The mode of action of acetylcholine and 5-hydroxytryptamine at the neuromuscular junctions in a molluscan muscle (radular protractor)

Comp. Biochem. Physiol. Vol. 60C, pp. 115-1 ~

0306-4492/78/I)701-0115502.00/0

Pergamon Press Ltd 1978. Printed in Great Britain

THE M O D E OF ACTION OF ACETYLCHOLINE A N D 5-HYDROXYTRYPTAMINE AT THE N E U R O M U S C U L A R J U N C T I O N S IN A M O L L U S C A N MUSCLE (RADULAR PROTRACTOR) MAKOTO KOBAYASHIand YOSHINOBU SHIGENAKA Laboratories of Physiology and Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan

(Received 6 September 1977) Abstract--1. In the radular protractor of Rapana thomasiana, the nerve endings among muscle fibers contained 2 types of synaptic vesicles; granular and agranular. In most nerve endings both types of vesicles were found together. 2. Electrical stimulations of the muscle showed elevation of excitability of the muscle by 5-hydroxytryptamine (5-HT). 3. A mixture of 5-HT and acetylcholine (ACh) induced repetitive activities of the muscle. These rhythmic responses were abolished in Ca 2 +-free solution or by Mn z+, indicating that the rhythmic responses depended o n C a 2 +. 4. Peak potential of ACh-induced depolarization was not affected by [Ca2+]o, though the resting potential was lowered at decreased [ C a 2 + ] o . At decreased [Na+]o, membrane hyperpolarized and ACh-induced depolarization was depressed remarkably. These results suggest that ACh-induced depolarization depends mainly on Na + inflow.

MATERIAL AND METHODS

INTRODUCTION

It is generally accepted that acetylcholine (ACh) is an excitatory substance in molluscan muscular systems. Especially, the mode of activation of the contractile mechanism-by ACh has been intensively studied by using the anterior byssal retractor muscle of Mytilus (Twarog, 1954, 1960, 1967; Twarog & Muneoka, 1973; Sugi & Yamaguchi, 1976). In contrast, little is known about the mechanisms of electrical responses to ACh in the neuromuscular systems. The significance of 5-hydroxytryptamine (5-HT) as a physiologically active substance in certain molluscan neuromuscular synapses is well known (Gerschenfeld, 1973; Heyer et al., 1973). Effects of 5-HT, however, appear to be variable depending on the kind of tissues (Hidaka et al., 1967; Hill et al, 1970; Kobayashi, 1974; Wabnitz & yon Wachtendonk, 1976). In the radular muscle of a gastropod, Busycon, Hill (1958, 1970) studied the pharmacological effects of several postulated neurohumoral transmitters, and found that the muscle was contracted by ACh and could then be relaxed rhythmically by 5-HT, tryptamine and adrenaline. One of the present authors (Kobayashi, 1974) preliminarily demonstrated the effects of a few neurohumors on the radular protractor of Rapana, but the mechanisms of action of these neurohumors still remain to be clarified. The present experiments were undertaken to investigate luther the mode of action of ACh and 5-HT on the radular protractor and to pursue the ionic mechanisms of electrical and mechanical responses of the muscle to these drugs. Some observations on the fine structure of the neuromuscular junctions were also made, providing a histological basis to the physiological findings.

Specimens of a gastropod, gapana thomasiana Crosse, freshly caught or kept for a few days in constantly circulating seawater were used.

Histology Immediately after pinning out the buccal mass and immobilizing the radular protractor, the muscle was prefixed in 3% glutaraldehyde and postfixed in 1% OsO, after isolation. Thin slices of the musculature were dehydrated with graded ethanol and embedded in Spurr's low-viscosity embedding medium (Sparr, 1969). Sections were stained with 3% uranyl acetate and Reynolds' lead citrate stain (Reynolds, 1963), and examined by a JEM-100S electron microscope.

