Neurogenic contractile activity of the penis retractor muscle of Helix pomatia L.

Using convential mechanical and electromyographic recording methods 2 distinct types of neurogenically elicited activity can be observed in the penis retractor muslce (PRM) of Helix pomatia: (1) rhythmic, phasic contractions correlated with single or a few compound action potentials and (2) intervening, strong, prolonged contractions accompanied by sustained, high frequency electrical muscle activity. The 2 distinct types of muscle activity which seem to play a part in the normal behaviour of the PRM in the intact animal are mediated by both the central nervous system and peripheral neurons. While central neuronal structures are involved in causing the strong, prolonged contractions, the phasic activity is initiated by peripheral neuronal structures located at the proximal end of the PRM. There is evidence that the transmission of the excitation at the neuromuscular level of central and peripheral origin is mediated by ACh.     

Calcium dependency of pregnant rat myometrium: comparison of circular and longitudinal muscle

The purpose of this study was to determine if the changes in spontaneous contractions of circular uterine muscle during pregnancy were related to alterations in calcium (Ca) sensitivity or dependence. Circular muscle (CM) and longitudinal muscle (LM) segments from rats on Days 16-17 of gestation and at term were compared with respect to: sensitivity of potassium (K)-induced contractions to changes in extracellular Ca, and rate and magnitude of decrease of K- and acetylcholine (ACh)-induced contractions in Ca-free medium and in methoxyverapamil (D-600). The effects of low Ca and D-600 on spontaneous electrical activity of CM were also studied. Ca sensitivity was no different in CM and LM and did not change between Day 16 and term. There was no difference in the Ca-dependence of K- or ACh-induced contractions during this time. Potassium contractions declined more rapidly than ACh contractions in Ca-free media, especially in CM. Spontaneous action potentials in CM were Ca-dependent and disappeared in low Ca or D-600 on Days 16-17 and at term. Therefore the changes in contractions of CM during pregnancy are not related directly to Ca sensitivity or dependence, but indirectly via Ca modulation of the action potentials.     

Role of M2 muscarinic receptors in airway smooth muscle contraction

Airway smooth muscle expresses both M2 and M3 muscarinic receptors with the majority of the receptors of the M2 subtype. Activation of M3 receptors, which couple to Gq, initiates contraction of airway smooth muscle while activation of M2 receptors, which couple to Gi, inhibits beta-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization by acetylcholine (ACh) and endothelin-1 (ET-1) in porcine tracheal smooth muscle by measuring contractions at constant [Ca2+] in strips permeabilized with Staphylococcal alpha-toxin. Both ACh and ET-1 contracted airway smooth muscle at constant [Ca2+]. Pretreatment with pertussis toxin for 18-20 hours reduced ACh contractions, but had no effect on those of ET-1 or GTPgammaS. We conclude that the M2 muscarinic receptor contributes to airway smooth muscle contraction at constant [Ca2+] via the heterotrimeric G-protein Gi.     

Caffeine effects on buccal muscles of Aplysia

1. Caffeine (35-70 mM) elicited contractions of Aplysia buccal muscle El. In a Ca2+-free medium, in which ACh-elicited contractions rapidly fail, caffeine elicited contractions of approximately the same size as in normal medium. 2. 5-HT (10(-8) M and 10(-7) M) did not enhance caffeine-elicited contractions. 3. Lower concentrations (1-10 mM) of caffeine inhibited ACh-elicited contractions. Caffeine (7 mM) reduced the contraction by 80%. 4. Caffeine (7 mM) reduced ACh-elicited depolarization by 60%. 5. Caffeine (7 mM) increased 45Ca2+ influx into Aplysia buccal muscle I5. The stimulation of influx of 45Ca2+ by 10(-3) M ACh was non-additive with the stimulation caused by caffeine, and 7 mM caffeine reduced the influx caused by 10(-3) M ACh.     

