The mechanisms by which high-K+ saline and acetylcholine (ACh) mobilize cellular calcium in molluscan muscle was studied in three proboscis muscles of Busycon canaliculatum. BAY K 8644, a calcium agonist, enhanced tonic force induced by exposure to 25 mmol l−1 K+ in the odontophore and radular retractor muscles but inhibited this response in the radular sac muscle. Its effect on the radular protractor muscle was concentration-dependent, excitatory at 10−7 mol l−1 and inhibitory at higher concentrations. The tonic response to 80 mmol l−1 K+ was enhanced by BAY K 8644 in the odontophore retractor and radular retractor muscles but inhibited in the radular sac muscle. In all muscles, BAY K 8644 eliminated the fast twitches induced by 80 mmol l−1 K+, and inhibited the tonic ACh responses. Sucrose-gap studies with the radular protractor muscle showed that the effects of BAY K 8644 on the depolarizations induced by K+ and ACh were similar. The effect on the ACh-induced depolarization suggested that this agent had inhibited sodium influx through the ACh receptor.

Mytelase enhanced ACh-induced tonic force, but at 10−4mol l−1 it inhibited the response to doses of ACh above 10−5mol l−1. Mytolon reduced the maximum ACh-induced tonic force and increased the response threshold to ACh. ACh and high-K+ depolarization pathways were not synergistic for force development.

The existence of two separate cellular calcium pools, independently released by high-K+ saline and ACh, seems improbable. We suggest that K+ and ACh act via separate mechanisms to release calcium from a single cellular calcium pool.

The proboscis muscles of Busycon canaliculatum require extracellular calcium for contraction (Huddart et al. 1990b). A recent study of three of these muscles, the odontophore retractor, radular retractor and radular sac muscle, has revealed a great diversity in their responses to exposure to potassium or acetylcholine (Huddart et al. 1990a). These muscles all showed large differences in the way in which they responded to some commonly employed calcium antagonist drugs of mammalian smooth muscle (Huddart et al. 1990b). The antagonists studied, diltiazem, nifedipine and verapamil, had little effect upon sustained tonic force induced by exposure to high-K+ saline but they appeared to enhance fast twitch activity induced by low-K+ saline. They had differential effects on ACh-induced activity and K+-induced activity in these muscles. All three antagonists reduced or eliminated ACh-induced tonic contractures but two, verapamil and nifedipine, like serotonin (Hill, 1958), induced fast twitches in the responses to ACh (Huddart et al. 1990b).

The ACh response of Busycon proboscis muscles is normally a smooth maintained tonic contracture without twitch activity (Hill et al. 1970; Hill and McDonald-Ordzie, 1979; Hill and Licis, 1981, 1985; Huddart and Hill, 1988; Huddart et al. 1990a). These previous studies suggest that K+-induced and ACh-induced depolarization of these muscles act through quite separate membrane transduction routes upon the cellular stores of calcium required for contraction. This might explain why the responses of some molluscan muscles to inorganic calcium antagonists such as manganese and calcium-free media (Reilly and Peretz, 1987) are different from the responses to the organic calcium antagonists used in our recent experiments.

A more radical explanation of the differences in reaction of ACh and K+ in molluscan muscle to these agents has been proposed which envisages that ACh and K+ may act to release calcium from two separate independent cellular calcium stores (Bloomquist and Curtis, 1975; Sugi and Yamaguchi, 1976; Reilly and Peretz, 1987). If this were so, it would explain why the responses of molluscan muscle to K+ and ACh are so different in the presence of agents modulating calcium movement across the muscle fibre membrane.

Here we have attempted to differentiate between K+-induced calcium influx and ACh-induced sodium influx as induction mechanisms for the release of cellular calcium. We have used the calcium agonist BAY K 8644, the anti-cholinesterase Mytelase and the anti-cholinergic Mytolon to differentiate between the two cellular calcium access pathways in ACh/K+ synergism studies. Our results do not support the view that ACh-and high-K+-induced depolarization gain access to independently modulated cellular calcium pools. Instead, they suggest that in these muscles high-K+ saline and ACh may act via separate transduction mechanisms upon a single cellular calcium pool.

