Endothelial cells are well known to play a vital role in regulating vascular function. This regulatory role is accomplished through several mechanisms. These cells extract and degrade circulating vasoactive substances, convert precursors to vasoactive products, secrete potent vasodilators (PGI2 and EDRF) and synthesize potent vasoconstrictors (endothelin). In fact it has only recently been appreciated that endothelium mediates vasoconstriction presumably via production of endothelin. The range of stimuli and controls operating on these endothelialdependent mechanisms are incompletely understood. Factors which control the synthesis of endothelial-dependent vasoactive mediators and the mechanisms involved are obviously relevant to understanding the physiological and biochemical factors regulating vascular tone. Our knowledge of these factors and mechanisms will be useful in designing appropriate pharmacologic strategies for regulating disease processes involving alteration m vascular reactivity. Unfortunately very little is known regarding regulating of endothelin release. Our preliminary data suggests that the peptide is synthesized progressively over 4-6 hours by cells in culture by a mechanism dependent on protein synthesis. The amount of peptide formed can be regulated by a number of factors. For example we have found the phorbol myristate acetate not only increases endothelin production but potentiates the action of thrombin as an agonist. The vasodilator peptide, bradykinin, and A23187 were found to potently inhibit release. Nothing is known regarding the second messenger systems and mechanisms involved in these effects. The goal of the present proposal is to investigate mechanisms regulating endothelin release from endothelial cells. We will study the response of cells in culture to a variety of vasoactive peptides both vasoconstrictors (angiotensin U, vasopressin, PAF) and vasodilators (bradykinin, atrial natriuretic peptide), investigate the response of cells to vasoactive mediators (thrombin, phorbol esters) and drugs (nitroprusside, A23187, forskolin) and examine the response of cells to environmental stimuli which are known to produce changes in vascular tone (hypoxia, hyperoxia).
