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EFFECTS OF ANTIBIOTICS ON DEPOLARIZATION-INDUCED

Ca2+ UPTAKE INTO SYNAPTOSOMES

BY

Carol M. Beaman

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Pharmacology and Toxicology

1986

ABSTRACT

EFFECTS OF AQIIBIOTICS ON DEPOLARIZATION-INDUCED
Ca UPTAKE INTO SYNAPTOSOMES

BY

Carol M. Beaman

Antibiotics of four classes block neuromuscular
transmission by a combination of pre- and postjunctional

effects. The prejunctional effects may involve competitive

2+

block of Ca entry into the nerve terminal. This has never

been tested directly, however. The goal of the present study
was to determine if antibiotics known to block neuromuscular
transmission would impair depolarization-dependent uptake of
calcium into isolated nerve terminals (synaptosomes). Drugs
were applied in concentrations ranging from 1 - 1000 uM.

45

Uptake of Ca was determined during K+-stimulated

depolarization (K+ = 77.5 mM). Both the fast and slow phases

2+

of Ca influx, along with total influx were measured.

Neomycin (500 and 1000 uM) decreased fast phase Ca2+ uptake.
Oxytetracycline (1000 uM) decreased total and both phases of

Ca2+ uptake. Polymyxin B (5 uM) increased fast phase uptake,

while 500 and 1000 uM decreased total and slow phase Ca2+

uptake. The decrease in fast phase Ca2+ uptake caused by

oxytetracycline and neomycin was reversed by increasing the

external calcium concentration. These results indicate that
several antibiotics which cause neuromuscular block can alter
depolarization-induced calcium uptake into synaptosomes at

high concentrations.

ACKNOWLEDGMENTS

I would like to thank the members of my committee, Drs.
Brody, Hume and Thornburg, for their time and guidance.

To all the people in the Pharmacology department,
especially, Roseann Vorce, Paul Levesque and Corie Pawloski,
who made the time I spent at MSU more enjoyable; Thanks! A
special thanks to Lonnie Dahm for being there when I needed
someone.

To Dr. Atchison, my advisor, I owe my deepest thanks for
believing in me and giving me a chance to realize my goals.

Finally, to my husband Mark, without whose love, patience
and understanding this would not have been possible, my

sincere appreciation.

1°1-

TABLE OF CONTENTS

List of Tables..................................... v
List of Figures.................................... vi
Introduction....................................... 1

Antibiotic Induced Neuromuscular Block........ 1

Actions, Uses and Side Effects of Antibiotics

That Cause Neuromuscular Block................ 2
Neuromuscular Transmission.................... 5
NeuromuSCUlar BIOCk...OOOOOOOOOOOOOOOOOOOOOOOO 7

A) Mechanism of Antibiotic Induced

Neuromuscular Block..................... 8

B) Postjunctional Neuromuscular Block...... 11

C) Prejunctional Neuromuscular Block....... 12
Calcium Channels.............................. 13

A) Multiple Types of Calcium Channels...... 14

B) Calcium Channel Block................... 15

C) Calcium Channel Inactivation............ 17
Competitive Antagonism -- a possible mechanism 18
Synaptosomes.................................. 21
Calcium Uptake into Synaptosomes.............. 23
Experimental Rationale........................ 26
Method and Materials............................... 29
Solutions and Chemicals....................... 33
Statistical Analysis.......................... 34

ReSUItSOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO000...... 35

Uptake of Ca2+ into Synaptosomes.............. 35

2+

Total ca UptakeOOCOOOCOOOOOOOOOOO0.0.00.0... 42

2+

Fast Phase of Ca Uptake..................... 51

2+

Slow Phase of Ca Uptake..................... 60

The Effectszof Antibiotics on Baseline
(Low K ) Ca uptake.......................... 69

Summary -- Concentration Response............. 70
calCium Reversal. O O O O O O O O O O O O O O O O O O O O I O O O O O O O O 77
DiscuSSion...OOOOOOOOOOOOOOOOO ..... OOOOOOOOOOOOO... 88

List Of ReferenceSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 100

iv

LIST OF TABLES

Significant effects of antibigtics on net K+-
stimulated (77.5 mM) total Ca uptake (10 sec
incubation) into synaptosomes..................

Significant effects of antibiotics on net K+-
stimulated (77.5 mM) fast (1 sec incubation)
and slow phase (10 seS+incubation after
predepolarization) Ca uptake into
synaptosomes...................................

Control values and values for each AB
concentration that caused a statistically
significant change 'n net K -stimulated

(77.5 mM) total Ca uptake (10 sec incubation)
into synaptosomes. The values are expressed as
fmoles Ca uptake/ug protein 1 SEM............

Control values and values for each AB
concentration that caused a statistically
significant change in net K -stimulated
(77.5 mM) fast phase (1 sec incubation) and
slow phase (10 sec inSubation after
predepolarization) Ca uptake into 2+
synaptosomes, expressed as fmoles Ca

uptake/ug protein : SEM........................

78

79

80

81

10.

ll.

12.

LIST OF FIGURES

Structures of four antibiotic that block
neuromuscular transmission.....................

Structures of some agents that modify calcium
Channel actiVitYOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOC

Preparation Of synaptosomes....COCOOCOOOOOOOOOO

Dependence of Ca2+ uptake by depolarized
synaptosomes, on duration of depolarization....

Potassium-stimulated (77.5 mM) Ca2+ uptake by
different concentrations of synaptosomes
(protein)......OOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOO
Ca2+ uptake by depolarized synaptosomes

(772; mM potassium solution) as a function of

[Ca ] ........................................

Ca2+ uptake by potassium-depolarized (77.5 mM)

synaptosomes in the presence of various
concentrations of lead.........................

Total Ca2+ uptake (10 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of neomycin (1 - 1000 uM).........

Total Ca2+ uptake (10 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of clindamycin (1 - 1000 uM)......
Total Ca2+ uptake (10 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of oxytetracycline (1 - 1000 uM)..
Total Ca2+ uptake (10 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of polymyxin (1 - 1000 uM)........

Fast phase Ca2+ uptake (1 sec incubation) by

potassium-depolarized (77.5 mM) synaptosomes in
the presence of neomycin (1 - 1000 uM).........

vi

16
30

36

38

39

41

44

46

48

50

53

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Fast phase Ca2+ uptake (1 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of oxytetracycline (l - 1000 uM)..

Fast phase Ca2+ uptake (1 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of polymyxin (1 - 1000 uM)........

Fast phase Ca2+ uptake (1 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of clindamycin (1 - 1000 uM)......

Slow phase Ca2+ upta e (10 sec incubation after
predepolarization; K = 77.5 mM, no calcium) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of neomycin (l - 1000 uM).........

Slow phase Ca2+ upta e (10 sec incubation after
predepolarization; K = 77.5 mM, no calcium) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of oxytetracycline (l - 1000 uM)..

Slow phase Ca2+ upta e (10 sec incubation after
predepolarization; K = 77.5 mM, no calcium) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of polymyxin (l - 1000 uM)........

Slow phase Ca2+ upta e (10 sec incubation after
predepolarization; K = 77.5 mM, no calcium) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of clindamycin (1 - 1000 uM)......

A comparison of baseline (K+ = 5 mM) Ca2+
uptake du ing a 10 sec incubation (solid barsi+
and net K -stimulated (K = 77.5 mM) total Ca
uptake (striped bars) into synaptosomes........

A comparison of baseline (K+ = 5 mM) Ca2+
uptake du ing a 1 sec incubation (solid bars)
an§+net K -stimulated (K = 77.5 mM) fast phase
Ca uptake (striped bars) into synaptosomes...
A comparison of baseline (K+ = 5 mM) Ca2+
uptake during a 10 sec incubation after
p edepolarization (solid bars) and net 2+
K -stimu1ated (K = 77.5 mM) slow phase Ca
uptake (striped bars) into synaptosomes........

vii

55

57

59

62

64

66

68

72

74

76

23.

24.

Fast phase Ca2+ uptake (1 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of 1000 uM oxytetracycline
(squares) or drug-free control (circles), at
various external calcium concentrations

(0.05 - 1.0 mM)................................

Fast phase Ca2+ uptake (1 sec incubation) by
potassium-depolarized (77.5 mM) synaptosomes in
the presence of 1000 uM neomycin (squares) or
drug-free control (circles) at various external
calcium concentrations (0.05 - 0.5 mM).........

viii

84

86

INTRODUCTION

Antibiotic-Induced Neuromuscular Block

 

Neuromuscular block is a recognized clinical side
effect when using some antibiotics (ABs) of the a)
aminoglycoside, b) lincosamide, c) polymyxin and d)
tetracycline classes (Pittinger gt gt, 1970; Pittinger and
Adamson, 1972). Prolonged respiratory depression may occur
when ABs are used in conjunction with general anesthetics
(eg. enflurane, halothane, isoflurane, nitrous oxide) or
neuromuscular blockers (eg. d-tubocurarine, pancuronium,
succinylcholine) (Pittinger gt gt, 1970; Pittinger and
Adamson, 1972; Fogdall and Miller, 1974), in patients with
pre-existing neuromuscular (Pittinger gt gt, 1970) or renal
disease, or in patients with electrolyte imbalance (eg.
hypocalcemia). Reversal of AB-induced neuromuscular block
clinically involves increasing the concentration of Ca2+ or
giving neostigmine, but sometimes these regimens are not
effective (Pittinger gt gt, 1970). Since it has been
reported that the death rate is approximately 9% in patients
experiencing respiratory problems related to AB-induced

neuromuscular block (Pittinger gt gt, 1970), it is important

to determine the mechanism by which ABs block neuromuscular

1

transmission, and thus find ways to prevent or counteract

their life threatening actions.