Effect of different calcium modulators on motilin-induced contractions of the rabbit duodenum. Comparison with acetylcholine

Motilin and acetylcholine (ACh) have a direct contractile effect on rabbit small intestinal smooth muscle. To explore the role of calcium influx in these contractions, we studied the effect of extracellular calcium concentration and of calcium antagonists on the response of longitudinal muscle preparations from rabbit duodenum. Motilin- (10(-7) M) and ACh- (10(-4) M)-induced contractions were abolished in Ca2+-depleted medium. ACh (10(-4) M) or motilin (10(-8) and 10(-7) M) increased the contractile response to added Ca2+ to 130 +/- 6%, 129 +/- 10% and 145 +/- 5% of the maximal response to Ca2+ added alone (10 mM in a cumulative concentration response curve). The sensitivity to Ca2+ was greater in the presence of ACh and motilin (EC50 = 1.0 and 1.1 mM Ca2+) than in the absence of any agonist (1.7 mM). In cumulative concentration response (CCR) curves for motilin and ACh, pD2'-values were 7.0 and 6.6 for diltiazem, 8.4 and 7.8 for verapamil (two calcium entry blockers), 5.6 and 5.2 for TMB-8 (an inhibitor of intracellular calcium), 5.3 and 5.2 for TFP (a calmodulin-antagonist). All CCR-curves showed metactoid-like action of the antagonistic drugs. We conclude that ACh and motilin cause calcium to enter the smooth muscle cell. They are probably operating via separate channels, and use a mechanism which differs from K+-induced influx. Intracellular calcium stores appear to play a minor role in these contractions.     

Neuroanatomical,immunocytochemical, and physiological studies of the pharyngeal retractor muscle and its putative regulatory neurons playing a role in withdrawal and feeding in the snail, <Emphasis Type="Italic">Helix pomatia</Emphasis>

We describe the neurons regulating two separate functions of the pharyngeal retractor muscle (PRM), namely sustained contraction during body withdrawal and rhythmic phasic contractions during feeding, in the snail, Helix pomatia. The distribution of central neurons innervating the PRM is organized into two main units; one in the buccal-cerebral ganglion complex, the other in the subesophageal ganglion complex. Serotonin- (5-HT-), FMRFamide- (FMRFa-), and tyrosine-hydroxylase-immunostained neurons are present among the PRM neurons that densely innervate the PRM. 5HT both decreases and increases the amplitude of the electrically evoked contraction between concentrations of 0.1 M and 1 M. Dopamine (DA) only decreases the amplitude of contraction at a 1-M threshold concentration. In contrast, FMRFa increases the amplitude of the contraction and slightly elevates the tone of the PRM but requires a higher threshold (10 M). Assay by high-performance liquid chromatography of 5HT and DA in the PRM has shown that the 5HT level decreases during locomotion but increases during feeding, whereas the DA level increases during locomotion but slightly decreases during feeding. Thus, different segments of the PRM are innervated by neurons from different loci within the central nervous system. The segments of the PRM distal to the pharynx are innervated from loci of the subesophageal ganglion complex suggesting that they mediate withdrawal. The proximal segment of the PRM is innervated from cerebral and buccal loci indicating that these neurons mediate the feeding rhythm produced by buccal and cerebral feeding central pattern generators to induce rhythmic phasic contractions in the PRM during feeding.This work was supported by Hungarian Scientific Research Fund (OTKA) grants (T034106, T037389, T037505), the Wellcome Trust CRIG Programme, and the Wellcome Trust Travel Grant.     

Involvement of Dihydropyridine-Sensitive Ca2+ Channels in Adenosine-Evoked Inhibition of Acetylcholine Release from Guinea Pig Ileal Preparation

The effects of adenosine and nifedipine on endogenous acetylcholine (ACh) release evoked by electrical stimulation from guinea pig ileal longitudinal muscle preparations exposed to physostigmine were evaluated using an HPLC with electrochemical detection (ECD) system. Resting ACh release, which was sensitive to tetrodotoxin (0.3 microM), was enhanced by Bay K 8644 (0.5 microM; a Ca2+ antagonist) or 4-aminopyridine (30 microM; a K+ channel blocker) but not by theophylline (100 microM; a P1 purinoceptor antagonist) or atropine (0.3 microM). The enhancement of the resting ACh release by Bay K 8644 was virtually unaffected by atropine. Electrically evoked ACh release was enhanced by around two- to fourfold in the presence of theophylline, atropine, Bay K 8644, 4-aminopyridine, or atropine. On the other hand, the evoked ACh release was reduced by adenosine (10-30 microM), nifedipine (0.1-0.3 microM; a dihydropyridine Ca2+ channel antagonist), or bethanechol (1-3 microM) in a concentration-related fashion. The reduction induced by adenosine or nifedipine was almost abolished by either theophylline or Bay K 8644, whereas that induced by bethanechol was virtually unaffected by these drugs. The inhibition by adenosine of ACh release was not influenced in the presence of 4-aminopyridine or atropine. However, this inhibition by adenosine was considerably enhanced by halving the Ca2+ concentration in the Krebs solution and was diminished by doubling the Ca2+ concentration. These findings suggest that adenosine produces a cholinergic neuromodulation presumably via modifying dihydropyridine-sensitive Ca2+ channel activities in the cholinergic neurons, and thus L-type Ca2+ channels may exist on the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta

ABSTRACT: BACKGROUND: Electrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments. RESULTS: Intracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644). CONCLUSION: The isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.     

Sources of activator calcium ions in the contraction of smooth muscles in Aplysia kurodai

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.     

A possible coupling of postjunctional ATP release and transmitters' receptor stimulation in smooth muscles.

The nature of ATP release from mainly smooth muscles of guinea-pig was evaluated with KCl and agonists for different kinds of receptors. In ileal longitudinal muscles, amounts of net ATP release by ACh and bethanechol (1-10 microM) were much larger (about 10 fold) than that by other drugs, e.g., histamine, 5-hydroxytryptamine, prostaglandin-F2 alpha, substance P and bradykinin, including KC1, although differences between contractions of the tissue evoked by test drugs were approximately 1.5 times at most. The ATP release, as well as the contraction, evoked by ACh or bethanechol was markedly reduced by atropine (0.3 microM), thus, indicating primarily postjunctional release of ATP. The remarkable ATP release from vas deferens by norepinephrine (NE), but not by substance P, was abolished almost completely by prazosin (0.3 microM). Increases in intracellular Ca2+ and subsequent contraction in the ileal tissue were produced by ATP and these responses were fully antagonized by nifedipine (0.1 microM). These findings provide evidence that the drugs-stimulated ATP release from smooth muscles does not result from contractility of muscles, but is substantially elicited only by stimulation of neurotransmitter (NE or ACh) receptors, suggesting the existence of the receptor-stimulus-postjunctional ATP release coupling. The released ATP may contribute, in part, to the muscle contractility via increase of Ca2(+)-influx, presumably, in a manner related to the voltage-gated Ca2(+)-channels.     

Ca2+o-Independent Veratridine-Evoked Acetylcholine Release from Striatal Slices Is Not Inhibited by Vesamicol (AH5183): Mobilization of Distinct Transmitter Pools

The effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol), a compound known to block the uptake of acetylcholine (ACh) into cholinergic synaptic vesicles, on the release of endogenous and [14C]ACh from slices of rat striatum was investigated. ACh release was evoked either by electrical stimulation or by veratridine. The effect of electrical stimulation was entirely dependent on external Ca2+. By contrast, veratridine (40 microM) also enhanced ACh release in the absence of Ca2+. Indeed, with veratridine two components were clearly distinguished: one dependent on external Ca2+ and the other not. Vesamicol inhibited [14C]ACh release evoked by both veratridine and electrical stimulation in the presence of external Ca2+, provided it was added to the tissue prior to loading with [14C]choline. With the same treatment vesamicol only slightly affected the release of endogenous ACh. Under the same conditions the Ca2(+)-independent [14C]ACh release evoked by veratridine was not prevented by vesamicol. The differential responsiveness to vesamicol suggests that ACh pools involved in Ca2+o-dependent ACh release are different from those mobilized during Ca2+o-independent ACh release.     

Efficacy of muscarinic stimulation and mode of excitation-contraction coupling in bovine trachealis muscle

