Inhibitory actions of cilostazol on electrical responses of smooth muscle isolated from the guinea-pig stomach antrum.

We have investigated the effects of cilostazol, a type III phosphodiesterase inhibitor, on the electrical responses of smooth muscle tissue isolated from the guinea-pig stomach antrum. Cilostazol (10(-5) M) inhibited slow waves recorded from circular muscle cells, but did not significantly alter the pacemaker potentials and follower potentials recorded from myenteric interstitial cells and longitudinal muscle cells respectively. Slow potentials generated in isolated circular muscle bundles without attached myenteric interstitial cells were inhibited by cilostazol (>10(-7) M), while all membrane activities were abolished by 10(-5) M cilostazol. In circular muscle bundles, the input resistance of smooth muscle cells and the refractory period for the generation of slow potentials were not altered during the inhibition of spontaneous activity with cilostazol. While cilostazol at 10(-7) and 10(-6) M did not elevate the tissue content of cyclic AMP, at 10(-5) M cyclic AMP was elevated by about 30%. A similar elevation was also produced by 10(-7) M forskolin. The content of cyclic AMP was not significantly increased in preparations stimulated with 10(-3) M caffeine. The potency for inhibiting slow waves was in the order caffeine (10(-3) M) > forskolin (10(-7) M) > cilostazol (10(-5) M). The frequency of slow waves was decreased by caffeine or forskolin but not by cilostazol, while the duration was reduced by caffeine but not by cilostazol or forskolin. Follower potentials were modulated by caffeine and forskolin, but not by cilostazol: the duration was reduced by caffeine, the frequency was reduced by caffeine or forskolin, and the amplitude was not significantly altered by any of them. The results indicate that cilostazol has high selectivity in inhibiting the activity of circular muscle much more than that of longitudinal muscle or pacemaker cells, with no causal relation to the tissue content of cyclic AMP as appears to be the case for the inhibitory actions of caffeine and forskolin.     

Effects of endogenous and exogenous nitric oxide on electrical responses of circular smooth muscle isolated from the guinea-pig stomach antrum.

The effects of endogenous and exogenous nitric oxide (NO) on electrical activity were investigated in circular smooth muscle preparations isolated from the guinea-pig stomach antrum. The actions of endogenous NO were evaluated from the effects of inhibition of NO synthesis by N(omega)-nitro-L-arginine (nitroarginine), while those of exogenous NO were assessed from the effects of SIN-1, an NO donor. Antral circular smooth muscle generated slow potentials periodically at a frequency of about 1 cycle per min (cpm), and unitary potentials were also generated in a random fashion in the interval between slow potentials. Application of nitroarginine (10(-5) M) increased the frequency of slow potentials, with no significant alteration of the resting membrane potential and amplitude of slow potentials. Frequency analysis of unitary potentials revealed that nitroarginine also increased the spectral density at 0.01-1 Hz frequency. The refractory period for the generation of slow potentials evoked by depolarizing pulses was about 10 s, but was decreased to 6 s by nitroarginine. In the presence of nitroarginine, SIN-1 (10(-9)-10(-7) M) reduced the amplitude and frequency of slow potentials: low concentrations (<10(-8) M) reduced only the frequency of slow potentials, while higher concentrations (10(-8)-10(-7) M) reduced both the amplitude and frequency of slow potentials, in a concentration-dependent manner, before abolishing the slow potentials. The power spectrum of the unitary potentials indicated that SIN-1 (>10(-8) M) reduced the spectral density at 0.01-1 Hz frequency. The refractory period for the generation of slow potentials was increased again to about 10 s by SIN-1. Thus, the excitatory effects of nitroarginine could be antagonized by SIN-1, suggesting that the inhibitory effects of endogenous NO are comparable to those of exogenous NO produced by SIN-1. The results also suggested that the effects of NO on smooth muscle are insignificant and NO selectively inhibits the activity of intramuscular interstitial cells of Cajal (ICC-IM).     