Electrophysiolooy Nerve-muscle preparations were prepared from the radular protractor as described previously (Kobayashi, 1972a). The electrical activity of the muscle was recorded by using the sucrose gap or intraceilular microelectrodes filled with 3 M KCI having resistance of 20-50 M~. The sucrose gap method used in this experiment was essentially the same as reported previously (Kobayashi, 1972a). Wheff the microelectrode recording method was employed, the preparation of a few muscle bundles was mounted over the indifferent electrode of silver-silver chloride wire with a diameter of 1 ram, as shown in Fig. 1. A small piece of cartilage attached to the proximal end of the muscle and that of the radular membrane attached to the distal end were connected through 2 pulleys to the strain gauge for tension recording. Both the electrical and mechanical activities were simultaneously displayed on a cathode-ray oscilloscope, and the tracings were photographed with a kymograph camera. Electrical stimulations were given to the muscle directly or indirectly via nerve lightly sucked into a suction electrode. The standard artificial seawater used in this experiment contained (in raM): NaCI 461.9, KCI 9.4, CaCI2 9.0, MgCI2 54.0, tris (hydroxymethyl) aminomethane (Tris) 20.0 (pH 115

MAKOTO KOBAYASHI and YOSHINOBU SHIGENAKA

116

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RESULTS

Histolooy It is known that the radular protractor is innervated by radular nerves originating from a pair of buccal ganglions. The radular nerve subdivides, as it proceeds anteriorly, until finally forming single axons. Histological examinations of the tissue have revealed many nerve endings surrounded by muscle fibers, thus supporting the suggestion that each fiber of the radular protractor receives multiple innervations (Hill et al., 1970). In Fig. 2, a bundle of nerve fibers between 2 muscle cells is shown, and some nerves seem to

be rather close to the terminal. The size of nerve fibers found in such places ranged usually from 0.5 to 2 ~m in diameter, and they contained many synaptic vesicles. In some nerve endings only one type of vesicle was found as illustrated by an ending in Fig. 3, but in most nerve endings both granular and agranular vesicles were observed together (Figs 3 and 4). Twentyfive nerve terminals were examined in the present study, and there was a significant difference in the size of two types of synaptic vesicles. The granular vesicles ranged in size from 70 to 110 nm with a mean diameter of 92 nm, while the agranular ones ranged from 40 to 60 nm with a mean diameter of 47 nm. The junctional gap was about 20-30 nm, and both pre- and post-junctional membranes appeared to be thicker and denser (arrow in Fig. 4).

Electrophysiotogy The effect of 5-hydroxytryptamine (5-HT) on the radular protractor muscle was examined in a previous paper (Kobayashi, 1974), in which 5-HT at lower concentrations up to 10- ~ M elicited no apparent responses. In the present experiment the excitability of the muscle during the application of 5-HT was further studied by using the sucrose gap method. Figure 5 shows the responses to electrical stimulations in fluids containing various concentrations of 5-HT. In each fluid, stimulations of four kinds of strength (1. 2, 3 and 5 V) were given stepwise to the muscle via nerve through a suction electrode. In the control (seawater without 5-HT) only graded junctional potentials were obtained. In the fluid containing 10-7 M 5-HT, bigger responses were elicited and

Fig. 2. An electron micrograph of a bundle of nerve fibers between 2 muscle cells x 41,000.

ACh and 5-HT at molluscan neuromuscular junction

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Fig. 3. Nerve endings in contact with a muscle cell One nerve ending including mitochondrion (m) is packed with agranular vesicles, but others have both granular and agranular vesicles, x 41,000. a hUmplike potential accompanied by a small tension occurred by maximal stimulation. At 1 0 - 6 M 5-HT. an action potential of more than 50 mV followed by a large contraction was produced. Increment of responses in solutions containing 5-HT was also

observed even when the muscle was directly stimulated after inactivating the nerves b~ tetrodotoxin (10 -~ g/ml). These results clearly show that the excitability of the muscle was elevated by 5-HT, although no direct response to the drug was demonstrated.

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Fig. 5. Effect of 5-HT on the excitability of the muscle. In each column of this figure and Fig. 6, upper tracings are electrical activities recorded by the sucrose gap method, and lower tracings mechanical activities by a strain gauge. Vertical calibrations, 20 mV (upper) and 200 mg (lower). Horizontal calibration, 200 msec.

