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

Hong-Nian Liu

Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Susumu Ohya

Shinji Furuzono

Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan

Jing Wang

Biochemical Engineering of Environment Center, Faculty of Chemical Science and Engineering, University of Petroleum, Beijing, China

Yuji Imaizumi

Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan

Shinsuke Nakayama

Department of Cell Physiology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan;h44673a{at}nucc.cc.nagoya-u.ac.jp

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 InsP3R1 and 2 in ICCs.

Key Words: stomach • pacemaker • calcium oscillation • c-Kit-immunopositive cells • interstitial cells of Cajal • InsP3 receptors • TRPC homologues

Journal of Biological Rhythms, Vol. 20, No. 1, 15-26 (2005)
DOI: 10.1177/0748730404269572
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