Co-contribution of IP₃R and Ca²⁺ Influx Pathways to Pacemaker Ca²⁺ Activity in Stomach ICC

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Abstract
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-µm 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-Kitimmunoreactivity, 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. Asmall 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 (10µM) significantly affected [Ca2+]i oscillations in the presence of nifedipine. Low concentrations (40µM) of Ni2+ had little effect on the spontaneous [Ca2+]i oscillation, but SK&F96365 (40µM) and Cd2+ (120µM) terminated it. Applications of either 2-aminoethoxydiphenyl borate (10µM) or xestosponginC(10µM) completely and rather rapidly (~2 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 (InsP3R) 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 Ins P3R1 and 2 in ICCs.