A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin.
A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin.
复制标题
蛋白质磷酸化在调节毒胡萝卜素处理的兔血小板 Ca2+ 升高中的作用。
作者：
C. Murphy;A. Bullock;J. Westwick
在兔血小板中研究了通过抑制Ca(2+)- atp酶活性来调节蛋白激酶和磷酸酶活性对Ca2+内流的影响。用phorbol 12-肉豆蔻酸13-乙酸酯（PMA）激活蛋白激酶C （PKC）或用calyculin A抑制磷酸酶1/2A型（PP1/2A）活性，可引起thapsigargin （Tg）处理的血小板中胞浆内Ca2+升高的剂量依赖性抑制，并减少Ca2+内流到血小板中的Ca2+，此时通过Tg预处理的细胞已经打开了Ca2+通道。此外，PKC的激活和PP1/2A活性的抑制都导致tg处理的血小板中二价阳离子（Mn2+）内流（作为Ca2+内流的替代品）的剂量依赖性抑制。环加氧酶活性的抑制导致tg处理的血小板中[Ca2+]i升高的小幅下降，但对PMA或calyculin a抑制tg诱导的[Ca2+]i升高的能力没有影响。出乎意料的是，PMA抑制tg诱导的[Ca2+]i升高，在没有细胞外Ca2+的情况下，calyculin a将[Ca2+]i升高几乎降低到基础水平。本研究的结果用另一种Ca(2+)- atp酶抑制剂，即2,5-二（叔丁基）对苯二酚（tBHQ）证实。因此，这些发现表明，丝氨酸/苏氨酸氨基酸残基上靶蛋白磷酸化的修饰在Tg处理的血小板中Ca2+内流和细胞内Ca2+池的填充状态的调节中起作用。
The effect of modifying protein kinase and phosphatase activity on Ca2+ influx induced by inhibition of Ca(2+)-ATPase activity has been investigated in rabbit platelets. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or inhibition of phosphatase type 1/2A (PP1/2A) activity with calyculin A caused a dose-dependent inhibition of cytosolic Ca2+ elevation in thapsigargin (Tg)-treated platelets and decreased Ca2+ influx into platelets at a time when Ca2+ channels had already been opened by pretreatment of cells with Tg. In addition, both activation of PKC and inhibition of PP1/2A activity caused a dose-dependent inhibition of bivalent cation (Mn2+) influx (acting as a surrogate for Ca2+ influx) in Tg-treated platelets. Inhibition of cyclo-oxygenase activity caused a small decrease in [Ca2+]i elevation in Tg-treated platelets, but had no effect on the ability of PMA or calyculin A to inhibit Tg-induced [Ca2+]i elevation Unexpectedly, PMA inhibited Tg-induced [Ca2+]i elevation in the absence of extracellular Ca2+, and in agreement calyculin A decreased [Ca2+]i elevation almost to basal levels. The results from this study were confirmed with another Ca(2+)-ATPase inhibitor, namely 2,5-di(tert-butyl)hydroquinone (tBHQ). These findings therefore suggest that modification of phosphorylation of target protein(s) on serine/threonine amino acid residues plays a role in the regulation of both Ca2+ influx and in the filling state of the intracellular Ca2+ pool in platelets treated with Tg.