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THESIS

' LIBRARY

Michigan State

University

 

 

This is to certify that the
thesis entitled

THE ROLE OF CALCIUM AND OTHER CATIONS IN SENSORY
TRANSDUCTION AND HABITUATION IN THE CILIATED
PROTOZOAN SPIROSTOMUM AMBIGUUM

 

presented by

DonaTd G. Brunder

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in B iQphys .ICS

 

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THE ROLE OF CALCIUM AND OTHER CATIONS IN SENSORY
TRANSDUCTION AND HABITUATION IN THE CILIATED
PROTOZOAN SPIROSTOMUM AMBIGUUM

 

By

Donald G. Brunder

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Biophysics Department

1981

THE ROLE OF CALCIUM AND OTHER CATIONS IN SENSORY
TRANSDUCTION AND HABITUATION IN THE CILIATED
PROTOZOAN SPIROSTOMUM AMBIGUUM

 

by

Donald G. Brunder

Repeated mechanical (vibratory) but not electrical stimulation

(rate 0.l Hz) of the ciliated protozoan §pirostomum ambiguum leads to

 

a decrement in the probability (habituation) of the contractile
response. Habituation is a simpler form of learning/memory. The

growth medium for §pirostomum contains the chloride salts of Na, K,

 

Mg, and Ca and a pH buffer. Calcium appears to be an important ion

in the stimulus transduction and habituation processes. The present
experiments were undertaken to investigate further the role of calcium
and other cations in these processes. The effects of the following

cations were studied: Na+, K+, Mg2+ a2+, Li+, Cs+, Rb+, Ni2+,

2 2 2+

, C

3+

Zn2+, Ba +, Sr +, Co , Mn2+, and La ; in addition pharmacological

agents which alter membrane permeability to calcium or affect calcium
binding (A23187, caffeine, and verapamil) were tested for behavioral

effects on Spirostomum.

 

Reducing extracellular calcium levels decreased responsive-
ness to both modes of stimulation. Responsiveness to electrical

stimulation was reduced by raising the concentrations of Na+, K+, or

Donald G. Brunder

Mg2+ in the culture medium while increased [K+] and replacement of

Ca2 2+ or Mn2+ reduced responsiveness to mechanical stimula-

+ with Ba
tion.
A fast Ca-45 labeling component was observed in stimulated

and unstimulated Spirostomum. Electrical stimulation resulted in

 

increased Ca-45 uptake compared to the control and the amount of
uptake was dependent on extracellular [Ca2+]. Some indication of
Ca-45 uptake in response to mechanical stimulation was observed but
this result was not statistically significant. The calcium ionophore
A23187 and P042” both increased Ca-45 uptake in unstimulated

§pjrostomum.

 

A response decrement to electrical stimulation was observed
in low [Ca2+] media; surprisingly no change in decrement level

occurred in response to mechanical stimulation in the same media.

2+, Sr2+, or Zn2+ also resulted in a

2+

Replacement of calcium with Ba
decrement to electrical stimulation even though Ba and Sr2+ often
substitute for Ca2+ in many physiological processes. The presence

2 2+ reduced habituation levels to mechanical stimulation;

of Ba + or Zn
caffeine also removed habituation. La3+ and verapamil, a calcium
blocking agent, tended to increase the amount of habituation to
mechanical stimulation.

The results show that calcium is necessary for the trans-
duction of mechanical and electrical stimuli and may play a role in
the response decrement process. The differing responses to mechani-
cal and electrical stimulation in the presence of the same ions sug-

gest that the mechano- and electro-transduction sites are separate.

Donald G. Brunder

with respect to the role of calcium it is not clear whether calcium
acts by a binding or an uptake mechanism; the present results give
support to both mechanisms. The results are compatible with a model
where two separate calcium channels are responsible for sensory
transduction and habituation is due to reduced calcium entry through
the mechanoreceptor channel. The habituation process may involve
regulation of calcium permeability by a metalloenzyme. This work
suggests habituation in neural and aneural systems may have similar

mechanisms.

TABLE OF CONTENTS

Page

LIST OF TABLES .............................................. iii
LIST OF FIGURES ............................................. iv
INTRODUCTION ................................................ 1
Background ....... ' ....................................... 4
METHODS ..................................................... 12
Protozoan Cultures ...................................... 12
Stimulation and Data Analysis ........................... 12
Effects of Ions ......................................... 14
Ca-45 Uptake ............................................ 15
Pharmacological Agents .................................. 17
Deciliation ............................................. 20
RESULTS ..................................................... 21
Effects of Calcium ...................................... 21
Effects of Monovalent and Other Divalent Cations ........ 24
Ca-45 Uptake Studies .................................... 30
Effects of Pharmacological Agents ....................... 32
DISCUSSION .................................................. 39
Transduction ............................................ 40
Response Decrement ...................................... 46
LIST OF REFERENCES .......................................... 54

ii

LIST OF TABLES

Table Page

1. Effects of Free Calcium Concentration on Responsive-
ness and Response Decrement to Mechanical and
Electrical Stimulation ............................... 22

2. Effects of Monovalent and Divalent Cations on
Responsiveness, Response Decrement, and Recovery
to Mechanical Stimulation ............................ 25

3. Effects of Monovalent and Divalent Cations on
Responsiveness, Response Decrement, and Recovery
to Electrical Stimulation ............................ 27

4. Uptake of Ca-45 in Stimulated and Unstimulated
Spirostomum .......................................... 30

 

5. Effects of A23187 and Lanthanum on Ca-45 Uptake in
Unstimulated Spirostomum ............................. 32

 

iii

LIST OF FIGURES

Figure

1. Drawing (not to scale) of the free-swimming and
contracted forms of Spirostomum ambiguum ............

 

2. The effects of monovalent and divalent cations on
responsiveness, response decrement, and recovery
to mechanical stimulation (0.1 Hz for 11 min.;
4 min. rest; 1 min. stimulation to test recovery) ...

. 3. The effects of monovalent cations on responsiveness,
response decrement, and recovery to electrical
stimulation .........................................

4. The effects of divalent cations on responsiveness,
response decrement, and recovery to electrical
stimulation .........................................

5. The effect of caffeine on responsiveness, response
decrement, and recovery to mechanical stimulation
(0.1 Hz for 10 min.; 4 min. rest; 1 min. stimulation
to test recovery) ...................................

6. The responsiveness to 2 minutes of mechanical stimula-
tion as a function of lanthanum concentration (uM) ..

7. The responsiveness to 2 minutes of electrical stimula-
tion as a function of lanthanum concentration (pM) ..

8. The percent decrement to mechanical stimulation as a
function of lanthanum concentration (pH) for the
data of Figure 6 ....................................

9. The percent decrement to mechanical stimulation as a
function of verapamil concentration (pg/ml) .........

10. The responsiveness to one minute of mechanical or

electrical stimulation as a function of incubation
time in 100 uM lanthanum ............................

iv

Page

26

28

29

33

34

35

36

37

38

INTRODUCTION

Although extensively studied, the molecular mechanisms of
learning have thus far eluded neuroscientists. In complex nervous
systems much of the problem has to do with localization of the event
and accessibility of the preparation; this has led to the use of
model systems. It is also advantageous to eliminate complexity in
the behavior to be studied. Thus the study of simpler forms of
learning and memory in model systems can be a useful approach to
understanding mechanisms of learning and memory.

The work described here aims at obtaining an understanding
of habituation, a simpler form of learning and memory, at a cellular
level. Habituation is defined as a response decrement to an inter-
mittent but repetitive stimulus. A major problem in studying the
molecular basis of any kind of learning and memory is in finding a
"simple" enough preparation in which one can locate and isolate the
relevant changes. Most nervous systems are too complex. Neural
elements involved in a particular learned behavioral act are not
easily identified and it is often difficult, if not impossible, to
assess the relative contributions to such behavior of intraneuronal
vs. interneuronal (i.e., connectivity) factors. If one views the
functions carried out by nervous systems as having both intracellular
as well as intercellular components then one might view such work on
aneural single cells as a strategy to focus on the intracellular

1

components uncomplicated by intercellular considerations. The
protozoa (in particular the ciliates) would appear very promising
for such studies. All of the activity to be observed occurs in one
cell, which contains all of the sensory, motor, and integrative
aspects of the behavior. Thus, in addition to being a single cell
it is also an entire organism. The single-celled organisms allow
unrestricted access to the cell and its immediate environment and
thus allow the use of techniques which require access to the cell,
e.g., being able to maintain and alter in a controlled fashion the
ionic milieu of the cell. Thus, a single-celled organism allows
cellular experiments on habituation to be performed more easily than
on nervous systems such as in Aplysia. This study examines habitua-

tion in the large ciliated protozoan, Spirostomum ambiguum. This

 

one-celled organism exhibits habituation of its contractile response
to repeated mechanical (vibratory) but not to electrical stimuli
(Osborn, Blair, Thomas, and Eisenstein 1973a).

One interpretation of the significance of habituation in
higher animals is that it represents a mechanism that the nervous
system has evolved for filtering out unimportant inputs. Compared
to associative and Pavlovian conditioning, habituation is probably
the simplest and certainly the most ubiquitous form of learning; it
occurs in all animal phyla from single-celled aneural organisms such
as the ciliated protozoa (Applewhite and Morowitz 1966; Applewhite
1968a; Applewhite, Gardner, and Lapan 1969; Wood 1970a, 1973; Osborn
et al. 1973a) through mammals (Groves and Thompson 1970; Thompson,

Groves, Teyler, and Roemer 1973). The characteristics of the

habituation phenomenon are also remarkably similar throughout
phylogeny (Thompson and Spencer 1966; Eisenstein and Peretz 1973).
Many of the phenomena studied in neurobiology are not unique
to nervous tissue but also occur in aneural systems (e.g., conduction
of potentials and sensory transduction in protozoa [see Eisenstein
1975]). There also are striking similarities across phylogeny of
many metabolic pathways as well as in membrane structure and function.
It is therefore reasonable to expect that there also may be similarity
in the mechanisms underlying related behavioral phenomena such as
habituation and sensory transduction in neural and aneural systems.
Previous work suggests that habituation is closely tied to

the mechanotransduction process in Spirostomum; accordingly this work

 

also attempts to describe molecular mechanisms involved in the trans-
duction of stimuli, especially mechanical stimuli. Studying cellular
aspects of mechanotransduction has been difficult in higher systems
due to a variety of problems, such as the small size of the receptor
cell and the relative inaccessibility of the receptor region.

