GLT‘QTR CYELETY EN 'E'HE CiLEATEB PRGTGZCEAN, SMRQSYCSMUM :WBEGUUM B‘fi DUSTAEV' OSEQRN MCH 36%?‘3 STATE {if-{WERSWX’ E971; - j v - ~ . 'VVW"$“..~'L'SM-,” 1““ ,3 .' I ~ . . r~ g). . . . u-“ _ L 1 —" " .. ‘2}, ' ""‘41-\ i o I. , ‘ Mia-again brace Unwersity This is to certify that the thesis entitled CONTRACTILITY IN THE CILIATED PROTOZOAN, SPIROSTOMUM AMBIGUUM presented by Dustan Osborn has been accepted towards fulfillment of the requirements for Ph. D. ;degree in Biophysics 6% we; Iflhknpumuun Date ”/7/77’; _ / / 0-7639 ABSTRACT CONTRACTILITY IN THE CILIATED PROTOZOAN, SPIROSTOMUM AMBIGUUM BY Dustan Osborn Contractility in the ciliated protozoan, Spirostomum ambiguum, has been investigated. The probability of contraction to repetitive mechanical and electrical stimulation has been characterized and a model proposed to account for the differences in effector response to each stimulus. The model suggests that the site of action of the mechanical stimulus is at a mechano-receptor, which in turn, activates the contractile system. The electrical stimulus is suggested to by- pass the sensory receptor and to directly activate the contractile fibers. Data have been collected consistent with the predictions of the model. The distribution of calcium and other elements in Spirostomum has been mapped intracellularly using the electron microprobe with special interest in the proximity of calcium to the contractile appa- ratus. Three calcium stores have been characterized: 1) endoplasmic hydroxyapatite deposits, 2) calcium contained within mitochondria, and 3) calcium associated with the microfilaments assumed to be the con- tractile fibers of the organism. Calcium involvement in the contractile process has been stud- ied. Using radio-labelled calcium, the binding of calcium to Dustan Osborn intracellular membranes has been characterized as a function of stimu- lation of the contractile system. Modifying the amount of bound cal- cium with a calcium chelator, EGTA, changes in the probability of con- traction to repetitive mechanical and electrical stimulation have been characterized. These data have been found to be consistent with the proposed model of mechano-receptor and contractile response systems. It is suggested that unicellular organisms may prove useful in the study of sensory receptors and their correlated effector response systems. CONTRACTILITY IN THE CILIATED PROTOZOAN, SPIROSTOMUM AMBIGUUM By a.( 1* fl DustanLOsborn A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1971 2;) 2 (a (" '/ L1 3 / Dedicated to the American taxpayer who financed this research. 11 ACKNOWLEDGMENTS A heart-felt thanks is acknowledged to the students of the laboratory of Professor Eisenstein: H. J. Blair, T. C. Hamilton, J. C. Hsung, N. Novelly, N. St. Pierre, J. Thomas, and J. Thompson. Also, thanks to Dr. D. Hoy. Technical assistance from.the following is acknowledged: Dr. Paul Rasmussen and Mr. Vivion Shull, Electron Microprobe Laboratory; Dr. Gordon Spink, Electron Microscopy. The members of the thesis committee were: Dr. Neil Band, Zoology, Dr. Gabor Kemeny, Biophysics, Dr. Eloise Kuntz, Biophysics, Dr. James Trosko, Human Development. Their assistance has been appreciated. The comments of Dr. David McConnell also were appreci- ated. The chairman of the committee and director of this thesis research has been Professor Edward M. Eisenstein. His vigilance, insight and kindness have allowed the fruition of this research. The author was supported by NIH training grant GM-OthZ-OG. The research was carried out under NIH general research support grant 5-SOl-RR-05656-04 to the College of Human Medicine, MH grant 1 R03 MH 18570-01 and NSF grant GB 23371 to E. M. Eisenstein. The author also ‘wishes to thank Dr. John Nellor of the Office of Research Development for providing funds for the use of the electron microprobe. 111 TABLE OF CONTENTS I. The Effects of Mechanical and Electrical Stimulation on Habituation in the Ciliated Protozoan, Spirostomumgambiguum page Introduction..................................... 1 Methods.......................................... 2 1. Incubation.................................. 2 2. Stimulation of Spirostomum.................. 5 5. Observation of Behavior..................... 4 Results.......................................... 5 Discussion.......................................11 Bibliography.....................................22 II. The Distribution of Calcium and Other Elements in the Ciliated Protozoan, Spirostomum ambiggum Introduction.................................... 24 Methods......................................... 24 Results......................................... 25 Discussion...................................... 28 Bibliography.................................... )6 III. The Involvement of Calcium in Contractility in the Cilieted Protozoan, Spirostomum ambiggg! IntrOdUCtioneeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee ’8 M.th°d.OOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOO... 39 iv R..u1t.........0000000......OOOOOOOOOOOOOOOOOOOO #0 Di.°u.s1°n.....OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 1+4 BibliographyOOOOOOOOOOOOOOOOOOOO0.00000000000000 57 LIST OF FIGURES I. The Effects of Mechanical and Electrical Stimulation on Habituation in the Ciliated Protozoan, Spirostomum ambiguum page Figuretf 1. Schematic diagram of stimulation ‘pparatu..............0...00.000.000.00... 14 2. The probability of contraction to mechanical stimulation (1 stimulus per 10 seconds) versus time of ltimulation.............o................. 15 5. The probability of contraction to electrical stimulation (1 2 ms. biphasic pulse per 10 seconds) versus time of stimulation....................... 16 4. The probability of contraction to electrical stimulation (1 2 ms. biphasic pulse per 5 seconds) versus time of stimulation....................... 17 5. The probability of contraction versus time for stimuli delivered once per 10 seconds (mechanical-electrical- m.cnan1°‘1)eeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 18 6. The probability of contraction versus time for stimuli delivered once per 10 seconds(electrical-mechanical- electrical)............................... 19 7. The probability of contraction to mechanical stimulation (1 stimulus per 10 seconds) versus time of stimulation (glucose & 3-O-methyl- glucose).................................. 20 8. Model for the activation of the contraCtil. .y.t.m...........O'COOOOOOOOCO 21 Vi II. The Distribution of Calcium and Other Elements in the Ciliated Protozoan, gpirostomum ambiggum . page Figure # 1. Whole animal scan of §pirostomum for c.101m.....0...0.COICOOOOOOCCOOCOOOOO 50 2. Cross section through Spirostomum......... 51 5. Cross section through the cortical and subcortical region of §pirostomum..... 51 4A. Transverse section through Spirostomum. SCCnI for C‘1c1UI Qnd ph°.ph°ru.eeeeeeeeee 52 AB. Line scans for calcium and phosphorus .CTO.‘ trIDU'Orl. .OCtioneeeeeeeeeeeeeeeee 55 5. Transverse section through the cortical region of Spirostomum. Scans for calcium and phosphorus.................... 54 5. Signal averaged line scans through cortical region for magnesium, calcium, ‘nd ironOOOOOOCOOOOOOCOOOOOOIOOOOOOOOOOOOO 55 III. The Involvement of Calcium in Contractility in the Ciliated Protozoan, Spirostomum ambiggum Figure2i 1. lbs per cent decrease in membrane bound 50a for A) mechanically stimulated and B) electrically stimulated §pirostomum.... 50 2. Per cent membrane bound 450a versus EGTA concentration for cellular homogenates of Spirostomum after )0 Minute. incub.t1°neeeeeeeeeeeeeeeeeeeee 51 5. The mean probability of contraction to mechanical stimuli (scra)................. 52 k. The mean probability of contraction to .IOCtr1°.1 Still-111 (EGT‘)eeeeeeeeeeeeeeeee 5) 5. Change in.prebability of contraction versus EGTA concentration................. 5“ 6. Photographs of Spirostomum before and after electrical sthmulation.............. 55 7. Line scans for calcium in transverse sections through the contractile vacuole........... 