Actions, Uses, and Side Effects gt Antibiotics That Cause

 

Neuromuscular Block

Although ABs of the four classes all cause
neuromuscular block, their structures (figure 1) and
antibacterial actions are different. The aminoglycosides
and tetracyclines prevent bacterial growth by inhibition of
protein synthesis. The aminoglycosides inhibit protein
synthesis by preventing peptide initiation, by binding to
the 30 S ribosomal subunit and they may also induce
misreading of the genetic code (Luzzatto gt gt, 1969;
Pestka, 1971; Hash 1972; Sanders and Sanders, 1979). The
tetracyclines inhibit protein synthesis by interfering with
binding of the aminoacyl-tRNA to the acceptor site, thus
inhibiting elongation of the peptide chain (Pestka, 1971;
Bash 1972; Sanders and Sanders, 1979). The lincosamides
produce their antibacterial action by binding to the 50 S
subunit of the bacterial ribosome, leading to inhibition of
the peptidyl transferase reaction (Hash, 1972; Sanders and
Sanders, 1979). Polymyxins produce their antibacterial
actions by competitively displacing Mg2+ or Ca2+ from
phosphate groups on membrane lipids, disrupting the normal
packing of these lipids and thus changing the membrane

permeability (Storm gt gt, 1977; Sanders and Sanders, 1979).

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The four classes of ABs that cause neuromuscular block
have widely divergent clinical uses (Gilman gt gt, 1985).
For example, aminoglycosides are used primarily for
treatment of infections due to gram-negative bacteria.
Examples of these uses include topical application for burns
and wounds, preparation of patients for bowel surgery and
treatment of patients with tuberculosis. Lincosamides can
be used for infections due to gram-positive cocci; examples
of their uses are treatment of abdominal and lung abscesses
and pneumonia. Polymyxins may be used for infections due to
gram-negative bacteria; examples of their uses include
urinary tract infections and topical use for infections of
the skin, eyes and ears. Examples of the clinical uses of
tetracycline include Rocky Mountain spotted fever, and
urinary and ocular infections.

Since these ABs have different structures, mechanisms of
action and uses, it is not surprising that they produce
different side effects as well (Gilman gt gt, 1985). The
primary side effects of the aminoglycosides include
vestibular, cochlear and renal toxicity. The tetracyclines
can cause hepatic and renal toxicity, and gastrointestinal
irritation. In addition, tetracyclines have effects on
calcified tissues (eg, deposition in teeth and bones).

Clindamycin may cause development of pgeudomembranous

 

colitis, which may be lethal. Renal toxicity is the most
significant side effect of polymyxin B. Other side effects

of polymyxin B include facial flushing, dizziness, slurred

speech and blurred vision. Despite the differences in
chemical structure, antibacterial mechanism, clinical uses
and side effects, these ABs all have one feature in common:

they block neuromuscular transmission.

Neuromuscular Transmission

 

 

Neuromuscular transmission is comprised of several
steps beginning with synthesis of the neurotransmitter
acetylcholine (ACh) and ending with its hydrolysis to stop
its effects on the postjunctional membrane and thus
terminate its action. ACh is synthesized in the nerve
terminal from choline and acetyl CoA in a reaction catalyzed
by the enzyme choline acetyltransferase (Dauterman and
Mehrotra, 1963; Currier and Mautner, 1974). In the
terminal, ACh is stored in synaptic vesicles, although there
may be a nonvesicular fraction as well (De Robertis and
Bennett, 1955; Collier and MacIntosh, 1969; Ritchie and
Goldberg, 1970). Neurotransmitters, including ACh, are
thought to be released in discrete amounts (quanta) (Del
Castillo and Katz, 1954). After the depolarization of the
action potential invades the presynaptic terminal, synaptic
vesicles fuse with the membrane to release neurotransmitter
by exocytosis (Heuser, 1977; Heuser, gt gt, 1974, 1979).

The release of neurotransmitter is dependent on

2+

extracellular Ca (Katz and Miledi 1965b; 1967; 1969; Dodge

and Rahamimoff, 1967); somehow depolarization-induced Ca2+

6

entry into the presynaptic nerve terminal leads to release
of neurotransmitter (Llinas and Nicholson, 1975), but the
exact mechanism is unknown. A proposed role of calcium is
to open Ca2+-activated k+ channels present in synaptic
vesicles, thus causing osmotic changes that would lead to
fussion of the vesicle with the membrane and exocytosis of
the vesicle contents (Stanley and Ehrenstein, 1985). The
estimated time required for calcium entry and
neurotransmitter release is approximately 200-500 usec
(Parsegian, 1977; Llinas gt gt, 1981b). After ACh is
released from the presynaptic terminal, it crosses the
synaptic cleft. When ACh reaches the postjunctional membrane
it binds to the acceptor sites on a macromolecule known as
the ACh receptor. This leads to a change in ion
permeabilities in the postsynaptic membrane (Takeuchi and
Takeuchi, 1960 Katz and Miledi, 1972). The changes in the
postsynaptic membrane potential, in turn, cause generation
of an action potential in the muscle, which leads to
contraction of the muscle. The action of ACh is terminated
by acetylcholinesterase which hydrolyzes the ACh to choline
and acetate (Collier, 1977). Choline re-enters the nerve
terminal by a carrier-mediated process and is reused to make
ACh (Marchbanks, 1968, 1982; Diamond and Kennedy, 1969).
The entire process of neurotransmitter release from
depolarization to the appearance of the postsynaptic end-
plate current occurs in milliseconds (msec) (Katz and

Miledi, 1965a). The times of each individual synaptic delay

7
are not equal but fall in the range of 0 - 4 msec with a

peak in the distribution at 0.75 msec (Katz and Miledi,

1965a).

Neuromuscular Block

 

ABs can theoretically block neuromuscular transmission
at several sites these include: 1) conduction of the action
potential into the nerve terminal, 2) synthesis,
mobilization and release of neurotransmitter, 3) activation
of the postsynaptic receptor, 4) generation and propagation
of the action potential in the muscle membrane, and 5)
excitation - contraction coupling in the muscle. The
mechanism(s) by which ABS produce neuromuscular block is/are
unknown even though numerous experimental studies have been
undertaken in an attempt to study this effect (for example,
Elmqvist and Josefsson, 1962; Brazil and Corrado, 1969;
Wright and Collier, l976a,b, 1977; Prado gt gt, 1978; Singh
gt gt, 1978, 1979, 1982; Fiekers gt gt, 1979, 1983; Fiekers,
1981, 1983a,b). The techniques that have been used include;
twitch tension measurements following electrical stimulation
of motor axons or myofibers from different nerve/muscle
preparations, (Becker and Miller, 1976; Wright and Collier,
1976a, 1977; Singh gt gt, 1978), intracellular recordings of
postsynaptic potentials and action potentials from isolated
nerve/muscle or nerve preparations, respectively (Singh gt

gt, 1979, 1982; Caputy gt gt, 1981) and measurements of

endplate ionic currents using voltage clamp techniques
(Fiekers gt gt, 1979, 1983; Fiekers, 1981, l983a,b; Farley
gt gt, 1982). These various studies have demonstrated two
types of neuromuscular block caused by ABS: 1)
postjunctional i.e. effects on the end-plate receptor or
ionic channel (with d-tubocurarine as the prototype), and 2)
prejunctional i.e. effects on the release of the
neurotransmitter (with magnesium as the prototype).
Postjunctional effects have been demonstrated as a decrease
in the amplitude of miniature endplate potentials (MEPPs) or
currents (MEPCs), a decrease in the amplitude of endplate
potentials (EPPs) or currents (EPCs), or non-linearity in
the endplate current-voltage relationship. Prejunctional
actions of ABs have been demonstrated as a decrease in the
amount of neurotransmitter released by a nerve impulse (mean
quantal content, 3). However for many ABs the effects
cannot be classified as solely ”curare - like“ or "magnesium
- like" (Singh gt gt, 1978); these ABs often produce a

combination of effects.

A) Mechanisms gt Antibiotic Induced Neuromuscular Block

 

 

The aminoglycosides are thought to possess a
combination of pre- and postjunctional blocking actions,
with prejunctional effects predominating (Elmqvist and
Josefsson, 1962; Brazil and Corrado, 1969; Pittinger and

Adamson 1972; Singh gt gt, 1979, 1982; Sokoll and Gergis,

1981; Farley gt gt, 1982; Fiekers, 1983a,b). This has been
shown in combined studies of pre- and postjunctional effects
of aminoglycosides using a two microelectrode voltage clamp
of normal or transected twitch fibers of frog (Farley gt gt,
1982) or snake (Fiekers, 1983a,b), respectively. Neomycin
and streptomycin both decreased m, as determined by the
ratio of EPC/MEPC amplitude, at concentrations much lower
than those which cause postjunctional reduction of MEPC
amplitude or alteration of decay kinetics of the EPC. The
effect of neomycin in particular was more prominent on
prejunctional processes (Fiekers, l983a).

The lincosamides, clindamycin and lincomycin, may cause
neuromuscular block by a variety of mechanisms including a
direct action on the muscle or by postjunctional block of
receptor channels. In addition clindamycin may cause
neuromuscular block by suppression of ACh release.
Lincomycin and clindamycin cause a parallel decrease in
muscle contractile response due to nerve stimulation and
direct muscle stimulation (Wright and Collier, 1976a). This
leads to the conclusion that a myogenic component of block
exists. Lincomycin and clindamycin also decrease EPP or EPC
amplitude and endplate sensitivity to exogenously applied
ACh (Tang and Schroeder, 1968; Becker and Miller, 1976;
Rubbo gt gt, 1977; Singh gt gt, 1979, 1982; Fiekers gt gt,
1983), leading to the conclusion that lincosamides act
postjunctionally. Although there is much disparity in

experimental data, there is evidence that clindamycin may

10

also have prejunctional effects. Clindamycin has variously
been described to increase ACh release induced by nerve
stimulation (Rubbo gt gt, 1977), decrease quantal content
(Fiekers gt gt, 1983), and increase frequency of occurrence
of spontaneous MEPPs (Rubbo gt gt, 1977).

Polymyxin B has both pre- and post-junctional effects,
but postjunctional effects predominate. This is shown by
reductions in quantal content similar to those caused by
Mg2+ at high concentrations, and decreases in MEPP or MEPC
amplitude at very low concentrations (Singh gt gt, 1979,
1982; Fiekers, 1981; Sokoll and Gergis, 1981).

The effects of tetracyclines on neuromuscular
transmission are less well studied. Wright and Collier
(1976b) showed that tetracyclines produced an initial
augmentation and subsequent inhibition of muscle contraction
in response to nerve stimulation. However, using a phrenic
nerve-diaphram preparation they showed that the
concentration required to block the response to ACh
injection (via the inferior vena cava) was considerably less
than the concentration required to block the response to
nerve stimulation, suggesting that tetracycline causes
neuromuscular block by acting postjunctionally. In
opposition to this, Singh gt gt (1982) suggested that
tetracycline may have predominantly prejunctional blocking
activity along with actions that block muscle contractility.

