may regulate the availability of synaptic vesicles for
release (Llinas et al., 1985). However, the exact role of this molecule in
neurotransmitter release has yet to be determined.

The released transmitter diffuses across the synaptic cleft and binds to
receptors located on the membrane of the post-synaptic cell, resulting in an electrical
or chemical response in the post-synaptic cell. Continuous activation of the receptor
is prevented by re-uptake of the transmitter into the pre-synaptic cell, as in

adrenergic synapses, or by enzymatic destruction of the transmitter, as in cholinergic

synapses (Figure 1.2).

4) Types of Ca2+ channel

Entry of extracellular Ca2+ into the nerve terminal is critical for release of
neurotransmitter; N a+ and K" do not support release, nor will signiﬁcant release
occur in Ca2*-fl'ee medium (Katz and Miledi, 1967a,b). Therefore, an understanding
of the nature of the Ca2+ channels that may regulate Ca2+ entry associated with
neurotransmitter release is important to understand the process of synaptic

transmission. However, it is not currently possible to characterize "active zone” Ca2+

l9

    

Reuotahe

A ‘2 [Hydroiysts

Messenger

  
    

9.Biochemical
Changes

  

Figure 1.2. Sequence of events during synaptic transmission. 1) Depolarization of the terminal region
by electrotonic spread of the action potential into the terminal region from pro-terminal nodes of
Ranvier. 2) Activation of voltage-sensitive Ca2+ channels and inﬂux of Ca2+ into the nerve terminal.
3) Oak-triggered fusion of synaptic vesicles with the terminal membrane. 4) Release of
neurotransmitter into the synaptic cleft and 5) diﬂ‘usion to the post-synaptic cell where 6) binding to
receptors occurs. Following binding, the transmitter is removed from the cleft by 7) hydrolysis and/or
carrier-mediated re-uptake into the presynaptic terminal. Binding of the neurotransmitter to receptor
activated channel complexes results in 8) conductance changes and 9) synaptic potentials, whereas
binding to other receptor types results in changes in 8) second messingcrs and 9) cellular biochemistry.

Modiﬁed from: Shepherd, G. H. In: Synaptic organization of the brain. (Shepherd, G. H., ed.). p. 5,
(1990).

20

channels using intracellular or patch electrodes, as this region accounts for only a
small portion of the nerve terminal. Therefore, the best method presently available
to determine which type or types of Ca2+ channel may be involved in transmitter
release is to characterize the types of somatic Ca2+ channel,and then infer which
types may be involved in release based on the properties and pharmacological
sensitivities of Ca2+ channels and the release process. However, a lack of
pharmacological effect on neurotransmitter release may result from inadequate
access of drugs or toxins to the channel rather than channel insensitivity. Thus,
caution must be observed when making inferences as to channel type(s) critical to
neurotransmitter release bases solely on pharmacological sensitivity.

Two types of Ca2+ channel have been consistently demonstrated in neurons
(Llinas and Yarom, 1981; Carbone and Lux, 1984). One type begins to activate
(opens) at membrane potentials between —60 and -40 mV, has a very small unitary
conductance, completely inactivates during a sustained voltage pulse and remains
inactivated if the membrane potential is held positive to -60 mV. Depending on the
nomenclature system used, these channels are termed "Low Voltage Activated"
(LVA), "Type I", or "T-type" channels. The second type of Ca2+ channel is activated
at membrane voltages of -20 to 0 mV, has a large unitary conductance, and generally
does not inactivate during sustained voltage pulses of 100-500 ms. These channels are
denoted as "High Voltage Activated" (HVA), "Type II", or "L-type" Ca2+ channels.

A third subtype has been described in avian (Nowycky er al., 1985a; Fox et aL,
1987a,b) and mammalian (Kostyuk et al., 1988) embryonic dorsal root ganglion

21
(DRG) cells and in nerve grth factor-differentiated rat pheochromocytoma (PC12)

cells (Plummer et al., 1989) as well as many other types of neuronal cells. It is also
a high voltage activated channel, but the unitary conductance of this channel is
smaller than the "L-type" channel and inactivation of this channel occurs during a
sustained voltage pulse. This channel has been termed "N-type". However, the
existence of this channel as a separate entity from the L channel has been questioned
on the basis of analysis of tail current recordings from chick DRG neurons
(Swandulla and Armstrong, 1987) and growth cone currents from PC12 cells (Streit
and Lux, 1990). The evidence supporting three separate Ca2+ channel entities has
been reviewed recently (Swandulla et al., 1991).

Recently, an additional Ca2+ channel type has been described in Purkinje cell
neurons of guinea pig cerebellum and in the presynaptic terminal of squid giant
synapse (Llinas et al., 1989). The biophysical characteristics of this channel differ
from those of T-, N- and L-type channels. In addition, this channel is not sensitive
to DHPS or to -conotoxin GVIA, but is sensitive to a polyamine-like toxin from venom
of funnel web spiders (Llinas et al., 1989). Using a synthetic funnel-web spider toxin
(FTX), proteins have been isolated from mammalian cerebellar tissue which formed
FIX-sensitive channels when incorporated into bilayers. Furthermore, the molecular
mass of this protein markedly differed from that of other characterized Ca2+ channel
proteins (Cherksey er al., 1990) This channel is referred to as the P channel since it
was ﬁrst described in Purkinje cells. Ca2+ current from rat brain mRN A expressed

by Xenopus oocytes has a single exponential time course of inactivation of

22

approximately 650 ms and is not sensitive to DHPS nor to -conotoxin GVIA (Leonard
et al., 1987), but has recently been reported to be partially sensitive to FTX (Lin et
al., 1990b). These results suggest that the channel type expressed from rat mRNA
may be similar to the P-channel. The biophysical and pharmacological proﬁles of the
different Ca2+ channel types are summarized in Table 1.

Pharmacological differences also exist between the Ca2+ channel subtypes and
have been instrumental in distinguishing among the putative subclasses. The
dihydropyridine (DI-IP)-class of organic Ca2+ channel agonists and antagonists
interact preferentially with L-type channels, whereas N - and T-type channels are
unaffected by nanomolar to low micromolar concentrations of these drugs. The
peptide, to -conotoxin GVIA, (Cng) acts on a speciﬁc population of sensitive N-type
channels in mammalian neurons. However, in amphibian and avian preparations,
Cng also blocks L-type channels, as deﬁned by voltage sensitivity and current
kinetics. In some cases, block of L channels by Cng may be reversible or
incomplete. Inorganic divalent cations block all three types of Ca2+ channels.
However, the relative potencies of block differ: N and L channels are blocked more
readily by Cd2+ than N iz", whereas T channels are blocked more readily by N i2+ than
Cd2+ (Tsien et al., 1988).

It should be noted that slight differences in electrophysiological properties
and / or pharmacological sensitivities, such as the voltage at which current by a
channel type increases, or the time constant of inactivation of whole-cell current exist

for given Ca2+ channel types from one cell type to the next. In addition, since Ca2+

23

Table 1.1

Biophysical and Pharmacological

Characteristics of Ca2+ channel Types

 

 

T N L P
Activation Range (mV) -70 -10 -10 -30 to ~20
Inactivation Kinetics fast moderate slow slow
r=20-50ms r=95ms r>.555 1:?
Divalent Cation NI2+ > Cd?“ Cd2+ > N17” Cd" > N?‘ Cdz“, <100pM
sensitivity
DHPS - - + -
to Cng - + + a _
FIX - - - +
Single channel
conductance (pS) 8 13 25 10-12
(110 mM Ba“) (80 mM Ba“)

 

Data for chick DRG N,L and T channels modiﬁed from Tsien er al., TINS 10:431—438, 1988.
P-channel data compiled from Llinas eta1., Proc. Natl. Acad. Sci. USA 86: 1689-1693, 1989 and
Lin et al., Pmc. Natl. Acad. Sci. USA 87:4538—4542, 1990b.

" Mammalian L-type channels are not sensitive to Cng (Plumber er al., Neuron 2:1453-1463,

1989).

24

channel function often depends on the presence of intracellular components (Bean,
1985), channel activty often changes or is lost during whole-cell or cell-free patch
recording. This has hampered charactization of Ca2+ channel function and types.
Thus, the characteristics attributed to Ca2+ channel types are more descriptive than
absolute, as Ca2+ channel characteristics do not always ﬁt neatly into one of the
above categories or nomenclature systems. However, it is possible that there is
considerable heterogeneity in Ca2+ channel function among different cell types or
that additional channel types exist. This is supported by the recent isolation from rat
brain of 4 different types of cDNA that code for putative Ca2+ channels; two of
which are highly homologous (75% identical residues) with the a 1 subunit of the
DI-IP-sensitive Ca2+ channel from skeletal muscle, whereas the other two are much
less homologous (50% identical residues) but nonetheless related in sequence
(Snutch et al., 1990). Characterization of the primary structure and expression of
these cDNAs suggest that several different Ca2+ channel gene families exist which
are related in amino acid sequence, but may have subtle differences in
pharmacological sensitivity (Starr et al., 1990; Dubel and Snutch, 1990).

Despite considerable research characterizing neuronal Ca2+ channels, the
channel type(s) involved in neurotransmitter release are still not deﬁned clearly. This
is largely due to the technical limitations of current techniques. Intracellular and
patch clamp recording require the use of microelectrodes whose diameter is almost
as large as the nerve terminal itself. Thus, biophysical characterization of channel

types at the active zone is not possible. Exceptions to this problem are the terminals

25

of the neurohypophyseal neurons, which are large enough to allow use of patch-
clamp methodology. It has been reported that both L and N type channels are
present in these terminals, and that both may contribute to Ca2+ entry associated
with transmitter release. Under normal conditions of release, L channels are more
likely to be involved in release than N channels, due to rapid inactivation of the
latter during trains of action potentials (Lemos and Nowycky, 1989). More recently,
however, it has been reported that both Ca2+ channel types in this preparation may
be involved in an initial burst of transmitters release, whereas L-type Ca2+ channels
are important for sustained release of peptide transmitters (Lim et al., 1990;
N owycky, 1991). Although they provide the only mammalian system in which Ca2+
currents can be examined in nerve terminals, these neurons are highly specialized
structures which secrete the neurohormones oxytocin and vasopressin. Release of
peptide neurotransmitters from these terminals may or may not be truly
representative of release of small, classical transmitters from other central and
peripheral neurons.

Indirect information on the types of Ca2+ channels which mediate Ca2+ inﬂux
associated with neurotransmitter release can be obtained by examining the
pharmacological sensitivity of neurotransmitter release. At the rat neuromuscular
junction, DHP-sensitive Ca2+ channels can have at best a modulatory role in
neurotransmitter release (Atchison and O’Leary, 1987), but do not contribute to
nerve-evoked release under normal conditions (Atchison, 1989). DHP-sensitive or L-

type channels have been implicated in release of substance P from DRG cells (Rane

26
et al., 1987; Holz et al., 1988) and 3H-norepinephrine release from undifferentiated

PC12 cells (Kongsamut and Miller, 1986). DHP-insensitive or N-type channels have
been implicated as the important channel involved in neurotransmitter release in rat
sympathetic neurons (Hirning et al., 1988), peripheral neurons (Perney et al., 1986),
nerve growth factor differentiated PC12 cells (Kongsamut and Miller, 1986), various
areas of the central nervous system (Dooley et al., 1987a).

These data would suggest that N- and/ or L-type Ca2+ channels are responsible
for Ca2+ inﬂux associated with neurotransmitter release at many terminals. However,
substantial data exists which suggests that another as yet uncharacterized Ca2+
channel subtype may also mediate Ca2+ entry associated with neurotransmitter
release. For example, the Ca2+ current associated with neurotransmitter release from
the presynaptic terminal of the squid giant synapse activates at relatively
hyperpolarized potentials, inactivates slowly, and is insensitive to DHPS and Cng
(Charlton and Augustine, 1990). This suggests the possibility that P-type channels
may also be involved in transmitter release. In mammalian preparations, Cng and
DHPS do not affect release of ACh from motor nerve terminals (Anderson and
Harvey, 1987) or aspartate release from hippocampal slices (Mangano et al., 1991).
Transmitter release from rat sympathetic ganglion cells is not impaired by DHPS or
Cng (Seabrook and Adams, 1989). Species and / or tissue differences in the type
Ca2+ channel involved in neurotransmitter release may also exist in mammals, as in
vitm Cng produces a greater eﬁect on transmitter release in smooth muscle

preparations from guinea pigs than from rats (Maggi et al., 1988). Wessler er al.,

27

(1990) demonstrated in rats that 3H-ACh release from myenteric plexus neurons and
the phrenic nerve is not affected by verapamil or diltiazem, whereas Cng reduces
release from the neocortex (ICso = 13 nM) and myenteric plexus (ICso = 0.7 nM),
but not the phrenic nerve (ICso 2 100 nM). These results suggest that channels which
are neither N - nor L-type are involved in neurotransmitter release from some
mammalian nerve terminals. Therefore, at many terminals it appears as though N -
and/ or L-type channels may mediate Ca2+ entry associated with neurotransmitter
release, although additional Ca2+ channel subtypes may also be involved in
neurotransmitter release. There is no evidence to date which indicates that the T-
type Ca2+ channel is involved in release in any system, but this does not preclude an

unknown role of this channel.

5) Environmental contaminants, natural toxins and pharmaceutical agents which
affect Ca2+ channels.

Ca2+ channel function in neurons is affected by a wide variety of chemical
agents including metals, peptides and organic compounds. Inorganic heavy metal
cations are effective Ca2+ channel blockers. Lead (Audesirk, 1987 ; Audesirk and
Audesirk, 1989) and cadmium, although the latter is not overtly neurotoxic, are
environmental contaminants which block Ca2+ channels at concentrations between
1 and 10 micromolar. The transition metals nickel, mercury, copper and zinc are also
capable of blocking Ca2+ channels at low micromolar concentrations (Nachshen,

1984). In isolated nerve terminals, block of 45Ca’“ inﬂux by divalent cations such as

28
Pb“, Ni“, Cd2+ and Hg2+ (Nachshen, 1984; Atchison et al., 1986) is overcome by

increasing [Ca2+]e. Thus, it is thought that these metals bind to a site in the pore of
the Ca2+ channel with high afﬁnity, impeding the ﬂow of Ca2+ through the channel
(Hagiwara and Takahashi, 1967; Hagiwara et al., 1974; Lansman et al., 1986; Hess
et al., 1986).

In addition to inorganic metals, the organic metals methylmercury (Atchison
et al., 1986; Shafer and Atchison, 1989) and ethylmercury (Hewett and Atchison,
1990) are capable of blocking 45Ca2+ inﬂux into nerve terminals. Both charge and
lipophilicity may be important in determining the characteristics of Ca2+ channel
disruption by mercurials, as only positively charged mercurials block Ca2+ channels,
and only organic mercurials blocked “Ca2+ inﬂux into synaptosomes in a voltage-
dependent manner (Hewett and Atchison, 1990).

Several natural toxins which interact Speciﬁcally with Ca2+ channels have been
isolated from venom preparations. A family of polypeptide toxins with Ca2+ channel
activity has been isolated from venom of marine snails of the Conus family (Olivera
er al. , 1987). to -Conotoxin GVIA, isolated from the venom of Conus geographus
(Olivera er al., 1985 ), has been thoroughly characterized with respect to its action on
Ca2+ channels. This compound is a potent inhibitor of non-mammalian Ca2+
channels, binding to and blocking N— and L-type Ca2+ channels irreversibly in sub-
picomolar concentrations (Cruz and Olivera, 1986). In mammalian preparations, the
action of to -conotoxin GVIA is less clear. It has been suggested that only the N-type

Ca“ channel is sensitive to Cng in several preparations (Tsien er al., 1988; Plummer

29
et al., 1989). This peptide blocks neurotransmitter release (Keith et al., 1989) and

synaptic transmission (Kamiya er al., 1988; Dutar et al., 1989) in the hippocampus.
However, Cng does not affect ACh release at the neuromuscular junction
(Anderson and Harvey, 1987), and only partially reduces 45Ca2+ inﬂux into
mammalian synaptosomes (Suszkiw et al., 1989). In addition, expression of rat brain
mRNA in oocytes results in Ca2+ currents which are insensitive to Cng (Leonard
et al., 1987). As discussed in the previous section, these results suggest a unique Ca2+
channel type in these preparations.

Maitotoxin, which is produced by the marine dinoﬂagellate Gambierdiscus
toxicus, increases Ca2+ channel function in cultured neuronal cells (Takahashi et al.,
1982; 1983). Spider venoms also contain peptides and polyamine-like compounds
which block Ca2+ channels. The to -aga-toxins are peptides that have been isolated
from the venom of funnel web spider (Agelenopsrll~ aperta) and are potent blockers
of Ca2+ channels (Adams et al., 1990). These toxins can be subdivided into three
classes based on the ability to inhibit Cng binding in chick synaptosomes; to -AgaI
toxins do not affect binding, whereas at -AgaII and to -AgaIII (Venema and Adams,
1990) toxins inhibit Cng binding. (1) -AgaIIIA blocks N- and L-type channels in rat
DRG cells (Minty et al., 1990). However, the pharmacological characterization of
these compounds has not been completed to date. A polyamine-like toxin which is
a potent Ca2+ channel blocker also has been puriﬁed from the venom of funnel-web
spiders (Llinas et al., 1989). The structure of this toxin has not yet been determined,

although several compounds with Similar activities have been synthesized (Cherksey

30

et al., 1989). Pending further characterization of Spider toxins, these compounds may
be useful tools in neurobiology. Finally, the endogenous peptide endothelin has been
demonstrated to bind to and depolarize dorsal horn neurons (Yoshizawa et al., 1989).
Endothelin is a vasoactive peptide which is presumed to increase function of L-type
Ca2+ channels in muscle cells. That it has actions in the spinal cord may indicate that
there are also endogenous regulators of Ca2+ channel activity in the nervous system
as well as the cardiovascular system.

Several therapeutic compounds also exert Ca2+ channel block as either a
predominant mechanism of therapeutic action, or at least one of several potential
mechanisms of therapeutic and/ or toxic action. Prominent among these compounds
are several agents used for treatment of cardiovascular disorders such as angina
pectoris, hypertension and arrythymias. These so called "Ca2+ channel blockers" also
bind readily to CNS preparations (Gould et al., 1985). Therapeutic Ca2+ channel
antagonists fall into three chemical classiﬁcations: the dihydropyridines (DI-1P) such
as nifedipine, the phenylalkylamines such as verapamil and the benzothiazopines,
such as diltiazem. DHPS with Ca2+ channel agonist properties have also been
synthesized. These are represented by (+ )-Bay K 8644 and (+ )-S-202-791. The
DHPS, and presumably the phenylalkylamines and benzothiazopines, interact
preferentially with btype Ca2+ channels as opposed to other Ca2+ channel types (Fox
et a1. , 1987a,b). Other therapeutic agents whose action is postulated to involve Ca2+
channel block include anticonvulsants such as phenytoin (Twombly et al., 1988), the
barbiturates (Blaustein and Ector, 1975; Werz and Mcdonald, 1985; Gross and

31
Mcdonald, 1988; Gunderson et al., 1988), as well as the benzodiazepines (Leslie,

1987) and certain general anesthetics such as halothane (Takenoshita and Steinbach,
1991). The neuroleptic chlorpromazine blocks Ca2+ inﬂux into synaptosomes (H085
and Forrnaniak, 1984) and Ca2+ currents in neuroblastoma cells (Ogata and
Narahashi, 1990). Finally, the aminoglycoside antibiotics may also interact with
neuronal Ca2+ channels and this interaction may contribute to the toxicity of these
therapeutic agents. In synaptosomes, neomycin and polymyxin block synaptosomal
Ca2+ channels in a manner which is reversed by increasing extracellular [Ca2+]
(Atchison et al., 1988) and aminoglycoside antibiotics compete with binding of
radiolabelled m-conotoxin GVIA (Knaus et al., 1987).

Low concentrations of ethanol (EtOH) decrease 4SCaz“ inﬂux into
synaptosomes (Friedman et al., 1980; Leslie et al., 1983; Skattebol and Rabin, 1987)
and block Ca2+ currents in neuroblastoma cells (Twombly et al., 1990). Chronic
exposure to low millimolar concentrations of EtOH results in an increase in
depolarization-induced 45Ca2" inﬂux into PC12 cells. This increase in Ca2+ inﬂux is
presumably channel mediated, as it is blocked by DHPS and inorganic divalent
cations (Greenberg et al., 1987a). In addition, chronic exposure to ethanol alters the

binding of Ca2+ channel ligands to PC12 cells (Marks et al., 1989).

CHAPTER TWO

SYNAPTOSOMES AND PHEOCHROMOCYTOMA CELLS

AS MODEL SYSTEMS FOR STUDY OF Ca2+ CHANNELS

32

33
A) SYNAPTOSOMES AND SYNAPTOSOMAL Ctr2+ CHANNELS

Because of the limitations imposed by the small size of most nerve terminals,
direct electrophysiological study of channel function is severely restricted.
Synaptosomal suspensions are preparations containing large numbers of pinched-off
nerve terminals which allow for biochemical study of channel function and
neurotransmitter release in vitro. Synaptosomes are derived by homogenization of
central nervous system tissue followed by differential centrifugation to separate
synaptosomal material from other CNS components. Synaptosomes are spherical
particles 1 - 2 pm in diameter and contain intracellular organelles including
mitochondria, endoplasmic reticulum and synaptic vesicles (Gray and Whittaker,
1962). Many metabolic pathways and synthetic pathways remain intact in
synaptosomes, including glycolysis, Kreb’s cycle and neurotransmitter synthesis
(Suszkiw and O’Leary, 1982; Eder-Colli et al., 1989). Synaptosomes when incubated
in glucose-containing medium remain viable for biochemical studies of metabolic
function, channel function and neruotransmitter release for several hours after
isolation (Bradford, 1975). The activity of Na"/K+ ATPase is very high in
synaptosomal preparations, allowing for maintenance of a membrane potential
between -30 and -80 mV (Blaustein and Goldring, 1975 ; Suszkiw et al., 1986).
Synaptosomes also contain voltage-sensitive Na‘, K+ and Ca2+ channels (Nelson et
al., 1983; Nelson, 1985; 1986) and release neurotransmitter in a Ca2*-dependent
manner following depolarization (Blaustein, 1975; Floor, 1983; Leslie, er al., 1985).

This preparation has been widely employed in studies of channel function (N achshen

34

and Blaustein, 1979; 1980; 1982), neurotransmitter synthesis and release, receptor
binding (see Chapter four), and regulation of metabolic processes (Blaustein et al.,
1978) at the nerve terminal. Results of experiments using this preparation have
contributed Signiﬁcantly to knowledge of processes that occur at this portion of the
neuron.

Depolarization-dependent Ca2+ inﬂux into synaptosomes occurs in two distinct
phases (Nachshen and Blaustein, 1980; Suszkiw and O’Leary, 1983; Nachshen, 1985):
a "fast" phase, which inactivates after 1-2 s of depolarization and a "Slow" phase which
remains active for 20-90 s after depolarization (Figure 2.1). The slow phase of inﬂux
may be mediated by a Ca2+ channel and a reversed N a+ / Ca2+ exchange mechanism;
the latter may predominate, as removal of Na+e largely reduces "slow" inﬂux
(Coutinho et al., 1984; Turner and Goldin, 1985; Suszkiw et al., 1986).

The fast phase of inﬂux, which is associated with neurotransmitter release
(Drapeau and Blaustein, 1983; Floor, 1983; Suszkiw and O’Leary, 1983; Leslie et al.,
1985), is presumably mediated by voltage-dependent Ca2+ channels and is not
dependent on Na“,. In many ways, 4sCa“ inﬂux into synaptosomes correlates with
Ca“ channel function in other systems. Depolarization of synaptosomes by K”
results in inﬂux of 45Ca“ in a manner which is related to the [10],. Preng that
[K+]e/ [10]i (Blaustein and Goldring, 1975) determines the membrane potential,
approximately 20-40 mV depolarization is required to initiate 45’Ca“ inﬂux
(Nachshen and Blaustein, 1980; Suszkiw er al., 1986; 1989; Adam-Vizi and Ligeti,

1986). Predepolarization of the synaptosomal suspension in the presence or absence

Ca“ 032+

 

Figure 2.1. Components of Ca2+ inﬂux into synaptosomes. Schematic diagram of a synaptosome
containing mitochondria (M; and synaptic vesicles (SV). Depolarization of the membrane (A EM) results
in fast, channel-mediated Ca + inﬂux (FAST) and slow inﬂux (SLOW). The slow component is mediated
by reversed Na” /Ca2+ exchange and slowly- or non-inactivating Ca "’ channels.

36
of Ca2+ eliminated the fast component (Nachshen and Blaustein, 1980). Ca2+

channels in synaptosomes also undergo both voltage- and Ca2+-dependent
inactivation (Suszkiw et al., 1986; 1989). In addition to Ca“, Sr2+ and Ba2+ are also
permeant through these Ca2+ channels; the relative inﬂux of Caz“:Sr2":Ba2+ for the
fast component of synaptosomal inﬂux is 6:3:2 (Nachshen and Blaustein, 1982).
Incorporation of synaptosomal Ca2+ channels into lipid bilayers indicated that the
single channel conductance was gBa2+ > gCa“ =gSr2+ (Nelson, 1986). This is similar
to cardiac Ca2+ channels, for which whole cell current indicated that the relative
premeability was Ca2+ > Sr2+ > Ba“, but single channel conductance measurements
indicated that gBa2+ >gCa2+ (Hess et al., 1986).

Like Ca2+ channels in other systems, fast 45Ca2+ inﬂux into synaptosomes is
sensitive to block by inorganic divalent and trivalent cations. The most potent
blockers of inﬂux are La“, Pb“ and Cd“, which have Ki values of less than 10 11M.
Other metals including Co“, Ni“, Hg2+ and Zn2+ also block the fast component of
infux, although higher concentrations (Ki = 10 - 100 nM) are required (Nachshen,
1985). The organic Ca2+ channel blocker verapamil also decreases 45Ca2+ inﬂux into
synaptosomes (N achshen, 1985). Single-channel recordings of synaptosomal Ca2+
channels in lipid bilayers also conﬁrm that La”, Cd2+ and verapamil are potent
blockers of synaptosomal Ca2+ channels (Nelson, 1985 ). Block of Ca2+ channels by
verapamil suggests the possibility that L-type channels may be involved in Ca2+
inﬂux. However, this is a subject of considerable controversy, as DHPS do not have

consistent effects on ”Ca2+ inﬂux in synaptosomal preparations (see below).

37

Fast inﬂux has been associated with release of Substance P (Floor, 1983),
acetylcholine (Suszkiw and O’Leary, 1983) and dopamine (Drapeau and Blaustein,
1983, Leslie et al., 1985) from synaptosomes. Thus, the fast phase couples Ca2+ entry
and subsequent neurotransmitter release from nerve terminals.

The slow component of ”Ca2+ inﬂux into synaptosomes differs in many
respects from the fast component. It does not inactivate for up to 90 5 during
depolarization and is much less sensitive to La3+ than the fast phase (N achshen and
Blaustein, 1980). Furthermore, the relative ﬂux of Caz“:Sr2“:Ba2+ for the slow
component is 6:3:1 (Nachshen and Blaustein, 1982), indicating that this component
has different properties of selectivity than the fast component. Finally, this
component is extremely sensitive to [N a"]e (Blaustein and Oborn, 1975). Inasmuch
as preparation and incubation of synaptosomes in Na”-free solutions drastically
reduces inﬂux via this component, it has been proposed that a reversed Na+/Ca2+
exchange contributes signiﬁcantly to this phase of inﬂux (Blaustein and Oborn, 1975;
Akerman and N icholls, 1981; Coutinho et al., 1984; Turner and Goldin, 1985; Suszkiw
et al., 1986). This does not preclude involvement of Ca2+ channels in this component,
as small ﬂuxes are measured in the absence of Na+ (Suszkiw et al., 1986; Shafer and
Atchison, 1989) and DHPS have been reported to decrease this component of inﬂux
in Na+-containing solutions (Martinez-Serrano et al., 1989).

Initial experiments in this dissertation examine effects of MeHg on
synaptosomal “Ca2+ inﬂux Thus, information regarding Ca2+ channel types in this

system is important. Synaptosomes are likely to contain more than one type of Ca2+

38

channel, but the types of Ca2+ channel present have not been identiﬁed. DHP
antagonists bind to synaptosomal preparations with high afﬁnity (Rampe, 1984,
Turner and Goldin, 1984; Suszkiw et al., 1986; Dooley et al., 1987b; Massieu and
Tapia, 1988). However, synaptosomal ”Ca2+ inﬂux and / or neurotransmitter release
have been variously reported to be sensitive (Turner and Goldin, 1985 ; Dunn, 1988;
Martinez-Serrano, 1989) or insensitive (Nachshen and Blaustein, 1979; Daniel] et al.,
1983; Reynolds et al., 1986; Rivier et al., 1987; Suszkiw et al., 1986; 1989; Massieu
and Tapia, 1988) to block by DHP antagonists. Additionally, in mammalian
synaptosomes, 4SCa“ inﬂux is only partially sensitive to block by to -conotoxin GVIA
(Suszkiw et al., 1989). Furthermore, the activation, inactivation (Suszkiw et al., 1989)
and pharmacological sensitivity (Suszkiw et al., 1989; Lundy et al., 1991) of 45Ca2+
inﬂux into synaptosomes suggest that these preparations contain a high voltage
activatid channel which is neither N- nor L-type. In some respects, Ca2+ inﬂux into
synaptosomes has characteristics similar to current mediated by P-channels. However,
further characterization is required to verify the type or types of Ca2+ channels

present in synaptosomal preparations.

B) Ca” CHANNEL FUNCTION IN PC12 CELLS

PC12 cells contain many voltage-sensitive channel types important to neuronal
function. These include tetrodotoxin- (TT'X) sensitive and -insensitive N a” channels
(Rudy ct al., 1987), voltage-sensitive K” channels (Pun and Behbehani, 1989), and
Ca2+ channels. PC12 cells have been utilized extensively to study the functions,

39

expression and regulation of Ca2+ channels. Furthermore, this cell line has been
employed for numerous studies of neuronal function. AS such, many biochemical and
phsyiological processes of this cell line are well understood. Neurotransmitter release
and receptor function in PC12 cells are reviewed in the Appendix.

By contrast to synaptosomes, the type of Ca2+ channels present in PC12 cells
are much better deﬁned. It is generally agreed that NGF-differentiated PC12 cells
contain both DHP-sensitive L- (Figure 2.2, top panel) and to -conotoxin GVIA-
sensitive N- (Figure 2.2, bottom panel) type Ca2+ channels (Plummer et al., 1989;
Janigro et al., 1989; Usowicz et al., 1990; Shafer and Atchison, 1991). The presence
of T-type Ca2+ channels in differentiated PC12 cells also has been reported (Garber
et al., 1989), although pharmacological characterization is required to conﬁrm the
presence of T-type channels in PC12 cells.

I The existence of DHP-sensitive (L-type) channels in both undifferentiated and
NGF-differentiated cells was established by both ion ﬂux assays (Stallcup, 1979; Toll,
1982; Takahashi and Ogura, 1983; Takahashi et al., 1985) and electrophysiological
studies (Streit and Lux, 1987; Plummer et al., 1989; Janigro er al., 1989; Usowicz gt
a], 1990; Shafer and Atchison, 1991). The presence of DHP-insensitive (N-type)
channels in PC12 cells has been more difﬁcult to establish. Release of 3I-I-NE from
PC12 cells grown in NGF was less sensitive to DHPS than was release from
undifferentiated PC12 cells (Takahashi er al., 1985; Kongsamut and Miller, 1986),
although 4"’Caz“ inﬂux remained largely DHP-sensitive (Kongsamut and Miller, 1986).

Depolarization of undifferentiated PC12 cells by K" resulted in Ca2+-dependent

4o

 

 

 

 

Figure 2.2. N and L type Ca2+ channels in PC12 cells. DHP—sensitive and ~insensitive single Ca2+
channel current in differentiated PC12 cells recorded using the cell-attached patch voltage-clamp
technique. (a) current records from a patch containing one 'L-type” and one "N-type" channel. Four
consecutive sweeps are shown for each set of depolarizations from two diﬁerent holding potentials, as
indicated in the voltage protocol above the sweeps. (b) Current traces from a patch containing a single
"N-type' channel In each case, the DHP agonist (+)-(S)-202-791 is present at 1 uM, and Ba2+ (110
mM) is the charge carrier.

From Plummet er at. M 2:1453-1463 (1989).

 

41

release of transmitter which is sensitive to DHP Ca2+ channel agonists and
antagonists (Figure 2.3). Differentiation with NGF resulted in decreased sensitivity
of neurotransmitter release to DHP drugs (Figure 2.3B,C). Indeed, in most 45Ca2+
ﬂux studies in PC12 cells, no DHP-insensitive components were reported with the
exception of a small (< 10%) component in one study (Shafer et al., 1990). Although
this latter study and studies of neurotransmitter release indicated expression of DHP-
insensitive or N-type Ca2+ channels in differentiated PC12 cells, they did not provide
conclusive evidence that N-type channels were indeed being expressed. By contrast
to radiolabel ﬂux experiments, expression of DHP-insensitive or N -type channels has
been demonstrated in NGF-differentiated PC 12 cells using both whole-cell (Plummer
et al., 1989; Usowicz _e_t al, 1990; Shafer and Atchison, 1991) and cell-attached
(Plummer et al., 1989) patch voltage-clamp techniques. In undifferentiated PC12
cells, small N -type current components have been reported (Plummer er al., 1989;
Janigro et al., 1989).

In addition to ﬂux and electrophysiological measurements on Ca“ channel
function, numerous studies of Ca2+ channel ligand binding have been performed to
identify and characterize pharmacologically the Ca2+ channel subtypes expressed in
PC12 cells. The most commonly used ligands are the DHP (+)-PN 200-110 and the
peptide to -conotoxin GVIA. Differentiation of PC 12 cells increases the number of
conotoxin binding sites by 2-3 fold and increases the number of DHP binding Sites
by less than 2 fold (Usowicz 51 a1, 1990). In PC12 cells, conotoxin is speciﬁc for N-

type channels, as there is no interaction between DHP binding and conotoxin binding

42

>

 

 

 

 

 

 

 

“g 20' 1
if?) 15'
< 5
‘5' 5" 10' i" 1'
: \E
g g :5”, “‘1.
I'- 5 ’ J’ \‘E.
I.
O 5 10 15 20 25
g TIME (min)
250[ B ’3 120[ C -e- -NGF
xi" \
I, \\
I ' \
_. zoo. / \\ 5‘ 80.
O r \ I
E “ "
‘1 a
O 150. o .
100 e g ~ ~4
0 0.010.1 1 10
BAY K8644 (III) NITRENDIPINE (I‘M)

Figure 2.3. Nore inephrine release from P012 cells. Release of [3H]-norepincphrine from PC12 cells.
A.) Release of [ -norepinephrine from PC12 cells expressed as a percentage of tissue stores present
at the beginning of the particular fraction. The ﬁrst two 5 min. fractions were release in non-depolarizing
K" solutions, with or without incubation of drugs. The third fraction was release induced by
depolarization with 70 mM K+ solution. The ﬁnal two fractions are release in non-depolarizing
solutions. (diamond) 70 mM K+ alone, (square) 70 mM K+ plus 10" M Bay K 8644, (circle) 70 mM
K+ plus 1045 M nitrendipine, (triangle) 70 mM K+ Ca2+-tree plus 02 mM EGTA. B. and C.) Dose-
response relationship for eﬂ’ects of Bay K 8644 (B) and nitrendipine (C) on release of NE from N GF-
differentiated (open circle) and undiﬁerentiated (solid circle) PC12 cells. Results are expressed as a
percentage of control-stimulated release in depolarizing solutions. Values are the meanzi: SEM of 6-12

quJeriments.
Modiﬁed from Kongsamut and Miller, Proc. Natl. Acad. Sci. (U.S.A.) 83:2243-2247 (1986).

43

(Usowicz et al., 1990), and conotoxin blocks only the inactivating component of N-
type current (Plummer er al., 1989). In an NGF-insensitive PC12 mutant line, no
changes in Ba2+ current or conotoxin binding were observed in response to NGF
(Usowicz et aL, 1990). In addition, high concentrations of ouabain (0.1 mM), which
did induce morphological differentiation of the mutant line, did not induce changes
in Ca2+ channel expression (Usowicz et al., 1990). These results suggest that NGF-
differentiation induces increased expression of N-type Ca2+ channels in PC12 cells,
and that morphological differentiation and changes in Ca2+ channel expression are
not necessarily co-requisite events.

As indicated by binding studies, the proportion of N- to L- type channels
changes with differentiation by N GF. PC12 cells grown in absence of N GF express
predominantly L-type Ca2+ channels, whereas those treated with N GF exhibit a
substantial increase in the expression of N-type Ca2+ channels without a concomitant
decrease in expression of L-type channels (Streit and Lux, 1987; Plummer et al., 1989;
Usowicz 31 a1, 1990). Ca2+ channels are regionally distributed in differentiating PC12
cells; growth cones contain a higher density of Ca2+ current than neurites and Ca2+
current Shows more inactivation in grth cones than in the soma (Streit and Lux,
1989). Streit and Lux conclude that the increase in the inactivating component of
current (which resembles currents mediated by N -type channels) with N GF treatment
may result from changes in the distribution, but not types (eg. N and L) of Ca2+

channels in PC12 cells (Streit and Lux, 1990). However, this conclusion is based on

44

whole cell recordings and does not consider pharmacological sensitivity to conotoxin
of Ca2+ currents in PC12 cells nor single channel current kinetics.

The apparent differential expression of Ca2+ channel subtypes in PC12 cells
is important for two reasons. First, N— and L-type Ca2+ channels have been suggested
to modulate or possibly even to mediate Ca2+ entry associated with neurotransmitter
release from several neuronal preparations (Atchison and O’Leary 1987; Rane et al.,
1987; Holz et al., 1988; Atchison, 1989; Lemos and Nowycky, 1989). Second, one can
control to some extent, by use or omission of NGF, the proportion and distribution
of N- vs L-type channels in PC12 cell cultures. This change induced by NGF may
help to deﬁne whether N- and L-type channels are indeed separate entities or
whether the biophysical and pharmacological differences arise from different
"functional states” of one channel type.

There is some evidence that L-type Ca2+ channels in PC12 cells are regulated
by second messengers, including protein kinase C (PKC) and G-proteins. Activators
of PKC reduce DHP-sensitive ”Caz" inﬂux into PC12 cells (Harris et al., 1986; Di
Virgilio et al., 1986), while inhibitors of PKC have little effect on inﬂux (Greenberg
er al. , 1987b). Calmidazolium and triﬂuoroperazine, inhibitors of calmodulin,
decrease inﬂux of 45Ca“ into PC12 cells, indicating that calmodulin or calmodulin-
dependent enzymes may also regulate Ca2+ channels (Greenberg et al., 1987b). G-
proteins may also be involved in Ca2+ channel regulation. Stable GTP analogs
increase the displacement PN 200-1 10 binding by the DHP agonist Bay K 8644.

Pertussis toxin inhibits this action of Bay K 8644, as well as Bay K 8644-induced

45

potentiations of depolarization-dependent 4SCaz“ inﬂux into PC 12 cells (Bergamaschi
et aL, 1990). Chronic K*-induced depolarization or treatment with ionomycin, a Ca2+
ionophore, reversibly decreased depolarization-induced 45Ca2“ inﬂux and the number
of [3H]-nitrendipine (a DHP antagonist) binding Sites (DeLorme et al., 1988).
Elevations in [Ca2+], may be involved in Ca2+ channel down regulation, although
[Caz‘i]i returned to normal levels while Ca2+ channel function remained depressed
(DeLorme and M‘Gee, 1988). One second messenger which does not appear to affect
Ca2+ channel function in PC12 cells is cAMP (Rabe et al., 1982; Nishizawa et al.,
1990). Further characterization of the regulation of Ca2+ channels by second
messengers is needed to understand more fully the role of Ca2+ channels in
neurotransmitter release and other cellular functions in PC12 cells.

In general, PC12 cells have not been used extensively as a tool in
neurotoxicological studies, although the interactions of several neurotoxic agents with
Ca2+ channels have been examined. PC12 cells have been used to characterize
interactions of the neurotoxicant methylmercury (MeHg) with Ca2+ channels. MeHg
blocks 4"Ca2+ inﬂux into undifferentiated and N GF-differentiated PC 12 cells. In
addition, MeHg alters the binding of conotoxin to intact PC12 cells (Shafer et al.,
1990). In more recent studies, acute effects of MeHg on current carried by Ba2+ (13,)
in NGF-differentiated PC12 cells have been examined. MeHg blocks both N- and L-
type Ca2+ current in PC12 cells in a manner which is voltage- but not State-
dependent (see chapter 5). The effects of MeHg on Ca2+ channels are not reversed

by washing with MeHg-free solutions, but are antagonized by increasing extracellular

I

Hi
ml

ll...

I' :-
"‘\
,r
the

46
[Bah] (Shafer and Atchison, 1991). These most recent results suggest that the effects

of MeHg on 13, in PC12 cells mirror the actions of MeHg on synaptosomal 4sCa“
inﬂux (Atchison et al., 1986; Shafer and Atchison, 1989; Shafer et al., 1990). EtOH
also alters Ca2+ channel function in PC12 cells. Chronic exposure to low millimolar
concentrations of EtOH results in an increase in depolarization-induced 45Ca2+ inﬂux
into PC12 cells. This increase in Ca2+ inﬂux is presumably channel mediated, as it
is blocked by DHPS and inorganic divalent cations (Greenberg et al., 1987a).
Furthermore, chronic exposure to EtOH increased the number of (+ )-PN 200-110
binding sites in PC12 cell membranes (Figure 2.4). This action was not reduced by
co-treatment with Bay K 8644, indicating that increased channel number was not the
result of decreased Ca“ influx into PC12 cells (Marks et al., 1989). Further study of
the effects of EtOH on Ca2+ channel numbers in PC12 cells may provide insight into
the mechanisms of regulation of expression of Ca2+ channels in PC12 cells. Chronic
exposure (3 days) to immunoglobulin G isolated from serum of patients with
Lambert-Eaton Myasthenic Syndrome has also been demonstrated to decrease 45Ca2“
inﬂux into PC12 cells (Lang, et al., 1989). The effects of volatile anesthetics (Kress
er al., 1987), maitotordn (Takahashi et al., 1982; 1983; Choi et al., 1990) and a-
latrotoxin (Vicentini and Meldolesi, 1984; Meldolesi et al., 1986) on Ca2+ channel
fUnction have also been examined using PC12 cells. Thus, PC12 cells are clearly
useful models for the study of interactions between neurotoxic compounds and Ca2+

channels.

47

.p

 

(A

N

-A

 

 

PN 200-110 bound (truer/10‘ cells)

0
O

 

l L I

 

l
100 200 300 400 500
PM zoo-110 (pM)

Figure 2.4. Effects of EtOH on Ca2+ channel density in PC12 cells. Saturable binding of the DHP
Antagonist (+)-[3H]PN 200-110 to intact PC12 cells grown for 6 days in the absence (open circles) or
presence (closed circles) of 200 mM ethanol. Data shown are the average of triplicate values from a
single representative experiment.

From: Marks er at. W 53:168-172 (1989).

