NO from smooth muscle cells decreases NOS expression in endothelial cells:role of TNF-alpha

Despite the evidence that cytokines stimulate nitric oxide (NO) production by inducible nitric oxide synthase (iNOS), several reports recently demonst rated that the hypotensive response related to endothelial nitric oxide syn thase (eNOS) activity could be inhibited by the same cytokines. The aim of the present work was to analyze whether NO generated by vascular smooth mus cle cells (VSMC) could modify eNOS protein expression in endothelial cells. Bovine aortic endothelial cells (BAEC) and bovine VSMC (BVSMC) in cocultur e were used for the study. Interleukin-1 beta (IL-1 beta, 10 ng/ml)-treated BVSMC, which expressed iNOS protein, decreased eNOS protein expression in BAEC. The presence of NO antagonists N-omega-nitro-L-arginine methyl ester (10(-3) mol/l) or N-G-monomethyl-L-arginine (10(-3) mol/l) prevented the de crease in eNOS protein expression induced by IL-1 beta-treated BVSMC. Surpr isingly, two different NO donors, 3-morpholinosydnonimine (10(-4) mol/l) an d S-nitroso-N-acetyl-D,L-pencillamine (10(-4) mol/l), failed to modify eNOS expression in BAEC, suggesting the existence of a diffusible mediator rele ased from IL-1 beta-treated BVSMC that acts on endothelial cells by reducin g eNOS expression. The presence of NO antagonists reduced tumor necrosis fa ctor-alpha (TNF-alpha) production by IL-1 beta-stimulated BVSMC. This effec t was also produced in the presence of a protein kinase G inhibitor, guanos ine-5'-O-(2-thiodiphosphate) trilithium salt. A polyclonal antibody against TNF-alpha prevented eNOS expression in the BAEC-BVSMC coculture. In conclu sion, NO by itself failed to modify eNOS protein expression in endothelial cells but increased TNF-alpha generation by IL-1 beta-stimulated BVSMC and, in this way, reduced eNOS expression in the endothelium.