Unusual responses of proboscis muscles ofBusycon canaliculatum to some calcium antagonist agents

Summary K- and ACh-induced responses of the radular sac, odontophore retractor, and radular retractor muscles ofBusycon canaliculatum were found to be strongly dependent upon [Ca]₀. Diltiazem had strong positive inotropic and chronotropic actions on fast twitch activity in the odontophore retractor and radular protractor muscles. K-induced tonic force in these muscles was partly inhibited by diltiazem but only at very high concentrations. ACh responses in all muscles were eliminated by diltiazem. Nifedipine enhanced fast twitches and tonic force in response to high K, and induced persistent spontaneous fast twitch discharges. Nifedipine inhibited ACh-induced tonic force, but induced rhythmic bursts of fast twitches persisting long after nifedipine washout. Verapamil strongly inhibited K- and ACh-induced tonic force in all three muscles at high concentration, but stimulated fast twitch responses and converted ACh contractures into fast twitch activity. Sucrose gap studies showed that nifedipine and diltiazem reduced K- and ACh-induced tension and depolarization. Paradoxically, verapamil reduced K- and ACh-induced tension but significantly enhanced their induced depolarizations. Diltiazem, nifedipine and verapamil did not act like slow Ca channel antagonists in these muscles. This may reflect differences in channel structure between molluscs and mammals, or differences in the cellular calcium release pathways operated by such channels in molluscan and mammalian muscle. These Ca-ant-agonists appeared to act as agonists of fast twitch activity in these muscles and antagonists of the ACh-induced calcium release pathway for tonic force development.     

Dependence upon external calcium for contractile activity in two molluscan proboscis muscles.

1. Both the radular sac and odontophore retractor muscles of Buccinum undatum depend upon [Ca]0 to raise the [Ca]i concentration of the contractile system to activation level. 2. The K-induced responses of the muscles depend mainly upon [Ca]0 for activator Ca while the ACh responses depend upon [Ca]0 to raise stored intracellular Ca to activation levels. 3. In the radular sac muscle, it is probable that the inward current is carried by Na+ or is Na(+)-dependent and this current may release [Ca]i for contraction since the muscle became spontaneously active during ACh- and K-contractures in Ca-free seawater containing 2 mM EGTA as a calcium chelator. 4. It is proposed that since calcium antagonists are more inhibitory on ACh responses than on K-contractures, ACh releases the activator calcium for the contractile system through a slow-type Ca channel while high K releases Ca through a fast-type calcium channel in these muscles.     

Calcium dependency and the effect of calcium antagonists on molluscan proboscis smooth muscles from the whelk,Buccinum undatum

In all four proboscis muscles of the whelk Buccinum undatum, the potassium-induced depolarization response was acutely dependent upon extracellular calcium, being eliminated in calcium-free conditions. The responses to acetylcholine were found to be partly dependent upon intracellular calcium. Responses to the peptides phenylalanine-methionine-arginine-phenylalanine-NH₂ and phenylalanine-leucine-arginine-phenylalanine-NH₂ were much more resistant to calcium-free conditions and appeared to engage the excitation-contraction coupling mechanism by mobilizing stored intracellular calcium. Sucrose-gap studies of radular retractor muscles showed that the organic calcium “antagonist” nifedipine enhanced potassium-induced depolarization responses, initiating spike-like action potentials and associated fast twitch activity. The inorganic calcium antagonist gadolinium exerted concentration-dependent inhibitory actions on these muscles. Basal tonus and fast twitch activity in response to potassium-induced depolarization were eliminated as were the spike-like action potentials of the membrane electrical response. The inorganic calcium “antagonist” cadmium greatly enhanced potassium-induced contractures in all four muscles, and on its own it induced tonic force and fast twitches in all the muscles. It seems likely that cadmium may have displaced stored intracellular calcium to induce myofilament activation. While these molluscan smooth muscles appear to possess calcium channels with fast and slow characteristics, their behaviour and pharmacological manipulation is very different from their more well known mammalian transient and long-lasting channel counterparts.     