Effects of 5-hydroxytryptamine on electrical responses of circular smooth muscle isolated from the guinea-pig gastric antrum.

The effects of 5-hydroxytryptamine (5-HT) on electrical responses of the membrane were investigated in circular smooth muscle isolated from the guinea-pig stomach antrum. Small segment of circular muscle tissue produced a periodical generation of slow potentials at frequency of 0.1-2 cycles min(-1), during random generation of unitary potentials. Application of 5-HT (10(-7)-10(-5) M) hyperpolarized the membrane and either increased or decreased the frequency of slow potentials, both with associated increase in amplitude of slow potential. These effects of 5-HT were abolished by methysergide. N(omega)-nitro-L-arginine (L-NA) increased the frequency of spontaneously generated slow potentials and also increased the frequency of slow potentials generated during stimulation with 5-HT, suggesting an involvement of the increased production of nitric oxide (NO) by 5-HT. Atropine did not alter spontaneous and 5-HT-induced electrical responses. The hyperpolarization produced by 5-HT was associated with a decrease in input resistance and time constant of the membrane. The amplitude of the 5-HT-induced hyperpolarization was increased in low [K(+)](o) solution and decreased in high [K(+)](o) solution or in the presence of glybenclamide, suggesting that the hyperpolarization was produced by activation of ATP-sensitive K-channels. The increase in amplitude of slow potentials by 5-HT may be secondary due to hyperpolarization of the membrane. The inhibition by 5-HT of the frequency of slow potentials may be partly due to the increased release of NO, however the mechanism by which dual effects of 5-HT on the frequency of slow potentials remains unsolved.     

Dual effects of cyclopiazonic acid on excitation of circular smooth muscle isolated from the guinea-pig gastric antrum.

The effects of cyclopiazonic acid (CPA), a known Ca2+-pump inhibitor at internal stores, were investigated on electrical responses of the membrane of smooth muscle cells in small segments (0.3-0.5 mm long) of circular smooth muscle isolated from the guinea-pig gastric antrum. In most preparations, the membrane was spontaneously active with the generation of unitary potentials and regenerative slow potentials. Low concentrations (< 1 microM) of CPA did not alter either the membrane potential or the amplitude and frequency of slow potentials. CPA at a concentration of 1 microM initially increased the frequency of slow potentials, but this was followed by a decrease in the frequency as a result of sustained exposure to CPA, with no alteration of either the membrane potential or the amplitude of slow potentials. Higher concentrations of CPA (2-5 microM) depolarized the membrane and decreased the amplitude and frequency of slow potentials. CPA at higher than 10 microM abolished slow potentials with depolarization of the membrane. Intracellular electrical responses recorded simultaneously from paired cells were synchronized, indicating electrical coupling of the cells. Depolarization of the membrane with current stimuli through one electrode evoked regenerative slow potentials superimposed on the electrotonic potentials. The evoked slow potential had a refractory period of about 7 s. CPA (up to 10 microM) did not prevent the synchronization of paired cells. The refractory period for slow potentials was reduced by low concentrations of CPA (< 1 microM) and increased by higher concentrations of CPA (2-10 microM). These results suggest that lower concentrations of CPA produce excitatory actions on gastric smooth muscles due to a secondary effect of increased intracellular [Ca2+], while higher concentrations of CPA produce inhibitory actions as a result of reduced release of Ca2+ from depleted internal stores.     

The effects of flufenamic acid on spontaneous activity of smooth muscle tissue isolated from the guinea-pig stomach antrum.