Singh gt gt (1982), used isolated frog sciatic nerve-

sartorius muscle preparations to show that tetracycline,

11

like Mgz+

, did not decrease the MEPP amplitude, suggesting
that tetracycline does not act postjunctionally. They also
showed that tetracycline and oxytetracycline reduced both

the maximum rate of rise and fall of action potentials in

the muscle, suggesting again a direct myogenic action.

B) Postjunctional Neuromuscular Block

 

Despite the fact that ABs act at both pre- and post-
junctional sites, the majority of experimental studies have
focused on the postjunctional actions. ABs may cause one or
more of the following postjunctional effects: block of
receptor occupation by ACh, block of ion permeation through
the receptor-activated ionic channels, inhibition of action
potential prOpagation in the muscle, or disruption of muscle
contractility. For example, lincomycin has been shown to
interfere with muscle membrane excitation or muscle
contraction (Wright and Collier, 1976a). Clindamycin and
lincomycin each interact with the open state of the endplate
receptor/channel complex in a noncompetitive manner (Fiekers
gt gt, 1983). Streptomycin has mixed competitive and
noncompetitive actions (Brown and Taylor, 1983), but the
predominate postjunctional action is competitive block of
the ACh receptor (Farley gt gt, 1982). Neomycin also exerts
mixed competitive and noncompetitive actions, with the
noncompetitive block of the ACh receptor predominating

(Brown and Taylor, 1983). At higher concentrations,

12

neomycin reacts with the receptor channel when it is in an
open configuration (Fiekers, 1983b). Polymyxin B interacts
in a noncompetitive manner (Brown and Taylor, 1983) with the
receptor/channel complex to produce a voltage-dependent

block of the open channel (Fiekers, 1981).

C) Prejunctional Neuromuscular Block

 

Whereas the postjunctional blocking actions of ABS
have been studied extensively, the prejunctional blocking
actions of ABS have been studied incompletely. Drugs that
have prejunctional effects can act on action potential
propagation, neurotransmitter synthesis or release. More
specifically ABs may ultimately block neurotransmission
presynaptically by any of several mechanisms, such as l)
decreasing the calcium concentration in the external medium
(by binding calcium, or altering the ionized to nonionized
ratio), 2) blocking calcium from entering the nerve terminal
(by blocking or inactivating the voltage-dependent calcium
channel, 3) altering stimulus/secretion coupling, or 4)
affecting neurotransmitter release (decreasing the size of a
quantum, or altering the permeability of the membrane).
There is evidence based on individual ABS for or against
some of these possibilities. However, the effects of none
of the ABS have been studied in sufficient detail to
determine their mechanism of action, nor has a general

mechanism responsible for prejunctional block been proven

13

for all ABS. For example, in the presence of streptomycin
(Pittinger, 1970; Tamaki, 1983) or neomycin (Elmqvist and
Josefsson, 1962; Wright and Collier, 1977) the concentration
of the ionized form of calcium is not changed. These
studies have excluded the possibility of calcium binding to
the ABS as the mechanism of neuromuscular block. However,
it has been postulated that tetracycline's mechanism of
action is calcium chelation, with a resultant decrease in
neurotransmitter release (Pittinger and Adamson, 1972).

This does not seem likely Since Wright and Collier (l976b)
have shown that rolitetracycline did not decrease evoked
release of ACh; a decrease would be expected if tetracycline
acted by chelating calcium. Furthermore, Bowen and McMullan
(1975) have shown (in horses) that calcium binding does not
seem to contribute to the neuromuscular block produced by
oxytetracycline. Clindamycin may have prejunctional effects
due to its local anesthetic-like actions (Wright and
Collier, l976a). However, these only occur at very high
concentrations and thus are unlikely to contribute

significantly to the clinically observed effect.

Calcium Channels

 

One potential prejunctional mechanism by which ABS may

act is to block Ca2+

entry through voltage-regulated calcium
channels. Calcium channels are found in all excitable

membranes, such as nerve and cardiac muscle membranes.

14

Calcium channels play a role in coupling membrane excitation
to cellular responses, eg. secretion or contraction.

Calcium channels are also essential for the rhythmic firing
of nerve cells (Llinas and Sugimori, 1980). Calcium entry
contributes to other neuronal functions such as neurite
extension (Anglister gt gt, 1981), generation of dendritic
calcium spikes (Llinas and Yarom, 1981) and calcium-
dependent potassium conductances (Barrett and Barrett, 1976;
Krnjevic gt gt, 1975, 1978; Llinas and Sugimori, 1980). In a
variety of cells, depolarization of the membrane increases
the membrane's permeability to calcium, i.e. these cells

have voltage-dependent calcium channels.

A) Multiple Types gt Calcium Channels

 

The different functions of Ca2+

suggest different or
multiple types of calcium channels may exist. Two distinct
populations of calcium channels have been demonstrated in a
clonal cell line derived from rat pituitaries (GH3 cells)
based on their closing kinetics (Armstrong and Matteson,
1985). Two distinct types of voltage-activated calcium
conductances have also been found in the unfertilized egg of
Neanthes arenaceodentata, (Fox and Krasne, 1981), and
Mediaster aequalis (Hagiwara gt gt, 1975), suggesting the
presence of two calcium channels. Fishman and Specter

(1981), working with neuroblastoma (NlE-115), have shown

that the decay of the calcium currents exhibits two time

15

constants, which again suggests the existence of two calcium
channels. Tsunoo gt gt (1984) have demonstrated two
different types of calcium channels in neuroblastoma cells
(NlE-llS), based on difference in gating properties and
insensitivity to cyclic AMP. Nowycky gt gt (1985), have
demonstrated three types of calcium channels in dorsal root
ganglion cells. Two types of calcium channels have been
proposed to exist in synaptosomes (Nachshen and Blaustein,
1980). Recently, Penner and Dreyer (1986) have presented
evidence suggesting the presence of multiple type of calcium
channels in the motor nerve terminal at the mouse
neuromuscular junction. Thus, results from many different
preparations suggest the existence of multiple Ca2+

channels.

B) Calcium Channel Blockers

 

Calcium channels are blocked by a number of agents
(figure 2). Note that these structures are markedly
different from those of the ABS (figure 1). A peptide,

isolated from Conus geographus and designated omega toxin,

 

blocks calcium entry into the nerve terminal during the
presynaptic action potential (Kerr and Yoshikami, 1984).

Inorganic cations such as Ni2+, Mn2+, and Coz+

can block
calcium influx in a competitive manner (Nachshen, 1984;
Drapeau and Nachshen, 1984; and see reviews by Hagiwara and

Byerly, 1981; Kostyuk, 1981; Edwards, 1982), while the

16

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17

organic cation, methylmercury, blocks calcium influx in an
apparently noncompetitive fashion (Atchison gt gt, 1986).
Other inorganic cations, La3+ (Nachshen and Blaustein, 1980,

1982) and Cd2+

(Nowycky gt gt, 1985; Narahashi gt gt, 1986)
preferentially block some calcium channels. Organic calcium
antagonists include verapamil and D-600; however, these
antagonists may not be calcium specific but may also affect
sodium channels (Van der Kloot and Kita, 1975; Nachshen and
Blaustein, 1979). Other calcium antagonists, the

2+ channel

dihydropyridines, have been shown to modify Ca
activity. For example, nifedipine and nitrendipine act as a
calcium antagonists whereas Bay K 8644 acts as a calcium
agonist (Hess gt gt, 1984). Calcium channels in neuronal
tissue appear to be much less sensitive to verapamil, D-600
and dihydropyridines than are calcium channels in cardiac or
vascular smooth muscle (Gotgil'f and Magazanik, 1977;
Nachshen and Blaustein, 1979; Daniell gt gt, 1983; Glossman
gt gt, 1984). This suggests that there may be differences

between calcium channels in excitable membranes of various

different tissues.

C) Calcium Channel Inactivation

 

Inward calcium currents are found in many cell
membranes, and have been shown to relax with time (Standen,
1974; Hencek and Zachar, 1977; Kostyuk and Krishtal, 1977;

Tillotson, 1979). In some cases the calcium currents relax

18

with time under a maintained depolarization (Tillotson,
1979; Eckert and Tillotson, 1981; Fox, 1981). Some calcium
channels (for example, those in egg cell membrane of
Neanthes, and in synaptosomes) have been shown to inactivate
in a voltage-dependent manner (Fox, 1981; Nachshen, 1985a;
Ashely, 1986). Other calcium channels (for example, neurons
of Aplygia californica, rabbit sino-atrial node and calf
Purkinje fibers) have been shown to inactivate in a calcium-
dependent manner (Tillotson, 1979; Brown gt gt, 1981; Eckert
and Tillotson, 1981; Marban and Tsien, 1981). It is also
possible that some channels may inactivate in both a
calcium- and voltage-dependent manner (possibly
synaptosomes, Suszkiw gt gt, 1986). Therefore, flow of
inward current through calcium channels in different tissues

may be terminated by different mechanisms.

Competitive Antagonism -- 5 Possible Mechanism

 

 

Several lines of evidence suggest that ABS act as
competitive antagonists of calcium at a common presynaptic
site. ABS with neuromuscular blocking activity tend to be
large, often charged molecules which suggests they should
act outside the nerve terminal. The effects of some of the
ABS occur rapidly, and are rapidly reversed by removing the
drug, (Wright and Collier, 1977; Fiekers, 1983a; Prado gt
gt, 1978; Tamaki, 1983), which also suggests they act

outside the nerve terminal. Twitch tension measurements

19

obtained using the isolated phrenic nerve-diaphragm
preparation of rats with bath application of aminoglycosides
showed that increasing CaCl2 in the bath caused a parallel
shift to the right in the log dose response curve, thus
demonstrating competitive antagonism (Prado gt gt, 1978).

