Yoshihiko Kito

Properties of Pacemaker Potentials Recorded from Myenteric Interstitial Cells of Cajal Distributed in the Mouse Small Intestine

Recording of electrical responses from isolated small intestine of mice using conventional microelectrodes revealed two types of potential, a pacemaker potential and a slow wave, both with rapid rising primary components and following plateau components. The rate of rise and peak amplitude were greater for pacemaker potentials than for slow waves, and the plateau component was smaller in slow waves than in pacemaker potentials. Both potentials oscillated at a similar frequency (20–30 min−1). Unitary potentials often discharged during the interval between pacemaker potentials. Infusion of Lucifer Yellow allowed visualization of the recorded cells; pacemaker potentials were recorded from myenteric interstitial cells of Cajal (ICC‐MY) while slow waves were recorded from circular smooth muscle cells. Pacemaker potentials were characterized as follows: the primary component was inhibited by Ni2+, Ca2+‐free solution or depolarization with high‐K+ solution, the plateau component was inhibited by 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS), an inhibitor of Ca2+‐activated Cl− channels, low [Cl−]o solution or Ca2+‐free solution, and the generation of potentials was abolished by co‐application of Ni2+and DIDS or by chelating intracellular Ca2+ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM). These results indicate that in the mouse small intestine ICC‐MY generate pacemaker potentials with two components in situ; the primary and plateau components may be generated by activation of voltage‐dependent Ca2+‐permeable channels and Ca2+‐activated Cl− channels, respectively. Slow waves are generated in circular smooth muscles via electrotonic spread of pacemaker potentials. These properties of intestinal pacemaker potentials are considered essentially similar to those of gastric pacemaker potentials.

Spontaneous electrical activity and associated changes in calcium concentration in guinea-pig gastric smooth muscle

Spontaneous electrical activity and internal Ca2+ concentration ([Ca2+]i) were measured simultaneously using conventional microelectrodes and fura‐2 fluorescence, respectively, in isolated circular smooth muscle bundles of the guinea‐pig gastric antrum. The smooth muscle bundles generated periodic slow potentials with accompanying spike potentials and associated transient increases in [Ca2+]i (Ca2+‐transients). Nifedipine abolished the spike potentials but not the slow potentials, and reduced the amplitude of associated Ca2+‐transients. Caffeine, in the absence or presence of ryanodine, reduced resting [Ca2+]i levels and abolished the slow potentials and associated Ca2+‐transients. Depolarization elevated and hyperpolarization reduced resting [Ca2+]i levels with associated changes in the frequency of slow potentials. The amplitude of Ca2+‐transients changed in a bell‐shaped manner with the membrane potential change. Slow potentials and associated Ca2+‐transients were abolished if [Ca2+]i levels were reduced by BAPTA‐AM or if the internal Ca2+ pump was inhibited by cyclopiazonic acid. 2‐Aminoethoxy‐diphenylborate (2‐APB), a known inhibitor of inositol trisphosphate (IP3)‐mediated Ca2+ release, also blocked slow potentials and Ca2+‐transients. Carbonyl cyanide m‐chlorophenyl hydrazone (CCCP), a mitochondrial protonophore, depolarized the membrane, elevated [Ca2+]i levels and abolished slow potentials and Ca2+‐transients. Inhibition of mitochondrial ATP‐sensitive K+ channels by glybenclamide and 5‐hydroxydecanoic acid (5‐HAD) abolished slow potentials and Ca2+‐transients, without altering the smooth muscle [Ca2+]i. It is concluded that in antrum circular muscles, the frequency of slow potentials is correlated with the level of [Ca2+]i. The slow potential is coupled to release of Ca2+ from an internal store, possibly through the activation of IP3 receptors; this may be initiated by the activation of ATP‐sensitive K+ channels in mitochondria following Ca2+ handling by mitochondria.