Accessibility may be more easily achieved in Spirostomum. Its large

 

size (1 to 3 mm in length) make it especially attractive for single
cell behavioral, molecular, and cellular studies.

In summary then, this study is concerned with the earliest
cellular representation of the transduction of mechanostimuli. The
goal is to understand how an external stimulus event is converted
into its biological representation (transduction) and then coupled
to the motor output and further how this coupling changes over time

(habituation). The focus is on the nature of both the transduction

and habituation mechanisms, i.e., what they are, where they are

located, and what changes occur with time and stimulation.

Background
Many ciliates, including Spirostomum, have two motor systems--

contractile and ciliary. The latter is involved in locomotory
behavior. Spirostomum ambiguum (Figure 1) contracts to approximately
50% of its resting length in less than 5 milliseconds when stimulated
electrically (Jones, Jahn, and Fonseca 1966; Hamilton and Eisenstein
1969; Hamilton and Osborn 1977). Similar rapid contractions occur in

response to mechanical (Hamilton and Osborn 1977) as well as photic

 

Figure 1.--Drawing (not to scale) of the free-swimming and contracted
forms of Spirostomum ambiguum. This ciliated protozoan
grows to lengths of 1-3 mm and can contract to less than
50% of this length in a few milliseconds.

stimuli (Borsellino, Cavazza, and Riani 1971). Habituation occurs

in response to repeated mechanical stimulation1 but not to repeated
electrical stimulation (Osborn et al. 1973a). This latter result
rules out the possibility that habituation occurs on the motor, i.e.,
contractile, side of the behavior since the same response apparatus

is used for both mechanical and electrical stimulation but only the
former leads to habituation; thus habituation is not fatigue of the
contractile apparatus. The habituation process must occur between”
the initial mechanotransduction step and the initiation of contraction
(Applewhite et al. 1969; Osborn et al. 1973a). The response of

Spirostomum to mechanical and electrical stimulation is stimulus spe-

 

cific (Osborn et al. 1973a), i.e., mechanical stimuli do not affect
the responsiveness to electrical stimuli while electrical stimulation
has only a small effect on responsiveness to mechanical stimulation
(Applewhite et al. 1969; Osborn et al. 1973a). Stimulus specific
habituation also has been observed in the closely related protozoans,

Stentor (Wood 1970a, 1973) and Vorticella (Patterson 1973), as well

 

as in higher organisms (Thompson et al. 1973). In addition, habitua-
tion shows generalization from the point at which the ciliate is
stimulated to its entire surface (Hood 1972).

Calcium, in contrast to most other ions in a cell's normal
environment.is often involved in control processes (Chang and Triggle

1973; Rubin 1974; Carafoli, Clementi, Drabikowski, and Margreth 1975;

 

1See Kinastowski (1963a,b) for extensive studies on the
effects of mechanical stimulus parameters (intensity, frequency,
number of trials) on habituation in Spirostomum.

Rasmussen 1975; Doughty and Diehn 1979). In higher systems calcium
is important in muscle contraction (Ebashi 1972; Smith 1977; Scarpa
and Carafoli 1978), vision (Hagins and Yoshikami 1974), synaptic
transmission (Katz and Miledi 1967; Triggle 1972; Miledi 1973;
Rahamimoff, Erulkar, Alnaes, Meire, Rotshenker, and Rahamimoff 1976;
Weller and Morgan 1977), and secretion (Matthews 1970; Rubin 1974;
Scarpa and Carafoli 1978). It also is involved in the regulation of
cyclic nucleotide synthesis (Rasmussen 1975). In lower systems
calcium has a regulatory role in bacterial chemotaxis (Ordal 1977)

and in the ciliary reversal process in Paramecium (Grebecki 1965;

 

Kuznicki 1966, 1970, 1973; Naitoh 1968; Naitoh and Eckert 1969;
Eckert 1972).
Using the calcium-sensitive photoprotein aequorin, Ettienne

(1970) showed that contraction of Spirostomum is preceded by an

 

increase of cytoplasmic free calcium concentration; contractions also
have been elicited by injection of ESTA-buffered calcium solutions at
concentrations greater than 10'5 M (Hawkes and Holberton 1974) and in
calcium activated models (Ettienne 1976). Although it appears that
ATP is not required for contraction (Seravin, Skoblo, and Bagnjuck
1965), some aspect of mitochondrial metabolism would appear to be
important in the regulation of intracellular calcium in Spirostomum,
based on the action of various pharmacological agents which inhibit
mitochondrial respiration and affect the contractility of Spirostomum
(Ettienne and Selitsky 1974; Holberton and Ogle 1975; Dikstein and
Hawkes 1976). The existence of calcium in the mitochondria of

Spirostomum has been confirmed by electron microprobe analysis

 

(Osborn, Hsung, and Eisenstein 1973b; Osborn and Hamilton 1977). In
higher forms mitochondria prefer calcium uptake to oxidative
phosphorylation; the calcium is apparently stored as a calcium-
phosphate compound (Lehninger 1970; Carafoli and Crompton 1976).

Based on studies utilizing Ca-45 and P-32, Spirostomum actively

 

accumulates both ions in the cytoplasm (Jones 1966, 1967; Bai and
Dilly 1976; Balcerzak 1978) and at least a portion is stored in the
form of hydroxyapatite (Pautard 1959; Bien and Preston 1968) which

is found in the endoplasmic reticulum of Spirostomum (Osborn et al.

 

1973a; Osborn and Hamilton 1977). Calcium control of contractility
also has been observed in other peritrich ciliates where intracellular
calcium is stored in the endoplasmic reticulum and possibly released
by a linkage structure between the reticulum and the myonemes (Allen
1973), the tightly packed bundles of microfilaments responsible for
contraction (Lehman and Rebhun 1971). Vesicles, possibly containing
calcium, have been observed with the electron microscope near the

myonemes in Spirostomum (Yagiu and Shigenaka 1963; Finley, Brown,

 

and Daniel 1964; Vivier, Legrand, and Petitpriz 1969; Legrand and
Prensier 1976), but no linkage structures between vesicles and
myonemes have been reported.

Calcium may be involved in the sensory transduction process
as well as the contractile process. For example, previous work shows
that the addition of EGTA to the medium in which Spirostomum swims
results in a decreased probability of contraction to mechanical but

not to electrical stimulation (Osborn et al. 1973b). In Paramecium

 

responses to changes in hydrostatic pressure (which is probably the

direct stimulus Spirostomum receives during mechanical [vibratory]

 

stimulation) are apparently mediated by calcium channels in the plasma
membrane (Otter and Salmon 1979). In addition, it appears there may
be cholinergic sites involved in sensory transduction in some ciliates
(Wood 1975, 1977; Doughty 1978).

Kandel (1976, l978, l979) suggests that habituation in the
gastropod mollusc Aplysia is due to decreased free calcium concentra-
tion in the sensory presynaptic terminals; he postulates that this
lowered calcium level is caused either by a decreased calcium influx
or increased mitochondrial calcium uptake. The sequence of steps
occurring at the site of habituation in a ciliate may be analogous
to the presynaptic steps occurring at the site of habituation in
nervous systems, e.g., Aplysia.

The normal culture medium of Spirostomum contains the chloride

 

salts of sodium, potassium, magnesium, and calcium (Carter 1957) and

a HEPES-PIPES pH buffer. Ions other than calcium also may be involved
in the transduction and habituation processes. For example, Apple-
white and Davis (l969) found that Mg2+ affects habituation as well as
responsiveness to mechanical stimulation. Various ions have been
found to increase spontaneous contractility (Sleigh 1969, 1970) and
increase the threshold to electrical stimulation (Fabczak 1974).
Possibly calcium is involved in the regulation of the permeability

of Spirostomum to other ions, as it is in a variety of other systems,

 

e.g., nerve and muscle tissue in the mollusc, arthropod, and verte-
brates (Frankenhaeuser and Hodgkin 1957; Triggle 1972; Rubin 1974;
Meech 1976; Ohki 1978; Schultz and Heil 1979).

The ciliary motor system of protozoa has received much study
in recent years. The role of calcium is well defined in the ciliary

reversal process of Paramecium (Naitoh 1968, 1974; Naitoh and Eckert

 

1969; Eckert 1972; Sakai and Hiramoto 1975; Machemer 1976; Machemer
and dePeyer 1977; Eckert and Brehm 1979). When the anterior end of

Paramecium is mechanically stimulated, calcium enters resulting in

 

ciliary reversal (Ogura and Machemer 1979). A stimulus to the pos-
terior end causes potassium to enter, triggering an increase in the
ciliary beat leading to forward movement (Naitoh and Eckert 1969;
Eckert 1972). The process seems to be controlled by an ion exchange

process on the membrane of Paramecium (Jahn 1962; Grebecki 1965;

 

Kuznicki 1966, l970; Naitoh and Yasamasu 1967; Naitoh 1968). Reversal
is activated when the intraciliary calcium level is greater than 10"8
to 10'6 M (Naitoh and Kaneko 1972; Sakai and Hiramoto 1975). Since
calcium-dependent electrical responses are eliminated by deciliation
(Ogura and Takahashi 1976; Dunlap 1977), calcium may enter via
channels in the ciliary membrane. Calcium binding sites also have
been localized at the base of the cilium in Paramecium (Fisher,

Kaneshiro, and Peters 1976; Tsuchiya and Takahashi 1976). Machemer

and Ogura (1979) found that mechanoreceptor channels in Paramecium

 

are distributed over the somatic (nonciliary) membrane. Kuznicki
(l973) provides evidence arguing against the involvement of extra-
cellular calcium in the ciliary reversal process, thus supporting the
possibility the calcium may come from a membrane-bound source.
Electrophysiological studies of Spirostomum have not been

particularly successful. Resting potentials of approximately -16 mv

10

(inside negative) have been reported by Ettienne (1970) and Hawkes
and Holberton (1974). Kokina (1972) claims to have measured resting
potentials varying from -37 mV to 47 mV. Jahn (1966) reported no
potential changes accompanying contraction. Intracellular recording
with glass micropipettes is very difficult with this animal; they are
far too agile and contractile. Without extensive development intra-
cellular electrophysiological studies are not likely to contribute
to the understanding of habituation and mechanotransduction in this
organism.

