The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium͞calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium͞calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, N G -hydroxy-L-arginine, L-thiocitrulline, and L-N G -monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH 4 ) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH 4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of f lavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH 4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Recently, we have shown that chronic exercise increases endothelium-derived relaxing factor (EDRF)/nitric oxide (NO)-mediated epicardial coronary artery dilation in response to brief occlusion and acetylcholine. This finding suggests that exercise can provide a stimulus for the enhanced production of EDRF/NO, thus possibly contributing to the beneficial effects of exercise on the cardiovascular system. Therefore, the purpose of the present study was to examine whether chronic exercise could influence the production of NO (measured as the stable degradation product, nitrite) and endothelial cell NO synthase (ECNOS) gene expression in vessels from dogs after chronic exercise. To this end, dogs were exercised by running on a treadmill (9.5 km/h for 1 hour, twice daily) for 10 days, and nitrite production in large coronary vessels and microvessels and ECNOS gene expression in aortic endothelial extracts were assessed. Acetylcholine (10`to mol/L) dose-dependently increased the release of nitrite (inhibited by nitro-L-arginine) from coronary arteries and microvessels in control and exercised dogs. Moreover, acetylcholine-stimulated nitrite production was markedly enhanced in large coronary arteries and microvessels prepared from hearts E a ndothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO)12 or a closely related molecule,3 is released by the endothelium in response to local hormones, changes in blood flow velocity, or endothelial shear stress.4 However, the physiologically relevant stimulus for EDRF/NO release in vivo is not known and may reflect the summation of local hormones and blood flow. Evidence supporting the contribution of basally released EDRF/NO to the regulation of vascular tone is derived from experiments showing that specific inhibitors of NO synthase (NOS) elicit a prolonged pressor response and reduce regional blood flows in vivo and inhibit endothelium-dependent relaxations in vitro.5

The balance of nitric oxide (⅐NO) and superoxide anion (O 2 . ) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ⅐NO. As eNOS is capable of generating both ⅐NO and O 2 . , we hypothesized that Hsp90 might also mediate eNOS-dependent O 2 . production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 g/ml; 17.8 M) and then stimulated with the calcium ionophore, A23187 (5 M). GA significantly decreased A23187-stimulated eNOS-dependent nitrite production (p < 0.001, n ‫؍‬ 4) and significantly increased A23187-stimulated eNOS-dependent O 2 . production (p < 0.001, n ‫؍‬ 8). A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ⅐NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O 2 . production.Nitric oxide (⅐NO) and superoxide anion (O 2 . ) play opposing roles in vascular biology. Nitric oxide generation is increased greatly when Hsp90 associates with eNOS 1 in endothelial cell cultures (1, 2). A decrease in the amount of Hsp90 co-precipitating with eNOS is associated with a decrease in ⅐NO production by pulmonary artery endothelial cells exposed to prolonged periods of hypoxia (3). Geldanamycin (GA) is an ansamycin antibiotic that binds to the ATP binding site of Hsp90, thereby inhibiting the ATP/ADP cycle required for the interaction with client proteins such as eNOS (2-4). GA has been used to demonstrate that ⅐NO production in mesentary arteries and rat aortas depends on Hsp90 activity, implying that factors adversely affecting this interaction between Hsp90 and eNOS may be one of the mechanisms for portal hypertension and increased vascular tone (2, 4). Taken together, these reports indicate that Hsp90 is critical for eNOS generation of ⅐NO.Emerging evidence suggests that under pathological conditions eNOS may also generate O 2. (5-

To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS)