Addition of spironolactone to angiotensin-converting enzyme inhibition in heart failure improves endothelial vasomotor dysfunction: role of vascular superoxide anion formation and endothelial nitric oxide synthase expression.

OBJECTIVES: We sought to investigate the effects of adding spironolactone (SP) to angiotensin-converting enzyme (ACE) inhibition on endothelium-dependent vasodilation in rats with chronic heart failure (CHF). BACKGROUND: Adding SP to ACE inhibitors reduces mortality and morbidity in CHF. Endothelial vasomotor dysfunction contributes to increased peripheral vascular resistance and reduced myocardial perfusion in CHF. METHODS: Seven days after extensive myocardial infarction (CHF) or sham operation, Wistar rats were treated with placebo, the ACE inhibitor trandolapril (TR, 0.3 mg/kg body weight per day), SP (10 mg/kg per day) or a combination of both for 11 weeks. RESULTS: Maximal acetylcholine-induced, nitric oxide (NO)-dependent relaxation was significantly attenuated in aortic rings from rats with CHF as compared with sham-operated animals (R(max) 44 +/- 3% vs. 63 +/- 3%). Spironolactone alone had no influence (46 +/- 5%) and TR improved NO-mediated relaxation (55 +/- 4%), whereas treatment with both completely restored endothelium-dependent vasorelaxation (64 +/- 4%). Aortic superoxide formation was significantly increased in rats with CHF as compared with sham-operated animals, but was normalized by treatment with SP or SP plus TR. In addition, aortic messenger ribonucleic acid expression of the oxidase subunit p22(phox) in rats with CHF was significantly reduced by SP or TR plus SP. Endothelial NO synthase expression was increased in TR-treated animals. Incubation of isolated porcine coronary arteries with SP dose-dependently attenuated superoxide formation. CONCLUSIONS: Spironolactone added to an ACE inhibitor normalizes NO-mediated relaxation in experimental CHF by beneficially modulating the balance of NO and superoxide anion formation.     

Vascular endothelial dysfunction and superoxide anion production in heart failure are p38 MAP kinase-dependent

OBJECTIVE: The mitogen-activated protein (MAP) kinase system, especially the p38 MAP kinase, is activated in chronic heart failure (CHF). However, the role of vascular p38 MAP kinase in CHF has not been analyzed yet. METHODS AND RESULTS: In aortic rings from rats with CHF 10 weeks after myocardial infarction, acetylcholine-induced relaxation was attenuated (maximum relaxation, Rmax: 54+/-5%) compared to sham-operated animals (Rmax: 77+/-5%, p<0.01), while endothelium-independent relaxation elicited by sodium nitroprusside was not significantly changed. Aortic levels of phosphorylated p38 MAP kinase protein were significantly elevated in rats with CHF. In addition, phosphorylation of MAP kinase-activated protein kinase-2 (MAPKAPK-2), an index of p38 MAP kinase activity, was increased. Aortic superoxide anion generation was significantly enhanced in rats with CHF accompanied by elevation of the NAD(P)H oxidase subunit p47phox protein expression. Inhibition of p38 MAP kinase by treatment with the p38 MAP kinase inhibitor SB239063 (800 ppm in standard rat chow) reduced MAPKAPK-2 phosphorylation, preserved acetylcholine-induced relaxation (Rmax: 80+/-4%, p<0.01), and reduced vascular superoxide formation. SB239063 treatment did not affect blood pressure and left ventricular enddiastolic pressure. In aortic tissue from CHF animals treated with the angiotensin-converting enzyme (ACE) inhibitor trandolapril, p38 MAP kinase phosphorylation was significantly reduced. CONCLUSIONS: Vascular p38 MAP kinase is markedly activated in rats with CHF. Chronic p38 MAP kinase inhibition with SB239063 prevented endothelial vasomotor dysfunction through reduction of superoxide anion production.     

