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 therole of the plasma membrane in the control oftentacle contraction in Heliophrya erhardi. Intra-cellular recordings gave membrane potentialsbetween —20 and — 30 mV. In a Ca2+-containingmedium mechanical stimulation induced ten-tacle contraction but no associated electricalevents were recorded. Intracellular stimulationwith 50 nA, 100 ms hyperpolarizing current in-duced contraction, but no significant changesin membrane potential, whereas up to 100 nA,100 ms depolarizing current had no effect. In aCa2+-free medium neither mechanical stimu-lation nor electrical stimulation induced contrac-tion. Extracellular stimulation of 15 V, 100 msinduced a Caz+-dependent, unilateral (anodal)contraction response with a threshold of 5x

10 9M-Ca2+. At concentrations above this neitherlatency to contraction nor contraction timeshowed significant variation. In a standard con-centration of 10~4M-Ca2+ the sensitivity to extra-cellular stimulation was increased and latency tocontraction was reduced in the presence of aphorbol ester (TPA), which mimics the secondmessenger diacylglycerol in stimulating the ac-tivity of protein kinase C. It is suggested thatcontrol of tentacle contraction is unlikely to bemediated by stimulus-activated ion channels inthe plasma membrane, and the possibility thatthe polyphosphoinositide signalling pathway isinvolved is discussed.

Key words: contraction, calcium, protozoon, phorbolester, inositol trisphosphate, stimulus-response.

Introduction

Suctoria are modified ciliates that have no cilia in theadult cell but have contractile tentacles specialized forprey capture. The tentacles of Heliophrya erhardi arecovered by a fibrous cortex and contain a central canal(axoneme) with a two-layered microtubular wall of thecommon suctorian pattern, which is surrounded bymicrofilaments, particularly at its base (Hauser & VanEyes, 1976; Mogensen, 1985). A similar distribution ofmicrofilaments has been described in other suctoria(Curry & Butler, 1976; Mogensen, 1985).

Suctorian tentacles contract spontaneously or inresponse to contact with prey organisms. Contractionmay be induced experimentally by mechanical orelectrical stimuli (Curry & Butler, 1976; Hackney &Butler, 1981a,c; Mogensen & Butler, unpublished).Suctorian tentacles also contract in response to addedextracellular cations, and are particularly sensitive to

Journal of Cell Science 88, 121-127 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

Ca2+ (Okajima, 1957; Hackney et al. 1982). Glycer-inated tentacles of H. erliardi and Discophrya collinicontract in a reaction mixture of Ca2+, Mg2+ and ATP(Hauser & Van Eyes, 1976; Hackney & Butler, 1981n).Cells of D. collini, contracted by high external levels ofCa2+, show subsequently elevated levels of boundcalcium in cytoplasmic, membrane-bound, elongatedense bodies (EDB) (structures found in all suctor-ians), indicating that these may function as intra-cellular Ca2+ reservoirs (Hackney & Butler, 19816).Subsequent investigation of cells subjected to combi-nations of Ca2+, 4:>Ca2+, the divalent cation ionophoreA23187 and Ruthenium Red, an inhibitor of Ca2+

membrane transport, confirm that contraction is ac-companied by reduction in EDB Ca2+ levels (Al-Khazzar et al. 1983), and that calcium enters the cellfrom the extracellular medium (Al-Khazzar et al.1984). As a result of these investigations, a model waspresented that proposes that tentacle contraction is

121

achieved by activation of an actin-based mechanismthrough an increase in cytosolic Ca2+, which itselfdepends upon Ca2+ fluxing through the plasma mem-brane and/or the EDB membrane (Al-Khazzar et al.1984). It is not clear how the induction stimuli aretranslated into changes in Ca2+ influx and efflux, orhow the activities of the plasma membrane and EDBmembrane are related.

This paper describes experiments directed towardsdetermining the role of the plasma membrane in thecontrol of Caz+-sensitive tentacle contraction in H.eHwrdi. Membrane potentials were recorded duringmechanically and electrically induced contraction, andthe threshold of extracellular Ca2+ concentrationrequired for electrically induced contraction was deter-mined. A possible role for Ca2+/phosphatidylserine/diacyglycerol-dependent protein kinase C in the gener-ation of intracellular signals associated with contractionwas investigated using a phorbol ester (12-O-tetradeca-noylphorbol 13-acetate). These experiments point tothe generation of a Ca2+-dependent, membrane-gener-ated signal, probably consequent upon hydrolysis ofmembrane phospholipids, which leads to the release ofintracellular Ca2+ and activation of the contractionmechanism.

