Contrasting effects of thiol-modulating agents on endothelial NO bioactivity

The bioactivity of endothelium-derived nitric oxide (NO) is an important co mponent of vascular homeostasis that is sensitive to intracellular redox st atus. Because glutathione (GSH) is a major determinant of intracellular red ox state, we sought to define its role in modulating endothelial NO bioacti vity. In porcine aortic endothelial cells (PAECs), we depleted intracellula r GSH (>70%) using 1) buthionine-(S, R)-sulfoximine (BSO), which inhibits G SH synthesis; 2) diamide, which oxidizes thiols; or 3) 1-chloro-2,4-dinitro benzene (CDNB), which putatively depletes GSH through glutathione S-transfe rase activity. Cellular GSH depletion with BSO had no effect on endothelial NO bioactivity measured as A-23187-induced cGMP accumulation. In contrast, oxidation of intracellular thiols with diamide inhibited both A-23187-indu ced cGMP accumulation and the cGMP response to exogenous NO. Diamide treatm ent of either PAECs, PAEC membrane fractions, or purified endothelial nitri c oxide synthase (eNOS) resulted in significant inhibition (similar to 75%) of eNOS catalytic activity measured as L-[H-3] arginine- to-L-[H-3] citrul line conversion. This effect appeared related to oxidation of eNOS thiols a s it was completely reversed by dithiothreitol. Glutathione depletion with CDNB inhibited A-23187-stimulated cGMP accumulation but not the cGMP respon se to exogenous NO. Rather, CDNB treatment impaired eNOS catalytic activity in intact PAECs, and this effect was reversed by excess NADPH in isolated purified eNOS assays. Consistent with these results, we found spectral evid ence that CDNB reacts with NADPH and renders it inactive as a cofactor for either eNOS or glutathione reductase. Thus thiol-modulating agents exert pl eiotropic effects on endothelial NO bioactivity, and these data may help to resolve a number of conflicting previous studies linking GSH status with e ndothelial cell NO bioactivity.