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Background and Purpose: Stimulation of muscarinic receptors in intestinal smooth muscle cells results in suppression of voltage-gated Ca2+ channel currents (I(Ca)). However, little is known about which receptor subtype(s) mediate this effect., Experimental Approach: The effect of carbachol on I(Ca) was studied in single intestinal myocytes from M2 or M3 muscarinic receptor knockout (KO) and wild-type (WT) mice., Key Results: In M2KO cells, carbachol (100 microM) induced a sustained I(Ca) suppression as seen in WT cells. However, this suppression was significantly smaller than that seen in WT cells. Carbachol also suppressed I(Ca) in M3KO cells, but with a phasic time course. In M2/M3-double KO cells, carbachol had no effect on I(Ca). The extent of the suppression in WT cells was greater than the sum of the I(Ca) suppressions in M2KO and M3KO cells, indicating that it is not a simple mixture of M2 and M3 receptor responses. The G(i/o) inhibitor, Pertussis toxin, abolished the I(Ca) suppression in M3KO cells, but not in M2KO cells. In contrast, the G(q/11) inhibitor YM-254890 strongly inhibited only the I(Ca) suppression in M2KO cells. Suppression of I(Ca) in WT cells was markedly reduced by either Pertussis toxin or YM-254890., Conclusion and Implications: In intestinal myocytes, M2 receptors mediate a phasic I(Ca) suppression via G(i/o) proteins, while M3 receptors mediate a sustained I(Ca) suppression via G(q/11) proteins. In addition, another pathway that requires both M2/G(i/o) and M3/G(q/11) systems may be operative in inducing a sustained I(Ca) suppression.

Background and Purpose: The functional roles of M(2) and M(3) muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M(2) or M(3) receptor subtypes., Experimental Approach: Contractile responses to transmural electrical (TE) stimulation were isometrically recorded in ileal segments from M(2)-knockout (KO), M(3)-KO, M(2)/M(3)-double KO, and wild-type mice., Key Results: TE stimulation at 2-50 Hz frequency-dependently evoked a fast, brief contraction followed by a slower, longer one in wild-type, M(2)-KO or M(3)-KO mouse preparations. Tetrodotoxin blocked both the initial and later contractions, while atropine only inhibited the initial contractions. The initial cholinergic contractions were significantly greater in wild-type than M(2)-KO or M(3)-KO mice; the respective mean amplitudes at 50 Hz were 91, 74 and 68 % of 70mM K(+)-induced contraction. Pretreatment with pertussis toxin blocked the cholinergic contractions in M(3)-KO but not in M(2)-KO mice. Cholinergic contractions also remained in wild-type preparations, but their sizes were reduced by 20-30 % at 10-50 Hz. In M(2)/M(3)-double KO mice, TE stimulation evoked only slow, noncholinergic contractions, which were significantly greater in sizes than in any of the other three mouse strains., Conclusion and Implications: These results demonstrate that M(2) and M(3) receptors participate in mediating cholinergic contractions in mouse ileum with the latter receptors assuming a greater role. Our data also suggest that the lack of both M(2) and M(3) receptors causes upregulation of noncholinergic excitatory innervation of the gut smooth muscle.

Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M(2) or M(3) muscarinic receptors, in order to characterize the mechanisms involved in M(2) and M(3) receptor-mediated contractile responses. Single applications of carbachol (0.1-100 microM) produced concentration-dependent contractions in preparations from M(2)-knockout (KO) and M(3)-KO mice, mediated via M(3) and M(2) receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M(2)-preferring antagonist methoctramine (300 nM) or the M(3)-preferring antagonist 4-DAMP (30 nM). The M(2)-mediated contractions were mimicked in shape by submaximal stimulation with high K(+) concentrations (up to 35 mM), almost abolished by voltage-dependent Ca(2+) channel (VDCC) antagonists or depolarization with 140 mM K(+) medium, and greatly reduced by pertussis toxin (PTX) treatment. The M(3)-mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K(+)-depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K(+) depolarization consisted of different components, either sensitive or insensitive to extracellular Ca(2+). The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M(2) receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca(2+) entry, especially associated with action potential discharge, and that the M(3) receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca(2+) entry and intracellular Ca(2+) release. In intact tissues coexpressing M(2) and M(3) receptors, M(2) receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

(1) The effects on the whole-cell carbachol-induced muscarinic cationic current (mIcat) of antibodies against the alpha-subunits of various G proteins, as well as the effect of a Gbetagamma subunit, were studied in single guinea-pig ileal smooth muscle cells voltage-clamped at -50 mV. Ionized intracellular calcium concentration, [Ca(2+)](i), was clamped at 100 nM using a 1,2-bis(2-aminophenoxyl-ethane-N,N,N',N'-tetraacetic acid)/Ca(2+) mixture. (2) Application of ascending concentrations of carbachol (1-300 micro M) activated mIcat (mean amplitude 0.83 nA at 300 micro M carbachol; EC(50) 8 micro M; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of G(i3)/G(o) or G(o) antibodies resulted in about a 70% depression of the maximum response without change in the EC(50) value. In contrast, antibodies against alpha-subunits of G(i1), G(i1)/G(i2), G(i3), G(q)/G(11) or G(s) protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gbeta or infusion of the Gbetagamma subunit itself had no effect on mIcat. (3) If cells were exposed briefly to carbachol (50 or 100 micro M) at early times (<3 min) after infusion of antibodies to Galpha(i3)/Galpha(o) or to Galpha(o) had begun, carbachol responses remained unchanged even after 20-60 min; that is, the depression of mIcat by these antibodies was prevented. (4) These data show that Galpha(o) protein couples the muscarinic receptor to the cationic channel in guinea-pig ileal longitudinal smooth muscle and that Gbetagamma is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long-lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.

