Fig. 1.
S-nitrosoglutathione (GSNO) reductase (GSNOR) inhibition, localization, and activity. A: N6338 caused dose-dependent and potent inhibition of human GSNOR activity in vitro. Graph represents one experiment using duplicate samples at each dose tested. mAU, milliabsorbance units. B: N6338 increased S-nitrosothiol (SNO) levels in mouse embryonic fibroblasts. Mean SNO levels ± SE are shown (n = 12 independent wells per group; P < 0.0001, two-tailed t-test). C–G: GSNOR protein was evident in human and rat cardiovascular tissue by immunohistochemistry: IgG negative control (C) or human-specific GSNOR antibody staining predominantly in the smooth muscle of a myocardial arteriole or small artery in human heart (D), and predominantly in the endothelium of a human myocardial venule (E); IgG-negative control (F) or rat-specific GSNOR antibody staining predominantly in the smooth muscle of an arteriole in rat heart (G). In all micrographs, scale bars = 50 μm, asterisks denote vessel lumens, blue arrows show smooth muscle, and red arrows show endothelium. H: GSNOR activity was present in rat heart and aorta and was inhibited by N6338 when this compound was added in vitro. Activity was measured in the absence (open bars) or presence of 1 μM (shaded bars) or 10 μM (solid bars) N6338. Bars are means ± SD of tissues from 4 rats. I: N6338 caused dose-dependent relaxation in rat aortic rings precontracted with phenylephrine. This relaxation by N6338 was significantly attenuated by pretreatment of rings with 10 μM 1H-[1,2,4]oxadiazolo [4,3-α]quinoxalin-1-one (ODQ), 100 μM NG-monomethyl-l-arginine (l-NMMA), or 300 μM 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy PTIO) for 30 min before N6338 administration. Values are means ± SE of 4 rings/group.