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 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.