Intracellular recordings have been made from both Paramecium

and Stentor (Paramecium shows little or no contraction and Stentor,

 

 

although contractile like Spirostomum, will attach to a site and

 

remain fixed when impaled). Eckert, Naitoh, and Friedman (1972) and
Ogura and Machemer (1979, 1980) found a receptor potential in

Paramecium associated with mechanical stimulation; Wood (1970b,c,

 

1980) recorded a receptor potential in Stentor in response to mechani-
cal stimulation which did not occur to electrical stimulation and
which became progressively smaller with repeated mechanostimulation

in correlation with habituation of the contractile response (no
habituation of the contractile response occurred to electrical

stimulation in Stentor just as it does not for Spirostomum). Receptor

 

potentials also have been observed during chemokinesis in Paramecium
(Van Houten 1979).

Several approaches have been used to study the basis of
habituation of the contractile response to mechanical stimulation in

Spirostomum. Applewhite and co-workers, for example, studied RNA and

11

protein synthesis and failed to find evidence that they were involved
in habituation to mechanical stimulation (Applewhite and Gardner

1968; Gardner and Applewhite 1970b; Applewhite 1970). Low tempera-
tures were found to improve retention of habituation but had no effect
on the rate or amount of habituation itself (Applewhite 1968b,c;
Gardner and Applewhite 1970a).

Specifically, this work involves studying the role(s) of
calcium in mechanotransduction and habituation. This is accomplished
by: (1) altering calcium levels in the media, including calcium
buffering with EGTA; (2) measuring calcium uptake in behaving

_§pirostomum using a radiolabeled calcium species (Ca-45);

 

(3) replacing calcium with or adding other divalent cations; and

(4) applying pharmacological agents which alter membrane permeability
to calcium or affect calcium binding, e.g., A23187, caffeine,
lanthanum, and verapamil. The effects of altering other ions com-
posing the Carter's medium in which the animals are cultured also is
investigated. In addition, deciliation is attempted to explore the
role of the cilia in the transduction process. The results of the
present study should aid in the characterization of the molecular
events occurring during sensory transduction and behavior modifica-

tion in the ciliated protozoan, Spirostomum ambiguum.

 

METHODS

Protozoan Cultures

 

Spirostomum were obtained from Connecticut Valley Biological

 

Supply Company (Southampton, Massachusetts) and cultured in an
aqueous medium containing the chloride salts of potassium, sodium,
magnesium, and calcium in glass doubly-distilled water; the concen-
trations used were: KCl - 0.5 mM, NaCl - 2.0 mM, MgCl
CaCl 2

2 - 0.2 mM,

- 0.5 mM, and a 2.5 mM HEPES-PIPES pH buffer (Carter 1957).

2
The pH of the medium was approximately 7.2. Heat-killed wheat seeds

were added to support bacteria upon which Spirostomum feed. The

 

protozoans were recultured every 7-21 days. All cultures were grown

at room temperature (23-25°C) with continual exposure to room light.

Stimulation and Data Analysis

 

For behavioral studies Spirostomum were tested in groups of

 

four and contractions observed through a dissecting microscope posi-
tioned over the slide in which the protozoans swim (Osborn et al.
1973a). Following transfer to the microscope slide a rest period of
at least 5 minutes was given prior to stimulation. Mechanical
stimuli were applied by dropping the core of a solenoid on the slide.
Electrical stimulation was provided by 2 millisecond biphasic pulses

delivered across two 1 cm lengths of .002" silver wire on each side

 

2HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;

PIPES: Piperazine-N,N'-bis-(2-ethanesulfonic acid).

12

13

of the slide well or across platinum plated on opposing sides of the
well; the pulses were generated by a Grass S-8 stimulator and STU-4678
isolation units. Voltages supplied to the slide well ranged from 10
to 75 volts; the stimulus delivered to the animal depends on the size,
shape, and contents of the slide well as well as the orientation of
the animal to the electrodes. Stimulus intensities were chosen to
obtain probabilities of contraction between 0.70 and 0.80 for one
minute of electrical or mechanical stimulation in the control Carter's
medium. Stimuli were presented at a rate of 0.l Hz for varying
periods of time (usually 2 or 10 minutes). To minimize variability
the number of contractions for 6 trials (one minute of stimulation)
was combined and expressed as a probability. The probability of
contraction for minute 1 of the stimulation period is considered an
index of the efficacy of transduction. The amount of habituation

was calculated by subtracting the probability of contraction for the
final minute of stimulation from that for minute 1; this difference
was then divided by the probability for minute 1 to reduce the
influence of the starting level of responsiveness and was expressed

as a percent. Recovery was measured by testing responsiveness for
one minute after a short (4 minute) rest period; it was expressed as
the percent of the decrement for which responsiveness has returned.
The data were examined by analysis of variance and the least signifi-
cant difference (LSD) multiple range test was used to locate signifi-
cant effects. By testing in groups of 4 and using a dichotomous
measure (contractions were scored yes-no) the assumptions of analysis

of variance can be met (J. L. Gill, personal communication).

14

Correlation and covariance analysis also were performed to evaluate
the relationship of the date and time of the experiment and the age
of the culture of Spirostomum to the responsiveness, response decre-
ment, and recovery parameters. All statistical analyses were per-
formed with the CDC Cyber 750 computer at Michigan State University
using the Statistical Package for the Social Sciences (SPSS).

Effects of Ions

 

Calcium
The effects of extracellular calcium on sensory transduction

and habituation were investigated by incubating Spirostomum for 15

 

minutes in media containing several different concentrations of cal-
cium and EGTA3 (a calcium buffer); following incubation the animals
were stimulated for 2 minutes to measure responsiveness and response
decrement. The following concentrations of calcium and EGTA were
used: (1) 0.5 mM Ca + 1.0 mM EGTA, (2) 0 Ca + 0.l mM EGTA, (3) 0.5
mM Ca + 0.5 mM EGTA, (4) 0 Ca + 0 EGTA, (5) 0.25 mM Ca + O EGTA,

(6) 0.5 mM Ca + 0.25 mM EGTA, (7) 0.5 mM Ca + 0 EGTA (control), and
(8) 0.75 mM Ca + 0.25 mM EGTA. Free calcium and magnesium concentra-
tions were calculated using the successive approximation method of
Portzehl, Caldwell, and Ruegg (1964) and assuming calcium and
magnesium contamination levels of 5 uM (Table 1). Atomic absorption
spectroscopy indicates calcium-free Carter's media contains about

1 uM Ca; this level does not include calcium contamination due to the

 

3Ethyleneglycol-bis-(B-amino-ethyl ether)-N,N'-tetra acetic
acid.

15

slide or calcium that is transferred with the animals. The calculated

free calcium concentrations can be considered maximal levels.

Other Ions

 

The effects of several monovalent and divalent cations were
investigated by the addition, removal, or replacement of the ion in

the Carter's medium. Non-toxic concentrations were chosen on the

2+

basis of 24 hour tests. Only Zn2+ and Ni were toxic at levels

equal to that of calcium (0.5 mM). Replacements were as follows:

2+ 2+ 2+

Li+ replaced Na+; Rb+ and Cs+ replaced K+; Sr , Ba2+, Mn

2

, and Co

2+

replaced Ca2+; Zn + and Ni2+ replaced a portion of the Ca in the

Carter's medium. Spirostomum were stimulated at a rate of 0.1 Hz

 

for 11 minutes followed by a 4 minute rest and another minute of

stimulation to test recovery.

Ca-45 Uptake

Uptake of calcium by behaving Spirostomum was measured using

 

Ca-45. The animals were first centrifuged, dialyzed, and starved for
four days in clean Carter's medium to remove bacteria (Jones 1966).
Ca-45 (New England Nuclear, Boston, Massachusetts) was added to a

large-welled slide containing either 50 or 500 Spirostomum; the final

 

Ca-45 activity was approximately 1 pC/ml. The organisms were then
stimulated or rested (controls) for periods of 2, 5, 10, 30, and 60
minutes. To increase the sensitivity of the technique the 60 minute
incubation was in calcium-free Carter's medium. Upon addition of the
Ca-45 the calcium concentration was approximately 6 uM. This allowed

an increase of the percentage of labeled calcium available for uptake.

16

Following stimulation the contents of the slide were filtered through
0.8 u Millipore or Gelman filters (Martonosi and Feretos 1964); the
filters were pre-rinsed with 10 ml of Carter's medium to reduce non-
specific Ca-45 binding (Browning and Nelson 1976). The filters were
post-rinsed with 10 ml of Carter's medium. Each filtration step
lasted 30 seconds. The filters were dissolved in 1 ml of 2-methoxy-
ethanol (cellosolve) for at least 15 minutes (Gelman filters do not
dissolve but rather become clear) and 10 ml of Aquasol (New England
Nudlear, Boston, Massachusetts) or tritosol (80 ml Fluoralloy, 320
ml 95% EtOH, 100 ml ethylene glycol, 760 ml triton-X, 1740 ml xylene)
was added. The suspension was mixed thoroughly and analyzed by
liquid scintillation counting. Quench corrections were not made.
However, the counting window was not restricted and self-adsorption
is not a problem for the energetic B of Ca-45 (Zarybnicky and Reich
1980). Counting samples as prepared above or after the addition of
EGTA or as gels had little effect on the relative Ca-45 uptake
measured. The use of EGTA (Van Breemen and Casteels 1974; Aaronson,
Van Breemen, Loutzenhiser, and Kelber 1979) or La3+ (Mayer, Van
Breemen, and Casteels I972; Neihe, Hartschuh, Metz, and Bruhl 1977;
Hellman 1978) to determine the contribution of cell surface binding
were not satisfactory (see discussion). Counts per minute (CPM)
data were analyzed statistically and the uptake expressed as pmole
calcium uptake per animal. This method does not distinguish between
uptake and binding of calcium and thus in the results the terms

uptake, binding, and incorporation will be used synonymously.

