Stimulus-response coupling in the contraction of tentacles of the suctorian protozoon Heliophrya erhardi

Stimulus-response coupling in the contraction of tentacles of the suctorian protozoon Heliophrya erhardi R. D. BUTLER* and C. R. McCROHAN Departments of Cell and Structural Biology and Physiological Sciences, University of Manchester, Manchester M13 9PL, L K Author for correspondence: at Department of Cell and Structural Biology Summary Experiments were carried out to determine the role of the plasma membrane in the control of tentacle contraction in Heliophrya erhardi. Intracellular recordings gave membrane potentials between 20 and 30 mv. In a Ca 2+ -containing medium mechanical stimulation induced tentacle contraction but no associated electrical events were recorded. Intracellular stimulation with 50 na, 100 ms hyperpolarizing current induced contraction, but no significant changes in membrane potential, whereas up to 100 na, 100 ms depolarizing current had no effect. In a Ca 2+ -free medium neither mechanical stimulation nor electrical stimulation induced contraction. Extracellular stimulation of 15 V, 100 ms induced a Ca z+ -dependent, unilateral (anodal) contraction response with a threshold of 5x 10 9 M-Ca 2+. At concentrations above this neither latency to contraction nor contraction time showed significant variation. In a standard concentration of 10~ 4 M-Ca 2+ the sensitivity to extracellular stimulation was increased and latency to contraction was reduced in the presence of a phorbol ester (TPA), which mimics the second messenger diacylglycerol in stimulating the activity of protein kinase C. It is suggested that control of tentacle contraction is unlikely to be mediated by stimulus-activated ion channels in the plasma membrane, and the possibility that the polyphosphoinositide signalling pathway is involved is discussed. Key words: contraction, calcium, protozoon, phorbol ester, inositol trisphosphate, stimulus-response. Introduction Suctoria are modified ciliates that have no cilia in the adult cell but have contractile tentacles specialized for prey capture. The tentacles of Heliophrya erhardi are covered by a fibrous cortex and contain a central canal (axoneme) with a two-layered microtubular wall of the common suctorian pattern, which is surrounded by microfilaments, particularly at its base (Hauser & Van Eyes, 1976; Mogensen, 1985). A similar distribution of microfilaments has been described in other suctoria (Curry & Butler, 1976; Mogensen, 1985). Suctorian tentacles contract spontaneously or in response to contact with prey organisms. Contraction may be induced experimentally by mechanical or electrical stimuli (Curry & Butler, 1976; Hackney & Butler, 1981a,c; Mogensen & Butler, unpublished). Suctorian tentacles also contract in response to added extracellular cations, and are particularly sensitive to Journal of Cell Science 88, 121-127 (1987) Printed in Great Britain The Company of Biologists Limited 1987 (Okajima, 1957; Hackney et al. 1982). Glycerinated tentacles of H. erliardi and Discophrya collini contract in a reaction mixture of Ca 2+, Mg 2+ and ATP (Hauser & Van Eyes, 1976; Hackney & Butler, 1981n). Cells of D. collini, contracted by high external levels of Ca 2+, show subsequently elevated levels of bound calcium in cytoplasmic, membrane-bound, elongate dense bodies (EDB) (structures found in all suctorians), indicating that these may function as intracellular Ca 2+ reservoirs (Hackney & Butler, 19816). Subsequent investigation of cells subjected to combinations of Ca 2+, 4:> Ca 2+, the divalent cation ionophore A23187 and Ruthenium Red, an inhibitor of Ca 2+ membrane transport, confirm that contraction is accompanied by reduction in EDB Ca 2+ levels (Al- Khazzar et al. 1983), and that calcium enters the cell from the extracellular medium (Al-Khazzar et al. 1984). As a result of these investigations, a model was presented that proposes that tentacle contraction is Ca 2+ 121