Two lines of evidence are consistent with a general
effect of ABS on calcium-dependent transmitter release.
First, neomycin blocks release of norepinephrine and ACh
(Wright and Collier, 1977) and streptomycin blocks release
of L-glutamate (Washio, 1984), thus the effect of the ABS is
not specific for cholinergic neurons. Second, increasing
bath calcium has been shown to reverse or partially reverse
the neuromuscular block caused by several ABS (Elmqvist and
Josefsson, 1962; Pittinger, 1970; Prado gt gt, 1978; Singh
gt gt, 1978, 1979, 1982; Fiekers, 1983a; Washio, 1984).
Based on these findings coupled with the decrease in 3,
caused by aminoglycosides and other ABS, it has been
proposed that ABS act as competitive inhibitors of calcium
influx.

Although the available evidence is consistent with this
notion, it is not sufficient to prove that blocking calcium
influx is the cause of the neuromuscular block. AB-induced
inhibition of calcium influx into the nerve terminal has not
been tested directly. Since it has not been determined that
the ABS block calcium influx, it is possible to explain the
experimental data, showing that an increase in the external

calcium concentration ([Ca2+]o) can reverse the AB-induced

20

neuromuscular block, based on functional antagonism. It is
possible that the ABS have their effects intracellularly and
that increasing the internal calcium concentration relieves
the AB-induced effect by noncompetitive functional
antagonism. For example agents such as 4-aminopyridine and
tetraethylammonium (TEA) which prolong the presynaptic
depolarization by blocking K+ efflux (Armstrong and
Binstock, 1965; Kusano gt gt, 1967; Yeh gt gt, 1976; Lundh,
1978) and thus increase the internal calcium concentration,
can relieve the block of neuromuscular transmission caused
by botulinum toxin (Cull-Candy gt gt, 1976; Lundh gt gt,

2+ channels but is

1977). Botulinum toxin does not block Ca
believed to affect ACh release by intracellular actions
(Wonnacott gt gt, 1978; and see reviews by Narahashi, 1974;
Howard and Gundersen, 1980). Moreover, the effects of
myasthenia gravis, which is a postjunctionally-directed
disease can also be relieved with aminopyridines (Kim and
Sanders, 1980; Kim, 1982). Since aminopyridines and TEA
cause functional antagonism by increasing the internal
calcium concentration they can reverse the neuromuscular
block caused by presynaptic intracellular events and/or
postjunctional events. Therefore, in order to determine
whether increasing [Ca2+]O reverses the AB-induced
neuromuscular block by competitive antagonism or by
functional antagonism it is necessary to know if ABS block
2+

Ca influx. The goal of this project is to determine

directly whether ABS reduce depolarization-induced calcium

21

influx into the nerve terminal.

Synaptosomes

 

The small size of the presynaptic motor nerve terminal
prohibits use of manipulations (eg, impalement with a
recording micropipette, Ca2+ selective microelectrode, or
injection of test agent) that would give direct information
about calcium influx. Thus, in order to measure calcium
influx another system must be used. Gray and Whittaker
(1962) developed a procedure for preparing pinched off nerve
terminals (synaptosomes) from mammalian brain tissue. Brain
tissue is used as the source of synaptosomes, since
isolation of nerve terminals from the neuromuscular junction
is not feasible due to the small number of nerve terminals
compared to the large quantity of muscle.

Synaptosomes provide a useful model for studying
physiological and biochemical processes which occur at
presynaptic nerve terminals (Blaustein, 1975; Blaustein gt
gt, 1981). Synaptosomes retain many morphological and
functional properties of intact neurons (Blaustein gt gt,
1977). For example, synaptosomes maintain a resting
membrane potential that responds to depolarizing agents
(Blaustein and Goldring, 1975). Synaptosomes retain a
functional choline uptake system (Haga and Noda, 1973), and
can synthesize and release ACh (Haga, 1971). Synaptosomes

also retain functional glycolytic and oxidative metabolic

22

pathways. Synaptosomes accumulate potassium, extrude sodium

(Blaustein gt gt, 1977), regulate Ca2+

influx (Blaustein,
1975; Nachshen and Blaustein, 1980) and release transmitter
after a depolarization—induced calcium influx (Blaustein,
1975).

Despite these advantages there are several problems
associated with the use of synaptosomal preparations
(Blaustein gt gt, 1977). First, synaptosomal preparations
may not contain only pure nerve terminals, but may also
contain other membranes or subcellular organelles such as
mitochondria. Second, damaged or nonfunctional nerve
terminals may be present. Third, in most cases the
terminals obtained from homogenates are heterogeneous with
respect to the transmitter they contain. Fourth, and most
important, the time resolution of radiolabel flux
measurement into synaptosomes is much slower than the time
course of synaptic transmission. Normal synaptic
transmission is completed in milliseconds (Katz and Miledi,
1965a, 1967), whereas depolarization-evoked influx of Ca2+
into synaptosomes is measured over periods of seconds. Use
of specialized rapid mixing techniques allow this
measurement period to be shortened and permits a time
resolution of 100 msec or less (Nachshen, 1985a; Suszkiw gt
gt, 1986). Using these techniques, Nachshen (1985a)
reported that results obtained during shorter time intervals

corresponded reasonably well to those obtained at 1 sec, and

that values obtained with the rapid mixer were similar to

23

values obtained by hand-pipetting at times of 1-10 sec.

Calcium Uptake Into Synaptosomes

 

Depolarization leads to an increase in calcium
permeability of the prejunctional membrane (Baker gt gt,
1971; Llinas gt gt, 1981a). Since the extraterminal calcium
concentration exceeds the intraterminal calcium
concentration (Nachshen, 1985b), the increased permeability
leads to calcium influx. This calcium influx is the trigger
that couples nerve terminal excitation and transmitter
release (Katz and Miledi, 1967, 1969). Calcium is known to
be the trigger because axonal depolarization in the absence
of calcium does not cause transmitter release (Katz and
Miledi, 1965b; Miledi and Slater, 1966). The properties of
calcium influx into synaptosomes have been studied
extensively. Backflux of radioactive calcium is negligible
during the first 10 seconds of incubation (Nachshen and

Blaustein, 1980), thus the 45

Ca movement measured during
this time represents influx, rather than net flux. Two
phases of calcium entry into synaptosomes have been
demonstrated (Gripenberg gt gt, 1980; Nachshen and
Blaustein, 1980; Nachshen, 1985a): a fast phase mediated by
a pathway that is inactivated after 1-2 seconds and is

3+ (< 1 uM), and a slow

inhibited by low concentrations of La
phase, mediated by a pathway that is not inactivated during

long-lasting depolarization (1-2 min) and is only blocked by

24

3+

high concentrations of La (> 0.1 mM) (Nachshen and

Blaustein, 1980, 1982; Suszkiw and O'Leary, 1983; Nachshen,
1984). Based on the differential sensitivity to La3+, the
two phases of calcium entry have been proposed to correspond
to two different calcium channels (Nachshen and Blaustein,
1980, 1982), although other interpretations have also been
cited (Wang gt gt, 1985; Suszkiw gt gt, 1986). The fast
pathway is of primary interest because it is associated with
voltage- and calcium-dependent release of dopamine from
striatal synaptosomes (Drapeau and Blaustein, 1983),
voltage- and calcium-dependent release of substance P from
lower brain synaptosomes (Floor, 1983), and voltage- and
calcium-dependent release of ACh from forebrain synaptosomes
(Suszkiw and O'Leary, 1983). Voltage-dependant

norepinephrine release also paralleled Ca2+

2+

uptake in a
synaptosome preparation; both Ca uptake and norepinephrine
release have a fast and slow phase (Daniell and Leslie,
1986). The initial rate of dOpamine release is close to the
rate of release evoked by nerve stimulation of intact tissue
(Drapeau and Blaustein, 1983) and the time course of
dopamine release parallels calcium entry through the fast
channel (Drapeau and Nachshen, 1984; Leslie gt gt, 1985).

As suggested by Nachshen and Blaustein (1980) the slow phase
of calcium uptake may be carrier mediated; Wang gt gt (1985)
have shown that under certain conditions the slow phase may
2+

be mediated by a membrane potential-sensitive Na+/Ca

exchange mechanism. Measurement of potassium-stimulated

25
calcium uptake by synaptosomes using rapid-mixing techniques
has shown that the initial rate of calcium influx is faster
than previously predicted, but that two phases of calcium
influx are still apparent at the times previously tested
(Nachshen, 1985a). These results imply that 1 and 10 second
incubation times used in manual mixing experiments are
useful time points for measure of the two phases of calcium
influx.

The potassium-stimulated calcium uptake in a variety of
preparations does not occur via the sodium channel as it is
not blocked by the Na+ channel blocker, tetrodotoxin (TTX)
(Baker gt gt, 1973b; Blaustein, 1975; Nachshen and
Blaustein, 1980) or by replacement of external sodium with
choline (Blaustein, 1975). Both phases of calcium influx
are insensitive to TTX (Nachshen and Blaustein, 1980), but

can be blocked by Ni2+, Mn2+, Mg2+ and Co2+

in a competitive
manner (Nachshen and Blaustein, 1980; Drapeau and Nachshen,
1984). Verapamil and D-600 also block calcium influx into
synaptosomes, but with a much lower potency than divalent
cations (Nachshen and Blaustein, 1979; Daniell gt gt, 1983).
The slow phase of calcium influx into squid axons is not
blocked by the K+ channel blocker TEA (Baker gt gt, 1973a).
There is a local electric response in squid neurons, in the
presence of external TTX and internal TEA, when there is a
high external calcium concentration. (Katz and Miledi,

1969). This evidence implied that the calcium does not

enter the nerve terminal by either sodium or potassium

26

channels, so a calcium channel was proposed (Baker gt gt,
1973a). As previously described, it is possible that
several calcium channels exist.

The increase in calcium permeability is depolarization-
dependent; this implies that calcium uptake is voltage—
dependent. Depolarization is normally caused by an
electrical impulse, however potassium may be used to cause a
depolarization and thus induce calcium influx (Blaustein,
1975). The potassium-induced change in calcium permeability
is not caused by making the synaptosome irreversibly leaky
to calcium since prior stimulation in a solution with a
high concentration of potassium does not change the rate of
calcium uptake in a solution with a low concentration of
potassium (Blaustein, 1975). Nor is the potassium-induced
increase in calcium permeability due to incorporation of
bulk extracellular fluid by pinocytosis since the
extracellular markers mannitol or inulin do not accompany
the calcium as it is taken up by the synaptosomes

(Blaustein, 1975).