The effects of flufenamic acid were investigated on slow waves, follower potentials and pacemaker potentials recorded respectively from circular smooth muscle cells, longitudinal smooth muscle cells and interstitial cells of Cajal distributed in the myenteric layers (ICC-MY) of the guinea-pig stomach antrum. Flufenamic acid (>10(-5) M) inhibited the amplitude and rate of rise of the upstroke phase of the slow waves, with no marked alteration in their frequency of occurrence. The inhibitory actions of flufenamic acid appeared to be mainly on slow potentials recorded from circular smooth muscle cells, but not on follower or pacemaker potentials. After abolishing spontaneous slow potentials with flufenamic acid, depolarizing current stimuli could evoke slow potentials with an amplitude that was much smaller than in the absence of flufenamic acid, with no significant alteration to the input resistance of the membrane. The time elapsed for the generation of the 2nd component of the slow waves or the slow potentials evoked during depolarizing current pulse stimulation was increased by flufenamic acid. The rate of rise of unitary potentials, but not the frequency of occurrence, was inhibited by flufenamic acid. These results indicate that the inhibitory actions of flufenamic acid appear to be mainly on the circular muscle layer including the interstitial cells of Cajal distributed within the muscle bundles (ICC-IM). Nifedipine-sensitive spike potentials were not inhibited by flufenamic acid. It is concluded that the selective inhibition of the 2nd component of slow waves by flufenamic acid may be mainly due to the inhibition of ion channels, possibly Ca2+-sensitive Cl--channels, activated during generation of slow potentials in the ICC-IM distributed in the circular muscle layer.     

Effects of inhibitors of nonselective cation channels on the acetylcholine-induced depolarization of circular smooth muscle from the guinea-pig stomach antrum.

In circular smooth muscle bundles isolated from the guinea-pig stomach antrum, the effects of quinidine, Ni2+, flufenamic acid, niflumic acid, La3+, SKF-96365 and 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on acetylcholine (ACh)-induced depolarization were investigated. Recording membrane potentials from smooth muscle cells with intracellular microelectrodes revealed that ACh (1 microM) depolarized the membrane by 5-8 mV and increased the amplitude and frequency of slow potentials. These effects were inhibited by atropine. Quinidine (10 microM) increased the amplitude of ACh-induced depolarization, with no alteration to the properties of slow potentials. Ni2+ (50 microM) transiently (5-10 min) depolarized the membrane by about 5 mV, with an associated increase in frequency and amplitude of slow potentials. In the stabilized condition with Ni2+, the amplitude of ACh-induced depolarization remained unchanged. Flufenamic acid (10 microM) inhibited the generation of slow potentials, with no change in either the amplitude of ACh-induced depolarization or of the amplitude and frequency of slow potentials generated during ACh stimulation. A high concentration of flufenamic acid (100 microM) depolarized the membrane and increased the amplitude of ACh-induced depolarization. Niflumic acid (10 microM) hyperpolarized the membrane and increased the amplitude and frequency of slow potentials and also the amplitude of ACh-induced depolarization. DIDS (100 microM) hyperpolarized the membrane and inhibited the amplitude and frequency of slow potentials, with no alteration to the amplitude of ACh-induced depolarization. SKF-96365 (3-50 microM) depolarized the membrane in a concentration-dependent manner, but did not change the level of ACh-induced depolarization. La3+ (50 microM) did not alter the properties of the slow potentials or the ACh-induced responses. These results provide evidence that ACh-induced depolarization is not inhibited by chemicals known to inhibit non-selective cation channels. We suggest that muscarinic receptor-mediated signal transduction may be different in smooth muscle and interstitial cells.     

Spontaneous activity and its cholinergic modulation in circular smooth muscle isolated from guinea-pig stomach antrum