It has been known that acetylcholine (ACh) induces in the radular protractor muscle a depolarization followed by a contraction, and a typical example of ACh effects is shown in Fig. 6-1. However, when the muscle was pretreated by 5-HT of lower concentrations ( 1 0 - T - 1 0 - S M ) , the application of ACh evoked remarkable oscillatory potentials superimposed on the rising and the plateau phases of the depolarization (Fig. 6-2). Concurrent with these electrical firings, re-

petitive contractions occurred. Oscillatory responses usually continued for about 10sec, but sometimes they were maintained for more than 1 rain. The frequency of the activities was from 0.8/sec to 1.3/sec with the mean value of about 1/sec. In Ca 2 +-free solution, these repetitive responses in the presence of both 5-HT and ACh were completely abolished leaving the slow depolarization (Fig. 6-3). Manganese ion of 10mM, which blocked the Ca-

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Fig. 6. Effect of a mixture of 5-HT and ACh on the muscle. 1. At the arrow. ACh of 10 -6 M was applied. 2. 5-HT of 10-6M was applied at the first arrow and ACh of 1 0 - 6 M was given at the second arrow. 3. Same procedure as in 2. performed in Ca-" "-free solution. Vertical calibrations. 20 mV (upperl and 500 mg (lower). Horizontal calibration. 10 sec.

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potential of the radular protractor (Kobayashi, 1972b), also eliminated repetitive responses. These results suggest that oscillatory responses are dependent on Ca 2 +, though ACh depolarization is related with the mechanism including one or more other ions. In the following experiments, the ionic meohanisms of ACh-induced depolarization were pursued by using intracellular microelectrodes. Figure 7 shows the procedure and the results of a series of experiments, where the resting potential and the peak potential of depolarization induced by a constant concentration of ACh in various kinds of solutions are illustrated. Mechanical tension is not plotted in this figure. In abscissa of the graph the kinds of solutions tested and the time in hours are shown. The muscle was immersed in each solution for at least 15 min before ACh application. The solid circles represent the results in the control seawater (N), and the open cirdes show the results in the test solutions (T1-Ts). The value of the resting potential and the magnitude of depolarization by ACh in the control seawater showed a tendency to decrease with lapse in time. Therefore, the control trial was performed in all 3 trials, and when either one or both of the resting potential and the magnitude of depolarization dropped to less than 80% of the values observed at the first control trial, the series of experiments was stopped. In the case cited in Fig. 7, the experiment was continued for 4 hr. It is evident from this experiment that the peak potential of ACh depolarization was little affected by the concentration of Ca 2+ in the fluid ([Ca'+]o) although the resting potential was affected. It is also clearly demonstrated that when Na + in the solution was replaced with Tris both the resting potential and the peak potential of depolarization became much more negative compared with the control values, but when Li + was substituted for Na ÷ both potentials were not so different from the controls.

Several examples of the actual oscillographic tracings are shown in Fig. 8, where the electrical and mechanical responses to ACh in solutions of three kinds of [Ca'+]o at both normal Na + and Na+-free are illustrated. The general contour of the depolarization curve was similar irrespective of [Ca~+]o, thotlgh the magnitude varied. It is noteworthy that only a very small contraction developed in Na+=free solution, even if it contained 90 mM Ca 2 +. Such an experiment as illustrated in Fig. 7 was performed many times with a combination of various kinds of solutions, and the results are summarized in Figs 9 and 10. In Fig. 9, the resting potential, the peak potential of ACh depolarization and the tension which developed with depolarization are plotted against [Ca'+]0. The mean value with standard error of the resting potential at 9 mM (control) and 90 mM (xl0) of [Ca'+]o were - 4 5 + l . l m V and - 4 8 + 3.1 mV, respectively. Although the difference between two values was not statistically significant, in a series of experiments using the same preparation a larger resting potential at higher [Ca'+]o was generally observed. When [Ca'+]o was lowered to 1 mM or in C a ' +-free, the resting potential was less than - 4 0 mV, and the values were significantly lower than the control. It is assumed that the permeability of Na + ,, s increased at lower [Ca2+]0 and greater Na + inflos, occurred to depolarize the membrane. On the other hand, the peak potentials of ACh depolarization were very similar to each other irrespective of [Ca z +'lo. The values measured at six different [Ca'+]o ranged from - 2 0 . 5 m Y to -23.5mV, but the differences between them were all insignificant. This suggests that ACh depolarization depends mainly on ions other than Ca z + and that the equilibrium potential for ACh is about - 2 0 to - 2 3 m V . The tension which developed following depolarization did not show any significant change when [Ca'+]o was varied from 4.5 to 90raM. At 1 mM