Mature Busycon canaliculatum, weighing 250–350 g, were obtained from Wood Hole Marine Biological Laboratory and kept in aerated sea water at 16°C. The radular protractor, the radular retractor, the odontophore retractor and the radular sac muscle were dissected from the proboscis. The location and activity of these muscles have been described elsewhere (Huddart et al. 1990a). The muscles were placed in aerated sea water, and ligated at their insertions with fine monofilament nylon. They were then suspended in glass-jacketed organ baths between a glass hook and the force plate of Grass FT. 03 force-displacement transducers. A passive load of 1 g was applied to the radular retractor muscles and a load of 0.5 g was applied to the other muscles. The transducer outputs were connected to four-channel Grass model 7 polygraphs to display tension developed. The organ baths were cooled to 14 °C with a closed-circuit cooler/circulator system and the bath contents were constantly aerated.

High-K+ depolarization of the muscles was achieved by direct addition of appropriate amounts of 3 mol l−1 KC1 stock solution. Responses obtained in this way do not differ from those obtained with equimolar K+/Na+ substitution (Huddart and Hill, 1988). Acetylcholine was applied to the preparations by direct addition to the baths of 10 mmol l−1 stock solution made up in sea water. After setting up, the preparations were allowed to equilibrate for 30 min and a standard time interval of 20 min was allowed between high-K+ and ACh exposures.

The calcium agonist BAY K 8644, a gift from Bayer Pharmaceuticals, was made up as a 25 mmol 1−1 stock solution in ethanol. The final ethanol level did not exceed 0.1%, which has no effect on the preparations (Huddart and Hill, 1988). Experiments with BAY K 8644 were carried out in low lighting because of the photosensitivity of this compound. The anti-cholinesterase Mytelase (amben-onium chloride) and the cholinoreceptor antagonist Mytolon (benzoquinonium chloride) were gifts from the Sterling-Winthrop Research Institute and were made up as 10 mmol l−1 stock solutions in sea water.

The electrophysiological effects of BAY K 8644 were recorded using a single sucrose-gap system described previously (Hill and Licis, 1985; Huddart and Hill, 1988). Briefly, the muscles were drawn through fine holes in the sucrose compartment rubber membranes and their ligatures attached to right and left tension transducers. Compound membrane potential differences between the two lateral compartments were monitored with IVM silver/silver chloride flat-tipped probes and displayed, along with the force transducer outputs, on a Grass model 7 polygraph. Through the central compartment flowed 0.75 mol l−1 sucrose, with sea water flowing through the lateral compartments. Each experiment was begun with a depolarization of the reference (right) end of the muscle using isotonic 0.56 mol l−1 KC1. This side of the muscle was kept depolarized throughout the experiment. The membrane potential difference between right and left compartments represented the compound resting potential of the left end of the muscle, which was usually maximal within 3–6 min of depolarization. Drugs and test agents in sea water were introduced to the left (test) compartment and the resulting membrane potential changes and tension developed were recorded simultaneously.

The effects of BAY K8644

BAY K 8644 is a dihydropyridine which is a very effective slow calcium channel agonist in several mammalian smooth muscles (Kanmura et al. 1984; Finet et al. 1985; Gopalakrishnan et al. 1985; Mikkelsen, 1985), although Langton and Huddart (1988) found that it also enhanced fast twitch activity in rat prostatic vas deferens, an activity thought to depend upon fast voltage-sensitive calcium channels.

BAY K 8644 had different effects on the different muscles. In the odontophore and radular retractor muscle, it enhanced tonic force in response to 25 mmol l−1 K+ without chronotropic effect on fast twitch activity, and recovery took about 2 h (Fig. 1A,B). In the radular sac muscle, BAY K 8644 caused slight inhibition of the tonic force in response to 25 mmol l−1 K+. At the highest concentration used (10−5mol l−1) BAY K 8644 itself caused a small drop in resting tone in the muscle. Recovery of the response after this treatment was poor, with over 40% force inhibition 2h after a washout in sea water (Fig. 1C).

In the radular protractor, the action of BAY K 8644 on the response to 150 mmol l−1 K+ was concentration-dependent, being excitatory at 10−7mol l−1 but inhibitory at higher concentrations (Fig. 2). This was mirrored in the sucrose-gap recordings which showed that the K+-induced depolarization was unaffected by 10−7mol l−1 BAY K 8644, but showed 25% inhibition at 10−6moll−1.