Circular smooth muscle isolated from the guinea-pig gastric antrum generated periodic slow potentials in the presence of nifedipine and nitroarginine to prevent the activity of voltage-gated L-type Ca-channels and endogenous production of NO respectively. Chelerythrine, an inhibitor of protein kinase C (PKC), in the concentration range 10^–7–3×10^–7 M reduced the frequency but not the amplitude of spontaneous slow potentials without altering the resting membrane potential. 2-Aminoethoxydiphenyl borate (2-APB, 3×10^–6 M), an inhibitor at inositol-1,4,5-trisphosphate (IP₃) receptors, depolarized the membrane, increased the frequency and reduced the amplitude of the slow potentials; the latter actions were independent of depolarization. Two different phorbol esters, phorbol 12,13-dibutyrate and phorbol-12-myristate-13-acetate, increased the frequency of slow potentials, without altering the amplitude or changing the resting membrane potential; the effects of phorbol esters were antagonized by chelerythrine. Stimulation of muscarinic receptors with acetylcholine (ACh), in concentrations below those causing membrane depolarization (3×10^–8–10^–7 M), increased the amplitude and frequency of slow potentials. Chelerythrine inhibited the ACh-induced increase in the frequency of slow potentials but did not prevent the increase in their amplitude. 2-APB inhibited the ACh-induced increase in the amplitude of slow potentials but did not prevent the increase in their frequency. These results suggest that the frequency of spontaneous slow potentials is regulated by PKC and their amplitude by IP₃ production. ACh increases both the amplitude and frequency of slow potentials; the former is related to the activation of PKC, while the latter is related to activation of IP₃-receptors.     

Role of protein kinase C in the excitatory action of cholinergic nerve stimulation on spontaneous activity of circular smooth muscle isolated from the guinea-pig stomach antrum

Following inhibition of NO production with nitroarginine, circular muscle isolated from the guinea-pig gastric antrum generated periodic slow potentials and unitary potentials. Transmural nerve stimulation (TNS) during the interval between slow potentials evoked an apamin-sensitive inhibitory junction potential (IJP) followed by an atropine-sensitive depolarization; the latter was either a transient depolarization with enhanced generation of unitary potentials or a slow potential. After inhibition of unitary potentials and slow potentials with 1 mM caffeine, TNS evoked an IJP and subsequent cholinergic depolarization, the latter developing slowly and lasting for about 10 s. TNS was unable to elicit a slow potential until a certain period of time had elapsed following the cessation of a slow potential. The period during which TNS could not evoke slow potentials (termed the high-threshold period) was about 10 s, and this period was increased by chelerythrine and decreased by phorbol esters. It is concluded that cholinergic nerve-mediated excitation of gastric muscle involves the activation of protein kinase C (PKC), and that the high-threshold period, during which the generation of slow potentials by TNS is inhibited, may be a consequence of reduced activity of PKC.     

Effect of phorbol 12,13-dibutyrate on smooth muscle tone in rat stomach fundus.

We investigated the effects of phorbol 12,13-dibutyrate (PDBu), a typical protein kinase C (PKC) activator, on smooth muscle tone in the rat stomach fundus. In 5-hydroxytriptamine (5-HT)-precontracted stomach fundus strips, PDBu induced dose-dependent relaxation, but 4alpha-phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not induce relaxation. A PDBu-induced dose-dependent relaxation was also observed in strips precontracted with platelet-activating factor (PAF), carbachol, or 60 mM K+. In stomach fundus strips pretreated with PDBu, the contractile responses to 5-HT and PAF were completely blocked, but those induced by carbachol and endothelin-1 (ET-1) were only partially inhibited. In stomach fundus strips preincubated with carbachol in Ca2+-free medium, the Ca2+-induced contraction was decreased by preincubation with PDBu. In strips preincubated with 5-HT, PAF, or ET-1 in Ca2+-free medium, Ca2+-induced contractions were greatly inhibited by pretreatment with PDBu. These results suggest that in rat stomach fundus strips, PDBu-induced relaxation is mediated by activation of PKC. We speculate that a major factor mediating the relaxant action of PDBu in rat stomach fundus smooth muscle is represented by a reduction in Ca2+ influx via an inhibition of Ca2+ channels.     