CHAPTER THREE

BLOCK OF 45C3“ INFLUX INTO SYNAPTOSOMES BY NIETHYLMERCURY:

Ca2+ AND Na+ DEPENDENCE

48

49
ABSTRACT

Block of Ca2+ inﬂux into isolated nerve terminals by the neurotoxicant
methylmercury (MeHg) was studied for its dependence on extracellular Ca2+ and
Na‘. Depolarization-independent entry of ”Ca“ was determined in rat forebrain
synaptosomes incubated in 5 mM K+ solution. 4SCaz” inﬂux was Similarily measured
following 1 ("fast" phase) or 10 5 ("total") of elevated K+ - (41.25 mM) induced
depolarization, or during 10 S of elevated K+-induced depolarization after
synaptosomes had been predepolarized for 10 s in Ca2+- and MeHg-free solutions
("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 11M and greater
Signiﬁcantly reduced synaptosomal 45Ca2+ accumulation measured during 1 or 10 S
of incubation. In K*-depolarized synaptosomes, the estimated [CS0 for block of
"total", "fast" and "slow" 45Ca2+ inﬂux was 75 nM: 250 uM MeHg reduced inﬂux by
approximately 90%. The ability of [Ca2+],: to antagonize block was tested by
increasing [Ca2+]e from 0.01 to 1.15 mM in the presence and absence of MeHg.
When compared to control, 50 11M MeHg reduced "total" inﬂux of 45Ca""' by 270%
and reduced "fast" inﬂux by 20-60% at all [Ca2+], tested. At [Ca2+], concentrations
of 0.01-0. 15 mM, MeHg (50 nM) reduced "slow" inﬂux by 75-90%, but did not affect
Slow inﬂux at higher [Ca2+]e (2 0.30 mM). When the dependence of block of 45Ca2+
inﬂux on [Na‘i]c was tested, equivalent levels of inhibition were caused by MeHg (25
nM) for fast inﬂux by synaptosomes in Na+-containing and NaI-free solutions.
Conversely, 25 11M MeHg reduced slow inﬂux by 26.1 14.5 and 96.01-4.0% in Nai-

containing and N a“-free solutions, respectively. In Na*-free solutions, block of the

50

slow phase of 45Ca2+ inﬂux was not overcome by increasing [Ca2+]c. Results of this
study indicate that 1) MeHg affects Ca2+ inﬂux into synaptosomes in the absence of
depolarization; 2) block of the fast phase of 45Ca2+ inﬂux by MeHg is only weakly
antagonized by Ca”; and 3) block of fast 45Ca2“ inﬂux was independent of [Na+]e;
4) block and reversal of block of the slow phase of 45Ca2+ inﬂux by MeHg are highly

Na+-dependent.

5 1
INTRODUCTION

The hypothesis states that effects of MeHg on voltage-sensitive Ca2+ channels
which are involved in neurotransmitter release at the motor nerve terminal may
contribute to disruption of neurotransmitter release produced by MeHg. This would
be best tested by simultaneously measuring Ca2+ entry and neurotransmitter release
in intact motor nerve terminals. However, several limitations discussed in Chapter
One preclude direct measurement of function of Ca2+ channels in intact motor nerve
terminals. Although Ca2+ channel function in intact nerve terminals is difﬁcult to
examine using conventional electrophysiological techniques, biochemical
measurement of Ca2+ channel function can be accomplished easily by measuring
45C2?“ ﬂux into isolated nerve terminals, or synaptosomes. Thus, experiments to
examine the ability and nature of MeHg interaction with nerve terminal Ca2+
channels were performed using synaptosomal preparations.

Heavy metals are known to disrupt Ca2+ inﬂux into nerve terminals leading to
decreased neurotransmitter release. Divalent heavy metal cations such as Co“, Cu“,
Zn“, Cd“, Pb2+ and Hg2+ block the inﬂux of “Ca2+ into K+-depolarized
synaptosomes in a reversible manner (N achshen, 1984; Suszkiw et al., 1985; Atchison
et al., 1986). Micromolar concentrations of monovalent MeHg also block 45Ca2+
inﬂux into Ki-depolarized synaptosomes (Atchison et al., 1986). In contrast to
inorganic divalent cations, MeHg blocked 45Ca2" inﬂux into synaptosomes during 10
s of depolarization in a manner which was not completely antagonized by increasing

[C324]: (Atchison er al., 1986). However, the ability of increasing [Ca2+], to

52

antagonize block by MeHg of fast and slow components of 45Ca“ inﬂux was not
examined. Studies at the neuromuscular junction demonstrated that after increasing
stimulus duration and/ or intensity, the subsequent block of nerve-evoked endplate
potentials (EPPS) by MeHg could be reversed by increasing [Caz‘i]e (Traxinger and
Atchison, 1987b). Since increasing [Ca2"]c can, under the conditions described above,
reverse the effects of MeHg at the neuromuscular junction, and antagonize block of
synaptosomal 45Ca2” inﬂux during 10 S of K"-induced depolarization (total inﬂux),
experiments were designed to examine the ability of increasing [Ca2+], to antagonize
block by MeHg of the fast and slow phases of Ca2+ inﬂux into synaptosomes.

When examining block by MeHg of the fast and slow phases of 45Ca2+ inﬂux
into synaptosomes, consideration must be given to the mechanisms mediating Ca2+
inﬂux during each of the two phases. As mentioned previously, the fast phase is
mediated by voltagesdependent Ca2+ channels, while the slow phase is mediated
largely by a reversed Na+ /Ca2+ exchange mechanism. Because the two phases differ
in their dependence on [Na*]c, it is possible that the block of ”Ca2+ inﬂux by MeHg
during the fast and slow phases may also exhibit a differential dependence on [Na+]¢.
Thus, experiments were designed to test the dependence of block of each phase by
MeHg on [Na+]e.

MeHg is also capable of forming mercaptides with sulfhydryl groups (Klaassen,
1985) and reacting with amino, carbonyl and hydroxyl groups (Bidstrup, 1964), all of
which can be constituents of membranes or membrane proteins. Because of its

reactivity, MeHg can affect a variety of membrane-associated functions. MeHg alters

53

the resting membrane potential and blocks the action potential in the squid giant
axon (Huneeus-Cox er al., 1966), and increases its threshold for excitation (Shrivastav
et al., 1976). In addition, MeHg alters Na+- and Ki-channel function (Quandt et al.,
1982; Shrager, 1977) and permeability (Passow and Rothstein, 1960). At rest,
permeability of the membrane to Ca2+ may be mediated in part by random openings
of Ca“ channels, active transport proteins or non-speciﬁc permeability. Therefore,
effects of MeHg on depolarization-independent entry of 45Ca2" into synaptosomes
were examined as a measure of the speciﬁcity of MeHg for Ca2+ channel-mediated

transport.

54
MATERIALS AND METHODS

Materials. Methylmercuric acetate was obtained from Pfaltz and Bauer, Inc.
(Stamford, CT) and was dissolved in 4% (v/v) glacial acetic acid to make a 2 mM
stock solution. Aliquots of this solution were diluted in the appropriate buffers and
adjusted to pH 7.4 at room temperature (25 °C) to yield the ﬁnal concentrations of
MeHg indicated in the text of chapters 3 and 4. N-methyl—glucamine, ethyleneglycol-
bis-(B-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), and choline chloride
were obtained from Sigma Chemical Co. (St. Louis, MO). N-2-
Hydroxyethylpiperazine—N’-2-ethanesulfonic acid (HEPES) was purchased from
United States Biochemical Corporation (Cleveland, OH). 4S’CaClz, 133BaCl2 and
858er (speciﬁc activities 15-47, 11 and 5.8 mCi/mg, respectively) were purchased
from New England Nuclear Co. (Boston, MA). For incubation of synaptosomes,
normal physiological saline contained (mM): NaCl, 145;KC1, 5; MgClz, 1; d-glucose,
10; and HEPES, 10. For 45Ca“ inﬂux experiments, elevated Ki solution contained
(mM): NaCl, 72.5; KCl, 77.5; MgClz, 1; CaClz, 0.04; d-glucose, 10; and HEPES, 10.
Normal K+ (5 mM) solution contained the same constituents and amounts as did
elevated K+ solution except that [K*] was lowered to 5 mM by replacement of KCl
with 72.5 mM choline chloride. Ca2+-free solutions contained no added CaClz, but
may have contained residual amounts of Ca2+ (1-2 I: M). In Na*-free experiments,
N aCl was replaced in all solutions by choline chloride on an equimolar basis.
Quenching solution contained (mM): KCl, 5; MgClz, 2; EGTA, 1; d-glucose, 10;

HEPES, 10; and N-methylglucamine, 145. N -methylglucamine, which replaces Na+

55

in the buffer solution, does not permeate the voltage-dependent N a“ channel (I-Iille,
1971). All solutions were adjusted to pH 7.4 at room temperature (25 °C).

Synaptosomal preparation. Synaptosomes were prepared from forebrains of
male Sprague-Dawley rats (Harlan, 175-200 gm) using a modiﬁcation of the method
of Gray and Whittaker, (1962) as described by Atchison et al. (1986, 1988).
Forebrains were homogenized in 0.32 M sucrose and centrifuged for 10 min at
1000xg. The pellet was discarded and the supernatant was centrifuged at 17,500xg for
20 min. This pellet was resuspended in fresh 0.32 M sucrose and layered onto a
discontinuous sucrose gradient of 0.8 and 1.2 M sucrose. After centrifuging for 2 h
at 69,000xg, the synaptosome-enriched fraction was removed from the interface
between the 0.8 and 1.2 M sucrose layers and diluted slowly 1:1 (v/v) with
physiological saline. This step facilitates return of the synaptosomal preparation to
physiological osmolarity. The synaptosomes were repelleted by centrifugation at
10,000xg for 10 min and then resuspended to an approximate concentration of 20—40
mg/ml in physiological saline (Lowry et al., 1951). This step returns the synpatosomal
preparation to physiological osmolarity and removes sucrose from the solution. As
the synaptosomal preparation was performed at 0-4 °C, synaptosomal suspensions
were incubated in a 37 °C water bath for 20 min prior to their use.

45Ca2+ inﬂux measurements. 4SCaz“ inﬂux was measured as described by
Nachshen and Blaustein (1980; 1982) by adding 501:1 of synaptosomal suspension
(approximately 20 mg protein/ ml) to 50 pl of either elevated (77.5 mM) or normal

(5 mM) [K‘] solution containing 0.06 mM 45Ca2+ and MeHg, when appropriate.

56

Actual volume of synaptosomes in the suspension is presumed to be negligable, as
protein concentrations are low. Therefore, the ﬁnal K+ concentration of the
depolarizing solution was 41.25 mM. After 1 s (fast phase) or 10 s (total inﬂux) of
incubation, 2 ml of ice-cold quench solution was added to stop 45Ca2+ inﬂux. A
metronome was used to facilitate measurements at 1 s. Predepolarized (slow phase)
inﬂux was measured by adding 25 pl of synaptosomal suspension (approximately 40
mg protein/ml) to 25 ul of Ca2+- and MeHg-free elevated [K*] solution for 10 s,
followed by the addition of 50 ul of elevated [K+] solution containing 0.06 mM of
“Ca2+ and MeHg (when appropriate). After an additional 10 s, the reaction was
quenched as above. A stopwatch was used to measure 10 5 intervals. The ﬁnal
extracellular concentration of Ca2+ plus 45Ca2“ ([Ca2*]c) was always 50 MM, unless
Otherwise noted. In experiments in which [Ca2+], was increased, the molar ratio of
4“(’Caz‘VCaz“ was maintained at a constant value.

Immediately following addition of quenching solution, samples were ﬁltered
through Millipore ﬁlters (0.45 pm) and washed with two 5 ml aliquots of ice-cold
quenching solution. Filters were placed into scintillation vials containing 1.5 ml of
Triton X-100/ HCl solubilizer and 10 ml of scintillation cocktail was added after 10
min. Radiotracer content was determined in a Searle Mark III scintillation counter
with a 70% efﬁciency for “Ca“, or in a Searle model 1197 gamma counter with
efﬁciencies of 47 and 53% for 85Sr2+ and 133Ba“, respectively. Depolarization-
induced “Ca“ inﬂux was determined by subtracting the average of three replicate

values obtained in 5 mM K“ solution from the average of three replicate values

57

obtained in 41.25 mM K” solutions and converting the net value to nmol ”Ca2+ /ug
of synaptosomal protein (Lowry et al., 1951).

Statistical analysis. All dose-response data were analyzed using a randomized
block analysis of variance (ANOVA) and Dunnett’s one-tailed t-test (Steele and
Torrie, 1980). Data from experiments in which [Ca2+], was increased were analyzed
using a split-plot ANOVA and, if signiﬁcant differences were found, comparisons
were made using the least signiﬁcant difference test. Comparisons between data
obtained in NaI-containing and Nai-free solutions were made using a paired

Student’s t-test. For all comparisons, statistical signiﬁcance was deﬁned by ps0.05.

58
RESULTS

Effects of MeHg on depolarization-independent entry of ”Ca2+ into
synaptosomes. Because of the ability of MeHg to alter a variety of membrane-
associated functions, its ability to disrupt those mechanisms which help to regulate
cytosolic Ca2+ in synaptosomes in the absence of depolarization was investigated.
Synaptosomes were incubated in normal (5 mM) K+ solutions in the presence of
MeHg and 45Ca“ for 1 or 10 s, and then assessed for 45Ca“ content. Compared to
untreated controls, MeHg had no signiﬁcant effect on depolarization-independent
4sCa2+ entry into synaptosomes at concentrations up to 50 I: M, but signiﬁcantly
reduced 45Ca2+ entry at concentrations of 125 and 250 p. M (Figure 3.1). Thus, during
exposures of 1 and 10 s, 45Ca2+ entry into synaptosomes is suppressed, but only by
high concentrations of MeHg. As noted in the Methods section, this component of
inﬂux is subtracted from depolarization-induced inﬂux. Thus, this effect did not
contribute to block of depolarization-induced inﬂux by MeHg.

Effects of MeHg on depolarization-induced 6Ca2+ inﬂux into synaptosomes.
Depolarization of synaptosomes with elevated [K*] solutions resulted in two
components of inﬂux (Figure 3.2). A rapid component which inactivated after
approximately 2 s and a slower component which saturated after approximately 20

3 Furthermore, inﬂux was linearly related to the concentration of protein in the
sYIlaptosomal preparation (Figure 3.3).

The effects of increasing concentrations of MeHg on total 45Ca2“ inﬂux into

sYnaptosomes are shown in Figure 3.4A. In contrast to the effects of MeHg described

 

 

 

:9” SiA
g
x D
.e i
a x
e 3- 1*
CL
0’
g ..
O
E
5 I 1 - e
B
m sol
§
E
*-
8 20-
9 . at-
'O 5 ‘5'0 ‘560

[MeHg] (1.1M)

Figure 3.1. MeHg blocks resting 4L5Ca2+ entry into synaptosomes. Eﬁects of MeHg on depolarization—
independent 45Ca2+ inﬂux into synaptosomes during 1 s (A) or 10 s (B). Synaptosomes were incubated
in 5 mM K" solutions for 1 or 10 s in the presence of 5Ca2+ and various concentrations of MeHg.
Values are the mean 1- SEM of three or ﬁve separate experiments, respectively. Data points not
connected indicate MeHg-free control values and the asterisks (’) indicate values which are signiﬁcantly
less than control (150.05).

INFLUX

20

“’Ca

312%:
it In,
lift] 1
“Win

6O

 

 

'o 70 .
>5 1; ./.\"O
.1
u. -' ,.
z 2
+- o. 35 ‘ ./
°‘ or
.8 g. 0’./
g 0,; -
t:
V O 5 10 30 45 60

TIME (sec)

Figure 3.2. Inﬂux of “Ca“ into synaptosomes vs duration of depolarization. 45Ca2“ inﬂux into
synaptosomes induced by depolarization in elevated [K+] solutions as a function of time. Two phases
are illustrated: a fast component which lasts approximately 2 s, and a slower component which lasts up
to 60 s. The values shown are the mean i SEM of three experiments, where the values for any one

experiment are the average of three replicates.

61

1500

1000

500 '

 

/ .

0 100 200 300

 

0

“Ca“lNFLUX (0pm x 10’)

pg PROTEIN/5 O uL

Figure 3.3. Inﬂux of ”Ca2+ into synaptosomes as a function of protein concentration. Values from a
single representative experiment comparing 45Ca2+ inﬂux with protein content of the synaptosomal
suspension. The line is the linear least-squares regression of inﬂux on protein content. Values for inﬂux
are the average of three replicates.

62

for depolarization-independent inﬂux of Ca2+, depolarization-induced inﬂux was
more prominently suppressed by MeHg. At concentrations above 5 [J M, MeHg
caused a concentration-dependent suppression of 45Ca2+ inﬂux which reached
approximately 100% inhibition with 250 11M MeHg. Concentrations of MeHg 2 25
11M signiﬁcantly depressed 45Ca“ inﬂux when compared to untreated controls. An
estimated 1C5o value for MeHg effects on total inﬂux is 75 BM. These results are
similar to those of Atchison et a1. (1986).

When effects of MeHg on the two distinct phases of depolarization-induced
45Ca2“ inﬂux were examined, tracer inﬂux was again reduced in a concentration-
dependent manner by MeHg. Increasing concentrations of MeHg caused a dose-
dependent suppression of 45Ca2+ inﬂux during the fast phase (Figure 3.4B) which
reached a maximal effect of approximately 90% with 250 [1. M MeHg. Nearly identical
effects of MeHg were observed on the slow phase of ”Ca2+ inﬂux (Figure 3.4C). The
estimated ICso value for MeHg is also approximately 75 u M for both the fast and
slow phases of 45Ca”’ inﬂux.

Antagonism or block of ”Ca2+ inﬂux in response to MeHg by increasing
[Ca2+],. The effect of increasing [Ca2+], from 0.01 to 1.15 mM on block of total
45Ca2“ inﬂux by MeHg is shown in Figure 3.5A. This concentration of MeHg
approximated the estimated 1C,0 value. Block of 45Ca2+ inﬂux by 50 u M MeHg was
decreased from approximately. 98% to 75% by increasing [Can]e from 0.01 to 1.15
mM. However, at each [Ca2+], tested, there was signiﬁcantly less inﬂux of ”Ca“ in

the presence of 50 11M MeHg than for the corresponding untreated control.

63

50'

 

 

 

45C0 UPTAKE (7: Control)

50'

 

 

5 50 500

MW

Figure 3. 4. Meng blocks depolarization-induced 6Ca2+ inﬂux into synaptosomes. Eﬁects of MeHg
on inﬂux of4 ”Ca into rat forebrain synaptosomes during 10 (TOTAL, A) and 1 (FAST, B) s of
depolarization with 41.25 mM KCl, or during 10 s of depolarization which was preceded by a 10 s
predepolarization in the absence of Ca2+ and MeHg to inactivate the fast phase (SLOW, C).
Synaptosomes In physiological saline solution (5 mM K+) were added to a reaction mixture which
contained 77.5 mM KCl, 40 ll M CaCl,, 0 .06 mM 45CaCl,, and MeHg. Final concentrations of MeHg
ranged from 2.5 to 2.50 nM. Inﬂux In 5 mM K+ solutions was subtracted from that m 41.25 mM K+
as depolarization-independent background. All values are the mean 1: SEM of four to six diﬁerent
experiments. The values for any individual experiment are the average of three replicates. When SE bars
are not shown, the SE is smaller than the size of the symbol. The asterisks (‘) indicate results that are

signiﬁcantly less than control values (1:50.05).

 

     
 

r“ '
.0.
K
5 L5
0
'2'
a.
01.0
E"
'2
50.5

45m: UPTAKE

 

mm

Figure 3.5. Block of Ca2+ channels by MeHg is not reversed by increasing [Ca2+]e. Dependence on
[Ca2+]e of 45Ca2+ inﬂux into synaptosomes in the absence (solid circles) and presence (open circles)
of 50 p. M MeHg during A) 10 s, or B) 1 s of K+-induced depolarization, or C) 10 s of depolarization
following a 10 s predepolarization in the absence of [Ca2+]e. 5Ca2+ inﬂux during TOTAL, FAST and
SLOW phases was measured as described in Figure 3.4. [Ca2+]e ranged from 0.01 to 1.2 mM, and the
mole to mole ratio of 45Ca2+ to Ca2+ was held constant at 0.22. Values are the mean: SEM of ﬁve to
seven different experiments. The values for any individual experiment are the average of three replicates.
When SE bars are not shown, the SE is smaller than the size of the symbol. The asterisks (‘) indicate
results that are signiﬁcantly less than control values (50.05).

65
Figure 3.5B depicts the effect of increasing [Ca2”]g on block of the fast phase

of 45Ca“ inﬂux by 50 11M MeHg. Once again, in the presence of MeHg, increasing
[Ca2+]: did not overcome the block of depolarization-dependent 4S’Ca“ inﬂux.MeHg
reduced 45Ca2+ inﬂux by approximately 50% at all [Ca2+]c tested, except 0.01 and
0.62 mM. With the exception of the value for 0.62 mM [Ca2+]c, 45Ca2+ inﬂux at each
[Caz‘i]e tested was signiﬁcantly lower in the presence of MeHg, when compared to
the respective control values.

In contrast to the results observed for both total and fast inﬂux, block of the
slow phase of 45Ca2+ inﬂux was reversed completely by increasing [Ca2+],3 (Figure
3.5C). At [Ca2+], below 0.3 mM, 50 11M MeHg caused signiﬁcantly less inﬂux of
45Ca2+ when compared to control values, reducing 45Ca2+ inﬂux by 50 to 90%.
However, above 0.3 mM [Ca2+]c, ”Ca2+ inﬂux returned to control levels despite the
presence of MeHg. It should be noted that the sum of the fast and slow components
does not equal the amount of total 45Ca2* inﬂux. Two factors account for this. The
ﬁrst is that the fast phase is largely inactivated by the 10 s predepolarization step
when Slow inﬂux is measured. However, the fast phase does contribute an unknown
amount to the 10 s of 45Ca“ inﬂux measured for the total component. The second
factor is that total inﬂux is measured over the ﬁrst 10 s of depolarization, whereas
the slow component of inﬂux is measured over seconds 10-20 of depolarization
following a 10 s predepolarization. Small differences in the slow component of inﬂux
may therefore exist in the two intervals during which total and slow inﬂux are

measured.

66
Dependence of MeHg-induced block or “Ca2+ inﬂux on [Nat], The

dependence of block of 45C3“ inﬂux on [Na‘i]c in each phase of inﬂux was examined
by using synaptosomes incubated in solutions in which NaCl had been replaced by
choline chloride on an equimolar basis. MeHg (50 11M) caused nearly complete
suppression of 45Ca2+ inﬂux into synaptosomes incubated in N ai-containing and N ai-
free solutions (data not Shown). Figure 3.6A depicts effects of 25 11M MeHg on the
fast phase of 45Ca“ inﬂux into synaptosomes incubated in Na”-containing and Na+-
free solutions. Synaptosomal 4'"Caz" inﬂux was signiﬁcantly reduced to approximately
50% of the MeHg-free control value in both Na*-containing and Nai-free solutions.

Figure 3.63 depicts effects of 25 pM MeHg on the slow phase of 45Ca2+ inﬂux
in Na+-containing and Na*-free solutions. In the absence of MeHg (open bars),
inﬂux of ”Ca2+ into synaptosomes incubated in Na+-free solutions is reduced greatly
compared to that in Na*-containing solution. Since Slow phase 45Ca2+ inﬂux into
synaptosomes may be due largely to reversal of electrogenic Na+ /Ca2+ exchange
(Coutinho et al., 1984; Turner and Goldin, 1985; Suszkiw et al., 1986), removal of
Na” from the incubation medium would deplete the synaptosomes of Na“,
eliminating or greatly reducing Ca2+ uptake via Na+ /Ca2+ exchange. In the presence
of 25 1: M MeHg, ”Ca2+ inﬂux into Na” containing and Na*-free synaptosomes was
reduced to values signiﬁcantly less than their respective MeHg-free controls.

The comparative blocking effect of 25 11M MeHg in both N a+-containing and
N a”-free solutions in shown for both fast and slow phases of 45Ca2+ inﬂux in Figure

3.7. In the slow phase, 25 BM MeHg inhibited 26.1245 and 96.0:4.0% of control

67

 

 

 

 

 

 

 

 

 

l5 UCONTROL
A 72 MeHg

it?
_C_>
X '0 ..
.E
2 ill *
O
E.
or 5 -
5:"
O
E
5

O

45
to B
x
E .
:1 3O '
O
o
5,?

I5 -

i
O

NORMAL No-Free

Figure 3.6. Fast and slow components of inﬂux have differential dependence on Nae. Dependence of
block of 4sea“ inﬂux into syna tosomes by MeHg on [Na+]e during A) 1 s (FAST) of K+-induced
depolarization or B) 10 s of K -induced depolarization following a 10 s predepolarization in the
absence of Ca2+ and MeHg (SLOW). Synaptosomes were incubated in normal Na+ (145 mM) or NaI-
free (NaCl replaced by choline chloride) solutions in the absence (open bars) or presence (striped bars)
of 25 [1M MeHg. Values shown are the mean 1 SEM of ﬁve individual experiments. Values for any
individual experiment are the average of three replicates. The asterisks (’) indicate results that are

signiﬁcantly less than control values (50.05).

68

IOO r

50 -

 

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ x
1.,

 

 

We

 

 

 

 

 

 

 

% REDUCTION FROM CONTROL

SLOW FAST

Figure 3.7. In the absence of Na, block of slow inﬂux by MeHg is much greater in magnitude.

Reduction of “Ca2+ inﬂux from control values by 25 u M MeHg during the slow and fast phases of
inﬂux in normal (open bar) and NaT-free (striped bars) solutions. All other conditions are the same as

those in Figure 3.4. The asterisk (‘) indicates a signiﬁcantly geater percent reduction by 25 u. M MeHg
in Na+-free solution than in normal Na+-containing solution (50.05).

69

tracer inﬂux in synaptosomes incubated in Na*-containing and Na*-free solutions,
respectively. Thus, block of the slow phase of 45Ca2+ inﬂux was signiﬁcantly more
pronounced when Na“c was deleted from the solutions. For the fast phase, 25 BM
MeHg inhibited control tracer inﬂux by 44.6:5.1% in Na*-containing and by
51.81-63% in Na+-free solutions, respectively. This result is consistent with the Na+-
independence of the fast phase of 45Ca2+ inﬂux.

Complete antagonism of block by MeHg of slow 45Ca2+ inﬂux by increasing
[Ca2+], may be due to competitive inhibition of the various components of inﬂux
associated with this phase, or to an irreversible block of one component by MeHg
which is counteracted by increasing [CaZi]e due to a lack of effect of MeHg on some
other component(s) of inﬂux. For example, MeHg may not affect the exchange-
mediated component of the slow phase of inﬂux, but may reduce or block the other
component(s) of this phase of inﬂux. This type of action by MeHg would enable an
operating exchange component to induce a functional antagonism of MeHg-induced
block of the other component(s) of inﬂux. Thus, the true efﬁcacy of the blocking
action of MeHg on the other component(s) of slow inﬂux can only be observed when
the exchange-mediated component is made inoperative, such as by the removal of
Na+ from the medium. As [Ca2+]c is increased, the amount of Ca2+ uptake by the
reversed Na+ /Ca2+ exchanger is greater due to the increased driving force. If [Ca2+],
is increased to a high enough level, the resulting uptake may completely mask the
block by MeHg of other component(s) of inﬂux. If MeHg blocks slow inﬂux by this

mechanism, block of 45Ca2+ inﬂux should not be reversed when Na+ is removed from

70

the medium. This hypothesis was tested and the results are illustrated in Figure 3.8.
At [Ca2+]e = 0.15 mM, “Ca2+ inﬂux was 24:07 x 10'5 and 06:06 x 10'5 nmol
4S‘Ca“ /ug protein in the absence and presence of 25 it M MeHg, respectively. When
[Caz‘i]e was increased to 1.15 mM, the values for 45’Ca2+ inﬂux in the absence and
presence of 25 11M MeHg were 16.1:4.9 x 10—5 and 05:05 x 105 nmol 4SCa“ /p. g
protein, respectively. Thus, MeHg-induced block of 45Ca2“ inﬂux was not antagonized

by increasing [Cay]e in the absence of Na+c.

71

[Céle (mM)

*
ZOi 8&015
. EJIJ5

4506 UPTAKE
§

(nmol/pg protein xIO-Si)

 

 

 

CON MeHg

 

Figure 3.8. In the absence of Nae, block of slow45 inﬂux by MeHg is not reversed by' Increasing [Ca2+].
Effects of Increasing [Ca +e] on eblock of slow 452+Ca inﬂux by MeHg (5 I1» in Na -free solutions.

452+Ca inﬂux was measured In solutions which contained 0.15 or 1.15 mM Ca + .All other conditions
are as in Figure 3. 4. Values shown are the mean: SEM of ﬁve individual experiments. Values for any
individual experiment are the average of+ three replicates. The asterisk (‘) indicates results that are
signiﬁcantly increased by Increasing [Ca2 +c] from 0.15 to 1.15 mM (50. 05).

72
DISCUSSION

This study conﬁrms previous results showing a dose-dependent suppression of
depolarization-induced ”Ca2+ inﬂux into synaptosomes by MeHg, and partial
antagonism of MeHg-induced block of total inﬂux by increasing [Ca2+]c (Atchison et
al., 1986). In addition, the present experiments characterize further the mechanisms
of blocking action of MeHg on Ca2+ inﬂux into nerve terminals. More speciﬁcally,
these results suggest that: 1) high concentrations of MeHg depress depolarization-
independent 45Ca2" entry into synaptosomes; 2) block of the fast and slow phases of
45Ca’" inﬂux by MeHg differ with respect to their sensitivity to antagonism by
increasing [Ca2+]e; 3) the magnitude of block of the Slow phase by MeHg is increased
in the absence of Na+,; and 4) block of the slow phase is not antagonized by
increasing [Caz‘i]e when synaptosomes are incubated in Na+-free solutions.

Incubation of synaptosomes in non-depolarizing solution containing MeHg
resulted in decreased 45Ca2“ entry after 1 and 10 s when compared to MeHg-free
controls. These results indicate that high concentrations of MeHg reduce the
permeability of synaptosomes for Ca“. MeHg increases K+ permeability in yeast
(Passow and Rothstein, 1960) and increases leakage conductance in squid axon
(Shrivastav er al., 1976). Interestingly, MeHg has been shown to increase Ca2+
permeability in synaptosomes (Komulainen and Bondy, 1987), but only after long
periods of incubation. The decrease in Ca2+ permeability produced by MeHg in the
present experiments may be due speciﬁcally to block of Ca2+ entry via random

openings of channels, a decrease in the frequency of random openings resulting from

 

73

a K*-induced hyperpolarization, or an effect on membrane Ca2+ transport systems.
Given the ability of MeHg to block depolarization-dependent Ca2+ inﬂux via Ca2+
channels, it is possible that MeHg may also block Ca2+ entry via random openings
of channels in nonodepolarized membranes. Alternatively, MeHg alters Na“ and K+
gating mechanisms in neuroblastoma cells (Quandt et aL, 1982) and may therefore
alter Ca2+ channel function indirectly. If MeHg were to increase K+ permeability in
synaptosomes as it does in yeast (Passow and Rothstein, 1960), then
hyperpolarization of the membrane would result in a decreased frequency of random
openings of Ca2+ channels, thus decreasing the permeability of synaptosomes for
Ca“. The lack of alteration by MeHg of decay of miniature endplate potential
(MEPP) frequency (Traxinger and Atchison, 1987a) and the ability of increasing
[Ca2+]: to reverse block of the Slow phase of depolarization-induced inﬂux indicate
that membrane Ca2+ transport systems, such as Na+ /Ca2+ exchange may be less
susceptible to disruption by MeHg. Finally, MeHg causes release of Ca2+ from
intraterminal stores, such as the mitochondria (Levesque and Atchison, 1987; 1988;
1990). Increases in intrasynaptosomal [Ca2+] may decrease random opening of Ca2+
channels resulting from Ca2"-dependent channel inactivation (Eckert and Chad,
1984). Thus, MeHg may reduce Ca2+ permeability by decreasing Ca2+ entry via
random openings of channels, or indirectly by decreasing the frequency of random
openings of Ca2+ channels.

In contrast to its effects on depolarization-independent “Ca2+ entry into

synaptosomes, these results indicate that MeHg is a potent and effective inhibitor of

74
depolarization-induced 4SCa“ inﬂux. Atchison et al., (1986) have shown previously

that MeHg reduces inﬂux of 45Ca2+ into synaptosomes and that increasing [Ca2+]c
only partially antagonizes block of total inﬂux of 45Ca2+ by MeHg. Inasmuch as total
inﬂux consists of at least two distinct components, the effect of increasing [Ca2+]c on
MeHg-induced block of each phase was examined to determine the mechanism of
interaction of MeHg with Ca2+ channels, as well as exchange processes.

Block of the fast phase of inﬂux by MeHg was not signiﬁcantly antagonized
when [Ca2+]e was increased. Thus, MeHg appears to block the voltage-dependent
Ca2+ channels which mediate fast inﬂux in a non-competitive, irreversible manner.
Fast inﬂux has been associated with release of Substance P (Floor, 1983),
acetylcholine (Suszkiw and O’Leary, 1983) and dopamine (Drapeau and Blaustein,
1983, Leslie et al., 1985) from synaptosomes. Thus, the fast phase couples Ca2+ entry
and subsequent neurotransmitter release from nerve terminals. The ability of
increasing [Ca2+]e to antagonize block of fast inﬂux by MeHg is consistent with the
ability of increased [Ca2"]e to cause partial restoration of EPPS at the neuromuscular
junction following block by MeHg (Traxinger and Atchison, 1987b). In isolated nerve
terminals, then, MeHg appears to block Ca2+ channels which are associated with
depolarization-dependent Ca2+ entry and subsequent neurotransmitter release.
Complete restoration of EPPS following block by MeHg is also dependent on
increased duration and / or intensity of stimulus, indicating that MeHg may exert
additional effects on membrane excitability at the nonmyelinated portion of the

neuron or along the axonal membrane (Traxinger and Atchison, 1987b). It is possible

75

that MeHg acts at both sites simultaneously or that block of Ca2+ channels follows
diminished membrane excitability by MeHg.

Increasing [Ca2+], resulted in reversal of block of the slow phase of inﬂux by
MeHg. This result is by comparison much different from those observed with total
or fast inﬂux. However, given the mechanism by which slow Ca2+ inﬂux occurs in
synaptosomes, this result is neither contradictory nor unexpected. Because the slow
phase of inﬂux may be mediated by both a channel component and a reversed
Na*/Ca2+ exchange, an explanation of the actions of MeHg on this phase of inﬂux
cannot be derived simply on the basis of the results of this experiment, as MeHg may
affect either non-inactivated Ca“ channels, or Na+ /Ca2+ exchange, or both.

Na+ /Ca2+ exchange in synaptosomes is extremely sensitive to the internal and
external concentrations of Na+ (Blaustein and Oborn, 1975; Akerman and Nicholls,
1981; Nachshen et al., 1986) and is thought to be reversed in synaptosomes due to
their tendency to become Na+-loaded (Coutinho et al., 1984). When the Na+-
dependence of the blocking action of MeHg was examined, MeHg block of fast
4'"Ca2“ inﬂux was unaﬁected by the absence of extracellular Na‘. This is consistent
with the Nai-independent, channel-mediated mechanism of fast inﬂux. The large
decrease in slow inﬂux observed in MeHg-free synaptosomes in the absence of Na”
is also consistent with previous results indicating that the slow phase is mediated
largely by reversed Na“ /Ca2+ exchange, which can be abolished by the removal of

Na“c (Coutinho et al., 1984; Turner and Goldin, 1985; Suszkiw et aL, 1986).

76
When Na+ /Ca2+ exchange is inactivated by removal of N a+, inhibition of the

remaining component of slow inﬂux by MeHg is increased greatly. Therefore, MeHg
may exert a preferential effect on the channel-mediated component of this phase,
which could be offset by an unaffected Nai /Ca2+ exchange in the presence of Na”.
At the neuromuscular junction, MeHg did not alter the rates of decay of frequency
of miniature endplate potentials (MEPPS) after repetitive Stimulation with Sr2+ and
Ba“ (T raxinger and Atchison, 1987a). MEPP frequency is directly related to free
intraterminal divalent cation concentration, and the inability of MeHg to alter the
rate of decay of MEPP frequency suggests that MeHg may not alter the ability of
nerve terminal buffering systems, including Na+ /Ca2+ exchange, to clear divalent
cations from the cytoplasm.

The inability of increasing [Ca2+] to reverse block of slow inﬂux in the

absence of N a: is consistent with an irreversible, non-competitive block of the
channel portion of slow inﬂux. In the presence of Naie this is relieved by a
functional, but reversed N a+ / Ca2+ exchange. Thus, block of both fast and slow inﬂux
by MeHg occurs via an irreversible block of the channel components which mediate
each phase. This is a unique action of MeHg when compared to block of Ca2+
channels by divalent cations such as Hg“, Pb“, Zn2+ and Cd“, in that block of
synaptosomal Ca2+ inﬂux by these ions is reversed by increasing [Ca2+], (Nachshen,
1984; Suszkiw et al., 1985). Whether this difference is due to the reactivity, increased

lipophilicity, or monovalent charge of MeHg is not presently known.

77

In summary, low concentrations of MeHg block channel-mediated 45Ca2+ inﬂux
into KI odepolarized synaptosomes in a manner which is only partially antagonized
by increasing [Ca2+]e, whereas only high concentrations of MeHg affect Na”/Ca2+
exchange. Furthermore, non-depolarized inﬂux of 45Ca2+ is only depressed by high
concentrations of MeHg. These results suggest a unique pattern of interaction of this

monovalent, neurotoxic organic cation with Ca2+ channels involved in neurosecretion.

CHAPTER FOUR

CHARACTERIZATION OF THE INTERACTIONS OF
METHYLMERCURY WITH Ctr2+ CHANNELS IN
SYNAPTOSOMES AND PHEOCHROMOCYTOMA CELLS:

RADIOTRACER FLUX AND BINDING STUDIES

78

79
ABSTRACT

The interaction of methylmercury (MeHg) with neuronal Ca2+ channels in rat
forebrain synaptosomes, and dihydropyridine (DHP)-sensitive Ca2+ channels in rat
pheochromocytoma (PC12) cells was examined using radiotracer ﬂux assays and
radioligand binding analyses. In synaptosomes, the inﬂux of 45Ca2+ was used to
examine the voltage- and state-dependence of block of Ca2+ channels by MeHg as
well as the effects of MeHg on apparent inactivation of 45Ca2+ inﬂux. In addition, the
differential inﬂux of 45Ca“, 85Sr“ and 133Ba2+ was used to examine the effect of
MeHg on the ionic selectivity of synaptosomal Ca2+ channels. The ability of MeHg
to block 45Ca2“ inﬂux via a DHP-sensitive Ca2+ channel was examined in PC12 cells.
Effects of MeHg on binding of [3H]-nitrendipine in synaptosomes and [mu-w -conoto-
xin GVIA (Cng) in synaptosomes and PC12 cells were measured. In synaptosomes,
MeHg blocked 45Ca2+ inﬂux in a voltage-dependent manner, as increasing the
extracellular K” concentration increased the magnitude of block by 100 1.1M MeHg.
When synaptosomes were incubated for 1 s in either a non-depolarizing or a
depolarizing solution prior to measuring 1 s of depolarization-induced 4SCaz“ inﬂux,
the potency and efﬁcacy of the block of 45Ca“ inﬂux by MeHg were similar. Thus,
block of Ca2+ channels by MeHg does not appear to be state-dependent. To
determine the kinetics of apparent inactivation of 45C3“ inﬂux, synaptosomes were
predepolarized in Ca2+-free, high [K‘] solution for intervals varying from 1-10 s prior
to measuring 1 s of KI-induced 45Ca“ inﬂux. When compared to control, MeHg (100

I: M) altered the rate consth for apparent inactivation and decreased the fraction

 

80

of 45Ca“ inﬂux which does not inactivate. Inﬂux of 45Ca“, 8SSrZ“ and 133Ba2+ during
1 S of depolarization was blocked in a dose-dependent manner by MeHg with
estimated 1C,0 values of 125, 150 and > 150 uM for ”Ca“, 8588* and 133m“,
respectively. In triple-label experiments MeHg decreased inﬂux of all three divalent
cations: the effect on 858T2+ inﬂux was signiﬁcantly greater than on 45Ca2“ and 1”Ba2+
inﬂux. The relative ﬂux of radiolabeled Ca2+:Sr2+:Ba2+ was altered from ap-
proximately 6:2:3 to 6:1:3 in the presence of 100 uM MeHg. In undifferentiated and
nerve grth factor (NGF)-differentiated PC12 cells, K*-induced 45Ca2“ inﬂux was
blocked by the DHP nifedipine with an approximate ICso value of 5 nM. MeHg
reduced 45Ca2+ inﬂux in PC12 cells with an estimated ICso value of 50 11M, and 125
RM MeHg reduced uptake by > 90%. [3H]-nitrendipine bound to synaptosomes with
high afﬁnity in normal and elevated [K+] solutions. In normal [K+] solutions,
Scatchard analysis of the binding data resulted in a KD value of 630: 160 pM and a
BM value of 130:40 frnol / mg protein. In the presence of 100 u. M MeHg, the values
for KD and BM were 25202630 pM and 200130 fmol/ mg protein, respectively.
Results obtained in the absence and presence of MeHg in elevated [K*] buffers were
not signiﬁcantly different from those obtained in normal [Ki] buffers. [mI]-m -
conotoxin GVIA bound to a single high afﬁnity site on synaptosomes and PC 12 cells
with a half saturation occurring in the subnanomolar range. 100 u M MeHg did not
signiﬁcantly alter binding of Cng in synaptosomes, but did alter Cng binding in
PC 12 cells by decreasing the amount of Cng bound at all concentrations of Cng

tested. These results indicate that MeHg: 1) blocks synaptosomal Ca2+ channels in

81

a voltage- but not state-dependent manner; 2) alters synaptosomal Ca2+ channel ionic
selectivity and inactivation kinetics; 3) blocks Ca2+ inﬂux via DHP-sensitive Ca2+
channels in PC12 cells; and 4) alters the binding properties of DHPS in synap-

tosomes, and Cng in PC12 cells, but not synaptosomes.

82
INTRODUCTION

Preliminary experiments demonstrate that MeHg does block Ca2+ channels in
isolated nerve terminals, and indicate that effects of MeHg on channels are difﬁcult
to overcome (Atchison et al., 1986; Shafer and Atchison, 1989). However, these
experiments provide little information regarding mechanisms by which MeHg
interferes with Ca2+ channel function, or the types of Ca2+ channels affected by
MeHg. However, results of preliminary experiments indicate that there may be
interesting differences in the interactions of MeHg and divalent metal cations with
neuronal Ca2+ channels.

