PDGFRα+ Interstitial Cells are Effector Cells of PACAP Signaling in Mouse and Human Colon.

BACKGROUND & AIMS: Platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance Ca2+ activated K+ channels 3 (SK3 channels) and transduce inhibitory inputs from purinergic and sympathetic nerves in mouse and human colon. We investigated whether PIC also express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, PAC1 (PAC1R), and are involved in mediating inhibitory regulation of colonic contractions by PACAP in mouse and human colons. METHODS: Gene expression analysis, Ca2+ imaging, and contractile experiments were performed on mouse colonic muscles. Ca2+ imaging, intracellular electrical recordings, and contractile experiments were performed on human colonic muscles. RESULTS: Adcyap1r1 (encoding PAC1R) is highly expressed in mouse PIC. Interstitial cells of Cajal (ICC) and SMCs expressed far lower levels of Adcyap1r. Vipr1 and Vipr2 were expressed at low levels in PIC, ICC, and SMCs. PACAP elicited Ca2+ transients in mouse PIC and inhibited spontaneous phasic contractions via SK channels. In human colonic muscles, PAC1R agonists elicited Ca2+ transients in PIC, hyperpolarized SMCs through SK channels and inhibited spontaneous phasic contractions. CONCLUSIONS: PIC of mouse and human colon utilize PAC1R-SK channel signal pathway to inhibit colonic contractions in response to PACAP. Effects of PACAP are in addition to the previously described purinergic and sympathetic inputs to PIC. Thus, PIC integrate inhibitory inputs from at least 3 neurotransmitters and utilize several types of receptors to activate SK channels and regulate colonic contractile behaviors.

Norepinephrine Has Dual Effects on Human Colonic Contractions Through Distinct Subtypes of Alpha 1 Adrenoceptors.

BACKGROUND & AIMS: Colonic musculature contain smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet-derived growth factor receptor α+ cells (PDGFRα+ cells), which are electrically coupled and operate together as the SIP syncytium. PDGFRα+ cells have enriched expression of small conductance Ca2+-activated K+ (SK) channels. Purinergic enteric neural input activates SK channels in PDGFRα+ cells, hyperpolarizes SMC, and inhibits colonic contractions. Recently we discovered that PDGFRα+ cells in mouse colon have enriched expression of α1A adrenoceptors (ARs), which coupled to activation of SK channels and inhibited colonic motility, and α1A ARs were principal targets for sympathetic regulation of colonic motility. Here we investigated whether PDGFRα+ cells in human colon express α1A ARs and share the roles as targets for sympathetic regulation of colonic motility. METHODS: Isometric tension recording, intracellular recording, and Ca2+ imaging were performed on muscles of the human colon. Responses to α1 ARs agonists or electric field stimulation with AR antagonists and neuroleptic reagents were studied. RESULTS: Exogenous or endogenous norepinephrine released from nerve fibers inhibited colonic contractions through binding to α1A ARs or enhanced colonic contractions by acting on α1D ARs. Inhibitory responses were blocked by apamin, an antagonist of SK channels. Phenylephrine, α1 AR agonists, or norepinephrine increased intracellular [Ca2+] in PDGFRα+ cells, but not in ICC, and hyperpolarized SMCs by binding to α1 ARs expressed by PDGFRα+ cells. CONCLUSIONS: Human colonic contractions are inhibited by α1A ARs expressed in PDGFRα+ cells and activated by α1D ARs expressed in SMC.

A novel postsynaptic signal pathway of sympathetic neural regulation of murine colonic motility.

