ACTIVATION OF ENDOTHELIN ETA RECEPTORS INDUCES PHOSPHORYLATION OF ALPHA(1B)-ADRENORECEPTORS IN RAT-1 FIBROBLASTS

The effect of endothelin-l on the phosphorylation of alpha(1b)-adrenor eceptors, transfected into rat-1 fibroblasts, was studied, Basal alpha (1b)-adrenoreceptor phosphorylation was markedly increased by endothel in-l, norepinephrine, and phorbol esters, The effect of endothelin-l w as dose dependent (EC50 approximate to 1 nM), reached its maximum 5 mi n after stimulation, and was inhibited by BQ-123, an antagonist select ive for ETA receptors. Endothelin-1-induced alpha(1b)-adrenoreceptor p hosphorylation was attenuated by staurosporine or genistein and essent ially abolished when both inhibitors were used together. The effect of norepinephrine. was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used togeth er. These data suggest the participation of protein kinase C and tyros ine kinase(s) in endothelin-l-induced receptor phosphorylation. Howeve r, phosphoaminoacid analysis revealed the presence of phosphoserine an d traces of phosphothreonine, but not of phosphotyrosine, suggesting t hat the putative tyrosine kinase(s), activated by endothelin, could ac t in a step previous to receptor phosphorylation. The effect of endoth elin-1 on alpha(1b)-adrenoreceptor phosphorylation was not mediated th rough pertussis toxin-sensitive G proteins. Calcium mobilization induc ed by norepinephrine was diminished by endothelin-l, Norepinephrine an d endothelin-1 increased [S-35]GTP gamma S binding to control membrane s. The effect of norepinephrine was abolished in membranes obtained fr om cells pretreated with endothelin-l. Interestingly, genistein plus s taurosporine inhibited this effect of the endothelial peptide, Endothe lin-l did not induce alpha(1b)-adrenoreceptor internalization, Our dat a indicate that activation of ETA receptors by endothelin-1 induces al pha(1b)-adrenoreceptor phosphorylation and alters G protein coupling.