The electrical activity recorded from smooth muscle of the circular layer of the human stomach

The membrane properties of circular muscles of 55 human stomachs were investigated by microelectrode and double sucrose gap methods. The membrane potential of the circular muscle of the corpus region was —57 mV and no regional difference was evident as compared with tissues from the antrum and cardia. The stomach muscle presented cable like properties, and the length constant measured in the corpus region was 1.34 mm. The circular muscle of all regions of the stomach exhibited slow waves. The amplitude and duration of slow waves varied markedly (the mean values were 18 mV and 6 s, respectively). TheQ ₁₀ value for the slow wave was 2.4. The slow wave could be divided into two different components (first and second component) by application of electrical current or by using solutions with various ionic environments. Na ions had more effect on the spike component and Ca ions on the second component. The generation of the first component of the slow wave was blocked by either Na-free, K-free, Ca-free, or Cl-deficient solution but this component reappeared by application of outward current pulse, except in Cl-deficient solution. These results suggest that the generation of slow wave depends on more than one type of ion and that metabolic factors do indeed play a role. Membrane properties of the human stomach were compared with those of the guinea-pig stomach.     

Some differences in contractile responses of isolated longitudinal and circular muscle from the guinea-pig stomach.

The mechanical and electrical properties of the longitudinal (fundus and corpus) and circular (antrum) muscle fibres of the guinea-pig stomach were investigated. 1. In the longitudinal but not in the circular muscle isotonic K Krebs and Na-free (sucrose) Krebs solutions produced a contracture with a tonic component. The different mechanical responses were not accompanied by different membrane responses. Verapamil abolished both phasic and tonic components of K-induced contracture. 2. During the tonic response of the K-induced contracture, repolarization of the membrane by current pulses relaxed the tissue; after cessation of the current pulse, rebound contracture occurred. In the circular muscle, the Q10 value for the rate of relaxation induced by inward current pulse was 3-1 and for the development of rebound contracture was 2-4. 3. After the tissue had been immersed in Ca-free isotonic K Krebs solution, application of Ca produced a large contracture in the longitudinal muscle, but contracture in the circular muscle was small or absent. However, the amplitude of subsequent carbachol-induced contracture in the above solution was enlarged in proportion to the durations of Ca treatment in both tissues. 4. Direct tetanic electrical stimulation could produce tension in both tissues. With low frequency of stimulation (0-1 Hz) a positive staircase was observed in the circular but not in the longitudinal muscle. 5. It is concluded from these results that topical differences of the motility in the stomach may be due not only to the activity of nervous elements, but also to differences in the properties of the muscle fibres themselves.     

Effects of iodoacetate on spontaneous electrical activity, slow wave, in the circular muscle of the guinea-pig gastric antrum

In the circular muscle of the guinea-pig gastric antrum, the contribution of glycolysis to spontaneous electrical activity, slow wave, was studied. The slow wave could be maintained without a marked change in glucose-free solution for more than 1 h even when treated with iodoacetic acid (IAA, 0.1-0.5 mM). However, reapplication of glucose following the IAA treatment produced clear inhibitory effects on the slow wave. Lactate release from the tissue was reduced to about 10% of the control by IAA (0.1 mM) in the absence of glucose and there was very slow recovery on glucose reapplication. This suggests that IAA did not block glycolysis completely and that the inhibition of slow wave was mainly due to the accumulation of some metabolites. Small electrical activity often remained during the inhibition by IAA and glucose. When the excitability of the smooth muscle was increased by Co(2+) application or Na(+) removal, slow wave-like activity could be generated under the condition in which the slow wave was strongly inhibited by IAA and glucose. These results may be explained by assuming that the accumulation of glycolytic metabolites decreases the excitability of smooth muscle cells and also reduces the driving potential generated in the interstitial cells of Cajal to a subthreshold level for the slow wave in the smooth muscle cells.     