Transcriptome data revealed α1 adrenoceptors (ARs) expression in platelet-derived growth factor receptor α+ cells (PDGFRα+ cells) in murine colonic musculature. The role of PDGFRα+ cells in sympathetic neural regulation of murine colonic motility was investigated. Norepinephrine (NE), via α1A ARs, activated a small conductance Ca2+ -activated K+ (SK) conductance, evoked outward currents and hyperpolarized PDGFRα+ cells (the α1A AR-SK channel signal pathway). α1 AR agonists increased intracellular Ca2+ transients in PDGFRα+ cells and inhibited spontaneous phasic contractions (SPCs) of colonic muscle through activation of a SK conductance. Sympathetic nerve stimulation inhibited both contractions of distal colon and propulsive contractions represented by the colonic migrating motor complexes (CMMCs) via the α1A AR-SK channel signal pathway. Postsynaptic signaling through α1A ARs in PDGFRα+ cells is a novel mechanism that conveys part of stress responses in the colon. PDGFRα+ cells appear to be a primary effector of sympathetic neural regulation of murine colonic motility.

Regulation of Gastrointestinal Smooth Muscle Function by Interstitial Cells.

Interstitial cells of mesenchymal origin form gap junctions with smooth muscle cells in visceral smooth muscles and provide important regulatory functions. In gastrointestinal (GI) muscles, there are two distinct classes of interstitial cells, c-Kit(+) interstitial cells of Cajal and PDGFRα(+) cells, that regulate motility patterns. Loss of these cells may contribute to symptoms in GI motility disorders.

Characterization of slow waves generated by myenteric interstitial cells of Cajal of the rabbit small intestine.

Slow waves (slow wavesICC) were recorded from myenteric interstitial cells of Cajal (ICC-MY) in situ in the rabbit small intestine, and their properties were compared with those of mouse small intestine. Rabbit slow wavesICC consisted of an upstroke depolarization followed by a distinct plateau component. Ni(2+) and nominally Ca(2+)-free solutions reduced the rate-of-rise and amplitude of the upstroke depolarization. Replacement of Ca(2+) with Sr(2+) enhanced the upstroke component but decreased the plateau component of rabbit slow wavesICC. In contrast, replacing Ca(2+) with Sr(2+) decreased both components of mouse slow wavesICC. The plateau component of rabbit slow wavesICC was inhibited in low-extracellular-Cl(-)-concentration (low-[Cl(-)]o) solutions and by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl(-) channels, cyclopiazonic acid (CPA), an inhibitor of internal Ca(2+) pumps, or bumetanide, an inhibitor of Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Bumetanide also inhibited the plateau component of mouse slow wavesICC. NKCC1-like immunoreactivity was observed mainly in ICC-MY in the rabbit small intestine. Membrane depolarization with a high-K(+) solution reduced the upstroke component of rabbit slow wavesICC. In cells depolarized with elevated external K(+), DIDS, CPA, and bumetanide blocked slow wavesICC. These results suggest that the upstroke component of rabbit slow wavesICC is partially mediated by voltage-dependent Ca(2+) influx, whereas the plateau component is dependent on Ca(2+)-activated Cl(-) efflux. NKCC1 is likely to be responsible for Cl(-) accumulation in ICC-MY. The results also suggest that the mechanism of the upstroke component differs in rabbit and mouse slow wavesICC in the small intestine.

Spontaneous transient hyperpolarizations in the rabbit small intestine.

Four types of electrical activity were recorded and related to cell structure by intracellular recording and dye injection into impaled cells in muscles of rabbit small intestine. The specific cell types from which recordings were made were longitudinal smooth muscle cells (LSMCs), circular smooth muscle cells (CSMCs), interstitial cells of Cajal distributed in the myenteric region (ICC-MY) and fibroblast-like cells (FLCs). Slow waves (slow wavesSMC) were recorded from LSMCs and CSMCs. Slow waves (slow wavesICC) were of greatest amplitude (>50 mV) and highest maximum rate of rise (>10 V s(-1)) in ICC-MY. The dominant activity in FLCs was spontaneous transient hyperpolarizations (STHs), with maximum amplitudes above 30 mV. STHs were often superimposed upon small amplitude slow waves (slow wavesFLC). STHs displayed a cyclical pattern of discharge irrespective of background slow wave activity. STHs were inhibited by MRS2500 (3 µm), a P2Y1 antagonist, and abolished by apamin (0.3 µm), a blocker of small conductance Ca(2+)-activated K(+) channels. Small amplitude STHs (<15 mV) were detected in smooth muscle layers, whereas STHs were not resolved in cells identified as ICC-MY. Electrical field stimulation evoked purinergic inhibitory junction potentials (IJPs) in CSMCs. Purinergic IJPs were not recorded from ICC-MY. These results suggest that FLCs may regulate smooth muscle excitability in the rabbit small intestine via generation of rhythmic apamin-sensitive STHs. Stimulation of P2Y1 receptors modulates the amplitudes of STHs. Our results also suggest that purinergic inhibitory motor neurons regulate the motility of the rabbit small intestine by causing IJPs in FLCs that conduct to CSMCs.