17

3+

The effects of A23187, La , and P042- on Ca-45 uptake in

unstimulated Spirostomum also were tested. A23187, a divalent cation

 

ionophore (Reed and Lardy 1972), was a gift from the Eli Lilly Co.

3

(Indianapolis, Indiana). A23187 and La + were added just prior to

the addition of Ca-45. One mM P042- was present in the pH buffer
(0.5 mM KH2P04 + 0.5 mM NazHPO4) of the Carter's medium replacing
HEPES-PIPES. Ca-45 uptake was measured during a one hour incubation
in Carter's medium in the presence of 7.5 mM La3+ or 2 pM A23187
(dissolved in EtOH); the uptake of Ca-45 in 2% EtOH-Carter's also

was monitored. Since the calcium and phosphate transport systems

may be coupled, uptake in phosphate-buffered and HEPES-PIPES-buffered

Carter's media were determined after a two week incubation with 50

Spirostomum; wheat seeds and bacteria were added to allow the

 

Spirostomum to feed. The filtration and counting procedures were

 

as above.

Pharmacological Agents

 

Several agents which affect calcium-dependent processes were

examined for behavioral effects on Spirostomum. These included

 

caffeine, lanthanum, verapamil, A23187, serotonin, tubocurarine, and

decamethonium.

Caffeine
Caffeine apparently facilitates release of calcium from
vesicles (Bianchi 1961; Weber and Herz 1968; Kitazawa and Endo 1976)

and increases spontaneous contraction rates in Spirostomum (Sleigh

 

1970). Spirostomum were incubated in 5 mM caffeine for 10 minutes

 

18

and then stimulated mechanically for 10 minutes and after a 4 minute

rest stimulated for an additional minute to test recovery.

Lanthanum

La3+ displaces Ca2+ from Ca2+ binding sites and blocks many
processes involving calcium (Takata, Pickard, Lettvin, and Moore
1966; Mela 1968; Weiss 1973; Reed and Bygrave 1974; Larsen and
Vincenzi 1977). Lanthanum in concentrations of 0, 1, 5, 10, 50, and
100 uM in Carter's medium was used to test the responsiveness of
Spirostomum to 2 minutes of mechanical or electrical stimulation
following a 15 minute incubation period. The media were prepared
such that the sum of the lanthanum and calcium concentrations was
always 0.5 mM, the normal calcium concentration of Carter's medium,
i.e., differing amounts of Ca2+ were replaced by La3+. Incubation
times ranging from 6 to 120 minutes in O, 5, 50, and 100 pM

lanthanum-Carter's also were tested.

Verapamil

Spirostomum were incubated for 15 minutes in Carter's medium

 

containing 0, 1, 10, 100, and 1000 ug/ml verapamil (Knoll Industries,
Whippany, New Jersey) (Haas and Haerthfelder 1962), a mammalian
calcium channel blocking agent. This was followed by a 2 minute
mechanical or electrical stimulation period. Also animals were
incubated for periods ranging from 6 to 180 minutes in 1, 25, 50 or

100 ug/ml verapamil-Carter's and tested as above.

19

.A2_3181

A23187, a calcium ionophore, facilitates Ca2+ entry across a
variety of membranes (Reed and Lardy 1972; Pfeiffer, Reed, and Lardy
1974; Desmedt and Hainaut 1976; Wulf and Pohl 1977). A stock solution
of 10 mM A23187 in ethanol was used to make experimental media by
adding aliquots of this solution to normal Carter's medium 5-10
minutes before use. Final A23187 concentrations ranged from 10'6 to

10'4

M; ETOH concentrations were always less than 1% (v/v). Control
media containing the same amount of EtOH but without A23187 also were
tested. After a 15 minute incubation in one of the above solutions

the responsiveness of Spirostomum to mechanical or electrical stimula-

 

tion was measured.

Serotonin

Serotonin may remove habituation in neural systems by facili-
tating calcium influx (Kandel 1976, 1977). One to three hour incuba-
tions in 0.2 mM to 2.0 mM serotonin were used to investigate the

effect of serotonin on responsiveness and habituation in Spirostomum.

 

Tubocurarine and Decamethonium

 

These compounds block mechanosensitive calcium channels in

the ciliate Stentor coeruleus (Wood 1977). Spirostomum were incu-

 

 

bated in 2.0 mM decamethonium Br or 0.l mM d-tubocurarine Cl for one
to three hours followed by 2 minutes of mechanical or electrical

stimulation.

20

Deciliation

 

Deciliation of Spirostomum was intended to supply information

 

on (1) the role of the cilia in mechanotransduction, and (2) the
possible location of voltage-sensitive or mechano-sensitive calcium
channels in the ciliary membrane (Ogura and Takahashi 1976; Dunlap
1977; Ogura and Machemer 1979). Several deciliation procedures were
tried including chloral hydrate (Kuznicki 1963; Kennedy and
Brittingham 1968; Dunlap 1977), dibucaine (Satir, Sale, and Satir
1976; Thompson, Baugh, and Walker 1974), ethanol (Ogura and Machemer
1979), and calcium shock (Watson and Hopkins 1962; Rosenbaum and
Carlson 1969; Everhart 1972). None of these techniques was satis-
factory. All weakened the membrane and caused lysis of Spirostomum
due to loss of membrane integrity. The results with chloral hydrate
confirmed those of Kuznicki (1963) who found that this agent causes

deciliation in Paramecium but not in Spirostomum.

 

 

RESULTS

Analysis of behavioral data focuses on (1) the initial
responsiveness, i.e., contractility during the first minute of stimu-
lation, and (2) the response decrement, i.e., the drop in responsive-
ness between the first and last minutes of stimulation. The initial
response level is interpreted as an indicator of the efficacy of
sensory transduction (when compared to the control). Recovery from
habituation also is studied when possible. Except where indicated
all data are analyzed by analysis of variance and tested with the
least significant difference (LSD) procedure; all significance levels

are for the p < .05 level.

Effects of Calcium

 

The effects of varying free calcium concentration on
responsiveness and response decrement to mechanical and electrical
stimulation are shown in Table 1. Free calcium and magnesium concen-
trations were calculated by successive approximation of the equations
describing the binding kinetics of EGTA, Ca, and Mg. Free calcium

-11

levels ranged from 0.83 x 10 mM to 0.505 mM; free magnesium levels

were depressed in media with excess EGTA (EGTA very selectively binds

Caz+ 2* with-

over MgZ+). The control group (*) contains 0.505 mM Ca
out the addition of EGTA. The decrements in Table 1 were calculated
from the differences in responsiveness between minutes 1 and 2 of

the stimulation period and are expressed as percents of the minute 1

21

 

22

m mm. om. mm «m. on. mom. mom. mm. mmn. mm.

 

 

 

 

 

 

 

m mm. mm. um um. om. mom. mom. i «mom. i
m mm. mm. mm Fm. on. mow. mmN. mm. mom. mm.
m No. mm. mm mm. oo. mom. mmm. i mmN. i
we mm. mm. Pm Ne. mo. mow. moo. u moo. n
mm mm. mm. pm Pm. Fe. mow. moo. om. mom. m.
Fe om. no. mm Fe. no. opp. miopxpp. op. moo. H.
mm mm. mm. mm mm. mm. N..opxom. F—iopxmo. Pm. mom. o.H
peeewmvuea em: ...“: peeswmwg .em: a“: Anny go “new“. Ago «WNW
”movcpumpm pmowcmgomz omen uczom Pouch
cowuomcucoo mo xpwpwnmnoca mcmumsocma Pmpcmspcmnxm

 

.cowpmpaswpm Fmowcuompm new Fmowcmsumz
op acmemguoo mmcoammm new mmmcm>wmcoammm co coppmcpcmocoo sapoqu mmem mo muommmmii.fi m4m<p

23

level. The data of Table 1 are from 12 groups of four Spirostomum

per group for each concentration.

Mechanical Stimulation

 

At the assumed calcium contamination level (5 HM) and below

responsiveness of Spirostomum to mechanical stimulation was lower

 

than to control calcium levels (0.5 mM). The probability of contrac-
tion was reduced for both minutes in these low calcium media. The
amount of habituation (percent decrement) was not affected. The high
level of habituation for the 0.5 mM EGTA + 0.505 mM Ca medium is

probably due to the low initial response level.

Electrical Stimulation.

 

The initial responsiveness of Spirostomum to electrical

 

stimulation in low calcium levels is reduced in the same manner as

for mechanical stimulation (Table 1). The four media with free Ca2+

less than 0.005 mM all decreased the probability of contraction

during the first minute of stimulation. In addition, these low Ca2+

media cause a large decrement; the decrement is as large as that
occurring in the mechanically stimulated control group.

2+ of the Carter's medium to

In summary, reducing free Ca
micromolar and below levels results in decreased initial responsive-
ness to mechanical or electrical stimulation. A large decrement
occurs in response to electrical stimulation that is not present in

media containing 0.255 mM or more (free) Ca2+.