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 onsupported nylon nets, were floated on aerated pond watermaintained at 18-22°C. Discs colonized by H. erhardi weretransferred to a Petn dish containing an inorganic medium(Stewart, 1972), maintained in darkness, and fed on Col-pidiuin twice weekly. The medium was replaced immediatelyafter feeding and stocks were subcultured at regular intervalsby the transfer of fresh, newly colonized discs to additionalPetri dishes.

For scanning electron microscopy (SEM), glass disc cul-tures 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 aCambridge S150 electron microscope.

Intracellular recordings were made from cells using glassmicroelectrodes filled with 3-0M-potassium acetate to giveresistances of 20-30 MQ. Penetration of cells with microelec-trodes was considerably easier for Heliophrya than in pre-vious studies using Discophrya (Hackney & Butler, 1981c),since the former species lacks a stalk and is firmly anchored tothe substrate. Signals were amplified, recorded on magnetictape and displayed using a pen recorder. Depolarizing andhyperpolarizing stimuli were applied through the same elec-trode via a bridge balance circuit in the preamplifier. Currentinjected was monitored by placing a current-to-voltage con-verter between the indifferent bath electrode and earth.

Extracellular electrical stimulation was applied usingpaired silver-wire electrodes placed 500 jUm apart on eitherside of the cell. Stimuli of varying voltage and duration were

supplied by a Grass stimulator. Mechanical stimulation wasearned out using a glass probe mounted on a micromanipu-lator.

Throughout the investigation cells were used 1 day afterfeeding. Colonized discs were transferred from stock culturesto appropriate test solutions and observed with transmittedlight under a binocular microscope. All experiments wereinitiated approximately 5 min after immersion. Tentacleswere considered to be fully contracted when they reached onequarter of their original length. For all treatments, latency(time to initial movement), contraction time (time from onsetof stimulus to completion of movement) and the proportionof tentacles showing full contraction were recorded from 10cells. Solutions covering the range 10~3M to 8x 10~MIM-Ca2+

were made using CaC^/EDTA mixtures. Solutions of thephorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate;Sigma) were made up by dissolving the solid in dimethylsulphoxide (DMSO) and diluting with culture medium togive final concentrations of 10 nM to 104nM-TPA in 0-0005 %DMSO.

Results

Scanning electron miavscopy

H. erhardi is a dorso-ventrally flattened cell withits capitate tentacles arranged in peripheral fascicles(Fig. 1A). Contraction is initiated in the central regionof the tentacle but full contraction involves a shorten-ing along the whole tentacle length (Fig. 1B,C).

Intracellular recording and stimulationNormal media. Intracellular recordings from cells

immersed in culture medium, which contained 10~4M-Ca2+, revealed membrane potentials between —20 and— 30 mV. Cells showed spontaneous brief hyperpolariz-ing 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 ofcontractile vacuoles and, in a given cell, were eitherhyperpolarizing or depolarizing. The difference intheir polarity between different cells was probablyrelated to the position of the electrode with respect toboth the contractile vacuoles and the three membranesthat are known to surround the cell. No other spon-taneous electrical event was recorded.

Repeated mechanical stimulation of a cell resulted inartefactual deflections of the recording trace, followedby tentacle contraction, but there were no associatedelectrical events (Fig. 3A). Extracellular stimulationwith 10 V, 5 ms produced no tentacle contraction; 20 V,5ms produced unilateral (anodal) contraction; 30V,5 ms produced overall contraction. In no case wasthere any associated electrical event apart from thestimulus artefact^Fig. 3B). Intracellular stimulationwith 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. Partiallycontracted 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 inmembrane potential (Fig. 3C). In contrast, intracellu-lar stimulation with up to 100 nA, 100 ms depolarizingcurrent had no effect.

Low-Caz+ media. In low levels of extracellular Ca2+

(10~' JM) the cells were unstable and disintegrated,either spontaneously or on contact with microelec-trodes. In 2x 10~y M-Ca2+ the cells remained intact andcontractile vacuole activity continued, with the normalelectrical events. However, no tentacle contractioncould be induced. The cells failed to respond tomechanical stimulation (Fig. 3D), extracellular electri-cal stimulation (10X normal threshold voltage), or to

intracellular stimulation up to 1000 nA, 1000 ins hypcr-polarizing or depolarizing current. These stimuli con-tinued to produce stimulus artefacts, which wereclearly, therefore, not associated with contraction.

Extracellular stimulation

Ca2+ dependence. T h e ability of cells to respond toa standard extracellular electrical stimulus over a rangeof external Ca 2 + (10~ l u to 1 0 ~ 3 M ) was determined.Cells were stimulated with 15 V, 100 ms pulses, whichwere well above the threshold for full contraction instandard culture medium. ( N . B . in these experimentsstimuli 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 areassociated with visual observation of contractile vacuole discharge (arrows). These potentials were hyperpolanzing (A), ordepolarizing (B).