Voltage dependency of the frequency of slow waves in antrum smooth muscle of the guinea-pig stomach

The effects of membrane depolarization on the frequency of spontaneous activities were investigated in circular smooth muscle of the guinea-pig antrum attached with (intact tissue) or without longitudinal muscles (circular tissue). Both types of tissue were spontaneously active; the intact tissues generated slow wave and circular tissues generated regenerative potential. The latter but not the former was abolished by caffeine. Increasing K(+) concentrations depolarized the membrane and reduced the amplitude and interval between spontaneous activities in both tissues; the amplitude was reduced linearly with depolarization and disappeared at about -35 mV; the interval was reduced successively with depolarization and reached a stable value (about 8 s) at about -45 mV. The depolarization and reduction in amplitude and interval of spontaneous activities induced by high K(+) solution were not altered by atropine, nitroarginine, or apamin in either tissue, suggesting that these changes did not involve the effects of neurotransmitters. The depolarization of the membrane by electrical stimulation also reduced the amplitude and interval of spontaneous activities in both tissues, in a potential-dependent way. The absolute refractory period for generation of the evoked regenerative potential was about 8 s, and the relative refractory period was 8--12 s. The results indicate that the frequency of slow waves increases with a depolarization of the membrane up to -45 mV, irrespective of the presence of caffeine-insensitive components. A depolarization of the membrane above -45 mV does not further increase the frequency of slow waves, possibly because of the refractory period for the generation of slow waves.     

Effects of RHC-80267, an inhibitor of diacylglycerol lipase, on excitation of circular smooth muscle of the guinea-pig gastric antrum.

In small segments of circular smooth muscle isolated from the guinea-pig gastric antrum, the effects of RHC-80267, an inhibitor of diacylglycerol lipase, were investigated both on regenerative slow potentials (either occurring spontaneously or as the result of a depolarizing intracellular current injection) and on the actions of acetylcholine (ACh). As diacylglycerol is a known activator of protein kinase C (PKC), it would therefore be expected that RHC-80267 would activate PKC indirectly. In circular smooth muscle bundles, spontaneously generating slow potentials recorded simultaneously from two given cells were synchronized, indicating that these two cells were electrically coupled. RHC-80267 (0.3-1 microM) increased the frequency of slow potential generation, with no alteration to the amplitude of either the slow potentials or the resting membrane potential. Synchronous electrical activity in a given pair of cells was also unchanged by RHC-80267, indicating that intercellular electrical coupling was not altered. The input resistance of smooth muscle cells calculated from the amplitude of electrotonic potentials produced by injection of current was not significantly altered by RHC-80267. The refractory period for the generation of slow potentials evoked by depolarizing stimuli was about 8 s, and it was decreased to about 5 s by RHC-80267, with no significant alteration to the amplitude of spontaneous or evoked slow potentials. ACh (0.5 microM) depolarized the membrane by about 5 mV and increased the amplitude and frequency of slow potentials. The actions of ACh on the frequency of slow potentials were enhanced by RHC-80267, with no alteration to the amplitudes of both the ACh-induced depolarization and slow potentials. These results support the idea that PKC is involved in determining the frequency of slow potentials, by shortening the refractory period for excitation of gastric smooth muscle cells.     

Properties of spontaneous electrical activity in smooth muscle of the guinea-pig renal pelvis

In the guinea-pig renal pelvis, most smooth muscle cells examined (>90%), using a conventional microelectrode, had a resting membrane potential of about -50 mV and produced spontaneous action potentials with initial fast spikes and following plateau potentials. The remainder (<10%) had a resting membrane potential of about -40 mV and produced periodical depolarization with slow rising and falling phases. Experiments were carried out to investigate the properties of spontaneous action potentials. The potentials were abolished by nifedipine, suggesting a possible contribution of voltage-gated Ca(2+) channels to the generation of these potentials. Niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), inhibitors of Ca(2+)-activated Cl(-) channels, showed different effects on the spontaneous action potentials, and the former but not the latter inhibited the activities, raised the question of an involvement of Cl(-) channels in the generation of these activities. Depleting internal Ca(2+) stores directly with caffeine or indirectly by inhibiting Ca(2+)-ATPase at the internal membrane with cyclopiazonic acid (CPA) prevented the generation of spontaneous activity. Chelating intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) increased the amplitude of the spike component of spontaneous activity. Indomethacin inhibited the spontaneous activity, whereas prostaglandin F(2 alpha) enhanced it. The results indicate that in smooth muscle of the renal pelvis, the generation of spontaneous activity is causally related to the activation of voltage-gated Ca(2+) channels through which the influx of Ca(2+) may trigger the release of Ca(2+) from the internal stores to activate a set of ion channels at the membrane. Endogenous prostaglandins may be involved in the initiation of spontaneous activity.     