Effects of Hange-shashin-to (TJ-14) and Keishi-ka-shakuyaku-to (TJ-60) on contractile activity of circular smooth muscle of the rat distal colon.

The Japanese Kampo medicines Hange-shashin-to (TJ-14) and Keishi-ka-shakuyaku-to (TJ-60) have been used to treat symptoms of human diarrhea on an empirical basis as Japanese traditional medicines. However, it remains unclear how these drugs affect smooth muscle tissues in the distal colon. The aim of the present study was to investigate the effects of TJ-14 and TJ-60 on the contractile activity of circular smooth muscle from the rat distal colon. TJ-14 and TJ-60 (both 1 mg/ml) inhibited spontaneous contractions of circumferentially cut preparations with the mucosa intact. Blockade of nitric oxide (NO) synthase or soluble guanylate cyclase activity abolished the inhibitory effects of TJ-60 but only attenuated the inhibitory effects of TJ-14. Apamin (1 µM), a blocker of small-conductance Ca(2+)-activated K(+) channels (SK channels), attenuated the inhibitory effects of 5 mg/ml TJ-60 but not those of 5 mg/ml TJ-14. TJ-14 suppressed contractile responses (phasic contractions and off-contractions) evoked by transmural nerve stimulation and increased basal tone, whereas TJ-60 had little effect on these parameters. These results suggest that 1 mg/ml TJ-14 or TJ-60 likely inhibits spontaneous contractions of the rat distal colon through the production of NO. Activation of SK channels seems to be involved in the inhibitory effects of 5 mg/ml TJ-60. Since TJ-14 has potent inhibitory effects on myogenic and neurogenic contractile activity, TJ-14 may be useful in suppressing gastrointestinal motility.

Factors which determine the duration of follower potentials in longitudinal smooth muscle isolated from the guinea-pig stomach antrum.

In isolated longitudinal muscle tissues of the guinea-pig stomach antrum, recording electrical responses from smooth muscle cells revealed a periodical generation of follower potentials with variable durations. The I-D relationship, made by plotting the duration as a function of the interval before generating follower potential, was linear. Experiments were carried out to investigate the effects of chemicals which had been known to modulate the release of Ca(2+) from the internal stores (2-aminoethoxy-diphenyl-borate, cyclopiazonic acid, caffeine), inhibit mitochondrial metabolic activity (m-chlorophenyl hydrazone, 2-deoxy-D-glucose, potassium cyanide, rotenone), inhibit ATP-sensitive K-channels distributed in mitochondria (glibenclamide, 5-hydroxydecanoic acid) and inhibit the activity of proteinkinase C (chelerythrine), on the I-D relationship of follower potentials. The effects of depolarization on follower potentials were assessed by stimulating tissues with high potassium solution. Experiments were carried out mainly in the presence of nifedipine which minimized the movements of muscles with no modulation of follower potentials. Cycropiazonic acid and caffeine reduced the slope of I-D relationship, with associated reduction of the duration and frequency of follower potentials. 2-Aminoethoxydiphenyl borate reduced the duration and amplitude and increased the frequency of follower potentials, with depolarization of the membrane, and the effects were simulated by high potassium solution. m-Chlorophenyl hydrazone, potassium cyanide, 2-deoxy-D-glucose, rotenone, 5-hydroxydecanoic acid and glibenclamide reduced the slope of I-D relationship, with associated reduction of the frequency of follower potentials. Chelerythrine did not modulate the slope of I-D relationship, with reduced frequency of follower potentials. It seemed likely that the amount of Ca(2+) released from the internal stores and also mitochondrial function had causal relationship to the duration of pacemaker potentials, suggesting that internal Ca-stores and mitochondria are taking the central role for determining the duration of the pacemaker activity. Proteinkinase C did not seem to participate to the function of mitochondria and internal Ca(2+) stores.