56 THE EFFECTS OF MECHANICAL AND ELECTRICAL STIMULATION ON HABITUATION IN THE CILIATED PROTOZOAN, SPIROSTOMUM AMBIGUUH INTRODUCTION: Severe limitations are imposed on the researcher of the chemistry of learning in higher organisms. The enormous number of neuronal elements, the complex circuitry through which sensory input must course within the central nervous system, the difficulty of establishing the exact behavior modification to be correlated with molecular changes combine to make research on the molecular aspects of learning one of the most refractory to understanding in the field of biology. One approach to the study of molecular changes associated with learning is to define a simpler system, one that is amenable to separation of variables yet still shows an acceptable repertoire of behavioral change (3, 6, 7). The protozoans appear promising for such studies since intercellular interactions are eliminated from consideration (1, 4, 8, 10, ll, 12, 17). In the study reported here, the ciliated protozoan, Spirostomun ambiggum, has been chosen. This cigar-shaped organism, one to three ‘millimeters in length, lives in an aqueous environment and locomotion through its surroundings is achieved by use of cilia functioning as cars to propel the Spirostomum along its long axis. Should the organism experience a noxious stimulus, the power stroke of the ciliary beat may be reversed to stop locomotion in a forward direction and backwards swimming ensues (10). Alternatively, Spirostomum may respond to the stimulus by contracting along its major axis to about one half of its extended length. We have chosen to investigate changes in the contrac- tion probability of Spirostonrm to repetitive stimuli known to produce contractions, i. e., electrical shocks and mechanical vibrations (2, 13, 14). The intent of this study is to segregate the behavioral manifestations of contraction to stimulation into those aspects associa- ted with activation of the contractile apparatus from those associated with stimulus transduction. METHODS: 1. Incubation of Spirostomum Spirostoma were purchased from Connecticut Valley Biologi- cal Supply Company, Southampton, Massachusetts. Animals'were cultured in an aqueous medium containing chloride salts of potassium, sodium, magnesium.and calcium.in glass doubly distilled water as shown (5): Carter's medium for culturing Spirostormsm ambiggum (modified by using a phosphate buffer to maintain constant pH): KCl 0.5 mM NaCl 2.0" MgCl2 0.2 " CaCl2 0.5 " I1 KHZPOA 1.0 NazHPOt, l . 0 " The pH of the medium‘was maintained at pH 7.2 by buffering with mono- and di-basic phosphate salts. The pH of the buffered medium remained constant over periods of several weeks. A single animal was placed in a four inch diameter Petri dish to which 40 mls. of medium had been added. Heat-killed wheat seeds were added to support bacteria upon which Spirostomum feeds. All cultures were grown at room temperature (24-260 C) with continual exposure to room light. Within two or three weeks a large colony of Spirostoma were thriving. All experiments reported are based upon cultures generated from this one animal. 2. Stimulation of Spirostomum Mechanical: The mechanical stimulation is defined as a transitory vibration of the aqueous medium produced by striking a micro- scope slide with the central metal core of a solenoid (Figure #1). The microscope slide contains a well 16 mm in diameter in which Spirostomum are placed. The metal core of the solenoid is spring driven to the slide when current flow to the solenoid is halted. The core is then electro- magnetically retracted. By varying the height from which the central core of the solenoid was dropped, the disturbance to the medium could be increased or decreased. The more intense the stimulus, the greater the probability of contraction. An intensity of disturbance was chosen that resulted in a high probability of contraction to the first several stimuli. Electrical: Platinum paint baked onto the walls of the slide well functioned as electrodes for electrical stimulation. A Grass 8-8 stimulator was employed to deliver a two ms. biphasic pulse at defined intervals. The probability of contraction to the electrical shock was proportional to the potential of the biphasic pulse. Potentials were empirically chosen to produce the desired initial probability of con- traction. Animals generally were stimulated for ten minutes once every ten seconds and the number of Spirostoma contracting to the stimulus was recorded. After this stimulation period the animals often were allowed to rest five minutes after which they were stimulated for an additional minute to obtain a measure of retention of any change in the probability of contraction to the previous ten minute stimulation period. Data from a number of separate runs for each particular experiment were combined to obtain group trends. 3. Observation of behavior Usually, one to four_§pirostoma were placed in the well of the microscope slide for stimulation. The contraction probability was observed through a stereo dissecting microscope at a magnification of about 20 diameters. For each stimulus delivered, it was observed whether the_§pirostomum contracted (9). In some cases, the position of the organism within the well was recorded. A typical experiment consisted of placing four_§pirostoma in the microscope slide well with enough medium to fill the well level to the top (approximately 0.4 m1). Transfer of animals from the culture bowl to the slide was accomplished using a Pasteur pipette and rubber bulb. Care was taken to minimize disturbance to the animals. Once placed in the slide, the stimulation period was not begun for five or ten minutes. This delay was required since the handling procedure during the transfer from culture bowl to stimula- tion slide lowered the initial probability of contraction. However, the initial probability of contraction increased ten to fifteen percent for animals allowed a five or ten minute rest period prior to mechanical stimulation. Allowing the animals to rest for longer periods than ten minutes did not further increase their initial probability of contraction to mechanical stimulation. The presence of a rest period before electri- cal stimulation had no effect on initial responsiveness compared to animals that were not given a rest period prior to electrical stimula- tion. RESULTS: Pour Spirostoma were placed in the stimulation slide. After a five minute rest period they were mechanically stimulated for ten mdnutes at a frequency of one stimulus per ten seconds. The mean probability of contraction in response to mechanical stimulation for ten groups of Spirostoma is presented in figure #2. During the first minute of stimulation Spirostoma contracted to 751 of the stimuli. During mdnute ten, they contracted to 17% of the stimuli. The decrease in probability of contraction from minute one to minute ten is termed habituation and for this group was 581 (p-0.01, Wilcoxon matched pairs signed, one tailed). To determine whether the decrease in probability of contraction was due to an irreversible damage to animals, one minute of stimulation was given after a five minute rest period. During this second period of stimulation, the animals contracted to 461 of the delivered stimuli, demonstrating that the decrement in probability of contraction is reversible. Habituation to mechanical stimulation also occurred in bacteria-free distilled water. Einastowski has reported a famdly of curves for the retention of the habituated state as a function of the interval between the initial stimulation period and a second stimulation period (13, 14). We have confirmed his published results that the greatest return occurs in the first few minutes after stimulation with little evidence of retention at 45 minutes. Using a 2 ms. 100 volt biphasic electric pulse, animals contracted in a manner and with a time course that are visually indistinguishable from the contraction elicited by mechanical stimulation. Figure #3 shows the mean contraction probability of five groups of four Spirostoma per group to electrical stimuli delivered once every ten sec- onds for ten minutes. By varying the potential of the biphasic pulse between 40 and 100 volts, the probability of contraction to the first minute of stimulation could be systematically lowered or elevated. For each stimulus potential of electric shock used, the animals did not show a decrease in the probability of contraction during the stimulation period. Increasing the stimulation period to 15 minutes did not result in a decrement in probability of contraction. However, if the frequency of stimulation was increased to one per five seconds and the duration of the stimulation period also extended to 15 minutes, a decrease in con- traction probability between minutes 1 and 15 is observed (Figure #4). (p-0.02, Wilcoxon matched pairs test, one tailed). The probability of contraction is greater when the long axis of the animal is perpendicular to the electrodes, than it is when this axis is parallel to the electrodes (15). However, no change in orientation was observed to account for the response decrement seen. These data show that it is possible to produce a decrement in probability of contraction using electrical stimuli but a greater fre- quency of stimulation is required for a decrement compared to mechanical stimulation. Also, the time course for the decrement varies for the two forms of stimulation. The greatest amount of response decrement to mechanical