Co-contribution of IP3R and Ca2+ influx pathways to pacemaker Ca2+ activity in stomach ICC

Intracellular Ca2+ oscillations in interstitial cells of Cajal (ICCs) are thought to be the primary pacemaker activity in the gut. In the present study, the authors prepared small tissues of 100- to 300-mum diameter (cell cluster preparation) from the stomach smooth muscle (including the myenteric plexus) of mice by enzymatic and mechanical treatments. After 2 to 4 days of culture, the intracellular Ca2+ concentration ([Ca2+](i)) was measured. In the presence of nifedipine, a dihydropyridine Ca2+ channel antagonist, spontaneous [Ca2+](i) oscillations were observed within limited regions showing positive c-Kit-immunoreactivity, a maker for ICCs. In the majority of cell cluster preparations with multiple regions of [Ca2+](i) oscillations, [Ca2+](i) oscillated synchronously in the same phase. A small number of cell clusters (8 of 53) showed multiple regions of [Ca2+](i) oscillations synchronized but with a considerable phase shift. Neither tetrodotoxin (250 nM) nor atropine (10 muM) significantly affected [Ca2+](i) oscillations in the presence of nifedipine. Low concentrations (40 muM) of Ni2+ had little effect on the spontaneous [Ca2+](i) oscillation, but SKF96365 (40 muM) and Cd2+ (120 muM) terminated it. Applications of either 2-aminoethoxydiphenyl borate (10 muM) or xestospongin C (10 muM) completely and rather rapidly (similar to2 min) abolished the spontaneous [Ca2+](i) oscillations. The results suggest that pacemaker [Ca2+](i) oscillations in ICCs are produced by close interaction of intracellular Ca2+ release channels, especially inositol 1,4,5-trisphosphate receptor (InsP(3)R) and Ca2+ influx pathways, presumably corresponding to store-operated type channels. Reverse transcription polymerase chain reaction examinations revealed expression of TRPC2,4, and 6, as well as InsP(3)R1 and 2 in ICCs.