achieved by activation of an actin-based mechanism through an increase in cytosolic Ca 2+, which itself depends upon Ca 2+ fluxing through the plasma membrane and/or the EDB membrane (Al-Khazzar et al. 1984). It is not clear how the induction stimuli are translated into changes in Ca 2+ influx and efflux, or how the activities of the plasma membrane and EDB membrane are related. This paper describes experiments directed towards determining the role of the plasma membrane in the control of Ca z+ -sensitive tentacle contraction in H. ehwrdi. Membrane potentials were recorded during mechanically and electrically induced contraction, and the threshold of extracellular Ca 2+ concentration required for electrically induced contraction was determined. A possible role for Ca 2+ /phosphatidylserine/ diacyglycerol-dependent protein kinase C in the generation of intracellular signals associated with contraction was investigated using a phorbol ester (12-O-tetradecanoylphorbol 13-acetate). These experiments point to the generation of a Ca 2+ -dependent, membrane-generated signal, probably consequent upon hydrolysis of membrane phospholipids, which leads to the release of intracellular Ca 2+ and activation of the contraction mechanism. Materials and methods H. erhardi was isolated from a pond at Castle Hill, Cheshire (O.S. sheet 109 750856). Discs of resin or glass, mounted on supported nylon nets, were floated on aerated pond water maintained at 18-22 C. Discs colonized by H. erhardi were transferred to a Petn dish containing an inorganic medium (Stewart, 1972), maintained in darkness, and fed on Colpidiuin twice weekly. The medium was replaced immediately after feeding and stocks were subcultured at regular intervals by the transfer of fresh, newly colonized discs to additional Petri dishes. For scanning electron microscopy (SEM), glass disc cultures were fixed in 2-5% (v/v) glutaraldehyde in 0-1 M- sodium cacodylate (ph7-2) for 25min, rinsed in buffer, freeze-dried, sputter-coated with gold and examined in a Cambridge S150 electron microscope. Intracellular recordings were made from cells using glass microelectrodes filled with 3-0M-potassium acetate to give resistances of 20-30 MQ. Penetration of cells with microelectrodes was considerably easier for Heliophrya than in previous studies using Discophrya (Hackney & Butler, 1981c), since the former species lacks a stalk and is firmly anchored to the substrate. Signals were amplified, recorded on magnetic tape and displayed using a pen recorder. Depolarizing and hyperpolarizing stimuli were applied through the same electrode via a bridge balance circuit in the preamplifier. Current injected was monitored by placing a current-to-voltage converter between the indifferent bath electrode and earth. Extracellular electrical stimulation was applied using paired silver-wire electrodes placed 500 jum apart on either side of the cell. Stimuli of varying voltage and duration were supplied by a Grass stimulator. Mechanical stimulation was earned out using a glass probe mounted on a micromanipulator. Throughout the investigation cells were used 1 day after feeding. Colonized discs were transferred from stock cultures to appropriate test solutions and observed with transmitted light under a binocular microscope. All experiments were initiated approximately 5 min after immersion. Tentacles were considered to be fully contracted when they reached one quarter of their original length. For all treatments, latency (time to initial movement), contraction time (time from onset of stimulus to completion of movement) and the proportion of tentacles showing full contraction were recorded from 10 cells. Solutions covering the range 10~ 3 M to 8x 10~ MI M-Ca 2+ were made using CaC^/EDTA mixtures. Solutions of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate; Sigma) were made up by dissolving the solid in dimethyl sulphoxide (DMSO) and diluting with culture medium to give final concentrations of 10 nm to 10 4 nm-tpa in 0-0005 % DMSO. Results Scanning electron miavscopy H. erhardi is a dorso-ventrally flattened cell with its capitate tentacles arranged in peripheral fascicles (Fig. 1A). Contraction is initiated in the central region of the tentacle but full contraction involves a shortening along the whole tentacle length (Fig. 1B,C). Intracellular recording and stimulation Normal media. Intracellular recordings from cells immersed in culture medium, which contained 10~ 4 M- Ca 2+, revealed membrane potentials between 20 and 30 mv. Cells showed spontaneous brief hyperpolarizing or depolarizing monophasic or biphasic potentials, of 0-2-2-0 s duration and up to 8mV amplitude (Fig. 2). These were