Electromechanical uncoupling in a molluscan muscle examined by the sucrose gap technique

Summary Membrane potential and tension ofBusycon radular protractor muscles were studied by sucrose gap methods.Excitation-contraction (EC) coupling was examined in response to acetylcholine (ACh) and high K which depolarized the fibres and induced tension, but without action potential firing. Potassium depolarization did not follow predictions expected from the Nernst equation at low and very high K levels, and maximum tension was found at about 100 mM K. EC coupling was very sensitive to [Ca]_o. Ca-free media eliminated K- and ACh-induced tension but with normal depolarization, showing full electromechanical uncoupling.Ionophore A23187 enhanced K- and ACh-induced responses and X-537A enhanced ACh responses, demonstrating acute dependence of activation on [Ca]_o in this muscle. The calcium antagonists nifedipine and nisoldipine reduced tension in the muscle only at very high concentrations, and both agents slightly reduced K- and ACh-induced depolarization.Verapamil reduced K- and ACh-induced tension but paradoxically it enhanced the depolarizing actions of these agents leading to electromechanical uncoupling. Abscisic acid (ABA) enhanced ACh- and K-induced tension and simultaneously enhanced their depolarizing actions. Ionophores and ABA appear to enhance calcium influx which may secondarily influence sodium influx.Calcium antagonists have no consistent actions on this muscle, suggesting that calcium channel activity of the radular protractor may be different from that seen in mammalian visceral muscles.Abbreviations ABRM Anterior byssus retractor muscle - ACh acetylcholine - ABA abscisic acid - EC excitation-contraction - SR sarcoplasmic reticulum - EGTA ethylene-diamine-tetraacetic acid     

The properties of the extraocular muscles of the frog. III. Morphological, mechanical and pharmacological properties of the isolated retractor bulbi muscle.

Some morphological, physiological, and pharmacological properties of the retractor bulbi muscle of the frog were tested. The enzyme-histochemical investigation shows that the retractor bulbi muscle contains twitch muscle fibres only. Two types of twitch muscle fibres, which are especially different in their diameter and in the content of mitochondria, build the muscle in an irregular arrangement; tonic muscle fibres were not observed. On the average, the isolated retractor bulbi muscle has at room temperature a contraction time of 26 ms, a half-relaxation time of 28 ms, a fusion frequency of 75 stimuli/s, and a twitch-tetanus ratio of 0.28. The fatigability of this muscle is higher than in oculorotatory eye muscles but lower than in skeletal muscles of the frog. An increase of the extracellular K+-concentration elicits in retractor bulbi muscles a quickly transient contracture; the mechanical threshold of the muscle fibres is found in a range between 20 and 25 mM K+ in Ringer solution. Similar short-lasting contractures, which are probably caused by twitch fibres, rich in mitochondria, are also evoked by application of depolarizing drugs like acetylcholine. The properties of the retractor bulbi muscle are compared with those of the sartorius muscle of the frog, which likewise contains twitch muscle fibres only.     

Glycerol treatment in mammalian skeletal muscle

Summary Potassium (K-) contractures were recorded from slow-twitch (mouse soleus) and fast-twitch (mouse extensor digitorum longus (EDL) and rat sternomastoid) muscles. The mouse limb muscles responded to a maintained increase in external potassium concentration with a rapid increase in tension (fast contracture) which inactivated and was followed by a slow contracture. Rat sternomatoid muscles responded with fast contractures only. The threshold potassium concentration for contraction was higher in fast-twitch muscles than in soleus muscles, at 22 and at 37°C. After corrections had been made for the more rapid depolarization of soleus fibers, the threshold potential for soleus fiber contraction was 15 mV closer to the resting membrane potential than the threshold for fast-twitch fiber contraction. The K-contracture results were confirmed by two microelectrode voltage-clamp experiments. Activation of fast twitch fibers required depolarizing pulses that were 15 to 20 mV greater than the pulses required to activate soleus fibers. When the time courses of K-contractures were compared it was evident that inactivation with prolonged depolarization was much faster in the fast-twitch muscles than in the soleus muscles. The results suggest that the voltage dependence and kinetics of the process coupling T-tubule depolarization with calcium release from the sarcoplasmic reticulum may depend on fiber type in mammalian skeletal muscle.     