We have compared the efficacy of cromakalim and nifedipine to inhibit acetylcholine (ACh) and pilocarpine-induced tonic contractions in control preparations and in tissues where a fraction of the muscarinic receptor population had been removed by alkylation with phenoxybenzamine (PBZ). Both agonists induced contractions by stimulating pharmacologically similar receptors, probably of the M3 muscarinic subtype. The receptor reserve was larger, and the coupling between stimulation and contraction (E-C coupling) more efficient when ACh was the stimulating agonist. For stimulations that produced equal levels of muscle response, cromakalim was more efficacious in inhibiting contractions induced by pilocarpine. The efficacy of cromakalim in relaxing contractions induced by ACh increased when the number of functional receptors decreased. Cromakalim and nifedipine decreased the efficiency of E-C coupling for ACh and pilocarpine. Cromakalim efficacy decreased in a sigmoid manner when stimulating concentrations of ACh (and receptor occupancy) increased, and there was an inverse relationship between receptor occupancy by ACh and cromakalim efficacy. In the presence of TEA, a K+ channel blocker, nifedipine almost completely inhibited contractions induced by the M3 muscarinic agonist bethanechol. These data indicate that in bovine tracheal smooth muscle, electro-mechanical coupling is an inherent part of muscarinic E-C coupling, but its functional expression is dependent upon the efficacy of stimulation. The data also suggest that the M3 receptor is coupled to a cellular pathway linked with the activation of K+ channels that exerts a potent functional antagonism against activation of voltage-dependent Ca2+ entry.     

Pharmacological identification of acetylcholine receptor subtypes in echinoderm smooth muscle (Sclerodactyla briareus)

Contractions of an echinoderm (sp. Sclerodactyla briareus) smooth muscle, the longitudinal muscle of the body wall (LMBW), were evoked by acetylcholine (ACh) and agonists: epibatidine, muscarine and nicotine (in order of force generation: ACh>muscarine=epibatidine>nicotine). ACh-induced contractions were blocked by atropine by 50%, and methoctramine, by 30%. ACh responses were also blocked by 25% by methyllycaconitine (MLA) but not by d-tubocurarine (dTC). Muscarine initiated large contractions that were completely blocked by atropine. To elucidate possible muscarinic ACh receptor (mAChR) subtypes, muscarinic agonists (oxotremorine, pilocarpine) and antagonists (methoctramine, pirenzepine) were tested. Oxotremorine, pilocarpine, and pirenzepine each enhanced resting tonus and potentiated ACh-induced contractions (order of potency: pilocarpine>oxotremorine=pirenzepine). Muscarine, oxotremorine or pirenzepine generated phasic, rhythmic contractions. Nicotine-induced contractions were almost completely blocked by dTC but were not altered by atropine. Large contractions evoked by epibatidine were potentiated by dTC whereas atropine had no effect on them. MLA blocked spontaneous rhythmicity. Cholinesterase inhibitors, neostigmine or physostigmine, caused marked potentiation of ACh-induced contractions and initiated rhythmic slow wave contractions in previously quiescent muscles. The present pharmacological evidence points to the co-existence of excitatory nicotinic ACh receptor (nAChRs) and mAChRs where nAChRs possibly modulate tone, and the mAChRs initiate and enhance rhythmicity.     

Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-gamma-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2(+)-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)-dependent Cl- current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP-gamma-S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of papaverine on the longitudinal and circular smooth muscle of the guinea-pig caecum.

Electrical and mechanical activity of smooth muscle of the guinea-pig caecum was recorded by means of the sucrose-gap technique. The responses of longitudinal and circular smooth muscle to acetycholine were differently affected by changes of extracellular calcium concentration (0.8, 2.5 and 7.5 mM). The contractions of both preparations were depressed at high Ca++ concentrations, whereas at low Ca++ concentrations only contractions of the circular smooth muscle were augmented. The stimulatory effect of acetylcholine was decreased by papaverine in both preparations at all three concentrations of Ca++. This inhibition was the greater the lower the concentration of extracellular Ca++ and this process was more pronounced in the circular muscle. The ability of papaverine to counteract the effect of lowered concentrations of extracellular Ca++ on membrane excitability may well explain its inhibitory effect upon intestinal smooth muscle.     

Mechanisms of the Ba2+-induced contraction in smooth muscle cells of the rabbit mesenteric artery

