BackgroundHyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs.MethodsWe performed whole-cell patch clamp, RT-PCR, and Ca2+-imaging in cultured ICCs from mouse mid colon.ResultsSQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca2+ [Ca2+]i oscillations, rolipram and 8-bromo-cAMP increased [Ca2+]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca2+]i oscillations.ConclusionsHCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

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.

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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