Responses of two proboscis muscles of Neptunea antiqua (mollusca) to some calcium antagonist drugs

1. Both the radular retractor (RR) and radular sac (RS) muscles of Neptunea antiqua depend upon [Ca]₀ to raise the internal calcium concentration of the contractile elements to activation level.2. The K- and ACh-induced responses of the muscles were strongly inhibited in calcium-free seawater.3. Calcium antagonist drugs were more inhibitory on ACh-induced responses than on K-responses suggesting a dichotomy of calcium channel activities modulated by these agonists.4. The calcium ionophore A23187 enhanced ACh-induced responses of both muscles but was without effect on K-induced responses.5. The responses of these Neptunea muscles to calcium antagonist drugs show some similarities but also differences to those of Buccinum muscles and are quite unlike the excitation induced by organic antagonists in similar muscles of the American whelk Busycon canaliculatum.     

Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens. II. Effects of denervation.

Neural regulation of mature normal fast twitch muscle of the chicken suppresses high activity, extrajunctional localization, and isozyme forms of acetylcholinesterase (AChE) characteristic of embryonic, denervated and dystrophic muscle. Normal adult slow tonic muscle ofthe chicken retains intermediate levels of activity and embryonic isozyme forms but not extrajunctional activity; it is not affected by muscular dystrophy. The hypothesis that neural regulation of the AChE system is lacking in slow tonic muscle and thus not affected by dystrophy was tested by denervating the fast twitch posterior latissimus dorsi and slow tonic anterior latissimus dorsi muscles of normal and dystrophic chickens. Extrajunctional AChE activity and embryonic isozyme forms increased, then declined, in both muscles. The results suggest that ocntrol of AChE is qualitatively similar in slow tonic and fast twitch muscle of the chicken.     

The relation between excitation-contraction coupling and fine structure of a molluscan muscle,the radular retractor of the whelk,Buccinum undatum

The Buccinum radula is of the rachiglossate type with two outer rows of fierce hook-like attack teeth and a medial row of straight sharp-pointed shredding teeth. Individual cells of the radular retractor muscle are 10–12 m in diameter and separated at the closest by gaps of only 40 nm, providing areas of potential electrical contact. The cell membranes are heavily invested with long finger-like invaginations, associated with sarcoplasmic reticular cisternae, and surface caveolae; the latter are associated with the numerous dense body membrane attachment plaques found in this muscle. The radular retractor muscle possesses a significant sarcoplasmic reticulum of peripheral cisternae and deeper vesicles associated with mitochondria. The surface caveolae may result from myofilament force exerted via attachment plaques at the cell membrane, while deeper invaginations may constitute a rudimentary transverse tubular system to relay surface depolarization to associated sarcoplasmic reticular cisternae inducing calcium release to effect excitation-contraction coupling. The radular retractor muscle possesses the usual thick paramyosin and thin actin myofilaments, the latter associated with dense bodies and attachment plaques presumably to transduce force to the cell membrane. The mitochondria are unusually large and packed into dense central clusters surrounded by large deposits of glycogen granules. The nerve endings on the radular retractor muscle fibres show four different types of transmitter vesicle, presumably related to the four kinds of agonist action in this muscle, cholinergic, serotonergic, peptidergic and purinergic. All nerve endings have mixed vesicle populations, clear evidence of co-transmission. In this muscle we see a modification of usual smooth muscle structure to effect fast sustained contractions, an ultrastructural configuration functionally designed for the muscle's central role in the feeding cycle.Abbreviations ABRM anterior byssus retractor muscle - EC coupling excitation-contraction coupling - RP radular protractor muscle - RR radular retractor muscle - SR sarcoplasmic reticulum - T-system transverse tubular system     

Effects of agonists and antagonists of rhyanodine receptors on potassium contractures in twitch and tonic frog skeletal muscle fibers

A comparative analysis of the effects of the concentrations of Ca2+ in external medium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of rhyanodine-sensitive Ca2+ channels of carcoplasmic reticulum on the characteristics of potassium contracture in frog twitch and tonic skeletal muscles has been performed. It was shown that the duration of contracture in tonic muscles is not restricted by the presence of Ca2+, as distinct from twitch muscles. Dandrolene does not practically affect the contractile responses of tonic fibres, and the concentration of cresol eliciting the contracture for tonic fibres is substantially higher (1 mM) than for twitch fibers (0.25 mM). In twitch fibers, the potassium contracture activated in the presence of cresol is comparable in amplitude and dynamics with the contracture under control conditions, and in tonic fibers a summing of responses without relaxation after the washing of excessive potassium is observed. This suggests that, in twitch fibers, the influx of Ca2+ can directly create the concentration sufficient for the maintenance of contraction, and in tonic fibers its involvement is mediated through the Ca(2+)-dependent activation of the beta-isoform of rhyanodine-sensitive channels.     