Components of pacemaker potentials recorded from the guinea-pig stomach antrum

Abstract. Pacemaker potentials recorded intracellularly from the guinea pig stomach consisted of initial primary and following plateau components. Inhibition of the internal Ca2+ store pump with cyclopiazonic acid depolarized the membrane and inhibited the plateau component of pacemaker potentials. 2-Aminoethoxydiphenyl borate (an inhibitor of IP3-induced Ca2+ release) and carbonyl cyanide m-chlorophenyl-hydrazone (a mitochondrial protonophore) depolarized the membrane and abolished pacemaker potentials. Low [Ca2+]o solution reduced the frequency and rate of rise of pacemaker potentials, and the effects were mimicked by BAPTA-AM (an intracellular Ca2+ chelator). 4,4′-Diisothiocyanatostilbene-2,2′-disulphonic acid and low [Cl–]o solution inhibited the plateau component of pacemaker potentials. Depolarization of the membrane with high [K+]o solutions increased the frequency and reduced the dV/dtmax of pacemaker potentials. During high-[K+]o-induced depolarization, cyclopiazonic acid abolished pacemaker potentials. Caffeine, forskolin, papaverine, 8-bromo-cGMP and (±)S-nitroso-N-acetylpenicillamine (SNAP) inhibited the plateau component, with no alteration of the primary component. It is concluded that the primary and plateau components of pacemaker potentials are related to voltage-gated Ca2+ influx and Ca2+-activated Cl– channels, respectively, and cyclic nucleotides inhibit mainly the latter. Pacemaker potentials may be generated by the release of Ca2+ from internal stores through excitation of inositol 1,4,5-trisphosphate receptors, coupled with Ca2+ uptake into mitochondria.

Excitation of smooth muscles isolated from the guinea‐pig gastric antrum in response to depolarization

In small segments of circular smooth muscle bundle isolated from the guinea‐pig gastric antrum, depolarization of the tissue with intracellular current stimuli evoked regenerative slow potentials after a refractory period of 5–10 s. The refractory period changed inversely with the amplitude and duration of the stimulating depolarization. Thapsigargin (an inhibitor of calcium‐ATPase at internal stores), 2‐aminoethoxydiphenyl borate (2‐APB, an inhibitor of inositol 1,4,5‐trisphosphate (IP3)‐receptor‐mediated Ca2+ release), and carbonyl cyanide m‐chlorophenyl‐hydrazone (a mitochondrial protonophore) reduced the amplitude of slow potentials, with no significant alteration of the refractory period. Bisindolylmaleimide I or chelerythrine (inhibitors of protein kinase C, PKC) increased the refractory period and inhibited the amplitude of slow potentials. These results indicate that the refractory period and amplitude of slow potentials are related to the activation of PKC and the amount of Ca2+ released from the internal stores through activation of IP3 receptors, respectively. Acetylcholine (ACh) reduced the refractory period and increased the amplitude of slow potentials: the former was antagonized by chelerythrine and the latter by 2‐APB. The results suggest that ACh has dual actions; stimulation of the metabolism of inositol phosphate and activation of PKC. Phorbol‐12‐myristate‐13‐acetate, a selective stimulant of PKC, at low concentrations (< 10 nm) mimicked the actions of ACh and at high concentrations reduced the frequency of slow potentials and increased the refractory period. The possible involvement of the concentration‐dependent differences in the actions of phorbol ester on the translocation of PKC was considered.

Pacemaker frequency is increased by sodium nitroprusside in the guinea pig gastric antrum

In the guinea pig gastric antrum, the effects of sodium nitroprusside (SNP), an NO donor, on pacemaker potentials were investigated in the presence of nifedipine. The pacemaker potentials consisted of primary and plateau components; SNP (> 1 μm) increased the frequency of occurrence of these pacemaker potentials, while inhibiting the plateau component. 1H‐[1,2,4]‐Oxadiazole [4,3‐a] quinoxalin‐1‐one, an inhibitor of guanylate cyclase, had no effect on the excitatory actions of SNP on the frequency of pacemaker potentials. Other types of NO donor, (±)‐S‐nitroso‐N‐acetylpenicillamine, 3‐morpholino‐sydnonimine and 8‐bromoguanosine 3′5’‐cyclic monophosphate had no excitatory effect on pacemaker activity. Forskolin, an activator of adenylate cyclase, or 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulphonic acid, an inhibitor of the Ca2+‐activated Cl− channel, strongly attenuated the generation of pacemaker potentials, and SNP added in the presence of these chemicals restored the generation of pacemaker potentials. The pacemaker potentials evoked by SNP were abolished in low‐Ca2+ solution or by membrane depolarization with high‐K+ solution. The SNP‐induced generation of pacemaker potentials was not prevented by cyclopiazonic acid, an inhibitor of internal Ca2+‐ATPase, but was limited to a transient burst by iodoacetic acid, an inhibitor of glycolysis, carbonyl cyanide m‐chlorophenyl‐hydrazone, a mitochondrial protonophore, or 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N‘,N‘‐tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator. These results suggest that the SNP‐induced increase in the frequency of pacemaker potentials is related to the elevated intracellular Ca2+ concentrations due to release from mitochondria, and these actions may be independent of the activation of guanylate cyclase.