MeHg and divalent metals produce similar disruptions of synaptic transmission
at the NMJ and impede the movement of Ca2+ through voltage—dependent Ca2+
channels. Micromolar concentrations of MeHg (Atchison et al., 1986; Shafer and
Atchison, 1989) or inorganic divalent cations (Pb2+, Ni2+ and Cd“) (Nachshen, 1984)
block 1 s (”fast" phase) of depolarization-induced inﬂux of 45Ca“ into synaptosomes.
Increasing [CaH]e concentration readily reverses block of "fast" inﬂux by divalent
cations (Nachshen, 1984), but not by MeHg (Atchison et al., 1986; Shafer and
Atchison, 1989).

MeHg differs in two important respects from divalent inorganic Ca2+ channel
blockers: enhanced lipophilicity imparted by the methyl group, and monovalent
rather than polyvalent charge. Because data suggest that MeHg disrupts Ca2+ entry
into the nerve terminal by interacting directly with Ca2+ channels, and because

chemical differences between MeHg and other heavy metal Ca2+ channel blockers

83

may give rise to differences in action on Ca2+ channels, experiments were designed
to characterize in detail the nature of the interaction of MeHg with nerve terminal
Ca2+ channels. Speciﬁcally, experiments were designed to determine whether MeHg
blocks Ca2+ channels in a voltage- and/or state-dependent manner, and whether
MeHg is capable of altering the kinetics of apparent inactivation and/or ionic
selectivity of synaptosomal Ca2+ channels.

The observation that multiple types of Ca2+ channels exist in nerve terminals
(Penner and Dreyer, 1986; Miller, 1987; Atchison and O’Leary, 1987) raises the
question of the identity of Ca2+ channels which are blocked by MeHg. As only some
types of Ca2+ channels have been associated with neurotransmitter release,
determination of the types of Ca2+ channels affected by MeHg is crucial to
determining whether or not effects on Ca2+ channels may contribute to the effects
of MeHg on transmission at the NMJ. Since the type or types of Ca2+ channels in
synaptosomal preparations are not well characterized, rat pheochromocytoma (PC 12)
cells were chosen as an alternative model system for studies of the effects of MeHg
on Ca2+ channels.

Under normal culture conditions, PC 12 cells express predominantly
dihydropyridine (DHP)-sensitive Ca2+ channels (Takahashi and Ogura, 1983) known
as "L" or "long opening" type (Nowycky et aL, 1985a,b). When cultured in the
presence of NGF, the expression of DHP-insensitive, N-type channels is greatly
increased without decreases in the expression of the DHP-sensitive channels. Thus,

PC12 cells provide a means by which to examine the effects of MeHg on a

84

homogenous and/ or deﬁned population of Ca2+ channel types which are associated
with neurotransmitter release.

Finally, the effects of MeHg on the binding of two well-characterized Ca2+
channel antagonists were examined. Although the pharmacological binding potency
of Ca2+ channel antagonists does not necessarily correlate with their functional
effects, competitive binding assays nevertheless provide a measure of the ability of
MeHg to compete with known Ca2+ channel antagonists for their binding sites on
nerve membranes. The DHP nitrendipine has been employed in numerous binding
and ﬂux Studies in synaptosomes (Rampe et al., 1984; Turner and Goldin, 1985;
Suszkiw et al., 1986) and PC12 cells (Toll, 1982) to examine the properties of binding
sites which are presumed to be located on or associated with "L"-type Ca2+ channels.
Therefore, the effects of MeHg on the binding of [3H]-nitrendipine could provide
additional information regarding the ability of MeHg to interact with binding sites
associated with this type of Ca2+ channel.

to -Conotoxin GVIA (Cng), a peptide isolated from the venom of the marine
snail Conus geographus, reduces Ca2+ inﬂux in chick synaptosomes (Rivier er al.,
1987) and binds to sites on synaptosomes and PC12 cells which appear to be distinct
from the DHP binding site (Abe et al., 1986; Cruz and Olivera, 1986; Marqueze et
al., 1988; Sher er al., 1988). Therefore, effects of MeHg on binding of Cng were
examined in synaptosomes and PC12 cells to determine whether MeHg may interact
with these sites instead of, or as well as, DHP binding sites. Inasmuch as Cng has

been reported to inhibit transmitter release from and 45Ca“ inﬂux into isolated nerve

85

endings, and to bind at distinct sites from the DHPS, its use would permit a
differential analysis of binding of MeHg to sites spatially associated with perhaps two

different Ca2+ channels, or two separate binding Sites on Ca2+ channels.

86
MATERIALS AND METHODS

The materials and solutions, procedure for isolation of synaptosomes and general
method for measuring radiotracer inﬂux are described in detail in Chapter three. The
methods described in this chapter are speciﬁc experimental procedures and
manipulations required for the experiments in Chapter four. Unless Speciﬁcally
stated, all solutions contained the same constituents and concentrations as those
described in Chapter three.

The K+ dependence of 45Ca2+ inﬂux was measured in the presence and
absence of MeHg (100 nM) by incubating synaptosomes for 2 s in Na-free K“
solutions containing 45Ca2+. Choline chloride in normal [K‘] solution was replaced
by KC] to achieve the various K+ concentrations used. To obtain a more accurate
estimate of inﬂux at the lower K+ concentrations used, inﬂux was measured for 2 s
at all K” concentrations.

The state-dependence of block of the "fast" phase of 4S’Caz" inﬂux by MeHg
was determined by measuring 1 s of 45Ca“ inﬂux in synaptosomes which had been
exposed to MeHg under conditions in which the population of Ca2+ channels would
be primarily in the "resting" state, or under conditions in which the population of
Ca2+ channels would be primarily in the "open" or "inactivated" State. To "open" or
"inactivate" Ca2+ channels, synaptosomes were predepolarized in 41.25 mM K”
solution for 10 s by adding 25 ill of synaptosomal suspension (5 mM K”) to 25 ul of
elevated [K‘] (77.5 mM) solution containing MeHg. Ca2+ channels in the resting

state were exposed to MeHg in 5 mM K” solution for 10 s by adding 25 ul of

87
synaptosomes (5 mM K") to 25 pl of normal (5 mM) [K*] solution containing MeHg.

Following the 10 s exposure to MeHg, 1 s of 4"Ca“ inﬂux was measured by addition
of 50 ul of K+ solution (77.5 or 113 mM K") containing 0.06 mM of 45Ca“ and
MeHg, so that the ﬁnal K“ concentration of all solutions was equal to 59.4 mM. One
s after the addition of K+ solution, 45Ca2" inﬂux was stopped by the addition of 2 ml
of quenching solution. In order to maintain osmolarity conditions equal to those in
other experiments and to increase [K‘] to 113 mM, [Na+] was lowered to 36 mM in
all K+ solutions, being replaced by KCl (113 mM K+ solution) or choline chloride
(physiological saline and 77.5 mM K“ solutions) on an equimolar basis.

The apparent inactivation of depolarization-induced Ca2+ inﬂux was
determined by measuring 1 s of 45Ca2+ inﬂux after various intervals of predepolariza-
tion (0-10 s) in nominally CaZI-free medium to inactivate Ca2+ channels (Suszkiw et
al., 1986). Brieﬂy, 200 pl of Ca2+-free, elevated (77.5 mM) [K”] solution with or
without MeHg were combined with 200 ul of synaptosomal suspension (5 mM K”).
Following 1 to 10 S of predepolarization, 400 pl elevated [K+] solution containing
0.02 mM “Ca2+ and MeHg were added to the mixture. After an additional 1 s, 3 ml
of ice-cold quenching solution were added to stop 4f’Ca“ inﬂux. The ﬁnal
concentration of MeHg before the addition of quenching solution was 100 u M.
Non-inactivated inﬂux (1 s) was measured by combining 200 ul of synaptosomal
suspension with 600 ill of elevated [K‘] solution containing MeHg (100 nM) and
”Ca2+. Synaptosomes were retained on Millipore ﬁlters and 4"‘Ca“ inﬂux was

determined as described in Chapter three.

 

88
PC12 Cell Experiments. PC12 cells were cultured at 37°C in a humidiﬁed 5%

CO2 atmosphere using Dulbecco’s modiﬁed Eagle medium (pH 7.4, GIBCO)
containing 10% horse serum, 5% fetal bovine serum, and 2 mM HEPES. PC12 cells
were differentiated by culturing for 7 days in medium containing 100 ng/ml nerve
growth factor (NGF) isolated from mouse submaxillary gland. The NGF was a gift
from Dr. Steven Heidemann at Michigan State University.

Cells were harvested by suspension in normal (5 mM) [K+] PC12 medium and
centrifugation for 10 min at 5000 x g. This Step was repeated twice to wash
completely the grth medium from the cells.

The effects of nifedipine (Sigma, St. Louis, MO) and MeHg on depolarization-
induced 45Ca2“ inﬂux were measured using the same method as for synaptosomes,
except that elevated [K‘] PC12 medium contained 100 mM K+ so that the ﬁnal
concentration of K“ used for depolarization of the PC12 cells was 52.5 mM. Elevated
(100 mM) and normal (5 mM) [K‘] PC12 medium used in inﬂux studies with PC12
cells have been described by Stallcup (1979) and are similar to those described for
synaptosomes in Chapter three. Inﬂux was measured over 2 min. PC 12 cells were not
preincubated with nifedipine or MeHg, therefore the only exposure of the PC12 cells
to MeHg or nifedipine was the 2 min during which 4SCa“ inﬂux was measured. The
ﬁnal concentration of Ca2+ was 1 mM. During ﬁltrations, the cells were retained on
Whatrnan GF/B glass ﬁber ﬁlters.

Binding Experiments. The equilibrium binding of [TU-nitrendipine (speciﬁc

activity, 70 Ci / mmol) was measured in synaptosomes in the following manner. For

 

89

any given experiment, 2 ml of a stock solution containing [3H]-nitrendipine and
MeHg (100 nM), when appropriate, was prepared in normal [K‘] solution. Binding
of [am-nitrendipine was initiated by addition of 20 [J] of synaptosomal suspension
(250-350 mg protein) to 500 pl of stock solution. After 1 hr of incubation at 25°C, the
solution was ﬁltered rapidly through Whatman GF/B glass ﬁber ﬁlters and rinsed
with three 5-ml aliquots of quenching solution. To determine total ligand concentra-
tion, a SO-ul aliquot was taken from the remaining 500 pl of stock solution. Non-
speciﬁc binding was measured in the presence of 1 11M nifedipine. Radioactivity
remaining on ﬁlters was determined as described for 45Ca2+ inﬂux experiments,
except that the scintillation counter contained a 3H quench curve and program to
convert cpm to dpm. The average of triplicate values were used for Scatchard
analysis (1949) to determine the equilibrium dissociation constant (KD) value for
binding of Pin-nitrendipine and the density of binding sites (Bum). As with all
experiments using DHPS, these procedures were carried out in the dark. To measure
the binding of 3Iii-nitrendipine under depolarizing conditions, the exact same
procedure was followed using elevated [K‘] solution in place of normal [K‘] solution.

Binding of [mu-to -conotoxin GVIA (speciﬁc activity 2200 Ci/mmol) to PC12
cells and synaptosomes was measured under non-depolarizing conditions by methods
similar to those described for nitrendipine, except that PC12 cell medium was used
for binding experiments in PC12 cells. In addition, the effects of pretreatment of
synaptosomes with MeHg on Cng binding were examined in the following manner.

Once isolated, synaptosomes were resuspended in 5 ml of physiological saline

90

containing 100 RM MeHg and incubated at room temperature for 10 min. Following
the incubation, synaptosomes were repelleted and washed in MeHg-free physiological
saline by centrifugation at 10,000 x g for 10 min and subsequent resuspension.
Synaptosomes were then incubated for 25 min at room temperature (25°C) and 10
Ill aliquots were used to measure the binding of [1”I]-<o-conotoxin GVIA as
described above for nitrendipine. In all Cng binding experiments, non-speciﬁc
binding was measured in the presence of 1 11M unlabeled Cng (Peninsula
Laboratories, Belmont, CA) and normal [K+] solution contained 0.1% albumin and
no added calcium. After 1 hr, solutions were ﬁltered rapidly over Whatman GF/B
ﬁlters which had been presoaked in quenching solution containing 0.1%
polyethyleneimine (Sigma). Radioactivity remaining on the ﬁlters was determined in
the Searle model 1197 gamma counter with an efﬁciency of 53% for 125’1. Protein
content of synaptosomal and PC12 cell suspensions was determined by the method
of Lowry et al. (1951).

Statistical analysis. Statistical analysis of results of the inactivation
experiments is described in the Results section. For experiments using nifedipine or
MeHg to block 45Ca“ inﬂux into PC12 cells or to measure the time-course of block
by MeHg in synaptosomes, data were analyzed using a randomized block analysis of
variance (AN OVA) and, if signiﬁcant differences were found, individual comparisons
were made using Dunnett’s one-tailed t-test. For Ki-dependence experiments, a
mixed design ANOVA was used to compare inﬂux in control and MeHg-treated

synaptosomes; individual comparisons were made using the least signiﬁcant

91
difference (lsd) test. To compare the efﬁciency of block by MeHg at different [K+],

a randomized block ANOVA and Dunnett’s one-tailed t-test were used. Data from
triple label studies were analyzed using a randomized block AN OVA; individual
comparisons were made using Scheffe’s test. Comparisons of KD values for
3H-nitrendipine binding to synaptosomes were made using a mixed design ANOVA
and the lsd test. Data from Cng binding experiments were analyzed using a
randomized complete block AN OVA and the lsd test. For all comparisons, statistical

signiﬁcance was deﬁned by ps .05.

92
RESULTS

Time Course of Block by MeHg. Block of “Ca2+ inﬂux into synaptosomes by
MeHg as a function of time is shown in Figure 4.1 (ﬁlled circles). MeHg (50 nM)
blocked approximately 50% of the 45Ca“ inﬂux during the ﬁrst 1 s of depolarization.
There was no statistically signiﬁcant change in the degree of block by MeHg as a
function of time. The inﬂux of 45Ca“ into synaptosomes consists of two distinct
phases (Nachshen and Blaustein, 1980; Suszkiw et al., 1986): a fast phase, which is
mediated by voltage-dependent Ca2+ channels, and a slow phase, which is mediated
largely by reversed Na+ /Ca2+ exchange, but may also contain a residual channel-m-
ediated component (Shafer and Atchison, 1989). As low concentrations of MeHg may
not affect reversed N a+ / Ca2+ exchange in nerve terminals (Traxinger and Atchison,
1987b; Shafer and Atchison, 1989), uptake via this system may offset the block by
MeHg of channel-mediated inﬂux. Therefore, the time-dependent effects of MeHg
were examined in medium in which NaCl was replaced by choline chloride on an
equimolar basis to prevent uptake via reversed Na+ /Ca2+ exchange. Under these
conditions, block of 45Ca2" inﬂux by MeHg increased as time of exposure during
depolarization increased. After 10 or more 5 of exposure to 50 11M MeHg, block of
”Ca2+ inﬂux was signiﬁcantly greater than after only 1 s of exposure (Figure 4.1.,
open circles). Thus, as the time of exposure to MeHg increased, MeHg blocked a
greater portion of channel-mediated, depolarization-induced 45Ca2“ inﬂux.
K*(voltage)-Dependence Experiments. In Figure 4.2A, the K+-dependence of 45Ca“

inﬂux into synaptosomes is shown in the absence (solid symbols) and presence

93

 

 

 

 

 

 

 

100 r _._ +Na __O__ -Na
3 '5 Ti/ \' I
_.I I— /
u. 'E ‘
E O50 '0
° :
3 32 ‘
If) V ' ‘~
v 25 TFONA.
“‘9 ------------------ >3; __________________ 8‘
0 IO 20 30

TIME OF DEPOLARIZATION (seC)

Figure 4.1. Block of 45Ca2+ inﬂux into synaptosomes vs. time. Block of 45Ca2+ inﬂux into synaptosomes
by MeHg as a function of time of exposure in Na-containing (solid symbols) or Na-free medium (Open
symbols). 45Ca2+ inﬂrrx was measured by the addition of 50 211.1 of synaptosomes to 50 pl of elevated
(77.5 mM) or normal (5 mM) [K ] solutions containing 45 C and 50 i4 M MeHg. Inﬂux was stopped
after various intervals of time by the addition of 2 ml of quenching solution. Inﬂux In normal [K ]
solutions was subtracted 2to determine depolarization-induced 452Ca inﬂux. The results are expressed
as a percentage of 452+Ca inﬁrnr In MeHg-free solutions, and are the mean :1: SEM of three or four
separate experiments, respectively. Values for any particular experiment are the average of three
replicates. The asterisk (‘) indicates values that are signiﬁcantly different than (p < .05, one-sided t-test)
the value at 1 s in Na-containing (signiﬁcantly greater) or N a-free (signiﬁcantly less) synaptosomes.
After 30 s of depolarization, the values for inﬂux of 45Ca2+ into synaptosomes in the absence of MeHg
were 1.99 x 058 and 7.85 1 2.22 nmol/pg protein x 10'4 in Na-free and Na-containing solutions,

respectively.

94
(open symbols) of 100 11M MeHg. Inﬂux in 5 mM K+ has not been subtracted from

these data. The magnitude of depolarization caused by each K+ concentration can
be estimated by the relationship AVm = 60 log [K+]H/[K+]L, where an is the change
in membrane potential and [K”] is the K+ concentration of the normal (L) and
elevated (H) [K*] solutions (Blaustein and Goldring, 1975). Thus, the
voltage-dependence of inﬂux can be estimated by this method. The K+
concentrations used in this experiment will result in depolarizations of 0 to 55 mV
from the resting membrane potential, which under Similar conditions has been
estimated to be approximately -80 mV (Suszkiw et al., 1986). In the absence of
MeHg, synaptosomal 45Ca2+ inﬂux activated at [K+] above 10 mM, and increased in
a linear manner as [K‘] was increased to 41.3 mM. In a separate experiment, higher
concentrations did not increase “Ca2+ inﬂux further (data not shown). Thus, 45Ca2+
inﬂux in synaptosomes activates at K“ concentrations above 10 mM and is maximal
at K+ concentrations of approximately 41 mM. These results are consistent with
those of Suszkiw et al. (1989). In the presence of 100 11M MeHg, ”Ca2+ inﬂux
increased only gradually as the [K‘] was increased, being signiﬁcantly less than
control values at each respective K+ concentration. In panel B of Figure 4.2, the
percent inhibition of 45Ca“ inﬂux by MeHg is plotted as a function of [Ki]. At low
K+ concentrations, MeHg blocked approximately 20% of the 45Ca2+ inﬂux. As the
[K*] was increased, block of uptake by 100 u M MeHg increased to approximately

50% at the highest K+ concentration tested. The block of 45Ca“ inﬂux in 36 mM or

95

ﬂ
-
O
-
-
O

3.50

 

 

 

2.25 " ' /
. “.

“Ca iNFLUx x10...
("moi/no protein)

 

 

 

 

; U
—/3 0 T __-66
2 x 1
. é); ___________ (I, ______ it 1‘
1.00 *" L_t , ‘ l ‘ 1 1 n I
5 10 50
60 r B
a a II
.1 it: _ I '
lz" :-§ 40 [/11
I E I 1
A
o L ‘ l l n . 1 a a c c
5 10 50
“(‘1 (mM)

Figure 4.2. Voltage-dependence of 45Ca2+ inﬂux into synaptosomes and reﬂects of MeHg. A) Effects
of [K ] on inﬂux of 4'SCa“ into synaptosomes in the absence (solid symbols) and presence of 100 uM
MeHg (open symbols). Syna ynaptosomes were incubated for 2 s in N a-free buﬁers containing ”Ca and
various concentrations of K. Inﬁrnr was stopped by the addition of 2 ml of quenching solution. The
asterisk (‘) indicates values of inﬂux In the presence of MeHg which are signiﬁcantly less than (p< .05)
their respective MeHg-free control value. B) Magnitude of block of 45Ca2+ inﬂux by 100 uM MeHg
at diﬁ’erent K+ concentrations used in part A. The values marked by an asterisk (‘) indicate signiﬁcantly
greater block (p<.0 5) by MeHg than the block in 5 mM KL All values are the mean: SEM of four
experiments, where each experimental value is the average of three replicates.

96

higher concentrations of K+ was signiﬁcantly greater than that observed in 5 mM K“
solutions.

State-dependence Experiments. Block of Ca2+ channels in excitable membranes
frequently exhibits so-called "state-dependence", wherein the blocking affinity of the
antagonist is dependent upon the conﬁguration of the channel. Inasmuch as the
blocking afﬁnity of certain divalent cations for 45Ca2+ inﬂux into synaptosomes has
been shown to be "state-dependent" (Nachshen, 1985), experiments were designed to
determine whether the degree of block by MeHg depended on prior utilization of the
channel. This was accomplished by incubating synaptosomes for 10 s in the presence
of MeHg in either 5 mM or 41.3 mM K” solutions, prior to measuring 1 S of
depolarization-induced ”Ca2+ inﬂux. These K+ concentrations are below and above
the threshold for activation of 4"Ca“ inﬂux into synaptosomes, respectively. Thus, in
synaptosomes incubated in 41.3 mM K+, MeHg had access to the open and
inactivated states of the Ca2+ channel, while in synaptosomes incubated in 5 mM K+
solution, MeHg had access to Ca2+ channels in the resting state. After incubation in
5 mM K+ solution, MeHg caused a dose-dependent suppression of 1 s of 45Ca2+
inﬂux which reached 84.5 15.0% (mean a SEM, n =6) reduction with 250 it M MeHg;
the estimated 1C5o value was 75 BM (Figure 4.3, ﬁlled circles). When MeHg had
access to open or inactivated Ca2+ channels prior to measuring 1 s of inﬂux, a
dose-dependent suppression of 45Ca’“ inﬂux was also observed (Figure 4.3, Open
circles). The estimated 1C,o value for MeHg was also 75 I: M and inhibition was

maximum (89.414.5%, mean a SEM, n=5) at 250 uM MeHg. Although ”Ca2+

97

 

 

 

 

 

 

120 r
—‘—' Resting --O-- Open
I
x ... _,
D .3 90 \I
E! :3 °‘~~ L.\’.
.2.. ‘0: 60 i ”(1) ------------ 13’
0
(U
«.0 ES 30 -
V
o - - - _
2 10

 

[MeHg] (pM)

Figure 4.3. State-dependence of the actions of Mei-lg on “Ca2+ inﬂux. Inﬂux measured during 1 s of
K+ -induced depolarization following predepolarization for 10 s in the presence of MeHg (open symbols)
or following 10s of incubation with MeHg in a non-depolarizing solution (5 mM K”) (solid symbols).
Values are the mean: SEM of six different emeriments. For any given experiment, the value was the

average of three replicates.

98

inﬂux at low concentrations of MeHg was reduced to a greater extent in predepolar-
ized synaptosomes than in non-depolarized synaptosomes, there was no signiﬁcant
difference in the reduction of 45Ca“ inﬂux under the two conditions. Thus, block of
45Ca“ inﬂux by MeHg was not altered when MeHg was allowed access to different
states of the Ca2+ channel.

Inactivation Experiments. Predepolarization of synaptosomes in the absence
of added Ca2+ resulted in a time-dependent decrease in 45Ca2“ inﬂux into
synaptosomes when 45Ca2+ -containing solutions were subsequently added (Figure 4.4).
This decrease is presumably due to voltage-dependent inactivation of synaptosomal
Ca2+ channels. The ability of MeHg to alter this process in a quantiﬁable manner
was examined using the following model for apparent inactivation in synaptosomes:
Q = qle'kit + qz; where Q is the fraction of Ca2+ inﬂux remaining after time t of
predepolarization, k is the rate constant for apparent inactivation, and q1 and qz are
the fractions of Ca2+ inﬂux which do and do not inactivate, respectively. This
relationship has been shown previously (Suszkiw et aL, 1986) to describe the apparent
inactivation of Ca“ inﬂux in synaptosomes under nearly identical experimental
conditions. To validate the use of this model, the goodness of ﬁt of these data to the
above model was examined by non-linear least squares regression analysis. For
control experiments, correlation coefﬁcient values ranged from 0.77 to 0.94. The
goodness of ﬁt of these data to several other potential models for inactivation was
also examined, including a model for the exponential decay of a voltage-activated

current (Belluzzi er al., 1985) which has been used recently to describe the apparent

99

inactivation of Ca2+ inﬂux in synaptosomes incubated in Na+-free medium (Suszkiw
et al., 1989). Correlation coefﬁcient values for goodness of ﬁt of these data to other
models were less satisfactory than those for the model chosen.

Using the CRUNCH statistical program, non-linear, least-squares regression
analysis was performed on the data ﬁom each individual experiment to determine
ki, ql and q2. The average values for ki, ql and q2 for control and MeHg-treated
synaptosomes are listed in Table 1 and yield the lines in Figure 4.4 when used in the
above function. Signiﬁcant differences in ki, ql and q2 in MeHg-treated (n=3) and
control (n = 5) synaptosomes were tested for using a one-sided Student’s t-test (pg .05).
When MeHg-treated synaptosomes were compared to untreated control
synaptosomes, the values for ki, ql and q2 were signiﬁcantly different. Thus, MeHg
alters the kinetics of apparent inactivation of 45Ca’“ inﬂux in synaptosomes by
altering the rate of apparent inactivation and increasing the fraction of inﬂux which
inactivates. It should be noted that in the presence of MeHg, inactivation of 45Ca2+
inﬂux was essentially complete after only 1 s of predepolarization, thus the value for
the rate constant ki only reﬂects the apparent constant for the rate of inactivation.

Ionic Selectivity Experiments. For dose-response curves, solutions contained
a ﬁnal concentration of 2.4 mM unlabelled Ca2+, Sr2+ or Ba“, and 1 uCi of ”Ca2+,
85Sr2+ or 133Ba“, respectively. MeHg reduced depolarization-induced inﬂux of 45Ca2“
and 8SSrz” into synaptosomes in a dose-dependent manner, with estimated ICso values
of 125 and 150 uM, respectively (Figure 4.5). Although the ICso value for block of
“Ca2+ uptake by MeHg is slightly higher than previously reported (Shafer and

100

 

 

 

 

 

 

 

 

X
D
.1
L
E
_.l
< .
Z \
Q . .
l- 2
g .2
i
m T T
LI. 0 O
O . l . L .
O 4 8 1 2

PREDEPOLARIZATION (seC)

Figure 4.4. Effects of MeHg on apparent inactivation of synaptosomal “Ca“ inﬂux. Effects of MeHg
(100 u. M) on the rate of apparent inactivation of depolarization-induced 45Ca2+ inﬂux. Synaptosomes
(200 #1) were predepolarized for varying intervals by addition of 200 #1 of Can-free, elevated
[KW-solution containing (open symbols) or free of (solid symbols) MeHg. Following predepolarization,
400 pl of elevated [K+] solution containing MeHg (if appropriate) and 1 uCi of 45Ca2+ were added.
Inﬂux was stopped after 1 s by the addition of 3 ml of quenching solution. Fractional inﬂux is the
amount of 45Ca + inﬂux remaining after a designated interval of predepolarization, relative to 1 s of
45Ca“ inﬂux in the absence of predepolarization. The values shown are the mean :1: SEM of ﬁve
(MeHg-free) and three (MeHg) different experiments. Values for any particular experiment are the
average of three replicates. Curves are drawn using the average non-linear least squares values listed
in Table 1.

101

Table 4.1

Non-Linear least squares regression values
for apparent inactivation of 45Ca2+ inﬂux into synaptosomesa

 

ki (5’1) (11 (12

 

Controlb 050: 0.10 0.66: 0.02 038: 0.04

MeHgc

6.33: 0.08‘ 0.74! 0.02‘ 0.26: 0.2‘

 

aNon-linear least squares regression was used to fit data to the function Q = qle'kit + 92
where k! is the rate constants, ql is the fraction of inﬂux which inactivates and 92 is the fraction
of inﬂux which does not inactivate.

bData from ﬁve diﬁerent experiments were used to calculate regression values which are given
as the mean :9: SEM.

cData from three different experiments using 100 [1M MeHg were used to calculate regression
values which are given as the mean: SEM.

‘Signiﬁcantly different from control values (1:50.05).

 

102

Atchison, 1989), the concentration of extracellular calcium used in this experiment
was 2.4 mM, compared to 0.05 mM Ca2+ in previous experiments. In contrast to Ca“
and Sr“, the inﬂux of 133Ba2+ was only slightly inhibited by concentrations of MeHg
as high as 250 uM (Figure 4.5). The differential sensitivity of 45Ca“, 8581'“, and
133Ba“ ﬂuxes to block by MeHg suggests that MeHg alters the ionic selectivity of the
Ca2+ channel for these ions. However, the decreased potency of MeHg to block Ba2+
inﬂux may not be due to a reduced blocking action of MeHg on Ba“ inﬂux, but to
an increased inﬂux of Ba“ into synaptosomes due to lack of Ca2+-induced Ca2+
channel inactivation by Ba2+ (Eckert and Chad, 1984). In order to control for
differences in Ca2+ channel inactivation, triple-label studies were designed in which
the inﬂux of 45Ca2+, 8SSr“ or 133Ba2+ into synaptosomes was measured in solutions
which contained 0.1 mM 811* and Ba“, 0.02 mM Ca2+ and 0.5 mM Mg“. The
effects of MeHg (100 nM) on the relative inﬂux of the three divalent cations are
shown in Figure 4.6. To correct for differences in concentration, the values have been
normalized by dividing the inﬂux of each ion by its concentration in the extracellular
medium (Nachshen and Blaustein, 1982). In the absence of MeHg, the ratio of inﬂux
of 4sCazi’SSrsza was approximately 6:2:3. When 100 uM MeHg was present in the
medium, the inﬂux of 45Ca“ and 1338a“ were 61129.4 and 72.6: 11.6% of their
respective control values, but the inﬂux of 8SSr“ was only 44.1 t 6.9% of its inﬂux
into synaptosomes in the absence of MeHg. This was a statistically signiﬁcant

difference when compared to the effects of MeHg on 45Ca“ and 1333a2+ inﬂux into

 

103

 

 

 

 

 

 

 

150 _ .._.‘_ 058'. _-O_- 10:88 _._ 4803

X g 100-

D C

.1 ° -

ll. 0

Z

"" 3‘: 50 -
o - AAA. - . n A A kLA. - -
5 ‘IO 50 100

[MeHg] (IJM)

Figure 4.5. Eﬂ'ects of MeHg on the inﬂux of 45Ca2+, ”Sr” and 1338a” into synaptosomes. Inﬂux was
measured by addition of 50 ul of synaptosomal suspension to 50 pl of elevated (77.5 mM) or normal
(5 mM) [Kl'] solution containing MeHg and 1 [.5 Ci of tracer. Inﬂux was stopped after 1 s by the addition
of 2 ml of quenching solution. Depolarization-induced inﬂux was determined by subtracting the inﬂux
of radiolabel in normal +] solution. The values shown are the mean: SEM of four (“Ca“), three

(8583+) and ﬁve (1338a +) different experiments. Values for any particular experiment are the average
of three replicates.

104

 

 

 

 

 

 

 

 

 

 

1.20 P
- CONTROL El 100 pM MeHg.

X
D
_l
[2'- 0.80 ~ 57%
UJ
..>_
E 0.40 ~
.J
UJ
(I *44%

0.00 ‘

4608 858'. 13388
RADIOISOTOPE

Figure 4.6. Effects of Meﬂgzg; the relative inﬂux of 45Ca“, ”Sr“ and 133Ba2+ into synaptosomes.
Inﬂux of 45Ca2"’, 8SSr“ or a2+ during 1 s of K+-induced inﬂux was measured in the absence (solid
bars) or presence (open bars) of 100uM MeHg. K+ solutions contained (mM): SrClz, 0.1; BaClz, 0.1;
CaClz, 0.02; M302, 0.05 and 1 u Ci of ”Ca“, 3539*, or 1333a“. The results have been normalized
by dividing the inﬂux of each cation by its external concentration. The values shown are the mean 3:
SEM of three to ﬁve separate experiments. Values for any particular experiment are the average of
three replicates. The asterisk (‘) indicates an effect of MeHg on 8581"?+ inﬂux which is signiﬁcantly
diﬂerent from the effect of MeHg on 45'01“ and 1338a2+ inﬂux (p< .05).

105
synaptosomes. Thus, MeHg altered the relative permeability of 4sCaz‘gssrsza to

6:1:3.

“Ca2+ Inﬂux into PC12 Cells. Inﬂux of 45Ca2+ into PC12 cells by depolariza-
tion in 52.5 mM K+ gradually increased as time of depolarization was increased,
reaching a maximal level after about 1.5 to 2 min of depolarization (Figure 4.7). In
addition, inﬂux was a linear function of protein content of the PC12 cell suspension
(Figure 4.8). The effects of nifedipine, a Ca2+ channel antagonist, on 2 min of 45C3“
inﬂux into PC12 cells are shown in Figure 4.9. In two experiments, the approximate
1C5o value for block of 45Ca2+ inﬂux by nifedipine in non-differentiated PC12 cells
was 5 nM, and 100 nM nifedipine reduced inﬂux of 45Ca2+ by greater than 95 % (solid
symbols). This is consistent with previous studies which have shown that Ca2+ inﬂux
in undifferentiated PC12 cells is largely DHP-sensitive (T011, 1982; Takahashi and
Ogura, 1983; Kongsamut and Miller, 1986), therefore further repetitions were
deemed unnecessary. In NGF-differentiated cells, nifedipine reduced
depolarization-dependent 45Ca2+ inﬂux with an estimated 1C50 of approximately 5 nM
(n=3) (Figure 4.9, open circles). When compared to untreated controls, nifedipine
signiﬁcantly reduced ”Ca“ inﬂux at all concentrations tested in differentiated PC12
cells. Although 45Ca2+ inﬂux was largely DHP-sensitive, 10.4 i 0.9% of control inﬂux
remained in cells treated with 100 nM nifedipine, possibly indicating a DHP-insensi-
tive component. Higher concentrations of nifedipine were not tested.

In undifferentiated PC12 cells, MeHg reduced 45Ca2+ inﬂux with an ICso of

approximately 50 u M, and 250 u M MeHg blocked 45C3“ inﬂux completely (Figure

106

 

 

 

X 3 20[
3 x
2E o/I\o

9- 10 ' o—O/l 1' .
t. a /11 1
0 = ,o
3 O ’*

E 0L - . 7,

O 100 200 300 600

TIME (sec.)

Figure 4.7. Inﬂux or ”Ca“ into PC12 cells vs duration of depolarization. ”Ca2+ inﬂux into PC12 cells
induced by depolarization in elevated [K’] solutions as a function of time. The values shown are the
mean 1 SEM of three experiments, where the values for any one experiment are the average of three

replicates.

   

107

 

 

 

O 250 500

4"Ca“INFLUX (nmolx 1 0")

pg PROTEIN/5 O |JL

Figure 4.3. Inﬂux of 4sea” into PC12 cells as a function or protein concentration. Values from a
single representative experiment comparing 45Ca2“ inﬂux with protein content of the PC12 cell
suspension. The line is the linear least-squares regression of inﬂux on protein content. Values for inﬂux
are the average of three replicates from a single representative experiment.

108

 

 

 

 

 

 

 

100 P
’ -0- -NGF
x :2 75 " --O-- +NGF
D O
E “E
z o 50 -
"" O
(U .
0 °\0 is
ID v 25 ' '0 ..... g
V ‘\
I \ \\‘*
ox?
0 A AA]. AA Al ; A-AAA.

[NIFEDIPINE] (mm

Figure 4.9. Eﬂ'ects of nifedipine on depolarization-induced inﬂux of 4E’Ca“ in PC12 cells. Inﬂux was
measured by the addition of 50 pl of nonodifferentiated (open symbols) and NGF-diﬁ'erentiated (solid
symbols) PC12 cell suspension in normal (5 mM) [10], PC12 medium to 50 pl of elevated (100 mM)
[K+] or normal [K+] PC12 medium containing 1 u. Ci of 4""Ca“ and various concentrations of
nifedipine. The values shown are the mean of two (non-differentiated) and mean 1 SEM of three
(NGF-differentiated) different experiments. Values for any particular experiment are the average of
three replicates. For experiments using NGF-differentiated cells, SE bars are not shown if the SEM was
smaller than the symbol size. The asterisk (‘) indicates results that are signiﬁcantly less than control
(p< .05).

 

 

109
4.10, ﬁlled circles). In NGF-differentiated PC12 cells, nearly identical results were

obtained (Figure 4.10, open circles). MeHg concentrations of 50 uM and above
signiﬁcantly reduced 45Ca2+ inﬂux in undifferentiated and NGF-differentiated PC12
cells when compared to untreated controls. Thus, MeHg blocks a DHP-sensitive Ca2+
channel in PC12 cells with nearly identical potency to its block of Ca2+ entry in
synaptosomes. In addition, in two out of three experiments, MeHg completely
blocked 45Ca2" inﬂux into NGF-differentiated PC 12 cells. This may indicate an action
of MeHg on a DHP-insensitive Ca2+ channel in PC12 cells, but would require
electrophysiological experiments to conﬁrm. MeHg did not alter the depolarization-
independent entry of ”(332+ into undifferentiated or N GF-differentiated PC12 cells.
In addition, concentrations of MeHg as high as 250 llM did not affect the viability
of PC12 cells as measured by Trypan Blue exclusion, even after 1 hr of exposure
(data not shown). Thus, the effects of MeHg on “Ca2+ inﬂux into PC12 cells appear
to be due to disruption of voltage-dependent, channel-mediated Ca2+ inﬂux and not
to overt cytotoxicity to PC12 cells.

Nitrendipine Binding. The binding of [3H]-nitrendipine to synaptosomes
reached equilibrium after 10-15 min and was stable for 90 min (results not shown).
When the binding of [am-nitrendipine (concentrations ranged from 0.18 to 3 nM) to
synaptosomes was measured in normal (non-depolarizing) [K‘] solutions, a single,
high-affinity binding site was observed (Figure 4.11). Non-speciﬁc binding accounted
for approximately 50% of total binding. Scatchard analysis of the data (n=3) yielded
a KD value of 630: 160 pM and an apparent BM value of 130140 fmol/mg of

 

110

 

 

 

 

 

 

 

100 -
P
:3 75 ” -O- -NGF
3‘ o
-l :5 ’ -°0--+NGF
a s
._ o 50 -
m I
0 e3
3 25 .
r \\*
. “5 ..... *-
0 “U' - --_-_-. X...”
5 10 50 100

[MeHg] (uM)

Figure 4.10. Effects or MeHg on depolarization-induced ”Ca2+ inﬂux into PC12 cells. 4Sea“ inﬂux
into non-diﬂ'erentiated (solid symbols) and N GF-diﬂ'erentiated (open symbols) PC12 cells was measured
as described in Figure 4.9. The values shown are the mean :9: SEM for three separate experiments.
Values for any particular experiment are the average of three replicates. When SE bars are not shown,
the SE is smaller than the size of the symbol. The asterisk (‘) indicates results that are signiﬁcantly less
than control (p< .05).

111

protein. Hill slope was approximately equal to 1. These values are consistent with
values reported by others (Rampe et al., 1984; Turner and Goldin, 1985; Suszkiw et
al., 1986) for [3H]-nitrendipine binding in synaptosomes. In the presence of 100 uM
MeHg, the binding of [3H]-nitrendipine was also linear, however, the value for K0
was increased signiﬁcantly over the control KD value to 25201630 pM (n=3). The
apparent Bum was 200130 fmol/mg of protein in the presence of 100 MM MeHg. A
valid determination of BM could not be made due to the nitrendipine concentration
range used, therefore the BM value obtained in the presence of MeHg is only an
estimate and little direct information about the effects of MeHg on the number of
binding sites can be gained by comparison to control Bmax values. Under depolarizing
conditions, similar results were obtained. In the absence of MeHg, the KO value for
the binding of [3H]-nitrendipine was 11001300 pM. Although KD was higher under
depolarizing conditions, it was not signiﬁcantly different from the KB value in normal
[K‘] solutions. In the presence of MeHg, the KB value in elevated [K+] solutions was
533012100 pM and was signiﬁcantly higher than the respective control value.
Apparent BM values in the absence and presence of 100 uM MeHg were 200123
and 320174 frnol/ mg, respectively. Thus, MeHg decreased the afﬁnity of binding of
[am-nitrendipine to synaptosomes, indicating that it may act at or near the
nitrendipine binding site. As depolarization did not alter the characteristics of
[3H]nitrendipine binding to synaptosomes, the results of these experiments are

reported here, but are not illustrated in Figure 4.11.

112
m-Conotoxin Binding. The speciﬁc binding of [mu-to -conotoxin GVIA to

synaptosomes in the absence and presence of MeHg is shown in Figure 4.12A. Non-
speciﬁc binding accounted for approximately 20-30% of total binding. In the absence
of MeHg, Cng bound to synaptosomes with an apparent half-saturation of 0.1 to 0.2
nM. The maximum density of binding sites estimated by extrapolation of the
saturated component to the Y-axis is between 300 and 450 frnol/ mg protein. These
results are similar to results for Cng binding to synaptosomal membranes from rat
and chick brain (Abe et al., 1986; Cruz and Olivera, 1986; Marqueze et al., 1988). In
the presence of 100 uM MeHg, binding of Cng to synaptosomes was not
signiﬁcantly different from that of control. Because the actions of MeHg at the
neuromuscular junction (Traxinger and Atchison, 1987b) and in synaptosomes are not
readily reversible (Shafer and Atchison, 1989), synaptosomes were pretreated with
MeHg prior to measuring Cng binding. This pretreatment did not change the
characteristics of Cng binding (results not shown).

In undifferentiated PC12 cells, speciﬁc binding of [mm-o -conotoxin in the
absence of MeHg was saturable (Figure 4.12B). The apparent half saturation was less
than 0.1 nM Cng. The apparent BM was approximately 25-50 pmol / mg protein. In
the presence of 100 llM MeHg, the amount of [mike -conotoxin speciﬁcally bound
was reduced signiﬁcantly when binding was measured at Cg'I‘x concentrations
between 10 and 500 pM. These results indicate that MeHg interacts with the receptor

for to -Cng on PC12 cells, but not on synaptosomes.

 

113

 

 

 

 

 

 

 

0.07
a A CONTROL

UJ
1
L 0.05
\
0
§
0. 0.02
CD

0.00

5 27 48 7o
BOUND (pM)

Figure 4.11. Effects of MeHg on nitrendipine binding to synaptosomes. Speciﬁc binding of [sin-nitren-
dipine to synaptosomes in the absence (solid triangles) and presence (Open circles) of 100 u. M MeHg
in normal [K+] solutions. Values shown are the average of three replicates from a single representative
experiment»

114

 

 

 

 

 

0-60 i o CONTROL
A 0 MeHg I
0.40 r (T --------- i
/o
O 20 l ”x 1
o g/x.’
A" 1
A“
0.00 '1’

 

0.0 0.1 0.2 0.3 0.4 0.5

B i
/—

i e
l

50'

i I
25 . _/
2

Cng BOUND
(pmol/mg protein)

0 . .
0.0 0.1 0.2 0.3 0.4 0.5

[Cng] (nM)

 

r0; *ﬁ-l

Figure 4.12. Effects of Mei-lg on binding of conotoxin to synaptosomes and PC12 cells. Speciﬁc binding
of [mun-conotoxin GVIA to synaptosomes (A) and PC12 cells (B) in the absence (open symbols) and
presence (solid symbols) of 100 p M MeHg. Values shown are the mean 1 SEM of three experiments,
where the values for any given experiment are the average of three replicates. When SE bars are not
shown, the SE is smaller than the size of the symbol. The asterisks (‘) indicate values which are
signiﬁcantly different from their respective MeHg-free control (p< .05).

