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 x 10(-9) M) increase of inositol-phosphate production with a much higher potency than phenylephrine (EC50 = 1.4 x 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 inositol-phosphate accumulation. However, when the uncompetitive inositol monophosphatase inhibitor Li+ was only present during the last 30 min of 150 min incubation, the inositol-phosphate 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-bisphosphate. A very rapid resynthesis of this substrate took place as its level remained almost constant during 45 min stimulation with 10(-8) M endothelin-1 while the accumulation of inositol-phosphates was at least 15-fold higher than the initial cellular phosphatidylinositol 4,5-bisphosphate content. After 120 min preincubation of cells with 10(-9) M 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 x 10(-6) M) also led to inhibition of the phenylephrine (10(-4) M)-mediated inositol-phosphate 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.