24

Effects of Monovalent and Other Divalent Cations

 

Mechanical Stimulation

 

Table 2 shows the effects of removal, addition, or replace-
ment of various monovalent and divalent cations on responsiveness,

response decrement, and recovery of Spirostomum to mechanical stimula-

 

tion. Increasing Na levels, replacing Na+ with Li+, or replacing K+

with Cs+ or Rb+ had no effect on these parameters for Spirostomum

 

exposed to 11 minutes of mechanical stimulation. Raising (to 10 mM)

or lowering (to 0 mM) the Mg2+ levels did not alter responsiveness of

2

Spirostomum. Replacing Ca2+ with Sr + or Co2+ and partial replacement

2+ 2+

 

of Ca with Ni , Zn2+, or Ba2+ also were without effect. Those
monovalent and divalent cations with significant effects on respon-

siveness of Spirostomum to mechanical stimulation are shown in

 

Figure 2. Increasing [K+] from 0.5 mM (control level) to 10 mM
reduced the initial probability of contraction from 0.76 to 0.30.
Habituation still occurred as responsiveness dropped to near zero

after 11 minutes of mechanical stimulation. Very little recovery

2 2+

occurred in the high [K+] medium. Replacing Ca2+ with Mn + or Ba

also reduced initial responsiveness of Spirostomum. Mn2+-treated

 

animals habituated and recovered to the same levels as the control

2+ 2

group. Spirostomum in 0.5 mM Ba and no Ca + habituated less than

 

the control groups, as well as exhibiting a depressed initial response

level; this did not occur in groups incubated in media containing

0.25 mM Ba2+ and 0.25 mM Ca2+ (partial replacement) indicating that
barium ions exert their effects only in the almost complete absence
of calcium. In contrast, replacing one-half of the Ca2+ with Zn2+

255

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o.mo p.om me. op. op. pm. me. m ++ou \++::p+v
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m.om p.po pm. op. mm. me. op. m ++uu \++mecv
o.vm p.mm pm. on. pm. me. om. m ++uo \++uopao
p.pm m.~m me. mo. op. me. op. m ++au i++aop+v
m.m~ m.op mm. no. em. pm. pp. m ++au i++c~ppv
e.om ¢.mo em. pp. mm. mm. cu. m ++au i++pzp+o
o.nm m.pp mm. op. mm. pm. op. m ++ox :5 op
o.mm N.mo me. pp. op. mm. mm. m mogwi++oz
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xcm>oomx pcoeocooo mp op m N p : ucosuuocp
accuse; ucmoema

 

h “a .l Itiriu .I . IN! “it!

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I.O- )——— Iocluiul Sit-elation % Issuer, ——-|
a u-
: \ 4-0 25.. l ‘3
Z =_fi — . I
g 0.0: \ \ / 0.5.. h.”
E .3. \i =/.::;:;' II“.

0.2-I \\ \:

\o A/OIODI It
I S I; I;

TIME (minutes)

Figure 2.--The effects of monovalent and divalent cations on
responsiveness, response decrement, and recovery to
mechanical stimulation (0.1 Hz for 11 min. 4 min. rest;
1 min. stimulation to test recovery). Responsiveness is
shown on the ordinate as probability of contraction.
Data are plotted from Table 2. Standard errors (SEM)
ranged from .01-.l2 responsiveness units.

(0.25 mM) almost completely eliminated habituation. Complete
replacement of calcium by zinc was not possible due to the toxicity

of higher [Zn2+].

Electrical Stimulation

 

The effects of monovalent and divalent cations on responsive-

ness, response decrement, and recovery of Spirostomum to electrical

 

stimulation are shown in Table 3. As with mechanical stimulation
replacing Na+ with Li+ or K+ with Cs+ or Rb+ had no effect on
responsiveness. Raising [Na+] or [K+] to 10 mM depressed initial
responsiveness and this reduced response level was maintained during

the 11 minute stimulation period (Figure 3). Initial response

227

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++uo :5 me. + ++pz IE mo.p+v

Espums m.coucoo up ucouom mouapnoc cop umcpp-\p o
*

 

 

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28

1.0 q

 

 

1
Electrical Sthelatlee 1 Recovery —4

i
Z

 

 

 

 

°"" \ ~10..- In:
:/
,__s:::::::::::°—-______‘-~_o
02- \oua v
o ' I. 1;

TIME (minutes)

Figure 3.--The effects of monovalent cations on responsiveness,
response decrement, and recovery to electrical stimula-
tion. Stimulation and response parameters are the same
as in Figure 2. Data are plotted from Table 3. Standard
errors (SEM) ranged from .04-.O7 units.

levels were not affected by partial or complete replacement of Ca2+

by Ni2+, Zn2+, Ba2+, Sr2+, C02+, or Mn2+; replacement of Ca2+ with

Ba2+ or Mn2+ reduced contractility of Spirostomum but not signifi-

 

cantly. Raising [MgZ+] to l0 mM depressed responsiveness to electri-
cal stimulation in a manner similar to Na+ and K+ (Figure 4).

Although they had no significant effect on initial probability

2 2+

of contraction, replacement of Ca + with Ba or Sr2+ and partial

2+ with Zn2+ or Ba2+ resulted in a response decre-

2+ +

replacement of Ca
ment to electrical stimulation (Figure 4; data for 0.25 mM Ba

0.25 mM Caz+ 2+J

not shown). This result also was found in low [Ca

Carter's media (Table 1). Spirostomum in control Carter's do not

 

show a decrement to repeated electrical stimulation (Table 3,

Figures 3 and 4).

29

p
o
O
J

 

Electrical Stilllatiu % louver, ——|
/>
‘ a

 

 

 

 

)5

9'
p
n

i
s.///
y/

o ..5 I. S!"
; II II II”
0.25 ml lo”

° 0.5 II In”

 

 

5
TIME (minutes)

Figure 4.--The effects of divalent cations on responsiveness,
response decrement, and recovery to electrical stimula-
tion. Stimulation and response parameters are the same
as in Figure 2. Data are plotted from Table 3. Standard
errors (SEM) ranged from .03-.11 units.

In summary, initial responsiveness to mechanical stimulation
was decreased by high [K+] and by replacing Ca2+ with Ba2+ or Mn2+.
For electrical stimulation raising [Na+], [K+], or [MgZ+] reduced
the initial probability of contraction. The amount of habituation
was reduced by partial replacement of Ca2+ with Zn2+ and by complete
replacement of Ca2+ with Ba2+; partial replacement with Ba2+ had no
effect. A response decrement occurred during electrical stimulation
in media where Ba2+ or Sr2+ completely replaced Ca2+ and in media

2 2+

with partial replacement of Ca + by Ba2+ or Zn This effect is

similar to that seen in low [Ca2+] media.

3O

Ca-45 Uptake Studies

 

Uptake of Ca-45 during 2 to 60 minutes of mechanical,
electrical, or no (control) stimulation is shown in Table 4. The
uptake is given as picomoles of calcium incorporated per animal.

Each figure is based on 2-8 repetitions. Both stimulated and unstimu-

lated Spirostomum show significant uptake (or binding) in the first

 

two minutes of exposure of Ca-45. This very fast binding also can be

seen by first filtering Spirostomum and then filtering 0.5 ml Ca-45

 

Carter's medium through the millipore apparatus; the uptake with
this procedure is not as much as the 2 minute uptake but is substan-
tially higher than the non-specific filter binding component. The

data for 2-30 minutes of incubation is for 50 Spirostomum per sample

 

and the 60 minute incubation data is from 500 animals per sample.
The 60 minute incubation was in low calcium Carter's medium (approxi-
mately 6 pM Ca) to increase sensitivity by increasing the labeled to

unlabeled calcium ratio. Only electrical stimulation for 30-60

TABLE 4.--Uptake of Ca-45 in Stimulated and Unstimulated Spirostomum.

 

 

Ca-45 UPTAKE (pmole/animal)
Incubation Time (minutes)

 

 

2 5 10 30 60
Control 0.64 0.58 0.58 1.02 0.50
Mechanical 0.54 0.38 0.65 1.42 0.53

Electrical 0.74 0.45 0.78 1.58 1.41

 

31

minutes produced Ca-45 uptake that was significantly higher than
control levels. Although the amount of Ca-45 uptake during 10-60
minutes of mechanical stimulation is higher than the control, the
difference is not statistically significant. Table 4 also shows that
lowering the extracellular calcium reduces total calcium uptake in

both stimulated and unstimulated Spirostomum.

 

Table 5 shows Ca-45 uptake by Spirostomum during one hour of

 

incubation in the calcium ionophore A23187 or in LaCl3. Each measure-
ment is the mean of 3-6 repetitions. Filter binding Ca-45 levels are
due to non-specific adsorption to the filter. Incubation in 2% EtOH
(the ionophore solvent) slightly but not significantly increases
uptake compared to control levels. Incubation in 2 uM A23187 sig-
nificantly increases Ca-45 incorporation during the one hour incuba—
tion period. Both 2% EtOH and 2 uM A23187 in 2% EtOH increase spon-

taneous contraction rates in Spirostomum. Incubation in 7.5 mM

 

LaCl-Carter's for one hour reduces Ca-45 uptake to a level lower than
the filter binding. However, this figure is not statistically dif-

ferent from the control. Calcium uptake in unstimulated Spirostomum

 

also depends on phosphate levels; a two week incubation in phosphate-
buffered Carter's medium resulted in three times the Ca-45 uptake as
a HEPES-PIPES-buffered medium (data not shown).

Thus calcium uptake or binding occurred very quickly in

stimulated and unstimulated Spirostomum. Electrical stimulation

 

resulted in increased calcium uptake compared to a control and the

amount of uptake was dependent on the extracellular [Ca2+]. A23187

 

and P042“ increased Ca-45 uptake in unstimulated Spirostomum while

32

TABLE 5.--Effects of A23187 and Lanthanum on Ca-45 Uptake in
Unstimulated Spirostomum.

 

 

 

Treatment Ca-45 Uptake (CPM i SEM)
Filter binding 600 t 64
Control 715 i 88
EtOH (2% v/v) 842 1 82
A23187 (2 pM in 2% EtOH) 1443 i 363
LaCl3 (7.5 mM) 463 i 143

 

La3+ may have affected non-specific filter binding as well as

incorporation into the animal.

