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 va scular homeostasis. The aim of the present study was to explore the po ssible role of cyclic nucleotides in the control of ET-1 production in human umbilical vein endothelial cells (HUVEC). ET-1 release was dete rmined 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 ser um-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 pr evented the response to FCS. In contrast, the addition of other PK inh ibitors had little effect on basal or serum- stimulated ET-1 release a t the concentrations used. N-6,2'-O-dibutyryladenosine 3',5'-cyclic mo nophosphate (DBcAMP) produced significant alterations in ET-1 release depending on the basal level of production. Under serum-free condition s 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-r esponse relationship for DBcAMP was potentiated by KT-5720 (0.1 mu M), an inhibitor of PKA, with a significant shift to 10-fold lower concen trations, whereas it was blocked by KT-5823 (4 mu M), which can inhibi t PKG. In contrast, in serum-stimulated conditions of high ET-1 produc tion, 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-d ependent protein kinases, H-8 (5 mu M), prevented both stimulation and inhibition of ET-1 production by DBcAMP. A similar pattern of respons e to DBcAMP and the PK inhibitors was observed in the presence of thro mbin (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. DB cAMP also suppressed bradykinin-stimulated [H-3]diacylglycerol generat ion in these cells by a PKA-dependent mechanism, pointing to the prese nce of inhibitory cross talk between the adenylate cyclase and phospho lipase C pathways. Finally, the expression of prepro-ET-1 mRNA in seru m-free conditions was selectively enhanced by DBcAMP, an action potent iated by inhibition of PKA. Therefore, these results suggest that inhi bition 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.