115
DISCUSSION

The goal of these experiments was to characterize the effects of MeHg on
nerve terminal Ca2+ channel properties using synaptosomes and to attempt to identify
the type or types of neuronal Ca2+ channels with which MeHg interacts in a deﬁned
population using PC12 cells. These results suggest that MeHg: 1) blocks 45Ca2+
inﬂux into synaptosomes in a voltage-dependent manner; 2) blocks Ca2+ inﬂux
through synaptosomal Ca2+ channels in a manner which does not depend on the
conﬁguration (open, resting or inactivated) of the Ca2+ channel; 3) acts on
synaptosomal Ca2+ channels to alter both the kinetics of apparent inactivation and
ionic selectivity; 4) is capable of blocking a DHP-sensitive Ca2+ channel in PC12
cells, and may also block a DHP-insensitive Ca2+ channel in NGF-differentiated
PC12 cells; and 5) alters nitrendipine and Cng binding to high-afﬁnity sites on
synaptosomes and PC 12 cells, respectively.

The block of 45Ca2“ inﬂux by MeHg increases as a function of time of
exposure in Na-free medium, but not in N a+-containing meduim. Under the former
conditions, Na+ /Ca2+ exchange does not contribute signiﬁcantly to 45Ca“ inﬂux;
therefore inﬂux occurs predominantly via Ca2+ channels (Suszkiw et al., 1986; Shafer
and Atchison, 1989). These results indicate that MeHg may block more Ca2+
channels as the time of exposure increases or alter the inactivation rate of Ca2+
channels or both.

MeHg differs from inorganic, divalent Ca2+ channel blockers such as Cd“,

Pb“, Hg2+ in that it is monovalent and has increased lipophilicity imparted by the

116
methyl group. The ability of MeHg to block 45Ca2+ inﬂux in a voltage-dependent

manner implies that the monovalent charge on MeHg may be important for the
interaction of this compound with Ca2+ channels. More recent results suggest
however, that lipophilicity may be critical for voltage-dependent actions of mercurials,
as only organic mercurials block synaptosomal 45Ca2+ inﬂux in a voltage-dependent
manner (Hewett and Atchison, 1990). Voltage-dependent block of cardiac Ca2+
channels by inorganic divalent cations is thought to arise due to binding of the cation
to sites within the pore of the channel (Hess et al., 1986; Lansman et al., 1986). Thus,
these results suggest that MeHg may act at a site within the channel.

It should be noted that the voltage-dependence of block could be due to non-
speciﬁc interactions of MeHg with the membrane rather than speciﬁc interaction
with Ca2+ channels. Sulfhydryl reactive agents such as mercuric chloride and
parahydroxymercuribenzoate depolarize the membrane (Huneeus-Cox et aL, 1966).
If MeHg were to cause signiﬁcant depolarization, it might also give rise to a "voltage-
dependent" block of inﬂux, as it may antagonize the block by MeHg at lower K+
concentrations by causing depolarization and Opening more Ca2+ channels than in
the control synaptosomes. In squid axon membranes, concentrations of MeHg less
than 100 p. M do not cause depolarization (Shrivastav et al., 1976) and in
neuroblastoma cells 40 u M MeHg results in only 4 mV of depolarization after 6 min
of exposure (Quandt et al., 1982). Thus, MeHg does not cause signiﬁcant
depolarization of the membrane in these preparations. However, in guinea pig

synaptosomes, 100 u M MeHg caused a depolarization of the membrane of 36 mV

117

after 1 min of exposure (Kauppinen et al., 1989). Lower concentrations of MeHg may
be less effective at depolarizing the synaptosomal membrane, as 10 llM MeHg
produces a maximal depolarization of 15 mV (Hare and Atchison, 1990). In the
experiments reported in the present study, exposure to MeHg was extremely short
(2 5). Therefore, although it is possible that MeHg causes some membrane
depolarization, it is unlikely that the magnitude of depolarization would be great
enough to affect the results signiﬁcantly.

Access to the resting or open and inactivated states of synaptosomal Ca2+
channels did not affect the potency or efﬁcacy of block of 45Ca2+ inﬂux by MeHg.
Different association and dissociation rates from the resting, open and inactivated
states of ionic channels is thought to underlie state-dependent block of IN, by local
anesthetics (Strichartz, 1973; Courtney, 1975 ; Hille, 1977) and the action of certain
Ca2+ channel blockers (Lee and Tsien, 1983). Results of the present study suggest
that MeHg does not have an increased afﬁnity for any particular state of the Ca2+
channel, as its blocking effects were equivalent following access to the channel in the
presence and absence of predepolarization. This is distinct from the blocking action
of DHPS which block 1c. in a state-dependent manner in cardiac myocytes (Uehara
and Hume, 1985) and the possible state-dependence of Ni“, La3+ and verapamil in
synaptosomes (Nachshen, 1985 ). It should be noted that synaptosomal preparations
may contain multiple types of Ca2+ channels which may have different rates of
inactivation. Thus, it is possible that these experiments have examined effects of

MeHg on two or more subpopulations of Ca“ channels and that MeHg has equal

118

afﬁnity for each subpopulation. However, apparent inactivation in these experiments
was best described by a single exponential process, which is consistent with previous
results (Suszkiw et al., 1986; 1989). Therefore, the results of the state-dependent
experiments have been interpreted as though the synaptosomal Ca2+ channel
population responded to depolarization in a homogeneous manner.
Predepolarization of synaptosomes in the presence of MeHg signiﬁcantly in-
creased the rate of apparent inactivation of 45Ca“ inﬂux. Inactivation of
synaptosomal Ca2+ channels with time results in decreased “Ca2+ inﬂux; MeHg may
decrease inﬂux further by blocking open/ active Ca2+ channels, giving rise to an
increase in the rate of apparent inactivation. Intuitively, one might expect that the
rate constants for normal inactivation and MeHg-induced block of Ca2+ channels
would differ, resulting in two exponential components of inactivation, unlike the case
observed in the present study. Nevertheless, the results of the present experiment
cannot rule out this hypothesis deﬁnitively. Alternatively, these results may indicate
an ability of MeHg to hasten the process of Ca2+ channel inactivation. MeHg may
act directly on the Ca2+ channel to increase the rate of apparent inactivation or may
act indirectly to increase inactivation of Ca2+ channels by causing release of Ca2+
from intracellular stores (Kauppinen et al., 1989), resulting in increased [Ca2+]i and
Ca2+-dependent inactivation of Ca2+ channels. This latter mechanism, although
possible, is unlikely to contribute largely to the increased rate of apparent inactiva-
tion of 45Ca2+ inﬂux in this experiment. It requires that MeHg diffuse through the

synaptosomal membrane and interact with the mitochondria to cause Ca2+ release.

119

Ca2+ must then diffuse back to the membrane and interact with Ca2+ channels to
cause inactivation. This sequence of events would have to take place extremely
rapidly, as apparent inactivation in the presence of MeHg occurs in less than 1 s. In
addition, the synaptosomes have been prepared, incubated in and predepolarized in
nominally Ca2+-free buffers. A redistribution of Ca2+ from the mitochondria would
be expected, lowering the overall mitochondrial Ca2+ content.

In addition to increasing the rate of apparent inactivation, MeHg also
decreases the ﬁaction of 45Ca2+ inﬂux which does not inactivate during the
predepolarization period. N on-inactivating 45Ca2“ inﬂux in synaptosomes is comprised
of both a channel-mediated component and a reversed Na‘ /Ca2+ exchange
component (Coutinho et al., 1984; Suszkiw et al., 1986; Shafer and Atchison, 1989).
Nerve terminal N a+ / Ca2 + exchange appears to have decreased sensitivity to block
by MeHg (T raxinger and Atchison, 1987b; Shafer and Atchison, 1989); therefore, if
MeHg caused signiﬁcant Ca2+ release from mitochondria, it could decrease this
phase of uptake by decreasing the driving force for exchange, or reversing exchange.
Alternatively, the effect of MeHg on the non-inactivating portion of Ca2+ inﬂux
suggests an action of MeHg on a non-inactivating population of Ca2+ channels. This
decrease may be the result of either a direct block of these channels, or increased
Ca2+-dependent inactivation due to MeHg-induced Ca2+ release from intracellular
stores.

MeHg also altered the relative inﬂux of ”Ca2+, 85Sr2+ and 133Ba2+ into

synaptosomes via Ca2+ channels. 85812+ inﬂux through Ca2+ channels was more

120

sensitive to block by MeHg than was inﬂux of 45Ca2“ or 133Ba“. This represents a
unique action by MeHg on Ca2+ channels when compared with other divalent cations
such as Ni“, which will block the inﬂux of Ca2+, Sr2+ and Ba2+ without altering the
relative inﬂux of these three cations (Nachshen and Blaustein, 1982). Although these
experiments do not deﬁne the site of action of MeHg, they suggest that MeHg may
affect the function of the selectivity ﬁlter of the Ca2+ channel. It has been suggested
that ionic selectivity in cardiac Ca2+ channels is due to differences in the afﬁnity of
divalent cations for binding sites within the pore of the channel, rather than a
physical structure which "ﬁlters" ions which can enter the channel (Hess et al., 1986;
Lansman et al., 1986). If this were the case for synaptosomal Ca2+ channels as well,
then these results suggest that Sr2+ is less effective at displacing MeHg from this site
than Ca2+ or Ba“. In addition, these results would be consistent with voltage-
dependent effects of MeHg.

The ability of nifedipine to block the inﬂux of 45Ca“ into PC12 cells is
consistent with the action of other DHP antagonists on Ca2+ inﬂux in this cell line
(Toll, 1982; Takahashi and Ogura, 1983; Kongsamut and Miller, 1986) and indicates
that depolarization-induced 45Ca2“ inﬂux into these cells is mediated largely by DHP-
sensitive or "L'Ltype Ca2+ channels. In addition, in the present study, approximately
10% of 45Ca’“ inﬂux in differentiated PC12 cells remained after exposure to 100 nM
nifedipine, indicating a DHP-insensitive portion of “Ca2+ inﬂux. Although it would
require electrophysiological experiments to conﬁrm, the remaining inﬂux may

represent inﬂux via an "N"-type conductance in PC12 cells. MeHg blocked 4sCaz“

121
inﬂux into both non-differentiated and NGF-differentiated PC 12 cells at

concentrations similar to those which block 45Ca2” inﬂux into synaptosomes (Shafer
and Atchison, 1989). In two out of three experiments, MeHg completely blocked
4S’Ca“ inﬂux into NGF-differentiated PC12 cells. This indicates that MeHg may be
capable of blocking a DHP-insensitive Ca2+ channel as well as a DHP-sensitive
channel. The ability of MeHg to block DHP-sensitive or "L"-type Ca2+ channels in
PC12 cells is important in light of recent studies which indicate that these channels
may be responsible for, or at least modulate Ca2+ entry associated with release of
some neurotransmitters from neurons (Perney et al., 1986; Atchison and O’Leary,
1987; Rane et al., 1987; Holz et al., 1988; Atchison, 1989) and PC12 cells (Takahashi
et al., 1985 ; Kongsamut and Miller, 1986). In addition, DHP-insensitive Ca2+
conductances have been reported to mediate in part release of [3H]-norepinephrine
from NGF-differentiated PC12 cells (Kongsamut and Miller, 1986). Thus, MeHg is
capable of blocking at least one and possibly two types of Ca2+ channels which are
associated with neurotransmitter release. This action may contribute in large
measure to the observation that MeHg depresses Caz+-dependent transmitter release
(Traxinger and Atchison, 1987b).

High afﬁnity binding of DHPs has been demonstrated in synaptosomes (Turner
and Goldin, 1985; Suszkiw er al., 1986; Dooley et al., 1987b; Dunn, 1988). However,
the relationship between binding of DI-IPs and effects of DHPs on 45Ca“ inﬂux into
synaptosomes is unclear. Several studies (Daniell et al., 1983; Suszkiw et al., 1986;
1989; Reynolds et al., 1986; Rivier et al., 1987) indicate that Ca2+ inﬂux into .

122

synaptosomes is insensitive to block by DHP antagonists, whereas, other studies
(Turner and Goldin, 1985; Dunn, 1989; Martinez-Serrano et al., 1989) indicate that
a portion of Ca2+ inﬂux in synaptosomes is sensitive to DHP antagonists. In either
case, the concentrations Of DHP required to block 45Ca2+ inﬂux are in great excess
of those at which DHPs bind speciﬁcally to synaptosomes. In present study,
[ﬁn-nitrendipine bound to a single class of high-afﬁnity sites in rat forebrain
synaptosomes. The values for KD and Bmu correlate well with values observed by
others for nitrendipine binding in synaptosomes (Turner and Goldin, 1985; Suszkiw
et al., 1986). DHP agonists and antagonists are thought to bind to Ca2+ channels
more readily when the channel is in the open or inactivated state, respectively
(Uehara and Hume, 1985 ). However, depolarization of the synaptosomes did not
readily affect the afﬁnity of nitrendipine binding or the maximum number of binding
sites. The binding afﬁnity of the DHP antagonist (+ )-PN200-110 to synaptosomes
was decreased by KI-induced depolarization (Dunn, 1989). Although other
explanations are possible, the lack of effect of depolarization on the afﬁnity of DHP
binding would be consistent with the suggestion that DHP binding sites in
synaptosomal preparations are associated with fragments of postsynaptic membrane
which remain attached to the synaptosomes (Suszkiw et al., 1989). These fragments
could not maintain a membrane potential and hence would not respond to changes
in K" concentration. The afﬁnity of nitrendipine binding to synaptosomes was
decreased in the presence of a concentration of MeHg which readily blocks ”Ca2+

inﬂux in synaptosomes (Shafer and Atchison, 1989) and DHP-sensitive inﬂux in PC12

123
cells. The ability of MeHg to compete with nitrendipine for a high-afﬁnity binding

site in synaptosomes is consistent with its ability to block DHP-sensitive channels in
PC12 cells. Experiments to demonstrate speciﬁc binding of nitrendipine to intact
PC12 cells were unsuccessful, as appreciable speciﬁc binding was not observed. The
author is unaware of any reports of speciﬁc binding of nitrendipine to intact PC12
cells.

Caution must be observed in correlating the results of binding studies in
synaptosomes with ﬂux studies in PC12 cells and concluding that MeHg blocks a
DHP-sensitive Ca“ channel in synaptosomes. According to one model for the
movement of Ca2+ through DHP-sensitive cardiac Ca2+ channels, higher concen-
trations of Ca2+ are necessary to observe a Ca“ current (Hess and Tsien, 1984) than
are normally employed in experiments with synaptosomes. Thus, ﬂuxes measured in
these experiments may not necessarily be mediated by DHP-sensitive Ca2+ channels.
As shown in Figure 4.5, MeHg will block 45Ca2+ inﬂux into synaptosomes at con-
centrations of Ca2+ high enough to mediate ﬂux via btype channels. However, the
contribution, if any, of “Ca“ ﬂux into synaptosomes mediated by L-type channels
under these conditions has not been determined.

The polypeptide, to -conotoxin GVIA, binds to Ca2+ channels at a site which is
distinct from the DHP binding site (Abe et al., 1987; Cruz and Olivera, 1986;
Marqueze et al.; 1988; Sher et al., 1988) and in some axon terminals is thought to be
associated with transmitter release. As opposed to nitrendipine, MeHg did not alter

the binding of Cng to synaptosomes. This indicates that MeHg does not interact

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124
with this site in synaptosomes, or that Cng displaces MeHg from this site. The

effects of MeHg on 45Ca2+ uptake in synaptosomes are only partially reversible by
increasing [Ca2+]c (Shafer and Atchison, 1989), and at neuromuscular junctions are
only reversible under certain conditions (Traxinger and Atchison, 1987b). Thus, it
seemed likely that MeHg is tightly or irreversibly bound to receptors on the cell
surface. Consequently, experiments were designed to test whether pretreatment with
MeHg might alter Cng binding to its receptor in synaptosomes. Results of these
experiments suggest that MeHg does not interact with the Cng binding site in
synaptosomes, as Cng binding was not altered by pretreatment with MeHg or in the
presence of MeHg.

In contrast to the results of synaptosomal experiments, MeHg did alter the
binding of Cng to PC12 cells. The amount of Cng bound to PC12 cells was
decreased signiﬁcantly in the presence of MeHg at concentrations which effectively
block 45Ca2“ entry into this cell line. Thus, MeHg apparently interacts with the Cng
receptor in PC12 cells but not on synaptosomes. This suggests that the Cng
receptor, and hence perhaps the Ca2+ channels, in synaptosomes and PC12 cells are
different. to -Conotoxin is thought to interact with "N" and possibly "L" type channels
in mammalian neurons (Reynolds et al., 1986). In both synaptosomes and PC12 cells,
experimental results indicate the presence of only a single class of conotoxin
receptor, thus it is possible that Cng binds to one channel type in synaptosomes and
the other type in PC12 cells. However, it is difﬁcult to interpret the results of

conotoxin binding experiments, as the type(s) of Ca2+ channel(s) in synaptosomes are

125
unknown, and, in PC12 cells, only "N" type conductances are sensitive to block by

Cng (Plummer et al., 1989). While the latter indicates that m -conotoxin GVIA may
not block "L"-type channels in PC12 cells, it does not preclude binding to this channel
type from taking place. Thus, it is to determine which Ca2+ channel subtype Cng is
binding to in these experiments. Alternatively, Cng may be binding to a site in
synaptosomes and/or PC12 cells which is not associated with Ca2+ channels. Much
higher concentrations of Cng are required to block 45Ca2+ inﬂux in mammalian
synaptosomes than in avian or amphibian synaptosomes (Suszkiw et al., 1987), in
spite of the presence of high afﬁnity binding sites for Cng. Thus, interpretation of
these data beyond the conclusion that MeHg interacts with the Cng receptor in
PC12 cells, but not synaptosomes, would be purely speculative.

MeHg is a very reactive heavy metal and alters a number of neuronal
functions. Although effects of MeHg on Ca2+ channels are probably not solely
responsible for the neurotoxic actions of MeHg, the rapid action of MeHg on Ca2+
inﬂux into synaptosomes indicates that nerve terminal Ca2+ channels may be an
initial site of interaction of MeHg with neurons. In summary, the results of this study
suggest that MeHg interacts with nerve terminal Ca2+ channels in a manner unique
from inorganic divalent cations or DHPs. MeHg clearly interacts with a
DHP-sensitive Ca2+ channel in PC12 cells, but the relationship of this interaction to
MeHg’s effects on 45Ca’“ inﬂux in synaptosomes, and synaptic transmission at the
neuromuscular junction is unclear. In addition, MeHg does not block Ca2+ channels

in a state-dependent manner as do DHPs. MeHg is also unique from other

126

neurotoxic heavy metals in its ability to alter both the apparent inactivation kinetics
and ionic selectivity of ﬂuxes through the Ca2+ channel. Differences in lipophilicity

and charge may underlie the unique blocking action of MeHg on Ca2+ channels.

EFFECTS OF THE NEUROTOXICANT METHYLMERCURY
ON NEURONAL on2+ CHANNELS:
EFFECTS ON on“ CHANNELS INVOLVED IN TRANSMITTER RELEASE
Volume H
By

Timothy Joseph Shafer

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHI]..OSOPHY
Department of Pharmacology and Toxicology
and Institute for Environmental Toxicology

1991

CHAPTERFIVE

METHYLMERCURY BLOCKS N- AND L-TYPE Ca“ CHANNELS

IN NERVE GROWTH F ACTOR-DIFFERENTIATED

PHEOCHROMOCYTOMA (PC12) CELLS

127

128
ABSTRACT

Effects of methylmercury (MeHg) on whole cell Ba2+ currents in rat
pheochromocytoma (PC12) cells were examined. Based on biophysical characteristics
and sensitivity to to -conotoxin GVIA (Cng) and dihydropyridine (DHP) agonists and
antagonists, voltage-activated Ba2+ currents in PC12 cells were mediated by N- and
L-type Ca2+ channels. Addition of MeHg (10p M) to the extracellular solution caused
a rapid and complete block of current carried by 20 mM Ba“. The rate of block of
1,3,I by MeHg increased in a concentration-dependent manner between 1 and 20 ll M.
Increasing the frequency of stimulation from 0.1 to 0.4 Hz facilitated block of IBa by
MeHg. A 2 min application of 10 pM MeHg in the absence of stimulation also
reduced 18, by approximately 80%. Thus, block of 13‘ by MeHg is not state-
dependent. Additionally, MeHg blocked 13. when the membrane holding potential
was -40, -70 and -90 mV, indicating that both N- and L—type Ca2+ channels are
blocked by MeHg. Block of In. by MeHg was voltage-dependent at a membrane
holding potential of -40 mV, but not at holding potentials of -70 and -90 mV.
Decreasing the extracellular concentration of Ba2+ ([Ba2*]e) from 20 mM to 10 mM
increased the magnitude of block by MeHg from 45.6 to 77.3%. Increasing [Ba2"]e
to 30 mM mused no further antagonism of block. Block of IE, by MeHg was not
reversed by washing with MeHg-free solution. The ionic permeability of PC12 cell
Ca2+ channels was Ca2+ = Sr2+ > Ba“. In the presence of MeHg, all three divalent
cations were equally permeant. These results indicate that: 1) MeHg blocks N - and

L-type Ca2+ channels in PC12 cells at low micromolar concentrations; 2) block of

129 '

Ca2+ channels by MeHg occurs regardless of the state of the Ca2+ channel or
membrane holding potential; 3) block of L-type Ca2+ channels by MeHg is voltage-
dependent; 4) decreasing the extracellular [Ba2*] exacerbates the block by MeHg, but
washing cells in MeHg-free solution did not reverse block of Ca2+ channels by

MeHg; 5) the ionic permeability of Ca2+ channels in PC12 cells is altered by MeHg.

130
INTRODUCTION

Results of experiments presented in previous chapters conﬁrm important
differences in the action of MeHg on synaptosomal 45Ca2+ inﬂux and neuromuscular
transmission when compared to divalent cations. While increasing [Ca2+]e readily
reverses block by divalent cations of 45Ca2+ inﬂux into synaptosomes (Nachshen,
1984; Suszkiw et al. , 1985 ) and nerve-evoked transmitter release at the neuromuscular
junction (Nilson and Voile, 1976; Kober and Cooper, 1976; Cooper and Manalis,
1983), the effects of MeHg on synaptosomes (Atchison et al., 1986; Shafer and
Atchison, 1989) and at the neuromuscular junction (Traxinger and Atchison, 1987b)
are only partially antagonized by increasing [Ca2+]e. MeHg also alters the ionic
selectivity and apparent rate of inactivation of 45Ca“ inﬂux in synaptosomes and
blocks synaptosomal 45Ca2" inﬂux in a manner which is voltage-, but not state-
dependent (Shafer et al., 1990). Such effects are not typically observed with inorganic
divalent cations.

Furthermore, MeHg blocks depolarization-induced inﬂux of 45Ca2+ into PC12
cells and synaptosomes with similar potency and efﬁcacy (Shafer et al., 1990) and
alters binding of speciﬁc Ca2+ channel antagonists to their receptors on synaptosomes
and PC12 cells. These data provide preliminary indications that MeHg interacts with
Ca2+ channel types which are associated with neurotransmitter release. However, as
noted in the previous chapter, the results of binding experiments do not allow for
unambiguous determinations of channel types due to occasional lack of correlation

of functional effects with speciﬁc binding. In addition, the kinetic resolution of

131

putative channel-mediated events using ﬂux measurements is markedly slower than
for electrophysiological measurements of channel activity in single cells. Thus,
experiments were designed to make unambiguous determinations of Ca2+ channel
types affected by MeHg and to examine the effects of MeHg on channel function
using techniques with more physiologically relevant time resolution.

Calcium or barium currents from PC12 cells cultured in nerve grth factor-
(NGF) containing medium are mediated by separate channel entities with distinct
kinetics and pharmacological sensitivities resembling the L- and N-type Ca2+
channels (Plummer et al., 1989; Usowicz et al., 1990) described by Fox et al.
(1987a,b). These two channel types have been associated with neurotransmitter
release in several different preparations (Atchison and O’Leary 1987; Rane et al.,
1987; Holz et al., 1988; Atchison, 1989; Lemos and Nowycky, 1989). In the present
study, PC12 cells and the patch voltage-clamp technique (Hamill et al., 1981) were
used to examine directly the ability of MeHg to interact with N - and/ or L-type Ca2+
channels and to examine characteristics of Ca2+ channel block by MeHg such as
reversibility of block, state and voltage-dependence of block, and effects of MeHg on

Ca2+ channel inactivation and ionic permeability.

132
MATERIALS AND METHODS

Chemicals and solutions. Stock solutions (500p M) of methylmercuric chloride
(ICN Biomedicals, Plainview, NY) were prepared weekly in extracellular solution
containing the proper concentration and species of divalent cation, from which test
solutions were prepared daily. Stock solutions of nifedipine (Sigma, St. Louis, MO)
and (+)-(S)-202-791 (generously donated by Sandoz Ltd., Switzerland) in 95 %
ethanol were diluted in extracellular solution to the required concentrations (less
than 0.05% ethanol after addition to extracellular solution). 0) -Conotoxin GVIA was
purchased from Peninsula Laboratories (Belmont, CA). Unless otherwise noted,
extracellular solution contained (mM): HEPES (20), MgCl2 (1.0), d-glucose (10),
NaCl (115), and TEACl (20) pH 7.3. This solution was mixed with appropriate
volumes of 100 mM BaClz, CaCl2 or SrCl2 to yield the ﬁnal concentration of divalent
cation indicated in the text (usually 20 mM BaClz). Tetrodotoxin (TTX, 5 nM)
purchased from Sigma (St. Louis, MO) was added to block voltage-sensitive Na+
channels (Narahashi et aL, 1964). Internal (pipette) solution contained (mM): I-IEPES
(10), MgC12(1), d-glucose (10), TEACl (10), EGTA (5), CsCl (125) and ATP (Sigma,
St. Louis, MO) (2), pH 73. TBA", Ba“ (Hagiwara et al., 1978; Armstrong et al.,
1982) and Cs+ were used in external and / or internal solutions to suppress K+ current
in PC 12 cells. ATP was added to internal solutions to prevent time-dependent

rundown of current amplitude (Bean, 1985).

133
Culture of PC12 cells. PC12 cells were cultured at 37°C in a humidiﬁed 10%

CO2 atmosphere using Dulbecco’s modiﬁed Eagle medium (pH 7.3, GIBCO)
containing 10% horse serum, 5% fetal bovine serum and 2 mM HEPES. Inasmuch
as certain antibiotics have been shown to be capable of blocking Ca2+ channels
(Atchison et al., 1988), antibiotics were omitted from the culture medium. PC12 cells
were differentiated by addition of 100 ng/ml of 7s NGF. To maintain consistency
from experiment to experiment, cells from the 15th passage from our receipt of the
cell line were used for recording Ba2+ currents.

Current recordings. Cells were plated into 35 mm polystyrene dishes and
cultured for 5-7 days in 2 ml of medium containing NGF. Prior to recording Ba2+
current, the culture medium was removed and cells were washed 2-3 times and then
covered with 1 ml of extracellular solution. Test compounds were prepared in
extracellular solutions such that addition of a 500 pl aliquot to the dish would result
in the ﬁnal concentrations of test compound indicated in the text and ﬁgure legends.
This method ensured rapid mixing of solutions and maintained osmolarity of the
extracellular solution. Voltage-activated Ba2+ currents in PC12 cells were recorded
using the whole cell patch voltage-clamp method (Hamill et al., 1981). Resistance of
patch electrodes (1.2 mm glass, World Precision Instruments, New Haven, CT) was
between 2-6 M0 . The patch clamp circuit consisted of an Axon Instruments CV-1
headstage and Axopatch 1B patch clamp. Pulse protocols were generated and current
responses recorded on-line using a Compaq 386 micro computer and the pClampm

program interfaced to the Axopatch 13 via a Axon Instruments TL-l interface board.

134

For analysis of current-voltage relationships, a 255 ms pulse to each indicated test
potential was applied from a holding potential of -40, -70 or -90 mV. Leak current
was measured at the appropriate holding potential by pulses which were equal in
duration, but opposite in polarity and V2 the magnitude of the test potentials. To
examine the reversibility of MeHg-induced block of In. by washing in MeHg-free
solutions, inward (holding potential -90 mV, test potential + 10 mV, 255 ms pulses)
and leak currents were recorded from PC 12 cells in the absence of MeHg. MeHg was
then added to the extracellular solution for 1 min and the currents were recorded
again. All but approximately 100 - 200 ll] of extracellular solution were then removed
from the dish and replaced with a 1 ml aliquot of MeHg-free extracellular solution.
This was repeated 3 times to wash the cells and current was recorded again. Current
responses were ﬁltered at 10 KHz using a 4-pole lowpass Bessel ﬁlter. Following
scaling, linear leak and capacitive currents were subtracted from gross current using
the pClampTM program. Pulse protocols and ﬁlter settings for other experimental
protocols are described in the Results section. Series resistance remaining after
compensation was less than 2 M0 , as 80% or greater compensation was commonly
achieved. Recordings which contained indications of inadequate space clamp, such
as pronounced shoulders during current activation, were not included in analysis. All
recordings were made at room temperature (22-25°C). A minimum of 3 cells were
recorded from for any given experiment and dishes of cells were discarded after

addition of test solutions.

135
RESULTS

Description of In. in PC12 cells. The pharmacological and biophysical
properties of Ba“ current in differentiated PC12 cells were characterized in order
to conﬁrm the identity of current components presumably mediated by N - and/ or L-
type Ca2+ channels (Plummer et al., 1989; Usowicz et al., 1990). Ba2+ currents elicited
by a test potential to + 10 mV from holding potentials of -40 (Figure 5.1A), -70
(Figure 5.1B), or -90 mV (Figure 5.1C) contained non-inactivating and inactivating
components (-70 and -90 mV only). The time constant, 1: , for the inactivating
component of currents at a test potential of + 10 mV averaged 225 1 86 ms (n= 8) and
175139 (n= 15) ms in cells held at -90 and -70 mV, respectively. A family of Ba2+
currents is illustrated in Figure 5.2. The holding potential was -90 mV and test
potentials began at ~50 mV and were increased in 10 mV increments to +70 mV.

Components of Ba2+ current in PC12 cells are sensitive to block by the N-
channel antagonist Cng (Figure 5.3A). The inactivating component of current was
apparently eliminated, while a non-inactivating component remained. Nifedipine (20
nM), a DHP Ca2+ channel antagonist, substantially reduced 18, (data not shown),
whereas (+)-(S)-202-791 (<5 uM), a pure DHP Ca2+ channel agonist (Hof et al.,
1985), enhanced 13, during voltage steps (Figure 53B) and induced tail currents upon
repolarization (Figure 5.3, inset). Thus, Ba“ currents recorded from differentiated
PC12 cells cultured in this laboratory have pharmacological and biophysical proﬁles

consistent with currents mediated by N- and L-type Ca2+ channels, respectively.

136

 

 

 

-40—
mV _70___

~90—’

 

_
l —
—

 

Figure 5.1. inactivating and non-inactivating components of Ba“ current in PC12 cells. Ba2+ current
in diﬂerentiated PC12 cells in response to a voltage step to + 10 mV from membrane holding potentials
of ~40 (A), ~70 (B) or ~90 (C) mV. Command potentials shown below current traces were 255 ms long.
linear components of leak and capacitance have been subtracted. Current responses were ﬁltered at

10 1012.

137

 

 

 

Figure 5.2. Current responses to a series of test potentials. Current responses in a differentiated PC12
cell to test potentials between ~50 and +70 mV when the magnitude of the test potential is increased
in 10 mV increments. Membrane holding potential was ~90 mV and test potentials were 255 ms in
duration. Current responses were ﬁltered at 10 KHz and linear components of leak and capacitance
current have been subtracted.

138

C ng

 

 

 

 

 

 

 

 

 

 

CON
100 pA L—
30 ms
[—
8202-791
B "i
CON

W

 

 

8202-791 1

3°PAL_

30ms

Figure 5.3. Effects oral-conotoxin and (+)~8202~791 on Ba2+ current in PC12 cells. A) Eﬁect of Cng
(16 MM) on 13. in a diﬁerentiated PC12 cell. The current response is to a 255 ms step to + 10 mV from
a holding potential of ~90 mV. Current response is ﬁltered at 10 KHz. B) Effect of (+)~(S)~202-791 (<
5 nM) on 13. in a diﬁerentiated PC12 cell. The current response is to a 755 ms step to + 10 mV from
a holding potential of ~70 mV. Inset: Tail currents recorded upon repolarization to ~70 mV from a 20
ms step to + 10 mV prior to and after addition of (+)~(S)~202-791. Scale bars are 400 pA and 7 ms. All
current responses were ﬁltered at 10 KHz and linear components of leak and capacitance were
subtracted. Responses shown are the average of 5 trials prior to and after addition of Cng or (+)~(S)~
202-791.

139
Eﬂ‘ect of MeHg on In. in PC12 cells. The effects of MeHg on membrane

properties and currents were examined using the pulse protocol shown in Figure
5.4A. A depolarizing pulse was delivered to elicit inward current, followed by a
hyperpolarizing pulse so that the leak current in the cell could be measured. Linear
components of leak and capacitive current were not subtracted ﬁ'om these records.
Thus, effects of MeHg on inward current, presumably mediated by Ba“, leak current
and capacitive current can be examined in consecutive current traces. Addition of
MeHg (10 nM) to the extracellular solution resulted in a rapid block of inward
current (Figure 5.4A), but did not alter membrane leak current nor membrane
capacitance, indicating that the effect of MeHg is not due simply to disruption of
membrane electrotonic properties. When this depolarizing pulse was repeated once
every 5 5, addition of increasing concentrations of MeHg to the extracellular solution
resulted in a concentration-dependent increase in the rate and magnitude of block
of 13,, following leak subtraction (Figure 5.4B).

Ba2+ current amplitude in PC12 cells often increased during the ﬁrst 1-1.5 min
after adjusting whole cell capacitance and series resistance compensation controls
(Figures 5.4B and 5.5, data prior to MeHg addition), after which time current
amplitude remained stable for beyond 10 min. This initial increase in current may
be associated with exchange of cytosolic constituents and pipette solutions, resulting
in block of K+ channels by Cs+ and TEA+ and/ or phosphorylation of Ca2+ channels
by ATP-dependent mechanisms. To ensure that effects of MeHg were examined on

stable Ba2+ current, cells were held at rest for 2 min before recording 13. in all

140

 

 

 

 

 

 

O)
O
o
E
C
_m
u.
0
Z
9.
l-
0
<
0:
LL 0 100 200 300
TIME (s)

Figure 5.4. Eﬂ'ects of MeHg on currents in PC12 cells. A) Effect of 10 u. M MeHg on gross inward and
leak current measured using whole cell patch voltage-clamp techniques on a diﬂerentiated PC12 cell.
As shown in the command potential protocol below the current traces, membrane potential was clamped
at ~70 mV and current responses to the designated voltage steps (100 ms ea.) were recorded. This
- voltage protocol was repeated once every 5 5. Current traces shown were recorded immediately before
addition of MeHg (CON), and 25, 50 and 100 s after addition of MeHg to the recording solution.
Current responses were ﬁltered at 1 KHz and leak current has not been subtracted. B) Eﬁect Of
increasing concentrations of MeHg on peak Ba2+ current measured in response to the pulse protocol
in A after leak subtraction. Pulses were repeated once every 5 s and recording medium containing
MeHg was added after 100 s, as indicated by the arrow. Final concentrations of MeHg were: 0 (upright
triangles), 1 (solid circles), 2.5 (solid diamonds), 5 (solid squares), 10 (open circles) or 20 (inverted
triangles) u. M. Peak current in each trace was normalized to peak current measured prior to the
addition of MeHg. Values shown are the average of peak meat from 4—8 different cells and average
standard error values were 23% of mean values throughout the experiment.

141

subsequent experiments, except for those designed to examine frequency-dependence
of block. In addition, all experiments were completed within 10 min of adjusting
patch circuit controls.

Use-dependence of block of In. by MeHg. The ability of MeHg to interact with
Ca2+ channels in PC12 cells in a use-dependent manner was assessed by examining
effects of MeHg on Ba2+ currents elicited by stimulating PC12 cells at frequencies
between 0.1 and 0.4 Hz. At the fastest rate of stimulation, the time required for
MeHg (5 ll M) to block approximately 70% of the current (approximately 3 min) was
less than the time required for MeHg to block current at slower rates (approximately
6 min) of stimulation (Figure 5.5). Thus, increasing the frequency of stimulation, and
hence use of Ca2+ channels, appears to facilitate slightly their block by MeHg.

Although increasing the frequency of stimulation appears to facilitate block of
Is. by MeHg, Ba2+ current was reduced appreciably in MeHg-containing solutions
even at relatively low rates of stimulation (0.1 Hz). In addition, block of
synaptosomal 45Ca2“ inﬂux by MeHg is not state-dependent (Shafer et al., 1990).
Thus, experiments were designed to determine if activation of Ca2+ channels is
essential for the blocking action of MeHg. Accordingly, effects of MeHg on In. in
PC12 cells were examined following a period of inactivity during which MeHg was
added to the extracellular solution. The protocol was as follows: a series of 10
voltage steps from ~70 to + 10 mV (255 ms duration) was delivered at 3 5 intervals.
Leak current, which was subtracted after scaling and averaging, was then measured

by a series of 20 steps from ~70 to ~110 mV. After measurement of leak current, 5

142

FRACTION OF I
at 100 s

 

 

O 200 400 600
TIME (s)

Figure 5.5. Use-dependent effects of MeHg on In. in PC12 cells. Effect of increasing the rate of
stimulation on peak inward current in the absence (solid symbols) or presence of 5 11M MeHg (open
symbols) in differentiated PC12 cells. The pulse protocol used in Figure 5.4A was repeated at
frequencies of 0.01 (squares), 0.2 (triangles) and 0.4 (circles) Hz. Peak current in each trace was
normalized to peak current measured prior to the addition of MeHg. Values shown are the mean of 4-8
different cells and average standard error values were 24% of mean values throughout the experiment.
Current responses were ﬁltered at 1 KHz and leak current has been subtracted. Control values for 0.2
and 0.4 Hz were not diﬂ'erent from values for 0.1 Hz and are not illustrated.

143

or 10 uM MeHg was added to the extracellular solution, and 2 min later the voltage
protocol described above was repeated. When no MeHg was added to the
extracellular solution, no signiﬁcant changes were observed in peak or end current
(results not shown). After a 2 min exposure to 5 uM MeHg in the absence of
stimulation, the average amplitude of the first current response was reduced when
compared with the average amplitude of the ﬁrst current response in the absence of
MeHg. Peak and end currents were reduced by an average of 32% and 26%,
respectively (Figure 5.6, bottom panel). Addition of 10 uM MeHg resulted in
signiﬁcant reductions (paired t-test, p < 0.05) of 77 and 70% for peak and end
currents, respectively, when compared to control values (Figure 5.6, bottom panel).
In addition, 10 u M MeHg caused complete elimination of the inactivating component
of whole-cell current (Figure 5.6, top panel). in none of four cells to which 10 uM
MeHg was added could an estimate of r be obtained for the ﬁrst current response
in the presence of MeHg, and no current responses containing inactivating
components were observed.

Holding Potential- and Voltage-Dependence of Block by MeHg. Current-
voltage relationships for 13, in PC12 cells were obtained from holding potentials of
~40, ~70, and ~90 mV in the presence and absence of 10 uM MeHg (Figure 5.7). At
all three holding potentials, a 2 min exposure to MeHg decreased the magnitude of
peak Ba2+ current elicited at each test potential. There was no readily apparent
holding potential-dependence of the action of MeHg. Currents elicited by

depolarizing steps from a holding potential of ~40 mV were blocked in a voltage-

144

AWWI‘

 

 

 

IBcr
M“ 200 pA L—
30ms
ICON .Mel-lg

"oi PEAK CURRENT {

390 -

 

600

 

300

INWARD Ba CURRENT AMP. (pMUJ

 

 

5pm 109“

Figure 5.6. Resting block of 13‘ by MeHg in PC12 cells. A) In“ in a differentiated PC12 cell recorded
prior to (CON) and after MeHg (10 u M) was present in the recording solution for 2 min. B) Amplitude
of inward Ba2 current recorded before (solid bars) and after (striped bars) a 2 min. exposure to 5 or
10 it M MeHg. The asterisk (‘) indicates a signiﬁcant reduction in current amplitude in the presence
of MeHg when compared to control values. (Paired t-test, P< .05). Values shown are the mean: SEM
of 7 and 4 diﬂerent cells exposed to 5 or 10 u. M MeHg, respectively. Current responses are to a 255
ms voltage step to + 10 mV from a membrane holding potential of ~70 mV and were ﬁltered at 10 KHz.
Linear components of leak and capacitance current have been subtracted.

145

TEST POT. (mV)

-4O -20 O 20 4O 60
Tl/‘o

—
UN- /

A \‘O‘N '0---o- L ‘0’ /;

 

A 4' T 3/'
< ‘75 3/
Q h.p.=-40
v
'150
-60-40 '20 O 20 4O 60 8O
’0
”k? \5. T 5’
B \3 \°‘ ' I T I,~I/’ /
‘ ,o’
\I‘ <,\\C"°’ '/

‘" °°“ \--200 /3/
-0- Mai-lg “\l. l/ /..hp =-70
./.

 

-400

-60-40 ~20 O 20 4O 60 so
- R /,_
‘k.\\‘a\ ’5/25
C “It. {,9 -/'

(V -/'
inv/ ;/

4501/

°\" T h.p.--90

. Ba -20mM
LI/ I I
-700

PEAK CURRENT

 

Figure 5.7. Eﬂ'ects of MeHg at different holding and test potentials in PC12 cells. Current-voltage
relationships for 13. in differentiated PC12 cells recorded in the absence (ﬁlled symbols) and presence
(open symbols) of 10 uM MeHg. Voltage steps (255 ms) to the test potentials (TEST POT.) indicated
in the ﬁgure were applied from holding potentials of ~40 (A), ~70 (B) and ~90 (C) mV. Current
responses were ﬁltered at 10 KHz and following subtraction of leak and capacitance current, peak
current was measured. Results shown are the mean: SEM of peak current from 4 to 8 cells, each cell
received all test potentials.

146
dependent manner by MeHg. The magnitude of block by MeHg increased from 23

to 98% as the test potential was increased from ~20 to +40 mV.Block of 18a by
MeHg was not strongly voltage-dependent at holding potentials of ~70 and ~90 mV.
At the holding potential of ~90 mV, the magnitude of reduction of 18a by MeHg at
the most weakly and most strongly depolarizing test potentials (~30 and + 70 mV) was
greater than the magnitude of reduction of IBa by MeHg at test potentials which elicit
peak IBa (0 and + 10 mV). MeHg did not alter the test potentials at which activation
of [Ba or peak IBa were observed. However, MeHg caused a negative shift in the
apparent reversal potential of In, at all holding potentials.