The functional role of intramuscular interstitial cells of Cajal in the stomach.

Intramuscular interstitial cells of Cajal (ICC-IM) are found within the smooth muscle layers of the stomach. ICC-IM are mainly spindle shaped cells with bipolar processes orientated along the long axis of surrounding smooth muscle cells. ICC-IM make close contacts with nerve varicosities and form gap junctions with neighbouring smooth muscle cells, indicating that ICC-IM mediate enteric motor neurotransmission. These morphological properties of ICC-IM are similar throughout the stomach. However, the electrical properties of these cells differ from region to region. In the fundus, ICC-IM generate spontaneous transient depolarizations (STDs), resulting in an ongoing discharge of unitary potentials in the smooth muscle cells. ICC-IM in the corpus generate slow waves and as they fire at the highest frequency they serve as the dominant pacemaker cells in the stomach. On the other hand, ICC-IM in the antrum generate the secondary component of slow waves triggered by the initial component that propagates passively from myenteric ICC (ICC-MY). Thus, the different electrical properties of ICC-IM play a critical role in creating the distinct functions of the proximal and distal regions of the stomach such that the fundus acts as a reservoir of food, the corpus as a dominant pacemaker region, while the antrum acts as a region for mixing and propulsion of food.

Properties of Rikkunshi-to (TJ-43)-induced relaxation of rat gastric fundus smooth muscles.

The relaxant effects of Rikkunshi-to (TJ-43), a gastroprotective herbal medicine, on rat gastric fundus were investigated. Experiments were carried out using standard tension and intracellular microelectrode recording techniques. During contraction induced by enprostil (0.5 microM), a prostaglandin E(2) analog, TJ-43, produced relaxation dose dependently (0.1-5.0 mg/ml) in the rat fundic circular smooth muscle (CSM) strips. The relaxant effects of TJ-43 were not affected by tetrodotoxin or 1 H[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (10 microM), an inhibitor of soluble guanylate cyclase. TJ-43 inhibited enprostil-induced membrane depolarization. Apamin (1 microM), a blocker of small-conductance Ca(2+)-activated K(+) (SK) channel, inhibited T-43-induced membrane repolarization. TJ-43-induced relaxation was biphasic, comprising of an initial fast followed by a second slow relaxation. The fast relaxation was abolished by apamin. Application of high K(+) (29.4 mM [K(+)](o)) also abolished the fast relaxation induced by TJ-43. In diabetic Goto-Kakizaki (GK) rat fundic CSM strips, the relaxant responses of TJ-43 during enprostil-induced contraction were increased compared with control rat strips. These results indicate that TJ-43 elicited fast muscle relaxation through membrane hyperpolarization induced by the activation of SK channels; the time-dependent slow relaxation reflects an additional direct of TJ-43 on CSM in the rat gastric fundus. Because TJ-43-evoked relaxation of fundic CSM strips was more potent in diabetic GK rat than in control rat, further analysis of this herb could lead to better treatments of diabetic gastroparesis.

Interstitial cells of Cajal generate spontaneous transient depolarizations in the rat gastric fundus.

Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were 15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 microM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 microM) also increased the amplitude of STDs and enhanced dV/dt(max). These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.

Role of K(+) channels in the regulation of electrical spontaneous activity of the mouse small intestine.