1 In guinea-pig ileal longitudinal muscle, muscarinic partial agonists, 4-(N-[3-chlorophenyl]-carbomoyloxy)-2-butynyl-trimethylammonium (McN-A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca(2+) concentration ([Ca(2+)]c) with a higher EC(50) than that of the full agonist carbachol. The maximum response of [Ca(2+)]c or tension was not much different among the three agonists. The Ca(2+) channel blocker nicardipine markedly inhibited the effects of all three agonists 2 The contractile response to any agonist was antagonized in a competitive manner by M(2) receptor selective antagonists (N,N'-bis[6-[[(2-methoyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride and 11-[[2-[(diethlamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one), and the apparent order of M(2) antagonist sensitivity was McN-A343>pilocarpine>carbachol. M(3) receptor selective antagonists, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN-A343, while darifenacin had a similar action in the case of pilocarpine. Both M(3) antagonists behaved in a competitive manner in the case of the carbachol response. 3 McN-A343 failed to release Ca(2+) from the intracellular stores, and the Ca(2+)-releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca(2+) sensitivity of the contractile proteins. 4 McN-A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. 5 The results suggest that muscarinic agonist-evoked contractions result primarily from the integration of Ca(2+) entry associated with the increased spike discharge and myofilaments Ca(2+) sensitization, and that Ca(2+) store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M(2) and M(3) receptors plays a crucial role in mediating the contraction response.

1. The abilities of muscarinic agonists (arecoline, bethanechol, carbachol, McN-A343, methacholine, pilocarpine) to inhibit isoprenaline-induced cyclic AMP production in chopped fragments (via M(2) receptors), and to evoke cationic current (I(cat)) (via M(2) receptors) or calcium store release (via M3 receptors) in enzyme-dispersed, single voltage-clamped cells from longitudinal smooth muscle of the guinea-pig small intestine were examined. 2. All muscarinic agonists (1 - 300 microM) examined inhibited isoprenaline (1 microM)-induced accumulation of cyclic AMP, the IC(50) varying from 52 to 248 microM. However, their relative potencies to evoke this M(2) effect were not significantly correlated with their ability to evoke I(cat), also a M(2) effect, whether or not calcium stores were depleted; pilocarpine and McN-A343 inhibited the I(cat) response to carbachol. 3. Muscarinic agonists (concentration 300 or 1000 microM), except pilocarpine and McN-A343 which were ineffective, evoked Ca(2+)-activated K(+) current (I(K-Ca)) resulting from Ca(2+) store release (M(3) effect). Their effectiveness was tested by estimating residual stored calcium by subsequent application of caffeine (10 mM). The relative potencies to evoke Ca(2+) store release (M(3)) and for I(cat) activation (M(2)) were closely correlated (P<0.001). 4. These data might be explained if M(2)-mediated adenylyl cyclase inhibition and I(cat) activation involve different G proteins, or involve different populations of M(2) receptors. The observed correlation of agonist potency between I(cat) activation and Ca(2+) store release supports the proposal (Zholos & Bolton, 1997) that M(3) activation can potentiate M(2)-cationic channel coupling through Ca(2+)-independent mechanisms.

1. Effects of agents, which affect microtubule polymerization-depolymerization cycle, on Ba2+ current (IBa) flowing through voltage-gated Ca2+ channels and carbachol (CCh)-induced sustained suppression of IBa were examined in whole-cell voltage-clamped smooth muscle cells of guinea-pig ileum. 2. offchicine (100 microM) and vinblastine (100 microM), microtubule depolymerizers, increased the ampitude of IBa. Lumicolchicine (100 microM), an inactive analogue of colchicine, had no effect on IBa. 3. Taxol (1 - 100 microM), a microtubule polymerizer, decreased IBa in a concentration-dependent manner and accelerated the rate of inactivation of IBa. Baccatin III (100 microM), an inactive analogue of taxol, had no effect on IBa. 4. Colchicine (100 microM) and vinblastine (100 microM), but not lumicolchicine (100 microM), decreased or abolished the sustained component of CCh (10 microM)-induced IBa suppression. 5. Pretreatment with taxol (10 - 100 microM) resulted in a concentration-dependent decrease in IBa and the action of CCh on IBa. The inhibitory effects of taxol and CCh on IBa were not additive. 6. Colchicine (100 microM) or taxol (100 microM) had no effect on voltage-gated K+ channel current or CCh-induced non-selective cationic channel current. 7. These results suggest that polymerization of microtubules leads to suppression of Ca2+ channel activity, and that muscarinic sustained suppression of Ca2+ channel current is mediated by a signal transduction element which involves microtubule cytoskeleton.

