TUMOR-NECROSIS-FACTOR-ALPHA DECREASES PULMONARY-ARTERY ENDOTHELIAL NITROVASODILATOR VIA PROTEIN-KINASE-C

We postulated that tumor necrosis factor-alpha (TNF) decreases endothe lium-derived nitrovasodilator(s) via a protein kinase C (PKC)-dependen t pathway. Calf pulmonary artery endothelial monolayers (PAEM) were tr eated with TNF (10, 100, and 1,000 U/ml) for 15 min or 18 h during an 18-h incubation. At the end of the incubation, the cell lysate and sup ernatant were harvested. Compared with controls, an 18-h incubation wi th TNF (100 and 1,000 U/ml) resulted in a decrease in NO2- [the oxidat ion product of nitric oxide (NO)] in PAEM lysate and supernatant. TNF (100 U/ml) treatment for 15 min did not suppress NO2- levels. The decr ease in NO2- and the increase in lipid peroxides in response to TNF we re prevented by pretreatment (15 min prior to and throughout the incub ation) with either calphostin C (1 mu M; a specific PKC inhibitor) or the antioxidants N-acetylcysteine (1 mM), 4,5-dihydroxy-1,3-benzene di sulfonic acid (Tiron) (10 mM), and superoxide dismutase (10 U/ml). Tre atment with phorbol 12-myristate 13-acetate (PMA, 1 mu M for 15 min), an activator of PKC, decreased NO2- similarly to TNF. Pretreatment wit h calphostin C or N-acetylcysteine prior to TNF (10 U/ml) revealed an increase in NO2- levels above control treatment. Treatment with the NO synthase antagonists N-G-monomethyl-L-arginine (1 mM) and N-nitroso-L -arginine (1 mM) induced an L-arginine (1 mM)-dependent decrease in NO 2- in control but not in TNF-treated PAEM. The induction of NO2- by ca lcium ionophore (A23187; 500 nM) was not affected by treatment with TN F. The data suggest that tumor necrosis factor-alpha decreases pulmona ry artery endothelial nitrovasodilator by the PKC-dependent generation of reactive oxidant species.