Experimental Rationale

 

The studies cited herein were designed to clarify the
mechanism by which ABs act prejunctionally. In particular I
sought to determine whether block of depolarization-induced

2+

Ca influx by ABS could be demonstrated in isolated nerve

terminals. The experiments utilized a synaptosome-enriched

27

preparation that was depolarized by potassium to induce
influx of calcium. Effects of ABs on calcium influx were
studied during both the fast (1 sec) and slow (10 sec)
phases, as well as for total influx over 10 sec. Several
ABS: clindamycin, neomycin, oxytetracycline, and polymyxin B
(one from each of the 4 groups known to block neuromuscular
transmission) were tested for their effects on calcium
uptake.

Previous experiments have not looked at calcium uptake
directly; instead they have used measures (ACh release, EPP,
MEPP, etc.) that are believed to be related to calcium
influx. I determined calcium uptake into synaptosomes
directly through the use of radioactive calcium. Based on
previous experiments with isolated nerve/muscle preparations
it was expected that the aminoglycoside and tetracycline
would decrease calcium influx, clindamycin might increase
calcium influx and polymyxin B might decrease calcium influx
or have no effect. One possible mechanism by which ABS may
decrease calcium influx is by competition with calcium for a
common binding site. If the decrease in calcium influx was

2+

]

competitive and reversible, then increasing the [Ca 0

should reverse the effects of the AB. Inorder to determine
if the ABS act as competitive antagonists to calcium entry,
the ability of calcium to reverse AB-induced block of Ca2+
influx ([Ca2+]o = 0.05mM - lmM) was tested. The attempt at
reversal was done using either the concentration of AB that

caused 50 percent inhibition (ICSO) or, if the maximal

28

percent inhibition was less than 50%, a concentration of AB
that caused maximal decrease of influx during the fast

phase.

METHOD AND MATERIALS

Synaptosomes were isolated, and partially purified,
from a homogenate of rat forebrain through the use of
several steps of centrifugation. The partial purification
was performed to isolate synaptosomes and attempt to remove
other cellular (brain) contaminants such as mitochondria and
axonal membranes. The mitochondria should be removed as
they are capable of calcium uptake and thus could confound
the measurements of tracer uptake. (However, there are data
that imply that potassium-stimulated calcium uptake is
probably associated with synaptosomes only and not with
other organelles which may contaminate the synaptosome
fraction (Blaustein, 1975)). Extra membranes and cellular
components must be eliminated to obtain an accurate
measurement of the amount of synaptosomal protein to
standardize presentation of results.

Synaptosomes were prepared from homogenates of rat
(Sprague-Dawley, 175-250 grams, male) forebrains, using a
modification of the method of Gray and Whittaker
(l962)(figure 3). A 10% (weight/volume) homogenate in 0.32
M sucrose was centrifuged at 1,000 x g for 10 min (Sorvall
RCZB; Ivan Sorvall Inc., Norwalk, CT.). The supernatant was

recentrifuged at 17,500 x g for 20 min (Sorvall). The

29

30

homogenization: 6 strokes x 550 rpm

centrifuge: 1,000 x g, 10 min

 

supernate pellet

centrifuge: 17,500 x g, 20 min

 

supernate pellet
suspend in 6-7 ml 0.32 M sucrose
layer on 1.2 M, 0.8 M discontinuous sucrose gradient
centrifuge: 61,900 x g, 2 hr
collect synaptosomes gbetween 1.2 M and 0.8 M sucrose)
wash with buffer

centrifuge: 10,000 x g, 10 min

 

supernate pellet
suspend in 3 ml buffer
homogenize: 6 strokes x 400 rpm

warm synaptosomes — 370 C

Figure 3. Preparation of synaptosomes. Flow chart shows the
method used for preparation of synaptosomes from rat
forebrain.

31

resulting pellet was resuspended in 6 - 7 ml 0.32 M sucrose,
and layered onto two discontinuous sucrose gradients that
were made with 10 m1 of 1.2 M sucrose (bottom) and about

17 ml of 0.8 M sucrose (middle). The sucrose gradient was
centrifuged (Beckman L8-55 ultracentrifuge; Beckman
Instruments, Palo Alto, CA.) at 61,900 x g for 120 min using
a SW-27 rotor. The synaptosome-rich fraction, between the
0.8 M and 1.2 M sucrose layers, was collected by
centrifuging at 10,000 x g for 10 min and then suspended in
Ca2+-free buffer. After preparation, the synaptosomes were
warmed to 370 C, and were always used within 3 hr. Bradford
(1975) has shown that synaptosomes remain viable for 3 - 4
hr, after preparation, at 370 C.

Uptake of 45

Ca was measured by incubating 50 ul of
synaptosomal suspension with 50 ul of "high-K+" ([K+] = 77.5
mM) or "low-K+” ([K+] = 5 mM) solution with tracer. The
high-K+ buffer was used to cause a depolarization-induced

Ca2+ influx and the low-K+ buffer was used to control for

Ca2+ influx under nonstimulated conditions. ABS were added
so as to give final concentrations ranging from 1 to 1000
uM. This range is larger than the dose range used

45Ca uptake was terminated by rapidly diluting

clinically.
the incubation mixture with a quench buffer containing
ethylene glycol bis-(beta-aminoethyl ether) N, N, N', N'-
tetraacetic acid (EGTA) and N-methylglucamine. EGTA was

added to chelate the calcium making it unavailable to the

synaptosomes. N—methylglucamine was included in the quench

32

buffer as a large ion that does not enter axonal sodium
channels. Hille (1971) showed that sodium channels are
impermeant to methylated ions. Because of this, N-
methylglucamine prevents further depolarization. The
incubation times were 1 sec (timed with a metronome) for the
fast channel and 10 sec (timed with a stop watch) for total
calcium uptake. Calcium influx during the slow phase was
measured by first inactivating the fast channel by pre-
depolarizing (10 sec) in high-K+ buffer before the 10 sec
incubation.

After 45

Ca uptake had been quenched, the reaction
mixtures were filtered through 0.45 um Millipore filters
(Millipore Corporation, Bedford, MA.). The filters were
then washed twice with 5 m1 aliquots of the quench buffer,
and then placed in scintillation vials with solubilizer (1%
Triton X-100 in 0.5 M HCL, weight/volume) for at least 10
min. Scintillation fluid, Formula 963 (New England Nuclear,
Boston, MA) (10 ml) was then added and the sample counted in
a liquid scintillation counter (Beckman LS 7000; Beckman
Instruments, Fullerton, CA.). The amount of calcium influx
was calculated as the difference between calcium uptake in
solutions containing high—K+ and low-K+ concentrations.
Appropriate blanks were run and their values were used to
adjust the results for nonspecific binding of calcium to the
filters. The results (calcium influx caused by

depolarization) of initial studies used to determine whether

the preparation was viable were expressed in terms of counts

33

per minute (cpm) per ug synaptosomal protein (Lowry gt gt,
1951). The results of the subsequent studies using ABS were

’15 moles)

expressed as femtomoles (fmoles; 1 fmole = 1 x 10
of calcium influx per ug synaptosomal protein, or as percent

of drug-free control.

Solutions and Chemicals

 

The buffer used to suspend the synaptosomes contained
(mM): NaCl 145, KCl 5, MgCl2 1, d-glucose 10, and
N-2-hydroxyethylpiperazine—N'-2-ethanesulfonic acid (HEPES)
10. The high potassium buffer contained (mM): NaCl 72.5,
KCl 77.5, MgCl2 1, CaCl2 0.04, d-glucose 10, and HEPES 10.
The low potassium buffer contained (mM): NaCl 72.5, KCl 5,
choline chloride 72.5, MgCl

1, CaCl 0.04, d-glucose 10,

2 2
and HEPES 10. The quench buffer used to stop the reaction

contained (mM): N-methylglucamine 145, KCl 5, MgCl 1, EGTA

2
l, d-glucose 10, and HEPES 10. The solubilizer was 1%
Triton X-100 in 0.5 M HCl (weight/volume). The
scintillation fluid used was Formula 963 (New England
Nuclear, Boston, MA). Sucrose concentrations (for
discontinuous sucrose gradient) were 1.2 M, 0.8 M and
0.32 M. All solutions were made with deionized water
(Milli-Q) and had an osmolality of 290-320 mOsm as
determined by freezing point depression (Micro osmette,

Precision Systems, Inc., Natick, MA). The pH of the

solutions (except sucrose) was adjusted to 7.4 using glacial

34

acetic acid or NaOH. Drugs tested include clindamycin
hydrochloride, neomycin sulfate, oxytetracycline dihydrate
and polymyxin B sulfate (Sigma Chemical Co., St. Louis, MO),
and lead acetate (J. T. Baker Chemical Co., Phillipsburg,
NJ). The radioactive calcium was used in the form of

45

CaCl2 (ICN Pharmaceuticals, Irvine, CA).

Statistical Analysis

 

The results of studies of uptake through the fast and
slow channel and of total uptake were analyzed using a
randomized block analysis of variance (ANOVA). A randomized
block ANOVA was performed for each drug and was used to
determine if the blocks (different days the experiments were
done on) were significantly different (Steel and Torrie,
1980). If the blocks were not significantly different, a
completely random ANOVA was used (Steel and Torrie, 1980).
In either case if the effects of AB treatment were
significant, Dunnett's t-test (Steel and Torrie, 1980) was
used to determine which concentrations caused significantly
different effects compared to drug-free control. The
results of the calcium reversal studies were analyzed using
a blocked factorial ANOVA, followed by the least significant
difference test (lsd) if significant differences were
detected (Steel and Torrie, 1980). The criterion for

significance was p t 0.05 for all studies.

RESULTS

Uptake gtgg2+ into Synaptosomes

 

Initial experiments were designed to test the effects
of altering several different parameters of this system
(under drug—free conditions) on calcium uptake, in order to
assess the viability of the synaptosomal preparation.

The dependence of calcium uptake on the duration of
potassium-induced depolarization was determined for periods
of depolarization between 1 and 60 sec. The results do not
extrapolate back to time zero, a result consistent with that
of Nachshen and Blaustein (1980). This has been taken as
evidence of multiple phases of uptake, a hypothesis which
was substantiated in more elaborate studies using rapid
quench, automated mixing techniques (Nachshen, 1985a;
Suszkiw gt gt, 1986). As shown in figure 4, two apparent
phases of uptake can be seen, a fast phase which appears to
end by 2 sec followed by a slower phase which does not reach
a plateau for at least 60 sec. This compares favorably with
the results of others (Nachshen and Blaustein, 1980;
Nachshen, 1985a), in which 1 and 10 sec incubations were
used to study the fast and slow channels respectively.