The response to 80 mmol l−1 K+ was also affected differently in different muscles by BAY K 8644. In the odontophore retractor and the radular retractor muscles, fast twitch activity was greatly reduced by BAY K 8644, but tonic force was slightly enhanced, not an effect expected from an agent acting upon slow calcium channels (Fig. 3). In contrast, the maintained tonic force in response to 80 mmol l−1 K+ of the radular sac muscle was inhibited in a dose-dependent manner, up to nearly 50% inhibition at 10−5mol l−1 BAY K 8644; initial tonic force was slightly enhanced with 10−6 mol 1−1 BAY K 8644 but was not maintained (Fig. 4). As with the other two muscles, fast twitch activity of the radular sac muscle was eliminated by BAY K 8644. While the odontophore and radular retractors recovered well from BAY K 8644 treatment, the radular sac muscle remained greatly inhibited in both phasic and tonic components even 1 h after washout (Fig. 4).

BAY K 8644 consistently inhibited the response of all four muscles to 5×10−6mol l−1 ACh. Sucrose-gap studies of the radular protractor muscle showed that BAY K 8644 at a concentration as low as 10−7mol l−1 reduced both ACh-induced depolarization and subsequent tonic force by over 60% (Fig. 2). The radular sac muscle was also strongly inhibited by BAY K 8644, the ACh-induced tonic force being reduced by 50% with 10−5mol l−1 BAY K 8644, and very poor recovery was seen even l h after washout with sea water (Fig. 5). In the odontophore retractor the ACh response was slightly inhibited by BAY K 8644, while in the radular retractor muscle the maintained tonic force in the presence of 10−5 mol l−1 BAYK8644 showed frequent transient relaxations. In all four muscles, tonic force was not well sustained in the presence of BAY K 8644 (Fig. 5).

The effects of Mytelase

In these muscles BAY K 8644 was not selective enough to differentiate well between K+-induced and ACh-induced membrane depolarization events. In the hope of selectively examining the ACh route leading to release of cellular calcium for contraction, we used Mytelase (ambenonium chloride), a potent selective antiacetylcholinesterase with prolonged stable action; it is not itself hydrolysed.

Mytelase enhanced the tonic force developed due to cumulative ACh-induced depolarization without altering receptor threshold (Fig. 6). However, at 10−4moll−1, Mytelase clearly enhanced the response to 10−5mol l−1 ACh but inhibited responses to higher doses of ACh, suggesting receptor desensitization, a phenomenon not seen in control responses up to 10−4mol l−1 ACh in the absence of Mytelase (Fig. 6E). There is evidence that Mytelase may have anti-cholino-receptor activity in the heart of Venus mercenaria (Luduena and Brown, 1952), which may also explain the sharp inhibition of responses to very high doses of ACh at high concentrations of Mytolon (Figs 6, 7B).

The three muscles were similar in ACh dose–response relationship, as shown in Fig. 7A for the radular sac muscle and the odontophore retractor muscle, and in Fig. 7B for the radular retractor muscle. The effect of Mytelase, shown in Fig. 7B for the radular retractor muscle, was similar in all the muscles.

The effect of Mytolon

To determine whether ACh-induced increases in cellular calcium level (Huddart and Hill, 1988) did indeed operate via the ACh/cholinoreceptor interaction, causing sodium-dependent depolarization (Hill and McDonald-Ordzie, 1979), we employed Mytolon (benzoquinonium chloride), a powerful anti-cholinoreceptor neuromuscular blocking drug. The effects of Mytolon on ACh dose–response relationships were examined in the odontophore retractor and radular retractor muscles. Mytolon not only shifted the threshold to higher ACh levels but, at 10−4mol l−1, it also significantly reduced the maximum response to ACh by 55 %. Using the standard pA2 determination method, Mytolon was found to show competitive antagonism to the cholinoreceptors in the muscles. A pA2 value of −6 was obtained, as to be expected if ACh were activating cellular calcium via cholinoreceptors. We used Mytolon to examine the relationship between ACh-and K+-operated pathways for the control of cellular calcium concentration (e.g. Fig. 8). The ACh dose-response curve for the radular retractor muscle in the presence of increasing concentrations of Mytolon shows the inhibitory action of this blocker (Fig. 9).

ACh/K+ synergism

The Mytelase experiments showed that anti-acetylcholinesterase action could enhance calcium release from the cellular pool normally accessed by ACh. To test whether this calcium pool was also accessed by K+-induced depolarization or whether a separate K+-accessed calcium pool was present and was independent of ACh action, we exposed all three muscles to 80 mmol l−1 K+ and compared the responses with the response to 80 mmol l−1 K+ followed by 10−5 mol l−1 ACh, and then with the response to ACh followed by high-K+ saline. Since these are concentrations of potassium and acetylcholine which induce on their own between 50 and 60 % of maximal force, any synergistic effects would be noticeable. In none of these experiments (e.g. Fig. 10) did we observe any synergism in the interaction of the two depolarization pathways.