1. Although Na+-H+ exchange (NHE) inhibitors are reported to protect the myocardium against ischaemic injury, NHE activation has also been proposed as a potential mechanism of ischaemic preconditioning-induced protection. This study was performed to test any modifiable effect of cariporide, an NHE inhibitor, on cardioprotective effects of preconditioning. 2. Anaesthetized rats were subjected to 30 min of coronary artery occlusion and 150 min of reperfusion. The preconditioning (PC) was induced by 3 min of ischaemia and 10 min of reperfusion (1PC) or three episodes of 3 min ischaemia and 5 min reperfusion (3PC). Cariporide (0.3 mg kg(-1)) an NHE inhibitor, was administered 30 min (cari(30)) or 45 min (cari(45)) before coronary ligation (n=8-11 for each group). 3. Ventricular arrhythmias during 30 min ischaemia and infarct size (measured by triphenyltetrazolium (TTC) and expressed as a per cent area at risk (%AAR)) were determined. Cari(30) reduced ventricular fibrillation (VF) incidence and infarct size (from 45 to 0% and 34+/-4 to 9+/-2%; each P<0.05), whereas cari(45) did not. Likewise, 3PC reduced these variables (to 0% and 10+/-2%; P<0.05 in each case) whereas 1PC did not. Moreover, subthreshold preconditioning (1PC) and cariporide (cari(45)), when combined, reduced VF incidence and infarct size (to 0% and 15+3%; each P<0.05 ). 4. In conclusion, changes in NHE activity do not seem to be responsible for the cardioprotective action of ischaemic preconditioning. Protective effects of NHE inhibition and subthreshold preconditioning appear to act additively.

1. The effect on membrane currents of infection of mouse neuroblastoma NA cells with rabies virus was studied by using the whole-cell patch clamp technique. 2. Three types of membrane currents, namely voltage-dependent Na+ current (I(Na)), delayed rectifier K+ current (I(K-DR)) and inward rectifier K+ current (I(K-IR)) were elicited in uninfected cells. 3. In cells 3 days after infection with the virus, no detectable change was observed in morphology and membrane capacitance, but I(Na) and I(K-IR) were significantly decreased in amplitude without any appreciable difference in the time course of current activation and inactivation. The voltage-dependence of I(Na) activation was significantly shifted in the positive direction along the voltage axis with a decreased slope. I(K-DR) remained almost unaltered after the viral infection. 4. The resting membrane potential, measured with a physiological K+ gradient across the cell membrane, was decreased (depolarized) after the viral infection. The depolarization was associated with the decreased amplitude of I(K-IR). 5. These results suggest that infection of mouse neuroblastoma NA cells with rabies virus causes reduction of functional expression of ion channels responsible for I(Na) and I(K-IR), and provide evidence for possible involvement of the change in membrane properties in the pathogenesis of rabies disease.

1. Whole-cell patch-clamp recordings were made from smooth muscle cells isolated from the longitudinal muscle layer of guinea-pig ileum. Carbachol (acting at muscarinic receptors) or histamine (acting at H1 histamine receptors) suppressed Ca2+ channel current. The effect of either agonist had an initial transient component followed by a sustained component. 2. Wortmannin inhibited transient and sustained components of carbachol-induced Ca2+ channel current suppression: half-effective inhibitory concentrations (IC50) were 1.1 microM and 0.6 microM for the two components respectively. Wortmannin also inhibited the transient phase of carbachol-induced cationic current (IC50 1.6 microM) and Ca2+-dependent K+-current (IC50 1.7 microM). Wortmannin did not appear to produce any direct block of cationic channels or Ca2+ channels. 3. Intracellular application of the phospholipase inhibitor D609 (tricyclodecan-9-ylxanthogenate) inhibited transient and sustained components of histamine action on the Ca2+ channel current: the IC50 was about 130 microM for both components. Carbachol action on Ca2+ channels was also inhibited by D609. D609 had no significant direct blocking effect on Ca2+ channels, cationic channels activated by carbachol, or Ca2+-activated K+-current in response to flash-photolysis of caged-inositol 1,4,5-trisphosphate. 4. Micromolar concentrations of wortmannin and D609 are inhibitors of both components of spasmogen-induced Ca2+ channel suppression. The data suggest that both components are mediated by a common, or similar, signal transduction element which is a phospholipase C (PLC) or phospholipase D (PLD) isoform.

1. To characterize increases in cytosolic free Ca2+ concentration ([Ca2+]i) associated with discharge of action potentials, membrane potential and [Ca2+]i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques. 2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca2+]i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca2+]i transients with an almost constant amplitude. 3. Nicardipine (1 microM) and La3+ (10 microM), blockers of voltage-dependent Ca2+ channels (VDCCs), abolished both the action potential discharge and the [Ca2+]i transient. 4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca2+-activated K+ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca2+]i transients. 5. Thapsigargin (1 microM), an inhibitor of SR Ca2+-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca2+]i transients without affecting action potentials. In addition. in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca2+ current but exerted a similar effect on its concomitant rise in [Ca2+]i to that on the action potential-triggered [Ca2+]i transient. 6. Similar action potential-triggered [Ca2+]i transients were induced by brief exposures to high-K+ solution. They were not decreased, but rather increased, after depletion of intracellular Ca2+ stores by a combination of ryanodine (30 microM) and caffeine (10 mM) through an open-lock of Ca2+-induced Ca2+ release (CICR)-related channels. 7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca2+]i. Intracellular Ca2+ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca2+]i transients by replenishing cytosolic Ca2+. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca2+]i transient.