Effects of Pharmacological Agents

 

Figure 5 gives the effects of incubating Spirostomum in 5 mM

 

caffeine on responsiveness to mechanical stimulation. Initial
responsiveness is not affected and the caffeine-treated animals do
not habituate during the 10 minute stimulation period. After 10
minutes of stimulation the control response is significantly lower
than the caffeine-treated level (p < .05, Mann-Whitney, U, two-
tailed). Caffeine also increases spontaneous contraction rates in

Spirostomum.

 

Spirostomum were unable to survive in Lanthanum concentrations

 

greater than 10 mM. A 15 minute incubation in concentrations ranging
from 10 to 100 uM lanthanum lowered the probability of contraction to

mechanical stimulation for both minutes 1 and 2 of the stimulation

33

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0.4 '
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—— lode-led Sit-elation 1' locum ——|
I l r
0 5 10 35

THE (minutes)

Figure 5.--The effect of caffeine on responsiveness, response decre-
ment, and recovery to mechanical stimulation (0.1 Hz for
10 min.; 4 min. rest; 1 min. stimulation to test recovery).
Spirostomum were incubated in 5 mM caffeine for 10
minutes prior to testing. Responsiveness is plotted as
probability of contraction. The vertical bars represent
the standard errors of the means. Control data are from
9 groups of 4 Spirostomum per group; caffeine data are
from 4 groups.

 

 

period (Figure 6); the responsiveness at 10 and 100 uM are signifi-
cantly lower than the control (0 uM La3+) for both minutes.

Responses in 1 or 5 uM lanthanum-Carter's were not significantly
different from control levels for either minute. Increasing lanthanum
concentration reduced initial responsiveness to electrical stimulation
only at the 10 pH level (Figure 7). The difference in responsiveness
between minutes 1 and 2 is not significant, i.e., no habituation or
decrement to electrical stimulation occurs at any La3+ concentration.

Figure 8 shows that the amount of habituation to 2 minutes of

34

 

 

125 - I f
In
on
3 1
m
5
5,- “ uncommon.
g srmuurlow W“- 2
25 -
' —'I-Yll l l— ' I |
o 1 10 100

LANTHANUM coucenrnnrlou (um)

Figure 6.--The responsiveness to 2 minutes of mechanical stimulation
as a function of lanthanum concentration (pM). The prob-
ability is expressed as a percent of the control group's
(0 uM La) response to the first minute of stimulation
(percent of control). Note that the abscissa has a
logarithmic scale. Data are from 5 groups of Spirostomum
per group for each concentration.

mechanical stimulation increased at lanthanum concentrations of 10-
100 uM. Increasing verapamil (a calcium channel blocker) concentra-
tion also increased habituation levels between minutes 1 and 2 of
mechanical stimulation (Figure 9); 100 pM verapamil produced signs
of toxicity in Spirostomum.

Only incubation in 100 uM lanthanum significantly affected
responsiveness during a 120 minute incubation. Figure 10 shows the
responsiveness during the first minute of mechanical or electrical

stimulation for Spirostomum in 100 pM La3+ at various incubation

 

35

 

 

 

 

125.
T \
1oo- inJ
W : 4
In ‘ '
2 .,// /\} min. 1
u: - .. ‘
a 75 /_\} min. 2
3 “sermon.
2 so- srmuurion
3
I
as-
o 1 10 100

LANTNANUM CONCENTRATION (AIM)

Figure 7.--The responsiveness to 2 minutes of electrical stimulation
as a function of lanthanum concentration (11M). The
ordinate units are the same as shown in Figure 6. Data
is from 5 groups of 4 Spirostomum per group for each
concentration.

 

times. The response to electrical stimulation decreased quickly and
recovered faster than the response to mechanical stimulation. The
response levels of mechanically and electrically stimulated

Spirostomum at 6, 60, and 90 minutes are significantly different.

 

No significant effects of tubocurarine, decamethonium, or

serotonin on responsiveness or response decrement in Spirostomum to

 

mechanical or electrical stimulation were observed after 1-3 hours

of incubation in each of these compounds.

36

 

i:
%40- I I] 1 l/\}/1
g... I " I I

 

 

0 J-‘r—IP . . .
OT 1 10
LANTNANUM CONCENTRATION (AIM)

'

l
10M)

Figure 8.--The percent decrement to mechanical stimulation as a
function of lanthanum concentration (uM) for the data
of Figure 6. The percent decrement is calculated by
subtracting the probability of contraction for minute 2
from that for minute 1 and expressing the result as a
percent of the response for minute 1.

In summary, caffeine eliminated habituation of Spirostomum

 

to mechanical stimulation. Lanthanum decreased initial responsive-
ness to mechanical stimulation and increased habituation levels.
Verapamil also increased habituation levels. Incubation in 100 uM
La3+ showed that response to electrical stimulation drops quickly
and recovers; responsiveness to mechanical stimulation dropped and

recovered more slowly.

37

 

 

goon
gco- /
5 /'I
gzo- {/1
o —.—f . H . -

I
O . 1 IO IOO
VERAPAMIL CONCENTRATION (AIS/ml)

Figure 9.--The percent decrement to mechanical stimulation as a
function of verapamil concentration (pg/ml). The percent
decrement is calculated as in Figure 8. Data is from 5
groups of 4 Spirostomum per group for each concentration.

 

38

 

 

 

 

120-
0.1 III" LANTNANUM (min. 1)

a 100 'l J ------- I ......... i “06;.
u: Inec .
2: I
In
2
2 so-
8
B

ICC)-

1i l l I I I
O 30 60 90 120

INCUBATION TIME (minutes)

Figure 10.--The responsiveness to one minute of mechanical or
electrical stimulation as a function of incubation time
in 100 uM lanthanum. The probability of contraction is
expressed as a percent of control as in Figure 6. Data
is from 9 groups of 4 Spirostomum per group for each
incubation time.

 

DISCUSSION

The results implicate calcium in both transduction and
response decrement to mechanical and electrical stimulation. It is
not surprising that calcium is involved in the excitability and

behavior of Spirostomum since calcium plays a role in the excita-

 

bility of many organisms, particularly invertebrates (Brink 1954;
Hagiwara and Naka 1964; Hagiwara 1975; Hidalgo, Luxoro, and ROjas
1979; Chiarandini, Sanchez, and Stefani 1980; Nelson, Young, and
Gardner 1980). A requirement for calcium by excitable tissue was
first noticed by Ringer (1883) in studies on heart muscle.

Based on studies in the ciliates Paramecium (Naitoh and

 

Eckert 1969; Machemer and Ogura 1979; Ogura and Machemer 1979, 1980)
and Stentor (Wood 1980, personal communication) it was expected that
in Spirostomum calcium would carry currents related to both mechano—
and electro-reception through separate channel structures in the
somatic and ciliary membranes respectively, and further, that habitua-
tion to repeated mechanical stimulation would be the result of a
decreased calcium entry through the mechanoreceptor channel. A model
was developed based on these and other studies (Eisenstein, Lovell,
Reep, Barraco, and Brunder 1980) and was subsequently used to design

the experiments reported here.

39

4O

Transduction

 

The presence of micromolar levels of Ca2+ in the extracellular
medium is necessary for the transduction of electrical and mechanical

stimuli in Spirostomum (Table 1). Responsiveness is reduced to both

 

modes of stimulation when the free calcium concentration is below
about 5 pH. The effects are not correlated with total [Ca] or [EGTA]
or with the ratio of [Ca] to [M9]; in some systems the Ca-EGTA complex

2+ and thus total calcium is an important vari-

may act like free Ca
able in such preparations (Sarkadi, Schubert, and Gardos 1979). The
presence of a 'threshold' effect suggests that calcium acts by a
membrane-binding mechanism rather than by passive channel conductance
since an influx mechanism would most likely show a greater dependence
on the calcium gradient present across the membrane. In disagreement

with the present study Fabczak (1974) reported responsiveness of

Spirostomum was unchanged after a short incubation in an EGTA-CaCl
7

 

solution with free [Ca2+] of 10' M. However, there were no other

cations present in the test solution while the media used in the
present experiments contained Na+, K+, and Mg2+ in addition to Ca2+;
this may account for the observed difference. Removal of excitability
by lowering extracellular calcium also is seen in other systems,
e.g., barnacle muscle (Hidalgo et al. 1979).

Since most membranes are impermeable to EGTA (Weber, Herz,
and Reiss 1966; Van Breemen and Van Breemen 1969; Baker 1972; Wheeler
and Weiss 1979), EGTA must exert its effect by buffering extracellular

calcium or by altering calcium binding to the membrane (Schlatz and

Marinetti 1972; Wheeler and Weiss 1979). Although the use of EGTA

41

to remove membrane bound calcium was not satisfactory as a quanti-

fiable technique with Spirostomum, the experiments did suggest that

 

EGTA was able to chelate membrane-bound Ca-45. If calcium binding

is important one might expect to see media with excess EGTA have a
greater effect on responsiveness than media without excess EGTA;

this result was not observed. In muscle studies EGTA levels of
80-90 mM are required to reduce excitability (Barrett and Barrett
1978); however, the presence of 'protected' extracellular calcium
compartments, e.g., the t-tubule system, must be considered in muscle
systems. Thus it appears that EGTA acts primarily by buffering
calcium levels and no behavioral effect of EGTA on membrane-bound
calcium was indicated.

The present resuls agree with those of Osborn et al. (1973b)
for the effects of EGTA on electrical but not mechanical stimulation.
Osborn et al. (1973b) found that 0.5 mM Ca + 0.25 mM EGTA reduced
initial responsiveness to mechanical but not to electrical stimulation
when compared to the control (0.5 mM Ca). The present experiment
showed no change to either stimulation mode (compare rows 6 and 7
in Table 1). However, Osborn's experiments were repeated and the
results reproduced by this author. The methods used differed slightly

for the two experiments in that Osborn stimulated Spirostomum just

 

prior to EGTA addition (as a pre-EGTA control) (see Figure 3 in

Osborn et al. 1973b).