Regulation of vascular guanylyl cyclase by endothelial nitric oxide-dependent posttranslational modification

In isolated cells, soluble guanylyl cyclase (sGC) activity is regulated by exogenous nitric oxide (NO) via downregulation of expression and posttranslational S-nitrosylation. The aim of this study was to investigate whether such regulatory mechanism impact on endothelium-dependent vasodilation in a newly developed mouse strain carrying an endothelial-specific overexpression of eNOS (eNOS⁺⁺). When compared with transgene negative controls (eNOSⁿ), eNOS⁺⁺-mice showed a 3.3-fold higher endothelial-specific aortic eNOS expression, increased vascular cGMP and VASP phosphorylation, a L-nitroarginine (L-NA)-inhibitable decrease in systolic blood pressure, but normal levels of peroxynitrite and nitrotyrosine formation, endothelium-dependent aortic vasodilation and vasodilation to NO donors. Western blot analysis for sGC showed similar protein levels of sGC-α1 and sGC-β1 subunits in eNOSⁿ and eNOS⁺⁺. In striking contrast, the activity of isolated sGC was strongly decreased in lungs of eNOS⁺⁺. Semiquantitative evaluation of sGC-β1-S-nitrosylation demonstrated that this loss of sGC activity is associated with increased nitrosylation of the enzyme in eNOS⁺⁺, a difference that disappeared after L-NA-treatment. Our data suggest the existence of a physiologic NO-dependent posttranslational regulation of vascular sGC in mammals involving S-nitrosylation as a key mechanism. Because this mechanism can compensate for reduction in vascular NO bioavailability, it may mask the development of endothelial dysfunction.     

Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation

Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester-stimulated superoxide anion (O(2)(-)) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 micromol/L). The HMG-CoA reductase product mevalonic acid (400 micromol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester-stimulated O(2)(-) formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced approximately 2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O(2)(-) formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O(2)(-) in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol.     

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.     

Glucocorticoid excess induces superoxide production in vascular endothelial cells and elicits vascular endothelial dysfunction

Glucocorticoid (GC) excess often elicits serious adverse effects on the vascular system, such as hypertension and atherosclerosis, and effective prophylaxis for these complications is limited. We sought to reveal the mechanism underlying GC-induced vascular complications. Responses in forearm blood flow to reactive hyperemia in 20 GC-treated patients were significantly decreased to 43+/-8.9% (mean+/-SEM) from the values obtained before GC therapy (130+/-14%). An administration of vitamin C almost normalized blood flow responses. In human umbilical vein endothelial cells (HUVECs), production of hydrogen peroxide was increased up to 166.5+/-3.3% of control values by 10(-7) mol/L dexamethasone (DEX) treatment (P<0.01). Concomitant with DEX-induced hydrogen peroxide production, intracellular amounts of peroxynitrite significantly increased and those of nitric oxide (NO) decreased, respectively (P<0.01). Immunoblotting analysis using anti-nitrotyrosine antibody showed that peroxynitrite formation was increased in DEX-treated HUVECs. Using inhibitors against metabolic pathways for generation of reactive oxygen species (ROS), we identified that the major production sources of ROS by DEX treatment were mitochondrial electron transport chain, NAD(P)H oxidase, and xanthine oxidase. These findings suggest that GC excess causes overproduction of ROS and thereby perturbs NO availability in the vascular endothelium, leading to vascular complications in patients with GC excess.     