stimuli occurs during the first few minutes of stimulation. However, in the case of electrical stimulation, the greatest decrement in probability of contraction occurs in the latter minutes of the stimula- tion period. In addition, recovery is much slower for electrical compared to mechanical stimulation. (50 percent in 15 minutes; Compare figures 2 and 4). Often, towards the and of a 15 mdnute period of electrical stim- ulation, the animals appeared to move more slowly and have enlarged con- tractile vacuoles. Some damage may occur with the higher frequency electrical stimulation. To examine further the contributions of repeated stimuli and repeated contractions on the response decrement observed to mechanical stimulation, the following experiment utilizing both mechanical and electrical stimulation was performed. One minute of mechanical stimulation was given to a group of four Spirostoma to determine their initial probability of contraction. Then electrical stimuli were delivered for minutes two through nine. Finally, one minute of mechanical stimulation was again delivered to note any change in the probability of contraction between minutes one and ten. All stimuli were presented at ten second intervals. A 100 V, 2 ms. biphasic electrical stimulus that produced a 70% probability of con- traction.was employed for curve A of Figure #5. Curve B of Figure #5 employed a 30 V electrical stimulus that produced a 10% probability of contraction. Each curve represents the mean probability of contraction for ten groups. In both cases, the probability of contraction at the tenth minute of stimulation was less than that at the first minute of stimulation (5). (p-0.05, Wilcoxon matched—pairs test, one tailed). A greater decrement occurred for the electrical stimulus that produced the greater number of contractions. (p-0.05, sign test, one tailed). However, neither shock group showed as much decrement in probability of contraction during the tenth minute of stimulation as those animals given only mechanical stimuli (dotted curve from Figure 5 for comparison). The response level in minute ten of the group shown in Figure #2 was significantly less than for the group in Figure #5 curve A. (p-0.01. sign test, one tailed). Animals given one minute of mechanical stimula- tion and then allowed to rest eight mdnutes before being given an additional minute of mechanical stimulation generally showed no change or an increase in responsiveness between the first and tenth minute. Thus the decrement seen between minutes one and ten was not due to time. These data indicate that the number of contractions may play a role in the magnitude of response decrement to mechanical stimulation (cf figures 5A and SB). This is somewhat confounded by the observation that different levels of electrical stimulation were also used to pro- duce the differences in number of contractions. What is definite though is that the nature of the stimulus and its site of action play a role in the magnitude of response decrement as shown by the fact that animals given ten minutes of mechanical stimulation (Figure #2) show an overall level of number of contractions for minutes two through nine between those of Figure #5A and SB yet show a signifi- cantly lower response level at minute ten. The previous experiment demonstrated that repeated electrical stimulation once per ten seconds, while not producing response decrement itself, nevertheless, led to a decrement in the response to a mechanical stirmrlus. A corollary of this experiment was to examine the effect of repeated mechanical stimulation on the responsiveness to the electrical stimulus. Five groups of four Spirostoma‘were each given one minute of electrical stimulation followed by ten minutes of mechanical stimulation. Immediately following mechanical stimulation, electrical stimuli were again delivered for one minute. All stimuli were presented ten seconds apart (Figure #6). A decrement in the probability of contraction to electrical stimulation did not occur following habituation to the mechanical stimulus. Although the contraction probability has been characterized for both mechanical and electrical stimulation and for combinations of the two, the mechanismm involved to produce habituation have not been elucidated. An initial investigation was undertaken to explore the possibility that habituation of contraction to mechanical stimulation resulted as a consequence of the depletion of metabolic stores (16). Spirostoma'were incubated in 10 mM glucose for thirty minutes before stimulation. Figure #7 curve B shows the mean contraction probability for ten groups of four Spirostoma per group incubated in 10 mM glucose. The probability of contraction to stimli in the first minute was 64% and was 361 in the tenth minute. (p80.02, Wilcoxon matched-pairs signed, one tailed). Compared to animals not incubated in glucose (curve A) glucose incubation resulted in less habituation, that is, a significantly smaller decrement between minutes one and ten in contraction probability with stimulation. (p-0.01 Mananhitney U test, one tailed). When the experiment was repeated other times, the initial level of contraction to the mechanical stimulus was the same or quite often elevated for animals incubated in glucose compared to those which were not. However, in all cases, the amount of habitua- tion (i.e., the decrement between minutes one and ten) to the mechani- cal stimulus was reduced with glucose incubation. To determine whether glucose was taken up by Spirostoma, groups of 100 animals were incubated in uniformly labeled 14-C-g1ucose for 15 mdnutes and collected on millipore filters with 0.3 micron pore size. Samples were counted using BBOT (2,5-bis [2¥(5-tert-Buty1bens- oxasoly117'Thiophene, 4 grams/liter of toluene) as the scintilator on a Packard Tri-Carb liquid scintillation counter. In 15 minutes 5 x 10'8 10 moles of glucose were taken up per Spirostomum. To insure that the label was not incorporated into bacteria in the incubation medium, the Spirostoma were first washed with three changes of fresh bacteria free medium. However, measuring the 14-C02 evolved from the incubation solution revealed no oxidative metabolism of 14-C-glucose by Spirostomum during the same time period. Since glucose was not oxidatively metabolized, it might be argued that the decrease in habituation for animals incubated in glu- cose was due to osmotic or viscosity changes. Therefore, a non- metabolizable substrate, 3-0-methyl glucose was substituted for glucose in the incubation medium. Figure #7 curve C shows the mean contraction probability for ten groups of four Spirostoma per group given mechanical stimuli once every ten seconds for ten minutes after incubating 30 minutes in 3-0-methyl glucose. The probability of contraction to stimuli delivered during the first minute is less than that delivered during the first minute to control animals (curve A), (p-0.01, sign test, one tailed). In the tenth minute, the stimulation produced a contraction probability the same as that for control animals. The results indicate that the lesser habituation in the presence of glucose compared to Spirostoma stimulated in the absence of glucose must not be due to an increase in viscosity of the medium or an increase in the osmolarity of the incubation medium preventing a decrement in responsive- ness since animals incubated in 3-0-methyl glucose showed the same probability of contraction as control animals in the tenth minute of stimulation. However, further work is required to elaborate the contri- bution of the depletion of metabolic stores to the behavioral changes observed to mechanical and electrical stimulation. 