2+

Raising the concentrations of Na+, K+, or Mg of the

Carter's medium decreased responsiveness of Spirostomum to electrical

 

stimulation (Table 3). This confirms studies by Fabczak (1974) who

42

found that increasing concentrations of Ba, Ni, Ca, Mg, K, or Na

from 0.5 to 10 mM reduced responsiveness of Spirostomum to electrical

 

stimulation. The present results also agree with Fabczak (1974) in
finding no evidence of Donnan equilibrium effects (between binding

2+ . . . . . .
of Ca and monovalent 1on5) as 15 seen 1n Paramec1um where c1l1ary

 

reversal is controlled by the ratio [Ca2+]§/[K+] (Jahn 1962; Naitoh
and Yasamasu 1967; Naitoh and Eckert 1968; Naitoh 1973). Fabczak
finds the effectiveness of cations in reducing responsiveness
follows Na < K < Mg while the present experiments order the effects
as Na < Mg < K. Fabczak (1974) proposes that the cations exert
their effect by acting on the electrical double layer at the cell
surface. Preliminary evidence suggests that reducing [Na] to 0.2 mM

(from 2.0 mM) increased responsiveness of Spirostomum to electrical

 

stimulation. Oubain also increases contractility in Spirostomum

 

(Sleigh 1970), perhaps through a [Na+] effect on the Na-Ca pump

leading to increased availability of intracellular calcium (Baker,
Blaustein, Hodgkin, and Steenhardt 1967). Reducing [K+] or [MgZ+]
had no effect on responsiveness to electrical stimulation. Extra-
cellular Cs+, which may block K+ channels (Gay and Stanfield 1977;

Adelman and French 1978), did not alter responsiveness of Spirostomum

 

suggesting no major role for K+ in electrotransduction. Perhaps a
better indication of the apparent role of each ion would be observed
if all ions, especially Cs+, Rb+, and Li+, also were tested at higher

levels.

2+ 2+ 2+ 2+

Partial or complete replacement of Ca with Ni , Zn , Ba ,

Sr2+, CoZ+, or Mn2+ had no effect on responsiveness to electrical

43

stimulation. In contrast to the present results Fabczak (1974)

found incubation of Spirostomum in tris buffer and BaCl greatly

 

2
decreased excitability. Again the lack of additional cations in

Fabczak's test media makes comparisons difficult. In the present
study Ba reduced response levels but not significantly. Sleigh
(1970) found that NiCl2 also reduced responsiveness of Spirostomum
to electrical stimulation; although similar toxicity was noted in

2* with

the two studies no effect of replacing a small amount of Ca
Ni2+ (0.05 mM) was observed. Reducing calcium concentrations
reduced responsiveness to electrical stimulation but no effects of
calcium replacement were observed; this is possibly due to a non-
specific ion effect on membrane potential, e.g., raising ion concen-
trations may hyperpolarize the membrane (Machemer 1976).
Responsiveness to mechanical stimulation behaves very dif-
ferently to changes in the ionic composition of Carter's medium
(Table 2). Raising [KI] to 10 mM still inhibits the contractile
response but neither high [Na+] nor [M92+] affect the contractility

of Spirostomum to mechanical stimulation. Replacing Ca2+ with Ba2+

2

 

or Mn + did lower responsiveness. Mn2+ (and C02+) often blocks

calcium currents (Baker, Meves, and Ridgeway 1973; Baker and Glitsch

2+, as well as Sr2+, is usually a good

1975; Kryshtal 1976). Ba
calcium substitute (Meves 1968; Hagiwara, Fukuda and Eaton 1974;

Hagiwara 1975; Adams and Gage 1980; Kerrick, Malenick, Hoar, Potter,
Coby, Pocinwong, and Fischer 1980; Miledi and Parker 1980). La3+
decreased responsiveness to mechanical stimulation and to a lesser

extent to electrical stimulation; the La3+ results do not discriminate

44

between uptake and binding since La3+ may block both processes (Van
Breemen and Deweer 1970; Van Breemen, Farinas, Gerba, and McNaughton
1972; Van Breemen, Farinas, Casteels, Gerba, Wuytack, and Deth 1973;
Weiss 1973; Freeman and Daniel 1973; Martin and Richardson 1979;
Kurzinger, Stadkus, and Hamprecht 1980). Responsiveness to either
mode of stimulation does not correlate with ionic radii or hydrated
ion sizes as might be expected for a channel-influx mechanism where
channel selectivity is based on size.

The results discussed above suggest that calcium and other

cations may affect the excitability of Spirostomum by altering the
2+

 

binding of Ca or by some other membrane-linked mechanism; passive
channel permeability also may involve ion binding. However, the

large uptake of Ca-45 during 60 minutes of electrical stimulation
(Table 4) supports a calcium influx mechanism. The uptake is much
higher than control levels and is much too large to represent membrane
binding. Mechanical stimulation did not result in significant Ca-45
uptake. However, the responsiveness in the Ca-45 test solutions was
very low due to limitations of the stimulation apparatus; the prob-
abilty of contraction was about 0.50 during the first minute and
habituated to a level of 0.15 by minute 10 of the mechanical stimula-
tion period. Response levels to electrical stimulation were around
0.70 for l0 minutes of testing. If a calcium current flows during
transduction then a much higher calcium influx would occur during
electrical stimulation in the apparatus used in this study.

2+

Electrical stimulation also causes the release of much more Ca

internally than mechanical stimulation (Osborn et al. 1973b) and

45

could possibly produce a larger calcium current. Entry of enough
calcium during transduction to elicit contraction is not necessary
since a calcium-induced calcium release (Endo, Tanaka, and Ogawa 1970;
Ford and Podolsky 1970) may occur; such an amplification mechanism in

Spirostomum has been suggested by Hawkes and Holberton (l974). Much

 

of the calcium entering during transduction may be expelled following

contraction; in Paramecium Ca-45 influx studies must be performed at

 

0°C to reduce Ca-45 efflux (Browning and Nelson 1976). It is diffi-
cult to imagine that the Ca-45 entering would not mix with and label

intracellularly sequestered calcium stores in Spirostomum; in

 

Paramecium, where uptake is into the cilia and no calcium stores are

 

present, complete efflux may be possible. In summary, calcium uptake
occurs during electrical stimulation and uptake associated with
mechanotransduction is not conclusively disproved.

Stimulus specificity and habituation to only one stimulation
mode (mechanical) support the separation of the electro- and mechano-
transducer sites (Osborn 1971). This separation is sustained by the
differential concentration effects (Figures 6 and 7) and differential
time effects (Figure l0) of lanthanum on responsiveness to electrical
and mechanical stimulation. Separate sites also are suggested by the
different effects of altering the ionic composition of the Carter's
medium, i.e., ion effects depend on the stimulation mode. The
mechano- and electro-transduction pathways may be completely separate

except for calcium activation of contraction.

46

Response Decrement
Before discussing the role of calcium in response decrement
in Spirostomum a comparison of the use of the terms 'response decre-
ment' and 'habituation' should be made. Although it is difficult to

separate habituation of Spirostomum to mechanical stimulation and

 

receptor fatigue (since no dishabituation occurs in Spirostomum

 

[Eisenstein and Peretz 1973]), the response to electrical stimulation
rules out effector or motor fatigue. Thus electrical stimulation acts
somewhat as a control to test the state of the effector. When a
decrement in responsiveness to electrical stimulation occurs a con-
trol situation is not as available. It would be advantageous to see
what response level would occur to mechanical stimulation after a
decrement-producing electrical stimulation period; this experiment
could be tried in the presence of Zn2+ where no decrease in initial
levels is seen and a response decrement to electrical stimulation
occurs. Because of the difficulty in assessing the state of the

contractile apparatus of Spirostomum after a decrement to electrical

 

stimulation, the term habituation is being reserved to describe the
response decrement to mechanical stimulation only and habituation is
hypothesized to occur at the mechanoreceptor site.

Although it was expected that habituation levels would be
very susceptible to low calcium concentrations, i.e., assuming that
habituation is due to decreased calcium entry (Kandel 1976, 1978;
Klein and Kandel 1980; Wood personal communication), reducing [Ca2+]
had little effect on habituation during 2 minutes of mechanical

stimulation (Table 1). Perhaps after 10 minutes of stimulation a

47

difference in the decrement levels would have appeared. Replacing

Caz 2+ 2+ 2+ 2

+ with Ni , Zn , Ba , Sr +, C02+, or Mn2+ was expected to

increase habituation levels since at least one or two of the ions

should block Ca2+ currents or interfere with calcium binding. Nearly

2+ and Zn2+, removed habitua-

2+

the opposite was observed—~two ions, Ba
tion. Applewhite and Davis (l969) report that Mg and Mn2+ block
habituation in the absence of Ca2+; this effect was not tested and
cannot be evaluated. Also, those ions that affected transduction
did not necessarily alter the presence or amount of the observed
response decrement suggesting that responsiveness and decrement
levels are not correlated.