1. Non-adrenergic, non-cholinergic (NANC) inhibitory transmission in chicken gizzard was studied by use of intracellular microelectrode techniques. Changes in membrane potential in response to NANC nerve stimulation were recorded in the gizzard smooth muscle pretreated with atropine (1 microM) and guanethidine (1 microM). 2. Field stimulation of the intramural nerves (FS) evoked inhibitory junction potentials (i.j.ps) which were abolished by tetrodotoxin (1 microM), but not inhibited at all by K+ channel blockers including apamin (0.5 microM), tetraethylammonium (TEA, 10 mM), charybdotoxin (0.2 microM) and glibenclamide (10 microM). 3. NG-nitro-L-arginine (3 mM), an inhibitor of nitric oxide (NO) synthase, inhibited i.j.ps. The effect was reversed by L-arginine (3 mM), but not by D-arginine (3 mM). 4. 8-Bromo cyclic GMP (100 microM), a membrane permeable analogue of cyclic GMP, produced a membrane hyperpolarization which was blocked by TEA (10 mM) or glibenclamide (10 microM). 5. NO at concentrations of up to 400 microM affected neither i.j.ps nor resting membrane potential. On the other hand, NO (80 microM) caused the membrane to hyperpolarize in the smooth muscle of guinea-pig ileum. 6. These results suggest that in the chicken gizzard, NANC i.j.ps may not arise from opening of conventional types of K+ channel and that NO seems unlikely to be involved in the generation of i.j.ps. A possible mechanism by which the inhibitory effect of NG-nitro-L-arginine on i.j.ps was brought about will be discussed.

1. The antiarrhythmic and haemodynamic effects of three class III antiarrhythmic drugs, MS-551, sematilide and dofetilide, were examined in the coronary artery, ligation-reperfusion model of pentobarbitone-anaesthetized rats, a species deficient in functional cardiac IK. MS-551 is a non-selective potassium channel blocker, while both sematilide and dofetilide are selective delayed rectifier potassium (K) channel (IK) blockers. 2. Before coronary ligation, 3 and 10 mg kg-1 MS-551 decreased the heart rate by 6% (P < 0.01) and 12% (P < 0.01), and increased mean arterial pressure (MAP) by 14% (P < 0.05) and 33% (P < 0.01), respectively. Sematilide at 10 and 30 mg kg-1 also decreased the heart rate by 4% (P < 0.01) and 9% (P < 0.01), respectively, and the higher dose of 30 mg kg-1 decreased MAP by 29% (P < 0.01). Dofetilide, 1 mg kg-1, decreased the heart rate (P < 0.01), but had no significant effect on MAP. 3. The QT interval was increased by 10% (P < 0.01) and 31% (P < 0.01), when 3 and 10 mg kg-1 MS-551 were given. Sematilide and dofetilide had no effect on the QT interval. 4. Immediately after reperfusion, lethal ventricular fibrillation (VF) was induced in 80% of the saline group. MS-551 at 3 and 10 mg kg-1, reduced the incidence of lethal VF to 50% and 20% (P < 0.05). Neither dofetilide 1 mg kg-1 nor sematilide (10 and 30 mg kg-1) decreased the incidence of lethal VF (70%, 80% and 50%, respectively). None of the three drugs had any effect on the occurrence of reperfusion-induced VT or the total incidence of VF. However, 10 mg kg-1 MS-551 delayed the onset of reperfusion-induced VF (27 +/- 5 s compared with 12 +/- 2 s of the control group, P < 0.05). 5. In conclusion, in rats which are deficient in cardiac IK MS-551 prolonged the QT interval and reduced the incidence of sustained VF after reperfusion. Blockade of channels other than IK might participate in the defibrillatory effect of MS-551. Sematilide and dofetilide, which are selective IK blockers, did not increase the QT interval nor did they show antiarrhythmic effects Mechanisms other than K channel block may be involved in the different effects of the three drugs on blood pressure.

1. To see if arachidonic acid (AA) plays a role in the sustained suppression of voltage-gated calcium channel currents produced by muscarinic receptor stimulation by carbachol (CCh), the effects of AA on membrane currents were examined in whole-cell voltage-clamped smooth muscle cells of the guinea-pig ileum. 2. In cells bathed in Ba2+ PSS and dialysed with Cs(+)-based low EGTA (0.05 mM) pipette solution, and in which Ba2+ current (IBa) flowing through voltage-gated calcium channels was evoked repeatedly by stepping to 0 mV from the holding potential of -60 mV, AA (1-30 microM), applied extracellularly, gradually suppressed IBa in a concentration-dependent manner. The IBa suppression was observed even with 20 mM EGTA in the pipette. 3. AA (3 microM) and CCh (10 microM) shifted the voltage-dependent inactivation curve of IBa in the negative potential direction, but the effect of AA differed from that of CCh in that an accompanying appreciable decrease in the slope was observed. 4. The sustained suppression of IBa induced by CCh (10 microM) remained almost unaltered after pretreatment with 4-bromophenacyl bromide (10 microM), an inhibitor of phospholipase A2, or a combination of indomethacin (10 microM), an inhibitor of the cyclo-oxygenase pathway, and nordihydroguaiaretic acid (10 microM), an inhibitor of the lipoxygenase pathway. 5. In cells bathed in Ca2+ PSS and dialysed with K(+)-based pCa 6.5 pipette solution, voltage-dependent Ca2+ current (ICa) and K+ current (IK) were recorded simultaneously. AA (3 microM) suppressed IK as well as ICa, whereas CCh (10 microM) suppressed ICa but not IK. 6. We conclude from these results that AA or its metabolite is unlikely to be involved in the sustained suppression of voltage-gated calcium channel current induced by muscarinic receptor stimulation in guinea-pig ileal smooth muscle cells.

