Thomas C P, Baldi E, Simonson M S, Kester M, Dunn M J
Department of Medicine, Case Western Reserve University, Cleveland, Ohio.
J Cardiovasc Pharmacol. 1991;17 Suppl 7:S79-84. doi: 10.1097/00005344-199100177-00022.
Endothelins (ETs) are a family of vasoactive peptides with profound biological actions in diverse cell systems. Among its varied actions, ET stimulates phospholipase C (PLC) in cultured mesangial cells. We investigated the presence of specific ET receptors in rat mesangial cells in culture, and studied the role of GTP-binding proteins (G proteins) in coupling PLC to the endothelin receptor. [125I]ET binding was time- and temperature-dependent, and Scatchard analysis of saturation data showed a single class of high-affinity binding sites. Heterologous displacement with two related peptides, ET-3 and sarafotoxin (SFTX), revealed the presence of two binding sites for these isopeptides. Preincubation of cells with ET-1 reduced the receptor number without affecting Kd, and this effect was not prevented by protein kinase C inhibition or downregulation. We confirmed the presence of a 41- to 43-kDa pertussis toxin substrate in rat mesangial cell membranes in an ADP ribosylation assay. ET-1 inhibits and GDP beta S enhances toxin-catalyzed transfer of ADP-ribose to this substrate. ET-1 potentiated GTP gamma S-induced phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. In addition, pertussis toxin partially inhibited ET-stimulated PI hydrolysis in intact mesangial cells. Pertussis toxin also reduced the magnitude of ET-stimulated intracellular free calcium [( Ca2+ )i]. Thus, ET-1 binds to specific receptors on rat mesangial cells and activates PLC, in part, through a pertussis toxin-sensitive G-protein.
内皮素（ETs）是一类血管活性肽家族，在多种细胞系统中具有深远的生物学作用。在其多种作用中，ET可刺激培养的系膜细胞中的磷脂酶C（PLC）。我们研究了培养的大鼠系膜细胞中特异性ET受体的存在情况，并研究了鸟苷三磷酸结合蛋白（G蛋白）在将PLC与内皮素受体偶联中的作用。[125I]ET结合具有时间和温度依赖性，对饱和数据的Scatchard分析显示存在一类单一的高亲和力结合位点。用两种相关肽ET-3和萨拉索毒素（SFTX）进行异源置换，揭示了这些异肽存在两个结合位点。用ET-1预孵育细胞可减少受体数量而不影响解离常数（Kd），并且这种作用不受蛋白激酶C抑制或下调的阻止。我们在ADP核糖基化试验中证实了大鼠系膜细胞膜中存在一种41至43 kDa的百日咳毒素底物。ET-1抑制，而GDPβS增强毒素催化的ADP核糖向该底物的转移。ET-1以浓度依赖性方式增强GTPγS诱导的磷脂酰肌醇（PI）水解。此外，百日咳毒素部分抑制完整系膜细胞中ET刺激的PI水解。百日咳毒素还降低了ET刺激的细胞内游离钙[（Ca2+）i]的幅度。因此，ET-1与大鼠系膜细胞上的特异性受体结合，并部分通过百日咳毒素敏感的G蛋白激活PLC。
