Imaging of Ca2+ release by caffeine and 9-methyl-7-bromoeudistomin D and the associated activation of large conductance Ca2+-dependent K+ channels in urinary bladder smooth muscle cells of the guinea pig

Ca2+ release by caffeine and 9-methyl-7-bromoeudistomin D (MBED) and the concomitant activation of large conductance Ca2+-dependent K+ (BK) channels were analyzed using confocal Ca2+ imaging and whole cell voltage-clamp methods in guinea pig urinary bladder smooth muscle cells. Puff application of 3 or 10 mM caffeine for several seconds (2 - 5 s) elicited a large increase in intracellular Ca2+ concentration ([Ca2+](i)) and induced a phasic outward current at a holding potential of -40 mV. The phasic outward current was the summation of spontaneous transient outward currents (STOCs) due to marked activation of BK channels and was followed by a short cessation of STOCs. Although the increase in superficial [Ca2+](i) by caffeine was faster than that in global [Ca2+](i), the peak [Ca2+](i) was identical in these areas. Puff application of 100 muM MEED also markedly enhanced STOCs for a few seconds. This response to MBED was not observed when stored Ca2+ was depleted by caffeine. The increase in [Ca2+](i) by MEED occurred mainly in superficial areas. Longer application of 100 muM MBED for 2 min did not induce significant global [Ca2+](i) increase but decreased the amount of Ca2+ release and eel shortening during the subsequent application of 10 mM caffeine. These results indicate that short application of MEED releases Ca2+ preferentially from superficial storage sites, presumably due to its slow approach to deeper sites. MBED may be a good pharmacological tool to manipulate selectively the superficial Ca2+ stores related to STOCs.