To determine whether irreversible, voltage-dependent block of Ca2+ channels
by MeHg occurred, block of IBa by 5 and 10 u M MeHg was examined using a holding
potential of ~40 mV; test pulses (255 ms) were increased by 10 mV increments from ~
30 to +50 mV and then decreased in 10 mV increments back to ~30 mV (Figure 5.8).
If MeHg blocks Ca2+ channels in an irreversible, voltage—dependent, manner, one
would expect a greater magnitude of block as test pulses are decreased from +50 to -
30 mV than that observed when test potentials were increased from ~30 to +50 mV.
Current amplitudes and effects of MeHg were similar in each case, indicating that
MeHg does not bind irreversibly to Ca2+ channels in a voltage-dependent manner.

Effects of [Ba’*]e on block of In. by MeHg. Previous studies in synaptosomes
indicated that at non-saturating [Ca2*]e, increasing [Ca2+]: antagonizes somewhat the
magnitude of block of “Ca2+ inﬂux by MeHg (Atchison et al., 1986; Shafer and

Atchison, 1989). Therefore, it seemed prudent to determine if the action of MeHg

147

on In. depended on [Ba2+]c. Decreasing [Baz‘i]¢ resulted in decreases in the control
current as well as currents measured after 2 min exposure to 10 uM MeHg (Figure
5.9). At a test potential of + 10 mV, MeHg caused a 77.3% reduction when
compared to control current for solutions containing 10 mM Ba2+ and a 45.6% for
those containing 20 mM Ba“. In 30 mM Ba2+ solutions, inward currents were not
signiﬁcantly larger than in 20 mM Ba2+ solutions, indicating that these concentrations
saturate Ca2+ channels in PC12 cells. In 30 mM Ba2+ solutions, exposure to 10 NM
MeHg reduced IBa by 49.8% (results not shown). Thus, [Ba2+]¢ antagonizes the
blocking action of MeHg, but is not capable of overcoming completely the effects of
MeHg. As in the above current-voltage relationships, MeHg also caused a negative
shift in the apparent reversal potential for 18., and an outward current was observed
in the presence of MeHg when [Ba2*]e= 10mM.

Reversibility of MeHg-induced Block by Wash. To determine if MeHg acted
on Ca2+ channels in a fashion similar to inorganic divalent cations or if MeHg acted
on Ca2+ channels in an irreversible fashion, the reversibility of MeHg-induced block
of In. in PC12 cells by washing cells with MeHg-free solutions was examined. Effects
on 13, of exposure to 10 uM MeHg were not reversed by washing with MeHg-free
solutions in 4 attempts (results not shown). In one cell, block of In. by 5 uM MeHg
was also not reversed by washing with MeHg-free solutions (results not shown). In
contrast, block of In, by 1 u M Cd2+ was reversed readily by washing with Cd2+-free
solutions (results not shown). The ability of micromolarconcentrations of Cd2+ to

block In. is consistent with previous results in cultured cells (Boland and Dingledine,

148

TEST POT.(mV)

-40‘-20 0 20 4O 60
.9
Q. 7§§ 7,6
\ \ V ' ,
Ntin‘L~l/rl I .

\

A)

 

   

 

l
.b
O

I
N
O
O
N
O

.5
O
O)
O

 

PEAK CURRENT (
J

\\7 Y ..
”£0171;pr may

 

Figure 5.8. Voltage-dependent block by MeHg of in. in PC12 cells is not irreversible. Current-voltage
relationship for In. in differentiated PC12 cells in the absence (ﬁlled symbols) and presence (open
symbols) of 5 or 10p. M Mei-lg. From a holding potential of ~40 mV, test pulses (255 ms) were delivered
in increasing increments (circles) of 10 mV from ~30 to +50 mV, followed by decreasing increments
(triangles) of 10 mV from +50 to ~30 mV. Current responses were ﬁltered at 10 KHz. Following
subtraction of leak and capacitance current, peak current was measured. Results shown are the mean
1 SEM ofpeakcurrentfrom4ceils.

1:
Q.
Ce.

 

149

TEST POT. (mV)
-60- 40 ~20 o 20 4o 60 30

 

 

 

 

_th._¢__Tr - O”°
0\._2 O 0 ”0,4; _/
A \3 \(Pho’ .0 _/o
< A \ /e/.
0.. 3 ~ .
V --o- CON - 3 5 of
./'
IE ‘0'” MeHg \. h.p.=-9 0
$ [Ba]=1 OmM
g - 7 00
O ~6£‘-:40 ~20 0 20 40 60 - 80'
x B 3 \\ ll’ﬁ’ O
< 1L 1 x i/
E \\ 1 i/ i 3/
l O
\ 2’ T/ '
- 3 5 O /I
’\ h. p. =-9 0
'i\ \l/ [Ba]=20mM
- 7 00

Figure 5.9. Ba2+ can antagonize block of Ca2+ channels by MeHg. Current-voltage relationships for
13. in diﬂ’erentiated PC12 cells recorded in the absence (solid circles) and presence (open circles) of
10 M MeHg in recording solutions containing 10 (A) or 20 mM BaC12 (B). Voltage steps (255 ms)
to the test potentials (TEST POT.) indicated in the ﬁgure were applied from a holding potential of ~90
mV. Current responses were ﬁltered at 10 IQ-Iz and following subtraction of leak and capacitance
current, peak current was measured. Results shown are the mean 1 SEM of peak current from 4 to 8
cells, each cell received all test potentials.

150
1990), as is the ability of washing with Cd2*~free solutions to reverse the block of IBa

in cultured cells (Boland and Dingledine, 1990) and block Of ”Ca“ inﬂux into
synaptosomes (Nachshen, 1984) by Cd“. Thus, effects of MeHg on Ca2+ channels
are not readily reversible.

Ionic permeability of Ca2+ channels in PC12 cells. Currents mediated by Ba2+
(Plummer et al., 1989; Usowicz et al., 1990) and Ca2+ (Garber et al., 1989) have been
measured in NGF-differentiated PC12 cells. However, the author is unaware of any
previous reports comparing the permeability of PC12 cell Ca2+ channels for divalent
cations (Ca2+, Ba2+ and Sr“). MeHg altered the ionic selectivity of synaptosomal
Ca2+ channels by decreasing inﬂux of 8SSr“ to a greater extent than inﬂux of
radiolabeled Ca2+ and/ or Ba2+ (Shafer et al., 1990). Thus, currents mediated by Ca2+
channels using 10 mM Ca“, Ba2+ or Sr2+ as the charge carrier in extracellular
solutions were examined and effects of MeHg on ionic permeability of PC12 cell
Ca2+ channels were characterized. To prevent differences in cell size ﬁom inﬂuencing
the results, recordings were made using solutions of Ca2+, Ba2+ or Sr2+ which were
labelled as unknowns, and results from recordings made from 46 cells were pooled.
Only after current records had been analyzed was the identity of the divalent cation
in the extracellular solution revealed. The permeability sequence of Ca2+ channels
in differentiated PC12 cells was Ca2+ = Sr“ > Ba2+ when the holding potential was
~90 mV (Figure 5.10). This observation was consistent throughout several cell passage
numbers (10, 11 and 15) and days in culture (4, 5, 7 and 12) in the presence of NGF.

MeHg (10 nM) reduced current mediated by all three divalent cations. However, the

 

151

apparent ionic selectivity of Ca2+ channels for Ca2+ and Sr2+ over Ba“ was

eliminated in the presence of MeHg.

152

TEST POT. (mV)

-60-40 -20 O 20 4O 60 80
'__I~.V

 

\\L\o I ‘I/5 .//
_o--1OIIIMB|-\\H I./O., [Kl

i\/ ’
-A-10mMCa ‘{ ‘ 441’
L
\ ./
---10nwlst . \ I

\.,

 

-60-4O ~20 O 20 40 60 80

 

 

PEAK CURRENT (pA)

 

-200

Figure 5.10. MeHg alters ionic permeability or Ca2+ channels in PC12 cells. Current-voltage
relationships for current mediated by 10 mM Ca2+ (triangles), Sr2+ (squares) or Ba2+ (circles) in the
absence (upper panel) or presence (lower panel) of 10 u. M MeHg. Voltage steps (255 ms) to the test
potentials (TEST POT.) indicated in the ﬁgure were applied from holding potentials of ~90 mV. Current
responses were ﬁltered at 10 KHz and following subtraction of leak and capacitance current, peak
current was measured. Results shown are the meant: SEM of peak current from 14-15 and 6 cells in the
absence and presence of MeHg, respectively, each cell received all test potentials.

153
DISCUSSION

Results of the present study indicate that 1) NGF-differentiated PC12 cells
cultured in this laboratory contain both N- and L-type Ca2+ channels; 2) low
micromolar concentrations of MeHg block IBa in PC12 cells and the rate of block is
concentration-dependent; 3) increasing the frequency of channel stimulation may
facilitate block of 13, by MeHg, but channel use is not required for the blocking
action of MeHg; 4) block of 13, by MeHg is not holding potential-dependent; 5)
block of 18a by MeHg is antagonized, but not completely overcome, by increasing
[Ba2+]c; 6) block of 13:: by MeHg is voltage-dependent when the holding potential is ~
40 mV, but is not strongly voltage-dependent when the holding potential is ~70 or ~90
mV, 7) block of In, by MeHg is not reversed by washing with MeHg-free solutions;
8) the apparent ionic selectivity of whole cell current mediated by Ca2+ channels in
PC12 cells is Ca2+ = Sr2+ > Ba“, and MeHg alters the apparent ionic selectivity of
Ca2+ channels in PC12 cells. These results suggest that block of 18a in differentiated
neuron-er cells mirrors block of radiotracer ﬂux into synaptosomes. Moreover, the
blocking properties of MeHg appear to be unique from inorganic divalent metals.

NGF~differentiated PC12 cells express both N- and L-type Ca2+ channels
(Plummer et al., 1989; Usowicz _e_t a1, 1990). The presence of T~type channels in PC12
cells has also been reported (Garber et al., 1990). Due to variability between PC12
subclones it is important to characterize the biophysical and pharmacological
properties of Ca2+ channel types present prior to making conclusions about effects

of uncharacterized chemicals on Ca2+ channels in these cells. Ba2+ current from cells

154

cultured in the presence of NGF in this laboratory contained no inactivating
component when held at ~40 mV, and contained inactivating and non-inactivating
components when held at more hyperpolarized membrane potentials. The average
time constant for decay of the inactivating portion of the current was somewhat
slower than that reported previously for N-type channels and current in PC12 cells
(Plummer et al., 1989), although many individual cells contained currents with r
values of around 95 ms. Furthermore, Ba2+ current from cells used in these
experiments exhibited characteristic responses to DHP L-channel type agonists and
antagonists and were blocked by to ~conotoxin GVIA, which affects exclusively N-type
channels in PC12 cells (Plummer, et al., 1989; Usowicz et al., 1990). Thus, based on
both the biophysical and pharmacological characteristics of whole cell current, these
results clearly indicate the presence of N- and L-type Ca2+ channels in PC12 cells
cultured in this laboratory.

Block of Ba2+ current in PC12 cells is rapid and complete upon addition of 10
ll M MeHg to the extracellular solution. Leak and capacitative currents in PC12 cells
were not affected by MeHg, indicating that effects of MeHg are relatively speciﬁc for
channel function. Both N~ and L-type channels in PC12 cells are affected by MeHg,
as MeHg blocks Ba2+ current at all holding potentials tested. These results conﬁrm
results Of previous binding experiments which indicated that MeHg interacts with N-
and Ltype channels in PC12 cells and synaptosomes by interfering with Cng and
DHP binding, respectively (Shafer et al., 1990). The concentration-dependency for

block of 13. occurred at lower concentrations (1-20 u M) of MeHg than did block of

155
4S’Ca“ inﬂux (50—100 nM) by MeHg into PC12 cells (Shafer gt .al, 1990) and

synaptosomes (Shafer and Atchison, 1989). Although this discrepancy does not
appear extremely large, one must consider differences in divalent cation
concentration used for electrophysiological (10-20 mM Ba“) compared to ﬂux (0.05 -
1 mM Ca2+) measurements. There are small but signiﬁcant differences in the
techniques used to measure Ca2+ inﬂux and Ba2+ current; namely continuous and
comparatively long (1-10 5) intervals of depolarization yet brief exposure (only 1-10
5) to MeHg for ﬂux studies as opposed to short (100-250 ms), repeated step
depolarizations during a several minute period of exposure to MeHg for Ba2+ current
measurements. Higher concentrations of MeHg may be necessary to block Ca2+
channels due to the much shorter overall lengths of exposure to MeHg during ﬂux
assays.
Block of Na+ channels by local anesthetics (Strichartz, 1973; Courtney, 1975;
Hille, 1977) and Ca2+ channels by organic Ca2+ channel antagonists (Uehara and
Hume, 1985) has been described as “use-dependent", indicating that these compounds
bind preferentially to the open or inactivated state of the channel. The results of the
present experiments suggest that MeHg effectively blocks Ca2+ channels in the
resting state, yet the blocking action can be facilitated by channel use. Thus, the
action of MeHg on Ca2+ channels is not state-dependent. This is consistent with
results obtained using synaptosomes (Shafer et al., 1990). DHP Ca2+ channel blockers
substantially reduce 10, in the absence of prior stimulation and have use—dependent

actions only during high-frequency stimulation (Lee and Tsien, 1983; Uehara and

156
Hume, 1985). By contrast, the actions of verapamil and D-600 are strongly use-

dependent, whereas diltiazem exhibits both resting and use-dependent block of ICa
(Lee and Tsien, 1983; Uehara and Hume, 1985). Furthermore, multivalent inorganic
cations (Nachshen, 1985) act on synaptosomal Ca2+ channels in a use-dependent
manner. MeHg, like the organic Ca2+ channel blockers, is lipophilic, but it is also
similar to divalent cations in size and is charged. Thus, MeHg may have access to the
Ca2+ channel by more than one route, including diffusion through the membrane and
entry through the channel pore. Perhaps the combination of charge and lipophilicity
of MeHg may account for its ability to block Ca2+ channels in the resting state, while
increasing frequency of stimulation facilitates the block.

Addition of MeHg in the absence of stimulation consistently eliminated the
inactivating component of current. This action of MeHg is unique compared to that
of Cd2+ and Ni“, which do not alter the time course of decay of 13, in a dorsal root
ganglion cell line (Boland and Dingledine, 1990). N—type Ca2+ channels in PC12 cells
may be more sensitive to block by MeHg than L-type channels. Alternatively, MeHg
may impair the ability of N-type channels to inactivate during the voltage step. This
latter mechanism would also require a simultaneous reduction in single channel
conductance or number of open Ca2+ channels (open probability) by MeHg to
account for the decreased current amplitude when MeHg is present. Either
mechanism is consistent with the results of the present experiments; single channel
recordings would be required to distinguish between them. MeHg altered the

inactivation kinetics of synaptosomal 45Ca2“ inﬂux; the rate constant for inactivation

157

was increased and the amount of non-inactivating inﬂux was decreased (Shafer et al.,
1990). Thus, MeHg produces analogous actions on inactivating and non-inactivating
current in PC12 cells and synaptosomes.

MeHg blocked Ba2+ currents elicited by depolarizing steps to positive
potentials from holding potentials of ~40, ~70 and -90 mV. The blocking action of
MeHg was not strongly voltage-dependent at holding potentials of ~70 and ~90 mV,
except that the degree of block was less at test potentials near the peak of the
current-voltage relationship when the holding potential was ~90 mV. This may be
related to the ability of Ba2+ to antagonize the blocking action of MeHg, as the
driving force for Ba2+ entry through Ca2+ channels and the number of channels in
the open state is greatest at these test potentials. Block of IBa was voltage-dependent
at ~40 mV. Presumably, there is very little N-type current at this holding potential
due to steady-state inactivation of N channels (Fox et al., 1987a,b). Thus, MeHg
appears to block L-type Ca2+ channels in a voltage-dependent manner, a result
consistent with voltage-dependent block of MeHg on synaptosomal ”Ca2+ inﬂux
(Shafer et aL, 1990). The hydrophobic nature of MeHg may be important in this
regard, as organic, but not inorganic mercurials block 45Ca2“ inﬂux into synaptosomes
in a voltage-dependent manner (Hewett and Atchison, 1990).

Additionally, MeHg caused a negative shift in the apparent reversal potential
of In, at all holding potentials and for all Ba2+ concentrations tested. Under the
experimental conditions used, the reversal potential for In, is theoretically positive

to + 80 mV. An actual outward Ba2+ current is not expected under the present

158

experimental conditions, due to the presence of EGTA in the internal solution. We
cannot eliminate the possibility that MeHg interferes with block of K+ channels by
TEA+ or Cs“, but does not block K+ channels itself, thus giving rise to outward
currents in the presence of MeHg. Outward currents have also been observed in
isolated cardiac myocytes (Lee and Tsien, 1982) and in bovine adrenal chromafﬁn
cells (Fenwick et al., 1982) when K+ or Cs+ permeate Ca2+ channels at highly
positive test potentials. However, Ca2+ channel blockers such as D600 or Cd2+
effectively block these outward currents (Lee and Tsien, 1982). Thus, any potential
outward Cs+ currents should be blocked by MeHg. The present data do not suggest
any particular mechanism underlying this effect of MeHg.

Washing PC12 cells in MeHg-free solutions did not reverse the block of 13, by
MeHg in any of the cells tested, although block of IBa by Cd2+ was reversed readily
by washing. Furthermore, extracellular Ba2+ can antagonize, but not completely
overcome the blocking actions of MeHg. This is consistent with effects of increasing
[Ca2+]c on the effects of MeHg on synaptosomal ”Ca2+ inﬂux (Shafer and Atchison,
1989). These results are also consistent with previous experiments at the
neuromuscular junction which indicate that the effects of MeHg on synaptic
transmission are not readily reversed by washing with MeHg-free solutions and can
only be partially reversed by increasing [Ca2“],3 (Traxinger and Atchison, 1987b).
Block of neuromuscular transmission by Pb“ (Cooper and Manalis, 1983; Atchison
and Narahashi, 1984; Pickett and Bornstein, 1984), Cd“ (Nilson and Voile, 1976;

Cooper and Manalis, 1984a,b), or Co2+ (Weakly, 1973) is reversed by increasing

159

[Ca2+]c or washing with normal, metal-free solutions. This represents a fundamental
difference in the action of MeHg when compared to divalent heavy metals. The
mechanism by which MeHg interacts with Ca2+ channels in an essentially irreversible
manner is unknown, although reactivity of MeHg with sulfhyde groups on
membrane proteins and/ or the enhanced lipophilicity of MeHg due to the presence
of the methyl group could be involved.

Ionic permeability ratios in PC12 cells have not been previously reported.
Based on whole cell currents, the permeability of PC12 cell Ca2+ channels was Ca2+
= Sr2+ > Ba“. This ionic permeability sequence was somewhat unexpected, as Ba2+
usually is not the least permeant of the three cations based on single channel
recordings (Tsien et al., 1988). Addition of MeHg to the extracellular solution
resulted in a loss of apparent ionic selectivity of Ca2+ channels, a result consistent
with the ability of MeHg to alter ionic selectivity of synaptosomal 45Ca2+ inﬂux
(Shafer et al., 1990).

In conclusion, MeHg interacts rapidly with N- and L-type Ca2+ channels in
PC12 cells at low micromolar concentrations. Given the ability of MeHg to interfere
with binding of Ca2+ channel antagonists (Shafer et al., 1990), pharmacological
methods alone cannot be used to examine the effects of MeHg on individual Ca2+
channels subtypes. Further characterization of the effects of MeHg on the individual
Ca2+ channel subtypes will require single channel recording techniques.
Concentrations of MeHg which block In, in PC12 cells are consistent with those

which block evoked endplate potentials at the neuromuscular junction (Atchison and

Natal
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160
Narahashi, 1982). The ability of MeHg to block both N- and L-type channels is

important in this respect. At the neuromuscular junction, L-type Ca2+ channels can
have at least a modulatory role in neurotransmitter release (Atchison and O’Leary,
1987), but do not contribute to nerve-evoked release under normal conditions
(Atchison, 1989). L-type channels have also been implicated in release of substance
P ﬁ'om chick dorsal root ganglion cells (Rane et al., 1987; Holz et al., 1988) and 3H~
norepinephrine release from undifferentiated PC12 cells (Kongsamut and Miller,
1986). In rat sympathetic neurons (Hirning et al., 1988), peripheral neurons (Perney
et al., 1986), NGF-differentiated PC12 cells (Kongsamut and Miller, 1986), various
areas of the central nervous system (Dooley et al., 1987a) and presumably rat motor
nerve terminals (Atchison, 1989), N-type Ca2+ channels are implicated as the
predominant type involved in neurotransmitter release. Given the ability of MeHg
to block channel types which are associated with neurotransmitter release, block of
Ca2+ channels at the nerve terminal may be an important component of its action to

disrupt synaptic transmission.

CHAPTER SIX

EFFECTS OF METHYLMERCURY ON Na+ CHANNELS IN

NEUROBLASTOMA CELLS

 

161

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162
ABSTRACT

Effects of MeHg on whole-cell Na+ currents am) in neuroblastoma NIB-115 and
NG~108 cells were examined using patch-voltage clamp techniques. In neuroblastoma
cells, 10 EM MeHg reduced INa by approximately 50% after a 2 min exposure. MeHg
did not alter the activation time Of Na+ current, nor the time course of whole-cell
current inactivation. Na+ currents could still be recorded after 8 min of exposure to
MeHg. Effects of MeHg on NG~108 cells did not differ from those of MeHg on N1E~
115 cells. These results suggest that MeHg interacts readily with Na+ channels in

neuroblastoma cells.

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163
INTRODUCTION

Results of Traxinger and Atchison (1987b) indicate that MeHg disrupts action
potential conduction as well as Ca2+ channel function at the N MI. The focus of this
dissertation has been directed towards possible effects of MeHg on Ca2+ channels.
However, propagation of the action potential in mammalian motor nerves, the
stimulus for Ca2+ channels to open, depends on the function of Na+ and K+ channels
in the plasma membrane. MeHg blocks the action potential in squid giant axon
(Huneeus-Cox et al., 1966) and increases its threshold for excitation (Shrivistav et al.,
1976). Na” currents in neuroblastoma cells are decreased by MeHg while only small
reductions in K“ current are observed (Quandt et al., 1982). Furthermore, the
sulfhydryl agents p-chloromercuriphenylsulfonic acid and inorganic mercury reduced
Na" current in crayﬁsh axons (Shrager, 1977). Thus, in addition to Ca2+ channels,
Na’r channels are also a potential target for MeHg action in mammalian motor
nerves. Alterations in Na+ channel function by MeHg may therefore indirectly affect
Ca2+ channel function by altering patterns of cellular excitability.

In previous investigations of the effects of MeHg on action potential
propagation and Na+ channel function, concentrations of MeHg between 25 and 100
uM were required to alter Na“ channel function (Shrivistav et al., 1976; Quandt et
al., 1982), whereas concentrations between 5 and 20 u M were required to block Ba2+
current in PC12 cells (Shafer and Atchison, 1991). However, studies of effects of
MeHg on N a“ channel function have employed primarily intracellular voltage-clamp

techniques, while effects of MeHg on Ca2+ channel function has been examined using

164

whole-cell patch voltage-clamp techniques. Thus, it is difﬁcult to compare these
results directly due to the differences in techniques employed to study channel
function. Therefore, experiments were designed to examine effects of MeHg on Na+
currents measured using the whole-cell conﬁguration of the patch voltage-clamp
technique. By doing so, effects of MeHg on Na+ channels will be described under
conditions very similar to those for which effects of MeHg on Ca2+ channels have
been well characterized. This will provide additional information concerning the

ability of MeHg to affect a second type Of voltage-gated channel in neuronal cells.

eh'ci

165
MATERIALS AND METHODS

Na+ current recordings. Voltage-activated Na“ currents were recorded ﬁ'om
undifferentiated mouse neuroblastoma NIB-1 15 cells (provided by Dr. P. Cobbett of
Michigan State University) or CAMP-differentiated (7 days) NG~108 cells using the
whole cell conﬁguration of the patch voltage-clamp technique (Hamill et al., 1981).
These cell lines were used to examine effects of MeHg on Na+ current rather than
PC12 cells because consistent expression of Na+ current could not be achieved from
available stocks of PC12 cells. External solution contained (mM): HEPES (20),
MgC12(1.0), d-glucose (10), NaCl (115), and TEACl (20) pH 7.3. Internal or pipette
solution contained (mM): HEPES (10), MgCl2 ( 1), d-glucose (10), TEACl (10),
EGTA (5), CsCl (125) and ATP (2), pH 7.3.

To record Na+ currents, resistance of patch electrodes (1.2 mm glass) was
between 2-6 megohms and the patch clamp circuit consisted of an Axon Instruments
CV-l headstage and Axopatch 1B patch clamp. Pulse protocols were generated and
current responses recorded on-line using a Compaq 386 personal computer and the
pClampTM program interfaced to the Axopatch 1B via an Axon Instruments TL-1
interface board. Current responses were ﬁltered at 10 kHz. Linear leak and
capacitive current components were subtracted using a computer-generated protocol.
This protocol functions by using a series of small amplitude subpulses to compute
leak and capacitive current expected during a larger test pulse. Ideally, the subpulses
elicit no voltage-gated current. Hence the leak and capacitive current measured

during the subpulses can be averaged and scaled to that expected from the test pulse.

166
The magnitude of the subpulses is determined by dividing the magnitude of the test

pulse by the number of subpulses. For these experiments, a series of 10 subpulses
was used to measure leak. MeHg was added to 1 ml of bath solution in 500 ill

aliquots to yield the ﬁnal concentration indicated in the text.

167
RESULTS

Effects of MeHg on Na+ current in neuroblastoma cells. Addition of MeHg to the
extracellular solution rapidly reduced Na+ current in neuroblastoma cells. A 2 min
resting exposure of neuroblastoma cells to 10 pM MeHg reduced peak inward Na”
current by 20 ~ 50% at all test potentials examined (Figure 6.1A). However, in
contrast to effects of MeHg on whole-cell Ba2+ current (Shafer and Atchison, 1991),
MeHg did not alter the inactivation kinetics of whole-cell Na+ current. This is clearly
apparent if current recordings obtained from the same cell in the absence and
presence of MeHg are compared after scaling the MeHg-treated current response to
the same amplitude as the control response. (Figure 6.1, inset). MeHg did not affect
t , the rate constant for inactivation, at test potentials between ~40 and +40 mV
(Figure 6.1B). Also, MeHg did not alter signiﬁcantly the time to peak amplitude of
Na+ current in neuroblastoma cells (Figure 6.2). This is consistent with effects of
other sulfhydryl reagents on Na” channels (Shrager, 1977). Na+ current could be
recorded from neuroblastoma cells after 8 min of exposure to 10 EM MeHg (not
shown). This concentration of MeHg completely blocks In. in PC12 cells after 3-5
min exposure. Effects of MeHg on Na+ currents in NG~108 cells were not different
from those on N1E~115 cells (results not shown). Blocking actions of MeHg may be

altered by administration of hyperpolarizing prepulses (results not shown).

168

A TEST POTENTIAL (mV)
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TEST POTENTIAL (mV)

Figure 6.1. Effects or MeHg on Na“ channels in N1E-115 cells. A. Peak inward Na+ current-voltage
relationship in N 1E~115 cells recorded prior to (ﬁlled circles) and following a 2 min exposure to 10 u. M
MeHg (open circles). B. Time constant (tau) for inactivation of whole-cell Na+ current at test potentials
used in A measured prior to (ﬁlled circles) and following a 2 min exposure to 10 p. M MeHg (open
circles). All current responses were recorded using 10 ms test potentials from a holding potential of ~90
mV. ReSponses were ﬁltered at 10 kHz and values are the mean: SEM of six cells, each of which was
treated with MeHg. Inset: Top pair of traces are current responses to a test potential of ~10 mV prior
to (ﬁlled circles) and following a 2 min exposure to 10 u M MeHg (open circles). The lower trace is the
MeHg-treated response scaled to the same magnitude as the control response. Note that the kinetics
of decay do not appear to be altered by Mel-lg. Scale bars for the tOp pair of traces are 200 pA and 1
ms. The current responses shown are the average of ten responses.

169

 

 

2.0 ‘
-50 -20 1O 40

TIME To PEAK I", (ms)

TEST POT. (mV)

Figure 6.2. Eﬂ’ects of MeHg on time to peak Na+ current. The time required for inward Na+ current
to reach peak amplitude following a depolarizing stimulus. The values are the mean: SEM of 5 (solid
circles, control) or 3 cells treated with 10 u M MeHg.

170
DISCUSSION

Results of these experiments indicate that low micromolar concentrations of
MeHg disrupts N a+ channel flmction in clonal cells. Several previous studies
indicated that MeHg disrupts action potential propagation and Na+ channel function
(Shrivistav et al., 1976; Quandt et al., 1982), but effects of MeHg on Na+ channel
function had not been examined using patch-voltage clamp techniques. Shrivistav et
al., (1976) reported that concentrations of 25-100 llM MeHg decreased N 3" current
by 50% in squid axons after 15 min of exposure in the bath solution. Quandt et al.,
(1982) reported that a 3 min exposure to 20 uM MeHg reduced Na+ current by
approximately 10% in N1E~115 cells. Higher concentrations of MeHg (60 nM)
produced greater reductions (80 ~ 100%) in Na+ current (Quandt et al., 1982).
Results of present experiments indicate that 10 RM MeHg rapidly and effectively
blocks Na+ current in neuroblastoma cells. It is possible that the lower temperatures
(10°C) employed by Quandt et al. (1982) may account for some of the differences in
the effects of MeHg on neuroblastoma cells. Changes in temperature may alter
channel gating (Hille, 1984) or may inﬂuence ﬂuidity of the cell membrane; should
target sites important for MeHg-disruption of Na‘ channel function be located within
the membrane, changes in ﬂuidity may alter the ability of MeHg to reach these sites.

In addition to Na+ current, MeHg also blocks Ca2+ current in PC12 cells at
concentrations between 1~20 uM (Shafer and Atchison, 1991). This is unique when
compared to inorganic Ca2+ channel antagonists, in that MeHg affects two different

channel types with equal avidity. Although the present data (including effects of

171

MeHg on Ca2+ channels) do not suggest any particular mechanism responsible for
this ability of MeHg, at least three mechanisms could account for this effect. First,
like Na+, MeHg is monovalent. However, the Na” channel is not permeanated to
positively charged methylated amines (Hille, 1984), and therefore may not be
permeanated by MeHg. In addition, mercury is a transition metal; this class of metals
includes Hg“, Cd“, Ni“, Pb2+ and Zn2+ (Nachshen, 1984), which block Ca2+
channels by interactions with binding sites within the channel pore (Lansman et al.,
1986; Hess et al., 1986). Thus, MeHg may enter the pore of both Na+ and Ca2+
channels, resulting in block. Second, other Ca2+ channel antagonists, including the
DHPS, have also been shown to block N a+ channels in cardiac myocytes (Yatani and
Brown, 1985), although the concentrations of DHP required to block Na+ channels
were higher than those which block Ca2+ channels. PD 122860, is a DHP compound
which stimuluates Na‘ channel function and blocks Ca2+ channel function (Haleen
et al., 1989). In addition, veratridine and grayanotoxin block Ca2+ and Na“ channels
in the same concentration range in N1E~115 cells (Romey and Lazdunski, 1982). This
has led to the suggestion that similarities in the molecular structures of Na+ and
Ca2+ channels may underlie these interactions (Hosey and Lazdunski, 1988). There
is a high degree of sequence homology between the a subunit of the voltage-
dependent Na+ channel and the a 1 subunit of the DHP-sensitive Ca2+ channel from
skeletal muscle. The greatest area of homology exists within four repeated domains,
each containing six transmembrane segments, one of which presumed to be

responsible for voltage-sensitivity (Campbell et al., 1988). Perhaps MeHg, which is

172

lipophilic, interacts with these regions in both Na” and Ca2+ channels. The possible
effect of MeHg on steady-state inactivation of Na+ channels is interesting in this
regard. Recently, four putative Ca2+ channel cDNA molecules which are homologous
to the a 1 subunit of the DHP-sensitive Ca2+ channel from heart and skeletal muscle
have been isolated from rat brain (Snutch et al., 1990). Finally, ethanol has also been
reported to block both Na+ (Harris and Bruno, 1985) and Ca2+ channels (Greenberg
et al., 1987a; Marks et al., 1989) in different systems. Thus, disruption of lipid
environment may also reduce channel function.

MeHg alters both Na+ and Ca2+ channel function, however, there appear to
be differences in the characteristics of block of Na+ and Ca2+ channels by MeHg.
The most striking difference is the lack of effect of MeHg on inactivation kinetics of
whole-cell Na” current. In PC12 cells, 10 11M MeHg consistently eliminated the
inactivating component of Ba2+ current (Shafer and Atchison, 1991). Second, the
time courses may be different; block of Na+ channels by MeHg was not complete
after 8 min, whereas block of Ca2+ channels by 1011M MeHg was complete after 3-5
min. Although this difference was not statistically signiﬁcant, the phenomenon was
consistent.

In conclusion, MeHg interacts readily with Na+ channels in neuroblastoma
cells at concentrations which also block Ca2+ channels under similar conditions. The
ability of MeHg to interact with Na+ channels at low micromolar concentrations is
consistent with previous reports (Huneeus-Cox et al., 1966; Shrivistav et al., 1976;

Quandt et al., 1982) as well as effects of MeHg at the neuromuscular junction

173
(Traxinger and Atchison, 1987b). The ability of MeHg to interact with more than one

channel type at similar concentrations is unique when compared to inorganic Ca2+
channel antagonists. Furthermore, in contrast to effects on whole-cell Ba2+ current,
MeHg reduced whole cell Na+ current without producing apparent alterations of N a”
channel inactivation kinetics. Thus, in addition to Ca2+ channels, Na“ channels may

also be an important site of MeHg action in neuronal membranes.

CHAPTER SEVEN

EFFECTS OF METHYLMERCURY ON Na'r AND Ca2+

CURRENTS IN INTACT NEUROMUSCULAR JUNCTIONS

174

175
ABSTRACT

The ability of MeHg to block presynaptic Ca2+ and N a+ channels in intact
neuromuscular junctions was examined in mouse triagularis stemi motor nerves.
Potential changes arising from Na+ and Ca2+ channel function could be recorded
from the perineurial sheath surrounding motor neurons when K+ channels were
blocked by TEA and 3, 4~diaminopyridine. Two components of the Ca2+~dependent
potential were observed; a fast component of approximately 60 ms duration and a
long-lasting component of approximately 100 ~ 800 ms duration. MeHg (100 nM)
blocked both Na+ and Ca2+ components rapidly. In 2 of 5 preparations exposed to
50 uM MeHg, the Ca2+ channel-mediated component was blocked prior to block of
the Na+ channel-mediated component. In the remaining three preparations, both
Na*~ and Ca2+~dependent potentials were blocked at similar times. Following block
by MeHg, perfusing the preparation in MeHg-free solutions did not result in recovery
of Na+ or Ca2+ channel activity; nor did increases in intensity and/or duration of
stimulus to the intercostal nerves. In the presence of K" channel blockers, repetitive
ﬁring of nerves in response to a single stimulus was observed in 20 ~ 30% of the
triangulan's preparations; in two preparations treated with MeHg, repetitive ﬁring
decreased prior to block of the stimulus-induced N a+ / Ca2+ potentials. These results
suggest that MeHg blocks Na+ and Ca2+ channels in intact neuromuscular

preparations.

176
INTRODUCTION

The results of experiments presented in previous chapters provide strong
evidence that MeHg disrupts function of Ca2+ channels in nerve terminals and blocks
Ca” channel types which are thought to be involved in neurotransmitter release.
Effects of MeHg on synaptosomal 4S’Caz“ inﬂux and on Ba2+ currents through somatic
N- and L-type Ca2+ channels in PC12 cells are comparable in many respects; MeHg
alters ionic selectivity and inactivation, blocks Ca2+ channels in a voltage- but not
state-dependent manner, and still exerts blocking effects in spite of increasing [Ca2+],
These results suggest either that the same types of Ca2+ channels are present in each
preparation, or that MeHg interacts with a variety of Ca2+ types in a similar fashion.
Regardless, these results support the hypothesis that disruption of nerve-evoked
neurotransmitter release by MeHg is in part the result of block of Ca2+ channels at
the presynaptic terminal by MeHg. However, experiments performed previously have
not measured effects of MeHg on Ca2+ channel function in intact motor nerve
terminals. Thus, one cannot be certain that MeHg is indeed blocking Ca2+ channels
in intact motor nerve terminals. Furthermore, MeHg also blocks Na+ channels in
Cllltured cells (Chapter 6, Quandt et al., 1982) and may alter Na” channel flmction
in intact nerves (Traxinger and Atchison, 1987b). Therefore, experiments were
designed to examine effects of MeHg on Na+ and Ca2+ channel function in intact
m0tor nerves by measuring effects of MeHg on Na*~ and Can-dependent potentials

in the perineurial sheath of mouse motor nerves.

177

The perineurium is a layer of ﬂattened cells which surrounds mammalian nerve
trunks, forming a relatively impermeant barrier to small molecules and ions. Because
of this property, the perineurium functions as an electrical insulator around the nerve
trunk (Figure 7.1). Axial currents which move from the most distal nodes of Ranvier
into the nerve terminal form local circuits in the perineurium by returning to their
node of origin via the perineurial space (Gundersen et al., 1982; Mallart, 1985). By
placing a microelectrode inside the perineurial sheath near motor nerve terminal
regions, voltage changes resulting from inward Na+ currents in preterminal regions
and inward Ca2+ currents in nerve terminals can be recorded simultaneously
(Mallart, 1985; Penner and Dreyer, 1986). Furthermore, since the perineurium
contains multiple nerve ﬁbers, the signal to noise ratio is increased over "loose patch"
recordings (Brigant and Mallart, 1982; Mallart and Brigant, 1982) of Na" and Ca2+
currents from individual terminals (Penner and Dreyer, 1986). Thus, effects of
MeHg on Na+ and Ca2+ channels were examined simultaneously by using perineurial
recording techniques in intact motor nerve terminals of mouse triangular-lls stemz'

muscle.

178

 

 

 

 

 

 

 

 

 

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a_’_'_.--i/ ______ jif‘ _ _______ T3 3.5:,
no; / n L / n L

 

 

 

Figure 7 .1. Local circuits in the perineurial sheath of motor neurons. Top, simpliﬁed diagram of a
motor nerve terminal, with the terminal on the left. The current pathways are indicated by the dashed
lines. hn, heminodal region; n, node of Ranvier; p, perineurium; my, myelin sheath; a, axon. Bottom,
simpliﬁed equivalent electrical circuit. A corresponds to the external medium, B indicates the perineurial
space. Rm, membrane transverse resistance of the terminal; Rm, membrane transverse resistance at
the nodes; R and R. are the longitudinal resistances of the perineurial space and the axon, respectively.
The arrows indicate direction of current ﬂow.

Modiﬁed from: Mallart, J. Physiol. 368565-575 (1985).

179
MATERIALS AND METHODS

Na“ and Ca2+ voltage recordings from the perineurial sheath. Potential changes
arising from Na+ and Ca2+ channel function were recorded from the perineurial
sheath of motor neurons in mouse triangulanlr stemi muscle (McArdle et al., 1981).
The muscle and intercostal nerves were dissected from the ribcage of adult male
Swiss-Webster mice (30-50 g). Following cervical dislocation, the ribcage was
separated from the spinal column and bathed in oxygenated physiological solution
consisting of (mM): NaCl(135); KCl (5); CaCl2 (2); MgCl2 (1); HEPES (14) and d-
glucose (11), pH 7.4. This solution was replaced completely every 3-5 min while the
overlying intercostal muscles and ribs were removed. Upon completion of the
dissection (Figure 7.2), the muscle was immediately transferred to a perfusion
chamber and perfused at a rate of 1~2 m1 / min with oxygenated physiological solution.

To measure Na+~ and Ca2+ (Bard-dependent potentials in the perineurial
sheath, the muscle was perfused with solution containing (mM): NaCl (118); TEACl
(20); BaCl2 (5); MgCl2 (1); HEPES (14); d-glucose (11); 3,4-diaminopyridine (0.5)
and d-tubocurarine chloride (0.05), pH 7.4. The solution was saturated with 95 /5%
Oz/COZ. TEACl and 3,4-diaminopyridine were added to suppress K” channel activity
and d-tubocurarine was added to block post-synaptic ACh receptors, thereby
eliminating muscle twitch. The intercostal nerves were stimulated at 0.1 Hz by a
suction electrode; typical threshold values were 15-30 mV and 15-30 its duration.
Potential changes in the perineurial sheath were measured by glass microelectrodes

(1.2 mm O.D., 10 MD) ﬁlled with physiological solution. Signals were recorded using

180

a CV~1 headstage and an Axopatch 1D patch clamp ampliﬁer in the current clamp
mode. Voltage responses were stored on FM tape (Vetter Instruments) for later
analysis using a chart recorder (Gould Instruments) and/ or by digitization at a rate

of 1 point every 20 us and subsequent storage on a Compaq personal computer.

181

0

'l‘l'i'-,:H-‘/ O
I' {In . W/ e

 
        
 

   

,

 

Figure 7.2. Diagram of mouse niangdan': and muscle. The muscle, located on the inner surface of the
ribcage inserts into the sternum and the intercalations of the ribs. The overlying ribs and intercostal
muscles have been removed. Branches of the 2nd, 3'“, and 4‘“ intercostal nerves, which innervate the
triangulalis, are visible.

182
RESULTS

Effects of MeHg on Na" and Ca2+(Ba2*)~dependent voltage changes in the
perineurial sheath. Placing a glass microelectrode inside the perineurial sheath of
nerves in the triangularis stemi muscle resulted in a negative potential of ~2 to -5 mV,
consistent with previous reports (Mallart, 1985 ; Penner and Dreyer, 1986; Anderson
and Harvey, 1988). When perfusing the muscle with normal saline solution,
stimulation of the nerve was followed closely (approximately 0.8 ms) by a negative
deﬂection in voltage (Figure 7.3A); a small fast component (arrow) slightly preceded
a larger component of longer duration. Previously, it has been demonstrated that the
ﬁrst component arises from Na+ currents in the nodal and heminodal portions of the
nerve, whereas the second component arises from outward K+ current in the nerve
terminal (Brigant and Mallart, 1982; Mallart, 1985; Penner and Dreyer, 1986;
Anderson and Harvey, 1988).

When the solution perfusing the muscle contained 20 mM TEA and 500 uM
3,4-diaminopyridine, the potential consisted of a fast negative deﬂection followed by
a longer-lasting positive deﬂection (Figure 7.3B): demonstrated to arise from nodal
Na+ and terminal Ca2+ currents, respectively (Brigant and Mallart, 1982; Mallart,
1985; Penner and Dreyer, 1986; Anderson and Harvey, 1988). Addition of TTX (5
11M) to the perfusing solution blocked the Na+ component of the potential (results
not shown). Two components of positive potential were observed: a fast transient of
approximately 60 ms in duration (Figure 7.3C), and a long-lasting (100 ~ 800 nls)

potential of varying duration (Figure 7.3D,E). These results are consistent with those

183

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 73. Voltage responses recorded from perineurial sheaths. A) Na+ /I(+ voltage potential
recorded from perineurial sheaths near nerve terminal regions. The arrow indicates the shoulder arising
from Na+ current in the nodal and heminodal regions. B) Na" /Ca2+ voltage potential recorded in the
presence of TEA and 3,4-diaminopyridine in 5 mM Ba2+ solutions. C-E) Na" Ca2+ voltage potentials
from the same preparation demonstrating rapid (C) and long-lasting (D, E) Ca + responses. Responses
are not averaged or summed and were recorded in response to intercostal nerve stimulation at a rate
of 0.1 Hz. Scale bars are 035 mV and 2 ms (A), 0.5 mV and 4 ms (B), 0.5 mV and 200 ms (C-E).