1. Oscillations of cytosolic Ca2+ concentration ([Ca2+]i) evoked by carbachol (CCh; 2 microM), a muscarinic agonist, were detected as oscillatory changes of muscarinic receptor-coupled cationic current (Icat) in guinea-pig ileal smooth muscle cells by the whole cell patch-clamp technique. 2. Reduction of extracellular Ca2+ from 2 mM to 0.2 or 0.05 mM, during CCh-induced Icat oscillations, caused them to disappear or to decrease markedly in frequency. A return to 2 mM Ca2+ concentration restored the initial Icat oscillations. 3. Application of nifedipine (1-3 microM) or D600 (2-5 microM) to block the voltage-gated Ca2+ channel (VGCC) decreased the frequency of the ongoing Icat oscillations in the cells held at -20 mV, but it was without effect in cells held at -60 mV. 4. Displacement of the holding potential of -20 mV to -60 mV to deactivate VGCC produced a decrease, an increase or no noticeable change in the frequency of the Icat oscillations in different cells. Displacement to 20 mV to inactivate VGCC invariably produced a decrease in the frequency. In nifedipine-treated cells, the Icat oscillations varied in frequency voltage-dependently in a reverse and linear way within the range -80 to 40 mV. 5. Application of thapsigargin (1 or 2 microM), an inhibitor of Ca(2+)-ATPase in the membrane of internal Ca2+ stores, caused CCh-induced Icat oscillations to disappear with a progressing phase during which their amplitude, but not frequency, declined. 6. The results suggest that membrane Ca2+ entry has a crucial role to play in regulation of the frequency of CCh-induced [Ca2+]i oscillations in addition to persistence of their generation, and that the effect is brought about by a potential mechanism independent of Ca2+ store replenishment. They also provide evidence that two types of Ca2+ permeant channels, VGCC and an as yet unidentified channel, are involved in the Ca2+ entry responsible for modulation of [Ca2+]i oscillations.

1. Membrane current responses to ATP in enzymically-dispersed single smooth muscle cells from the chicken rectum were investigated by the whole-cell voltage clamp technique. 2. In cells dialysed with a KCl-rich solution under voltage clamp at a holding potential of -40 mV, ATP (10 microM) produced an inward current followed by an outward current. When the holding potential was changed to 0 mV and -80 mV, the biphasic current response to ATP was converted to an outward current alone and an inward current alone, respectively. 3. External application of tetraethylammonium (TEA, 5 mM), intracellular dialysis with a CsCl-rich solution, or inclusion of EGTA (10 mM) in the pipette abolished the outward current response to ATP. 4. Neither depletion of Ca2+ store with caffeine (10 mM) nor block of voltage-gated Ca2+ channels with nifedipine (10 microM) affected the biphasic current response to ATP. After removal of the extracellular Ca2+ the outward current response to ATP was abolished. 5. alpha,beta-methylene ATP (100 microM) elicited a current similar to the ATP-induced current. In the presence of alpha,beta-methylene ATP (100 microM), application of ATP (100 microM) was without effect. 6. In CsCl-filled cells, ATP analogues elicited an inward current and the order of potency was ATP not equal to alpha, beta-methylene ATP > ADP >> AMP. 7. Inclusion of GTP gamma S (0.2 mM) or GDP beta S (2 mM) in the pipette did not affect the ATP-induced inward current in CsCl-filled cells. The reversal potential of the ATP-induced inward current was about 0 mV and was completely inhibited after replacement of the cations in the bath solution by Tris. The reversal potential remained almost unchanged after replacement of Na+ in the bath solution with 1 10 mM Ca2+, but shifted in the negative direction after replacement of Na+ or both Na+ and Ca2+ with glucosamine.8. The results suggest that ATP acts on P2 purinoceptors to cause activation of cation channels with selectivity for Ca2+ over Na+. Moreover, it appears that no G-protein-mediated mechanism is involved and increased Ca2+ entry through the cation channels causes activation of Ca2+-activated K+ channels.