The Ba2+ and Zn2+ results suggest a possible habituation

mechanism. Calcium current in Paramecium and in Aplysia inactivates

 

itself through an effect of the intracellar [Ca2+] on the calcium
permeability system (Brehm and Eckert 1978, 1979; Eckert and Brehm
1979; Eckert and Tillotson 1979; Tillotson and Eckert 1979; Brehm,

2+ blocks Ca2+

Eckert and Tillotson 1980; Marban and Tsien 1981). Ba
inactivation of the Ca2+ channel, possibly by binding to the channel
macromolecule (Eckert and Brehm 1979; Adams and Gage 1980; Tsuda,
Morimoto, and Brown 1981). Thus, if calcium-induced calcium inactiva-
tion was the mechanism of habituation, Ba2+ would be expected to
remove habituation and this was observed. However, Ba2+ may block

K leakage currents which would result in the same effect; in such
experiments K+ currents are minimized by the use of K+-channel

blockers. Ba2+ is known to block K+ channels (Bezanilla and Armstrong

1972; Satow and Kung 1976; Valeev, Magura, and Zamekhovskii 1977;

48

Hagiwara et al. 1974; Standen and Stanfield 1978; Armstrong and
Taylor 1980; Eaton and Brodwick 1980; Schwindt and Grill 1980).
Zn2+ is a powerful enzyme activator (Cotton and Wilkinson 1972) and
could very easily regulate permeability controlling proteins; Zn2+
is often involved in the regulation of phosphorylating reactions
(Cotton and Wilkinson 1972) which may mediate ionic permeability
(Hirata and Axelrod 1980). Zn2+ also appears to facilitate Ca2+
binding to sperm cells based on chlortetracycline fluorescence
measurements (Nelson et al. 1980). Naitoh (1969) proposes that Zn2+
may interact with ATP and the contractile system of the ciliary

apparatus in Paramecium. The action of Zn2+ in sperm cells and

 

possibly in Spirostomum may be related to the activation of calcium-

 

dependent metalloenzymes which may control permeability. Applewhite
and Davis (l969) also suggest that metalloenzymes are involved in
habituation. Zn2+ may be a useful tool in the further molecular
characterization of habituation since Zn2+ (1) is available as an
isotope, (2) can be chemically identified and estimated, and (3) can
be removed from membranes by EDTA (Brierley, Knight, and Settlemire
1967).

Caffeine, which facilitates calcium release in muscle (Frank
1962; Weber and Herz 1968; Thorpe and Seeman 1971; Langer 1973;
Kitazawa and Endo 1976; Fujimoto, Vanamoto, Kuba, Morita, and Kate

1980), removed habituation in Spirostomum suggesting that decreased

 

availability of intracellular calcium may be the basis of habituation.
The actions of La3+ and verapamil, which increased habituation levels

(Figures 8 and 9), also support a decreased calcium entry mechanism

49

for habituation. If not for the possible toxicity of verapamil at
100 ug/ml (Figure 9) a calcium flux mechanism would be strongly
supported since verapamil blocks calcium channels (Crankshaw, Janis,
and Daniel 1977; Hoeschen 1977; Bondi 1978) and does not affect
calcium binding. Similarly, caffeine probably has little effect on
membrane binding of calcium.

Another surprising result was that reducing extracellular
calcium levels caused a large decrement to electrical stimulation
within the second minute of stimulation (Table 1). Thus, extra-
cellular calcium is necessary to maintain responsiveness to electrical

2+ w

stimulation. A decrement to electrical stimulation in 5 uM Ca as

observed by Sleigh (l970) by applying approximately 20 stimuli during
200 minutes of incubation. Forty micromolar [Ca2+] and above did

not result in a decrement (Sleigh 1970) suggesting that the 'cutoff'

for this effect is between 5 and 40 OM free Ca2+.

2+ 2+ 2+

The partial replacement of Ca with Zn or Ba or the

2+

2+
complete replacement of Ca2+ with Ba or Sr also resulted in a

response decrement to electrical stimulation. The reasons why these

2+, C02+, and Mn2+ (normally Ca2+

ions cause decrements, and why Ni
blockers) did not, are not clear. These decrements to electrical
stimulation may be caused by a reduced availability of extracellular
calcium.

The above results are compatible with a model where separate

mechanoreceptor and voltage-sensitive calcium channels exist in the

membrane of Spirostomum and where habituation is due to a decrease

 

in the mechanoreceptor calcium current. However, the experiments

50

performed here do not conclusively discriminate between calcium
fluxes, i.e., uptake, and calcium binding as the mechanism. The
binding mechanism is suggested by (l) the effects of low [Ca2+] on
transduction, (2) the inability to observe Ca-45 uptake during
mechanical stimulation, and (3) the effects of ions are not based

on ionic sizes. Calcium uptake mechanisms are strongly supported

by (l) the Ca-45 uptake during electrical stimulation, (2) data from

the ciliates Paramecium and Stentor, and (3) the presence of a

 

mechanism (Ca-induced Ca inactivation) which appears to fulfill the
requirements of a model for habituation. Mechanoreception is based

on calcium influx in the related ciliates Paramecium and Stentor

 

(Ogura and Machemer 1979, 1980; Machemer and Ogura 1979; Wood 1980);
however, membrane binding is also extremely important in the regula-

tion of ciliary reversal in Paramecium (Jahn 1962; Naitoh and

 

Yasamasu 1967; Naitoh and Eckert 1968; Naitoh 1973). More work will
be necessary to discriminate between these mechanisms. For example,
chlortetracycline fluorescence measurements would be an excellent
initial experiment to assess the role of calcium binding in the
effects observed. Calcium may affect membrane potential by binding
to the membrane surface (McLaughin, Szabo, and Eisenman 1971;
DiFrancesco and McNaughton 1979); this is also a possible transducer
mechanism (Gingell 1971) and could be involved in controlling the
membrane permeability to calcium and other ions (Kim and Sanders
1979).

Although Ca2+ appears to be the controlling ion it is impor-

tant to identify the excitation current carrying ion because calcium

51

may activate the conductance of other ions, i.e., K+, Na+, or MgZ+.

Ca2+-dependent Na+ and K+ currents have been reported in a variety of
organisms (Romero and Whittam 1971; Meech 1972; Peach 1975; Lew and
Ferreira 1976; Kostyuk, Kryshtal, and Tsyndrenko 1977; Schulz and Heil
1979; Hotson and Prince 1980; Woolum and Gorman 1981) including

Paramecium (Satow and Kung 1978; Saimi and Kung 1980). Mechanorecep-

 

tion in Aplysia statocyst cells is apparently mediated by sodium cur-
rents (Gallin and Wiederhold 1977). The present study does not rule
out the possibility that Na+ or K+ ions are directly involved in
habituation or transduction.

In evaluation of Ca-45 uptake studies co-transport of P042-

also must be considered. Spirostomum actively accumulates both ions

 

(Jones 1967; Balcerzak 1978). The present results show that Ca-45
uptake was three times higher in media containing PO42'. Another

ciliate Tetrahymena pyriformis also actively accumulates Ca2+ in the

 

presence of P042' (Rosenberg and Munk 1969). The calcium and phos-
phate apparently are stored as hydroxyapatite which is not likely to
be readily available for the activation of the contractile filaments.
Such non-exciting calcium uptake components must be considered in
interpreting the role of calcium in studies involving Ca-45.

The use of La3+ and EGTA to estimate calcium binding was not

3+

completely successful with Spirostomum. The action of La and EGTA

 

on non-specific filter binding (see Table 5) interfere with the

3* and EGTA did appear

ability to observe a decrease in binding. La
to reduce binding of Ca-45 but not in a quantifiable manner with the

filtration technique used.

52

Many differences were noted in comparing the results of the

present studies with studies on other ciliates, i.e., Paramecium and

 

Stentor. Differences with Paramecium are not surprising since the

 

calcium currents and mechanoreceptor potentials are related to a

different behavior from that observed in Spirostomum--in Paramecium

 

 

the behavior studied is ciliary reversal while in Spirostomum we

 

study contraction and habituation. However, Stentor exhibits the same

behavior pattern as Spirostomum, i.e., habituation of the contractile

 

response to mechanical but not electrical stimulation. One major

 

difference between Spirostomum and Stentor is that responsiveness to
mechanical stimulation in Stentor is blocked by tubocurarine and
decamethonium (Wood 1975, 1977)--a result that was not observed for

Spirostomum. Also, Co2+ blocks mechanoreceptor calcium currents in

 

Stentor (Wood 1980) but has no effect on responsiveness in

Spirostomum. Thus, the likelihood of identical habituation mechan-

 

isms in these two ciliates is doubtful.

Finally, in recent years it has become apparent that many of
the regulatory functions carried out by calcium ions may be mediated
by calmodulin (Cheung 1980; Roufogalis 1980; Klee, Crouch, and
Richman 1980; O'Callaghan, Dunn, and Lovenberg 1980; Blum, Hayes,
Jamieson, and Vanaman 1980; Wolff, Cook, Goldhammer, and Berkowitz
1980), a small protein whose structure is highly conserved throughout

2+ and translates Ca2+

phylogeny (Vanaman 1980). Calmodulin binds Ca
levels into a large variety of cellular responses. Calmodulin and
calcium may control the cyclic nucleotide dependent kinases that

have been isolated from protozoans (Kuznicki, Kuznicki, and

53

Drabikowski 1979; Blum et al. 1980; Lewis and Nelson 1980); these
kinases could be involved in regulating membrane permeability.
Interestingly, Zn2+ may act by inhibiting calmodulin (Brewer, Aster,
Knutsen, and Kruckeberg 1979); removal of habituation by Zn2+ could
be due to inhibition of calmodulin action on a calcium permeability-
controlling protein, suggesting that habituation is regulated by
calmodulin.

In summary, the results indicate:

1. Extracellular Ca2+ is necessary for transduction of
mechanical and electrical stimuli.

2. Various effects of ions on transduction were observed;
these effects were not related to hydrated or non-
hydrated ionic radii. The different ion effects suggest
different sites for mechano- and electro-transduction.

3. Remova of habituation to mechanical stimulation by Ba2+
and Zn + suggests a role for calcium in the habituation
process. However, reducing extracellular calcium levels
had little effect on the amount of habituation during
two minutes of stimulation.

4. Maintenance of responsiveness to electrical stimulation
requires the presence of extracellular calcium; a response
decrement occurs when the availability of extracellular
calcium is reduced. ‘

5. The experiments provide support for both binding and
uptake as mechanisms for calcium action.

6. The results are compatible with a heuristic model in
which two separate calcium channels are responsible for
sensory transduction and habituation is due to a reduced
calcium current through the mechanoreceptor channel.

The habituation process may involve regulation of calcium
permeability by a metalloenzyme which regulates mechano-
receptor current based on intracellular calcium levels.

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54

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