The buccal muscles of Busycon show an acute dependence upon extracellular calcium for contraction (Huddart and Hill, 1988; Huddart et al. 1990b) but contain a cellular pool of calcium, like other molluscan muscles. This pool needs continuous re-priming from the external medium. Such a pool has been shown in molluscs by oxalate and pyroantimonate calcium deposition studies (Heumann, 1969; Sugi and Atsumi, 1973; Atsumi et al. 1974; Suzuki and Sugi, 1978), by energy-dispersive X-ray microanalysis (Huddart et al. 1977) and by radiocalcium exchange (Hagiwara and Nagai, 1970; Tameyasu and Sugi, 1976). In several of these studies, cellular calcium translocation has been observed in muscles fixed during ACh-and K+-induced contracture and the consensus view is that at rest the calcium pool is peripherally located just below the plasma membrane whereas during mechanical activity it becomes diffusely distributed throughout the myoplasm (Heumann, 1969; Sugi and Atsumi, 1973; Huddart et al. 1977; Suzuki and Sugi, 1978). In addition, isolated submembrane vesicles have been shows in vitro to be able to take up radiocalcium (Gogjian and Bloomquist, 1977; Huddart et al. 1977).

Submembrane vesicles are common in molluscan muscle (Dorsett and Roberts, 1980; Hunt, 1981) and the radular protractor of Busycon, the only proboscis muscle examined in this respect, also shows abundant submembrane vesicles (Sanger and Hill, 1972, 1973). These vesicles may form the loci of calcium exchange with the external medium and may also be the source of activator calcium for ACh-induced contractures as well as the point of calcium–induced release of calcium (CIRC) in K+-induced contractures (Sugi and Atsumi, 1973; Tameyasu and Sugi, 1976). In many excitation–contraction (EC) coupling systems ACh releases calcium from the sarcoplasmic reticulum (SR) either by generating secondary messengers or by voltage-mediated calcium release from the SR (Berridge, 1988). However, molluscan muscles do not possess the elaborate T-system and SR array of insect or vertebrate muscles. It is, however, possible that ACh may cause the release of calcium from the submembrane vesicle system seen in molluscan muscle by induction of a secondary messenger, whereas high-K+ saline may cause the release of calcium from the same vesicles by CIRC. While fine structural and radiocalcium studies establish the existence of a cellular calcium pool which is mobilized during K+-induced and ACh-induced contractures, they give no clue as to whether the pool has independent fractions mobilized separately by ACh and by high-K+ saline.

Recently Reilly and Peretz (1987) observed in Aplysia gill efferent vein muscle that manganese ions abolished ACh-induced contractures but only partly inhibited K+-induced contractures. In this muscle, nominally calcium-free media abolished the ACh-induced contracture and reduced the K+-induced contracture to 16% of control. Reilly and Peretz (1987) concluded that ACh and high-K+ salines mobilized different cellular calcium stores to initiate EC coupling. Studies on the EC coupling mechanism of the anterior byssus retractor muscle (ABRM) of Mytilus had earlier suggested that ACh-induced contractures were mediated by calcium from cellular stores while K+-induced contractures were mediated by calcium influx from the medium (Bloomquist and Curtis, 1975; Twarog, 1976; Sugi and Yamaguchi, 1976; Tameyasu and Sugi, 1976). There is evidence that ACh-induced depolarization results from a receptor-operated ion channel inducing sodium influx (Wilkens and Greenberg, 1973) and that K+-induced depolarization induces calcium influx (Reilly and Peretz, 1987). Supporting this view is the observation of Tameyasu and Sugi (1976) that radiocalcium uptake was markedly increased by high-K+ solutions while salines containing ACh did not significantly increase radiocalcium uptake in Mytilus ABRM.

Our experiments here were designed to answer two questions. First, are there two separate mechanisms for initiating the rise in cellular calcium level needed for EC coupling, one mechanism operated by ACh-induced depolarization, the other by K+-induced depolarization? Second, are there two separate cellular calcium pools which can be independently mobilized by ACh on the one hand and high-K+ salines on the other?