1. The effect of trimebutine on the voltage-dependent inward Ca2+ current was investigated by the whole-cell voltage-clamp technique in single smooth muscle cells from rabbit ileum. 2. Trimebutine (3-100 microM) reduced the Ca2+ current in a concentration-dependent manner. The inhibitory effect on the Ca2+ current was also dependent on the holding potential. The Ca2+ current after a low holding potential was inhibited to a greater extent than that after a high membrane potential: the IC50 values were 7 microM and 36 microM at holding potentials of -40 mV and -60 mV, respectively. The Ca2+ current elicited from a holding potential of -80 mV could not be reduced by as much as 50% of the control by trimebutine at concentrations as high as 100 microM. 3. Trimebutine (30 microM) shifted the voltage-dependent inactivation curve for the Ca2+ current by 18 mV in the negative direction. The affinity of the drug for Ca2+ channels was calculated to be 36 times higher in the inactivated state than in the closed-available state. 4. Blockade of the Ca2+ current by trimebutine, unlike verapamil, was not use-dependent. 5. The results suggest that trimebutine inhibits the voltage-dependent inward Ca2+ current through a preferential binding to Ca2+ channels in the inactivated state in the smooth muscle cell from rabbit ileum. The inhibitory effect of trimebutine on gastrointestinal motility is discussed in the light of the present findings.

1. The effects of neurotensin (NT) on membrane potential and membrane current of the longitudinal smooth muscle of chicken rectum were investigated by intracellular recording and whole-cell voltage clamp. 2. NT (3 nM-1.2 microM), when applied via the bathing medium, produced a concentration-dependent membrane depolarization with an EC50 of 18 +/- 2 nM (n = 7) which was accompanied by an increase in the membrane conductance. The effect was biphasic: an initial, rapid depolarization reached a peak within 2-3 min and then declined to a lower but still elevated level which was sustained until washout. 3. Excitatory junction potentials (e.j.ps), which were non-adrenergic non-cholinergic (NANC) in nature, were decreased in amplitude and total duration in the presence of NT (0.6 microM). The depression of the e.j.p. was due mainly to the reduction of the membrane resistance. 4. When NT was applied locally by means of pressure ejection from a micropipette containing NT, some cells responded with a membrane depolarization and some failed to respond, whereas e.j.ps could invariably be elicited from all of them. 5. In single muscle cells enzymatically isolated from the muscle and dialyzed under voltage clamp at -50 mV with a CsCl-rich solution, NT (5 or 10 microM) produced an inward current. NT-induced inward currents were obtained with inclusion of 10 mM EGTA in the pipette solution and their reversal potential was around 0 mV. In cells dialyzed under voltage clamp at 0 mV with a KCl-rich solution, NT (5 microM) produced a brief outward current followed by abolition of spontaneous transient outward currents.6. The present results suggest that the membrane depolarization, which may arise from activation of non-selective cation channels, and release of calcium from internal stores produced by neurotensin are responsible for its contractile activity in the longitudinal smooth muscle of chicken rectum. Further, the depolarizing effect may provide support for the involvement of NT in the NANC transmission in this preparation.

1. Single smooth muscle cells were obtained from the rabbit portal vein by enzymic digestion and membrane currents under voltage clamp measured by whole-cell patch clamp technique. 2. When held at depolarized potentials, spontaneous outward currents (STOCs) were discharged; it is likely that these represent the cyclical storage and release within the cell of calcium in relation to Ca-activated K-channels. 3. Application of lower concentrations of carbachol (10(-5)M) or caffeine (10(-3)M) accelerated STOC discharge. Higher concentrations of caffeine (10(-2)M) or carbachol (10(-4)M), or noradrenaline (10(-5)M), produced an outward current of 1-5 nA which disappeared within 5-15s and which was considered to result from the discharge of calcium stores; STOC discharge was abolished for a period. 4. Ryanodine (10(-5)-10(-4)M) or a non-hydrolysable GTP analogue, GTP gamma S (10(-5)-10(-3)M) introduced into the cell abolished STOC discharge within 2-5 min. STOCs were large in cells filled with GDP beta S (10(-3)M) and the action of GTP gamma S introduced at various concentrations was antagonized. 5. GTP gamma S (10(-4)-10(-3)M) in the cell reduced or abolished outward current to caffeine (10(-2)M) noradrenaline (10(-5)M) or carbachol (10(-4)M); the effect on caffeine outward current was antagonized by GDP beta S (10(-3)M) introduced into the cell. GDP beta S reduced noradrenaline outward current but not caffeine outward current implying the existence of a G-protein step in noradrenaline-evoked Ca-store release, possibly regulating phospholipase C enzyme activity and D-myo inositol 1,4,5 trisphosphate formation. 6. If cyclic AMP (10(-3)M) or cyclic GMP (10(-3)M) was introduced into the cell, or 8-bromo cyclic AMP (0.5 x 10(-3)M) or 8-bromo cyclic GMP (0.5 x 10(-3)M) applied to the cell in the bathing solution, STOC discharge was only slightly affected. However, the outward current to caffeine applied after noradrenaline was much enhanced. 7. The results could be explained if cyclic GMP and cyclic AMP enhance calcium storage whereas GTP gamma S depletes calcium stores, an action antagonized by GDP beta S.