The dependence of total calcium uptake on protein

35

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37

concentration was also determined. Calcium uptake was
measured using incubation mixtures that contained different
amounts of synaptosomal protein. The concentration of
protein ranged from approximately 100 - 500 ug. In order to
be able to compare results from different synaptosomal
preparations the data must be expressed per ug of
synaptosomal protein. This is only possible if a linear
relationship exists between the concentration of protein in
the incubation mixture and calcium uptake. As shown in
figure 5 the relationship between protein concentration and

2+

Ca uptake was linear over the range tested. During

subsequent experiments with ABS the concentration of

synaptosomes used was maintained within this range.

2+

Effects of increasing the [Ca 10 on depolarization-

dependent Ca2+ uptake were determined (figure 6). The

[Ca2+

45Ca2+ proportional to the total [Ca2+]o. The [Ca2+]

ranged from 0.02 to 1.68 mM. Ca2+ uptake increased rapidly

10 was changed in a manner that kept the amount of

O

as the [Ca2+]o was increased up to 0.42 mM; at this
concentration the preparation began to exhibit saturation of
depolarization-evoked uptake. In a viable preparation, Ca2+
uptake should increase initially due to the increased
concentration driving force, however uptake should
eventually saturate. The presence of saturation suggests

the membrane is not leaky to calcium. Based on these

results, subsequent experiments using ABS were performed

38

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39

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(cud bfi/de) 3xv1dn +303

 

40

with a [Ca2+]o that was equal to 0.05 mM. This
concentration was in the rapidly-rising portion of the Ca2+
uptake curve.

The effect of lead was determined as a test of our

ability to antagonize potassium-stimulated Ca2+

uptake.
Lead is known to inhibit calcium uptake into synaptosomes
(Nachshen, 1984; Suszkiw gt gt, 1984), and to block
transmission at the vertebrate neuromuscular junction
(Manalis and Cooper 1973; Atchison and Narahashi, 1984).

Total Ca2+

uptake was not altered by lead at concentrations
ranging from 0.1 - 10.0 uM. At a concentration of 100 uM
lead caused a statistically significant decrease in total
calcium uptake (figure 7). This decrease was at a higher
concentration than that reported by other investigators
(Suszkiw gt gt, 1984). Concentrations of 20 - 100 uM lead
have been shown to cause a decrease in nerve-evoked release
of ACh at the neuromuscular junction (Atchison and
Narahashi, 1984), an effect that can be overcome by
increasing extracellular calcium.

Based on the above results it appeared that the
synaptosomal preparations were viable, were capable of

2+ during depolarization and of having this

2+

taking up Ca

effect blocked by a putative Ca channel blocker that also

caused neuromuscular block. Subsequent experiments were

designed to test the effects of ABS on depolarization-

2+

dependent and -independent Ca uptake.

41

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(baud no saga soup accumuuqc >~a2oouuucouu a. o=~c> oz» couaouomu M.~.x”w“wuww
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42

2+

Total 9g Uptake

The first goal was to determine if the ABS had any
effect on total uptake of Ca2+. In subsequent experiments
the two phases of Ca2+ uptake were examined separately to
determine if ABS blocked one or the other phase
preferentially. It should be noted that it is possible for

ABS to alter Ca2+

uptake via the fast channel even in the
absence of an apparent effect on total uptake.
Neither neomycin (n = 7), nor clindamycin (n = 9) had

2+ over the entire

any effect on total uptake of Ca
concentration ranged tested (1 - 1000 uM). As shown in
figure 8, neomycin may cause slight deviations from control
but does not cause a statistically significant alteration of
depolarization-evoked uptake. As shown in figure 9,
clindamycin does not cause any significant deviations from
its drug—free control value.

Oxytetracycline and polymyxin B each decreased total
Ca2+ uptake. The results of seven experiments with
oxytetracycline and eight experiments with polymyxin are
shown in figures 10 and 11 respectively, and are expressed
as percent of drug-free control. Oxytetracycline at
concentrations of l - 100 uM caused a slight increase in
Ca2+ uptake, but this effect was not statistically
significant. The concentration of 500 uM caused a slight

2+

decrease in Ca uptake, but again this effect was not

statistically significant. At the highest concentration

 

43

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48

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ma osam> wnu mmumoflpcfl .20 xmwumumm c4 .cfimuOHQ Hmsomoummcwm md\wxmums m0
mmHOEw hm.m H mm.hm magnum Houucou one .zmm H Houucoo mmHMImsup mo ucmwwmm

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sauna mane ..29 coca I av cwxwfimaoa mo oocmmwum map cw mmEOmoummcwm .25 m.nnv
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51

2+

(1000 uM) oxytetracycline reduced Ca uptake to 63% of

control. Similarly, polymyxin B at low concentrations (1 -

10 uM) caused a small, but statistically insignificant

2+ uptake, and at concentrations of 50 and 100

2+

increase in Ca
uM caused a statistically insignificant decrease in Ca
uptake. At concentrations of 500 and 1000 uM, polymyxin B
caused reductions to 75% and 74% of control respectively, in
Ca2+ uptake. Both of these were significantly less than
control.

Fast Phase gt gg2+ Uptake

 

The fast phase of Ca2+

uptake occurs via a putative
calcium channel (Nachshen and Blaustein, 1980), and is
thought to be associated with depolarization-induced release
of neurotransmitter (Drapeau and Blaustein, 1983; Suszkiw
and O'Leary, 1983; Drapeau and Nachshen, 1984; Leslie gt gt,
1985; Daniell and Leslie, 1986). Since the fast phase of
Ca2+ uptake is associated with neurotransmitter release it
is the phase that is of most interest to the neuromuscular
block caused by ABS.

2+

Neomycin (n = 7) caused a decrease in Ca influx via

the fast channel (figure 12). Ca2+

uptake was decreased by
neomycin over the entire concentration range tested (1 -
1000 uM), however only the results at 500 and 1000 uM were

significantly lower than control. These concentrations

caused reductions to 66% and 54% of control respectively.

 

52

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ma msHm> on» mmumowocw .«. xmwumumm ad .cflmuoum HmEOmoummcmm m5\mxmums m0
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mass ..2: oooa I H. onwaomomuumumxo mo mocmmoum on“ Ca mmEOmouchwm .25 m.>w.

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55

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54

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59

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6O

Oxytetracycline (n = 7) caused a statistically

I O O 0 +
Significant decrease in Ca2

uptake, to 35% of control, at a
concentration of 1000 uM. The other concentrations ranging
from 5 - 500 uM caused slight, though statistically
insignificant decreases. The results of these experiments
are shown in figure 13.

At concentrations ranging from 1 to 500 uM, polymyxin
(n = 8) caused an increase in Ca2+ uptake (figure 14).
However, only the results at the 5 uM concentration were
significantly different from drug-free control. At 5 uM

2+ uptake via the fast

polymyxin caused an 85% increase in Ca
phase. Polymyxin did not decrease fast phase Ca2+ uptake at
any concentration tested.

Similarly, clindamycin (n = 9) does not decrease
calcium uptake (figure 15). Rather, clindamycin causes a

2+

very small, yet non-significant, increase in Ca uptake

over the entire concentration range tested (1 - 1000 uM).

2+

Slow Phase gt 9g Uptake

 

Neomycin (n = 6) does not cause a significant decrease

in slow phase Ca2+ uptake. However, as depicted in figure

16, neomycin causes a slight decrease in slow phase Ca2+

uptake at the highest concentration (1000 uM).
Oxytetracycline (n = 6), at concentrations of 500 and
1000 uM, caused a significant decrease, to 70% and 36% of

2+

control respectively, in slow phase Ca uptake (figure 17).

 

61

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The lower concentrations did not decrease slow phase Ca2+

uptake.

2+

Polymyxin B caused a decrease in total Ca uptake but

not in uptake via the fast channel; this would imply that

polymyxin affects the slow phase of Ca2+

2+

uptake. Polymyxin
did cause a decrease in Ca uptake via the slow phase over
the entire concentration range tested (5 - 1000 uM). The
results of four experiments with polymyxin (figure 18)
showed a decrease from drug-free control, but the decrease
was statistically significant only for concentrations of
100, 500 and 1000 uM. These concentrations caused
reductions to 61%, 57% and 44% of control, respectively.
Clindamycin which had no effect on either total or fast

2+

channel Ca uptake, also had no effect on the slow phase of

calcium uptake (n = 6), as shown in figure 19.

The Effects of Antibiotics on Baseline (Low §fl_932+ Uptake

 

 

 

Each of the ABs caused a statistically significant

2+

change in Ca uptake under baseline conditions. Neomycin

and polymyxin (5 uM) caused a statistically significant

I I +
increase in Ca2

uptake during 1 sec of incubation, under
non-depolarized conditions. Neomycin, oxytetracycline and
polymyxin, at high concentrations, each caused a
statistically significant decrease in depolarization-

2+

independent Ca uptake during 10 sec of incubation both in

the absence and presence of predepolarization. Clindamycin,

70

at high concentrations, caused a statistically significant
decrease in baseline Ca2+ uptake following

predepolarization. The concentrations of ABs that caused
changes in baseline Ca2+ uptake do not correspond exactly
with the concentrations of ABs that caused changes in net

2+ uptake. Although baseline Ca2+ uptake was

2+

stimulated Ca

changed, the amount of Ca uptake in the absence of

stimulation is very small compared to that under K+

-stimulated conditions. Figures 20 - 22 depict the

difference in baseline Ca2+ uptake and net stimulated Ca2+

2+ 2+

uptake, for total Ca uptake, fast and slow phase Ca

uptake, respectively. The values depicted are for drug—free
control and the drug concentrations that cause a
statistically significant difference in baseline Ca2+ uptake
as compared to drug-free control. The baseline amounts of

Ca2+ uptake is in fact smaller than the standard errors of

2+

the means of the net stimulated Ca uptake.

