Yan W, Tiruppathi C, Lum H, Qiao R, Malik A B
Department of Pharmacology, College of Medicine, University of Illinois, Chicago 60612, USA.
Am J Physiol. 1998 Feb;274(2):C387-95. doi: 10.1152/ajpcell.1998.274.2.C387.
We studied the effects of protein kinase C (PKC) activation on endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration in human dermal microvascular endothelial cells (HMEC) exposed to alpha-thrombin or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TPA reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicating heterologous PAR-1 desensitization. Treatment of cells with staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC beta isozymes (PKC beta I and PKC beta II) by transducing cells with antisense cDNA of PKC beta I prevented the TPA-induced decrease in cell surface PAR-1 expression and restored approximately 60% of the cytosolic Ca2+ signal in response to thrombin. In contrast, depletion of PKC beta isozymes did not affect the loss of cell surface PAR-1 and induction of homologous PAR-1 desensitization by thrombin. Therefore, homologous PAR-1 desensitization by thrombin occurs independently of PKC beta isozymes, whereas the PKC beta-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.
我们研究了蛋白激酶C（PKC）激活对蛋白水解激活的凝血酶受体1（PAR-1）在内皮细胞表面表达及功能的影响。通过免疫荧光法（使用抗PAR-1单克隆抗体）评估细胞表面PAR-1的表达，并通过测量暴露于α-凝血酶或佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）的人真皮微血管内皮细胞（HMEC）中胞质Ca2+浓度的增加来评估受体激活情况。免疫荧光显示，凝血酶和TPA降低了PAR-1的细胞表面表达。HMEC预先暴露于凝血酶5分钟会使其对凝血酶脱敏，表明存在同源PAR-1脱敏。相反，预先用TPA激活PKC会导致对凝血酶和组胺脱敏，表明存在异源PAR-1脱敏。用PKC抑制剂星形孢菌素处理细胞可完全防止异源脱敏，而凝血酶诱导的同源脱敏仍然存在。通过用PKCβI的反义cDNA转导细胞来耗尽PKCβ同工酶（PKCβI和PKCβII），可防止TPA诱导的细胞表面PAR-1表达下降，并恢复约60%的对凝血酶的胞质Ca2+信号。相反，耗尽PKCβ同工酶并不影响细胞表面PAR-1的丧失以及凝血酶诱导的同源PAR-1脱敏。因此，凝血酶诱导的同源PAR-1脱敏独立于PKCβ同工酶发生，而PKCβ激活途径在内皮细胞异源PAR-1脱敏信号传导中起重要作用。