1. Changes in membrane potential in response to local application of ATP by pressure ejection from a micropipette were recorded intracellularly from smooth muscle cells of the longitudinal muscle strip of chicken rectum. 2. The local application of ATP produced a membrane depolarization. The depolarizing response increased to a plateau of 33 mV with prolongation of the duration of pressure pulses which determines the amount of ATP ejected. The latency and the time required to reach a peak depolarization were not related to the pulse duration, and the shortest latency was 50 ms. 3. When the application of ATP was repeated at a short interval, the second and subsequent depolarizing responses were suppressed, and their latency and time to reach a peak were also increased; the muscle cells became desensitized to ATP. Recovery from the desensitization occurred slowly over a period of 60 s. 4. Electrotonic potentials decreased in amplitude and time course during an ATP-induced depolarization, indicating a decrease in membrane resistance. 5. The ATP-induced depolarization was longer in the latency than an excitatory junction potential (e.j.p.) elicited by electrical field stimulation of the intramural nerves. The other variables such as amplitude, time to reach the peak and duration could not be matched with those of the e.j.p. at the same time. 6. The e.j.p. decreased in amplitude and duration during the ATP-induced depolarization, and its initial amplitude and duration were restored immediately after termination of the ATP-induced depolarization, as in the case of electrotonic potentials. The ej.ps were recorded in the same manner as the control from the muscle cell which was in a state of desensitization to ATP. 7. The present results provide an argument against the hypothesis that ATP mediates the ej.p. in the rectum of the chicken.

1. Effects of prolonged exposure to alpha,beta-methylene ATP (alpha,beta-Me ATP) on contractions and excitatory junction potentials (e.j.ps) evoked by non-adrenergic, non-cholinergic (NANC) excitatory nerve stimulation have been investigated in the chicken isolated rectum and longitudinal muscle strip from chicken rectum pretreated with atropine (0.5 microM), methysergide (2 microM) and pyrilamine (3 microM). 2. Alpha,beta-Me ATP (20 nM-4 microM) caused a rapid rise in tension of the longitudinal muscle of the isolated rectum preparation which returned to the baseline levels after a few minutes. The magnitude of the contractile response to NANC nerve stimulation was reduced after exposure to the drug. The inhibitory effect was related to the drug concentration; at 4 microM the nerve-mediated contraction was abolished and frequently converted to a relaxation. 3. Adenosine 5'-triphosphate (ATP, 100 microM), bovine neurotensin (2.5 nM) and K+-rich solutions (30 nM and 60 nM) all produced a transient contraction of the isolated rectum preparation. The exposure to alpha,beta-Me ATP (0.2 and 4 microM) also rendered the preparation less sensitive to these stimulant substances. 4. Alpha,beta-Me ATP (0.2 and 4 microM) caused a membrane depolarization in cells of the longitudinal muscle strip. The depolarization reached a peak within 2-3 min after application and then decayed to a steady level that was still more positive than the baseline level. The electrotonic potentials were reduced in amplitude to 44 +/- 8% (n = 7) of the normal amplitude if measured at the peak depolarization produced with 0.2 microM alpha,beta-Me ATP, and to 62 +/- 10% (n = 7) if measured at the steady-state depolarization. With 4 microM, the corresponding percentages were 33 +/- 7% (n = 8) and 55 +/- 7% (n = 8), indicating a decrease in membrane resistance. 5. The e.j.ps in response to field stimulation of the intramural nerves and Remak's nerve stimulation were decreased in amplitude and duration during exposure to alpha,beta-Me ATP (0.2 and 4 microM). 6. The smooth muscle cells regained normal membrane resistance and sensitivity to ATP on washout of alpha,beta-Me ATP (4 microM) more rapidly than the responses to NANC nerve stimulation. 7. It can be argued from the results that the suppression by alpha,beta-Me ATP of the contraction and e.j.p. evoked by NANC nerve stimulation in the chicken rectum, unlike the mammalian preparation described previously, is due mainly to a change in the electrical properties of the membrane of the smooth muscle cells, rather than being due, or only partly due, to desensitization of the purine receptor.

1. Development of the excitatory innervation to the rectal region of the intestine via Remak's nerve has been investigated in the rectum with Remak's nerve supply isolated from chicken embryos and young chicks aged less than two weeks. 2. Electrical stimulation of Remak's nerve produced a small contraction of the rectum isolated from chicken embryos on the 14th day of incubation (the earliest time examined). The contractile response was inhibited partially or totally by atropine (0.1 micrograms ml-1) but enhanced by physostigmine (0.01 to 0.05 micrograms ml-1), indicating its cholinergic nature. 3. During the embryonic stage, the proportion of the atropine-resistant component in the contractile response increased, and the contractile response became almost entirely atropine-resistant within the first week after hatching. 4. Later after hatching, the contractile response was increased in magnitude by atropine and reduced by physostigmine. 5. It is concluded that the excitatory innervation to the chicken rectum via Remak's nerve is cholinergic at the 14-16th day of incubation and is gradually replaced by a non-cholinergic innervation during embryonic development.

