title: Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase.
abstract: OBJECTIVE
  To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS).
  
  
  RESULTS
  A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 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 BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 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 BH4 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.
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Tobias
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Alice
authors.initials: A
authors.lastname: Habermeier
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Siuda
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Gisela
authors.initials: G
authors.lastname: Reifenberg
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Ning
authors.initials: N
authors.lastname: Xia
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Ellen I
authors.initials: EI
authors.lastname: Closs
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Förstermann
authors.affiliation: Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany.
authors.forename: Huige
authors.initials: H
authors.lastname: Li
keywords: Dexamethasone; Endothelial cells; Nitric oxide synthase; Reactive oxygen species; Tetrahydrobiopterin; eNOS uncoupling