Rosado J A, Sage S O
Department of Physiology, University of Cambridge, Downing St., Cambridge CB2 3EG, United Kingdom.
J Biol Chem. 2000 Mar 31;275(13):9110-3. doi: 10.1074/jbc.275.13.9110.
We have recently observed that small GTP-binding proteins are important for mediation of store-mediated Ca(2+) entry in human platelets through the reorganization of the actin cytoskeleton. Because it has been shown in platelets and other cells that small GTP-binding proteins regulate the activity of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, whose products, phosphoinositides, play a key role in the reorganization of the actin cytoskeleton, we have investigated the role of these lipid kinases in store-mediated Ca(2+) entry. Treatment of platelets with LY294002, an inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinases, resulted in a concentration-dependent inhibition of Ca(2+) entry stimulated by thapsigargin or the physiological agonist, thrombin. In addition, wortmannin, another inhibitor of these kinases, which is structurally unrelated to LY294002, significantly reduced store-mediated Ca(2+) entry. The inhibitory effect of LY294002 was not mediated either by blockage of Ca(2+) channels or by modification of membrane potential. LY294002 inhibited actin polymerization stimulated by thrombin or thapsigargin. These results indicate that both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase are required for activation of store-mediated Ca(2+) entry in human platelets and that the mechanism could involve the reorganization of the actin cytoskeleton.
我们最近观察到，小GTP结合蛋白通过肌动蛋白细胞骨架的重组，在介导人血小板中储存介导的Ca(2+)内流方面起着重要作用。由于在血小板和其他细胞中已表明，小GTP结合蛋白调节磷脂酰肌醇3激酶和磷脂酰肌醇4激酶的活性，其产物磷酸肌醇在肌动蛋白细胞骨架的重组中起关键作用，因此我们研究了这些脂质激酶在储存介导的Ca(2+)内流中的作用。用磷脂酰肌醇3和磷脂酰肌醇4激酶的抑制剂LY294002处理血小板，导致毒胡萝卜素或生理激动剂凝血酶刺激的Ca(2+)内流呈浓度依赖性抑制。此外，渥曼青霉素，这些激酶的另一种抑制剂，其结构与LY294002无关，显著降低了储存介导的Ca(2+)内流。LY294002的抑制作用既不是通过阻断Ca(2+)通道也不是通过改变膜电位介导的。LY294002抑制凝血酶或毒胡萝卜素刺激的肌动蛋白聚合。这些结果表明，磷脂酰肌醇3激酶和磷脂酰肌醇4激酶都是人血小板中储存介导的Ca(2+)内流激活所必需的，其机制可能涉及肌动蛋白细胞骨架的重组。