The mechanism of the Ba2+-induced contraction was investigated using intact and saponin-treated skinned smooth muscle (skinned muscle) strips of the rabbit mesenteric artery. After depletion of Ca2+ stored in the caffeine-sensitive site, greater than 0.65 mM Ba2+ evoked contraction in muscle strips depolarized with 128 mM K+ in Ca2+-free solution in a dose-dependent fashion, and the ED50 values for Ca2+ and Ba2+ were 0.5 mM and 1.2 mM in intact muscle strips, respectively. Nisoldipine (10 nM) blocked the contraction evoked by high K+ or 10 microM norepinephrine (NE) in the presence of 2.6 mM Ba2+, but did not block the contraction evoked in the presence of 2.6 mM Ca2+. These results may indicate that Ba2+ permeates the voltage-dependent Ca2+ channel. In skinned muscle strips, the ED50 values for Ca2+ and Ba2+ were 0.34 and 90 microM, respectively, as estimated from the pCa- and pBa-tension relationships. Calmodulin enhanced and trifluoperazine inhibited the Ba2+- and Ca2+-induced contractions. After the application of Ba2+ or Ca2+ with ATP gamma S in rigor solution, myosin light chain (MLC) was irreversibly thiophosphorylated, as estimated from the Ba2+- or Ca2+-independent contraction. Furthermore, both divalent cations phosphorylated MLC, as measured using two-dimensional gel electrophoresis, to the extent expected from the amplitudes of the contraction evoked by these cations. Thus, Ba2+ is capable of activating the contractile proteins as Ca2+ does. The amount of Ca2+ or Ba2+ stored in cells was estimated from the caffeine response evoked in Ca2+-free solution in intact and skinned muscle strips. After the application of 0.3 microM Ca2+ or 0.1 mM Ba2+ for 60 s to skinned muscle strips after the depletion of Ca2+ stored in cells, caffeine produced a contraction only upon pretreatment with Ca2+ but not with Ba2+. When Ba2+ was applied successively just after the application of Ca2+, the subsequently evoked caffeine-induced contraction was much smaller than that evoked by pretreatment with Ca2+ alone. The above results indicate that Ba2+ permeates the voltage-dependent Ca2+ channel but may not permeate the receptor-operated Ca2+ channel, it releases Ca2+ from store sites but is not accumulated into the store site, and it directly activates the contractile proteins via formation of a Ba2+-calmodulin complex.     

Effects of cholinergic drugs on muscle contraction in Moniliformis moniliformis (Acanthocephala)

In whole Moniliformis moniliformis spontaneous muscle contractions were rhythmic; longitudinal contractions were measured with a force transducer. The cholinergic agonists levamisole and nicotine significantly increased muscle tension in whole worms; these contractions were tonic and were antagonised by the ganglionic blocker pentolinium and by piperazine. In addition, levamisole-induced contractions were inhibited by gallamine, hexamethonium, and norepinephrine. In worm segments, where drugs in solution were injected through the worms, acetylcholine (ACh) and nicotinic agonists were effective in causing contractions, whereas muscarinic agonists in concentrations up to 1 mM had no effect. Although muscle contraction in M. moniliformis was induced by nicotinic agonists, these contractions were effectively antagonised by a range of chemicals that block ganglionic, skeletal, and muscarinic sites in vertebrates. The presence of ACh in M. moniliformis and the effects of nicotinic agonists on muscle contraction suggest that ACh is a putative excitatory neurotransmitter.     

Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage- independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3- muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.     

Neurotoxic effects evoked by cyanobacterial extracts suggest multiple receptors involved in electrophysiological responses of molluscan (CNS, heart) models

The responses of the snail central neurons (Helix pomatia, Lymnaea stagnalis) and the isolated Helix heart were characterized evoked by cyanobacterial extracts (Cylindrospermopsis raciborskii ACT strains) isolated from Lake Balaton (Hungary). The nicotinergic acetylcholine (ACh) receptors in the CNS (both excitatory and inhibitory) were blocked by the extracts of ACT 9502 and ACT 9505 strains and the anatoxin- a (homoanatoxin-a) producing reference strain of Oscillatoria sp. (PCC 6506), similar to the inhibitory effects of the pure anatoxin-a. The enhancement of the ACh responses by the ACT 9504 extract suggests additional, probably acetylcholine esterase inhibitory mechanisms. On the isolated Helix heart the crude ACT 9505 and PCC 6506 extracts evoked frequency increase and transient twitch contraction, opposite to the ACh evoked heart relaxation. Anatoxin-a similarly contracted the heart but did not increase its contration frequency. These data suggest the involvement of some non-cholinergic mechanisms, acting very likely by direct modulation of the electrical or contractile system of the isolated heart. Diversity of the effects evoked by the cyanobacterial extracts in the CNS and heart suggest pharmacologically different neuroactive components among the secondary metabolites of the cyanobacteria acting on both (anatoxin-a like) cholinergic and (unidentified) non-cholinergic receptors.