THROMBIN-MEDIATED DOWN-REGULATION OF ENDOTHELIN RECEPTORS IN MESANGIAL CELLS COINCIDES WITH THE DOWN-REGULATION OF NEUTRAL ENDOPEPTIDASE ACTIVITY

Thrombin-mediated down-regulation of endothelin (ET) receptors was stu died in rat glomerular mesangial cells. Overnight incubation of mesang ial 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 ET(A) receptor message. Such a decrease in ET receptors could result from an increase in ET levels caused by an increase in synthesi s and/or a decrease in degradation. It has been previously reported th at thrombin stimulates ET production in endothelial and mesangial cell s. Because ET is known to be degraded by neutral endopeptidase (NEP), which is present at high levels in the kidney, the potential effects o f 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 n M thrombin. This effect was specific for thrombin, because NEP activit y was not altered after treatment with thrombin in the presence of hir udin, an inhibitor of thrombin activity. The thrombin-mediated decreas e in NEP activity correlated with a decrease in NEP protein and mRNA l evels, as determined by Western and Northern analyses, respectively. T o determine whether the thrombin-mediated decrease in ET receptors had a functional corollary, ET-1-stimulated intracellular calcium mobiliz ation was measured. Overnight incubation with 10 nM thrombin resulted in a significant inhibition of ET-stimulated intracellular calcium mob ilization. This effect was specific for ET, because thrombin pretreatm ent did not affect vasopressin-stimulated intracellular calcium mobili zation in mesangial cells. These results indicate that the thrombin-me diated down-regulation of ET receptors is due, in part, to a thrombin- stimulated increase in ET resulting from the down-regulation of NEP an d the reported increase in ET synthesis. In addition, pretreatment of mesangial cells with ET-1 caused a significant decrease (85%) in ET re ceptor number and ET-1-mediated intracellular calcium release (84%), w ithout affecting vasopressin- or thrombin-mediated responses.