The roles of K(+) channels in the regulation of slow waves and pacemaker potentials recorded from mouse small intestine were investigated using intracellular recording techniques in the presence of nifedipine. Iberiotoxin (0.1 microM) and charybdotoxin (0.1 microM) had no effect on the generation of slow waves recorded from circular smooth muscle cells. Apamin (0.3 microM) depolarized the membrane and decreased the amplitude of early, rapid repolarization of slow waves, without altering the amplitude, frequency, duration, or maximum rate of rise of the initial upstroke phase (dV/dt(max)). The early, rapid repolarization was enhanced by phenylephrine (15 microM). 4-Aminopyridine (4-AP, 5 mM) depolarized the membrane and increased the amplitude and dV/dt(max) of slow waves. Both apamin and 4-AP depolarized the membrane and decreased the amplitude and dV/dt(max) of pacemaker potentials recorded from interstitial cells of Cajal distributed in the myenteric region (ICC-MY). Membrane depolarization with a high-K(+) solution decreased the amplitude and dV/dt(max) of slow waves. These results suggest that apamin-sensitive K(+) conductance and 4-AP-sensitive K(+) conductance may contribute to the resting membrane potential of circular smooth muscle cells. The early, rapid repolarization of slow waves appears to result from the opening of apamin-sensitive K(+) conductance. 4-AP-sensitive K(+) conductance is likely to be activated in the initial upstroke component (primary component) of slow waves. In ICC-MY, membrane depolarization induced by apamin or 4-AP may result from electrotonic spread from smooth muscle cells.

Effects of temperature on pacemaker potentials in the mouse small intestine.

The effects of temperature on the generation of pacemaker potentials recorded from myenteric interstitial cells of Cajal (ICC-MY) distributed in the mouse small intestine were investigated using intracellular recording techniques. In response to increasing temperatures in the range of 26-40 degrees C, the frequency and maximum rate of rise (dV/dt (max)) of pacemaker potentials were increased while their duration was decreased. The resting membrane potential and amplitude of the pacemaker potentials were not affected by change in temperature. Elevation of temperature decreased the amplitude, duration, and rise time of unitary potentials generated spontaneously during intervals between the pacemaker potentials. Metabolic inhibition (KCN and iodoacetic acid) decreased the frequency of pacemaker potentials with no alteration to the amplitude and dV/dt (max). Cyclopiazonic acid (3 muM), an inhibitor of the internal Ca(2+) pump, abolished pacemaker potentials in low-temperature conditions (<29 degrees C) but not at high-temperature conditions (>38 degrees C). These results suggest that the primary and plateau components of pacemaker potentials have different temperature sensitivities: the primary component is highly temperature-sensitive and is activated at higher temperatures, while the plateau component is formed by activation of temperature-insensitive mechanisms. The results also suggest that the mitochondria-induced intracellular Ca(2+) handling system seems to be involved in the initiation of the generation of pacemaker potentials but not in their configuration.

Mechanical and electrical responses modulated by excitation of inhibitory nerves during stimulation with high-potassium solutions in circular smooth muscle of the rabbit rectum.

The properties of the mechanical responses produced by solutions containing high concentrations of potassium ion (high-K solution, [K(+)](o) = 9-27 mM) were investigated in circular smooth muscle preparations isolated from the rabbit rectum. Isometric recording of mechanical responses of the muscle revealed spontaneous contractions, which successively decreased and finally disappeared in most preparations. Stimulation of the smooth muscle with high-K solutions elicited an increase in both amplitude and frequency of twitch contractions (sustained component), with about a 2 min delay in the beginning (initial inhibition), and a transient large contraction shortly after the cessation of stimulation (after contraction). Transmural nerve stimulation (TNS) with electrical pulses for 1 min at 1 Hz frequency produced a sustained inhibition, but a transient contraction followed after termination of TNS. In the presence of tetrodotoxin (TTX), the TNS-induced responses were abolished, while a high-K solution elicited increased twitch contractions with a short delay and abolished the after contraction. Suramin produced effects similar to TTX on the responses produced by high-K solutions or TNS, but this was not the case for atropine, guanethidine or N(omega)-nitro-L-arginine (L-NA). Recording membrane potentials with microelectrodes revealed that TNS evoked an inhibitory junction potential (i.j.p.) which was non-adrenergic, non-cholinergic and non-nitrergic in nature. High-K solutions elicited a tri-phasic change in the membrane potential; an initial hyperpolarization, followed by a sustained depolarization and finally a transient depolarization on cessation of high-K stimulation. TTX or suramin inhibited the i.j.p.s and altered the tri-phasic change in the membrane potential produced by a high-K solution to a mono-phasic depolarization. No significant modulation of electrical responses of the membrane induced by TNS or high-K solution was elicited by atropine, guanethidine or L-NA. The results indicated that the circular smooth muscle of the rabbit rectum is innervated by inhibitory nerves, and that stimulation with high-K solutions caused inhibitory neuronal modulation of both electrical and mechanical responses of the smooth muscle, in a suramin-sensitive way.