Effects of removal of Na(+) and Cl(-) on spontaneous electrical activity, slow wave, in the circular muscle of the guinea-pig gastric antrum

In the circular muscle of the guinea-pig gastric antrum, a decrease in the external Na(+) to less than 20 mM produced depolarization of the membrane with transient prolongation of the slow wave. This was followed by a high rhythmic activity. The activity was inhibited by reapplication of Na(+) before recovery. The depolarization in Na(+)-deficient solution was prevented and rhythmic activity continued at about 5/min for at least 6 min by simultaneous removal of K(+), Ca(2+), or Cl(-). After exposure to a Na(+)- and Cl(-)-deficient solution for a few minutes, reapplication of the Na(+) in Cl(-)-deficient solution inhibited generation of the slow wave until Cl(-) reapplication. Similar results were obtained when Na(+) and Cl(-) were reapplied in the absence of K(+) after exposure to a Na(+)-, K(+)-free, and Cl(-)-deficient solution, although the inhibition was weaker than Na(+) reapplication in a Cl(-)-deficient solution. In the presence of furosemide or bumetanide, a strong inhibition of activity was produced by the reapplication of Na(+) and Cl(-) after exposure to an Na(+)- and Cl(-)-deficient solution. A hypothesis is presented that intracellular Ca(2+) concentration ([Ca(2+)](i)) is the most important factor determining the generation and frequency of the slow wave and that [Ca(2+)](i) is regulated by the Na(+) concentration gradient across the plasma membrane. The recovery of the Na(+) concentration gradient by Na(+) reapplication after removal of Na(+) and Cl(-) is mainly controlled by a Na(+)-K(+)-Cl(-) co-transport.     

Inhibitory actions of indomethacin on electrical and mechanical responses produced by nerve stimulation in circular smooth muscle of the guinea-pig gastric fundus.

The effects of indomethacin on electrical and mechanical responses produced by transmural nerve stimulation (TNS) were investigated in isolated circular smooth muscle of the guinea-pig gastric fundus. TNS evoked a cholinergic excitatory junction potential (e.j.p.). The e.j.p.s were inhibited by 1-10 microM indomethacin, in a concentration-dependent manner, with no marked alteration of the resting membrane potential. Exogenously applied acetylcholine caused a depolarization of the membrane that was not altered by indomethacin. TNS evoked a cholinergic twitch contraction at low frequencies (0.1 Hz). A train of TNS's at high frequency (1 Hz) produced a transient contraction with a subsequent sustained relaxation. Indomethacin reduced the resting tension and inhibited these TNS-induced contractions. Application of Nomega-nitro-L-arginine (NOLA), an inhibitor of nitric oxide (NO) synthesis, increased the amplitude of twitch contractions, and altered transient contractions to tetanic contractions during TNS at a frequency of 1 Hz, also with an increased amplitude. In the presence of NOLA, indomethacin (5 microM) again reduced the resting tension and inhibited TNS-induced contractions. This inhibition was greater for twitch contractions than for tetanic contractions. Nifedipine reduced the TNS-induced contractions, while addition of indomethacin further reduced the amplitude of contractions. Contractions produced by low concentrations of acetylcholine (0.1 microM) were inhibited by indomethacin, while those produced by 1 microM were not. These results indicate that the inhibitory actions of indomethacin on TNS-induced contractions do not involve enhanced production of NO or selective inhibition of voltage-gated Ca-channels. Prejunctional autoregulatory mechanisms may also not be altered by indomethacin. As indomethacin inhibits the enzyme cyclooxygenase, it is speculated that endogenously produced prostaglandins exert excitatory actions on gastric smooth muscle, and act mainly postjunctionally to facilitate spontaneous and neurogenic electrical and mechanical activity.