Neural control exploits changing mechanical advantage and context dependence to generate different feeding responses in <Emphasis Type="Italic">Aplysia</Emphasis>

How does neural control reflect changes in mechanical advantage and muscle function? In the Aplysia feeding system a protractor muscle's mechanical advantage decreases as it moves the structure that grasps food (the radula/odontophore) in an anterior direction. In contrast, as the radula/odontophore is moved forward, the jaw musculature's mechanical advantage shifts so that it may act to assist forward movement of the radula/odontophore instead of pushing it posteriorly. To test whether the jaw musculature's context-dependent function can compensate for the falling mechanical advantage of the protractor muscle, we created a kinetic model of Aplysia's feeding apparatus. During biting, the model predicts that the reduction of the force in the protractor muscle I2 will prevent it from overcoming passive forces that resist the large anterior radula/odontophore displacements observed during biting. To produce protractions of the magnitude observed during biting behaviors, the nervous system could increase I2's contractile strength by neuromodulating I2, or it could recruit the I1/I3 jaw muscle complex. Driving the kinetic model with in vivo EMG and ENG predicts that, during biting, early activation of the context-dependent jaw muscle I1/I3 may assist in moving the radula/odontophore anteriorly during the final phase of protraction. In contrast, during swallowing, later activation of I1/I3 causes it to act purely as a retractor. Shifting the timing of onset of I1/I3 activation allows the nervous system to use a mechanical equilibrium point that allows I1/I3 to act as a protractor rather than an equilibrium point that allows I1/I3 to act as a retractor. This use of equilibrium points may be similar to that proposed for vertebrate control of movement.     

Further pharmacological study on the cholinergic and glutaminergic properties of the radula muscles of a mollusc,Rapana thomasiana

1. In the radula protractor of the prosobranch mollusc Rapana thomasiana, both twitch contractions and acetylcholine contractions were markedly depressed or blocked by propantheline (10⁻⁵ M) and strychnine (10⁻⁵ M), but in the radula retractor, only acetylcholine contraction was markedly affected by the antagonists,2. Glutamate contractions of both of the muscles were little or slightly affected by the drugs.3. Twitch contraction of the protractor was slowly depressed when the muscle was immersed in concanavalin A (0.3 mg/ml), while that of the retractor was first potentiated and then slowly depressed in it.4. In both of the muscles, glutamate contractions were markedly enhanced by the lectin, but acetylcholine contractions were not affected.5. These results support the notion that the principal excitatory neurotransmitter in the protractor is acetylcholine, whereas that in the retractor is glutamate.     

The ionic basis of cardiac activity in the bivalve mollusc Perna perna

The ionic basis of cardiac activity and aspects of excitation-contraction (E-C) coupling were investigated in the isolated heart of the bivalve mollusc Perna perna, using the sucrose-gap technique. The role of the principal ions was established employing artificial seawater, in which specific ion concentrations were modified, and ion channel blockers. The mean membrane resting potential (MP) and the action potential (AP) were -33+/-0.7 mV (n=89) and 13+/-0.3 mV (n=71), respectively. The MP potential was primarily dependent on K(+) ions. Three types of cardiac APs were identified: fast, slow and spike-plateau potentials. Cardiac activity was maintained in Na(+)- or Ca(2+)-free salines but ceased when either Cd(2+) or EDTA was added to these salines. Other Ca(2+) channel blockers reduced the amplitude and increased duration of the cardiac APs. Tetrodotoxin (TTX) and procaine did not alter the AP. The data showed that the depolarizing phase of the AP was dependent on Ca(2+) influx while the plateau phase, when present, resulted from Na(+) influx that was modulated by Ca(2+). The mechanical responses were more sensitive to changes in extracellular Ca(2+) concentration than were the electrical responses.     

