Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase

OBJECTIVE: To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). METHODS & RESULTS: A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH(4)). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH(4)-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH(4), dihydrobiopterin (BH(2)) as well as BH(4): BH(2) ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH(4): eNOS molar ratio in dexamethasone-treated cells. Because the BH(4)-eNOS stoichiometry rather than the absolute BH(4) amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. CONCLUSIONS: Although dexamethasone reduces BH(4) levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.