Summary —— Concentration Response

 

Tables 1 and 2 summarize respectively the significant

2 2+

effects of the various ABs on total Ca + uptake, and Ca

uptake via the fast and slow phases. Neomycin and

2+

clindamycin had no effect on total Ca uptake, while

oxytetracycline (1000 uM) and polymyxin (500 and 1000 uM)

both caused a decrease in total uptake of Ca2+. Neomycin

(500 and 1000 uM) and oxytetracycline (1000 uM) caused a

71

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77

decrease in fast phase Ca2+ uptake, while polymyxin (5 uM)
caused an increase in fast phase Ca2+ uptake and clindamycin
had no effect. Neomycin and clindamycin had no effect on
slow phase Ca2+ uptake, while oxytetracycline (500 and 1000
uM) and polymyxin (100, 500 and 1000 uM) caused a decrease
in slow phase Ca2+ uptake.

Since the control values varied between experiments the
control values are listed in Table 3 and Table 4, expressed
as fmoles Ca2+/ug synaptosomal protein. Table 3 (total
uptake) and Table 4 (fast and slow phase uptake) also list

2+

the amount of Ca uptake at all points that are

significantly different from control. These results

demonstrate that clindamycin has no effect on Ca2+ uptake

into synaptosomes. Neomycin and oxytetracycline, the two

ABs that act prejunctionally to cause neuromuscular block,

decrease Ca2+ uptake during the fast phase of Ca2+

(the Ca2+ uptake presumably related to release of

uptake

neurotransmitter). Based on these results the possibility

that ABS act prejunctionally to cause neuromuscular block by

2+

decreasing Ca uptake has not been ruled out.

Calcium Reversal

 

Since ABs may have actions that block neuromuscular
transmission by prejunctional competitive antagonism of Ca2+

entry into the terminal, I tested the hypothesis that

increasing the [Ca2+]O could reverse the neuromuscular block

78

T ble 1. Significant effects ofzantibiotics on net

K -stimu1ated (77.5 mM) total Ca uptake (10 sec incubation)
into synaptosomes.

 

 

 

TOTAL, DOSE

ANTBIOTIC n (’lochange) (9M)
NEOMYCIN 7 No effect

OXYTETRACYCLINE 7 ‘37 1000

POLYMYXIN 8 '25 500

' '26 1006
CLl-NDAMYCIN 9 No effect

 

 

 

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+

80

Table 3. Control values and values for each AB concentration
t at caused a statistically significant change in net

K -stimulated (77.5 mM) total Ca uptake (10 sec incubation)
into synaptosomes. The values are expressed as fmoles Ca
uptake/ug protein t'SEM.

 

 

TOTAL DOSE

ANTIBIOTIC n (JIM)
NEOMYCIN 7 74.|436.77 O
OXYTETRACYCLI N E 7' 35.43 £8.72 0

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82

caused by an AB. The ABs tested were neomycin and

oxytetracycline, because they caused a decrease in Ca2+

uptake via the fast channel. The concentration of AB that

was used was the one that caused the greatest decrease in

Ca2+ influx.

Oxytetracycline (1000 uM) did not significantly alter

Ca2+ uptake via the fast phase as compared to drug-free

2+

control, with [Ca ]0 ranging from 0.05 - 1.0 mM. Figure 23

shows both the control and oxytetracycline results (n = 5),

2+

expressed as fmoles Ca uptake/ug synaptosomal protein.

When the [Ca2+]o was 0.05 mM (the concentration used for the

2+ 2+

fast phase of Ca uptake) oxytetracycline decreased Ca

uptake to 39.8 i 7.4 percent of drug-free control. Even

though oxytetracycline caused a slight reduction in Ca2+

uptake at all [Ca2+]o the lines are not statistically

different.

2+

Neomycin (1000 uM) did not alter Ca uptake via the

2+1

fast phase as compared to drug-free control, with [Ca 0

ranging from 0.05 - 1.0 mM. Figure 24 compares the drug-

2+

free control values with Ca uptake in the presence of

neomycin (n = 5), these results are expressed as fmoles Ca2+

uptake/ug synaptosomal protein. When the [Ca2+

2+

was
10

0.05 mM neomycin decreased Ca uptake to 68.7 i 7.4 percent

of drug-free control.

2+ uptake produced by either

The decrease in Ca
oxytetracycline (1000 uM) or neomycin (1000 uM) was reversed

. . 2+ .
by rais1ng the [Ca 10. If these ABs cause competitive

83

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87

2+ 2+

antagonism of Ca influx raising the [Ca

2+

10 should have

reversed the AB-induced decrease in Ca influx in a

concentration dependent manner. Over the concentration

range of [Ca2+

Ca2+ uptake did not appear to be strictly concentration

10 tested, reversal of AB-induced block of

dependent.

DISCUSSION

One of the proposed mechanisms for AB-induced

2+ influx

neuromuscular block is competitive antagonism of Ca
into the presynaptic nerve terminal. Although many studies
have been done in attempts to determine the mechanism by
which ABs cause neuromuscular block, none of the experiments
have examined calcium influx, directly. Therefore the
experiments contained herein were designed to assess this
problem. Because of methodological constraints limiting the
use of the vertebrate neuromuscular junction, an alternate
assay was used. The assay consisted of measuring Ca2+
uptake into synaptosomes in the presence of each of several
ABs that cause neuromuscular block. Some of the ABs did

2+ influx in this system. Since some

2+

cause a decrease in Ca
of the ABs caused a decrease in Ca influx via the fast
phase it is feasible that this decreased calcium uptake can
contribute to neuromuscular block.

The experiments contained herein used synaptosomes as a
model for the neuromuscular junction. Some evidence for the
accuracy of synaptosomes as a model for the neuromuscular
junction, with respect to presumed calcium channels, was

provided by Nachshen and Blaustein (1979). Both

synaptosomes and the neuromuscular junction are relatively

88

89

insensitive to the calcium antagonists verapamil and D-600,
whereas other tissues are more sensitive to these drugs.
Nachshen (1984) demonstrated that a number of polyvalent

2+ uptake into synaptosomes.

cations block fast phase Ca
These metal cations also block calcium-dependent transmitter
release at the neuromuscular junction (Weakly, 1973; Forshaw
1977; Cooper and Manalis, 1983).

All of the ABs except clindamycin had the expected

2+ 2+

effects on Ca uptake. Neomycin caused a decrease in Ca
uptake via the putative fast channel. This was expected
since neomycin in particular and aminoglycosides in general
have been reported to exert predominantly prejunctional
neuromuscular blocking actions (Elmqvist and Josefsson,
1962; Brazil and Corrado, 1969; Pittinger and Adamson, 1972;
Singh et El: 1979, 1982; Farley gt El, 1982; Fiekers,
1983a). Oxytetracycline also caused a decrease in total and

2+

fast phase Ca uptake. This decrease is in agreement with

some of the proposed mechanisms of action for

oxytetracycline (Singh et El: 1982). Polymyxin B caused a

2+ 2+ influx via the

2+

decrease in total Ca uptake and in Ca

slow phase. In addition to the decrease in Ca influx, low

concentrations of polymyxin (5 uM) caused an increase in

2+

fast phase Ca influx. Polymyxin is thought to cause

neuromuscular block predominately by postjunctional actions
(Singh gt El, 1979, 1982; Fiekers, 1981), thus it was not

2+

expected to cause a decrease in Ca influx. Although

clindamycin has been shown to have prejunctional effects

90

(Rubbo gt gt, 1977; Fiekers gt gt, 1983), it did not cause

2+ influx. The fast phase of Ca2+ uptake

any change in Ca
appears to be associated with neurotransmitter release
(Drapeau and Blaustein, 1983; Suszkiw and O'Leary, 1983;
Daniell and Leslie, 1986), thus it would be the component of

2+ influx was the mechanism of AB-

concern if decreased Ca
induced neuromuscular block. From these results it can be
seen that neomycin, which causes neuromuscular block by
predominantly prejunctional actions, and oxytetracycline,
whose mechanism of neuromuscular block is unknown, decrease

2+

Ca influx during 1 sec of depolarization, whereas ABs

(polymyxin and clindamycin) that produce neuromuscular block
by predominantly postjunctional actions or for which Ca2+
does not provide effective reversal (Singh gt gt, 1982) do

not decrease fast phase of Ca2+

uptake into synaptosomes.
Clindamycin did not have the predicted effect on
calcium uptake; this may be due to the disparate nature of
the data upon which the prediction was based. Rubbo gt gt
(1977) and Singh gt gt (1982) have reported that clindamycin
leads to an increase in spontaneous MEPP frequency and an
increase in ACh release. Based on those results it was
expected that clindamycin would cause an increase in Ca2+
uptake. However, Fiekers gt gt (1983) have shown that
clindamycin altered EPC amplitude, increased EPC decay rate
and decreased EPC quantal content. If the decrease in EPC

quantal content is due to a decrease in the number of quanta

released and not a decrease in the size of the quanta

91

released, then this would be consistent with the fact that

2+

clindamycin did not cause an increase in Ca uptake into

synaptosomes. Clindamycin did not alter Ca2+

uptake via the
fast phase as would be expected (based on some results).
This could have occurred if there is not a general mechanism
for all ABs, but if more than one mechanism of prejunctional
block exists. For example, neomycin and oxytetracycline

2+ influx while

could block by competitive antagonism of Ca
clindamycin might have some other prejunctional effect. In
order to determine if there is more than one mechanism of
prejunctional block, other ABs that are known to block
neuromuscular transmission by a prejunctional mechanism will
have to be tested.

Polymyxin caused paradoxical apparent increase in fast

phase Ca2+

uptake. Polymyxin has both pre— and post-
junctional neuromuscular blocking actions (Singh gt gt,
1982) with the postjunctional actions predominating (Singh
gt gt, 1979; Fiekers, 1981). Polymyxin-induced
neuromuscular block is poorly reversed by calcium (Singh gt
gt, 1978). Even though synaptic transmission is dependent
on Ca2+ (Katz and Miledi, 1965b, 1967, 1969; Dodge and
Rahamimoff, 1966; Miledi and Slater, 1966) it has been
demonstrated that high levels of ionized calcium in the
presynaptic terminal can act to block synaptic transmission
(Miledi and Slater, 1966; Kusano, 1970; Adams gt gt, 1985).

Perhaps an increase in the intracellular calcium

concentration may contribute to neuromuscular block produced

92

by polymyxin.