All four proboscis muscles of Busycon differ from the ABRM and Aplysia gill vein muscle in that both ACh-induced and K+-induced contractures are eliminated in calcium-free sea water (Huddart and Hill, 1988; Huddart et al. 1990b), but the depolarizations induced by these agonists persist in calcium-free conditions. Furthermore Hill and Licis (1985) showed that lanthanum blocked the responses to both K+ and ACh in Busycon radular protractor muscle, in clear contrast to the findings of Reilly and Peretz (1987).

As suggested by Reilly and Peretz (1987), K+-induced depolarization leads to calcium entry while ACh-induced depolarization (by sodium entry) leads to intracellular calcium release. We expected BAYK8644 to augment K+-induced responses and be without effect on ACh-induced responses. Although BAY K 8644 did consistently inhibit responses to ACh it exerted a variety of effects on responses to K+. A surprising finding was that BAY K 8644 consistently eliminated the fast twitches of the 80 mmol l−1 K+ response in a manner similar to inactivation of fast twitches in these muscles by cumulative K+ depolarization (Huddart et al. 1990a).

We did not expect BAY K 8644 to affect fast twitch activity directly, but the enhancement of tonic force induced by 80 mmol l−1 K+ seen here with this agent suggests that two types of calcium channel might be coupled in this muscle. That is, the mechanism enhancing tonic force may inactivate fast channel activity without further depolarization. It is difficult to determine where BAY K 8644 acts. Its only consistent action in all four muscles was inhibition of responses to ACh; hence it may selectively affect some part of the ACh-induced depolarization pathway for cellular calcium release. Our sucrose-gap studies showed that BAY K 8644 not only inhibited ACh-induced contractures but also inhibited the ACh-induced depolarization which initiates them. Since ACh-induced depolarization is an event associated with sodium influx, BAY K 8644 may have inhibited membrane sodium influx, and thus have secondarily reduced calcium mobilization from the cellular calcium pool.

It is unlikely that BAY K 8644 entered the muscle fibres to affect the cellular calcium pool directly since it would then have similar effects on both K+-and ACh-induced responses. Possibly BAY K 8644 may have inhibited ACh-operated sodium-gating ion channels of the muscle fibres. Although molluscs seem to possess fast and slow types of calcium channel, they are not pharmacologically separable with BAY K 8644 as they are in mammals. In our experiments, K+-induced depolarization probably caused a steady influx of calcium. The time course over which this occurs (from seconds to minutes) will depend upon the driving force for calcium influx. This may account for the differences in the rate of force development seen with different levels of external potassium, reflecting differences in the rate of activation of CIRC (see Berridge and Galione, 1988).

Since BAY K 8644 failed to separate the K+ and ACh pathways that initiate EC coupling, we tried to isolate these with Mytelase and Mytolon, with more success. Mytolon inhibited ACh-induced force, showing that ACh acted only to release cell calcium through its receptor-operated sodium channel. Mytelase enhanced ACh-induced responses, without affecting receptor threshold, and at high concentrations it led to symptoms of either receptor desensitization or direct cholino-receptor antagonism (Fig. 6). However, these experiments did not tell us whether the two separate pathways released cellular calcium from a common pool or from two separate pools. To test this we used ACh/high-K+ synergism experiments.

Sequential exposure of muscles to ACh and high-K+ saline did not lead in any case to enhanced force development. The non-additive effect of these agonists indicated that they did not operate on two separate calcium pools but merely released, by different mechanisms, calcium from a single common pool.

It is most unlikely that ACh-and K+-induced depolarization operate via cyclic AMP and cyclic GMP as separate secondary messenger systems, since Ishikawa et al. (1981) have shown that neither ACh nor high-K+ saline causes any changes in cyclic AMP and cyclic GMP levels in the AB RM.

The data we have on Busycon proboscis muscles do not support the hypothesis that ACh and high-K+ saline induce the release of cellular calcium for EC coupling from two separate calcium pools. However, ACh and high-K+ saline do release cellular calcium with differing efficiencies. Maximal ACh-induced contracture is achieved at far lower levels of membrane depolarization than are required for maximal K+-induced contractures (Reilly and Peretz, 1987; Huddart and Hill, 1988), indicating that the in vivo agonist is more efficient at triggering EC coupling than is depolarization induced by high-K+ saline.

HH is most grateful to the Royal Society and the Wellcome Trust, DDB to the Wellcome Trust and RL to the Natural Environment Research Council for grants to support this work. This study was carried out while RL was in receipt of an NERC Research Studentship (GT4/85/ALS/20).

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