correlated with discharge of contractile vacuoles and, in a given cell, were either hyperpolarizing or depolarizing. The difference in their polarity between different cells was probably related to the position of the electrode with respect to both the contractile vacuoles and the three membranes that are known to surround the cell. No other spontaneous electrical event was recorded. Repeated mechanical stimulation of a cell resulted in artefactual deflections of the recording trace, followed by tentacle contraction, but there were no associated electrical events (Fig. 3A). Extracellular stimulation with 10 V, 5 ms produced no tentacle contraction; 20 V, 5ms produced unilateral (anodal) contraction; 30V, 5 ms produced overall contraction. In no case was there any associated electrical event apart from the stimulus artefact^fig. 3B). Intracellular stimulation with 50 na, 100 ms hyperpolarizing current induced 122 R. D. Butler and C. R. McCrohan

Fig. 1. Scanning electron micrographs of H. eiiiarcli. A. Whole cell showing extended tentacles in fascicles. B. Partially contracted tentacle. The proximal and distal regions are still extended. C. Fully contracted tentacles. A. X1200; B, X4800; C, X4000. tentacle contraction, but no significant changes in membrane potential (Fig. 3C). In contrast, intracellular stimulation with up to 100 na, 100 ms depolarizing current had no effect. Low-Caz+ media. In low levels of extracellular Ca 2+ ( 1 0 ~ ' J M ) the cells were unstable and disintegrated, either spontaneously or on contact with microelectrodes. In 2x 10~y M-Ca2+ the cells remained intact and contractile vacuole activity continued, with the normal electrical events. However, no tentacle contraction could be induced. The cells failed to respond to mechanical stimulation (Fig. 3D), extracellular electrical stimulation (10X normal threshold voltage), or to intracellular stimulation up to 1000 na, 1000 ins hypcrpolarizing or depolarizing current. These stimuli continued to produce stimulus artefacts, which were clearly, therefore, not associated with contraction. Extracellular stimulation Ca2+ dependence. T h e ability of cells to respond to a standard extracellular electrical stimulus over a range of external C a 2 + ( 1 0 ~ l u to 1 0 ~ 3 M ) was determined. Cells were stimulated with 15 V, 100 ms pulses, which were well above the threshold for full contraction in standard culture m e d i u m. ( N. B. in these experiments stimuli were longer in duration than those used in 10 mv 5s Fig. 2. Intracellular recordings from two H. erhardi (A,B). Spontaneous, brief potential changes occur, which are associated with visual observation of contractile vacuole discharge (arrows). These potentials were hyperpolanzing (A), or depolarizing (B). Tentacle contraction in Heliophrya 123

20 mv 30i L 10 mv 20-.3 10-10 mv D 2xl0" 9 M-Ca 2+ 20mV Fig. 3. Intracellular recordings from H. erliardi. Responses to mechanical, and to extracellular or intracellular electrical stimulation. A. Repeated mechanical stimuli (arrows), leading to full tentacle contraction, do not induce electrical events across the membrane. B. Extracellular electrical stimulation (20 V, 5 ms, arrow) produces a stimulus artefact but no electrical response. C. Intracellular stimulation (-50nA, 100ms), leading to tentacle contraction, evokes no regenerative membrane events. D. In medium containing 2x 10~ 9 M-Ca 2+, repeated mechanical stimulation (arrows) leads to neither electrical activity nor tentacle contraction. conjunction with intracellular recording (Fig. 3), where a prolonged stimulus might mask electrical events across the membrane. However, 100 ms stimuli permitted the use of much lower stimulus intensities.) Full contraction occurred only at 3xlO~ 8 M-Ca 2+ and above, and in this range latency was constant and contraction time showed no significant variation (Fig. 4). At concentrations of 10~ 8 M-Ca 2+ and below, no cells achieved full contraction; latency increased dramatically as Ca 2+ concentration decreased, although contraction time showed no significant change (Fig. 4). No response occurred at concentrations below 5xlO~ 9 M-Ca 2+. In 2xl0~ 9 M-Ca 2+ the cells remained intact but unresponsive, and in 8xlO~ lu M-Ca 2+ the cells disintegrated spontaneously after about 2 min in the test solution, or on stimulation. Effect