11 DISCUSSION: Two stimuli, mechanical and electrical, have quite different effects upon the probability of contraction of Spirostomum over time. Electrical shock stimuli from 40 to 100 volts delivered ten seconds apart do not produce habituation over a ten minute stimulation period. Mechanical stimulation results in a decrement in the probability of contraction with time. The mode of the stimulus may activate the con- tractile processes in different fashions for the two types of stimuli as suggested by the data in Figures #2 and #3. It is possible to observe a decrement in the probability of contraction to electrical stimuli if the stimulation frequency is increased to one stimulus per five seconds for 15 minutes. Comparing the form of the decrement over time for mechanically stimulated Spirostoma (Figure #2) with that for electrical stimulation at the stimulation frequency of one per five seconds (Figure #4), the greatest decrement to mechanical stimu- lation occurs early in the stimulation period, then asymptotes to a nearly constant probability of contraction during minutes seven through ten. To electrical stimulation, the probability of contraction remains essentially constant for the first several minutes, with the greatest amount of response decrement from minutes 10 to 15. Whatever mechanism is responsible for the decrement in probability of contraction to the mechanical stimulus, that mechanism manifests itself near the beginning of the stimulation period, while the mechanism responsible for the decrement to the electric shock delivered once every five seconds is not evidenced until much later in the stimulation period. A model to account for the data is shown in Figure #8 (17). A sensory receptor and contractile system are assumed to be linked in a linear and unidirectional manner. When the receptor is activated, it 12 may lead to contraction. The different modes of stimulation are suggested to activate difference sites: the mechanical stimulus activates a receptor sensitive to vibratory stimuli which in turn initiates contraction; the electrical stimulus activates the con- tractile mechanism. The data in Figure #5 suggest that the electri- cal stimulus may also directly affect the mechano-receptor (arrow a, Figure #8). Alternatively, the contraction elicited by electrical stimulation.mmy physically distort the mechano-receptor which in turn becomes less excitable to subsequent mechanical stimuli (arrow b, Figure #8). The data in Figure #6 indicate that mechanical stimulation does not affect the site of electrical activation. The decrease in probability of contraction to the mechanical stimulus could then be attributed to an increasing refractoriness of the sensory receptor. The asymptotic level of probability of contraction observed from minute seven to minute ten (Figure 2) reflects the maximal refractori- ness of that receptor for the given frequency and intensity of stimu- lation used. The eventual decrement in contraction probability to the electric shock delivered once per five seconds for 15 minutes probably indicates changes associated with the contractile processes-- perhaps even some damage. Incubation of Spirostoma in glucose prior to mechanical stimulation partially inhibits the final habituation level achieved in‘minute ten. However, substituting a nonwmetabolizable substrate, 3-0-methyl glucose, does not inhibit the habituation level achieved, although the initial probability of contraction is reduced. An interpretation consistent with these data is that glucose provides 15 the metabolic requirements necessary to maintain the receptor mechanism in the active state, that is, reduces the refractoriness of the receptor. Changes in the probability of contraction to mechanical and electrical stimulation have been explained by a model postulating a set of processes associated with mechano-transduction (receptor) and a set of processes associated with contraction (effector). The study shows that a unicellular organism can be used to investigate both sensory receptor and effector mechanisms, and changes in the response characteristics of each. 14 2d 5 Figure #1. Schematic diagram of stimulation apparatus. The mechanical stirmrlus is achieved by spring driving the central metal core (B) of a solenoid (A) to the edge of a microscope slide C containing a well (D). The slide rests upon rubber supports (E) and is held in place by a Plexiglas frame (0) and post (F). For electrical stimlation, electrodes of platinum paint have been baked onto the walls of the well. 15 5 o .75 in :5 Mean Probability of Contraction io u: 12345678910 16 Minutes Figure #2. The probability of contraction to mechanical stimu- lation (1 stimulus per 10 seconds) versus time of stimulation. Data from 10 groups of 4 Spirostomum per group are plotted. Probability of con- traction is plotted as the mean average for stimuli delivered any given minute. One minute of stimulation was given after a 5 minute rest period following the 10 minute stimulation period. 16 1.00 d .50 .25 Mean Probability of Contraction 12345678910 Minutes Figure #3. The probability of contraction to electrical stimulation (one 2 ms. biphasic pulse per 10 seconds) versus time of stimulation. The mean average of 5 groups of 4 Spirostoma per group are plotted. Electrical potential - 100 volts. 17 ‘5 o .75 .50 Mean Probability of Contract. io u- 5 10 15 30 Minutes Figure #4. The probability of contraction to electrical stimulation (one 2 ms. biphasic pulse per 5 seconds) versus time of stimlation. The mean average of 5 groups of 4 Spirostoma per group are plotted. Electrical potential - 100 volts. One minute of stimu- lation was given after a 15 minute rest period following the 15 minute stimulation period. 18 1.00 N u: .50 .25 Mean Probability of Contraction 12345678910 16 Minutes Figure #5. The probability of contraction versus time for stimuli delivered once per 10 seconds. The first minute gives the contraction probability to mechanical stimli (M). Minutes 2 through 9 show the contraction probability to electrical stimuli (E) (curve A, 2 mm. biphasic pulse at 100 volts; curve B at 40 volts). The tenth minute plots the contraction probability to mechanical stimuli (M). One minute of mechanical stimuli were delivered 5 minutes after the 10 minute stimulation period (Minute 16). Each curve is the mean average for 10 groups of 4 Spirostoma per group. For comparison, the data from figure 2 (10 minutes of mechanical stimulation) are shown by the dotted line. 19 1.00 N In in o .25 Mean Probability of Contraction 123456789101112 Minutes Figure #6. The probability of contraction versus time for stimuli delivered once per 10 seconds. The first minute gives the contraction probability for electrical stimuli. Minutes 2 through 11 show the contraction probability for mechanical stimuli and minute 12 is a final minute of electrical stimulation. The plot is the mean average for 5 groups of 4 Spirostoma per group. The electrical stimulus consisted of a 2 mm. 100 volt shock. 20 1.00 N u: in o .25 Mean Probability of Contraction 12345678910 16 Minutes Figure #7. The probability of contraction to mechanical stirmrlation (l stimulus per 10 seconds) versus time of stimlation. Curve A control (data from figure 2); Curve B, following a 30 minute incurbation period in 10 mM glucose: Curve C, following a 30 minute incurbation period in 10 mM 3-0-mthy1 glucose. Each curve is the mean average of 10 groups with 4 Spirostoma per group. One minute of stirmrlation was given after a 5 minute rest period following the stimulation periods. 21 T/ 2 «b- - Stimulus T Contraction Figure #8. Model for the activation of the contractile system. T is the mechano-sensory receptor and C the effector mechanism, the contractile process. Mechanical stimuli are shown to directly affect the T component while electrical stimuli are represented to activate the C component directly with a residual effect on the T component, via a or b. BIBLIOGRAPHY 3. 7. 10. ll. 12. BIBLIOGRAPHY Andrivon, C. Le Phenome du Renversement Ciliare chez les Protozo- aires. Annee Biologigu_e 8, 99, 1969. Applewhite, P. B., F. T. Gardner, and E. Lapan. Physiology of Habituation Learning in a Protozoan. 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A Technique for Continuous Recording of Behavior of a Single Protozoan. American Zoologist 2 (4), 1969. Jennings, H. S. The Psychology of a Protozoan. Ag. g. Psychol. Q, 503, 1899. Jensen, D. D. More on "Learning" in Paramecia. Science E5, 191, 1957. Katz, M. S. and W. A. Deterline. Apparent Learning in the Parame- cium. J. Cg. Physiol. Psychol. 51, 243, 1958. l3. 14. 15. 16. 17. 