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

Abstract

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 μM MBED 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 MBED occurred mainly in superficial areas. Longer application of 100 μM MBED for 2 min did not induce significant global [Ca²⁺]_i increase but decreased the amount of Ca²⁺ release and cell shortening during the subsequent application of 10 mM caffeine. These results indicate that short application of MBED 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.