of phorbol ester. Cells were immersed in 10, 10 2, 10 3 or 10 4 nm-tpa in culture medium plus DMSO and stimulated extracellularly with 5, 10 or 15 V for 100 ms. These stimuli included sub- and suprathreshold levels for tentacle contraction. Controls for these experiments were carried out in culture medium plus 0-0005 % DMSO, and produced similar responses to those recorded in culture medium alone. Fig. 5 10s 60n I 20' 10" 10" 8 10" 6 Ca 2+ (M) 10" 10" 2 Fig. 4. Latency to first movement (A), and contraction time (B) for cells stimulated extracellularly with 15 V, 100 ms, in varying concentrations of extracellular Ca +. Each point shows mean and standard deviation for 10 cells. Contraction time in 5xlO~ 9 M-Ca 2+ could not be measured since movement was so slight. No response at all was obtained in Ca z+ concentrations less than 5xlO~ y M. shows results obtained for latency and contraction time with the standard 15 V, 100 ms stimulus used in the Ca z+ -dependence experiments. Both latency and contraction time fell to values well below control values in 10 2 nm-tpa. Both parameters were higher again in 10 3 nm-tpa, and approached control values in 10 4 nm- TPA. Analysis of variance was carried out and revealed significant differences in both latency and contraction time between the treatment means (latency, F= 3-99, /><0-01; contraction time, F= 14-57, / ) <0-01). The least significant range (l.s.r.) was also calculated (latency, l.s.r. =2-27; contraction time, l.s.r. =7-12; both at 0-05 probability level), and showed that latency differed significantly from controls for 10 2 nm-tpa, and that contraction time was significantly different from the control in both 10 2 and 10 3 nm-tpa. The potentiating effect of TPA on stimulus-evoked contraction was also demonstrated by the proportion of cells in which all tentacles showed full contraction using the different stimulus voltages (5, 10 and 15 V; Fig. 6). In controls, full contraction was achieved by the majority of cells (90%) only when 15 V stimuli 124 R. D. Butler and C. R. McCrohan

T A 6-2- I 30 " raction 1 120" U 40-10- B I \ ; \, / \ \ * 10 10 2 TPA (nm) V 10 3 Iff 1 Fig. 5. Latency (A), and contraction times (B) following 15 V, 100 ms extracellular stimulation in different concentrations of TPA. Each point shows mean and standard deviation for 10 cells. Points marked with stars are significantly different from the control (P<005). were used. However, in the presence of TPA a high proportion of cells achieved full contraction; with 10 V stimuli in 10 2 nm-tpa (100%) and with only 5 V in 10 3 nm-tpa (90%) (Fig. 6). Discussion H. erliardi resembles other protozoa in that the membrane potential is approximately 30 mv. This corresponds to similar values found for the suctorians D. collini (Hackney & Butler, 1981c) and Trichophrya collini (Mogensen & Butler, unpublished), for ciliates (Eckert et al. 1976; Pape & Machemer, 1986) and for amoebae (Bruce & Marshall, 1965). The only electrical events recorded from H. erhardi were hyperpolarizing or depolarizing signals related to discharge of the contractile vacuoles. These contractile vacuole-related discharges in//, erhardi have been described (Eagleset al. 1980) and are probably the consequence of a momentary rupture of the plasma membrane. However, their presence demonstrates the validity of the intracellular recording techniques employed in this investigation, and emphasizes the fact that tentacle contraction, whether induced by mechanical or electrical stimuli, is not accompanied by any regenerative changes in membrane potential, at least in the region of the recording electrode. The role of extracellular Ca 2+ in the induction of tentacle contraction is not clear. The anodal contraction response (i.e. contraction in response to hyperpolarization rather than depolarization of the plasma membrane) shown by the tentacles of H. erliardi and other suctorians (Hackney & Butler, 1981c; Mogensen & Butler, unpublished) makes influx of Ca 2+ through voltage-dependent ion channels an unlikely candidate mechanism for production of the intracellular Ca + signal. Jahn (1967) suggested that the anodal responses exhibited by a large variety of protozoa with nonexcitable membranes were due to changes in the amount of Ca 2+ associated with both sides of the plasma membrane, rather