23 Kinastowski, W. The Problem of "Learning" in Spirostomum.ambiguum. Acta Protogoologica l_(24), 223, 1963. (trans. from.German) Kinastowski, W. The Influence of Mechanical Stimuli on the Con- tractility of Spirostomum.ambiggum. Acta Protozoologica‘l (23), 201, 1963. Kinosita, Haruo. Effect of Change in Orientation on the Electrical Excitability in Paramecium. g, Fac. Sci. Imp. 2, Tokyo, 4_(4), 189, 1936. Vivier, E., B. Legrand, and A. Petitprez. Recherches Cytochimiques et Ultrastructurales sur des Inclusions Polysaccharidiques et Caliques du Spirostome; leurs Relations avec la Contractilite. Protistologiza l, 145, 1969. Wood, D. C. Behavioral and Electrophysiological Studies of the Response Decrement Produced by Repeated Mechanical Stimulation in the Protozoan, Stentor coeruleus. Ph.D. Dissertation, U. Michigan, 1968. THE DISTRIBUTION OF CALCIUM AND OTHER ELEMENTS IN THE CILIATED PEOTOZOAN, SPIROSTOMUM AMBIGUUM INTRODUCTION: The presence of high concentrations of calcium in the ciliated protozoan, Spirostomum ambm, has been reported by Pautard, Jones, and Bien & Preston (10, 8, 1). Calcium is accumulated and then stored as the phosphate salt, hydroxyapatite (3 Cac(P04)2.Ca(OH)2) (10). The stored calcium has been suggested to provide greater rigidity for burrowing and to be reversibly solubilized as metabolic needs may require (1). Spirostomum.possesses the ability to respond to mechanical, electrical, or chemdcal stimuli, by contracting along its long axis to about 8 of its resting body length. Ettienne has reported that calcium is released from a stored form for the initiation of contraction (6). The analogy with mscle is apparent since calcium is a necessary pre- requisite for contraction in both systems. This study reports on the distribution of calcium and its relationship to the contractile apparatus using the electron mdcroprobe analyzer (4, 12, 13) in conjunction with transmission electron microscopy. METHODS: Spirostoma, purchased from Connecticut Valley Biological Supply, were cultured at room temperature in Carter’s medium buffered to pH 7.2 (3). Boiled wheat seeds were added to the medium to support 24 25 bacterial growth upon which Spirostomum feeds. Subcultures were begun from single cells and division and growth allowed until the population reached a maximal level. Cells were fixed in 2% glutaraldehyde in Carter's medium, or cacodylate buffer, pH 7.4, for two hours followed by post-fixation in 12 osmium tetroxide for thirty minutes. Cacodylate buffer was used to avoid precipitation of phosphate salts of calcium which often occurs in alkaline conditions. Dehydration was carried out using increasing concentrations of ethanol in water followed by propylene oxide. The cells were embedded in Epon and sectioned on a Sorval MT-2 ultramicro- tome. Sections were stained with uranyl acetate and lead citrate. Electron microscopy was carried out on a Phillips 100. The two micron thick sections through Spirostomum were mounted on quartz glass for observation with an Applied Research Laboratory electron microprobe analyzer. Thick sections were required rather than silver thin sections to insure that enough calcium was present to be detected by the microprobe analyzer. Whole cells were also prepared for electron microprobe analysis by freezing in liquid nitrogen followed by 1yophy- lization and mounting on carbon discs (2). The microprobe was operated at 15 kV with a 0.5 micron beam diameter. RESULTS: Scans of the whole animal for calcium reveal a greater con- centration in the anterior portion of Spirostomum than in the posterior region which contains the contractile vacuole (Figure la). Figure #1b shows the secondary electron image of a 1yophylized Spirostomum. The freeze drying technique preserves the organism in the contracted state with the outline of the contractile vacuole apparent. Scans of the 26 same animal for other cations gave detectable amounts of magnesium and potassium, but sodium was not present in high enough concentrations to be observed. These data show that calcium is present in sufficient concentrations to be detected by microprobe analysis. Yagiu and Shigenaka (15) suggest from electron microscOpic studies that the contractile material consists of a subcortical layer of microfilaments (5, 7, 9). Figure #2 shows a cross section through Spirostomum. The subcortical microfilaments are apparent central to the somatic grooves of the cortex and adjacent to a distribution of mitochondria. The fused cilia comprising the membranelle are present with a series of mictotubules coursing from the ciliary basal bodies into the endoplasm. Figure #3 shows the cortical region at greater magnification. The large number of mitochondria associated with this region are presumably related to the high metabolic activity associated with contraction and ciliary beating. A cross section two microns thick was scanned for calcium with the electron microprobe (Figure #4) and a punctate distribution of calcium was evident. If calcium is sequestered as the hydroxyapatite, phosphorus should show a coincident distribution. Within the resolving limits of the microprobe, the co-distribution was complete. A line scan across the section (Figure #4b) shows coincident peaks for calcium and phosphorus. The punctate distribution of calcium is endoplasmic and has been reported by Pautard, and Bien 8 Preston, to be deposited as the phosphate salt, hydroxyapatite (10, 1). However, endoplasmic hydroxy- apatite is probably not the only source of calcium. To determine the most lateral appearance of calcium, figure #5a shows an enlarged section 27 of a two micron thick transverse section through the organism. The distribution of calcium can be superimposed on the secondary electron image (Figure #5b). The punctate distribution is central to the somatic grooves by a distance equivalent to the position of the beginning of the endoplasm. The second scan is that for phosphorus which shows the same distribution as for calcium (Figure #5c). While the dense calcium deposits are endoplasmic, a fainter distribution of calcium and phosphorus is apparent peripheral to the dense deposits. This latter distribution is coincident with the zone containing the microfilaments. A transverse section taken through the posterior portion of the organism transects the contractile vacuole. This region con- tains no endoplasm. yet the cortical region resembles that in more anterior sections. Somatic grooves, mitochondria and the microfila- ments are present. Scanning this region for calcium reveals a faint distribution at the cortical region, but no calcium within the con- tractile vacuole. This observation further supports the contention that calcium can be detected at the region of subcortical microfilaments. As seen in Figures #2 and 3 large numbers of mitochondria are densely distributed lateral to the microfilaments. They occur two to three microns inward from the periphery. The microfilaments form a band central to the mitochondria about four microns inward from the periphery. If the analogy with muscle in higher systems is made, magnesium should be found at the microfilaments since magnesium as well as calcium is involved in the mechanism of muscle function. Since magnesium is not present in a sufficient concentration in the cortical region to be detected by single sweeps of the electron beam, 28 it was necessary to use a signal averager and sum the signals obtained for a number of sweeps of the beam. Both magnesium and calcium peaks are found in the region of the microfilaments (i.e., about 4 mm in from the periphery, Figure #6). Iron was observed in the region of the mitochondria. This is to be expected since they contain large quantities of both heme and non-heme iron associated with the electron transport chain. A profile for iron super-impos.able with that for calcium and magnesium distinguishes that portion of cortical calcium and magnesium contained within mitochondria from that associated with the contractile apparatus. DISCUSSION: The distribution of calcium within Spirostomum is not diffuse, but particulate, supporting the observations reported in the literature that there are calcium sequestering regions within the cytoplasm (l, 10, 14). These endoplasmic inclusions contain calcium as the phosphate salt, hydroxyapatite. Therefore, phosphorus shows the same distribu- as calcium. The suggestion that these calcium stores play a functional role in eliciting muscle contraction led us to investigate the presence of calcium in the vicinity of the contractile fibers. Although the dense punctate distribution of calcium and phosphorus observed in the endoplasm is not seen at the region of the microfilaments, the presence of calcium is detectable. In addition, magnesium was also observed. The involvement of calcium and magnesium in the contractile mechanism would necessarily depend upon their presence near the contractile apparatus. The endoplasmic stores may provide a calcium reserve for contraction, but more immediately accessible calcium is suspected. 29 Large numbers of mitochondria are adjacent to the microfila- ments. They contain iron, calcium and magnesium. Iron was used as the spectroscopic indicator of mitochondria because of the high concentra- tion of cytochromes known to be present in the electron transport chain. The highest calcium peak is not associated with a coincident peak for iron. It occurred at the position of the contractile microfilaments. This study has suggested three calcium stores within Spiros- tomum: l.) endOplasmic hydroxyapatite, 2.) calcium within mitochondria, and 3.) calcium in the vicinity of the contractile microfilaments. Endoplasmic calcium vesicles have been suggested to provide a primitive endoskeleton. These stores may not provide a direct source of calcium for contraction. Instead, it is thought that the cortical distribution of calcium functions as a calcium source for contraction. 