ROLE OF CYCLIC-NUCLEOTIDES IN THE REGULATION OF ENDOTHELIN-1 PRODUCTION BY HUMAN ENDOTHELIAL-CELLS

The regulation of endothelin-1 (ET-1) production by endothelial cells is likely of crucial physiological importance in the maintenance of vascular homeostasis. The aim of the present study was to explore the possible role of cyclic nucleotides in the control of ET-1 production in human umbilical vein endothelial cells (HUVEC). ET-1 release was determined by measuring levels of immunoreactive ET-1 in HUVEC-conditioned media after 6-h incubations. In the presence of 10% fetal calf serum (FCS) there was a threefold increase in ET-1 release compared with serum-free conditions (1.96 +/- 0.17 vs. 0.56 +/- 0.06 pg/mu g protein), respectively. Inhibition of protein kinase (PK) C using staurosporine (10 nM) reduced basal ET-1 release by similar to 50% and completely prevented the response to FCS. In contrast, the addition of other PK inhibitors had little effect on basal or serum- stimulated ET-1 release at the concentrations used. N-6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) produced significant alterations in ET-1 release depending on the basal level of production. Under serum-free conditions of low basal ET-1 production, DBcAMP increased ET-1 release by 68 +/- 22% but only at the highest concentration studied (1 mM). The dose-response relationship for DBcAMP was potentiated by KT-5720 (0.1 mu M), an inhibitor of PKA, with a significant shift to 10-fold lower concentrations, whereas it was blocked by KT-5823 (4 mu M), which can inhibit PKG. In contrast, in serum-stimulated conditions of high ET-1 production, a concentration-dependent inhibition of ET-1 release in response to DBcAMP was observed, which was amplified by KT-5823 and blocked by KT-5720. The relatively nonselective inhibitor of cyclic nucleotide-dependent protein kinases, H-8 (5 mu M), prevented both stimulation and inhibition of ET-1 production by DBcAMP. A similar pattern of response to DBcAMP and the PK inhibitors was observed in the presence of thrombin (1 U/ml), which produced an intermediate level of stimulation of ET-1 release. In contrast, 8-bromoguanosine 3',5'-cyclic monophosphate had no effect on ET-1 release under any of the conditions studied. DBcAMP also suppressed bradykinin-stimulated [H-3]diacylglycerol generation in these cells by a PKA-dependent mechanism, pointing to the presence of inhibitory cross talk between the adenylate cyclase and phospholipase C pathways. Finally, the expression of prepro-ET-1 mRNA in serum-free conditions was selectively enhanced by DBcAMP, an action potentiated by inhibition of PKA. Therefore, these results suggest that inhibition of stimulated ET-1 secretion may result from cross talk between the adenylate cyclase and phospholipase C pathways, whereas the cAMP-induced increase in production by nonstimulated cells may result from the direct increase in the expression of prepro-ET-1 mRNA.