Requirement of ryanodine receptors for pacemaker Ca2+ activity in ICC and HEK293 cells

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Abstract
Intracellular Ca2+ ([Ca2+]i) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca2+ release from intracellular Ca2+ stores produces [Ca2+]i oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca2+]i oscillations in ICCs are preserved in the presence of dihydropyridine Ca2+ antagonists, which suppress Ca2+ activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca2+]i oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca2+]i oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP3Rs. The reconstituted [Ca2+]i oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.