50 Figure # 1. Whole animal scan of S irostomum for calcium. A.) x-ray scan for calcium. B.) Secondary electron image. Note less dense distribution of calcium in the region of the contractile vacuole (cv) and the membranelle (m). 3C>a Figure 2. Cross section through §pirostcmum ambiggum showing the subcortical microfilaments (mt) and the membranellar mictotubules (mm) emanating from the endoplasm and approaching the basal bodies of the membranelle. 50001. Figure 5. Cross section through the cortical and sub- cortical region of §pirostomum ambiguum. Cilia (c) emanating from somatic grooves (ejand their basal bodies ch) along with the lateral microtubules (mt) form the kinetosomal system. Mitochondria (M) and found distributed immediately beneath the ectoplasmic ridges (er) and adjacent to the microfilamentous bundles (mf) believed to be the contractile apparatus. 10,000X. 51 32 Figure f 4 A. Transverse section through Spirostanum. Scans for calcium and phosphorus. Arrow indicates position of line scans shown in Figure # h B. 55 Figure 4 B Figure 4 B. Line scans for calcium and phosphorus across transverse section. Height of peaks represents relative magnitude of element scanned. 0) 0.) Bi Figure # 5. Transverse section through the cortical region of gpirostomum. Scans for calcium and phosphorus. Note denser distribution of both in endoplasm (END) and fainter distribution in cortical region (0R). 35 pmn AA AA nmf CM: A V Figure # 6 Signal averaged line scans through cortical region for magnesium, calcium and iron. pm, plasma membrane; M, mitochondrion; mf, microfilaments. Height of peaks for a given element represents relative magnitude. However, relative peak heights of the different elements cannot be compared. BIBLI OGRAPHY BIBLIOGRAPHY Bien, Saul M., and Francis B. Preston, Calcification of Spirostomum ambiguum. .l° Protozool.-l§(2), 251, 1968. Boyde, A. and V. C. Barber, Freeze-drying Methods for the Scanning F1 Electron-microscopical Study of the Protozoan Spirostomum ambiguum and the Statocyst of the Cephalopod Mollusc Loligo ' ‘ vulgaris. .l- Cell Sci. 4, 223, 1969. Carter, L., Ionic Regulation in the Ciliate Spirostomum ambiguum. Iggp. Biol. géfil), 71, 1956. '3 Coleman, J. R., S. M. DeWitt, P. Batt and A. R. Terepka, Electron Probe Analysis of Calcium Distribution During Active Transport in Chick Chorioallantois Membrane. _ Exptl. Cell Res. 6;, 216, 1970. Daniel, Wendell A. and Carl F. T. Mattern, Some Observations of the Structure of the Peristomial Membranelle of Spirostomum ambiguum. .g. Protozool..lg(1), 14, 1965. Ettienne, Earl M., Control of Contractility in_§pirostomum by Dissociated Calcium Ions. g, Gen. Physiol., 29, 168, 1970. 7. Finley, Harold E., Charles A. Brown and Wendell A. Daniel, Electron Microscopy of the Ectoplasm and Infraciliature of §pirostomum ambiguum. .1. Protosool.'ll(2), 264, 1964. 8. Jones, Alick R., Uptake of 45-Calcium by Spirostomum ambiguum. g, Protozool.,'l§(3), 422, 1966. 9. Lehman, W. J., The Structural Elements Responsible for Contraction in the Ciliate Spirostomum. Ph.D. thesis, Princeton University, I969. 10. Pautard, F. G. E., Mydroxyapatite as a Develoymental Feature of Spirostomum ambiguum. Biochemica gt Biophysica Acta, 2;, 33, 1959. ll. Pautard, F. G. E., Calcification in Unicellular Organisms, Biological Calcification, Cellular and Molecular Aspects. ed. Harold Schraer, 105, 1970. 56 12. 13. 14. 15. 57 Schopf, T. J. M. and J. R. Allan, Phylum Ectoprocta, Order Cheilostomata: Microprobe Analysis of Calcium, Magnesium, Strontium, and Phosphorus in Skeletons. Science 169, 289, 1970. Solomon, J. S. and W. L. Baun, Content Mapping with an Electron Microbeam Probe. Ag. Lab. 19, Dec., 1970. Vivier, E., B. Legrand at A. Petitprez, Recherches Cytochimiques et Ultrastructurales sur des Inclusions Polysaccharidiques et Calciques du Spirostome; leurs Relations avec la Con- tractilite. Protistologica l, 145, 1969. Yagiu, Ryoao and Yoshinobu Shigenaka, Electron Microscopy of the Logitudinal Fibrillar Bundle and the Contractile Fibrallar System in Spirostomum ambiguum. ‘J. Protozool. .1913), 364-369, 1963. THE INVOLVEMENT OF CALCIUM IN CONTRACTILITY IN THE CILIATED PROTOZOAN, SPIROSTOMUM AMBIGUUM INTRODUCTION: : {‘m’nj Calcium has been implicated in the contractile system of the ciliated protozoan, Spirostomum ambiguum.(4, ll, 13, 24). Ettienne has demonstrated that calcium is released from a storage form at the onset of contraction (8). Other laboratories have observed the presence of large stores of calcium in the organism (4, 12, 13, 21, 22). We have shown the distribution of calcium at three different sites within Spirostomum: l.) endoplasmic hydroxyapatite, 2.) calcium within mitochondria, and 3.) calcium in the vicinity of the contractile micro- filaments (20). Characteristic of the contractile response of Spirostomum is the initiation of contraction within 10 to 20 msec after stimulation and contraction along the long axis of the organism to about one half of the resting length within 5 msec (9, l4). Re-extension takes place ' during the next k to 2 seconds. When Spirostomum is stimulated by vibrating the aqueous medium in which it swims, the organism responds by the magnitude of the intensity of the stimulus (15, 16). When a train of mechanical stimuli is given, there is a waning in the proba- bility of contraction. This phenomenon is termed habituation and is well documented in the literature (2, 3, 15, 16). Likewise, the probability of contraction to electrical stimuli is determined by the 58 59 intensity (potential) of the shock. We have shown that the sites of action of these two stimuli can be separated behaviorally (19). A model has been proposed that suggests that the mechanical stimulus activates a sensory receptor which in turn, activates the contractile apparatus, while the electrical stimulus bypasses the sensory receptor and directly activates the contractile processes. Since calcium has been implicated at the contractile site, we have studied the involve- ment of calcium in contraction responses elicited by mechanical and electrical stimuli. METHODS: Spirostoma were purchased from Connecticut Valley Biological Supply, Southampton, Massachusetts and cultured in Carter's medium as has previously been described (5, 19). The organisms were stimulated to contract either by agitating the medium in which they were swimming (mechanical stimulation) or by applying a two msec biphasic electric pulse across platinum electrodes on opposite sides of the well containing the organisms. A detailed description of the stimulation procedure appears elsewhere (19). Control and electrically stimulated animals were fixed for electron microprobe analysis in 21 glutaraledhyde in Carter's medium followed by post-fixation in 1% osmium tetroxide. Dehydration was carried out in increasing concentrations of alcohol followed by embedding in Epon. Two micron thick sections were cut with glass knives on a Sorval MT-Z ultra-microtome and mounted on quartz glass for microprobe analysis. To incorporate 45-Ca into Spirostoma, animals were grown in Carter's medium to which 4S-Ca was added to give an activity of 0.25 micro-curies/ml of medium. Organisms were grown for two weeks in the [+0 labelled medium before they were used for experimentation (12). The total incorporated label was measured by removing groups of 50 Spirostoma from the incubation medium, washing with unlabelled medium and collecting the cells on a millipore filter with 0.3 micron sized pores. The label retained by the filter was counted on a Packard Tri-Carb liquid scintillation counter using BBOT (2,5-bisZS-tert- butylbenzoxazolyl)7 thiophene, 4 grams/liter of toluene) as the 5 scintillator. A second group of 50 Spirostoma were homogenized in a sintered glass homogenizer and the homogenate collected by filtration as above. The calcium retained by the filter after homogenizing Spirostoma is considered the membrane bound calcium while that passing ‘2'!" through the filter the soluble portion. RESULTS: Spirostoma incubated for two weeks in 45-Ca incorporated approximately 280 epm per cell. Approximately 20% of the total incorporated calcium label was retained on the millipore filter after homogenizing Spirostomum. Varying the pore size of the millipore filters from 0.05 microns to 8.0 microns did not alter the amount of labelled calcium retained, indicating the size of the calcium binding membranes to be large enough not to pass through an 8.0 micron pore. Groups of 50 Spirostoma were mechanically stimulated once per ten seconds for ten minutes, then homogenized. Likewise, groups of SO Spirostoma were electrically stimulated either once per ten seconds for ten minutes or once per five seconds for 15 minutes, then homogenized. Spirostoma mechanically stimulated for ten minutes showed the same amount of label retained by the millipore filter