Factors modifying the frequency of spontaneous activity in gastric muscle

The cellular mechanisms that determine the frequency of spontaneous activity were investigated in gastric smooth muscles isolated from the guinea‐pig. Intact antral muscle generated slow waves periodically; the interval between slow waves was decreased exponentially by depolarization of the membrane to reach a steady interval value of about 7 s. Isolated circular muscle bundles produced slow potentials spontaneously or were evoked by depolarizing current stimuli. Evoked slow potentials appeared in an all‐or‐none fashion, with a refractory period of ∼2–3 s. Low concentrations of chemicals that modify intracellular signalling revealed that the refractory period was causally related to the activity of protein kinase C (PKC). Activation of PKC increased and inhibition of PKC activity decreased the frequency of slow potentials. Chemicals that inhibit mitochondrial functions reduced the frequency of slow waves. Inhibition of internal Ca2+‐store activity decreased the amplitude, but not the frequency of slow potentials, suggesting that the amplitude is causally related to Ca2+ release from the internal store. The results suggest that changes in [Ca2+]i caused by the activity of mitochondria may play a key role in determining the frequency of spontaneous activity in gastric pacemaker cells.

Modulation of slow waves by hyperpolarization with potassium channel openers in antral smooth muscle of the guinea-pig stomach

Modulation of spontaneous electrical activities (slow waves, pacemaker potentials and follower potentials) in response to hyperpolarization produced by the ATP‐sensitive K+ channel openers (KCOs) pinacidil or nicorandil was investigated in smooth muscle tissues of the guinea‐pig stomach antrum. With hyperpolarization, the amplitude of slow waves and follower potentials was reduced and that of pacemaker potentials was increased, with a minor modulation of their frequency. The attenuation of slow waves was associated with an inhibition of the 1st component and abolition of the 2nd component. All these actions of KCOs were antagonized by glibenclamide. An increase in the extracellular K+ concentration prevented the KCO‐induced hyperpolarization with partial restoration of slow waves, suggesting that the inhibition was produced mainly by a decrease in membrane resistance. Exposure of tissues to KCOs for a long period of time (> 20 min) resulted in the reappearance of slow waves displaying both 1st and 2nd components. The 2nd component of the slow wave, which displayed a slower recovery, was inhibited again by 5‐hydroxydecanoic acid, an inhibitor of mitochondrial ATP‐sensitive K+ channels. Noradrenaline hyperpolarized the membrane by activating apamin‐sensitive K+ channels and increased the amplitude and frequency of slow waves through activation of α1‐adrenoceptors, actions different from those of KCOs. Thus, inhibition of slow waves by KCOs may be primarily related to the decrease in amplitude of a passive electrotonic component, possibly due to a reduction of the input resistance. The hyperpolarization shifted the threshold potential for generation of the 2nd component of slow waves to negative levels, presumably due to modulation of mitochondrial functions.

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 <or=10 mV in amplitude (unitary potentials). A second pattern of electrical activity was recorded in less than 1% of all impalements. This electrical activity was characterized by high frequency, large amplitude spontaneous transient depolarizations (STDs) with a maximum rate of rise (dV/dt(max)) of 0.5 V/s. Injection of the fluorescent dye propidium iodide into cells and double labeling with an antibody against the Kit receptor revealed that unitary potentials were recorded from circular smooth muscle cells (CSMC), whereas STDs were generated by intramuscular interstitial cells of Cajal (ICC-IM). Sustained injection periods (>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.

Spontaneous transient hyperpolarizations in the rabbit small intestine

Recently, it was shown that fibroblast‐like cells (FLCs) possess the apparatus to mediate purinergic motor neurotransmission in the gastrointestinal tract. However, the electrophysiological properties of FLCs in situ have not been determined. We recorded two patterns of slow waves from longitudinal smooth muscle cells and circular smooth muscle cells, large amplitude slow waves from interstitial cells of Cajal, and spontaneous transient hyperpolarizations (STHs) from FLCs in the rabbit small intestine using intracellular recording combined with dye injection to identify the cellular morphology of impaled cells. Drugs that inhibit the signalling pathway involved in purinergic neurotransmission inhibited STHs in FLCs. Small amplitude STHs were recorded in smooth muscle cells but not in interstitial cells of Cajal, suggesting that STHs from FLCs were conducted passively to smooth muscle cells. We conclude that FLCs display the molecular apparatus necessary to mediate purinergic neurotransmission and may tonically dampen smooth muscle excitability in the rabbit small intestine by an ongoing discharge of STHs.