NITRIC-OXIDE EXPOSURE INHIBITS ENDOTHELIAL NOS ACTIVITY BUT NOT GENE-EXPRESSION - A ROLE FOR SUPEROXIDE

Recent studies have characterized a rebound pulmonary vasoconstriction with abrupt withdrawal of inhaled nitric oxide (NO) during therapy fo r pulmonary hypertension, suggesting that inhaled NO may downregulate basal NO production. However, the exact mechanism of this rebound pulm onary hypertension remains unclear. The objectives of these studies we re to determine the effect of NO exposure on endothelial NO synthase ( eNOS) gene expression, enzyme activity, and posttranslational modifica tion in cultured pulmonary arterial endothelial cells. Sodium nitropru sside (SNP) treatment had no effect on eNOS mRNA or protein levels but did produce a significant decrease in enzyme activity. Furthermore, a lthough SNP treatment induced protein kinase C (PKC)-dependent eNOS ph osphorylation, blockade of PKC activity did not protect against the ef fects of SNP. When the xanthine oxidase inhibitor allopurinol or the s uperoxide scavenger 4,5-dihydroxy-1-benzene-disulfonic acid were co-in cubated with SNP, the inhibitory effects on eNOS activity could be par tially alleviated. Also, the levels of superoxide were found to be ele vated 4.5-fold when cultured pulmonary arterial endothelial cells were exposed to the NO donor spermine/NO This suggests that NO can stimula te xanthine oxidase to cause an increase in cellular superoxide genera tion. A reaction between NO and superoxide would produce peroxynitrite , which could then react with the eNOS protein, resulting in enzyme in activation. This mechanism may explain, at least in part, how NO produ ces NOS inhibition in vivo and may delineate, in part, the mechanism o f rebound pulmonary hypertension after withdrawal of inhaled NO.