1 Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous inward currents (pacemaker currents) responsible for the production of slow waves in gastrointestinal smooth muscle. The effects of noradrenaline on the pacemaker currents in cultured ICCs from murine small intestine were investigated by using whole-cell patch-clamp techniques at 301C. 2 Under current clamping, ICCs had a mean resting membrane potential of À5875 mV and produced electrical slow waves. Under voltage clamping, ICCs produced pacemaker currents with a mean amplitude of À410757 pA and a mean frequency of 1672 cycles min À1 . 3 Under voltage clamping, noradrenaline inhibited the amplitude and frequency of pacemaker currents and increased resting currents in the outward direction in a dose-dependent manner. These effects were reduced by intracellular GDPbS. 4 Noradrenaline-induced effects were blocked by propranolol (b-adrenoceptor antagonist). However, neither prazosin (a 1 -adrenoceptor antagonist) nor yohimbine (a 2 -adrenoceptor antagonist) blocked the noradrenaline-induced effects. Phenylephrine (a 1 -adrenoceptor agonist) had no effect on the pacemaker currents, whereas isoprenaline (b-adrenoceptor agonist) mimicked the effect of noradrenaline. Atenolol (b 1 -adrenoceptor antagonist) blocked the noradrenaline-induced effects, but butoxamine (b 2 -adrenoceptor antagonist) did not. In addition, BRL37344 (b 3 -adrenoceptor agonist) had no effect on pacemaker currents. 5 9-(Tetrahydro-2-furanyl)-9H-purine-6-amine (SQ-22536; adenylate cyclase inhibitor) and a myristoylated protein kinase A inhibitor did not inhibit the noradrenaline-induced effects and 8-bromo-cAMP had no effects on pacemaker currents. 8-Bromo-cGMP and SNAP inhibited pacemaker currents and these effects of SNAP were blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor). However, ODQ did not block the noradrenaline-induced effects. 6 Neither tetraethylammonium (a voltage-dependent K þ channel blocker), apamin (a Ca 2 þ -dependent K þ channel blocker) nor glibenclamide (an ATP-sensitive K þ channel blocker) blocked the noradrenaline-induced effects. 7 The results suggest that noradrenaline-induced stimulation of b 1 -adrenoceptors in the ICCs inhibits pacemaker currents, and that this is mediated by the activation of G-protein. Neither adenylate cyclase, guanylate cyclase nor a K þ channel-dependent pathway are involved in this effect of noradrenaline.

Serotonin (5-HT, 5-hydroxytryptamine) is a mitogen in vascular smooth muscle and vascular reactivity to 5-HT is significantly enhanced in hypertension and atherosclerosis. We have tested the hypothesis that tyrosine kinases, enzymes important for mitogenesis, may play a role in 5-HT-induced vascular smooth muscle contractility. Helical strips of rat carotid artery and aorta denuded of endothelium were mounted in tissue baths for measurement of contractile force. The tyrosine kinase inhibitor genistein (5 × 10^-6M) decreased the potency of 5-HT approximately 4-fold and reduced maximal contraction to 5-HT in carotid arterial strips denuded of endothelium (58% control). Genistein’s inactive congener daidzein (5 × 10^-6M) did not reduce maximal contraction to 5-HT in carotid arteries but did shift the 5-HT concentration response curve 3-fold to the right. Tyrphostin 23 (5 × 10^–5M), another tyrosine kinase inhibitor, decreased the potency of 5-HT 4-fold and reduced the maximal contraction to 5-HT in the carotid artery (10% control). Contractions induced by phorbol-12,13-dibutyrate (10^-9 to 10^-5M) were not reduced or shifted by either tyrosine kinase inhibitor, indicating that phorbol-ester-sensitive protein kinase C isoforms were not affected. KCl-induced contraction was shifted 2-fold and the maximum significantly inhibited by tyrphostin 23 (38.6% control) but not genistein or daidzein, indicating that tyrphostin 23 but not genistein may inhibit voltage-gated calcium channels to reduce contractility. Western blot analysis using antiphosphotyrosine antibody confirmed that 5-HT produced a time- and concentration-dependent increase in the phosphotyrosine immunoreactivity of a 42-kD protein in cultured aortic smooth muscle cells. Lysate immunoprecipitation with an anti-mitogen-activated-protein (MAP)-kinase antibody indicated that the 42-kD protein was most likely a MAP kinase. 5-HT (10^-5M) stimulated contraction and increased antiphosphotyrosine immunoreactivity in whole aorta mounted in tissue baths. Importantly, aortic contraction to 5-HT was shifted (5-fold rightward) and reduced (69% control) by genistein but not daidzein. These findings demonstrate that (1) tyrosine kinase activation may partially mediate contractility to 5-HT in arterial smooth muscle, (2) tyrphostin 23 is somewhat nonselective and (3) 5-HT stimulates tyrosine kinase as documented by increased tyrosyl phosphorylation of proteins in cultured aortic smooth muscle cells and aortic tissue in active contraction of 5-HT. These findings have significant implications not only in understanding a novel pathway of 5-HT signal transduction but also in vascular diseases in which growth and/or contractility to 5-HT is increased (e.g. hypertension, atherosclerosis).

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In this study, we studied whether hydrogen sulfide (H2S) has an effect on the pacemaker activity of interstitial cells of Cajal (ICC), in the small intestine of mice. The actions of H2S on pacemaker activity were investigated using whole-cell patch-clamp technique, intracellular Ca 2＋ analysis at 30 o C and RT-PCR in cultured mouse intestinal ICC. Exogenously applied sodium hydrogen sulfide (NaHS), a donor of hydrogen sulfide, caused a slight tonic inward current on pacemaker activity in ICC at low concentrations (50 and 100 μM), but at high concentration (500 μM and 1 mM) it seemed to cause light tonic inward currents and then inhibited pacemaker amplitude and pacemaker frequency, and also an increase in the resting currents in the outward direction. Glibenclamide or other potassium channel blockers (TEA, BaCl2, apamin or 4-aminopydirine) did not have an effect on NaHS-induced action in ICC. The exogenous application of carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and thapsigargin also inhibited the pacemaker activity of ICC as NaHS. Also, we found NaHS inhibited the spontaneous intracellular Ca 2＋ ([Ca 2＋ ]i) oscillations in cultured ICC. In doing an RT-PCR experiment, we found that ICC enriched population lacked mRNA for both CSE and CBS, but was prominently detected in unsorted muscle. In conclusion, H2S inhibited the pacemaker activity of ICC by modulating intracellular Ca 2＋ . These results can serve as evidence of the physiological action of H2S as acting on the ICC in gastrointestinal (GI) motility.

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