Heller R, Bussolino F, Ghigo D, Garbarino G, Schröder H, Pescarmona G, Till U, Bosia A
Department of Pathological Biochemistry, Medical Academy, Erfurt, F.R.G.
Biochim Biophys Acta. 1991 Jun 7;1093(1):55-64. doi: 10.1016/0167-4889(91)90138-n.
Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.
凝血酶刺激人内皮细胞（EC）会引发细胞内游离钙离子（[Ca2+]i）迅速增加、血小板活化因子（PAF）生成以及1-O-烷基-2-溶血-sn-甘油-3-磷酸胆碱（溶血PAF）：乙酰辅酶A乙酰转移酶（EC 2.3.1.67）活性增强。用凝血酶处理内皮细胞会导致胞质蛋白激酶C（PKC）活性降低90%；这种显著下降伴随着微粒体部分酶活性的增加。已评估PKC在凝血酶介导的PAF合成中的作用：（1）用部分选择性抑制剂（棕榈酰肉碱、鞘氨醇和H-7）阻断PKC活性；（2）使内皮细胞长期暴露于佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA），这会导致PKC下调。在这两种情况下，均观察到凝血酶诱导的PAF生成受到强烈抑制，表明PAF合成必须有PKC活性。有人提出PKC调节内皮细胞磷脂酶A2（PLA2）活性，因为在PKC缺失的细胞中，凝血酶诱导的花生四烯酸（AA）释放受到90%的抑制。内皮细胞短暂暴露于PMA会强烈抑制凝血酶诱导的[Ca2+]i升高、乙酰转移酶活化和PAF生成，这表明，除了正向作用外，PKC还对凝血酶作用于内皮细胞的膜信号通路提供负反馈控制。福斯高林和伊洛前列素这两种可增加内皮细胞中细胞cAMP水平的药物，在抑制凝血酶诱发的胞质Ca2+升高、乙酰转移酶活化和PAF生成方面非常有效；这表明内源性生成的前列环素（PGI2）可能调节人内皮细胞中PAF的合成。