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.

Effects of Dai-kenchu-to on spontaneous activity in the mouse small intestine.

The effects of Dai-kenchu-to (DKT), a Chinese medicine, on spontaneous activity of mouse small intestine were investigated. Experiments were carried out with tension recording and intracellular recording. DKT contracted mouse longitudinal smooth muscles in a dose dependent manner (0.1-10 mg/ml). Low concentration of DKT (0.1 mg/ml) did not contract the longitudinal muscles of mouse small intestine. DKT (0.1 mg/ml) inhibited contraction elicited by transmural nerve stimulation (TNS). DKT (1 mg/ml) evoked relaxation before contraction. The initial relaxation was abolished by Nomega-nitro-L-arginine (L-NNA). DKT (10 mg/ml)-induced contraction had two components: a transient rapid contraction and a following slow contraction. Atropine inhibited DKT (1 mg/ml)-induced contraction to about 50% of control. In the presence of atropine, tetrodotoxin (TTX) inhibited the contraction elicited by DKT (1 mg/ml) to about 80%. DKT depolarized the membrane and decreased the amplitude of pacemaker potentials recorded from in situ myenteric interstitial cells of Cajal (ICC-MY) with no alteration to the frequency, duration and maximum rates of rise in the presence of nifedipine and TTX. The same results were obtained in slow waves recorded from circular smooth muscle cells. These results indicate that DKT evoked both contraction and relaxation by releasing acetylcholine, nitric oxide and other excitatory neurotransmitters in mouse small intestine. DKT had no effects on pacemaker mechanisms and electrical coupling between ICC-MY and smooth muscle cells in mouse small intestine. The results also suggest that DKT may contract smooth muscles by depolarizing the membrane directly.

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.

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.

Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine.

Pacemaker potentials were recorded in situ from myenteric interstitial cells of Cajal (ICC-MY) in the murine small intestine. The nature of the two components of pacemaker potentials (upstroke and plateau) were investigated and compared with slow waves recorded from circular muscle cells. Pacemaker potentials and slow waves were not blocked by nifedipine (3 microM). In the presence of nifedipine, mibefradil, a voltage-dependent Ca(2+) channel blocker, reduced the amplitude, frequency, and rate of rise of upstroke depolarization (dV/dt(max)) of pacemaker potentials and slow waves in a dose-dependent manner (1-30 microM). Mibefradil (30 microM) changed the pattern of pacemaker potentials from rapidly rising, high-frequency events to slowly depolarizing, low-frequency events with considerable membrane noise (unitary potentials) between pacemaker potentials. Caffeine (3 mM) abolished pacemaker potentials in the presence of mibefradil. Pinacidil (10 microM), an ATP-sensitive K(+) channel opener, hyperpolarized ICC-MY and increased the amplitude and dV/dt(max) without affecting frequency. Pinacidil hyperpolarized smooth muscle cells and attenuated the amplitude and dV/dt(max) of slow waves without affecting frequency. The effects of pinacidil were blocked by glibenclamide (10 microM). These data suggest that slow waves are electrotonic potentials driven by pacemaker potentials. The upstroke component of pacemaker potentials is due to activation of dihydropyridine-resistant Ca(2+) channels, and this depolarization entrains pacemaker activity to create the plateau potential. The plateau potential may be due to summation of unitary potentials generated by individual or small groups of pacemaker units in ICC-MY. Entrainment of unitary potentials appears to depend on Ca(2+) entry during upstroke depolarization.