MAKOTO KOBAYASHIand YOSHINOBUSHIGENAKA

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[-Ca2 ~']o, however, the tension decreased remarkably, and in CaZ+-free solution no tension developed in most cases. Results in solution free from Ca z + and Na + by replacing with Tris are inserted in Fig. 9 for comparison. Figure 10 shows the resting potential, peak potential of ACh depolarization and tension at various [Na+]o. When [Na+]o was decreased by replacing Na + with Tris, the membrane hyperpolarized and the peak potential of ACh depolarization was remarkably depressed. At 46.2mM [Na+]0 (1/10 of normal) or less, the peak potentials were - 4 6 to - 4 8 mV and these values were about 25 mV more negative than the value at normal [Na+]0. Moreover, the peak potential in Na+-free solution ( - 4 7 m V ) was not appreciably changed even if [Ca 2 +]o was varied from 9 0 m M to 0 r a M . The magnitude of ACh depolarization at lower [Na+]o was reduced to less than half of that in normal [Na+]o. The tension following the

depolarization was further depressed at low [Na+]o and Na+-free; they were only about 50 mg compared to 600 mg at the control. However, when Na + was replaced by Li + instead of Tris, the resting potential, peak potential of ACh depolarization and developed tension were all about the same as the control values in normal seawater. The concentration of Mg 2+ in the fluid did not have any effects on the resting potential nor on the peak potential of ACh depolarization. These results suggest that depolarization by ACh in normal seawater is induced mainly by Na + inflow and that Li ÷ can be substituted for Na + when Na + is lacking. DISCUSSION In the present, study, the existence of 2 types of synaptic vesicles was demonstrated electron microsco-

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ACh and 5-HT at molluscan neuromuscular junction