than fluxing of Ca 2+ across the membrane. We have shown that tentacle contraction will occur in extremely low levels of extracellular Ca 2+ (5X10~ 9 M), indicating that this is not the primary source of the intracellular Ca 2+ signal. It seems likely therefore that the cytosolic Ca 2+ signal results from release of Ca 2+ from intracellular stores such as 10- Control 10riM-TPA 10 2 nm-tpa 10 3 nm-tpa 10 15 10 15 5 10 Voltage (V) 15 10 15 Fig. 6. Proportion of cells achieving full tentacle contraction using varying extracellular stimulus intensities (5, 10, 15 V; 100ms) and in varying concentrations of TPA. 10 z and 10 3 nm- TPA lower the threshold voltage for full contraction. Tentacle contraction in Heliophrya 125

the elongate dense bodies (Al-Khazzar et al. 1984). A similar control mechanism involving sequestered intracellular Ca 2+ is used by a number of cell types, including muscle (Barrit, 1981), and has recently been proposed for tentacle motility in the dinoflagellate Noctiluca scintillans (Metivier & Soyer-Gobillard, 1986). In this species, a low level of extracellular Ca z+ is necessary, but the source of cytosolic Ca 2+ is thought to be pools in the mitochondria and endoplasmic reticulum. If Ca 2+ influx is not essential for contraction in H. erfiardi, then the necessity for extracellular Ca 2+ could lie in its role either in maintaining the integrity of the membrane, or as a permissive agent in the production of a membrane-generated intracellular signal that activates the contraction mechanism. As suctorian tentacle contraction is known to be Ca """-activated (Hackney & Butler, \%\a,b,c\ Al-Khazzar et al. 1983, 1984; Mogensen & Butler, unpublished), the function of this signal is likely to be mobilization of Ca 2+ from intracellular stores. It has been shown using a variety of cell types that the hydrolysis of membrane polyphosphoinositides plays an important role in Ca 2+ signalling (Berridge, 1986). One product of this hydrolysis, inositol 1,4,5- trisphosphate, appears to be the primary second messenger responsible for mobilization of Ca 2+ from intracellular stores, particularly endoplasmic reticulum (Berridge, 1986). The second product, diacylglycerol, exerts its major effects via activation of Ca 2+ /phosphatidylserine-dependent protein kinase C. The present study demonstrates that tentacle contraction is potentiated in the presence of TPA, a phorbol ester that mimics diacylglycerol by activating protein kinase C. This strongly implicates inositol phospholipid metabolism in stimulus-contraction coupling in H. erliardi. The initial hydrolysis of phosphatidylinositol 4,5-bisphosphate in the plasma membrane would presumably be triggered by a mechano/chemostimulant such as prey cilia (Sundermann et al. 1986), or experimentally by extracellular cations, mechanical or electrical stimuli. Here, low levels of extracellular Ca might be required for the initial membrane response. Indeed, in some tissues, Ca + itself is thought to stimulate phosphatidylinositol 4,5-bisphosphate breakdown (Best, 1986). The results shown in Fig. 5 indicate an optimum concentration for the potentiating effect of TPA. The occurrence of a bell-shaped dose response curve of this sort following application of a pharmacological agent is not unusual. In this case it may be due to a feedback inhibition of phosphatidylinositol 4,5-bisphosphate breakdown by the activated protein kinase C in the higher TPA concentrations, as shown for other phorbol ester-treated cells (Berridge, 1986). Alternatively, higher concentrations of TPA may have a toxic effect on the cells. References AL-KHAZZAR, A. R., BUTLER, R. D. & EARNSHAW, M. J. (1983). The effect of ionophore A23187 and ruthenium red on tentacle contraction and ultrastructure in Discophrya collini: Evidence of calcium fluxing from intracellular reservoirs. Pmtoplasma 117, 158-166. AL-KHAZZAR, A. R., EARNSHAW, M. J., BUTLER, R. D., EMES, M. J. & SIGEE, D. C. (1984). Tentacle contraction in Discopluya collini: The effects of ionophore A23187 and ruthenium red on Ca z+ -induced contraction and uptake of extracellular calcium. Pmtoplasma 122, 125-131. BARRIT, G. J. (1981). Calcium transport across cell membranes: progress towards molecular mechanisms. Trends Biochem. Sci. 6, 322-325. 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