Ben-Amor Nidhal, Redondo Pedro C, Bartegi Aghleb, Pariente José A, Salido Ginés M, Rosado Juan A
Unité de Recherche de Biochimie, Institute Superieur de Biotechnologie, Monastir, Tunisia.
J Physiol. 2006 Jan 15;570(Pt 2):309-23. doi: 10.1113/jphysiol.2005.100800. Epub 2005 Nov 24.
A major pathway for Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. A de novo conformational coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this capacitative Ca(2+) entry (CCE) in several cell types, including platelets. Here we report that a cytochrome P450 metabolite, 5,6-EET, might be a component of the de novo conformational coupling in human platelets. In these cells, 5,6-EET induces divalent cation entry without having any detectable effect on Ca(2+) store depletion. 5,6-EET-induced Ca(2+) entry was sensitive to the CCE blockers 2-APB, lanthanum, SKF-96365 and nickel and impaired by incubation with anti-hTRPC1 antibody. Ca(2+) entry stimulated by low concentrations of thapsigargin, which selectively depletes the dense tubular system and induces EET production, was impaired by the cytochrome P450 inhibitor 17-ODYA, which has no effect on CCE mediated by depletion of the acidic stores using 2,5-di-(tert-butyl)-1,4-hydroquinone. We have found that 5,6-EET-induced Ca(2+) entry requires basal levels of H(2)O(2), which might maintain a redox state favourable for this event. Finally, our results indicate that 5,6-EET induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton, which might provide a support for the transport of portions of the Ca(2+) store towards the PM to facilitate de novo coupling between IP(3)R type II and hTRPC1 detected by coimmunoprecipitation. We propose that the involvement of 5,6-EET in TG-induced coupling between IP(3)R type II and hTRPC1 and subsequently CCE is compatible with the de novo conformational coupling in human platelets.
细胞内钙库耗竭后，非兴奋性细胞中钙离子内流的一条主要途径被激活。质膜（PM）中的成分与钙库之间的一种全新构象偶联被认为是在包括血小板在内的几种细胞类型中激活这种容量性钙内流（CCE）的最可能机制。在此我们报告，细胞色素P450代谢产物5,6 -环氧二十碳三烯酸（5,6 - EET）可能是人类血小板中这种全新构象偶联的一个组成部分。在这些细胞中，5,6 - EET诱导二价阳离子内流，而对钙库耗竭没有任何可检测到的影响。5,6 - EET诱导的钙离子内流对CCE阻滞剂2 -氨基乙氧基二苯硼酸（2 - APB）、镧、SKF - 96365和镍敏感，并因与抗hTRPC1抗体孵育而受损。低浓度毒胡萝卜素刺激的钙离子内流，毒胡萝卜素选择性地耗尽致密管状系统并诱导EET生成，被细胞色素P450抑制剂17 -十八碳二烯酸（17 - ODA）削弱，而17 - ODA对使用2,5 -二（叔丁基）- 1,4 -对苯二酚耗尽酸性钙库介导的CCE没有影响。我们发现5,6 - EET诱导的钙离子内流需要基础水平的过氧化氢（H₂O₂），这可能维持有利于此过程的氧化还原状态。最后，我们的结果表明5,6 - EET诱导酪氨酸激酶蛋白的激活和肌动蛋白细胞骨架的重组，这可能为钙库的部分向质膜的转运提供支持，以促进通过共免疫沉淀检测到的II型肌醇1,4,5 -三磷酸受体（IP₃R）与hTRPC1之间的全新偶联。我们提出5,6 - EET参与毒胡萝卜素诱导的II型IP₃R与hTRPC1之间的偶联以及随后的CCE与人类血小板中的全新构象偶联是相符的。
