Xu Y, Ware J A
Cardiovascular Division, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1995 Oct 13;270(41):23887-90. doi: 10.1074/jbc.270.41.23887.
Thrombin initiates many physiological processes in platelets and other megakaryocyte-lineage cells by interacting with surface receptors and generating rises in cytoplasmic Ca2+; these rises result from both Ca2+ release from intracellular stores and receptor-mediated Ca2+ entry. Regulators that limit Ca2+ entry after its initiation by thrombin have not been identified. In this study, prevention of expression of a single protein kinase C isoenzyme (PKC beta) by antisense cDNA overexpressed in HEL cells, a human megakaryoblastic cell line that expresses thrombin receptors, promotes thrombin receptor-mediated Ca2+ entry without altering thrombin-induced intracellular release of Ca2+. The cytoplasmic Ca2+ rise initiated by endoperoxide analogs was not affected by inhibiting PKC beta. Overexpression of a cDNA encoding wild-type PKC beta mutated to prevent recognition by the antisense cDNA abolished the enhancement of Ca2+ influx following thrombin. Thus, PKC beta appears to be a specific negative regulator of thrombin receptor-mediated Ca2+ entry.
凝血酶通过与表面受体相互作用并使细胞质钙离子浓度升高，从而引发血小板和其他巨核细胞系细胞中的多种生理过程；这些钙离子浓度的升高既源于细胞内储存库释放钙离子，也源于受体介导的钙离子内流。目前尚未发现凝血酶引发钙离子内流后限制其进入的调节因子。在本研究中，在表达凝血酶受体的人巨核母细胞系HEL细胞中，通过反义cDNA过表达来阻止单一蛋白激酶C同工酶（PKCβ）的表达，可促进凝血酶受体介导的钙离子内流，而不改变凝血酶诱导的细胞内钙离子释放。内过氧化物类似物引发的细胞质钙离子浓度升高不受抑制PKCβ的影响。编码野生型PKCβ的cDNA经突变后可防止被反义cDNA识别，其过表达消除了凝血酶作用后钙离子内流的增强。因此，PKCβ似乎是凝血酶受体介导的钙离子内流的特异性负调节因子。