as unstimulated Spirostoma (Figure #1 curve A), while Spirostoma stimulated 41 electrically showed 25% less label retained by the filter (Figure #1 curve B). (difference significant at p=0.01, sign test, one tailed), compared to unstimulated organisms electrical stimulation reduces the amount of bound calcium. Stimulating electrically at a frequency of one stimulus per ten seconds for ten minutes or one stimulus per five seconds for 15 minutes produced the same change in bound calcium. The total bound and unbound calcium within Spirostoma, that is, the label .. rm retained by the millipore filter after collecting unhomogenized cells was not reduced by electrical or mechanical stimulation (p-0.11, sign test, one tailed). The data show that measureable amounts of calcium do not leak out of Spirostomum when the organism is stimulated to contract. Incubating previously 45-Ca labelled Spirostoma in varying concentrations of EGTA (ethylene bis(oxyethylene-nitrilo) tetraacetic acid) a calcium chelator, reduces the amount of bound 45-Ca (Figure #2). Groups of ten Spirostoma were homogenized after 30 minutes incubation and the homogenate collected on a millipore filter. Measuring the time course of chelation demonstrated that the decrease in amount of bound calcium was complete within five minutes of incubation. Increasing the concentration of EGTA from 0.1 to 0.5 mM led to a decrease in the amount of bound calcium. Further increasing the EGTA concentration above 0.5 mM did not further reduce the amount of bound calcium. Incubation of Spirostoma in 0.25 mM EGTA reduces their proba- bility of contraction to mechanical stimulation delivered once per five seconds for five minutes (Figure #3) (10). Curve A is the probability of contraction for animals before EGTA incubation. These same animals were left undisturbed for 30 minutes then placed in 0.25 mM EGTA in \. 4-2 Carter's medium for an additional 15 minutes before stimulating a second time. Curve B shows the probability of contraction after such EGTA incubation. The difference in probability of contraction in minute one is significant at p-0.02, sign test, one tailed. Although the initial probability of contraction was reduced in the presence of EGTA both curves tend to the same limit. Control animals not incubated in EGTA were stimulated a second time as described above. The probability of contraction did not differ significantly between the groups; therefore, use of the same animals for both experimental and control groups was justified. The same experiment was repeated using electrical instead of mechanical stimuli. Figure #4 shows the effect of incubation in 0.25 mM EGTA on the probability of contraction to electrical stimuli. For both control and EGTA incubated groups, no habituation was observed, nor was the difference between the curves in minute one significant. The effect of varying the EGTA concentration from 0.1 mM to 1.0 mM on the probability of contraction to the mechanical stimulus was measured. Groups of four Spirostoma were stimulated once per five seconds for a total of 20 stimuli and the probability of contraction recorded. The Spirostoma were then left undisturbed for 30 minutes after which time they were transferred to the appropriate concentration of EGTA in Carter's medium. After an additional 15 minutes the organisms were again mechanically stimulated 20 times and the probability of contraction recorded. Figure #5 plots the difference in probability of contraction between pre- and post- incubation stimulation periods as a function of EGTA concentration. The higher the value on the ordinate, the fewer responses the animals gave following EGTA incubation. For each EGTA concentration tested, nine groups of four Spirostoma per 45 group were used. From 0.1 to 0.5 mM EGTA there is a linear relationship between the decrease in probability of contraction and the concentration of EGTA. However, further increasing the EGTA concentration from 0.5 to 0.75 mM results in a decrease in the difference in contraction probability between pre- and post- incubation stimulation periods. Below 0.75 mM EGTA, no alteration in the animals appearance was visually noted, but above 0.75 mM, ciliary activity and swimming ability were impaired. If Spirostoma are electrically stimulated after incubating in varying concentrations of EGTA, no change in the probability of contraction occurs up to 0.5 mM EGTA. However, at or above 0.75 mM EGTA, the first electrical shock delivered results in the contraction of the organism and maintenance of the cell in the contracted state. Subsequent stimuli result in the disintegration of the organism, usually within 15 to 20 stimuli. If Spirostoma in Carter's medium are stimulated electrically once per five seconds they begin to show an enlargement of the contractile vacuole after ten minutes. Figure #6A shows the posterior region of Spirostomum before electrical stimulation and B shows the same animal after stimulating for ten minutes once per five seconds. After cessation of stimulation the vacuole returned to the normal size in about five mdnutes. Since our data show that calcium is released from the bound state with electrical stimulation but does not leak out of the organism, greater concentrations of soluble calcium may be contained within the vacuole. To test the possibility that calcium was removed from the cytoplasm to the contractile vacuole, the presence of calcium within the contractile vacuole was compared for unstimulated and electrically 1,4 stimulated Spirostoma using the electron microprobe. Longitudinal sections through the contractile vacuole were cut after fixing the cells and embedding them in Epon. Figure #7 compares the line scan representation of calcium through the contractile vacuole for a con- trol and an electrically stimulated organism. The electrically stimu- lated Spirostomum shows the presence of more calcium than the unstimu- lated cell. The observed swelling of the contractile vacuole with electrical stimulation may be due to the displacement of calcium from cytoplasm to contractile vacuole with a concomitant increase in water (27). Spirostoma electrically stimulated in the presence of 0.5 mM EGTA do not show as pronounced a swelling of the contractile vacuole as do those electrically stimulated without EGTA present. This obser- vation further argues that calcium may be responsible for the vacuolar swelling. The EGTA chelates the soluble calcium which might impede its movement from the cytoplasm to the contractile vacuole. Further support for this argument is the observation that the swelling of the contractile vacuole is not observed as a consequence of mechanical stimulation. The mechanical stimulus has not been shown to release calcium from the bound state in the magnitude witnessed with electri- cal stimulation. DISCUSSION: Electron diffraction studies of Spirostomum suggest the endo- plasmic calcium is deposited as hydroxyapatite (21) which provides a primative endo-skeleton enabling the organism to withstand the greater hydrostatic pressures encountered during the burrowing phase of life (4). However, these endoplasmic calcium deposits described by Pautard, 45 Bien, and Vivier may not be involved in the release of calcium for the initiation of contraction (4, 21, 24). We have reported the presence of s subcortical distribution of calcium coincident with the location of the microfilaments which are assumed to be the contractile fibers (20). Calcium identified in this region is thought to provide the calcium reported to be involved in the contractile process (7, 20). When Spirostoma are homogenized, only 20% of the total label is retained on the millipore filter. The bulk of the label passes through the filter. The same amount of label is retained when pore sizes from 0.05 to 8.0 microns are used. Therefore, the calcium retained in filtration is probably associated with large membranes. Electron micrographs of the subcortical microfilaments reveal a tortuous arrangement of reticular membranes (6, 8, 18, 20, 25). We believe that the labelled calcium retained by the filter is associated with these reticular membranes. However, it is possible that the endoplasmic hydroxyapatite deposits are also retained by the filter. Jones has reported that the equilibrium time for incorporation of 45-calcium is at least 14 days (12). Should the two week incubation period employed for our experiments not result in a homogeneous distribution throughout the calcium stores of Spirostomum, the endoplasmic calcium would fail to contribute to the labelled calcium retained by the filter. Therefore, the 202 value of membrane bound calcium may be low. Cellular homogenates of mechanically stimulated Spirostoma did not show a decrease in labelled calcium retained by the filter. However, the amount of calcium necessary for contraction may be so small that its release could not be measured by the technique employed. 