Hill T D, Campos-Gonzalez R, Kindmark H, Boynton A L
Cancer Research Center of Hawaii, University of Hawaii, Honolulu 96813.
J Biol Chem. 1988 Nov 5;263(31):16479-84.
Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), an intracellular second messenger produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, interacts with cytoplasmic membrane structures to elicit the release of stored Ca2+. Ins(1,4,5)P3-induced Ca2+ mobilization is mediated through high affinity receptor binding sites; however, the biochemical mechanism coupling receptor occupation with Ca2+ channel opening has not been identified. In studies presented here, we examined the effects of naphthalenesulfonamide calmodulin antagonists, W7 and W13, and a new selective antagonist, CGS 9343B, on Ca2+ mobilization stimulated by Ins(1,4,5)P3 in neoplastic rat liver epithelial (261B) cells. Intact fura-2 loaded cells stimulated by thrombin, a physiological agent that causes phosphatidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)P3 release, responded with a rise in cytoplasmic free Ca2+ levels that was dose dependently inhibited by W7(Ki = 25 microM), W13 (Ki = 45 microM), and CGS 9343B (Ki = 110 microM). Intracellular Ca2+ release stimulated by the addition of Ins(1,4,5)P3 directly to electropermeabilized 261B cells was similarly inhibited by pretreatment with anti-calmodulin agents. W7 and CGS 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on protein kinase A or C in dose range required for complete inhibition of Ca2+ mobilization. Ca2+ release channels and Ca2+-ATPase pump activity were also unaffected by calmodulin antagonist treatment. These results indicate that calmodulin is tightly associated with the intracellular membrane mechanism coupling Ins(1,4,5)P3 receptors to Ca2+ release channels
肌醇1,4,5-三磷酸（Ins(1,4,5)P3）是由磷脂酰肌醇4,5-二磷酸水解产生的一种细胞内第二信使，它与细胞质膜结构相互作用，引发储存Ca2+的释放。Ins(1,4,5)P3诱导的Ca2+动员是通过高亲和力受体结合位点介导的；然而，将受体占据与Ca2+通道开放偶联的生化机制尚未明确。在本文所述的研究中，我们研究了萘磺酰胺钙调蛋白拮抗剂W7和W13以及一种新的选择性拮抗剂CGS 9343B对肿瘤大鼠肝上皮（261B）细胞中Ins(1,4,5)P3刺激的Ca2+动员的影响。完整的、负载fura-2的细胞受到凝血酶刺激，凝血酶是一种导致磷脂酰肌醇4,5-二磷酸水解和Ins(1,4,5)P3释放的生理因子，细胞质游离Ca2+水平升高，W7（Ki = 25 microM）、W13（Ki = 45 microM）和CGS 9343B（Ki = 110 microM）对其有剂量依赖性抑制作用。直接向电穿孔的261B细胞中添加Ins(1,4,5)P3刺激的细胞内Ca2+释放，用抗钙调蛋白药物预处理也同样受到抑制。W7和CGS 9343B能有效阻断Ca2+/钙调蛋白依赖性蛋白激酶，在完全抑制Ca2+动员所需的剂量范围内，对蛋白激酶A或C没有显著影响。钙调蛋白拮抗剂处理也不影响Ca2+释放通道和Ca2+-ATP酶泵活性。这些结果表明，钙调蛋白与将Ins(1,4,5)P3受体与Ca2+释放通道偶联的细胞内膜机制紧密相关。
