Tanaka Y, Tashjian A H
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
J Biol Chem. 1994 Apr 15;269(15):11247-53.
Two intracellular Ca2+ release pathways are modulated by Ca2+ itself, namely Ca(2+)-induced Ca2+ release (classical CICR), and Ca(2+)-sensitized inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release (CSIICR). Both of these pathways are thought to be important in generating cytosolic Ca2+ oscillations. Caffeine and the sulfhydryl reagent thimerosal (TMS) are frequently used as selective modulators of CICR and CSIICR, respectively. We have studied the modes of action of caffeine and TMS in sea urchin egg homogenates using the Ca2+ indicator fluo-3. Cyclic ADP-ribose (cADPR), the only known endogenous compound to cause Ca2+ release via the ryanodine receptor, was used as an agonist for the classical CICR pathway. cADPR and InsP3 each alone caused release of sequestered Ca2+. Coaddition of caffeine dose-dependently and dramatically enhanced Ca2+ release induced by cADPR. InsP3-induced Ca2+ release was not affected by caffeine, but it was amplified by TMS, indicating the existence of the CSIICR pathway in this preparation. Surprisingly, cADPR-induced Ca2+ release was also markedly potentiated by TMS. TMS alone caused a slow increase in Ca2+ release which was not inhibited by heparin and/or procaine, indicating that TMS-induced Ca2+ release was not due to the actions of endogenous agonists (InsP3 or cADPR) on their receptors. All observed actions of TMS were abolished by dithiothreitol. We conclude: 1) both the InsP3- and cADPR-controlled Ca2+ release pathways are potentiated by TMS, while caffeine is specific for the cADPR pathway, indicating that TMS cannot be used as a selective reagent for probing the CSIICR mechanism; and 2) the action of TMS on intracellular Ca2+ release is independent of endogenous Ca2+ channel agonists, suggesting a novel action of TMS possibly on intravesicular Ca2+ handling.
两种细胞内钙离子释放途径受钙离子自身调节，即钙诱导的钙离子释放（经典的钙诱导钙释放，CICR）和钙敏化的肌醇1,4,5 -三磷酸（InsP3）诱导的钙离子释放（钙敏化InsP3诱导钙释放，CSIICR）。这两种途径都被认为在产生胞质钙离子振荡中起重要作用。咖啡因和巯基试剂硫柳汞（TMS）常分别用作CICR和CSIICR的选择性调节剂。我们使用钙离子指示剂Fluo - 3研究了咖啡因和TMS在海胆卵匀浆中的作用模式。环ADP -核糖（cADPR）是唯一已知的通过ryanodine受体引起钙离子释放的内源性化合物，用作经典CICR途径的激动剂。单独的cADPR和InsP3各自都能引起储存钙离子的释放。咖啡因的共添加剂量依赖性地且显著增强了cADPR诱导的钙离子释放。InsP3诱导的钙离子释放不受咖啡因影响，但被TMS放大，表明该制剂中存在CSIICR途径。令人惊讶的是，TMS也显著增强了cADPR诱导的钙离子释放。单独的TMS引起钙离子释放缓慢增加，这不受肝素和/或普鲁卡因抑制，表明TMS诱导的钙离子释放不是由于内源性激动剂（InsP3或cADPR）对其受体的作用。TMS的所有观察到的作用都被二硫苏糖醇消除。我们得出结论：1）InsP3和cADPR控制的钙离子释放途径都被TMS增强，而咖啡因对cADPR途径具有特异性，表明TMS不能用作探测CSIICR机制的选择性试剂；2）TMS对细胞内钙离子释放的作用独立于内源性钙离子通道激动剂，提示TMS可能对囊泡内钙离子处理有新的作用。
