Little is known concerning the signal transduction involved in the release of endothelium-derived relaxing factor(s). A variety of G-proteins are known to have key roles in coupling receptors for neurotransmitters and hormones to intracellular effector systems. Pertussis toxin, which inactivates certain G-proteins, inhibits the endothelium-dependent relaxations evoked by alpha2-adrenergic and serotonergic receptor stimulation, and also those evoked by aggregating platelets in porcine coronary arteries. Relaxations to nitric oxide and endothelium-dependent relaxations to bradykinin, adenosine diphosphate or A23187 were unaffected by the toxin. In coronary arteries with regenerated endothelium (following in vivo denudation), the endothelium-dependent relaxations to alpha2- adrenergic and serotonergic stimulation, and also to aggregating platelets are reduced, whereas responses to the other agonists are unchanged. Moreover, in these arteries, pertussis toxin is without effect on the remaining relaxations. These results suggest that certain endothelium- dependent relaxations are mediated by activation of a pertussis toxin- sensitive G-protein in the endothelial cells. Furthermore, in regenerating endothelial cells there may be a selective loss of this G-protein-dependent mechanism, which may predispose the blood vessel to vasospasm, atherosclerosis and vascular occlusion. The aim of the present proposal is to determine in isolated porcine coronary arteries, and in isolated porcine endothelial cells (native, cultured and regenerated): 1) the identify of the pertussis toxin-sensitive G-protein coupled to endothelial serotonergic and alpha2-adrenergic receptors (using SDS-PAGE and Western blot analysis), 2) characterize the subcellular effects arisen from activation of this G- protein (by analyzing alterations in enzyme activity, calcium fluxes, and release of endothelial mediators), 3) identify the dysfunction in this G- protein-dependent mechanism that occurs in proliferating endothelial cells (by analyzing alterations in the function of the G-protein, or in the endothelial receptors, or in the ability of the G-protein to couple to the receptors or to activate the endothelium), and 4) analyze the possible cause of the dysfunction (by analyzing potential mechanisms known to disrupt G-protein function). These experiments will further our understanding of the normal physiology of vascular function and hopefully provide important insight into the mechanisms that may initiate vascular disease.