1 The rectum isolated from the fowl was perfused with Tyrode solution via the caudal mesenteric artery. Noradrenaline and adrenaline were biologically or fluorimetrically assayed in perfusates collected before and during stimulation of Remak's nerve or of the periarterial nerves.2 Perfusates collected during nerve stimulation relaxed the chick rectum and rat stomach strips which served as assay tissues. This effect was attributed to the action of noradrenaline or adrenaline released from adrenergic nerve endings which appeared in the perfusates.3 Perfusates obtained during stimulation (30 Hz for 60 s) of Remak's nerve contained both noradrenaline and adrenaline when measured fluorimetrically. The mean output per stimulus train was 0.8 +/- 0.2 ng/g wet wt. tissue for noradrenaline and 1.7 +/- 0.2 ng/g wet wt. tissue for adrenaline (n = 7). Perfusates obtained during stimulation (30 Hz for 60 s) of the periarterial nerves contained noradrenaline in a concentration of 1.6 +/- 0.3 ng/g wet wt. tissue per stimulus train, but almost no adrenaline (n = 7).4 Neither stimulation of Remak's nerve nor the periarterial nerves liberated catecholamines when the rectum was perfused with Tyrode solution containing low Ca(2+) (0.1 mM) and high Mg(2+) (10 mM).5 Infusion of high potassium solution (50 mM) increased markedly the output of noradrenaline and adrenaline.6 Adrenaline as well as noradrenaline may function as the adrenergic neurotransmitter in the rectum of the fowl.

Cholinergic inhibition of the non-adrenergic, non-cholinergic ( NANC ) transmission was investigated in the chicken isolated rectum with Remak's nerve attached. Stimulation of Remak's nerve (RT stimulation) at frequencies higher than 5 Hz elicited a late, slow contraction of the rectum in addition to an initial, fast NANC contraction. The late, slow contraction was blocked by atropine (0.25 microgram ml-1), potentiated by physostigmine (50 ng ml-1) and accompanied by an overflow of acetylcholine into the vascular perfusate, indicating the existence of cholinergic innervation to the rectum via Remak's nerve. RT stimulation (10 pulses at 0.5-1.0 Hz) elicited NANC -mediated excitatory junction potentials (e.j.ps). The e.j.p. amplitude declined at the second stimulus and then increased to reach a plateau. Atropine, by abolishing this decrease in amplitude, increased the mean amplitude of the e.j.ps during trains of stimuli but atropine did not affect the amplitude of the first e.j.p. Physostigmine reduced the mean e.j.p. amplitude, and this action was readily antagonized by atropine. A single intramural nerve stimulation delivered 2s or less before RT stimulation with trains of stimuli, suppressed the amplitude of the first e.j.p. of the train. This effect was abolished by atropine. Atropine in concentrations high enough to affect the e.j.p. amplitude had no effect on the resting membrane potential, the threshold for generating an action potential, or membrane resistance of the smooth muscle. It is concluded that RT stimulation at low frequencies causes the release of acetylcholine simultaneously with the NANC transmitter. The released acetylcholine acts mainly on prejunctional muscarinic receptors and mediates an inhibitory effect on the release of the NANC transmitter.

1. Isolated longitudinal muscle strips from the chicken rectum responded to isoprenaline, adrenaline and noradrenaline with a prolonged relaxation. The concentrations required to produce 50% of the maximum relaxation were 1.3 x 10(-8) M for isoprenaline, 1.7 x 10(-8) M for adrenaline and 10(-6) M for noradrenaline. The relaxing potency of isoprenaline is about equal to that of adrenaline, but more than 50 times that of noradrenaline. 2. Propranolol, 3.4 x 10(-6) M, blocked the isoprenaline-induced relaxation, and in the presence of this drug the responses to adrenaline and noradrenaline were converted into small, transient relaxations. The residual relaxation was blocked by phentolamine, 2.6 x 10(-6) M. 3. These catecholamines suppressed spontaneous spike discharge and produced membrane hyperpolarization. Propranolol, 3.4 x 10(-6) M, prevented the inhibitory effects of isoprenaline, and reduced but did not completely abolish those of adrenaline and noradrenaline. 4. Adrenaline and noradrenaline, but not isoprenaline, reduced membrane resistance in some preparations. 5. In the rectal muscle of the chicken, the beta-adrenoceptor mediates a prolonged relaxation and the alpha-adrenoceptor a fast and short-lasting relaxation which is usually obscured by the beta-response and unmasked only after blockade of the beta-adrenoceptors. The alpha- and beta-mediated relaxations are each associated with the suppression of spontaneous spike activity.

Peripheral nerve pathways responsible for adrenergic inhibition of mechanical and electrical activities in the chicken rectum and receptors mediating the adrenergic inhibition were investigated in isolated extrinsically-innervated rectum of the chicken. Electrical stimulation of the anal end (Ra) or the ileal cut end (Ri) of Remak's nerve, or perivascular nerves (P) elicited relaxation of the rectum pretreated with atropine (0.5 microM) and hexamethonium (0.3 mM) to block the cholinergic and non-cholinergic, non-adrenergic excitatory innervations. Ri stimulation was much less effective than Ra and P stimulations. The relaxation was shown to be related to cessation of spontaneous spike discharge of the longitudinal muscle which was accompanied by membrane hyperpolarization. The inhibitory effects elicited by Ra and P stimulations, which were prolonged beyond the period of the stimulation, were converted to transient ones by propranolol (3.4 microM). Phentolamine (2.6 microM) reduced effectively the residual effects. In contrast, the effects of Ri stimulation were little affected by these drugs. The present results provide evidence for the existence of two nerve pathways responsible for direct adrenergic inhibitory innervation to the chicken rectum, one running orally in Remak's nerve trunk, leaving it and descending in the branches to the rectum, and the other running as the perivascular nerves along the arterial supplies of the rectum. The direct innervation is mediated predominantly by beta-adrenoceptors.