184

of Penner and Dreyer (1986) in which two types of Ca2+ potentials were
demonstrated in mouse motor nerve terminals; presumably due to the presence of
two different Ca2+ channel subtypes. Microscopic resolution was limited (15OX) to
an extent which prevented placement of electrodes within the ﬁnest branches of the
perineurial sheath, which contain 3-4 nerves (Penner and Dreyer, 1986). Under these
conditions the number of nerves contributing to potentials cannot be determined.
Therefore, quantitative assessment of effects of MeHg on potential amplitude
provide little definitive information concerning effects of MeHg on individual nerves
or terminals. Amplitude scale bars have been included in some ﬁgures to provide
information concerning relative amplitudes. The amplitude of the Ca2+ channel-
dependent potential typically was between 0.5 and 1.5 mV in the absence of MeHg,
depending on the preparation.

The positive portion of the potential was dependent on the presence of
divalent cations in the perfusing solution. Preparations which were dissected in 2 mM
Ca2+ and then perfused with nominally Ca2+~ and Ban-free solution exhibited small
and short duration Ca2+ potentials, presumably due to residual Ca“ in the muscle
tissue (Figure 7.4A,B, left trace). Addition of 5 mM Ba2+ to the perfusing solution
increased both the magnitude and duration of the Ca2+ potential (Figure 7.4A,B,
right trace). Furthermore, "pufﬁng" 1 mM cobalt into the perfusion chamber
decreased the amplitude and duration of only the positive potential in a reversible
manner (Figure 7.4B). These results are consistent with results of Mallart (1985) and

Penner and Dreyer (1986) in which these potentials were demonstrated to arise from

185
Ca2+ channel function in nerve terminals. Addition of MeHg (100 nM) to the

perfusing solution caused a reduction in amplitude and eventual block of both the
Na” and fast Ca2+ potentials after a delay of 3-4 min (Figure 7.5). Both Na” and fast
Ca2+ potentials were decreased simultaneously in three preparations. In contrast to
the results of Traxinger and Atchison (1987b), washing with MeHg-free solution at
the time of block did not reverse effects of MeHg; even after 15-20 min. In addition,
increasing stimulus duration (up to 5 times threshold) and/or intensity (up to 10
times threshold) at the time of block and/or during wash also were ineffective in
reversing block of either the Na+ or the Ca2+ channel-mediated response by MeHg.

50 11M MeHg also rapidly decreased both Na+ and fast Ca2+ potentials in 4
out of 5 preparations. However, in 2 preparations, the fast Ca2+ component appeared
to be affected prior to the Na+ component. In three preparations (one had been
exposed to 100 p M MeHg), the long-lasting component of the positive potential was
decreased prior to block of Na” and fast Ca2+ potentials. These results suggest a
somewhat earlier or more speciﬁc effect of MeHg on nerve terminal Ca2+ channels.
Thus, MeHg is capable of blocking Ca2+ currents in intact motor nerve terminals. In
addition, effects of MeHg on speciﬁc components of perineurial waveforms indicate
that the decreases in signal amplitude are not due to disruption of the insulating
properties of the sheath by MeHg. As with 100 uM MeHg, these effects were not
reversible by washing the preparation in MeHg-free solutions or by increasing
stimulus intensity and/ or duration. Thus, block by MeHg of Na+ channels, and

possibly of Ca“ channels, appears to be irreversible under these conditions.

186

 

Figure 7.4. Ca2+ channel-mediated component of vol potentials in the perineurial sheath. When
perfusing with solutions containing no added Ca2+ or Ba +, the Ca2+ channel-mediated component of
the potential is small and of short duration (A and B, left traces). When 5 mM Ba“ is added to the
perfusing solution (A), or "puffed" into the perfusion chamber (B), the Ca2+ channel-mediated
component of the potential increases in amplitude as well as duration ( ' t traces). Scale bars are 9
msand025mVinAand0.125mVinB. C) WhileperfusinginSmMBa + solutionilrnMCo2+ was
"puffed“ over the muscle. The voltage traces were recorded prior to addition of Co I (CON), in the
presence of Co2+ (Co), and during washout of C02” (WASH). Scale bars are 025 mV and 20 ms.

187

 

 

 

Figure 7.5. Eﬂ'ects or MeHg on NaVCa“ potentials in the perineurial sheath. Nat /Ca2+ potentials
recorded prior to (A) and 0.16 (B), 1 (C), 2 (D), 3 (E) 4 (F) and 5 (G) min after addition of 100 pM
MeHg. Scale bars are 0.5 mV and 40 ms, except for B; for which the temporal bar is 80 ms. Note the
loss of the long-lasting component of the Ca2+ potential after 1 min.

188

When using K+ channel blocking agents to unmask Ca2+ channel activity in the
nerve terminal, repetitive ﬁring of neurons often occurs due to lack of repolarization
of the neuronal membrane by outward K+ currents. Other investigators have used
small concentrations of local anesthetics fo prevent repetitive ﬁring (Brigant and
Mallart, 1982; Mallart, 1985 ; Penner and Dreyer, 1986; Anderson and Harvey, 1987;
1988). Since MeHg may affect axonal Na‘ channels and decrease excitability
(Shrivistav et al., 1976; Quandt et al., 1982; Traxinger and Atchison, 1987b), use of
local anesthetics might mask or confound possible effects of MeHg on Na+ channel
function in intact nerves. Thus, no pharmacological manipulations were undertaken
in these experiments to suppress repetitive ﬁring of motor neurons. Repetitive ﬁring
was observed in approximately 20 to 30% of triangularis stemi preparations (Figure
7.6A,B), only 2 of which were treated with MeHg. Addition of MeHg (50 nM) to
these preparations resulted in a suppression of repetitive ﬁring prior to block of

stimulus-evoked N a” and fast Ca2+ potentials (Figure 7.6C-F).

189

 

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Figure 7.6. Eﬂ'ects of Mei-lg on repetitive ﬁring of motor nerves. An example of repetitive ﬁring in a
motor nerve recorded prior to (A,B) and 20 (C), 70 (D), 80 (E), and 90 (F) s after the addition of 50
#M MeHg. Note also the loss of the long-lasting portion of the Ca2+ response in this example. Scale
bar is 700 ms.

190
DISCUSSION

Results of these experiments indicate that MeHg: 1) appears to block both
N a” and Ca2+ currents in intact neuromuscular preparations, and 2) under the
conditions used in this study, blocks presynaptic Na+ currents in a manner which is
not reversible by increasing stimulus intensity or duration.

At concentrations of 50 and 100 NM, MeHg blocked the inward Na+~
dependent component of the voltage potential. In addition, MeHg suppressed
repetitive ﬁring of motor nerves prior to signiﬁcant reductions of stimulus-induced
Na+ or Ca“ potentials. Repetitive ﬁring of the nerve is a function of membrane
excitability, and may occur under conditions of these experiments due to lack of
repolarization of the nerve by outward K+ current before Na+ channels recover ﬁom
inactivation. The ability of MeHg to suppress repetitive ﬁring is consistent with the
results of Traxinger and Atchison (1987b) which indicated that MeHg decreased
neuronal excitability. Block of a portion of the population of N a+ channels by MeHg
may prevent repetitive ﬁring by decreasing the number of N 3+ channels available for
opening immediately after an action potential propagates through a given region of
the nerve. Alternatively, MeHg has been demonstrated to depolarize neuronal
membranes (Shrivistav et “al., 1976; Hare and Atchison, 1990). Should this occur in
intact axons as a result of MeHg exposure, one might expect a decrease in nerve
excitability; depolarization might result in steady-state inactivation of Na‘ channels.

Therefore, fewer channels would be available for activation.

191
In addition to blocking the Na” potential, MeHg also reduced the fast Ca2+

potential recorded from the perineurial sheath. Because of effects on Na+ potentials,
decreases in Ca2+ potentials might simply have been due to failure of the action
potential to invade the terminal and activate Ca2+ channels. However, in two
preparations, the Ca2+ channel-mediated potentials were reduced prior to block of
the Na+ channel-mediated potentials. In addition, the long-lasting component of the
Ca2+ potential was blocked prior to block of the Na+ and fast Ca2+ potential in three
preparations. Under these conditions, the action potential successfully invaded nerve
terminals, since the fast Ca2+ potential was unaffected. These results indicate that in
addition to N a” channels, Ca2+ channels in the presynaptic terminal are blocked by
MeHg. Effects of MeHg on neurotransmitter release may be due to effects on both
Na+ and Ca2+ channels in the presynaptic nerve; consistent with predictions of
Traxinger and Atchison (1987b).

The fast component of the Ca2+ potential is presumed to be associated with
neurotransmitter release (Mallart, 1985; Penner and Dreyer, 1986), although the
Ca2+ channel type(s) which contribute to this component have not yet been
determined. This component is not affected by organic Ca2+ channel blockers
(Penner and Dreyer, 1986) or to ~conotoxin GVIA (Anderson and Harvey, 1987), but
is blocked by millimolar concentrations of divalent cations (Penner and Dreyer,
1986). Thus, the pharmacological characteristics of this component cannot be
classiﬁed by the T, N, and L nomenclature system (N owycky et al., 1985a). The long-

lasting component of the potential is not thought to be coupled directly to

192

neurotransmitter release (Penner and Dreyer, 1986). However, no evidence exists to
preclude this mechanism from contributing to nerve-evoked release. This component
is sensitive to verapamil and diltiazem but not DHPS (Penner and Dreyer, 1986).
Thus, the Ca2+ channel types mediating this component are unknown. Although
MeHg blocks both N- and L-type Ca2+ channels in PC12 cells, the ambiguity over the
types of Ca2+ channels present in the motor nerve terminal makes it difficult to
determine whether or not MeHg is acting on N- and L-type channels in this
preparation.

Traxinger and Atchison (1987b) reported that block of EPPS in rat
hemidiaphram preparations could be reversed by washing in MeHg-free solutions and
increasing the intensity and/or duration of the stimulus to the phrenic nerve. In
addition, following reversal of initial MeHg-induced block, a subsequent block by
MeHg was relieved by increasing [Ca2+]c. In the present experiments, the effects of
MeHg on Na” and Ca2+ potentials were not relieved by washing in MeHg-free
solutions and / or by. increasing stimulus intensity or duration. The disparity of results
from this study with those of Traxinger and Atchison (1987b) may be due to the
conditions employed in this study to unmask Ca2+ channel-mediated components of
voltage changes in the perineurial sheath. Block of K+ channels by TEA and 3,4-
diaminopyridine will result in depolarization of the axonal membrane; should MeHg
further depolarize the membrane, decreases in excitability may result from Na+
channel inactivation. In addition, the high concentration of Ba“ used to measure

Ca2+ channel function may also stabilize the axonal membrane (Frankenheuser and

193
Hodgkin, 1957; reviewed by Hille, 1984). Indeed, Traxinger and Atchison (1987b)

reported that increasing [Ca2+]: along with addition of 4-aminopyridine prevented
reversal of MeHg-induced block of EPPS when stimulus intensity was increased.
Under the conditions of this study, therefore, it might be expected that MeHg-
induced decreases in nerve excitability may be difﬁcult to overcome.

In conclusion, effects of MeHg on Na+ and Ca2+ channel function in intact
motor nerves and terminals have been examined for the ﬁrst time. Consistent with
predictions from in vitro systems, the results indicate that MeHg disrupts function of
both channel types in intact nerve terminals. The ability of MeHg to block Ca2+
channels in intact motor nerve terminals supports the hypothesis that effects of
MeHg on Ca2+ channel function may contribute to block of neurotransmitter release

by MeHg.

CHAPTER EIGHT

SUMMARY AND CONCLUSIONS

194

195

This dissertation has presented results of experiments designed to examine the
following hypothesis: block of Ca2+ channels by MeHg may contribute to MeHg~
induced block of synaptic transmission at the motor nerve terminal. This hypothesis
was based on the experimental observations that block of synaptic transmission at the
neuromuscular junction (Traxinger and Atchison, 1987b), and of depolarization-
induced 45Ca2” inﬂux into synaptosomes by MeHg could, under certain conditions,
be antagonized by extracellular calcium (Atchison et al., 1986). Speciﬁcally,
experiments were designed to examine how MeHg disrupts Ca2+ channel function
and to determine types of Ca2+ channels affected by MeHg: particular emphasis has
been placed on Ca2+ channels located in nerve terminals and on Ca2+ channel types
which are presumed to mediate neurotransmitter release from central and/ or
peripheral terminals. The small size and close anatomical association of the motor
nerve terminal with muscle ﬁbers severely restrict the ability to measure directly Ca2+
channel function in intact NMJ preparations. Thus, out of necessity, some
experiments were performed on isolated nerve terminals or cultured cell lines. Based
on results of these experiments, one putative mechanism by which MeHg effects
neurotransmitter release at the N MJ is now better understood.

Preliminary experiments characterized the ability and nature of block by MeHg
of various components of Ca“ inﬂux into isolated nerve terminals. MeHg blocked
voltage-sensitive Ca2+ channels in synaptosomes; the component of ”Ca“ inﬂux
which has been associated speciﬁcally with neurotransmitter release from

synaptosomes (Blaustein, 1975; Floor, 1983; Leslie et al., 1985) was dramatically

196
reduced by MeHg (Atchison et al., 1986; Shafer and Atchison, 1989) Block of this

component was only partially relieved by increasing [Ca2+]: (Shafer and Atchison,
1989). Thus, in isolated nerve terminals, MeHg disrupts, in a potent and efﬁcacious
manner, function of Ca2+ channels demonstrated to mediate Ca2+ inﬂux responsible
for neurotransmitter release. The ability of MeHg to block Ca2+ channels in a
heterogeneous preparation may represent a common action of MeHg at all nerve
terminals. Furthermore, elucidation of the mechanisms underlying MeHg
neurotoxicity will require more information regarding effects of MeHg in the central
nervous system. Inasmuch as alterations in Ca2+inﬂux may contribute to acute effects
of MeHg on neurotransmitter release at the N M], these data suggest that alterations
of Ca2+ channel function should be considered in future studies of effects of MeHg
in the central nervous system.

Further characterization of MeHg effects on synaptosomal Ca2+ inﬂux
demonstrated that MeHg altered Ca2+ channel functions including ionic selectivity
and inactivation. In addition, block of Ca2+ channels by MeHg was demonstrated to
be voltage~ but not state-dependent. MeHg also altered binding of speciﬁc Ca2+
channel ligands to their receptor sites in synaptosomes and PC12 cells (Shafer et al.,
1990). These effects of MeHg were unique when compared to effects of divalent
inorganic cations such as Cd“, Co2+ and Pb2+ (Nachshen, 1984; Suszkiw et al., 1985).
The ability of MeHg to alter binding of to ~conotoxin GVIA and the DHP nitrendipine
suggested that MeHg may interact with receptor sites associated with N ~ and L-type

Ca2+ channels, respectively.

197

The role of voltage-sensitive Ca2+ channels in mediating neurotransmitter
release is crucial. To date, there are three well characterized Ca2+ channel types; N,
L and T (Tsien, 1988). The N- and L-type channels have been proposed to regulate
neurotransmitter release from a variety of central, peripheral and clonal nerve cells.
The patch-voltage clamp recording technique was used to examine the ability of
MeHg to block N- and L-type Ca2+ channels in the clonal cell line PC12; both
channel types were blocked rapidly by low micromolar concentrations of MeHg
(Shafer and Atchison, 1991). These experiments demonstrated two important aspects
of block of Ca2+ channels by MeHg; that MeHg blocks Ca2+ channel types which are
thought to mediate neurotransmitter release ﬁom central (Dooley et al., 1987a) and
peripheral nerve terminals (Rane et al., 1987; Holz et al., 1988; Atchison, 1989), and
that characteristics of block of somatic and synaptosomal Ca2+ channels by MeHg are
similar in many respects.

The ability of MeHg to affect Ca2+ channels in intact motor nerve terminals
was conﬁrmed by examining effects of MeHg on voltage changes in the perineurial
sheath arising from Ca2+ channel function in mouse motor nerve terminals. MeHg
blocked Ca2+ potentials as well as N a" potentials recorded from the perineurium.
This result conﬁrms directly that MeHg disrupts Ca2+ channel function in intact
motor nerve terminals, consistent with predictions from experiments in synaptosomes
and PC 12 cells. Thus, results of experiments within this dissertation clearly support
the hypothesis: MeHg disrupts function of Ca2+ channel types which are associated

with neurotransmitter release (Shafer and Atchison, 1991) and alters function of Ca2+

198
channels located in the terminals of central (Atchison et al., 1986; Shafer and

Atchison, 1989; Shafer et al., 1990) and motor neurons (chapter 7).

While no direct evidence has been presented htat Mehg disrupts channel
function by entering the channel pore, the facilitation of block of In, by MeHg by
increasing the frequency of stimuluation as well as the voltage-dependence of block
suggest that MeHg may interact within the channel pore. The ionic radii of ions
which carry current through Ca2+ channels range from 1.06 A for Ca2+ to 1.43 A for
Ba2+ while Hg2+ has an ionic radius of 0.93 A The hydration energies are 382, 316
and 441 Kcal/mol, respectively (Cooper et al., 1984). Divalent mercury (Hg2+) has
an atomic raduis of 1.49 A (Miller, 1984). Given that ionic radius will decrease by
addition of a positive charge and hydration energy will decrease, one might expect
that the ~Hg+ portion of MeHg would be on the order of ionic size and hydration to
allow it to enter the Ca2+ channel pore. The N a” channel is permeable to organic
ions such as guanidium and aminoguanidium, which are much larger in ionic radii
than N a“. Interestingly, methylated ions cannot pass through the Na+ channel (Hille,
1984). Thus, it is not inconceivable that MeHg may enter the pore of the Ca2+
channel, resulting in block of Ca2+ ﬂux through the channel.

These results also provide direction for future investigations of the neurotoxic
effects of MeHg. This includes but is not limited to: studies of effects of MeHg on
neurotransmitter release, further examination of effects on channel function, effects

of MeHg on Ca2+ channel functions other than neurotransmitter release, and how

199

effects on Ca2+ channels may relate to neurotoxicity observed during chronic
exposure to MeHg.

Although effects of MeHg on neurotransmitter release have been
demonstrated clearly at the neuromuscular junction, relatively little information exists
concerning effects of MeHg on neurotransmitter release from the central nervous
system (See: McKay et aL, 1986). Since effects of MeHg on Ca2+ channels in both
central and peripheral terminals have been characterized, the ramiﬁcations of these
effects on neurotransmitter release can be examined in more detail. Of particular
importance is how alterations in Ca2+ channel function produced by MeHg impact
MeHg-induced effects on neurotransmitter release. Release of neurotransmitters
from the nerve terminal is thought to follow transient, localized elevations of [Ca2"]i
to near micromolar or micromolar levels (see: Smith and Augustine, 1988). Effects
of MeHg on the rate and magnitude of this increase would be expected to result
from block of Ca2+ channels. However, MeHg may have additional effects on
neurotransmitter release independent of its effects on Ca2+ channel function
(Atchison et al. , 1986; Traxinger and Atchison, 1987a). It has also been suggested that
in addition to Ca2+ entry into the nerve terminal, voltage changes (beyond those
which evoke Ca2+ entry) may be required for neurotransmitter release (Parnas et al.,
1986; 1989). Therefore alterations of membrane potential by MeHg (Hare and
Atchison, 1990) may also contribute to block of the exocytotic process. Future studies
of effects of MeHg on the release processes which occur after Ca2+ entry into the

nerve terminal will provide additional information concerning the role of Ca2+

200
channel block by MeHg, as well as possibly providing a link between effects of MeHg

on Ca2+ channels and other neuronal systems.

Also important with respect to neurotransmitter release is the ability of MeHg
to block both N ~ and L~type Ca2+ channels. Present interpretation of pharmacological
and electrophysiological studies of Ca2+ channels involved in transmitter release
suggest that N-type channels mediate release of small clear vesicles containing
classical transmitters whereas L~type channels mediate release of dense-core vesicles
containing peptide neurotransmitters (Hirning et al., 1988; Smith and Augustine,
1988). Neuropeptides often are involved in modulation of neuronal activity, resulting
in long term changes. Thus, the ability of MeHg to block L~type Ca2+ channels may
be an effect that has important ramiﬁcations during chronic exposures to MeHg.
Effects of MeHg on T~type Ca2+ channels have not been examined; this channel has
been associated with dendritic action potentials and pacemaker activity in the CNS
(Tsien, et al., 1988). The ability of MeHg to affect the N- and L~type Ca2+ channels,
as well as the ability of MeHg to affect Na” channel function and neuronal
excitability (Traxinger and Atchison, 1987b), suggest that possible effects of MeHg
on T~type channels be considered in studies of MeHg effects in the CNS.

In addition to the role of Ca2+ channels in neurotransmitter release, effects of
MeHg on channel function suggest topics for further study. The actions of MeHg on
Ca2+ channels are unique in many ways from the actions of heavy metals such as
Cd“, Co“, Pb“, Hg2+ and Ni“; which also block Ca2+ channels (Nachshen, 1984).

One subject of interest is how diﬁerences in charge and lipophilicity affect the ability

201

of mercurials to alter channel function. Recent studies suggest that only positively
charged organic and inorganic mercurials are able to block Ca2+ channels, and only
lipophilic mercurials block Ca2+ channels in a voltage-dependent manner (Hewett
and Atchison, 1990). MeHg also alters Ca2+ channel ionic selectivity and inactivation
(Shafer et al., 1990; Shafer and Atchison, 1991), which suggests that interactions
between MeHg and Ca2+ channels differ from those of inorganic Ca2+ channel
blockers. Further characterization of effects of MeHg on Ca2+ channel function at
the single channel and biochemical level will provide additional information
regarding the mechanisms by which MeHg produces these effects.

In addition to effects on Ca2+ channels, this dissertation has presented
evidence that MeHg produces effects on Na” channels in clonal cell lines and in
motor nerve axons at similar concentrations to effects on Ca2+ channels. These
results are consistent with previous studies of MeHg action (Quandt et al., 1982;
Traxinger and Atchison, 1987b). Alterations of N a+ channel function by MeHg would
certainly affect synaptic transmission in intact NMJ preparations (T raxinger and
Atchison, 1987b; chapter 8), as block of impulse conduction by MeHg would prevent
neurotransmitter release. However, due to the importance of Na“ channels in
neuronal excitability, these effects of MeHg may also have implications for other
aspects of MeHg toxicity. Thus, future experiments on ionic channel function may
include examinations of common mechanisms of interaction of MeHg with different
channel types as well as how effects on different ionic channels in the membrane may

contribute to disruption of cellular function by MeHg.

202

MeHg alters a number of cellular processes including Ca2+ channel function.
It has been established that MeHg also alters mitochondrial function (Levesque and
Atchison, 1987; 1988; 1991) as well as Na+ channel function (Quandt et al., 1982;
chapters 7 and 8) and nerve terminal membrane potential (Hare and Atchison, 1990).
The reactivity of MeHg with sulfhydryl groups as well as amino and carboxyl groups
makes it unlikely that block of synaptic transmission, or other neurotoxic effects of
MeHg, are the result of disruption of a single process. Thus, it is important to
characterize all of the effects of MeHg and try to synthesize sequences of events
which contribute to toxicity. In addition to effects on neurotransmitter release,
MeHg-induced block of Ca2+ channels in neuronal cells may have other effects on
cellular function. For example, Ca2+ channels provide for transient, localized
elevations in [Ca2+]i involved in grth cone elongation (Streit and Lux, 1987; 1989;
1990) or activation of Can-dependent enzymes (Kennedy, 1989). Regardless, Ca2+
channels are one cellular component involved in regulation of [Ca2+]i; other
components include mitochondria, endoplasmic reticulum and other proteins and
organelles (Meldolesi et al., 1988). Thus, effects of MeHg on Ca2+ channels
(Atchison et al., 1986; Shafer and Atchison, 1989; 1991; Shafer et al., 1990), coupled
with eﬁects on mitochondrial function (Levesque and Atchison, 1987; 1988; 1990)
and membrane depolarization (Hare and Atchison, 1990) suggest the possibility that
alterations in Ca2+ regulation may occur in response to MeHg exposure. Disruption
of Ca2+ regulation might produce a cascade of events leading ultimately to cell death

(Choi, 1988).

203

In conclusion, interactions of MeHg with voltage-dependent Ca2+ channels
have been examined by the experiments contained in this thesis. Disruption of Ca“
channel function by MeHg has been observed in isolated and intact nerve terminals.
In addition, MeHg alterats speciﬁc Ca2+ channel functions and disrupts funtion of
two Ca2+ channel types which are presently associated with neurotransmitter release.
Although further study of the effects of MeHg on neurotransmitter release are
needed, evidence has been presented which supports the hypothesis that block of
neurotransmitter release at the NMJ is, in part, the result of block of voltage-
dependent Ca2+ channels which mediate Ca2+ entry into the nerve terminal.
Furthermore, results of these experiments provide additional knowledge concerning
interaction of MeHg with these critical membrane-associated molecules; which may
contribute to our understanding of the mechanisms of neurotoxicity of this important

environmental compound.

APPENDIX

APPENDIX

204
APPENDIX

TRANSMITTER, ION CHANNEL AND RECEPTOR PROPERTIES
OF PHEOCHROMOCYTOMA (PC12) CELLS:
A MODEL FOR NEUROTOXICOLOGICAL STUDIES

A) INTRODUCTION

The use of isolated nerve terminals (synaptosomes) and intact nerve-muscle
preparations to measure effects of MeHg on nerve terminal Ca2+ channels represent
model systems in which have been extensively used to study neuronal Ca2+ channel
channel function and therefore have been well characterized with respect to their
strengths and limitations. By contrast, pheochromocytoma (PC12) cells are not of
neuronal origin, and measurement of Ca2+ channel function in this cell line using
patch voltage-clamp techniques assesses function of somatic or growth cone Ca2+
channels. However, because of the unique properties of PC12 cells, data collected
under deﬁned conditions may be applicable to neurons. PC12 cells have been a
popular tool for neurobiological studies and represent a potentially useful tool for
neurotoxicological studies as well. This chapter will focus on use of PC12 cells as
model systems for studies of channel function as well as neurotransmitter release and
receptor function.

PC12 is a clonal cell line derived from a catecholamine-secreting tumor
(pheochromocytoma) of rat adrenal medulla (Greene and Tischler, 1976). When

cultured under normal conditions, these cells resemble adrenal chromafﬁn cells in

205

morphology as well as many aspects of physiology and biochemistry. However, when
cultured in the presence of nerve growth factor (NGF), PC12 cells differentiate to
resemble sympathetic neurons morphologically. The process of differentiation is
accompanied by physiological and biochemical changes which ultimately result in
neuron-like function in PC12 cells. In the absence or presence of NGF, PC12 cells
release dopamine (DA), norepinephrine (NE) and/ or acetylcholine (ACh) in
response to depolarization or activation of nicotinic or muscarinic ACh receptors. In
addition, PC12 cells possess membrane—bound receptors, including receptors coupled
to guanine nucleotide-binding proteins (G-proteins) and Na”, K+ and Ca2+ channels.
As such, PC12 cells provide a useful model for studying: 1) processes associated with
neuronal differentiation, 2) synthesis, storage and release of neurotransmitters, 3)
flmction and regulation of ion channels and 4) interactions of compounds with
membrane-bound receptors. The ability of PC12 cells to differentiate in response to
NGF allows for selective expression of certain proteins and for comparisons of

responses in undifferentiated, chromafﬁn-like and differentiated, neuron-like cells.

B) PC12 CELL DIFFERENTIATION. Inclusion of NGF in the culture medium elicits
a characteristic response from PC12 cells. Within 24 hours after its addition, the cells
Stop dividing and begin forming neuronal processes (Greene and Tischler, 1976).
After 4 to 14 days, the cell bodies have increased in size and grown long processes
which form extensive networks. This process of morphological differentiation is

accompanied by changes in the expression of a number of proteins including Na+ /K+

206
ATPase (Inoue et al., 1988), voltage-sensitive N a+ channels (Dichter et al., 1977;

Reed and England, 1986; Rudy et al., 1987) and increased choline acetyltransferase
(CAT) activity resulting in increased ACh content (Greene and Rein, 1977b).
Membrane electrical excitability and response of PC12 cells to ACh are increased by
N GF-induced differentiation (Dichter et al., 1977). In addition, Changes in Ca2+
channel expression (Takahashi et al., 1985) decrease the susceptibility of
neurotransmitter release to blockade by dihydropyridine-(DHP) type Ca2+ channel
antagonists (Kongsamut and Miller, 1986). Thus, the differential expression and/ or
function of proteins, including ion channels and receptors, can be induced by NGF-
treatment of PC 12 cells. Other substances such as ﬁbroblast growth factor (FGF) also
induce differentiation of PC12 cells (for review see Fujita et al., 1989). However, the
changes in enzyme and channel activity induced by FGF and other differentiating
agents differ from those induced by NGF (Garber et al., 1989; Matsuoka et al., 1989;
Damon et al., 1990; Pollock et al., 1990). Removal of NGF from the medium results
in a rapid reversal of the process of differentiation (Greene and Tischler, 1976). The
ability of PC12 cells to differentiate and de—differentiate in the presence or absence
of NGF has made these cells a popular model for studying processes associated with
N GF receptor activation and neuronal development and maturation. This use of
PC12 cells has been reviewed elsewhere (Guroff, 1985; Fujita, et al., 1989). Clearly,
there is great potential for use of this cell line in examining how neurotoxic

compounds affect critical processes during neuronal development.

207

One other aspect of PC12 cell differentiation deserves mention here. Both
adrenal chromafﬁn cells and sympathetic neurons are derived from embryonic neural
crest cells. By contrast to embryonic neural crest cells (Doupe et aL, 1985; Anderson
and Axel, 1986; Naranjo et al., 1986), Greene and Tischler (1976) reported that
glucocorticoids such as dexamethasone (DEX) do not prevent the actions of N GF on
PC12 cells. However, more recently, PC12 cells have been reported to respond to
sodium butyrate (Byrd et al., 1987) by differentiating to an adrenal chromafﬁn-like
phenotype. Upon butyrate treatment, PC12 cells undergo slight morphological
changes without neurite extension and produce greater amounts of opioid peptide
neurotransmitters (Byrd et al.,1987). Co-culture of PC12 cells with adrenal medullary
endothelial cells induces PC12 cells to differentiate into an adrenal chromafﬁn-like
phenotype which is unresponsive to N GF (Mizrachi, et al., 1990). Thus, PC12 cells
respond to some stimuli by differentiating to an adrenal chromafﬁn-like phenotype,

and other stimuli by differentiating to a neuron-like phenotype.

C) 'I'RANSMI'ITERS, ION CHANNELS AND RECEPTORS IN PC12 CELLS.

l) Neurotransmitter Release from PC 12 cells.

As previously mentioned, PC12 cells synthesize and release DA, NE (Greene and
Tischler, 1976; Greene and Rein, 1977a) and ACh (Greene and Rein, 1977b), but
not epinephrine (Greene and Tischler, 1976). These three transmitters are all
released in response to depolarization (Table 1). In addition, undifferentiated and

NGF-differentiated PC12 cells release catecholamines in a Ca2+~dependent fashion

208

in response to nicotinic (Greene and Rein, 1977c) and muscarinic (Rabe et al., 1987)
cholinergic agonists.

The apparent decrease in activity of enzymes and amounts of
neurotransmitters following treatment with NGF is most likely due to increased
expression of new proteins induced by NGF, not to a decrease in the expression of
enzymes associated with catecholamine synthesis (Greene and Tischler, 1976). The
membranes of PC 12 cells contain a saturable NE transporter which is sensitive to
cocaine and the tricyclic antidepressant desmethylimiprarnine (Greene and Rein,
1977a). NE and DA are most likely stored in vesicles, as exposure to reserpine
depletes cellular catecholamine levels (Greene and Rein, 1977a). Thus, the
mechanisms of synthesis, storage and re-uptake of catecholamines in PC 12 cells are
similar to those of catecholaminergic neurons.

In undifferentiated PC 12 cells, synthesis of NE, but not DA, changes with the
cell cycle, being maximal during the G2 phase. Release of catecholamines by
depolarization is maximal during G1, whereas carbamylcholine-induced release is
maximal during the S and G2 phases. Cell cycle-speciﬁc changes in 45Ca“ inﬂux
induced by K+ depolarization and muscarinic receptor activation correlate with
neurotransmitter release (Koike and Takashima, 1986). This characteristic of
undifferentiated PC12 cells is clearly different from neurons, which do not divide.

Permeabilization of PC 12 cells provides more direct access to release
mechanisms. In staphylococcal a ~toxin permeabilized cells, Mg-ATP, calmodan or

proteins containing ~SH or ~OH groups are not required for Ca2+~dependent release

209
(Ahnert-Hilger and Gratzl, 1987). Furthermore, release of catecholamines from a ~

toxin or digitonin-permeabilized cells is enhanced by phorbol esters (Pozzan et al.,
1984; Ahnert-Hilger et al., 1987; Carroll et al., 1990) and reduced in a pertussis toxin~
sensitive fashion by an activator of guanine binding proteins, GTPy S (Ahnert-Hilger
et al., 1987). These results indicate that protein kinase C (PKC) and G-proteins,
respectively, may modulate catecholamine release from PC 12 cells. However, the role
of G-proteins in modifying catecholamine release from PC12 cells is not yet clearly
deﬁned. GTPy S stimulated only Ca2+~independent NE release from digitonin-
permeabilized PC12 cells, whereas an inhibitor of guanine binding proteins cyclase,
GTPBS, reduced only the GTPy S component of release. Thus, the component of
release affected by cyclic guanine nucleotides is not essential to NE release (Carroll
et al., 1990). Furthermore, the opposite actions of GTPyS in a ~toxin and digitonin-
permeabilized cells indicate that the agent used to permeabilize cells may inﬂuence
G-protein-dependent pathways.

DA release evoked by depolarization and / or nicotinic ACh receptor activation
is accompanied by increases in cyclic AMP (CAMP). Moreover, forskolin, an activator
of adenylate-cyclase, enhances depolarization-(Rabe et al., 1982; Baizer and Weiner,
1985) or carbachol-induced release (Baizer and Weiner, 1985). Thus, CAMP may also
modulate catecholamine release in PC12 cells. However, the action of CAMP
presumably is on a component of the release mechanism which functions after Ca2+

entry, as forskolin and/or its analogs either do not affect 4sCa2+ entry (Rabe et al.,

210

1982), or block voltage-dependent Ca2+ channels in a CAMP-independent manner
(Nishizawa et al., 1990)

Effects of neurotoxic compounds on catecholamine synthesis and release have
been studied in PC12 cells. Chronic exposure to ethanol (EtOH) decreased
(muscarinic) ACh-induced release of [3H]-NE from PC12 cells without affecting NE
uptake or ACh binding. However, EtOH did decrease the rise in [Ca2+]i induced by
stimulation of muscarinic receptors on PC12 cells (Rabe and Weight, 1988).
Concentrations of DA and NE in PC12 cells were decreased by a 6 h. treatment with
1 to 100 ppm Arochlor 1254 (Seegal et al., 1989). Thus, PC12 cells may provide a
useful in vitro system for examining mechanisms of chemical action on catecholamine
levels and/or release processes in neurons.

In addition to catecholamine neurotransmitters, PC12 cells also synthesize and
release ACh. This ability to synthesize and release two distinct classes of transmitters
(ACh and catecholamines) permits PC12 cells to be used for differential studies of
effects of neurotoxicants on the release of unique transmitters. In contrast to its
effect on biosynthetic enzymes for catecholamines, NGF-differentiation increased
CAT activity (Table 1). Treatment with retinoic acid instead of NGF resulted in an
increase in CAT activity without morphological differentiation (Matsuoka et a1. ,
1989). Undifferentiated PC12 cells have a high-afﬁnity (Km = 12 u ), Na+~dependent
choline transporter which is relatively insensitive to hemicholinium~3 (Melega and
Howard, 1981). This differs from normal cholinergic neurons for which the high~

affinity choline transporter is sensitive to

211

Table A1

Activity and Content of Noradrenergic and Cholinergic
Synthetic Enzymes and N eurotransmitters in PC12 Cells

 

 

 

 

 

 

Undifferentiated NGF-Diﬂ'erentiated

Enzymes Enzyme Activity (pmol/min/ mg protein)
Tyrosine Hydroxylase 39 1 5 10 1 1
Dopa decarboxylase 770 1 99 130 1 15
Dopamine ﬂ ~hydroxylase 806 1 84 161 1 19
Choline Acetyltransferase‘ 440 1 17 520 :I: 20
N eurotransmitters Neurotransmitter Content (pmol/mg protein)
Dopamine 16.61 1.7 4.41 0.4
Norepinephrine 6.11 6 1.51 0.2
Epinephrine <0.15 <0.15
Acetylcholine‘ 1335 1 77 5594 1 637

 

PC12 cells were treated with 50 mg/ml of NGF for 14 (noradrenergic properties) or 22
(cholinergic properties) days prior to measuring enzyme activity and neurotransmitter content.
All values are expressed as mean 1 SEM and protein content is total cellular protein content.

.Values from: Greene and Rein, Nature (Land) 268: 349-352, 1977b.

All other values from: Greene and Tischler, PIOC. Natl. Acad. Sci. (U.S.A.) 73: 2424—2428, 1976.

212

hemicholinium~3. In undifferentiated PC12 cells, ACh and catecholamines are stored
in dense-core vesicles, but based on reserpine sensitivity and electron microscopy, the
two transmitters are not co~localized (Schubert and Kleir, 1977). Evidence that
release of ACh from PC12 cells is quantal was demonstrated by co~culture of PC12
cells with a skeletal muscle clonal cell line, L~6. After as few as 3 days of co~culture
in NGF-containing medium, a ~bungarotoxin~ and d-tubocurarine-sensitive miniature
endplate-potentials could be recorded from L-6 myotubes (Schubert et al., 1977).
Howard and co~workers loaded PC12 cells with 2H4~choline and used combined gas
chromatography-mass spectrometry to measure levels of endogenous 2ITO-ACh and
newly synthesized 2H4-ACh in the presence and absence of vesamicol, which inhibits
uptake of ACh into vesicles (Marshall, 1970). Their results clearly indicate that newly
synthesized ACh in PC12 cells is stored in vesicles (Figure A.1A) and evoked-release
of 2H4~ACh (Figure A.1B)is predominantly vesicular in nature (Melega and Howard,
1984; Howard at al., 1986). Thus, in these respects, release of ACh from PC12 cells
mirrors normal release from native cholinergic terminals.

Effects of neurotoxins on cholinergic properties of PC12 cells have not been
widely studied. The effects of tetanus toxin on ACh release from PC12 cells indicate
that this neurotoxin inhibits ACh release at subnanomolar concentrations (Sandberg
et al., 1989a) and that tetanus toxin may inhibit ACh release by interfering with

cyclic-GMP metabolism (Sandberg, et al., 1989b).

213

 

 

120 l
. (Eek,
g \ \k, \t x
E ‘, f\i
o
o \
\
* 4o - \
I
!\
\
\
0
.01 1 Io
[VESAMICOL] (uti)

Specific

E] I'HJAChII‘HJAChI .cnvay
0 r

P

w

18

% of VESAMICOL-FREE
CONTROLS

 

 

 

 

 

 

TOTAL ACh EVOKED
CONTENT RELEASE

Figure A.1. Vesicular nature of ACh release from PC12 cells. Effects of vesamicol (AH5183) on the
cellular and vesicular content of acetylcholine and on acetylcholine release in PC12 cells. A.) PC12 cells
were incubated for 30 min with 10 p. M [3H]choline in the presence of vesamicol. Acetylcholine (ACh)
was extracted from the cells (triangles) and from a vesicle fraction (circles) of a cell homogenate, and
the levels of [2H4]ACh (solid symbols) and native [ZHOJACh (open symbols) were measured. The results
are expressed as the percentage of control values. Values shown are the mean 1 SD for triplicate
incubations. B.) Release of ACh from vesamicol-treated cells during 5 min incubations. Total cellular
ACh content is the sum of the [2H01ACh or the [2H4]ACh released to the buffers during a 5 min
incubation and the amount of ACh remaining in the cells after the incubations. Speciﬁc activity is the
ratio of [2H4]-ACh/ total ACh. The results are expressed as a percentage of control values for cells that
were incubated in the absence of vesamicol. The values are the mean 1 SD for quadruplicate
incubations.

Modiﬁed from: Howard er al., In: Calcium, Neumnal Function and Transmitter Release. (R.
Rahamimoﬁ and B. Katz, eds). p. 282 (1986).

214
2) Ion channels in PC12 cells.

K+ Channels. PC12 cells also express multiple K+ conductances and channel
subtypes. Several groups have reported the presence of a large Ca2*~activated K+
conductance in PC12 cells (Hoshi and Aldrich, 1988a; Auguste et al., 1989; Pun and
Behbehani, 1990). This current is blocked readily by agents which decrease Ca2+
inﬂux into PC12 cells, such as inorganic divalent cations (Pun and Behbehani, 1990).
More than one type of Ca2+~activated K+ channel may be present in PC12 cells, as
86Rb+ efﬂux is only completely blocked by a combination of tetraethylammonium and
apamin (Schmid-Antomarchi et al., 1986), a polypeptide toxin from bee venom which
blocks some types of CaZI-activated K+ channels. PC12 cells express large amounts
of the apamin receptor after as little as 4 days in culture. Interestingly, after 7 days
in culture in the presence of NGF, the number of apamin receptors/ mg protein was
10 fold less than in undifferentiated cells (Schmid-Antomarchi et al., 1986). Thus, by
contrast to N a“ and Ca2+ channels, N GF treatment does not increase the expression
of this channel type. Apamin was used in afﬁnity labelling experiments in an attempt
to identify components of the apamin receptor in PC12 cells. 12$I~labeled derivatives
of apamin bound to a 30 kilodalton receptor in PC12 cell membranes (Auguste er al.,
1989). Since this cell line expresses large amounts of the apamin receptor, it may be
useful in isolating the genetic sequences which code for this type of Ca2+-activated
K+ channel.

Whole-cell currents similar to those mediated by rapidly inactivating (IA)

(Rogawski et al., 1988; Pun and Behbehani, 1990) and delayed rectiﬁer (Ix) K+

215
channels (Rogawski, et al., 1988; Hoshi and Aldrich, 1988a) have also been reported

for PC12 cells. Phencyclidine (1~100 n M) and tetraethylammonium chloride (20
mM) block IK (Rogawski, et al., 1988), whereas IA is inhibited by 2 mM 4-
aminopyridine (Rogawski et al., 1988). Hoshi and Aldrich (1988a,b) have
characterized four voltage-dependent K+ channels which may underlie whole-cell K”
currents in PC12 cells. The kinetics of three of these channels are similar to those
of delayed rectiﬁer K“ channels, whereas the kinetics of the fourth channel are
similar to kinetics of the inactivating K+ channel. However, the channels in PC12
cells also have characteristics which are unique from those of channels which mediate
IK and I A (Hoshi and Aldrich, 1988a,b). Thus, it cannot be assumed that K+ channels
in PC12 cells behave in a fashion identical to those described elsewhere.

