Yoshiaki Ohi1, Kaoru Atsuki1, Yuichi Torii1, Yasushi Ohizumi2, Minoru Watanabe1 and Yuji Imaizumi1,*
1Department of Molecular and Cellular Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan
2Department of Pharmaceutical Molecular Biology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
*Corresponding author. FAX: +81-52-836-3431, E-mail: email@example.com
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 supficial [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 supficial 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 supficial storage sites, presumably due to its slow approach to deeper sites. MBED may be a good pharmacological tool to manipulate selectively the supficial Ca2+ stores related to STOCs.
Keywords: Caffeine, 9-Methyl-7-bromoeudistomin D, Ca2+-dependent K+ channel, Smooth muscle, Spontaneous transient outward current
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