Tentacle contraction in Heliophrya 123

20 mV

L 10 mV

10 mV

30i

20-

.310-

D

2xl0"9

M-Ca2+

60n20mV

10s

Fig. 3. Intracellular recordings from H. erliardi.Responses to mechanical, and to extracellular orintracellular electrical stimulation. A. Repeated mechanicalstimuli (arrows), leading to full tentacle contraction, do notinduce 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 totentacle contraction, evokes no regenerative membraneevents. D. In medium containing 2x 10~9M-Ca2+, repeatedmechanical stimulation (arrows) leads to neither electricalactivity nor tentacle contraction.

conjunction with intracellular recording (Fig. 3),where a prolonged stimulus might mask electricalevents across the membrane. However, 100 ms stimulipermitted the use of much lower stimulus intensities.)Full contraction occurred only at 3xlO~8M-Ca2+ andabove, and in this range latency was constant andcontraction time showed no significant variation(Fig. 4). At concentrations of 10~8M-Ca2+ and below,no cells achieved full contraction; latency increaseddramatically as Ca2+ concentration decreased,although contraction time showed no significantchange (Fig. 4). No response occurred at concen-trations below 5xlO~9M-Ca2+. In 2xl0~9M-Ca2+

the cells remained intact but unresponsive, and in8xlO~luM-Ca2+ the cells disintegrated spontaneouslyafter about 2 min in the test solution, or on stimulation.

Effect of phorbol ester. Cells were immersed in 10,102, 103 or 104 nM-TPA in culture medium plus DMSOand stimulated extracellularly with 5, 10 or 15 V for100 ms. These stimuli included sub- and supra-threshold levels for tentacle contraction. Controls forthese experiments were carried out in culture mediumplus 0-0005 % DMSO, and produced similar responsesto those recorded in culture medium alone. Fig. 5

20'

I10" 10"8 10"6

Ca2+ (M)10" 10"2

Fig. 4. Latency to first movement (A), and contractiontime (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~9M-Ca2+ could not be measuredsince movement was so slight. No response at all wasobtained in Caz+ concentrations less than 5xlO~yM.

shows results obtained for latency and contraction timewith the standard 15 V, 100 ms stimulus used in theCaz+-dependence experiments. Both latency and con-traction time fell to values well below control values in102 nM-TPA. Both parameters were higher again in103 nM-TPA, and approached control values in 104 nM-TPA. Analysis of variance was carried out and revealedsignificant differences in both latency and contractiontime between the treatment means (latency, F= 3-99,/><0-01; contraction time, F= 14-57, / )<0-01) . Theleast 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 latencydiffered significantly from controls for 102nM-TPA,and that contraction time was significantly differentfrom the control in both 102 and 103 nM-TPA.

The potentiating effect of TPA on stimulus-evokedcontraction was also demonstrated by the proportion ofcells in which all tentacles showed full contractionusing the different stimulus voltages (5, 10 and 15 V;Fig. 6). In controls, full contraction was achieved bythe majority of cells (90%) only when 15 V stimuli

124 R. D. Butler and C. R. McCrohan

T A

6-

2-

40-

I30"

ion

1ra

ct

1 2 0 "U

10-

B

I

\ ;

\ , /

\

\

V

*

10 102

TPA (nM)103 Iff1

Fig. 5. Latency (A), and contraction times (B) following15 V, 100 ms extracellular stimulation in differentconcentrations of TPA. Each point shows mean andstandard deviation for 10 cells. Points marked with starsare significantly different from the control ( P < 0 0 5 ) .

were used. However, in the presence of TPA a highproportion of cells achieved full contraction; with 10 Vstimuli in 102nM-TPA (100%) and with only 5 V in103nM-TPA (90%) (Fig. 6).

Discussion

H. erliardi resembles other protozoa in that the mem-brane potential is approximately — 30 mV. This corre-sponds to similar values found for the suctorians D.collini (Hackney & Butler, 1981c) and Trichophryacollini (Mogensen & Butler, unpublished), for ciliates(Eckert et al. 1976; Pape & Machemer, 1986) and foramoebae (Bruce & Marshall, 1965). The only electricalevents recorded from H. erhardi were hyperpolarizingor depolarizing signals related to discharge of thecontractile vacuoles. These contractile vacuole-relateddischarges in// , erhardi have been described (Eaglesetal. 1980) and are probably the consequence of amomentary rupture of the plasma membrane. How-ever, their presence demonstrates the validity of theintracellular recording techniques employed in thisinvestigation, and emphasizes the fact that tentaclecontraction, whether induced by mechanical or electri-cal stimuli, is not accompanied by any regenerativechanges in membrane potential, at least in the region ofthe recording electrode.