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pically. The granular vesicles, similar to those found similar experiments on the molluscan neuromuscular in serotoninergic nerve endings, may be 5-HT storage systems except for a recent study on ABRM of sites, while the agranular vesicles, similar to those in Mytilus by Hidaka and Twarog (1977). In the present cholinergic ones. may be ACh storage sites. It is note- study, it was demonstrated that ACh depolarization worthy that both granular and agranular synaptic depended mainly on Na + inflow by means of the invesicles are being found in the same nerve endings. tracellular microelectrodes, although the voltage Similar observations are not rare in molluscan clamp technique was not employed. Since the equilibneuromuscular systems, that is, the mixing of both rium potential for ACh was - 2 0 to - 2 3 mV. the intypes of vesicles in nerve endings has been reported crease of conductance of Na + and possibly K + in the anterior byssal retractor muscle (ABRM) of and/or Cl- also may be included. Mytilus, lamellibranch (McKenna & Rosenbluth. It is rather difficult to interpret the mechanisms 1973), the radular muscle of Busycon, prosobranch of some depolarization induced by ACh in Na+-free (Hill & Sanger, 1974), the buccal muscle of Aplysia, solution. The peak potential of ACh depolarization opisthobranch (Orkand & Orkand, 1975) and the ten- was not affected by changing [Ca2+]o, and Mg 2÷ had tacle muscle of Va#inula, pulmonate (Barrantes, 1970). actually no effect on the membrane potential. Thus, Thus, it remains possible that there may be two kinds one plausible possibility is that Na +, which flowed of neuro-transmitter in nerve endings, and it can be out of the cell and remained in the vicinity outside said that such nerve endings have been rather of the cell, might be utilized as charge carriers. As frequently demonstrated in molluscs. another possibility, the influence of change in C l ' One of the striking findings in the present experi- conductance may be considered. ments is that rhythmical contractions were induced As shown in the present experiment, in the radular in the radular protractor, a phasic muscle, by a mix- protractor of Rapana, variation of [Ca2+]o between ture of 5-HT and ACh. As indicated in the present 90 mM and 4.5 mM had no effect on the magnitude study 5-HT even at lower concentrations of 10 -6 or of ACh-induced contraction, whereas in Ca2+-free 10-~M raised the excitability of muscle, and more- solution the contraction was depressed to zero. On over, at higher concentrations it directly evoked exci- the ACh-contracture of ABRM in Mytilus, Sugi and tatory responses (Kobayashi, 1974). Thus, 5-HT seems Yamaguchi (1976) obtained similar results and to be a physiologically active substance at the neuro- thought that ACh-contracture was mainly due to muscular junctions in the radular protractor of release of intracellularly stored Ca 2+. On the conRapana. Welsh and Moorhead (1959) have actually trary, Muneoka (1973) has postulated that the inward identified the presence of 5-HT in the radular muscle movement of external Ca 2 + is a source of ACh-conof Busycon. ACh has not yet been identified in radular tracture of ABRM, since the contracture in Na+-free muscles or nerves. However, it is plausible that ACh solution depended on ['Ca'+']o. In the radular prois an excitatory neuro-transmitter in the radular tractor of Rapana, however, before conclusion on the protractor, since ACh depolarized the muscle even origin of Ca' ÷ for ACh contraction can be drawn, in the concentration of 10 - s M, curare diminished the some histological evidences, as presented by Atsumi amplitude of junctional potential while eserine and Sugi (1976) in ABRM, would be necessary. It enhanced it, and Ca ' + and Mg '÷ showed antagonis- may be possible that some inward flow of external tic action at the neuromuscular junction (Kobayashi, Ca 2+ is required for the release of stored Ca 2÷. 1972a, 1974). These physiological findings as well as histological Acknowledgement--This research was supported in part observations may suggest the possibility of the pres- by a grant-in-aid from the Japanese Ministry of Education to M. K. ence of two types of excitatory synapses. Although rhythmic activities in spontaneously firing muscles have been well investigated from the REFERENCES point of membrane physiology, the ionic mechanism of the induced rhythmicity in the phasic muscle such ATSUMI S. & SUGI H. (1976) Localization of calcium-accumulating structures in the anterior byssal retractor as that in the radular protractor has hardly been studmuscle of Mytilus edulis and their role in the regulation ied at alL In the present study, rhythmic activities of active and catch contractions. J. Physiol., Lond. 257, are shown to be Ca2+-dependent. It was suggested 549-560. that the internal free Ca 2÷ concentration was lowered BARRAWrES F. J. (1970) The neuromuscular junctions of by 5-HT in ABRM of Mytilus (Hidaka et al., 1967; a puimon~tte mollusc I. UItrastructural study. Z. ZellMuneoka et al., 1977). This might be true in the raduforsch, microsk. Anat. 104, 205-212. lar muscle, too, and the Ca 2+ conductance might be CHIARANDINI O. J., STEFANI E. ,R, GERSCHENFELD H. M. increased by 5-HT. It is plausible that rhythmic re(1967) Ionic mechanisms of cholinergic excitation in sponses were elicited by ACh depolarization, when the molluscan neurons. Science, N.Y. 156, 1597-1599. GEV,SCH~Nt~t.D H. M. (1973) Chemical transmission in inpermeability of Ca 2+ had been elevated. vertebrate central nervous systems and neuromuscular The ionic mechanisms of postsynaptic actions of junctions. Physiol. Ret,. 53, 1-119. ACh have been studied in molluscan neurons and cardiac muscles, and the depolarizing response of D cells HEVERC. B., KA'IT~.S. B. & KARLSSO~U. L. (1973) Neuromuscular systems in molluscs. Am. Zool. 13, 247-270. of Helix or Aplysia (Kerkut & Meech, 1966; Sato HIDAKA T. ,~' Tw~OG B. M. (1977) Neurotransmitter et al., 1968), CILDA cells of Cryptomphallus (Chiaranaction on the membrane of Mytilus smooth muscle---I. dini et al., 1967), and the heart muscle cells of Mytilus Acetylcholine. Gen. Pharmac. 8, 83-86. (Shigeto, 1970) and of Modiolus (Wilkens & Green- HIDAKAT.. Os^ T, & TWAROGB. M. (1967) The action berg, 1973) has been proven to be induced by the of 5-hydroxytryptamine of Mytilus smooth muscle. J. Physiol., Lond. 192, 869-877. increase of Na + conductance. There is a paucity of c.n,P. 60'2c--~