Effects of Na-octanoate on potassium contractures in normal and denervated frog tonic fibres

In frog twitch muscle fibres, Na-octanoate (NaC8) shifted the relation between potassium induced tension and membrane potential to the right. The present study has been carried out to investigate the effect of this fatty acid on frog tonic fibres. Potassium contractures measured on bundles of 30-40 fibres of ileofibularis muscles were less decreased by NaC8 (2.5-10 mmol/l) than those of twitch fibre bundles. In denervated muscles the sensitivity to NaC8 was increased, probably due to the development of sodium channels in the membranes. Experiments with mixed fibre bundles also showed a lower influence of NaC8 on potassium contracture of tonic fibres. On the other hand, tonic fibres showed a lower threshold of the potassium induced tension as well as a lower K+ concentration for maximal activation. This lower threshold was further lowered by NaC8, corresponding to a shift of the relation between potassium concentration and tension to the left. The membrane resting potentials were -58 +/- 9 mV in tonic fibres and -83 +/- 5 mV in twitch fibres. Five mmol/l NaC8 only induced depolarization of the membrane of tonic fibres. This depolarization (by about 20 mV) may be responsible for the threshold shift to lower K+ concentration in NaC8-exposed tonic fibres. In addition to the effects of NaC8 on sodium channels, interactions with Ca2+ binding sites are discussed.     

Electrophysiological and pharmacological characteristics of buccal smooth muscle of the pest slug Deroceras reticulatum

Buccal mass muscle of the pest slug Deroceras reticulatum was examined by conventional tension recording and the sucrose-gap electrophysiological technique. Elevated potassium salines induced dose-dependent depolarisations accompanied by tonic contractures with superimposed rapid twitch contractions. The latter were suppressed at over 40 mmol · l⁻¹ external potassium, where depolarisation-induced inactivation of voltage-sensitive calcium channels may have occurred. Acetylcholine caused significant dose-dependent depolarisations and tonic contractures, while 5-hydroxy tryptamine induced lower depolarisations accompanied by phasic contractile activity superimposed on low level tonic force. Of the purines examined only guanosine triphosphate caused significant mechanical activity above a threshold of 0.1 μmol · l⁻¹. The tetrapeptides inhibited buccal muscle spontaneous activity, but the related small cardioactive peptide B was weakly excitatory. The amino acids glutamate and gamma-aminobutyric acid were weakly excitatory on buccal muscle while the molluscicides metaldehyde and methiocarb disrupted normal mechanical activity of the feeding musculature. Acetylcholine and 5-hydroxytryptamine appear to have major roles in regulating feeding muscle activity, seemingly modulated by guanosine triphosphate and inhibited by phenylalanine-methionine-arginine-phenylalanine-NH₂ and phenylalanine-leucine-arginine-phenylalanine-NH₂. Accepted: 22 July 1999     

The molluscan neurosecretory peptide FMRFamide: comparative pharmacology and relationship to the enkephalins

The molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) has diverse actions on excitable tissues of molluscs, including hearts, noncardiac muscles, complex organs, and neurons. The intracellular transducing mechanisms are also diverse and are not readily correlated with particular responses. FMRFamide increases cyclic AMP levels concomitant with both cardioexcitation and inhibition, but not with muscle contraction. In the same tissues, the effects of 5-hydroxytryptamine are dissimilar and are always accompanied by a cyclic AMP increase. FMRFamide and acetylcholine cause similar tonic contractions of the Busycon radula protractor muscle and identical catch contractures of the mytilid anterior byssus retractor muscle, but the ionic basis of excitation and the sources of activator calcium for contraction are not the same for the two agonists. A comparative study of structure-activity relations showed that FMRFamide receptors are heterogeneous. Helix aspersa ganglia contain no FMRFamide, but a close analog occurs and has been tentatively identified. Evidence supporting a proposed homology between FMRFamide-like and opioid peptides is summarized. The effects of the amphiactive heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 on the venus clam rectum support this hypothesis.     

