Nakashima Y, Ono K
Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Am J Physiol. 1994 Oct;267(4 Pt 2):H1514-22. doi: 10.1152/ajpheart.1994.267.4.H1514.
Single myocytes were obtained from guinea-pig ventricles, and the catecholamine-induced Cl- current (ICl) was recorded by the patch-clamp method combined with the use of caged adenosine 3',5'-cyclic monophosphate (cAMP) and the concentration jump technique. The rapid bath application of isoprenaline was followed by a marked latency of approximately 1.7 s before a sigmoidal onset of the ICl activation, while the photolysis of caged cAMP was followed by no measurable latency. The ICl conductance was increased by increasing the concentration of caged cAMP with a threshold of approximately 5 microM and a one-half effective concentration of 25 microM. On the other hand, the rate of the activation increased in proportion to the logarithm of the concentration of caged cAMP. When the ultraviolet flash was repeated or the myocytes were pretreated with either forskolin, 3-isobutyl-1-methylxanthine, or okadaic acid, the rate of the ICl activation accelerated, and the whole time course of the ICl activation became monoexponential. These findings suggest that both the production of cAMP and reactions on the multiple phosphorylation sites of the channel protein are major rate-limiting steps in the ICl activation.
从豚鼠心室获取单个心肌细胞，采用膜片钳技术结合使用笼锁型腺苷3',5'-环磷酸（cAMP）和浓度阶跃技术记录儿茶酚胺诱导的氯离子电流（ICl）。快速向浴槽中加入异丙肾上腺素后，在ICl激活呈S形开始之前有大约1.7秒的明显延迟，而笼锁型cAMP光解后没有可测量的延迟。通过增加笼锁型cAMP的浓度来增加ICl电导，阈值约为5 microM，半效浓度为25 microM。另一方面,激活速率与笼锁型cAMP浓度的对数成正比。当重复紫外线闪光或用福斯可林、3-异丁基-1-甲基黄嘌呤或冈田酸预处理心肌细胞时,ICl激活速率加快,ICl激活的整个时间进程变为单指数形式。这些发现表明,cAMP的产生和通道蛋白多个磷酸化位点上发生反应均是ICl激活过程中的主要限速步骤。
