Homologous Desensitization of the Endothelin-1 Receptor Mediated Phosphoinositide Response in Cultured Neonatal Rat Cardiomyocytes

The goal of the present study was to identify the molecular mechanism underlying desensitization of endothelin-1 receptor-mediated phosphoinositide response in cultured neonatal rat heart cells. Endothelin elicited a concentration-dependent (EC50=2.2 × 10-9 M) increase of inositolphosphate production with a much higher potency then phenylephrine (EC50=1.4 × 10-6 M). Endothelin-1 (10-8 M) evoked phosphoinositide turnover in the presence of 10 mM LiCl, which was greatly attenuated after 30-45 min of continuous stimulation with agonist, apparently resulting in a total absence of further inositolphosphate accumulation. However, when the uncompetitive inositol monophosphatase inhibitor Li+ was only present during the last 30 min of 150 min incubation, the inositolphosphate accumulation was decreased to a steady state of 33% of the initial rate. The loss of responsiveness of cardiomyocytes to endothelin-1 was not brought about by a limiting supply of phospholipase C substrate phosphatidylinositol 4,5-bishosphate. A very rapid resynthesis of this substrate took place as its level remained almost constant during 45 min stimulation with 10-8M endothelin-1 while the accumulation of inositolphosphates was at least 15-fold higher than the initial cellular phosphatidylinositol 4,5-bisphosphate content. After 120 min preincubation of cells with 10-9M endothelin-1 the activation of phospholipase C by a second higher dose (10-8 M) was severely (67%) inhibited at the same time leaving the induction of phosphoinositide turnover by phenylephrine (10-4 M) virtually intact. Preincubation with phenylephrine (3 × 10-6 M) also led to inhibition of the phenylephrine (10-4 M) mediated inositolphosphate response (36% inhibition) while the endothelin-1 (10-8 M) response was not affected. Addition of a direct activator of protein kinase C, phorbol 12-myristate 13-acetate, led to inhibition of the endothelin-1 evoked phosphoinositide turnover but the rate of desensitization was not affected. Inhibition of protein kinase C with staurosporine did not alter the time course of desensitization. In conclusion, the activity of the phosphoinositide cycle in cardiomyocytes is homologously desensitized after stimulation with endothelin-1. The desensitization is not likely to be due to either depletion of phospholipase C substrate or to the activation of protein kinase C by inositol 1,4,5-trisphosphate-mobilized Ca2+ and elevated 1,2-diacylglycerol levels.