Regulation of muscarinic cationic current in myocytes from guinea-pig ileum by intracellular Ca2+ release: a central role of inositol 1,4,5-trisphosphate receptors.

The dynamics of carbachol (CCh)-induced Ca(2+) changes was related to the kinetics of muscarinic cationic current (mI(cat)) and the effect of Ca(2+) release through ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) on mI(cat) was evaluated by fast x-y or line-scan confocal imaging of Ca(2+) combined with simultaneous recording of mI(cat) under whole-cell voltage clamp. When myocytes freshly isolated from the longitudinal layer of the guinea-pig ileum were loaded with the Ca(2+)-sensitive indicator fluo-3, x-y confocal imaging revealed CCh (10 microM)-induced Ca(2+) waves, which propagated from the cell ends towards the myocyte centre at 45.9 +/- 8.8 microms(-1) (n = 13). Initiation of the Ca(2+) wave preceded the appearance of any measurable mI(cat) by 229 +/- 55 ms (n = 7). Furthermore, CCh-induced Ca(2+) transients peaked 1.22 +/- 0.11s (n = 17) before mI(cat) reached peak amplitude. At -50 mV, spontaneous release of Ca(2+) through RyRs, resulting in Ca(2+) sparks, had no effect on CCh-induced mI(cat) but activated BK channels leading to spontaneous transient outward currents (STOCs). In addition, Ca(2+) release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI(cat) (n = 14). This was not due to an inhibitory effect of caffeine on muscarinic cationic channels (since application of 5 mM caffeine did not inhibit mI(cat) when Ca(2+) was strongly buffered with Ca(2+)/BAPTA buffer) nor was it due to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca(2+) sensitivity of muscarinic cationic channels (since in the presence of 5 mM caffeine, photorelease of Ca(2+) upon cell dialysis with 5 mM NP-EGTA/3.8 mM Ca(2+) potentiated mI(cat) in the same way as in control). In contrast, IP(3)R-mediated Ca(2+) release upon flash photolysis of "caged" IP(3) (30 microM in the pipette solution) augmented mI(cat) (n = 15), even though Ca(2+) did not reach the level required for potentiation of mI(cat) during photorelease of Ca(2+) (n = 10). Intracellular calcium stores were visualised by loading of the myocytes with the low-affinity Ca(2+) indicator fluo-3FF AM and consisted of a superficial sarcoplasmic reticulum (SR) network and some perinuclear formation, which appeared to be continuous with the superficial SR. Immunostaining of the myocytes with antibodies to IP(3)R type 1 and to RyRs revealed that IP(3)Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP(3)R-mediated Ca(2+) release plays a central role in the modulation of mI(cat) in the guinea-pig ileum and that IP(3) may sensitise the regulatory mechanisms of the muscarinic cationic channels gating to Ca(2+).