Two Ca current components of the receptor current in the electroreceptors of the marine catfish Plotosus

In the isolated sensory epithelium of the Plotosus electroreceptor, the receptor current has been dissected into inward Ca current, ICa, and superimposed outward transient of Ca-gated K current, IK(Ca). In control saline (170 mM/liter Na), with IK(Ca) abolished by K blockers, ICa declined in two successive exponential phases with voltage-dependent time constants. Double-pulse experiments revealed that the test ICa was partially depressed by prepulses, maximally near voltage levels for the control ICa maximum, which suggests current-dependent inactivation. In low Na saline (80 mM/liter), ICa declined in a single phase with time constants similar to those of the slower phase in control saline. The test ICa was then unaffected by prepulses. The implied presence of two Ca current components, the fast and slow ICa's, were further examined. In control saline, the PSP externally recorded from the afferent nerve showed a fast peak and a slow tonic phase. The double-pulse experiments revealed that IK(Ca) and the peak PSP were similarly depressed, i.e., secondarily to inactivation of the peak current. The steady inward current, however, was unaffected by prolonged prepulses that were stepped to 0 mV, the in situ DC level. Therefore, the fast ICa seems to initiate IK(Ca) and phasic release of transmitter, which serves for phasic receptor responses. The slow ICa may provide persistent active current, which has been shown to maintain tonic receptor operation.     

Purinergic modulation of spontaneous activity and of responses to high potassium and acetylcholine in rat ileal smooth muscle

1. In rat ileal smooth muscle both adenosine and ATP at 10⁻⁴ M significantly enhanced spontaneous mechanical activity. The excitatory actions of adenosine were blocked by the P₁ receptor antagonist 8-phenyltheophylline and the excitatory effects of ATP were significantly reduced by the P₂ receptor antagonist quinidine.2. The P₂ receptor desensitizer α,β-methylene-ATP was without effect on ACh responses nor did the stable analogue β,gg-methylene-ATP exert any effect on spontaneous mechanical activity.3. Pretreatment with adenosine caused a dose-dependent enhancement of K-induced contractures in the ileum. Low adenosine concentrations slightly inhibited and high concentrations slightly enhanced ACh-induced contractures in the ileum.4. ATP potentiated the phasic component of the ileal K-induced contracture but strongly inhibited tonic force at high concentrations. This agent slightly inhibited the phasic component of the ACh-induced contracture while strongly inhibiting ACh-induced tonic force.5. α,β-methylene-ATP inhibited ileal muscle ACh induced contractures while it potentiated both phasic and tonic K-induced contractures. β, γ-methylene ATP inhibited ACh-induced contractures but it enhanced K-induced phasic contractures while inhibiting K-induced tonic force.6. The results of this study suggest that rat ileum may contain the A₁ subtype of the P₁ receptor but the evidence for a P₂ receptor subtype is conflicting despite the inhibition of ATP actions by quinidine.7. The inhibition of K- and ACh-induced tonic force suggests that adenosine and ATP interactions with ileal smooth muscle may inactivate slow voltage-dependent calcium channels leading to EC uncoupling.     

Transient release of acetylcholine from Torpedo synaptosomes in response to prolonged depolarization

The release of ACh (acetylcholine) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay. A maintained depolarization by high KCl in the presence of Ca2+ triggered only a transient ACh release. It was shown that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response. The termination of release was probably caused by inactivation of voltage-dependent Ca2+ entry and rapid removal of intraterminal Ca2+ by a (Na+)0 dependent mechanism. It was found that exposure of the synaptosomes for a short period to low Ca2+-high K+ solutions greatly reduced the responses to Ca2+ reintroduction, as compared to the control release obtained when high K+ was applied in the presence of normal Ca2+. The response to Ca2+ reintroduction was measured following various times of preincubation with high K+ and low Ca2+; thus, an estimate of the time course of the inactivation of Ca2+ permeability during a depolarization could be made. A two component exponential kinetic was observed, with a rapid (tau = 3.6 s) and a slow phase (tau = 77 s). This inactivation was more pronounced when a higher KCl concentration was used to induce a greater depolarization. The presence of EGTA during the preincubation with high KCl greatly increased the response provoked by Ca2+ reintroduction, whereas increases in Ca2+ during the preincubation period caused proportional reduction in the subsequent response to Ca2+ reintroduction, indicating that the Ca2+ influx itself was involved in the inactivation process.     

Analysis of the negative inotropic effect of acetylcholine on frog atrial fibres

Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.