Albrightson C R, Pullen M, Wu H L, Dytko G, Hersh L B, Nambi P
Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939, USA.
Mol Pharmacol. 1995 Jun;47(6):1156-63.
Thrombin-mediated down-regulation of endothelin (ET) receptors was studied in rat glomerular mesangial cells. Overnight incubation of mesangial cells with thrombin (10 nM) resulted in a significant decrease (67%) in the number of ET receptors, with no change in affinity. Northern analysis of the mRNA from these cells showed a corresponding decrease in the ETA receptor message. Such a decrease in ET receptors could result from an increase in ET levels caused by an increase in synthesis and/or a decrease in degradation. It has been previously reported that thrombin stimulates ET production in endothelial and mesangial cells. Because ET is known to be degraded by neutral endopeptidase (NEP), which is present at high levels in the kidney, the potential effects of thrombin on NEP activity were evaluated. There was a decrease of NEP activity in mesangial cells at 16 and 24 hr after treatment with 10 nM thrombin. This effect was specific for thrombin, because NEP activity was not altered after treatment with thrombin in the presence of hirudin, an inhibitor of thrombin activity. The thrombin-mediated decrease in NEP activity correlated with a decrease in NEP protein and mRNA levels, as determined by Western and Northern analyses, respectively. To determine whether the thrombin-mediated decrease in ET receptors had a functional corollary, ET-1-stimulated intracellular calcium mobilization was measured. Overnight incubation with 10 nM thrombin resulted in a significant inhibition of ET-stimulated intracellular calcium mobilization. This effect was specific for ET, because thrombin pretreatment did not affect vasopressin-stimulated intracellular calcium mobilization in mesangial cells. These results indicate that the thrombin-mediated down-regulation of ET receptors is due, in part, to a thrombin-stimulated increase in ET resulting from the down-regulation of NEP and the reported increase in ET synthesis. In addition, pretreatment of mesangial cells with ET-1 caused a significant decrease (85%) in ET receptor number and ET-1-mediated intracellular calcium release (84%), without affecting vasopressin- or thrombin-mediated responses.
在大鼠肾小球系膜细胞中研究了凝血酶介导的内皮素（ET）受体下调情况。系膜细胞与凝血酶（10 nM）过夜孵育导致ET受体数量显著减少（67%），亲和力无变化。对这些细胞的mRNA进行Northern分析显示，ETA受体信息相应减少。ET受体的这种减少可能是由于合成增加和/或降解减少导致ET水平升高所致。先前已有报道称，凝血酶可刺激内皮细胞和系膜细胞产生ET。由于已知ET可被中性内肽酶（NEP）降解，而NEP在肾脏中含量很高，因此评估了凝血酶对NEP活性的潜在影响。用10 nM凝血酶处理后16小时和24小时，系膜细胞中的NEP活性降低。这种作用对凝血酶具有特异性，因为在存在凝血酶活性抑制剂水蛭素的情况下，用凝血酶处理后NEP活性未改变。分别通过Western和Northern分析确定，凝血酶介导的NEP活性降低与NEP蛋白和mRNA水平降低相关。为了确定凝血酶介导的ET受体减少是否具有功能上的必然结果，测量了ET-1刺激的细胞内钙动员情况。与10 nM凝血酶过夜孵育导致ET刺激的细胞内钙动员受到显著抑制。这种作用对ET具有特异性，因为凝血酶预处理不影响系膜细胞中血管加压素刺激的细胞内钙动员。这些结果表明，凝血酶介导的ET受体下调部分是由于NEP下调导致凝血酶刺激ET增加以及报道的ET合成增加所致。此外，用ET-1预处理系膜细胞导致ET受体数量显著减少（85%）和ET-1介导的细胞内钙释放减少（84%），而不影响血管加压素或凝血酶介导的反应。