45 Incubating Spirostoma in EGTA reduces the membrane bound as well as free calcium in homogenates of the cells. The chelation of these calcium stores may be responsible for the reduced probability of contraction to the mechanical stimulus of cells incubated in EGTA. Amos has reported that calcium must be present in a critical concen- tration for contraction of the stalk in another protozoan, Vorticella (1). At sub-threshold concentrations, contraction does not occur in Vorticella. In Spirostomum as the EGTA concentration is increased from 0.1 to 0.5 mM, less calcium is available for contraction and the probability of contraction is proportionately reduced. However, above 0.5 mM EGTA, the relationship between EGTA concentration and reduction in the probability of contraction is no longer linear. At 0.75 mM EGTA, the effect of EGTA is almost completely reversed. A possible explanation is as follows: The medium in which Spirostomum is grown contains 0.5 mM calcium. At EGTA concentrations below 0.5 mM free EGTA is present in very low concentrations. The greater binding constant for calcium insures that essentially no magnesium is chelated. The ratio of the binding constants is (17): KCa-EGTA KHz-EGTA _ 105.8 However, above 0.5 mM EGTA, appreciable amounts of magnesium are bound, since EGTA is now in excess of the calcium concentration. Within the organism, magnesium is probably also chelated. At concentrations of EGTA below which magnesium is chelated, the decrease in probabil- ity of contraction is proportional to the EGTA concentration. Above 0.5 mM EGTA, magnesium is chelated and the probability of contraction increases. By analogy with muscle, magnesium may be involved in maintenance of the resting state, or more 0a 47 likely, the ratio of calcium and magnesium may govern the contractile response (3). At EGTA concentrations between 0.5 and 0.75 mM, reducing available magnesium may reactivate the contractile fiber, or else, a ratio of calcium to magnesium may be approached that again promotes a higher probability of contraction. At concentrations of 0.75 mM EGTA and above, the chelator concentration exceeds that for the sum of the concentrations of calcium (0.5 mM) and magnesium (0.2. mM) in the incu- bation medium. At these concentrations of EGTA the animal undergoes deterioration. Swimming is impaired and often the animal appears in contorted positions with bulges in the body membrane. When electrical stimuli are delivered the release of much larger amounts of calcium is evidenced than when mechanical stimuli are delivered. Perhaps the electrical stimulus itself electrophoreti- cally drives calcium away from storage binding sites. The released calcium is probably far in excess of that required to initiate con- traction. Consequently, in the presence of EGTA, enough calcium is released with electrical stimulation to exceed the threshold of calcium necessary for contraction. This interpretation is consistent with the observation that EGTA does not inhibit the probability of contraction to electrical stimulation in the first minute of stimulation. Electri- cal stimulation at EGTA concentrations of 0.75 mM and above results in a prolonged contracted state. Re-extension occurs much more slowly than it does in normal animals. This phenomenon may be due to the chelation of magnesium by the excess EGTA. With a reduction in available magne- sium re-extension is impaired. Further stimulation probably results in the removal of most or all of the membrane bound calcium with a resulting instability of the cell membranes. Consequently, the subsequent electrical 1,3 stimuli electrophoretically disaggregate membrane subunits and the organism disintegrates. The swelling of the contractile vacuole that occurs with electrical stimulation and the observation of increased calcium in that region probably reflects a homeostatic mechanism within Spirostomum to maintain a defined soluble calcium concentration within the cytoplasm (27). It is suggested that the calcium released with electrical stimu- lation is transported to the contractile vacuole. However, since the swelling is reduced within five minutes after cessation of electrical stimulation, a mechanism may exist to return calcium from the con- tractile vacuole to the cytoplasm. However.the loss of label to the medium may also occur. It was not measured. The technique of preparation of cells for microprobe analysis necessitates the dehydration of the specimen. The shape of the line scans are artefactual in showing that soluble calcium is not uniformly distributed throughout the contractile vacuole. The calcium has precipitated against the cortical membranes of the cell and the increased soluble calcium due to electrical stimulation is observed as an increase in the height of cortical peaks. Our results suggest that the release of calcium from a bound form is necessary for contraction to occur and further that much more is released by electrical than by mechanical stimulation. That mechani- cal stimulation leads to habituation of the contractile response and electrical stimulation does not indicate that the site(s) of action of these two stimuli in the cell are different. A model we have suggested (19) postulates a sensory transducer site for the mechanical stimulus. In addition to its role in contraction, calcium may be a crucial mediator 49 in this sensory transduction. (Rasmassen has discussed possible mechanisms that could govern calcium release (25).) The lack of habituation to the electrical stimulus delivered at a frequency of 1 stimulus per 10 seconds may result as a consequence of the stimulus bypassing the sensory receptor assumed to be involved in the response to the mechanical stimulus. The electrical stimulus may directly release calcium which initiates contraction. O U “5 Q 825 on I! G) L. U C) '0 a! so- pre- post- stimulation Figure #l. The per cent decrease in membrane bound 45Ca is shown for A) mechanically stimulated and B) electrically stimulated Spirostoma. Mechanically stimulated cells were given either 1 stimulus per 5 seconds for 15 minutes or 1 stimulus per 10 seconds for 10 minutes, each producing the same decrease in membrane bound calcium. Curve A is based on 6 replications. Curve B is the mean change for 15 replications (p-0.0l, sign test, 1 tailed). 51 % BOUND C045 to 0: In 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 EGTA Concentration (mM) Figure #2. Percent membrane bound 45c; versus EGTA concen- tration is plotted for cellular homogenates of Spirostoma after 30 minutes incubation. V. ’e 1.00 Mean Probability of Contraction 52 'V on .50 .25 Minutes Figure #3. The mean probability of contraction to mechanical stimuli delivered once per 5 seconds for 5 minutes is plotted for Spirostoma before incubation in EGTA (curve A) and after a 30 minute rest period followed by incubation for 15 minutes in 0.25 mM EGTA in Carter's medium (curve B). After a 2 minute rest period following the 5 minute stimulation period, an additional one minute of stimulation was delivered. Nine replications of the experiment were performed. The difference in level of contraction in minute 1 is significant at p80.02, sign test, 1 tailed . Q. ’0 Mean Probability of Contraction 55 1.00 .75 Minutes Figure #4. The mean probability of contraction to electrical stimuli delivered once per 5 seconds for 5 minutes is plotted for Spirostoma before incubation in EGTA (curve A) and after a 30 minute rest period followed by incubation for 15 minutes in 0.25 mM EGTA in Carter's medium (curve B). Nine replications of the experiment were performed. The difference in level of contraction in minute 1 is not significant. 54 'u iL_ / o \ o A Probability of Contract. I I I I I I I .l .2 .3 .4 .5 .6 .7 .8 .9 1.0 EGTA Concentration (mM) Figure #5. Spirostoma were mechanically stimulated once per 5 seconds 20 times to obtain an initial probability of contraction. Follow- ing a 30 minute rest period, they were incubated in varying concentrations of EGTA for 15 minutes, then stimulated again once per 5 seconds 20 times. The difference between the initial and final probability of contraction (initial minus final) is plotted as a function of EGTA concentration in millimoles. The higher the value on the ordinate, the few responses the animal gave following EGTA incubation. 55 Figure #6. Photographs of Spirostomum before (A) and after (B) electrical stimulation once per 5 seconds for 15 minutes. Note the enlarged contractile vacuole after stimulation. Figure #7. Line scans for calcium in transverse sections through the contractile vacuole. 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