The role of extracellular Ca2+ in the induction oftentacle contraction is not clear. The anodal contrac-tion response (i.e. contraction in response to hyper-polarization rather than depolarization of the plasmamembrane) shown by the tentacles of H. erliardi andother suctorians (Hackney & Butler, 1981c; Mogensen& Butler, unpublished) makes influx of Ca2+ throughvoltage-dependent ion channels an unlikely candidatemechanism for production of the intracellular Ca +

signal. Jahn (1967) suggested that the anodal responsesexhibited by a large variety of protozoa with non-excitable membranes were due to changes in theamount of Ca2+ associated with both sides of theplasma membrane, rather than fluxing of Ca2+ acrossthe membrane. We have shown that tentacle contrac-tion will occur in extremely low levels of extracellularCa2+

( 5 X 1 0 ~ 9 M ) , indicating that this is not the pri-mary source of the intracellular Ca2+ signal. It seemslikely therefore that the cytosolic Ca2+ signal resultsfrom release of Ca2+ from intracellular stores such as

Control 10riM-TPA 102nM-TPA 103nM-TPA

10-|

10 15 10 15 5 10Voltage (V)

15 10 15

Fig. 6. Proportion of cells achievingfull tentacle contraction using varyingextracellular stimulus intensities (5, 10,15 V; 100ms) and in varyingconcentrations of TPA. 10z and 103 nM-TPA lower the threshold voltage for fullcontraction.

Tentacle contraction in Heliophrya 125

the elongate dense bodies (Al-Khazzar et al. 1984). Asimilar control mechanism involving sequestered intra-cellular Ca2+ is used by a number of cell types,including muscle (Barrit, 1981), and has recently beenproposed for tentacle motility in the dinoflagellateNoctiluca scintillans (Metivier & Soyer-Gobillard,1986). In this species, a low level of extracellular Caz+

is necessary, but the source of cytosolic Ca2+ is thoughtto be pools in the mitochondria and endoplasmicreticulum. If Ca2+ influx is not essential for contractionin H. erfiardi, then the necessity for extracellular Ca2+

could lie in its role either in maintaining the integrity ofthe membrane, or as a permissive agent in the pro-duction of a membrane-generated intracellular signalthat activates the contraction mechanism. As suctoriantentacle contraction is known to be Ca """-activated(Hackney & Butler, \%\a,b,c\ Al-Khazzar et al. 1983,1984; Mogensen & Butler, unpublished), the functionof this signal is likely to be mobilization of Ca2+ fromintracellular stores.

It has been shown using a variety of cell types thatthe hydrolysis of membrane polyphosphoinositidesplays an important role in Ca2+ signalling (Berridge,1986). One product of this hydrolysis, inositol 1,4,5-trisphosphate, appears to be the primary second mess-enger responsible for mobilization of Ca2+ from intra-cellular stores, particularly endoplasmic reticulum(Berridge, 1986). The second product, diacylglycerol,exerts its major effects via activation of Ca2+/phospha-tidylserine-dependent protein kinase C. The presentstudy demonstrates that tentacle contraction is poten-tiated in the presence of TPA, a phorbol ester thatmimics diacylglycerol by activating protein kinase C.This strongly implicates inositol phospholipid metab-olism in stimulus-contraction coupling in H. erliardi.The initial hydrolysis of phosphatidylinositol 4,5-bis-phosphate in the plasma membrane would presumablybe triggered by a mechano/chemostimulant such asprey cilia (Sundermann et al. 1986), or experimentallyby extracellular cations, mechanical or electrical stim-uli. Here, low levels of extracellular Ca might berequired for the initial membrane response. Indeed, insome tissues, Ca + itself is thought to stimulate phos-phatidylinositol 4,5-bisphosphate breakdown (Best,1986).

The results shown in Fig. 5 indicate an optimumconcentration for the potentiating effect of TPA. Theoccurrence of a bell-shaped dose—response curve of thissort following application of a pharmacological agent isnot unusual. In this case it may be due to a feedbackinhibition of phosphatidylinositol 4,5-bisphosphatebreakdown by the activated protein kinase C in thehigher TPA concentrations, as shown for other phorbolester-treated cells (Berridge, 1986). Alternatively,higher concentrations of TPA may have a toxic effecton the cells.

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(Received 22 April 1987 -Accepted 22 May 19S7)

Tentacle contraction in Heliophrya 127