Muscarinic stimulation increases basal Ca(2+) and inhibits spontaneous Ca(2+) transients in murine colonic myocytes

Localized Ca(2+) transients in isolated murine colonic myocytes depend on Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3)) receptors. Localized Ca(2+) transients couple to spontaneous transient outward currents (STOCs) and mediate hyperpolarization responses in these cells. We used confocal microscopy and whole cell patch-clamp recording to investigate how muscarinic stimulation, which causes formation of IP(3), can suppress Ca(2+) transients and STOCs that might override the excitatory nature of cholinergic responses. ACh (10 microM) reduced localized Ca(2+) transients and STOCs, and these effects were associated with a rise in basal cytosolic Ca(2+). These effects of ACh were mimicked by generalized rises in basal Ca(2+) caused by ionomycin (250-500 nM) or elevated external Ca(2+) (6 mM). Atropine (10 microM) abolished the effects of ACh. Pretreatment of cells with nicardipine (1 microM), or Cd(2+) (200 microM) had no effect on responses to ACh. An inhibitor of phospholipase C, U-73122, blocked Ca(2+) transients and STOCs but did not affect the increase in basal Ca(2+) after ACh stimulation. Xestospongin C (Xe-C; 5 microM), a membrane-permeable antagonist of IP(3) receptors, blocked spontaneous Ca(2+) transients but did not prevent the increase of basal Ca(2+) in response to ACh. Gd(3+) (10 microM), a nonselective cation channel inhibitor, prevented the increase in basal Ca(2+) after ACh and increased the frequency and amplitude of Ca(2+) transients and waves. Another inhibitor of receptor-mediated Ca(2+) influx channels, SKF-96365, also prevented the rise in basal Ca(2+) after ACh and increased Ca(2+) transients and development of Ca(2+) waves. FK-506, an inhibitor of FKBP12/IP(3) receptor interactions, had no effect on the rise in basal Ca(2+) but blocked the inhibitory effects of increased basal Ca(2+) and ACh on Ca(2+) transients. These results suggest that the rise in basal Ca(2+) that accompanies muscarinic stimulation of colonic muscles inhibits localized Ca(2+) transients that could couple to activation of Ca(2+)-activated K(+) channels and reduce the excitatory effects of ACh.