The two ABS (oxytetracycline and neomycin) that

decreased fast phase Ca2+

Ca2+ uptake when the incubation was carried out in a

2+

uptake did not alter fast phase

solution containing a [Ca 10 ranging from 0.05 - 1.0 mM.

Therefore the AB-induced block of Ca2+

uptake can be
reversed. However this reversal did not appear to be
concentration dependent over the range of concentrations
tested. Perhaps lower [Ca2+]o are needed to demonstrate a
strict concentration-dependent competitive antagonism of
Ca2+. My results are comparable to the recent results of
Yoshii gt gt (1986) in which they demonstrated that
streptomycin (an aminoglycoside) can block Ba2+-induced

2+ channels into neuroblastoma NlE-llS

currents through Ca
cells and that increasing the external Ba2+ concentration
overcame the AB-induced block of Ba2+ currents.

One problem with the results of the experiments

contained herein is that the amount of Ca2+

uptake is about
33 - 35% of that obtained by others (Blaustein gt gt, 1977;
Nachshen and Blaustein, 1980; Nachshen, 1984, l985a,b;
Leslie gt gt, 1983). This may be due to the fact that the
other authors used different methods of synaptosome
preparation, either the method of Hajos (1975) or the
modification of Hajos's method by Krueger gt gt (1979).
These other methods for preparing synaptosomes are reported
2+

to produce a purer synaptosomal fraction. Since the Ca

uptake results are expressed per ug of protein (the

93
assumption being all of the protein is synaptosomal) a purer
preparation would give greater uptake values.

The physiological correlate for the Slow phase of Ca2+

uptake is unknown. The Slow phase of Ca2+

uptake may be
associated with a channel (Nachshen and Blaustein, 1980) or
Na+/Ca2+ exchange (Wang gt gt, 1985; Suszkiw gt gt, 1986).
Sheu gt gt (1986) have demonstrated, using rat ventricular

2+

myocytes, that both voltage-sensitive Ca channels and

voltage-sensitive Na+/Ca2+

exchange contribute to the
increase in the internal calcium concentration during
membrane depolarization. If the slow phase of calcium

uptake into synaptosomes is due to Na+/Ca2+

exchange then
there could be ion movements that would alter the membrane
potential (an electrogenic pump). It is also possible that
calcium flowing down its concentration gradient could
provide energy to pump Na+ against its concentration
gradient.

The intracellular calcium concentration is buffered by
calcium binding or sequestration in mitochondria and other
organelles (Lehninger, 1970; Alnaes and Rahamimoff, 1975;
Blaustein gt gt, 1977; Kendrick gt gt, 1977; Scott gt gt,
1980). The internal calcium concentration is very low
104 t 8 nM (based on experiments with quin-2, a Ca2+
specific fluorescent indicator) while the external calcium
concentration is 1.2 mM (Richards gt gt, 1984).

Depolarization causes a 2-fold increase in the internal

calcium concentration (Richards gt gt, 1984). Thus the

94

internal calcium concentration remains well below the
external calcium concentration. Calcium entry into
synaptosomes is dependent on the ratio of the internal and
external Na+ concentrations (Coutinho gt gt, 1984; Nikezie
and Metlas, 1985). Either increasing the internal Na+
concentration or decreasing the external Na+ concentration

leads to greater Ca2+ 2+

uptake. The slow phase of Ca uptake
may be included in the mechanism by which the nerve terminal
removes Na+ that has entered during depolarization.

Therefore the decrease in slow phase Ca2+

uptake probably
does not contribute directly to the neuromuscular blocking
actions of the ABS.

A wide range of concentrations for the ABs was used for
the Ca2+ uptake experiments, yet in order to observe a
decrease in Ca2+ influx high concentrations of the ABS were
needed. The concentrations of ABS needed to cause a

C +
decrease in Ca2

influx are considerably higher than the
normal peak plasma levels of these ABS given clinically
(neomycin, polymyxin and oxytetracycline < 10 uM;
clindamycin < 40 uM). However, neuromuscular block usually
does not occur at these plasma levels unless other agents
are present (eg, general anesthetic or neuromuscular
blockers) that compromise neuromuscular function. AB-
induced neuromuscular block can also occur in patients with
hepatic or renal dysfunction that cause an increase in the

concentration of AB in the plasma.

The concentrations of ABS needed to cause a decrease in

95

Ca2+ influx are generally higher than those needed to cause

neuromuscular block in isolated nerve-muscle preparations.
Depending on what effect is measured, different
concentrations of the ABS are needed to cause a Significant
effect on transmission. These effects occur at the
following concentration ranges: neomycin 1-600 uM (Wright
and Collier, 1977; Fiekers, l983a), polymyxin 2 uM (Durant
and Lambert, 1981), clindamycin 200-800 uM (Wright and
Collier, 1976a; Fiekers gt gt, 1983), oxytetracycline 2,000-
10,000 uM for Z 80% reduction (Singh gt gt, 1978, 1982).
Thus the concentrations needed in synaptosomes are often,
but not always higher than those needed in other systems.
Several reasons could explain why higher concentrations are
needed to see an effect in synaptosomes as compared to the
isolated neuromuscular junction. First, synaptosomes are
heterogeneous with respect to transmitter; this means that
if the effects of ABS that cause neuromuscular block are
specific for cholinergic nerve terminals (such as those
found at the neuromuscular junction), the effects of the ABS
may be obscured. For example the ABS might act only at

2+ channels. As described earlier there

2+

certain types of Ca
are multiple types of Ca channels. If, as hypothesized by
Yu and Nelson (1986), neurons that utilize different types
of neurotransmitters have different Ca2+ channels, the ABS
may not act the same at all types of neurons (eg. the ABS
may have different affinities for or efficacy at different

2+

types of Ca channels). Second, synaptosome preparations

96

are not pure nerve terminals; they contain free

mitochondria, membrane fragments and other cellular debris.

2+

The mitochondria can also take up Ca and thus confound the

2+

results. The Ca uptake in these experiments was measured

based on the amount of 45

2+

Ca trapped by the filter. This

that entered mitochondria was also
2+

means that any Ca

included in the measurement of Ca influx. This problem

was reduced by partial purification of the synaptosomes to

remove mitochondria. Also the change in potassium

. , . +
concentration that caused an increase in Ca2

2+

uptake into
the synaptosomes, does not alter Ca uptake into
mitochondria (Blaustein, 1975), so the experimental design

2+

Should eliminate mitochondrial Ca uptake as a potential

variable from the final results (net stimulated Ca2+
uptake). Third, synaptosomes may not represent an accurate
model of the neuromuscular junction. For example, different
tissues have different types of Ca2+ channels, as shown by
differences in the effects of verapamil, D-600 and
dihydropyridines (Gotgil'f and Magazanik, 1977; Nachshen and
Blaustein, 1979; Daniell gt gt, 1983; Glossman gt gt, 1984),
thus synaptosomes may not have the same type of calcium
channel as do the presynaptic motor nerve terminals. Even
though evidence has been presented that shows synaptosomes
and the neuromuscular junction respond the same way in the
presence of organic calcium antagonists and multivalent

cations (Nachshen and Blaustein, 1979; Nachshen, 1984), this

does not mean that synaptosomes and the neuromuscular

97

junction will necessarily respond the same way in the
presence of antibiotics. Fourth and finally, block of Ca2+
uptake may not be the only mechanism by which ABS cause

2+ influx

neuromuscular block. Competitive antagonism of Ca
may be one of many factors involved in neuromuscular block,
or it may not be involved at all. If ABS cause

2+

neuromuscular block by blocking the Ca channel, it is not

necessary that the ABS act as competitive inhibitors of
Ca2+. Also because of the multiple steps involved in
synaptic transmission the ABS may also act at a step(s)

other than Ca2+

influx to cause neuromuscular block.

Some experiments could be done in order to address some
of these problems. The first problem could be avoided if
the studies were repeated with a pure cholinergic
preparation, such as the electric organ of Torpedo. If
cholinergic synaptosomes could be used this would more
closely resemble the neuromuscular junction than the
transmitter—heterogenous synaptosomes which characterize
those derived from the forebrain. The second problem can be
addressed by using methods that isolate a synaptosome
fraction of higher purity (Hajos, 1975; Krueger gt gt,
1979). The problem of determining if the calcium channels
of the two tissues are identical is unavoidable, until more
is known about the calcium channels of the respective
preparations. The fourth issue could be addressed by

testing ABS that do not cause neuromuscular block. If those

ABS decrease Ca2+ influx then decreasing Ca2+ influx may not

98
cause neuromuscular block.

Some Ca2+ channel blockers may Show preferential

2* channels. Nachshen (1985a) has

2

binding to inactivated Ca

demonstrated that inhibition of K+-stimulated Ca

Ni2+, La3+

+ uptake by
and verapamil was enhanced in synaptosomes that
were pre-depolarized for brief intervals in the presence of
Ca2+ channel blockers. Pre—depolarization Should cause Ca2+
channel inactivation in synaptosomes as the inactivation is
voltage, and not Ca2+-dependent (Nachshen, 1985a). This
implies that some Ca2+ channel blockers may bind
preferentially to inactivated Ca2+ channels. This
possibility should also be tested for ABS, since this could
help explain why ABS that cause neuromuscular block had no

2+

effect on Ca uptake into synaptosomes. Synaptosomes

should be depolarized in the presence of an AB before Ca2+
influx is measured. Experiments should also be done in
which the synaptosomes are pre-incubated with the ABS, in
case the ABS have their effect by inhibiting activation

2+ channel. Pre-incubation with ABS

(Opening) of the Ca
would resemble physiological conditions more closely than
the conditions of the experiments contained herein. If the

ABS had their effect by inhibiting opening of the Ca2+

2+ channel the

channel or by binding to the inactivated Ca
effects would not be seen in the experiments contained
herein.

The results described herein do not disprove the idea

that ABS can cause neuromuscular block by block of

99

depolarization-induced Ca2+ entry, but rather provide
evidence which though weak, is at least consistent with the
idea that some ABS may cause prejunctional neuromuscular
block by decreasing Ca2+ uptake. Further study, especially

with regards to the fast phase of Ca2+

uptake, is necessary
to elucidate the mechanism(s) by which ABS cause

neuromuscular block.

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LIST OF REFERENCES

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