Cavallini L, Coassin M, Borean A, Alexandre A
Department of Biological Chemistry, CNR Centro di Studio delle Biomembrane, University of Padova, Italy.
J Biol Chem. 1996 Mar 8;271(10):5545-51. doi: 10.1074/jbc.271.10.5545.
Prostaglandin I2 (PGI2) and sodium nitroprusside (SNP) induce a rapid decay of the thrombin-promoted increase of [Ca2+]i in aspirin-treated platelets incubated in the absence of external Ca2+. The mechanism of their effect was studied with a new method which utilizes ionomycin to increase [Ca2+]i, followed by bovine serum albumin (BSA) to remove the Ca2+ ionophore. The rapid decay of [Ca2+]i after BSA is mostly due to the reuptake into the stores, since it is strongly inhibited by the endomembrane Ca2+-ATPase inhibitor thapsigargin. PGI2 and SNP are without effect on the BSA-promoted decay both with and without thapsigargin, showing that they do not affect the activity of the Ca2+-ATPases. The fast decay of [Ca2+]i after BSA is decreased by thrombin which produces the Ca2+ releaser inositol 1,4,5-trisphosphate (InsP3), thus counteracting the activity of the endomembrane Ca2+ pump. When added after thrombin, PGI2 and SNP accelerate the BSA-activated decay of [Ca2+]i. However, under the same conditions, they do not decrease the concentration of InsP3. In saponin-permeabilized platelets, cAMP and cGMP counteract the Ca2+ release induced by exogenous InsP3. Their inhibitory effect disappears at high InsP3 concentrations. This demonstrates that PGI2 and SNP potentiate Ca2+ reuptake by inhibiting the InsP3 receptor. Two bands of approximately 260 kDa are recognized by a monoclonal antibody recognizing the C-terminal region of the InsP3 receptor. Both are phosphorylated rapidly, the heavier more intensely, in the presence of PGI2 and SNP. The phosphorylation of the InsP3 receptor is fast enough to be compatible with its involvement in the inhibition of the receptor by cyclic nucleotides.
前列腺素I2（PGI2）和硝普钠（SNP）可使在无细胞外钙的条件下孵育的阿司匹林处理的血小板中，凝血酶促进的细胞内钙离子浓度（[Ca2+]i）升高迅速衰减。采用一种新方法研究了它们的作用机制，该方法利用离子霉素升高[Ca2+]i，随后用牛血清白蛋白（BSA）去除钙离子载体。BSA作用后[Ca2+]i的快速衰减主要是由于其重新摄取到储存库中，因为它受到内膜钙离子ATP酶抑制剂毒胡萝卜素的强烈抑制。无论有无毒胡萝卜素，PGI2和SNP对BSA促进的衰减均无影响，表明它们不影响钙离子ATP酶的活性。BSA作用后[Ca2+]i的快速衰减被凝血酶降低，凝血酶产生钙离子释放剂肌醇1,4,5-三磷酸（InsP3），从而抵消内膜钙离子泵的活性。在凝血酶作用后添加时，PGI2和SNP加速了BSA激活的[Ca2+]i衰减。然而，在相同条件下，它们不会降低InsP3的浓度。在皂素通透的血小板中，环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）可抵消外源性InsP3诱导的钙离子释放。在高InsP3浓度下它们的抑制作用消失。这表明PGI2和SNP通过抑制InsP3受体增强钙离子的重新摄取。一种识别InsP3受体C末端区域的单克隆抗体识别出两条约260 kDa的条带。在PGI2和SNP存在的情况下，两者均迅速被磷酸化，较重的条带磷酸化更强烈。InsP3受体的磷酸化速度足够快，足以与其参与环核苷酸对受体的抑制作用相匹配。