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HILL R. B. (1958) The effects of certain neurohumors and of other drugs on the ventricle and radula protractor of Busycon canaliculatum and on the ventricle of Stromhus 9ig/as. Biol. Bull. 115, 471-482. HILL R. B. (1970) Effects of postulated neurohumoral transmitters on the isolated radular protractor of Busycon canaliculatum. Comp. Biochem. Physiol. 33, 249-258. HILL R. B. & SANGERJ. W. (1974) Anatomy of the innervation and neuromuscular junctions of the radular protractor muscle of the whelk, Busycon canaliculatura (L.). Biol. Bull. 147, 369-385. HILL R. B.. GREENBERG M. J.. IRISAWA H. & NOMURA H. (1970) Electromechanical coupling in a molluscan muscle, the radula protractor of Busycon canaliculatum. J. exp. Zool. 174, 331-348. KERKUT G. A. & MEECH R. W. (1966) The internal chloride concentration of H and D cells in the snail brain. Comp. Biochem. Physiol. 19, 819-832. KOBAYASm M. (1972a) Electrical and mechanical activities in the radula protractor of a mollusc, Rapana thomasiana. J. comp. Physiol. 78, 1-10. KOBAVASHIM. (1972b) Prolonged depolarization of a molluscan muscle in Ca-free solution. J. comp. Physiol. 78, 11-19. KOBAVASHi M. (1974) Antagonistic action of Ca and Mg on the neuromuscular transmission and excitation in a molluscan muscle (radula protractor). J. comp. Physiol. 94, 17-24. MCKENNA O. C. & ROSENBLUTHJ. (1973) Myoneural and intermuscular junctions in a molluscan smooth muscle. J. Ultrastruct. Res. 42, 434--450. MUNEOKA Y. (1973) Calcium-dependent acetylcholine contracture of a molluscan catch muscle in sodium-free solution. Comp. g/en. Pharmac. 4, 277-284. MUNEOKA Y., COTTRELL G. A. & TWAROG B. M. (1977) Neurotransmitter action on the membrane of Mytilus smooth muscle--Ill. Serotonin. Gen. Pharmac. 8, 93-96. ORKAND P. M. & ORKAND P. K. (1975) Neuromuscular junctions in the buccal mass of Aplysia: Fine structure and electrophysiology of excitatory transmission. J. Neurobiol. 6, 531-548.

REYNOLDS E. S. (1963) The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212. SATO M, AUSTIN G., YAI H. & MARUHASHIJ. (1968) The ionic permeability changes during acetylcholine-induced responses of Aplysia ganglion cells. J. yen. Physiol. 51. 321-345. SHIGETO N. (1970) Excitatory and inhibitory actions of acetylcholine on hearts of oyster and mussel. Am. J. Physiol. 218, 1773-1779. SPURR A. R. (1969) A low-viscosity Epoxy resin embedding medium for electron microscopy. J. UItrastruct. Res. 26, 31-43. SUGI H. & YAMAGUCHIT. (1976) Activation of the contractile mechanism in the anterior byssal retractor muscle of Mytilus edulis. J. Physiol., Lond. 257, 531-547. TWAROG B. M. (1954) Responses of a molluscan smooth muscle to acetylcholine and 5-hydroxytryptamine. J. cell. comp. Physiol. 44, 141-163. Twarog B. M. (1960) Effects of acetylcholine and 5-hydroxytryptamine on the contraction of a molluscan smooth muscle. J. Physiol., Lond. 152, 236-242. TWAROG B. M. (1967) Excitation of Mytilus smooth muscle. J. Physiol., Lond. 192, 857-868. TWAROG B. M. & MUNEOKA Y. (1973) Calcium and the control of contraction and relaxation in a molluscan catch muscle. In The Mechanism of Muscle Contraction. Cold Spring/ Harb. Syrup. quant. Biol. 37, 489-504. WABNITZ R. W. & WACHTENDONKD. YON (1976) Evidence for serotonin (5-hydroxytryptamine) as transmitter in the penis retractor muscle of Helix pomatia L. Experientia 32, 707-709. WELSH J. H. & MOORHEAD M. (1959) Identification and assay of 5-hydroxytryptamine in molluscan tissues by fluorescence method. Science. N.Y. 129, 1491-1492. WlLKENS L. A. & GREENBERG M. J. (1973) Effects of acetylcholine and 5-hydroxytryptamine and their ionic mechanisms of action on the electrical and mechanical activity of molluscan heart smooth muscle. Comp. Biochem. Physiol. 45A, 637-651.