Finally, it has been demonstrated that the peptide galanin inhibits DA
secretion from PC12 cells by activating a K" conductance (de Weille et al., 1989).
Galanin had no effect on L~type Ca2+ channels, but its action on K+ conductances
was inhibited by pretreatment with pertussis toxin. These results suggest that K+
channels and the galanin receptor may be linked by a G-protein in PC12 cells (de
Weille et al., 1989). Whether or not the galanin response is mediated by a voltage-
sensitive or receptor operated channel is presently unknown.

By contrast to Ca2+ channels, the effects of N GF ~differentiation on voltage-
sensitive K+ channels have not yet been widely studied. Thus, it is unclear at this

point whether “or not changes in distribution or composition of K+ channel types play

216

an important role in, or are affected by the process of differentiation of PC12 cells.
Toxicological studies utilizing K+ channels in PC12 cells are also lacking.

Na+ channels. Like Ca2+ channels, expression of voltage-gated Na+ channels
is increased by differentiation of PC12 cells with NGF. TTX-sensitive, Ca2+-
independent action potentials in PC12 cells differentiated with NGF for 2 weeks
were observed by Dichter et al. (1977). Undifferentiated PC12 cells express functional
Na+ channels, but differentiation with N GF increased TTX-sensitive inﬂux of ”Na”
by approximately 12 fold (Reed and England, 1986). Induction of a TTX-insensitive
N a+ ﬂux by NGF has also been reported (Rudy et al., 1987).

The mechanisms underlying N GF-induced increases in Na“ channel function
are presently unclear. Increases in Na+ channel function by NGF were mimicked by
increases in cAMP, and responses to N GF and CAMP were blocked by an inhibitor
of CAMP-dependent protein kinase (PKA), suggesting that the expression of Na+
channels in response to NGF is in part mediated by PKA (Kalman et al., 1990).
However, Pollock et al. (1990) reported that CAMP did not stimulate neurite
outgrowth and decreased INa in PC12 cells, whereas FGF was as effective as NGF
in increasing IN, In addition to NGF, treatment with DEX also increased the
expression of Na” current in PC12 cells (Figure A.2, Garber et al., 1989), although
this response appears to be inconsistent. Treatment with DEX and NGF produced
less of an increase in Na+ current than treatment with N GF alone, and despite
treatment with NGF and / or DEX, some PC12 cells failed to express measurable

whole-cell N a“ conductances (Figure A.2) (Garber et al., 1989). Furthermore, Pollock

217

A serum 3. ~50toOmV C.

 

 

 

 

m 1.33:

Figure A.2. Voltage-dependent Na+ channels in PC12 cells. Representative families of the voltage-
dependent Na+ currents from untreated, dexamethasone-(DEX), nerve gowth factor- (N GF), and
DEX/NGF-treated cells recorded using the whole-cell patch voltage-clamp technique. In each treatment
group, some cells (A) expressed no or little Na+ current while others (B) expressed appreciable amounts
of N a+ current. C, Peak current-voltage (I / V) curves were obtained from cells which did not express
the Na+ current (black squares) and those which expressed the Na+ current (circles). As K+ was often
used in internal solutions, outward K" currents are present in some families of current records.

From: Garber et al. Mm 9:3976-3987 (1989).

218
et al., (1990) reported that DEX failed to increase IN“, and inhibited NGF-induced

increases in IN, in PC12 cells. Finally, several other treatments including DEX,
mitotic inhibitors and dimethyl sulfoxide, which induced physiological differentiation
in neuroblastoma cell lines, were less effective at increasing N a+ current than N GF .
In addition, ACh responses induced by N GP in PC12 cells were not necessarily
coupled to increases in N a“ channel function (Ifune and Steinbach, 1990). Further
study is needed to understand clearly the regulation of Na“ channel expression in

PC 12 cells.

3) Receptors on PC12 cell membranes.
Another potential use of PC12 cells in neurotoxicology is for the study of interactions
of compounds with neurotransmitter receptors or receptor / ligand-activated channels.
Both nicotinic (Greene and Rein, 1977b) and muscarinic ACh receptors (Jumblatt
and Tischler, 1982; Cross at al., 1984) are expressed in PC12 cell membranes, as well
as adenosine receptors (Guroff et aL, 1981). A recent report also suggests that PC12
cell membranes may also contain N-methyl-D-aspartate receptors which stimulate
phosphoinositide hydrolysis, increased [Ca2+], and NE release (Kurozumi et al., 1990).
Adenosine or adenosine receptor agonists stimulate adenylate cyclase resulting
in increased tyrosine hydroxylase activity in PC12 cells (Rabe and M‘Gee, 1983;
Noronha~Blob et al., 1986). Radioligand binding studies have demonstrated that
adenosine receptors in PC12 cells are of the A2 subtype (Williams et al., 1987). Short

pretreatment of PC12 cells with adenosine increased depolarization-evoked release

219

of [3H]-NE and [3H]~ACh without altering 45Ca2“ inﬂux or depolarization as
measured by “RE efﬂux through nicotinic ACh receptor channels (Rabe and
M‘Gee, 1983). It has been suggested that adenosine or adenosine nucleotides may
act via A2 receptors in a paracrine or autocrine fashion (Roskoski and Roskoski,
1989). ATP released during exocytosis of NE— or ACh-containing vesicles may be
degraded to adenosine and stimulate A2 receptors. In PC12 cells, the activity of
adenosine is terminated by re-uptake (Roskoski and Roskoski, 1989).

Activation of muscarinic ACh receptors in undifferentiated PC12 cells results
in elevations in inositol triphosphate (1P3) and free [Ca2+]i (Vincentini et al., 1985),
followed by neurotransmitter release (Rabe et al., 1987). Inoue and Kenimer (1988)
have suggested that a receptor-activated Ca“ channel may mediate muscarinic
receptor-induced neurotransmitter release. Muscarinic agonists increased 45Ca2+
inﬂux, and neurotransmitter release was not blocked by Ca2+ channel blockers.
Moreover, the inﬂux of 4“"Ca2" and neurotransmitter release were inhibited by
pertussis toxin, indicating the possible involvement of G-proteins (Inoue and
Kenimer, 1988). However, neurotransmitter release and phosphoinositide (PI)
hydrolysis stimulated by muscarinic receptor activation appear to be separate events.
In Ca2+~free solutions, methacholine stimulated PI hydrolysis but not NE release. By
contrast, activators of PKC inhibited PI turnover but not NE release (Takashima and
Kenimer, 1989). Similarly, activation of muscarinic receptors in PC12 cells also
results in an inhibition of CAMP which is a separate event from P1 hydrolysis

(Horwitz, 1989). However, there is no evidence that these actions are mediated by

220

different receptor subtypes (Horwitz, 1989). Thus, stimulation of the muscarinic ACh
receptor in PC12 cells may also stimulate cellular processes in addition to
neurotransmitter release.

Characterization of muscarinic receptor subtypes in undifferentiated PC12 cells
by pharmacological sensitivity of [3H]~NE release and [3H]~telenzipine (a selective
antagonist for the M1~type muscarinic receptor) binding indicates that PC12 cells
contain more than one type of muscarinic receptor (Bt'inish et al., 1990). However,
the predominant receptor subtype clearly is not of the M2 or M3 category, and also
may not be of the M1 subtype (Michel et al., 1989; Bdnish et al., 1990). It has been
proposed that the muscarinic ACh receptor in PC12 cells may be of the M4 subtype
(Michel et al., 1989). Further study is needed to deﬁne better the type of muscarinic
ACh receptor(s) present on PC12 cells.

Effects of organophosphate insecticides on muscarinic ACh receptors in PC12
cells indicate that 50 uM soman decreased the number of muscarinic receptors after
24 h. of exposure. However, the insecticides did not inhibit binding of radiolabeled
muscarinic antagonists, nor did atropine prevent organophosphate-induced decreases
in muscarinic receptor numbers. Furthermore, decreases in muscarinic receptors
induced by organophosphates were qualitatively different ﬁom those produced by the
ACh agonist carbamylcholine. Thus, organophosphate insecticides decrease the
numbers of muscarinic ACh receptors by a mechanism other than agonist-induced

receptor desensitization (Viana et aL, 1988).

221

Stimulation of nicotinic and muscarinic ACh receptors results in a rapid
increase in PKC activity. However, this response appears to be largely the result of
nicotinic receptor activation, as muscarinic antagonists only Caused small reductions
of carbachol-induced PKC activity (Messing et al., 1989). Furthermore, the nicotinic
response was dependent on Ca2+ inﬂux via voltage-dependent Ca2+ channels, as the
DHP antagonist nifedipine reduced PKC activity to the level observed during
muscarinic receptor activation (Messing et al., 1989). Recent binding studies of
nicotinic ACh receptors in PC12 cells using [3H]-ACh and a ~bungarotoxin indicate
that there may be heterogeneous expression of nicotinic receptor subtypes (Lukas,
1990).

M°Gee and co~workers have used PC 12 cells to examine agonist-induced down~
regulation of the nicotinic ACh receptor (Robinson and McGee, 1985). The nicotinic
ACh receptor is not the target of CAMP-dependent phosphorylation in PC12 cells
(M‘Gee and Liepe, 1984), but high concentrations of forskolin can impair receptor
function by anesthetic-like effects (M°Hugh and McGee, 1986). Furthermore, nicotinic
ACh receptors are not inﬂuenced by prolonged depOlarization or elevations in [Ca2+]i
(DeLorme and McGee, 1988). Adenylate cyclase does not appear to be involved in
desensitization of nicotinic ACh receptors in PC12 cells, as forskolin and its analogs
do not markedly alter agonist-induced desensitization of nicotinic ACh receptors in
PC12 cells (Nishizawa et al., 1990). Further study of agonist-induced desensitization
of nicotinic ACh receptors is needed to elucidate the mechanisms responsible for

desensitization in PC12 cells. However, enough information exists about the types

222

and functions of ACh receptors in PC12 cells to allow examination of interactions

between neurotoxins and these receptor types.

D) CLONAL VARIABILITY AND SUBCLONES OF PC12

When using any model system, one must know the weaknesses and limitations
of the model in order to maximize its usefulness while minimizing the likelihood of
error or overinterpretation of results. One limitation of the PC12 cell line is that the
number of passages or subcultures from the original isolation of the line is unknown.
Second, PC12 cell cultures have a tendency to become heterogenous with respect to
cell morphology after approximately 20 passages (Guroff, 1985). Thus, it is often
difﬁcult to compare directly results from different laboratories due to differences in
passage number. This is most apparent for studies of the regulation of Na+ channel
expression, and undoubtedly limits the use of this line for screening and/ or regulatory
purposes. However, PC12 cells remain an excellent model for mechanistic studies of
disruption of neuronal function. By routinely keeping track of the number of passages
from receipt of the cell line, one can maintain internal consistency by using the same
"passage number" for experiments and can begin a new culture from a frozen stock
when the number of passages approaches 20.

A number of subclones of PC12 exist due to spontaneous and/ or induced
mutations. Heterogeneity in NE and DA synthesis and responses to glucocorticoids
has been reported (Koike and Takashima, 1984). Other clones vary in their ability

to respond to N GP or in the type of response induced by N GF (see Guroff, 1985).

223
Presently, the subclones of PC12 are used primarily in studies of the action of NGF.

However, they may also represent a useful tool in neurotoxicology as well.

E) SUMMARY

PC12 cells have been widely used for studies of neuronal biochemistry and
physiology. In addition to responses to NGF, the neurotransmitter, ion channel and
receptor properties of this cell line have been well characterized. A well
characterized and understood model system is a prerequisite for mechanistic studies
of the action of toxic compounds. PC12 cells represent a potentially useful model for
the study of mechanisms of action of neurotoxic compounds on neuronal
differentiation and maturation, synthesis, storage and release of DA, NE and ACh,
as well as the function of voltage-sensitive Na”, K+ and Ca2+ channels. With respect
to Ca2+ channels, the subtypes of Ca2+ channels expressed in this cell line are
extremely well characterized by ion ﬂux, binding and electrophysiological techniques.
In addition, the composition of Ca2+ channel subtypes can be altered predictably by
NGF-differentiation, permitting accurate electrophysiological assessment of effects
of putative toxicants on these important membrane macromolecules. Several receptor
types, including nicotinic and muscarinic ACh receptors and adenosine receptors are
also becoming well characterized in PC12 cells. Thus, this cell line, which has been
used only sparingly in neurotoxicological studies to date, may in the future become

an extremely useful model system.

REFERENCES

REFERENCES
ABE, T., HAGA, T. and KUROKAWA, M. (1975). Blockage of axonal transport and

depolymerization of assembled microtubules by methylmercury. Brain Res.

86:504-508.

ABE, T., KOYANO, K., SAISU, H., NASHIUCHI, Y. and SAKAKIBARA, S.
(1986). Binding of Q'COIIOIOXIII to receptors associated with the

voltage-sensitive calcium channel. Neurosci. Lett. 71:203-208.

ADAMS, M. E., BINDOKAS, V. P., HASEGAWA, L. and VENEMA, V. J. (1990).
(t) ~Agatoxins: Novel calcium channel antagonists of two subtypes from funnel

web spider (Agelenopsis aperra) venom. J. Biol. Chem. 265:861-867.

ADAM-VIZI, V. and LIGETI, E. (1986). Calcium uptake of rat brain synaptosomes
as a function of membrane potential under different depolarizing conditions.

J. Physiol. 372:363-377.

AHNERT—I-IILGER, G., BRAUT'IGAM, M. and GRATZL, M. (1987).
Ca2*~stimulated catecholamine release from alpha-toxin-permeabilized PC12
cells: biochemical evidence for exocytosis and its modulation by protein kinase

C and G proteins. Biochem. 26:7842-7848.

225
AHNERT-HILGER, G. and GRATZL, M. (1987). Further characterization of

dOpamine release by permeabilized PC12 cells. J. Neurochem. 49:764~770.

AKERMAN, K. E. 0. and NICHOLLS, D. G. (1981). Ca2+ transport by intact
synaptosomes. The voltage-dependent Ca 2+ channel; re-evaluation of the role

of sodium/ calcium exchange. Eur. J. Biochem. 117:491-497.

ANDERSON, D. J. and AXEL, R. (1986). A bipotential neuroendocrine precursor
whose choice of cell fate is determined by N GF and glucocorticoids. Cell

47: 1079-1090.

ANDERSON, A. J. and HARVEY, A. M. (1987). m-Conotoxin does not block the
verapamil-sensitive calcium channels at mouse motor nerve terminals.

Neurosci. Lett. 82:177-180.

ARMSTRONG, C. M., SWENSON, R. P. and TAYLOR, S. R. (1982). Block of
squid axon K channels by internally and externally applied barium ions. J.

Gen. Physiol. 80:663-682.

ATCHISON, W. D. (1986). Calcium-dependent and-independent effects of
methylmercury on spontaneous and potassium-evoked release of acetylcholine

at the neuromuscular junction. J. Pharmacol. Exp. Ther. 237 :672-680.

226
ATCHISON, W. D. (1989). Dihydropyridine-sensitive and ~inscnsitive components of

acetylcholine release from rat motor nerve terminals]. Pharmacol. Exp. Then

251:672-678.

ATCHISON, W. D. and NARAHASHI, T. (1982). Methylmercury induced

depression of neuromuscular transmission in the rat. Neurotoxicology 3:37-50.

ATCHISON, W. D., CLARK, A. W. and NARAHASHI, T. (1984). Presynaptic

effects of methylmercury at the mammalian neuromuscular junction. In:
Cellular and molecular neurotoxicology (Narahashi, T., ed.). Raven Press,

New York, pp. 23-43.

ATCHISON, W. D. and NARAHASHI, T. (1984). Mechanism of action of lead.

Neurotoxicology 5:267-284.

ATCHISON, W. D., JOSHI, U. and THORNBURG, J. E. (1986). Irreversible
supression of calcium entry into nerve terminals by methylmercury. J.

Pharmacol. Exp. Ther. 238:618-624.

ATCHISON, W. D. and O’LEARY, S. M. (1987). Bay K 8644 increases release of

acetylcholine at the murine neuromuscular junction. Brain Res. 419:315-319.

227
ATCHISON, W. D., ADGATE, L. and BBAMAN, C. M. (1988). Effects of

antibiotics on uptake of calcium into isolated nerve terminals]. Pharmacol.

Exp. Ther. 245:394-401.

AUDESIRK, G. (1987). Effects of in vitro and in vivo lead exposure on voltage-

dependent calcium channels in Lymnaea stagnalis. Neurotoxicol. 8:579-592.

AUDESIRK, G. and AUDESIRK, T. (1989). Effects of in vitro lead exposure on
voltage-sensitive calcium channels differ among cell types in Lymnaea

stagnalis. Neurotoxicol. 10:659-670.

AUGUSTE, P., HUGUES, M. and LAZDUNSKI, M. (1989). Polypeptide
constitution of receptors for apamin, a neurotoxin which blocks a class of

Can-activated K+ channels. FEBS Lett. 243350154.

AUGUSTINE, G. J., CHARLTON, M. P. and SMITH, S. J. (1987). Calcium action

in synaptic transmitter release. Ann. Rev. Neurosci. 10:633-693.

BAKIR, F., DAMLUJI, S. F., AMIN-ZAKI, L., MURTADHA, M., KHALIDI, A.,
AL-RAWI, N. Y., TIKRITI, S., DHAHIR, H. 1., CLARKSON, T. W., SMITH,
J. C. and DOHERTY, R. A. (1973). Methylmercury poisoning in Iraq.

Science 181:230-241.

228
BAIZER, L. and WEINER, N. (1985). Regulation of dopamine release from PC12

pheochromocytoma cell cultures during stimulation with elevated potassium

or carbachol. J. Neurochem. 44:495-501.

BARRETT, J ., BOTZ, D. and CHANG, D. B. (1974). Block of neuromuscular
transmission by methylmercury. Behavioral toxicology. In: Early detection of
occupational hazards. (Zintaras, C., Johnson, B. L. and de Groot, I., eds). US.

Dept. of Health, Education, and Welfare. 5:277-287.

BEAN, B. P. (1985). Two kinds of calcium channels in canine atrial cells. J. Gen.

PhysioL 86:1-30.

BELLUZZI, 0., SACCHE, O. and WANKE, E. (1985). A fast transient outward
current in rat sympathetic neurone studied under voltage-clamp conditions.

J. Physiol. (Lona) 358:91-108.

BERGAMASCHI, S., TRABUCCHI, M., BA'ITAINI, F., PARENTI, M.,
SCHETTINI, G., MEUCCI, O. and GOVONI, S. (1990). Modulation of

dihydropyridine-sensitive calcium channels: A role for G proteins. Eur. Neurol.

30:16-20.

229
BERGLUND, F., BERLIN, M., BIRKE, G., VON FULER, U., FRIBERG, L.,

HOLMSTEDT, B., JONSSON, E., RAMELC., SKERFVING, S.,
SWENSSEN, A. and TEJNING, S. (1971). Methylmercury in ﬁsh. A
toxicologic epidemiologic evaluation of risks. Report from an expert group.

Nord. Hyg. Tidskr. Suppl. 4.

BIDSTRUP, P. L. (1964). Toxicity of mercury and its compounds. Elsevier Publishing

Co., Amsterdam-London-New York. p.74.

BLAUSTEIN, M. P. (1975). Effects of potassium, veratridine and scorpion venum on

calcium accumulation and transmitter release by nerve terminals in vitm. J.

Physiol. 247:617-655.

BLAUSTEIN, M. P. and GOLDRING, B. M. (1975). Membrane potentials in
pinched-off presynaptic nerve terminals monitored with a ﬂourescent probe:
Evidence that synaptosomes have potassium diffusion potentials. J. Physiol.

(Land) 247:589-615.

BLAUSTEIN, M. P. and ECTOR, A. C. (1975). Barbiturate inhibition of calcium

uptake by depolarized nerve terminals. Mol. Pharmacol. 11:369-378.

230
BLAUSTEIN, M. P. and OBORN, C. J. (1975). The inﬂuence of sodium on calcium

ﬂuxes in pinched-off nerve terminals in vitro. J. PhysioL (Land) 247:657-686.

BLAUSTEIN, M. P., RATZLAFF, R. W. and KENDRICK, N. K. (1978). The
regulation of intracellular calcium in presynaptic nerve terminals. Ann. NY.

Acad. Sci. 307:195-211.

BOLAND, L. M. and DINGLEDINE, R. (1990). Multiple components of both
transient and sustained barium currents in a rat dorsal root ganglion cell line.

J. PhysioL (Land. ) 420223-245.

BONDY, S. C., ANDERSON, C. L, HARRINGTON, M. E. and PRASAD, K. N.
(1979). The effects of organic and inorganic lead and mercury on
neurotransmitter high-afﬁnity transport and release mechanisms Environ. Res.

19:102-111.

BONISCH, H., BOER, R., DOBLER, M. and SCHUDT, C. (1990). Pharmacological
characterization of muscarine receptors of PC12 (rat phaeochromocytoma)

cells. Naunyn-Schmiedeberg. Arch Pharmacol. 341:158-164.

BOROWI'IZ, J. L. (1974). Mechanism of adrenal catecholamine release by divalent

mercury. Toxicol. Appl. Pharmacol. 28:82-87.

231
BRADFORD, H. F . (1975). Isolated nerve terminals as an in vitna preparation for

study of dynamic aspects of transmitter metabolism and release. In-Handbook
of psychopharmacology. (Iverson, L. L, Iverson, S. D. and Snyder, S. H., eds.)

Plenum Press, New York, pp 191-252.

BRIGANT, J. L and MALLART, A. (1982). Presynaptic currents in mouse motor

nerve endings. J. Physiol. (Land. ) 333:619-636.

BUSSELBERG, D., EVANS, M. L, RAHMANN, H. and CARPENTER, D. O.
(1990). Lead inhibits the voltage-activated calcium current of Aplysia neurons.

Toxicol. Lett. 5:51-57.

BYRD, J. C., NARANJO, J. R. and LINDBERG, I. (1987). Proenkephalin gene
expression in the PC12 pheochromocytoma cell line. Endocrinology 121:1299-

1305.

CAMPBELL, K., P., LEUNG, A. T. and SHARP, A. H. (1988). The biochemistry
and molecular biology of the dihydropyridine-sensitive calcium channel TINS.

11:425-430.

CARBONE, E. and LUX, L D. (1984). A low voltage-activated, fully inactivating Ca

channel in vertebrate sensory neurones. Nature (Land. ) 310:501-503.

232
CARROLL, A. G., RHOADS, A. R. and WAGNER, P. D. (1990). Hydrolysis-

resistant GTP analogs stimulate catecholamine release from digitonin-

permeabilized PC12 cells. J. Neurachem. 55:930-936.

CHARLTON, M. P. and AUGUSTINE, G. J. (1990). Classiﬁcation of presynaptic
calcium channels at the squid giant synapse: neither T-, L- nor N-type. Brain

Res. 525:133-139.

CHANG, L W. (1977). Neurotoxic effects of mercury. A review. Env. Res. 14:329-

373.

CHANG, L W. (1979). Pathological effects of mercury poisoning. In: The
biochemistry of mercury in the environment. (Nriagu, ed.) Elsevier/North

Holland Biomedical Press.

CHANG, L W. (1980). Mercury. In: Experimental and Clinical Neurotoxicology
(Spencer, P. S. and Schaumburg, H. H., eds.). The Williams and Wilkins Co.,

Baltimore. pp. 508-526.

CHANG, L W. and HARTMANN, H. A. (1972a). Ultrastructural studies of the
nervous system after mercury intoxication. 1. Pathological changes in the nerve

cell bodies. Acta Neuropath. 20:122-138.

233
CHANG, L W. and HARTMANN, H. A. (1972b). Ultrastructural studies of the

nervous system after mercury intoxication. 11. Calcium ion-induced swelling.

Biochem. J. 95:378-386.

CHANG, L W. and HARTMANN, H. A. (1972c). Blood-brain barrier dysftmction

in experimental mercury intoxication. Acta Neurapath. 21:179-184.

CHERKSEY, B. D., SUGIMORI, M. and LLINAS, R. R. (1990). Isolation of a
calcium channel from mammalian cerebellar tissue using synthetic FTX.

Neurasci. Abstr. 16:956.

CHOI, D. W. (1988). Calcium-mediated neurotoxicity: relationship to speciﬁc channel

types and role in ischemic damage. TINS. 11:465-468.

CHOI, O. H., PADGETI‘, W. L, NISHIZAWA, Y., GUSOVSKY, F ., YASUMOTO,
T. and DALY, J. W. (1990). Maitotoxin: Effects on calcium channels,
phosphoinositide breakdown, and arachidonate release in pheochromocytoma

PC12 cells. Mol. Pharmacol. 37:222-230.

COHEN, M. w. JONES, o. T. and ANGELIDES, K. J. (1991). Distribution of Ca2+
channels in frog motor nerve terminals revealed by ﬂuorescent m -conotoxin.

J. Neurosci. 11:1032-1039.

234
COOPER, G. P. and MANALIS, R. S. (1983). Inﬂuence of heavy metals on synaptic

transmission: A Review. Neurotoxicology. 4:69-84.

COOPER, G. P. and MANALIS, R. S. (1984a). Interactions of lead and cadmium on
acetylcholine release at the frog neuromuscular junction. Taxical. App. Pharm.

74:411-416.

COOPER, G. P. and MANALIS, R. S. (1984b). Cadmium: effects on transmitter

release at the frog neuromuscular junction. Eur. J. Pharmacol. 99:251-256.

COOPER, G. P., SUSZKIW, J. B. and MANALIS, R. S. (1984). Presynaptic effects
of heavy metals. In: Cellular and molecular neurotoxicology. (Narahashi, T.,

ed.). Raven Press, New York. pp. 1-21.

COURTNEY, K. R. (1975). Mechanism of frequency dependent inhibition of sodium
currents in frog myelinated nerve by the lidocaine derivative GEA 968. J.

Pharmacol. Exp. Ther. 195:225-236.

COUTINHO, O. P., CARVALHO, C. A. M. and CARVALHO, A. P. (1984),
Calcium uptake related to K*-depolarization and Na+ /Ca2" exchange in

sheep brain synaptosomes. Brain Res. 290:261-271.

235
CROSS, A. J ., JOHNSON, J. A., FRITH, C. and TAYLOR, G. R. (1984). Muscarinic

cholinergic receptor in a rat phaeochromocytoma cell line. Biochem. Biophys.

Res. Comm. 119:163-167.

CRUZ, L J. and OLIVERA, B. M. (1986). Calcium channel antagonists. m -Conotoxin

deﬁnes a new high afﬁnity site. J. Biol. Chem. 261:6230-6233.

DAMON, D. H., D’AMORE, P. A. and WAGNER, J. A. (1990). Nerve growth factor
and ﬁbroblast growth factor regulate neurite outgrowth and gene expression

in PC12 cells via both protein kinase C- and CAMP-independent mechanisms.

J. Cell Biol. 110:1333-1339.

DANIELL, 1. c., BARR E. M. and LESLIE, s. w. (1983). 45Ca2+ uptake into rat
whole brain synaptosomes unaltered by dihydropyridine calcium antagonists.

J. Neurachem. 41:1455-1459.

DELORME, E. M. and MCGEE, R. (1988). Effects of prolonged depolarization on
the nicotinic acetylcholine receptors of PC12 cells. J. Neurochem. 50:1248-

1252.

236
DELORME, E. M., RABE, C. S. and MCGEE, R. (1988). Regulation of the number

of functional voltage-sensitive Ca+ + channels on PC12 cells by chronic

changes in membrane potential. J. Phannacal Exp. Ther. 244:838-843.

DE WEILLE, J ., FOSSET, M., LAZDUNSKI, M. and SCHMID-ANTOMARCHI,
H. (1989). Galanin inhibits dopamine secretion and activates a potassium

channel in pheochromocytoma cells. Brain Res. 485:199-203.

DICHTER, M. A., TISCHLER, A. S. and GREENE, L A. (1977). Nerve growth
factor-induced increase in electrical excitability and acetylcholine sensitivity

of a rat pheochromocytoma cell. Nature (Land) 268:501-504.

D’ITRI. F. Concentrations of contaminants if ﬁshes of the great lakes. In: Toxic
contamination in large lakes. Volume 2: Impact of toxic contamination on
ﬁsheries management. (Schmidtke, N. W., ed.). Lewis Publishers, Chelsea. pp

51-84.

DI VIRGILIO, F., POZZAN, T., WOLLHEIM, C. B., VICENTINI, L M. and
MELDOLESI, J. (1986). Tumor promoter phorbol myristate acetate inhibits
Ca2+ inﬂux through voltage-gated Ca2+ channels in two secretory cell lines,

PC12 and RINmSF. J. Biol. Chem. 261:32-35.

237
DOOLEY, D. J., LUPP, A. and HERTI‘ING, G. (1987a). Inhibition of central

neurotransmitter release by m-conotoxin GVIA, a peptide modulator of the

N-type voltage-sensitive calcium channel. Naunyn-Schmiedeberg Arch.

Pharmacol. 336:467-470.

DOOLEY, D. J., MAHLMANN, H., BRENNER, O. and OSSWALD, H. (1987b).
Characterization of the dihydropyridine binding sites of rat neocortical

synaptosomes and microvessels. J. Neurochem. 49:900-904.

DOUPE, A J., LANDIS, S. C. and PATTERSON, P. H. (1985). Environmental
inﬂuences in the development of neural crest derivatives: Glucocorticoids,

growth factors and chromafﬁn cell plasticity. J. Neurosci. 5:2119-2142.

DRAPEAU, P. and BLAUSTEIN, M. P. (1983). Initial release of [3Hldopamine from
rat striatal synaptosomes: Correlation with calcium entry. J. Neurosci.

3:703-713.

DUBEL S. J. and SNUTCH, T. P. (1990). Primary structure and expression of the

rat brain class-B calcium channel. Neurasci. Abstr. 16:1174.

238
DUTAR, P., PASCOL O. and LAMOUR, Y. (1989). m-Conotoxin GVIA blocks

synaptic transmission in the CA1 ﬁeld of the hippocampus. Eur. J. Pharmacol.

174:261-266.

DUNN, S. M. J. (1988). Multiple calcium channels in synaptosomes: Voltage
dependence of 1,4-dihydropyridine binding and effects on function.

Biochemistry 27:5275-5281.

ECKERT, R. and CHAD, J. E. (1984). Inactivation of Ca channels. Prog. Biophys.

Molec. Biol. 44:215-269.

EDER-COLLI, L, BRIAND, P. A., PELLEGRINELLI, N. and DURANT, Y.
(1989). A monoclonal antibody raised against plasma membrane of cholinergic
nerve terminals of the Torpedo inhibits choline acetyltransferase activity and

acetylcholine release. J. Neurochem. 53:1419-1427.

FATT, P. and KATZ. B. (1952). Spontaneous subthreshold activity at motor nerve

endings. J. Physiol. (Land) 117:109-128.

FENWICK, E. M., MARTY, A. and NEHER, E. (1982). Sodium and calcium

channels in bovine chromafﬁn cells. J. Physiol. 331:599-635.

239
FLOOR, E. (1983). Substance P release from K+-depolarlzed synaptosomes at one

second resolution. Brain Res. 279:321-324.

FORSHAW, P. J. (1977). The inhibitory effect of cadmium on neuromuscular

transmission in the rat. Eur. J. Pharmacol. 42:371-377.

FOX, A. P., NOWYCKY, M. C. and TSIEN, R. W. (1987a). Kinetic and
pharmacological properties distinguishing three types of calcium currents in

chick sensory neurones. J. Physiol. (Land) 394:149- 172.

FOX, A. P., NOWYCKY, M. C. and TSIEN, R. W. (1987b). Single channel
recordings of three types of calcium channels in chick sensory neurons. J.

Physiol. (Land) 394: 173-200.

FRANKENHEUSER, B. and HODGKIN, A. L. (1957). The action of calcium on the

electrical properties of squid axons. J. Physiol. (Land). 137:218-244

FRIEDMAN, M. B., ERIKSON, C. K. and LESLIE, S. W. (1980). Effects of acute
and chronic ethanol administration on whole mouse brain synaptosomal

calcium inﬂux. Biochem. Pharmacol. VOL:1903-1908.

24o
FUJITA, K., LAZAROVICI, P. and GUROFF, G. (1989). Regulation of the

differentiation of PC12 pheochromocytoma cells. Environ. Health Perspective

1989; 80: 127-142.

GARBER, S. S., HOSHI, T. and ALDRICH, R. W. (1989). Regulation of ionic
currents in pheochromocytoma cells by nerve growth factor and

dexamethasone. J. Neurosci. 9:3976-3987.

GOULD, R. J ., MURPHY, K. M. and SNYDER, S. H. (1985). Autoradiographic
localization of calcium channel antagonist receptors in rat brain with 3H-

nitrendipine. Brain Res. 330:217-223.

GRAY, E. G. and WHITAKER, V. P. (1962). The isolation of nerve endings from
brain: An electron microscopic study of cell fragments derived by

homogenization and centrifugation. J. Anat. (Land) 96:79-87.

GREENBERG, D. A., CARPENTER, C. L and MESSING, R. O. (1987a)
Ethanol-induced component of 45Ca2+ uptake in PC12 cells is sensitive to

Ca2+ channel modulating drugs. Brain Res. 410: 143-146.

241
GREENBERG, D. A., CARPENTER, C. L. and MESSING, R. O. (1987b).

Lectin-induced enhancement of voltage-dependent calcium ﬂux and calcium

channel antagonist binding. J. Neurochem. 48:888-894.

GREENE, L. A. and TISCHLER, A. S. (1976). Establishment of a noradrenergic
clonal line of rat adrenal pheochromocytoma cells which respond to nerve

growth factor. Proc. Natl. Acad Sci. (USA) 73:2424-2428.

GREENE, L. A. and REIN, G. (1977a). Release, storage and uptake of

catecholamines by a clonal cell line of nerve growth factor (NGF) responsive

pheochromocytoma cells. Brain Res. 129:247-263.

GREENE, L. A. and REIN, G. (1977b). Synthesis, storage and release of
acetylcholine by a noradrenergic pheochromocytoma cell line. Nature (Land)

268:349-351.

GREENE, L A and REIN, G. (1977c). Release of [3H]Norepinepherine from a
clonal line of pheochromocytoma cells (PC12) by nicotinic cholinergic

stimulation. Brain Res. 138:521-528.

242
GROSS, R. A. and MACDONALD, R. L (1988). Barbiturates and nifedipine have

different and selective effects on calcium currents of mouse DRG neurons in

culture: A possible basis for differing clinical actions. Neurology 38:443-451.

GUNDERSEN, C. B., KATZ, B. and MILEDI, R. (1982). The antagonism between
botulinum toxin and calcium in motor nerve terminals. Proc. R. Soc. B.

216:369-376.

GUNDERSON, C. B., UMBACH, J. A. and SCHWARTZ, B. E. (1988). Barbiturates
depress currents through human brain calcium channels studied in Xenopus

oocytes. J. Pharmacol. Exp. Ther. 247:824-829.

GUROFF, G., DICKENS, G., END, D. and LONDOS, C. (1981). The action of

adenosine analogs on PC12 cells. J. Neurochem. 37:1431-1439.

GUROFF, G.‘ (1985). PC12 cells as a model of neuronal differentiation. In: Cell
culture in the neurosciences. (Bottenstein, J. E. and Sato, G, eds). Plenum

Press, New York. pp 245-272.

HAGIWARA, S. and TAKASHIMA, K. (1967). Surface density of calcium ions and
calcium spikes in the barnacle muscle ﬁber membrane]. Gen. Physiol. 50:583-

601.

243
HAGIWARA, S., FUKUDA, J. and EATON, D. (1974). Membrane currents carried

by Ca, Sr and Ba in barnacle muscle during voltage clamp. J. Gen. Physiol.

63:564-578.

HAGIWARA, S., MIYAZAKI, S., MOODY, W. and PATTAK, J. (1978). Blocking
effects of barium and hydrogen ions on the potassium current during

anomalous rectiﬁcation in the starﬁsh egg. J. Physiol. (Land) 279:167-185.

HALEEN, S. J., STEFFEN, R. P., SIRCAR, I., MAJOR, T. C., TAYLOR, M. D.,
PUGSLEY, T. A. and WEISHARR, R. E. (1989). PD122860: A novel
dihydropyridine with sodium channel stimulating and calcium channel blocking

properties. J. Pharmacol. Exp. Ther. 250:22-30.

HAMILL, O. P., MARTY, A. SAKMANN, B. and SIGWORTH, F. J. (1981).
Improved patch-clamp techniques for high resolution current recording from

cell and cell-free membrane patches. Pﬂﬁgers Arch. 391:85-100.

HANSEN, J. C. (1990). Human exposure to metals through consumption of marine
food: A case study of exceptionally high intake among Greenlanders. In:
Heavy metals in the marine environment (Furness. A. W. and Rainbow, P. S.,

eds). CRC Press, New York. pp 227-243.

244
HARE, M. F. and ATCHISON, W. D. (1990). Differential effects of mercurials on

synaptosomal membrane potentials. Neurosci. Abstr. 16:445.

HARRIS, R. A. and BRUNO, P. (1985). Effects of ethanol and other intoxicant-
anesthetics on voltage-dependent sodium channels of brain synaptosomes. J.

Pharmacol. Exp. Ther. 232:401-406.

HARRIS, K. M., KONGSAMUT, S. and MILLER, R. J. (1986). Protein kinase C
mediated regulation of calcium channels in PC-12 pheochromocytoma cells.

Biochem. Biophys. Res. Commun. 134: 1298- 1305.

HESS, P. and TSIEN, R. w. (1984). Mechanism of ion permeation through Ca2+

channels. Nature (Land) 309:453-456.

HESS, P., LANSMAN, J. B. and TSIEN, R. W. (1986). Calcium channel selectivity
for divalent and monovalent cations. Voltage and concentration dependence

of single channel current in ventricular heart cells. J. Gen. Physiol. 88:293-319.

HEWE'IT, S. J. and ATCHISON, W. D. (1990). Calcium channel blocking actions
of charged and uncharged mercurials in rat forebrain synaptosomes. Neurosci.

Abstr. 16:512.

245
HILLE, B. (1971). The permeability of the sodium channel to organic cations in

myelinated nerve. J. Gen. Physiol. 58:599-619.

HILLE, B. (1977). Local anesthetics: Hydrophilic and hydrophobic pathways for the

drug-receptor reaction. J. Gen. Physiol. 69:497-515.

HILLE, B. (1984). Ionic channels of excitable membranes. Sinauer Associates Inc.,

Sunderland, MA.

HIRNING, L D., FOX, A. P., MCCLESKEY, E. W., OLIVERA, B. M., THAYER,
S. A., MILLER, R. J. and TSIEN, R. W. (1988). Dominant role of N-type
Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Science 239:57-61.

HODGKIN, A. L and HUXLEY, A. F. (1952). A quantative description of
membrane current and its application to conduction and excitation in the

nerve. J. Physiol. (Land) 117:500-544.

HOF, R. P., RUEGG, U. T., HOF, A. and VOGEL, A. (1985). Stereoselectivity at
the calcium channel: Opposite action of the enantiomers of a 1,4-

dihydropryidine. J. Cardiovasc. Phann. 7 :689-693.

 

246
HOLZ, G. G., DUNLAP, K. and KREAM, R. M. (1988). Characterization of the

electrically-evoked release of substance P from dorsal root ganglion neurones:

Methods and dihydropyridine sensitivity. J. Neurosci. 8:463-471.

HORWITZ, J. (1989). Muscarinic receptor stimulation increases inositol-phospholipid
metabolism and inhibits cyclic AMP accumulation in PC12 cells. J.

Neurochem. 53:197-204.

HOSEY, M. M. and LAZDUNSKI. (1988). Calcium channels: Molecular

pharmacology, structure and regulation. J. Membrane Biol. 104:81-105.

HOSHI, T. and ALDRICH, R. W. (1988a). Voltage-dependent K" currents and
underlying single K+ channels in pheochromocytoma cells. J. Gen. Physiol.

91:73-106.

HOSHI, T. and ALDRICH, R. W. (1988b). Gating kinetics of four classes of voltage-
dependent K+ channels in pheochromocytoma cells. J. Gen. Physiol. 91:107-

131.

HOSS, W. and FORMANIAK, M. (1984). Calcium channel activity in rat brain
synaptosomes: Effects of neuroleptics and other factors regulating

phosphorylation and transmitter release. Neurochem. Res. 9:109-120.

 

247
HOWARD, B. D., MELEGA, W. P., KOIDE, M., BUTLER, C. M., ZHANG, M.

and KHAW, M. (1986). Neurotransmitter release from PC12 cells. In:
Calcium, neuronal function and neurotransmitter release. (Rahamimoff, R.

and Katz, B, eds.) Martinus-Nijhoff Inc., Boston. pp. 279-297.

HUBBARD, J. I., JONES, S. F. and LANDAU, E. M. (1968). On the mechanism by
which calcium and magnesium affect the spontaneous release of transmitter

from mammalian motor nerve terminals. J. PhysioL (Land) 194:353-380.

HUNEEUS-COX, F., FERNANDEZ, H. L and SMITH, B. H. (1966). Effects of
redox and sulﬂiydryl reagents on the bioelectric properties of the giant axon

of the squid. Biophys. J. 6:675-689.

IFUNE, C. K. and STEINBACH, J. H. (1990). Regulation of sodium currents and

acetylcholine responses in PC12 cells. Brain Res. 506:243-248.

INOUE, K. and KENIMER, J. G. (1988). Muscarinic stimulation of calcium inﬂux

and norepinepherine release in PC12 cells. J. Biol. Chem 263:8157-8161.

INOUE, N., MATSUI, H. and HATANAKA, H. (1988). Nerve growth factor induces

a Na+,K*-ATPase in a nerve cell line. J. Neurochem. 50:230-236.

248
JANIGRO, D., MACCAFERRI, G. and MELDOLESI, J. (1989). Calcium channels

in undifferentiated PC 12 rat pheochromocytoma cells. FEBS Lett. 255:398-400.

JUAN G, M. S. (1976). An electrophysiological study of the action of methylmercury
chloride and mercuric chloride on the sciatic nerve-sartorius muscle

preparation of the frog. Toxicol. Appl. Pharmacol. 35:339-348.

JUMBLATI‘, J. E. and TISCHLER, A. S. (1982). Regulation of muscarinic ligand
binding sites by nerve growth factor in PC12 pheochromocytoma cells. Nature

(Land) 297:152-154.

KALMAN, D., WONG, B., HORVAI, A. E., CLINE, M. J. and O’LAGUE, P. H.
(1990). Nerve grth factor acts through CAMP-dependent protein kinase to

increase the number of sodium channels in PC12 cells. Neuron 2:355-366.

KATZ, B. and MILEDI, R. (1967a). The timing of calcium action during

neuromuscular transmission. J. Physiol. (Land) 189:535-544.

KATZ, B. and MILEDI, R. (1967b). A study of synaptic transmission in the absence

of nerve impulses. J. Physiol. (Land) 192:407-436.

