Co-contribution of IP sub(3)R and Ca super(2+) Influx Pathways to Pacemaker Ca super(2+) Activity in Stomach ICC

Intracellular Ca super(2+) 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- mu m 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 Ca super(2+) concentration ([Ca super(2+)] sub(i)) was measured. In the presence of nifedipine, a dihydropyridine Ca super(2+) channel antagonist, spontaneous [Ca super(2+)] sub(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 [Ca super(2+)] sub(i) oscillations, [Ca super(2+)] sub(i) oscillated synchronously in the same phase. Asmall number of cell clusters (8 of 53) showed multiple regions of [Ca super(2+)] sub(i) oscillations synchronized but with a considerable phase shift. Neither tetrodotoxin (250 nM) nor atropine (10 mu M) significantly affected [Ca super(2+)] sub(i) oscillations in the presence of nifedipine. Low concentrations (40 mu M) of Ni2+ had little effect on the spontaneous [Ca super(2+)] sub(i) oscillation, but SK&F96365 (40 mu M) and Cd sub(2+) (120 mu M) terminated it. Applications of either 2-aminoethoxydiphenyl borate (10 mu M) or xestosponginC(10 mu M) completely and rather rapidly (approximately 2 min) abolished the spontaneous [Ca super(2+)] sub(i) oscillations. The results suggest that pacemaker [Ca super(2+)] sub(i) oscillations in ICCs are produced by close interaction of intracellular Ca super(2+) release channels, especially inositol 1,4,5- trisphosphate receptor (InsP sub(3)R) and Ca super(2+) 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 sub(3)R1 and 2 in ICCs.