Endothelial Nitric Oxide Synthase–Derived Nitric Oxide Prevents Dihydrofolate Reductase Degradation via Promoting S-Nitrosylation

Objective—Dihydrofolate reductase (DHFR) is a key protein involved in tetrahydrobiopterin (BH4) regeneration from 7,8-dihydrobiopterin (BH2). Dysfunctional DHFR may induce endothelial nitric oxide (NO) synthase (eNOS) uncoupling resulting in enzyme production of superoxide anions instead of NO. The mechanism by which DHFR is regulated is unknown. Here, we investigate whether eNOS-derived NO maintains DHFR stability. Approach and Results—DHFR activity, BH4 content, eNOS activity, and S-nitrosylation were assessed in human umbilical vein endothelial cells and in aortas isolated from wild-type and eNOS knockout mice. In human umbilical vein endothelial cells, depletion of intracellular NO by transfection with eNOS-specific siRNA or by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)—both of which had no effect on DHFR mRNA levels—markedly reduced DHFR protein levels in parallel with increased DHFR polyubiquitination. Supplementation of S-nitroso-l-glutathione (GSNO), ...