 

249
KAUPPINEN, R. A., KOMULAINEN, H. and TAIPALE, H. (1989). Cellular

mechanisms underlying the increase in cytosolic free calcium concentration

induced by methylmercury in cerebrocortical synaptosomes from guinea pig.

J. Pharmacol. Exp. Ther. 248:1248-1254.

KAMIYA, H., SWADA, S. and YAMAMOTO, C. (1988). Synthetic to -conotoxin
blocks synaptic transmission in the hippocampus in vitro. Neurosci. Lett. 91:84-

88.

KEITH, R. A., MANGANO, T. J. and SALAMA, A. I. (1989). Inhibition of N-
methyl-D-aspartate- and kaniac acid-induced neurotransmitter release by to -

conotoxin GVIA. Br. J. Pharmacol. 98:767-772.

KENNEDY, M. B. (1989). Regulation of neuronal function by calcium. TINS 12:417-

419.

KITA, H. and VAN DER KLOOT, W. (1973). Action of Co and Ni at the frog

neuromuscular junction. Nature (Land) 245:52-53.

KLAASSEN, C. D. (1985). Heavy metals and heavy metal antagonists. In: The

pharmacological basis of therapeutics. (Gilman, A. G., Goodman, L. 8.,

250
Randall, T. W., and Murad, F., eds.). MacMillan Publishing Co., New York,

pp. 1611-1614.

KN AUS, H., STRIESSNIG, J ., KOZA, A. and GLOSSMANN, H. (1987). Neurotoxic
aminoglycoside antibiotics are potent inhibitors of [mI]-omega-conotoxin
GVIA binding to guinea-pig cerebral cortex membranes. Naunyn-

Schmiedeberg. Arch. Pharmacol. 336:583-586.

KOBER, T. E. and COOPER, G. P. (1976). Lead competitively inhibits
calcium-dependent synaptic transmission in the bullﬁ'og sympathetic ganglion.

Nature (Land) 262:704-705.

KOH(E, T. and TAKASHIMA, A. (1984). Clonal variability of PC12
pheochromocytoma cells with respect to catecholamine biosynthesis. J.

Neurochem. 42:1472-1475.

KOIKE, T. and TAKASHIMA, A. (1986). Cell cycle-dependent modulation of
biosynthesis and stimulus-evoked release of catecholamines in PC12

pheochromocytoma cells. J. Neurochem. 46: 1493-1500.

251
KOMULAINEN, A. and BONDY, S. C. (1987). Increased free intrasynaptosomal

Ca2+ by neurotoxic organometals: Distinctive mechanisms. T oxicoL Appl.

Pharmacol. 88:77-86.

KONISHI, T. (1985). Electrical exciatbility of motor nerve terminals in the mouse.

J. Physiol. (Land). 366:411-421.

KONGSAMUT, S. and MILLER, R. J. (1986). Nerve grth factor modulates the
drug sensitivity of neurotransmitter release from PC12 cells. Proc. Natl. Acad

Sci. (U.S.A.) 83:2243-2247.

KOSTIAL, K. and VOUK, V. B. (1957). Lead ions and synaptic transmission in the

superior cervical ganglion of the cat. Br. J. Pharmacol. Chemother. 12:219-222.

KOSTYUK, P. G., SHUBA, Y. M. and SAVCHENKO, A. N. (1988). Three types of
calcium channels in the membrane of mouse sensory neurones. Pﬂr’igers Arch.

411:661-669.

KRESS, H. G, ECKHARDT-WALLASCH, E., TAS, P. W. L and KOSCHEL K
(1987). Volatile anesthetics depress the depolarization-induced cytoplasmic

calcium rise in PC12 cells. FEBS Lett. 221:28-32.

_r ‘J‘L‘r-nl“

 

252
KUROZUMI, K., MURAYAMA, T. and NOMURA, Y. (1990). Generation of

inositol phosphates, cytosolic Ca2+ and secretion of noradrenaline in PC12

cells treated with glutamate. FEBS Lett. 270:225-228.

LANG, B., VINCENT, A., MURRAY., N. M. F. and NEWSOM-DAVIS, J. (1989).
Lambert-Eaton myasthenic syndrome immunogloban G inhibition of Ca2+

ﬂux in tumor cells correlates with disease severity. Ann. Neurol. 25:265-271.

LANSMAN, J. B., HESS, P. and TSIEN, R. W. (1986). Blockade of current through
single calcium channels by Cdz”, Mg“ and Ca2+. Voltage and concentration

dependence of calcium entry into the pore. J. Gen. Physiol. 88:321-347.

LEE, K. S. and TSIEN, R. W. (1982). Reversal of current through calcium channels

in dialysed single heart cells. Nature (Land) 297:498-501.

LEE, K. S. and TSIEN, R. W. (1983). Mechanism of calcium channel blockade by
verapamil, D-600, diltiazem and nitrendipine in single dialyzed heart cells.

Nature (Land) 302:790-794.

LEMOS, J. R. and NOWYCKY, M. C. (1989). Two types of calcium channels coexist

in peptide-releasing vertebrate nerve terminals. Neuron 2:1419-1426.

 

253
LEONARD, J. P., NARGIOT, J ., SNUTCH, T. P., DAVIDSON, N. and LESTER,

H. A. (1987). Ca channels induced in Xenopus oocytes by rat brain mRNA.

J. Neurosci. 7 :875-881.

LESLIE, S. W. (1987). Calcium channels. Interactions with ethanol and other
sedative-hypnotic drugs. In: Recent developments in alcoholism. Volume 5.

(Galanter, M., ed.). Plenum Publishing, New York. pp 289-302.

LESLIE, S. W., BARR, E., CHANDLER, J. and FARRAR, R. P. (1983). Inhibition
of fast- and slow-phase depolarization-dependent synaptosomal calcium

uptake by ethanol. J. Pharmacol. Exp. Ther. 225:571-575.

LESLIE, S. W., WOODWARD, J. J. and WILCOX, R. E. (1985). Correlation of
rates of calcium entry and endogenous dopamine release in mouse striatal

synaptosomes. Brain Res. 325:99-105.

LEVESQUE, P. C. and ATCHISON, W. D. (1987). Interactions of mitochondrial
inhibitors with methylmercury on spontaneous quantal release of

acetylcholine. ToxicoL App. Pharmacol. 87:315-324.

254
LEVESQUE, P. C. and ATCHISON, W. D. (1988). Effect of alteration of nerve

terminal Ca2+ regulation on increased spontaneous quantal release of

acetylcholine by methylmercury. Toxicol. Appl. Pharmacol. 95:55-65.

LEVESQUE, P. C. and ATCHISON, W. D. (1991). Disruption of brain
mitochondrial calcium sequestration by methylmercury. J. Pharmacol. Exp.

Ther. 256:236-242.

LIM, N. F., NOWYCKY, M. C. and BOOKMAN, R. J. (1990). Direct measurement
of exocytosis and calcium currents in single vertebrate nerve terminals Nature

(Land) 334:449-451.

LIN, Y. W., SUGIMORI, M., LLINAS, R. R., MCGUINNESS, T. L and
GREENGARD, P. (19903). Effects of synapsin I and calcium/calmodulin-

dependent protein kinase H on spontaneous neurotransmitter release in the

squid giant synapse. Proc. Natl. Acad Sci. (U.S.A.). 87:8257-8261.

LIN, Y. W., RUDY, B. and LLINAS, R. R. (1990b). Funnel-web spider venom and
a toxin fraction block calcium current expressed from rat brain mRNA in

Xenopus oocytes. Proc. Natl. Acad Sci. (U.S.A.). 87:4538-4542.

255
LLINAS, R. and YAROM, Y. (1981). Properties and distribution of ionic

conductances generating electroresponsiveness of mammalian inferior olivary

neurones in vitro. J. Physiol. (Land) 315:569-584.

LLINAS, R., STEINBERG, I. Z. and WALTON, K. (1981). Presynaptic calcium

currents in squid giant synapse. Biophys. J. 33:289-322.

LLINAS, R., MCGUINNESS, T. L, LEONARD, C. S., SUGIMORI, M. and
GREENGARD, P. (1985). Intraterminal injection of synapsin I or
calcium/calmodulin—dependent protein kinase II alters neurotransmitter

release at the squid gaint synapse. Proc. Natl. Acad Sci. USA 82:3035-3039.

LLINAS, R., SUGUMORI, M., LIN, J. W. and CHERKSEY, B. (1989). Blocking
and isolation of a calcium channel from neurons in mammals and cephalopods
utilizing a toxin fraction (FI‘X) from funnel-web spider poison. Proc. Natl.

Acad Sci. (U.S.A.) 86:1689-1693.

LOWRY, O. H., ROSEBROUGH, N. J ., FARR, A. L. and RANDALL, R. J. (1951).
Protein measurement with the Folin phenol reagent. J. Biol. Chem.

193:265-275.

 

256
LUKAS, R. J. (1990). Heterogeniety of high-afﬁnity nicotinic [3H]-acetylcholine

binding sites. J. Pharmacol. Exp. Ther. 253:51-57.

LUNDY, P. M., FREW, R., FULLER T. W. and HAMILTON, M. G. (1991).
Pharmacological evidence for an w‘CODOtOXIn, dihydropyridine-insensitive

neuronal Ca2+ channel. Eur. J. Pharmacol. 206:61-68.

MAGGI, C. A., PATACCHINI, R., SANTICIOLI, P., LIPPE, I. T., GIULIAMI, S.,
GEPPETI‘I, P., DEL BIANCO, E., SELLERI, S. and MELI, A. (1988). The
effect of omega conotoxin GVIA, a peptide modulator of the N-type voltage
sensitive calcium channels, on motor responses produced by activation of
efferent and sensory nerves in mammalian smooth muscle. Naunyn-

Schmiedeberg Arch. Pharmacol. 338:107-113.

MAGOS, L. (1975). Mercury and mercurials. Br. Med Bull. 33:241-245.

MALLART, A. (1985). Electric current ﬂow inside perineurial sheaths of mouse

motor nerves. J. Physiol. (Land) 368:565-575.

MALLART, A. and BRIGANT, J. L (1982). Presynaptic currents in mouse motor

endings. J. Physiol. (Land) 333:619-636.

257
MANALIS, R. S. and COOPER, G. P. (1973). Presynaptic and postsynaptic effects

of lead at the neuromuscular junction. Nature (Land) 243:354-356.

MANGAN OS, T. J .,PATEL, J ., SALAMA, A. I. and KEITH, R. A. (1991). Inhibition
of K+-evoked [3H]D-aspartate release and neuronal calcium inﬂux by
verapamil, diltiazem and dextromethorphan: evidence for non-L/non-N

voltage-sensitive calcium channels.

MARKS, S. S., WATSON, D. L, CARPENTER, C. L, MESSING, R. O. and
GREENBERG, D. A. (1989). Comparative effects of chronic exposure to

ethanol and calcium channel antagonists in calcium channel antagonist

receptors in cultured neural (PC12) cells. J. Neurochem. 53:168-172.

MARQUEZE, B., MARTIN—MOUTOT, N., LLEVESQUE, C. and COURAND, F.
(1988). Characterization of the cti-conotoxin binding molecule in rat brain

synaptosomes and cultured neurons. Mol. Pharmacol. 34:87-90.

MARSHALL, I. G. (1970). Studies on the blocking action of 2-(4-phenyl piperidino)

cyclohexanol (AH5183). Br. J. Pharmacol. 38:503-516.

MARTINEZ-SERRANO, A., BOGONEZ, E., VITORICA, J. and SATRUSTEGUI,

J. (1989). Reduction of K+-stimulated 4sCa inﬂux in synaptosomes with age

258

involves inactivating and non-inactivating calcium channels and is correlated
with temporal modiﬁcations in protein dephosphorylation. J. Neurochem.

52:576-584.

MASSIEU, L and TAPIA, R. (1988). Relationship of dihydropyridine binding sites
with calcium-dependent neurotransmitter release in synaptosomes. J.

Neurochem. 51:1184-1189.

MATSUOKA, l., MIZUNO, N. and KURIHARA, K. (1989). Cholenergic
differentiation of clonal rat pheochromocytoma cells (PC12) induced by
retanoic acid: Increase of choline acetyltransferase activity and decrease of

tyrosine hydroxylase activity. Brain Res. 502:53-60.

MCARDLE, J ., AN GAUT-PETIT, D., MALLART, A., FAILLE, L and BRIGANT,
J. L (1981). Advantage of the triangularis sterni muscle of the mouse for

investigations of synaptic phenomena. J. Neurosci. Meth. 4: 109-113.

MCHUGH, E. M. and MCGEE, R. (1986). Direct anesthetic-like effects of forskolin
on the nicotinic acetylcholine receptors of PC12 cells. J. Biol. Chem. 261:3103-

3106.

259
MCGEE, R. and LEIPE, B. (1984). Acute elevation of cyclic AMP does not alter the

ion-conducting properties of the neuronal nicotinic acetylcholine receptor of

PC12 cells. Mol. Pharmacol. 26:51-56.

MCKAY, S. J., REYNOLDS, J. N. and RACZ, W. J. (1986). Effects of mercury
compounds on the spontaneous and potassium-evoked release of [3H]-

dopamine from mouse striatial slices. Can. J. Physiol. Pharmacol. 64: 1507-1514.

MELDOLESI, J ., SCHEER, H., MADEDDU, L and WANKE, E. (1986).
Mechanism of action of a -latrotoxin: the presynaptic stimulatory toxin of the

black widow spider venom. Trends in Pharmacological Sci. 7: 151-155.

MELDOLESI, J ., VOLPE, P. and POZZAN, T. (1988). The intracellular distribution

of calcium. TINS 11:449-452.

MELEGA, W. P. and HOWARD, B. D. (1981) Choline and acetylcholine

metabolism in PC12 secretory cells. Biochem. 20:4477-4483.

MELEGA, W. P. and HOWARD, B. D. (1984). Biochemical evidence that vesicles
are the source of the acetylcholine released from stimulated PC12 cells. Proc.

Natl. Acad Sci. (U.S.A.) 81:6535-6538.

260
MESSING, R. O., STEVENS, A. M., KIYASU, E. and SNEADE, A. B. (1989).

Nicotinic and muscarinic agonists stimulate rapid protein kinase C

translocation in PC12 cells. J. Neurosci. 9:507-512.

MICHEL, A. D., STEFANICH, E. and WHITING, R. L (1989). PC12
phaeochromocytoma cells contain an atypical muscarinic receptor binding site.

Br. J. Pharmacol. 97:914-920.

MILLER, F. M. (1984). Chemistry, Structure and dynamics. M‘Graw-Hill Book
Company, St. Louis. pp 177, 748.
MILLER, R. J. (1987). Multiple calcium channels and neuronal function. Science

235:46-52.

MINTZ, J. M., VENEMA, V. J., ADAMS, M. B. and BEAN, B. P. (1990). T h e
funnel-web speder toxin in -Aga-IIIA blocks N- and L-type calcium channels in

neurons and cardiac muscle. Neurosci Abstr. 16:956.

MIYAMATO, M. D. (1983). Hg2+ causes neurotoxicity at an intracellular site

following entry through Na and Ca channels. Brain Res. 267:375-379.

MIZRACHI, Y., NARANJO, J. R., LEVI, B., POLLARD, H. B. and LELKES, P.

I. (1990). PC12 cells differentiate into chromafﬁn-cell like phenotype in

261
coculture with adrenal medullary endothelial cells. Proc. Natl. Acad Sci.

(U.S.A.) 87:6161-6165.

NACHSHEN, D. A. (1984). Selectivity of the Ca binding site in synaptosome Ca

channels. Inhibition by multivalent metal cations. J. Gen. Physiol. 83:941-967.

NACHSHEN, D. A. (1985). The early time course of potassium-simulated calcium
uptake in presynaptic terminals isolated from rat brain. J. Physiol. (Land)

361:25 1-268.

NACHSHEN, D. A. and BLAUSTEIN, M. P. (1979). The effects of some organic
"calcium antagonists" on calcium inﬂux in presynaptic nerve terminals. Mol.

Pharmacol. 16:579-586.

NACHSHEN, D. A. and BLAUSTEIN, M. P. (1980). Some properties of
potassium-stimulated calcium inﬂux in presynaptic nerve endings. J. Gen.

Physiol. 76:709-728.

NACHSHEN, D. A. and BLAUSTEIN, M. P. (1982). Inﬂux of calcium, strontium,

and barium in presynaptic nerve endings. J. Gen. Physiol. 79: 1065-1087.

 

262
NACHSHEN, D. A., SANCHEZ-ARMASS, S. and WEINSTEIN, A. M. (1986). The

regulation of cytosolic calcium in rat brain synaptosomes by sodium-dependent

calcium efﬂux. J. Physiol. (Land) 381:17-28.

NAKAZATO, Y., ASANO, T. and OHGA, A. (1979). The in vitro effect of mercury
compounds of noradrenaline output from guinea pig vas deferens. Toxicol.

Appl. Pharmacol. 48:171-177.

NARAHASHI, T. (1980). Nerve membrane as a target of environmental
neurotoxicants. In: Experimental and clinical neurotoxicology. (Spencer, P. S.,

Schaumburg, H. H., eds.). Williams and Wilkins, Inc. Baltimore. pp. 225-238.

NARAHASIH, T., MOORE, J. W. and SCOTT, W. R. (1964). Tetrodotoxin blockage
of sodium conductance increase in lobster giant axons. J Gen. Physiol.

47:965-974.

NARANJO, J. R., MOCCHETTI, I., SCHWARTZ, J. P. and COSTA, E. (1986).
Permissive effect of dexamethasone on the increase of proenkephalin mRNA
induced by depolarization of chromafﬁn cells. Proc. Natl. Acad Sci. (U.S.A.)

83:1513-1517.

 

263
NELSON, M. T., ROUDNA, M. and BAMBERG, E. (1983). Single channel K+-

current measurements from brain synaptosomes in lipid bilayers. Am. J.

Physiol. 245:C151-C156.

NELSON, M. T. (1985). Effects of blockers and permeant ions on the properties of
single calcium channels from brain. In: proteins of excitable membranes.
(Hille, B. and Fambrough, D., eds.). John Wiley and Sons, New York. pp 215-

230.

NELSON, M. T. (1986). Interactions of divalent cations with single calcium channels

from rat brain synaptosomes. J. Gen. Physiol. 87:201-222.

NILSON, R. and VOLLE, R. L (1976). Blockade by cadmium (CdH) at the frog

neuromuscular junction. Fed Proc. 35:696.

NISHIZAWA, Y., SEAMON, K. B., DALY, J. W. and ARONSTAM, R. S. (1990).
Effects of forskolin and analogues on nicotinic receptor-mediated sodium ﬂux,
voltage-dependent calcium ﬂux and voltage-dependent rubidium efﬂux in

pheochromocytoma cells. Cellular Molec. Neurobiol. 10:351-368.

264
NORONHA-BLOB, L, MARSHALL, R. P., KINNIER, W. J. and U’PRICHARD,

D. C. (1986). Pharmacological proﬁle of adensoine A2 receptor in PC12 cells.

Life Sci. 39:1059-1067.

NOWYCKY, M. C. (1991). Kinetic properties of L and Nt-type Ca2+ channels and
their ability to promote exocytosis in neurohypophysial terminals. Biophys. J.

59:276a.

N OWYCKY, M. C., FOX, A. P. and TSIEN, R. W. (1985a). Three types of neuronal
calcium channels with different calcium agonist sensitivity. Nature (Land)

316:440-443.

NOWYCKY, M. C., FOX, A. P. and TSIEN, R. W. (1985b). Long opening mode of
gating of neuronal calcium channels and its promotion by the dihydropyridine
calcium channel agonist Bay K 8644. Proc. Natl. Acad Sci. (U.S.A.)

82:2178-2182.

OGATA, N. and NARAHASHI, T. (1990). Potent blocking action of chlorpromazine
on two types of calcium channels in cultured neuroblastoma cells. J.

Pharmacol. Exp. Ther. 252:1142-1149.

 

265
OLIVERA, B. M., GRAY, W. R., ZEIKUS, R., MCINTOSH, J. M., VARGA, J.,

RIVIER, J., DESANT OS, V. and CRUZ, L J. (1985). Peptide neurotoxicns

from fish-hunting cone snails. Science (Wash. D. C.) 230:1338-1343.

OLIVERA, B. M., CRUZ, L J., DESANTOS, V., LECI-IEMINANT, G. W.,
GRIFFIN, D., ZEIKUS, R., MCINTOSH, J. M., GALYEAN, R., VARGA,
J ., GRAY, W. R. and RIVIER, J. (1987). Neuronal calcium channel
antagonists. Discrimination between calcium channel subtypes using to-

conotoxin from Conus magnus venom. Biochemistry 26:2086-2090.

PARNAS, H., DUDEI, J. and PARNAS, I. (1986). Neurotransmitter release and its
facilitation in crayﬁsh. VII. Another voltage dependent process beside Ca

entry controls the time course of phasic release. Pﬂugers Arch. 406:121-130.

PARNAS, H., HOVAV, G. and PARNAS, I. (1989). Effect of Ca2+ diffusion on the

time course of neurotransmitter release. Biophys. J. 55:859-874.

PASSOW, H. and ROTHSTEIN, A. (1960). The binding of mercury by the yeast cell

in relation to changes in permeability. J. Gen. Physiol. 43:621-633.

PENNER, R. and DREYER. F. (1986). Two different presynaptic calcium currents

in mouse motor nerve terminals. Pﬂu'gers Arch. 406: 190-197.

 

266
PERNEY, T. M., HIRNING, L D., LEEMAN, S. E. and MILLER, R. J. (1986).

Multiple calcium channels mediate neurotransmitter release from peripheral

neurons. Proc. Natl. Acad Sci. (U.SA.) 83:6656-6659.

PICKETT, J. B. and BORNSTEIN, J. C. (1978). Lead competitively inhibits
calcium-dependent evoked release at rat neuromuscular junctions. Neurology

28:335.

PICKETT, J. B. and BORNSTEIN, J. C. (1984). Some effects of lead at mammalian

neuromuscular junction. Am. J. Physiol. 246:C271-C276.

PLUMMER, M. R., LOGOTHETIS, D. E. and HESS, P. (1989). Elememtary
properties and pharmacological sensitivities of calcium charmels in

mammalian peripheral neurons. Neuron 2:1453-1463.

POLLOCK, J. D., KREMPIN, M. and RUDY, B. (1990). Differential effects of N GF ,
FGF, ECF, cAMP, and dexamethasone on neurite outgrowth and sodium

channel expression in PC12 cells. J. Neurosci. 8:2626-2637.

POZZAN, T., GATTI, G., DOZIO, N., VICENTINI, L M. and MELDOLESI, J.
(1984). Ca2*-dependent and independent release of neurotransmitters from

PC12 cells: A role for protein kinase C activation? J. Cell Biol. 99:628-638.

 

267
PUMPLIN, D. W., REESE, T. S. and LLINAS, R. (1981). Are the presynaptic

membrane particles the calcium channels? Proc. Natl. Acad Sci. (U.S.A.)

78:7210-7213.

PUN, R. Y. K. and BEHBEHANI, M. M. (1989). A rapidly inactivating
Ca2+ -dependent K+ current in phenochromocytoma cells (PC12) of the rat.

Eur. J. PhysioL 415:425432.

QUANDT, F. N., KATO, E. and NARAHASHI, T. (1982). Effects of methylmercury

on electrical responses of neuroblastoma cells. Neurotoxicology 3:205-220.

RABE, C. S., SCHNEIDER, J. and MCGEE, R. (1982). Enhancement of
depolarization-dependent neurosecretion from PC 12 cells by forskolin-induced

elevation of cyclic AMP. J. Cyclic Nucl. Res. 8:371-384.

RABE, C. S. and MCGEE, R. (1983). Regulation of depolarization-dependent
release of neurotransmitters by adenosine: Cyclic AMP-dependent

enhancement of release from PC12 cells. J. Neurochem. 41:1623-1634.

RABE, C. S., DELORME, E. and WEIGHT, F. F. (1987). Muscarine-stimulated
neurotransmitter release from PC12 cells. J. Pharmacol. Exp. Ther. 243:534-

541.

 

268
RABE, C. S. and WEIGHT, F. F. (1988). Effects of ethanol on neurotransmitter

release and intracellular free calcium in PC12 cells. J. Pharmacol. Exp. Ther.

243:417-422.

RAMPE, D., JANIS, R. A. and TRIGGLE, D. J. (1984). Bay K 8644, a
1,4-dihydropyridine Ca2+ channel activator: Dissociation of binding and

fimctional effects in brain synaptosomes. J. Neurochem. 43:1688-1692.

RANE, s. G., HOLTZ, G. G. and DUNLAP, K (1987). Dihydropyridine inhibition

of neuronal calcium current and substance P release. Pﬂiigers Arch.

409:361-366.

REED, LR. and D. ENGLAND. (1986). The effect of nerve grth factor on the

development of sodium channels in PC12 cells. Biochem. Cell Biol.

64:1153-1159.

REUHL, K. R. (1987). Neuropathology of organometallic compounds: Review of
selected literature. In: N eurotoxicants and neurobiological function: Effects

of organoheavy metals. (Tilson, H. A. and Sparber, S. B., eds.) John Wiley

and Sons, Inc., New York. pp 117-136.

rm 'fﬂp

269
REYNOLDS, I. J ., WAGNER, J. A., SNYDER, S. H., THAYER, S. A., OLIVERA,

B. M. and MILLER, R. J. (1986). Brain voltage-sensitive calcium channels
- differentiated by Q'COHOLOXIH fraction GVIA. Proc. Natl. Acad Sci. (U.S.A.)

83:8804-8807.

RIDLEY, W. P. DIZIKES, L. J. and WOOD, J. M. (1977). Biomethylation of toxic

elements in the environment. Science 197:329-332.

RIVIER, J ., GAIﬁEAN, R., GRAY, W. R., AZIMI-ZONOOZ, A., MCINTOSH, J.
M., CRUZ, L J. and OLIVERA, B. M. (1987). Neuronal calcium channel

inhibitors. Synthesis of to -conotoxin 45Ca uptake by synaptosomes. J. Biol.

Chem. 262:1194-1198.

ROBINSON, D. and MCGEE, R. (1985). Agonist-induced regulation of the neuronal

nicotinic acetylcholine receptor of PC12 cells. Mol. Pharmacol. 27:409-417.

ROBITAILLE, R., ADLER, E. M. and CHARLTON, M. P. (1990). Strategic
location of calcium channels at transmitter release sites of frog neuromuscular

synapses. Neuron 5:733-739.

 

270
ROGAWSKI, M. A., PIENIEK, M., SUZUKI, S. and FFRENCH-MULLEN, J. M.

H. (1988). Phencyclidine selectively blocks the sustained voltage-dependent

potassium conductance in PC12 cells. Brain Res. 456:38-48.

ROSKOSKI, R. and ROSKOSKI, L. M. (1989). Adenosine receptor activation and
the regulation of tyrosine hydroxylase activity in PC12 and PC18 cells. J.

Neurochem. 53:1934-1940.

ROMEY, G. and LAZDUNSKI, M. (1982). Lipid-soluble toxins thought to be
speciﬁc for Na“ channels block Ca2+ channels in neuronal cells. Science

297:79-80.

RUDY, B., KTRSCHENBAUM, B., RUBENSTEIN, A. and GREENE, L A. (1987).
Nerve growth factor increases the number of functional Na channels and
induces TTX-resistant Na channels in PC12 pheochromocytoma cells. J.

Neurosci. 7 :1613-1625.

RUSTAM, H., VON BURG, R., AMIN-ZAKI, L and EL HASSANI, S. (1975).
Evidence for a neuromuscular disorder in methylmercury poisioning. Clinical
and electrophysiological ﬁndings in moderate to severe cases. Arch. Environ.

Health 30:190-195.

 

271
SAGER, P. R., DOHERTY, R. A. and OLMS'IED, J. B. (1982). Interaction of

methylmercury with microtubules in cultured cells and in vitro. Exp. Cell Res.

146: 127-137.

SANDBERG, K., BERRY, C. J. and ROGERS, T. B. (1989a). Studies on the
intoxication pathway of tetanus toxin in the rat pheochromocytoma (PC12)
cell line. Binding, internalization, and inhibition of acetylcholine release. J.

Biol. Chem. 264:5679-5686.

SANDBERG, K., BERRY, C. J. and ROGERS, T. B. (1989b). A role for cGMP
during tetanus toxin blockade of acetylcholine release in the rat

pheochromocytoma (PC12) cell line. J. Neurosci. 9:3946-3954.

SCATCHARD, G. (1949). The attractions of proteins for small molecules and ions.

Ann. NY. Acad Sci. 51:660-672.

SCHMID-ANTOMARCHI, H., HUGUES, M. and LAZDUNSKI, M. (1986)
Properties of the apamin-sensitive Ca2+-activated K+ channel in PC12
pheochromocytoma cells which hyper-produce the apamin receptor. J. Biol.

Chem. 261:8633-8637.

. 9

272
SCHUBERT, D. and KLIER, F. G. (1977). Storage and release of acetylcholine by

a clonal cell line. Proc. Natl. Acad Sci. (U.S.A.) 74:5184-5188.

SCHUBERT, D., HEINEMANN, S. and KIDOKORO, Y. (1977). Cholenergic
metabolism and synapse formation by a rat nerve cell line. Proc. Natl. Acad

Sci. (U.S.A.) 74:2579-2583.

SEABROOK, G. R. and ADAMS D. J. (1989). Inhibition of neurally-evoked
transmitter release by calcium channel antagonists in rat parasympathetic

ganglia. Br. J. Pharmacol. 97:1125-1136.

SEEGAL, R. F., BROSCH, K., BUSH, B., RI'I‘Z, M. and SHAIN, W. (1989). Effects
of Arochlor 1254 on dopamine and norepinephrine concentrations in

pheochromocytoma (PC-12) cells. Neurotoxicology 10:757-764.

SHAFER, T. J. and ATCHISON, W. D. (1989). Block of 45Ca uptake into
synaptosomes by methylmercury: Ca2+- and N a+-dependence. J. Pharmacol

Exp. Ther. 248:696-702.

SHAFER, T. J. and ATCHISON, W. D. (1991). Methylmercury blocks of N- and L-
type Ca2+ channels in nerve growth factor-differentiated pheochromocytoma

cells. J. Pharrhacol. Exp. Ther. 258:697-702.

 

 

273
SHAFER, T. J., CONTRERAS, M. L and ATCHISON, W. D. (1990).

Characterization of interactions of methylmercury with Ca2+ channels in
synaptosomes and pheochromocytoma cells: Radiotracer ﬂux and binding

studies. Mol. Pharmacol. 38:102-113.

SHEPHARD, G. M. (1990). Introduction to synaptic circuits. In: The synaptic
organization of the brain. (Shepherd, G. M., ed.). Oxford University Press,
New York. p 5.

SHER, E., PANDIELLA, A. and CLEMENTI, F. (1988). to-Conotoxin binding and
effects on calcium channel function in human neuroblastoma and rat

pheochromocytoma cell lines. FEBS Lett. 235:178-182.

SIHRA, T. S., WANG, J. K., GORELICK, F. S. and GREENGARD, P. (1989).
Translocation of synapsin I in response to depolarization of isolated nerve

terminals. Proc. Natl. Acad Sci. (U.S.A.) 86:8108-8112.

SHRAGER, P. (1977). Slow sodium inactivation in nerve after exposure to sulfhydryl

blocking reagents. J. Gen. Physiol. 69:183-202.

SHRIVASTAV, B. B., BRODWICK, M. S. and NARAHASHI, T. (1976).
Methylmercury: Effects on electrical properties of squid axon membranes. Life

Sci. 18:1077-1082.

 

 

274
SILBERGELD, E. K., FALES, J. T. and GOLDBERG, A. M. (1974a). Evidence for

a junctional effect of lead on neuromuscular function. Nature (Land)

247:49-50.

SILBERGELD, E. K., FALES, J. T. and GOLDBERG, A. M. (1974b). The effects

of inorganic lead on the neuromuscular junction. Neuropharmacal. 13:795-801.

SKATTEBOL, A. and RABIN, R. A. (1987). Effects of ethanol on 45Ca2+ uptake in

synaptosomes and PC12 cells. Biochem. Pharmacol. 36:2227-2229.

SMITH, S. J. and AUGUSTINE, G. J. (1988). Calcium ions, active zones and

synaptic transmitter release. TINS 11:458-464.

SNUTCH, T. P., LEONARD, J. P., GILBERT, M. M., LESTER, H. A. and
DAVIDSON, N. (1990). Rat brain expressis a heterogeneous family of

calcium channels. Proc. Natl. Acad Sci. (U.S.A.). 87:3391-3395.

SOMJEN, G. G., HERMAN, S. P., KLEIN, R., BRUBAKER, P. E., BRINER, W.
H., GOODRICH, J. K., KRIGMAN, M. R.,and MESEMAN, J. K. (1973).
The uptake of methylmercury (”Hg) in different tissues rleated to its

neurotoxic effects. J. Pharmacol. Exp. Ther. 187:602-611.

 

275
STALLCUP, W. B. (1979). Sodium and calcium ﬂuxes in a clonal nerve cell line. J.

Physiol. (Land) 286:525-540.

STARR, T. V., PRYSTAY, W. A. and SNUTCH, T. P. (1990). Primary structure and

expression of class-A calcium channel. J. Neurosci. 16:1174.

STEELE, R. G. D. and TORRIE, J. H. (1980). Principles and Procedures of

Statistics. McGraw-Hill. New York. pp. 99-160.

STEINWALL, O. and KLATZO, I. (1966). Selective vulnerability of the blood brain
barrier in chemically induced lesions. Acta Neuropathol. Exp. Neurol. 25:542-

559.

STEINWALL O. and OLSSON, Y. (1969). Selective vulnerability of the blood brain

barrier in chemically induced lesions. Acta Neurol. Scand 45:351-361.

STREIT, J. and LUX, H. D. (1987). Voltage dependent calcium currents in PC12
growth cones and cells during N GF-induced cell growth. Pﬂu'gers Arch.

408:634-641.

STREIT, J. and LUX, H. D. (1989). Distribution of calcium currents in sprouting

PC12 cells. J. Neurosci. 9:4190-4199.

 

276
STREIT, J. and LUX, H. D. (1990). Calcium current inactivation during nerve-

growth-factor-induced differentiation of PC12 cells. Pﬂiigers Arch. 416:368-374.

STRICHARTZ, G. R. (1973). The inhibition of sodium currents in myelinated nerve

by quaternary derivatives of lidocaine. J. Gen. Physiol. 62:37-57.

SUSZKIW, J. B. and O’LEARY, M. E. (1982). Differential labeling of depot and
active acetylcholine pools in nondepolarized and potassium-depolarized rat

brain synaptosomes. J. Neurochem. 38:1668-1675.

SUSZKIW, J. B. and O’LEARY, M. E. (1983). Temporal characteristics of
potassium-stimulated acetlycholing release and inactivation of calcium inﬂux

in rat brain synaptosomes. J. Neurochem. 41:868-873.

SUSZKIW, J. B., TOTH, G., MURAWSKY, M. M. and COOPER, G. P. (1985).
Effects of Pb2+ and Cd2+ on acetylcholine release and Ca movements in
synaptosomes and subcellular fractions from rat brain and Torpedo electric

organ. Brain Res. 323:34-46.

SUSZKIW, J. B., O’LEARY, M. E., MURAWSKY, M. M. and WANG, T. (1986).

Presynaptic calcium channels in rat cortical synaptosomes: Fast-kinetics of

277

phasic calcium channel inactivation, and relationship to nitrendipine receptors.

J. Neurosci. 6: 1349-1357.

SUSZKIW, J. B., MURAWSKY, M. M. and FORTNER, R. C. (1987).
Heterogeneity of presynaptic calcium channels revealed by species differences
in the sensitivity of synaptosomal 45Ca entry to to -conotoxin. Biochem. Biophys.

Res. Commun. 145:1283-1286.

SUSZKIW, J. B., MURAWSKY, M. M. and SHI, M. (1989). Further characterization
of phasic calcium inﬂux in rat cerebrocortical synaptosomes: Inferences
regarding the calcium channel type(s) in nerve endings. J. Neurochem.

52:1260- 1269.

SWANDULLA, D. and ARMSTROMG, C. M. (1987). Fast-deactivating calcium

channels in chick sensory neurons. J. Gen. PhysioL 92:197-218.

SWANDULLA, D., CARBONE, E. and LUX, H. D. (1991). Do calcium channel

classiﬁcations account for neuronal calcium channel diversity. TINS 14:46-51.

TAKAHASHI, M., OHIZUMI, Y. and YASUMOTO, T. (1982). Maitotoxin, a Ca2+

channel activator candidate. J. Biol. Chem. 257:7287-7289.

278
TAKAHASHI, M. and OGURA, A. (1983). Dihydropyridines as potent calcium

channel blockers in neuronal cells. FEBS Let. 152:191-194.

TAKAHASHI, M., TATSUMI, M., OHIZUMI, Y. and YASUMOTO, T. (1983)
Ca2+ channel activating function of maitotoxin, the most potent marine toxin

known, in clonal rat pheochromocytoma cells.J. Biol. Chem. 258:10944-10949.

TAKAHASHI, M., TSUKUI, H. and HATANAKA, H. (1985). Neuronal
differentiation of Ca2+ channel by nerve growth factor. Brain Res.

341:381-384.

TAKASHIMA, A. and KENIMER, J. G. (1989). Muscarinic-stimulated
norepinephrine release and phosphoinositide hydrolysis in PC12 cells are

independent events. J. Biol. Chem. 264:10654-10659.

TAKENOSHITA, M. and STEINBACH, J. H. (1991). Halothane blocks low-voltage-

activated calcium current in rat sensory neurons. J. Neurosci. 11:1404-1412.

TAKEUCHI, T., NORIKAWA, N., MATSUMOTO, H. and SHRIAISHI. Y. (1962).

Pathological study of minamata disease in Japan. Acta Neuropathol. 2:40-57.

279
TAKEUCHI, T., MATSUMOTO, H., SASHI, M., KAMBARA, T., SHIRAISHI, Y.,

HIRATA, Y., NOBUKIRO, M. and ITO, H. (1968). Pathology of Minimata

disease. Kumamato Med J. 34:521-524.

TOLL, L (1982). Calcium antagonists; high-afﬁnity binding and inhibition of calcium

transport in a clonal cell line. J. Biol. Chem 257: 13189-13192.

TRAXINGER, D. L. and ATCHISON, W. D. (1987a). Comparative effects of
divalent cations on the methylmercury-induced alterations of acetylcholine

release. J. Pharmacol. Exp. Ther. 240:451-459.

TRAXINGER, D. L and ATCHISON, W. D. (1987b). Reversal of methylmercury-
induced block of nerve-evoked release of acetylcholine at the neuromuscular

junction. Toxicol. Appl. Pharmacol. 90:23-33.

TSIEN, R. W., LIPSCOMBE, D., MADISON, D. V., BLEY, K. R. and FOX, AP.
(1988). Multiple types of neuronal calcium channels and their selective

modulation. Trends in Neurosci. 10:431-438.

TUOMISTO, J. and KOMULAINEN, H. (1983). Release and inhibition of uptake
of 5-hydroxytryptamine in blood platelets in vitro by copper and methyl

mercury. Acta Pharmacol. Et Toxicol. 52:292-297.

280

TURNER, T. J. and GOLDIN, S. M. (1985). Calcium channels: Identiﬁcation and

pharmacological characterization. High afﬁnity blockade by organic Ca

channel blockers. J. Neurosci. 5:841-849.

TWOMBLY, D. A, YOSHII, M. and NARAHASHI, T. (1988). Mechanisms of

calcium channel block by phenytoin. J. Pharmacol. Etp. Ther. 246:189-195.

TWOMBLY, D. A, HERMAN, M. D., KYE, c. H. and NARAHASHI, T. (1990).

Ethanol effects on two types of voltage-dependent calcium channels. J.

Pharmacol. Exp. Ther. 254:1029-1037.

UEHARA, A. and HUME, J. R. (1985). Interactions of organic calcium channel

antagonists with calcium channels in single frog atrial cells. J. Gen. Physiol.

85:621-647.

USOWICZ, M. M., PORZIG, H. BECKER, C. and REUTER, H. (1990).
Differential expression by nerve grth factor of two types of Ca2+ channels

in rat phaeochromocytoma cell lines. J. Physiol. 426:95-116.

281
VENEMA, V. J. and ADAMS, M. E. (1990). New to-Agatoxin calcium channel

antagonists from funnel web spider venom active in the avain and mammalian

brain. Neurosci Abstr. 16:956.

VIANA, G. B., DAVIS, L H. and KAUFFMANN, F. C. (1988). Effects of
organophosphates and nerve grth factor on muscarinic receptor binding

number in rat pheochromocyotoma PC12 cells. Toxicol. Appl. Pharmacol
93:257-266.

VICENTINI, L M. and MELDOLESI, J. (1984). Alpha-latrotoxin of black widow
spider venom binds to a speciﬁc receptor coupled to phosphoinositide

breakdown in PC12 cells. Biochem. Biophysical. Res. Comm. 121:538-544.

VICENTINI, L M., AMBROSINI, A., DI VIRGILIO, F., POZZAN, T. and
MELDOLESI, J. (1985). Muscarinic receptor-induced phosphoinositide
hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells. J. Cell Biol.

100: 1330- 1333.

WEAKLY, J. N. (1973). The action of cobalt ions on neuromuscular transmission in

the frog. J. Physiol. (Land) 234:597-612.

282
WERZ, MA. and MACDONALD, R. L (1985). Barbiturates decrease voltage-

dependent calcium conductance of mouse neurons in dissociated cell culture.

Mol. Pharmacol. 28:269-277.

WESSLER, 1., DOOLEY, D. J., WERHAND, J. and SHHLEMMER, F. (1990).
Differential effects of calcium channel antagonists (on -conotoxin GVIA,
nifedipine, verapamil) on the electrically-evoked release of[3H] acetlycholine
from the myenteric plexus, phrenic nerve and neocortex of rats. Naunyn-

Schmiedeburg Arch. Pharmacol. 341:288-294.

WILLIAMS, M., ABREU, M., JARVIS, M. F. and NORONHA-BIDB, L (1987).
Characterization of adenosine receptors in the PC12 pheochromocytoma cell
line using radioligand binding: Evidence for A2 selectivity. J. Neurochem.

48:498-502.

WOOD, J. M., KENNEDY, F. S. and ROSEN, C. G. (1968). Synthesis of methly-
mercury compounds by extracts of a methanogenic bacterium. Nature (Land)

220:173-174.

YATANI, A. and BROWN, A. M. (1985) The calcium channel blocker nitrendipine
blocks sodium channels in neonatal rat cardiac myocytes. Circulation Res.

56:868-875.

283
YOSHINO, Y., MOZAI, T. and NAKAO, K. (1966). Biochemical changes in the

brain of rats poisoned with an alkyl mercuric compound, with special
reference to the inhibition of protein synthesis in brain cortex slice. J.

Neurochem. 13:1223-1230.

YOSHIZAWA, T., KIMURA, S., KANAZAWA, 1., UCHIYAMA, Y.,
YANAGISAWA, M. and Y. MASAKI. (1989). Endothelin localizes in the
dorsal horn and acts on the spinal neurones: Possible involvement of

dihydropyridine-sensitive calcium channels and substance P release. Neurosci.

Lett. 102:179-184.

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