EVIDENCE FOR A TYROSINE KINASE-DEPENDENT ACTIVATION OF THE ADENYLYL CYCLASE PKA CASCADE DOWNSTREAM FROM THE G-PROTEIN-LINKED ENDOTHELIN-ETARECEPTOR IN VASCULAR SMOOTH-MUSCLE/

Endothelin (ET-1), a contractor and mitogen in the vasculature, enhanc ed cAMP production (t(1/2), 2.2 min; EC50, 89 +/- 6.3 nM) and stimulat ed activity of the cAMP-dependent protein kinase (PKA) in pig coronary arteries. These responses were blunted by the protein tyrosine kinase (PTK) inhibitors genistein and herbimycin-A, but not by inhibitors of protein kinase C or cyclooxygenase. In contrast, forskolin-stimulated cAMP production was unaffected by PTK inhibition. Immunoblot analysis revealed that ET-1 induced a concentration-dependent protein tyrosine (PT) phosphorylation. Sarafotoxin-c, a selective ETB receptor agonist , had no effect on either cAMP levels or PT phosphorylation. Moreover, pervanadate (PV), a potent inhibitor of PT phosphatases, enhanced bot h cAMP formation and PT phosphorylation, both of which were blocked by PTK inhibitors. The effects of ET-1 and PV were not additive, suggest ing a similar mode of activation, whereas responses to ET-1 and forsko lin were synergistic. These findings indicate that AC and PKA are acti vatable via a nonreceptor PTK-dependent pathway downstream from the G- protein-linked ETA receptor. Because cAMP is a dilator and antimitogen in smooth muscle, stimulation of AC activity may be a negative feedba ck mechanism regulating ET-1-induced vasoconstriction and/or mitogenes is, (C) 1998 Academic Press.