Effects of sodium, potassium and calcium ions on the slow wave in the circular muscle of the guinea-pig stomach

1. The contribution of Na, K and Ca ions to the generation of slow waves in the circular muscle of the guinea-pig stomach was studied.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

Spike-free activation mechanism in smooth muscle of guinea-pig stomach

Electrical and mechanical activity of circular muscle strips of guinea-pigs stomach, taken from the distal corpus/proximal antrum region, were recorded. Spontaneous activity consisting of phasic contractions combined with bursts of spike potentials was suppressed by verapamil (5-10-6 - 2-10-5 mol/l). Under these conditions acetylcholine produced a spike-free tonic activation. Under normal conditions phasic contractions were superimposed on this tonic activation. The acetylcholine-induced activation, therefore, consists of two different components, one of which can be selectively blocked with verapamil. Both components disappear quickly in calcium-free solution. It can be concluded that two different calcium activation systems are responsible for the two components of activation. In comparative studies with taenia coli preparations a comparable spike-free tonic activation was not found.     

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

In the circular muscle of guinea-pig gastric antrum, the effects of removal and reapplication of K(+) and Cl(-) were studied on the slow wave, which consists of the lower, first and upper, second components. The first component appeared to be triggered by the driving potential generated in the interstitial cells. K(+) removal slightly depolarized the membrane, increased frequency, and shortened the first component and driving potential, and K(+) reapplication hyperpolarized and prolonged these potentials transiently. Ouabain abolished the K(+)-induced hyper-polarization but had no inhibitory effect on the K(+)-induced potentiation. The K(+)-induced prolongation was much reduced in Ca(2+)-deficient and increased in Ca(2+)-excess solution. BAPTA-AM, thapsigargin, and cyclopiazonic acid shortened the slow wave and inhibited the K(+)-induced prolongation but did not block the slow wave. Effects of Cl(-) removal were stronger than K(+) removal in shortening and increasing the frequency. In Cl(-)-deficient solution, no prolongation was observed on K(+) reapplication. Although no conclusive evidence was obtained as to the ionic mechanism involved in the effects of K(+) or Cl(-) removal and reapplication, a possibility is considered that the sarcoplasmic reticulum is involved in determining the duration of the driving potential and the first component of the slow wave.     

Metabolic component of the temperature-sensitivity of slow waves recorded from gastric muscle of the guinea-pig.

The effects of changes in temperature on slow waves were investigated in smooth muscle tissues isolated from the guinea-pig gastric antrum. Within the range 24 degrees C to 42 degrees C, elevation of temperature increased the frequency and maximum rate of rise of the upstroke phase (dV/dt) of slow waves and decreased their duration, with no alteration to amplitude or resting membrane potential. These observations also applied to follower potentials and pacemaker potentials recorded from longitudinal muscle and myenteric interstitial cells, respectively. Slow waves were comprised of 1st and 2nd components, and the latency for generating the 2nd component was decreased exponentially by elevating temperature, reaching a stable value of about 1 s above 32 degrees C. The temperature coefficient was >2 for the frequency, dV/dt and latency of the 2nd component, about 1.7 for the duration and about 1 for amplitude. Potassium cyanide (KCN), an inhibitor of mitochondrial metabolic activity, reduced the frequency and duration of slow waves, with no alteration to other parameters (amplitude, dV/dt, latency). In the presence of 30 microM KCN, the temperature-dependency of the frequency of slow waves was diminished or abolished, while other parameters of slow waves remained unaltered. These results indicate that in slow waves the frequency may be related to metabolic activities, while the temperature-dependent changes in the dV/dt, latency for the 2nd component and duration of slow waves are produced largely by mechanisms other than metabolic activity.     

花生四烯酸抑制豚鼠胃窦环行肌细胞容积敏感氯电流

利用全细胞膜片钳技术 ,在用胶原酶急性分离的豚鼠胃窦环行肌细胞上观察了外源性花生四烯酸 (arachidonicacid ,AA)及其它不饱和脂肪酸对容积敏感氯电流 (volume sensitivechloridecurrent,VSCC)的影响。细胞膜电位钳制在- 6 0mV条件下 ,低渗性细胞膨胀激活VSCC。当给予按 2 0mV间隔的去极化阶跃性方波刺激时 ,VSCC的电流 电压曲线具有外向性整流的特点 ,并表现出去极化到 +40mV以上时呈现时间依赖性衰减的特点。AA呈齐量依赖性地抑制VSCC ,而对VSCC的随时间衰减部分的抑制效应更明显。其它不饱和脂肪酸 ,如含有 2个双键的亚油酸 (Linoleicacid,LA)、含有 1个双键的油酸 (Oleicacid ,OA)也抑制VSCC ,抑制强度顺序为AA >LA >OA ,但饱和脂肪酸 (stearicacid,SA)对VSCC无抑制作用。提示外源性不饱和脂肪酸抑制VSCC ,其抑制效应在VSCC的随时间衰减部分更明显。外源性不饱和脂肪酸对VSCC的抑制作用可能与脂肪酸链中的双键数目有关