Secondary endothelial dysfunction: hypertension and heart failure

The endothelium is a major regulator of vascular tone, releasing vasoactive substances such as endothelium-derived nitric oxide (EDRF), endothelium-derived hyperpolarizing factor(s), cycloxygenase metabolites, endothelin and other endothelium-derived contracting factors (EDCF). In a number of cardiovascular pathologies, such as hypertension or heart failure, the balance in the endothelial production of vasodilating and vasoconstricting mediators is altered. The resulting apparent decrease in endothelium-dependent relaxations is termed 'endothelial dysfunction'. In hypertensive patients and in animal models of hypertension, endothelium-dependent relaxations are impaired. However, this endothelial dysfunction presents different characteristics depending on the model studied. In Dahl-salt-sensitive rats, the decrease in endothelium-dependent relaxations is associated with impaired constitutive nitric oxide synthase activity. The presence of an endogenous nitric oxide synthase inhibitor and a decreased response of vascular smooth muscle to the mediator may contribute also to the dysfunction observed in this model. In other animal models of hypertension (such as spontaneous hypertension). the contribution of the L-arginine nitric oxide pathway to endothelium-dependent responses appears normal or impaired despite reports of increased nitric oxide synthase activity or expression. In large arteries from SHR, endothelium-dependent relaxations are impaired mainly because of the concomitant augmented release of endoperoxides activating thromboxane-endoperoxide receptors. Superoxide anions may also play a role in some models, but only in the early phase of the disease: whether or not these species contribute to further development of endothelial dysfunction or to increases in blood pressure remains to be examined. The endothelial dysfunction observed in hypertension is likely to be a consequence of high blood pressure. but it could facilitate the maintenance of elevated peripheral resistance at a later stage in the disease and favour the occurrence of complications, such as atherosclerosis.     

内皮功能障碍在心力衰竭中的作用

在心力衰竭的病理生理学中,内皮功能障碍的作用尚不完全清楚。氧化应激引起的内皮功能障碍会诱发心力衰竭。内皮功能和一氧化氮（NO）-环磷酸鸟苷(cGMP)途径参与心力衰竭的病理生理过程。内皮依赖性的血管舒张改变引起反复的缺血/再灌注,可诱发心肌的收缩与舒张功能障碍,NO-cGMP通路的改变直接影响心肌稳态。内皮功能障碍与心力衰竭更差的预后密切相关,与心血管事件更高的发生率密切相关。心力衰竭患者NO-cGMP途径提供了潜在的治疗策略基础,尽管目前相关临床数据尚无明确定论,但NO-cGMP途径是一个有希望的潜在治疗靶标。本文将对内皮功能障碍的病理生理机制、在心衰发生发展中的作用以及在心衰治疗的潜在价值进行综述。     

Oxidative stress and endothelial dysfunction in heart failure

The clinical syndrome of congestive heart failure (CHF) is characterized by abnormalities of left ventricular function and neurohormonal regulation, which are accompanied by effort intolerance, fluid retention, and decreased longevity. While an increased sympathetic tone and an activated renin-angiotensin system may contribute to the reduced vasodilatory capacity in patients with CHF, the important role of the endothelium in coordinating tissue perfusion has now been recognized. CHF is associated with endothelial dysfunction, as demonstrated by impaired endothelium-mediated vasodilation. Endothelial dysfunction in patients with CHF is a critical component in the systemic vasoconstriction and reduced peripheral perfusion that characterizes these patients. Endothelial regulation of vascular tone is mediated mainly by nitric oxide. Increased oxidative stress in patients with CHF is likely caused by decreased bioavailability of nitric oxide due to reduced expression of endothelial nitric oxide synthase and increased generation of reactive oxygen species. These react with nitric oxide in the setting of decreased antioxidant defenses that would normally clear these radicals, culminating in attenuated endothelium-dependent vasodilation in patients with CHF. Therapies that improve endothelial function have been shown to improve exercise tolerance and outcomes in patients with CHF. Endothelial dysfunction is thus an important target for future therapy in patients with CHF.     

Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats

Nebivolol is a beta(1)-receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of angiotensin II-induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin-NO concentrations. Treatment with the beta-receptor blocker nebivolol but not metoprolol (10 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH oxidase, as well as membrane association of NADPH oxidase subunits (Rac1 and p67(phox)). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67(phox) and Rac1, as well as an inhibition of NADPH oxidase activity assessed in heart membranes from angiotensin II-infused animals, as well as in homogenates of Nox1 and cytosolic subunit-transfected and phorbol ester-stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH oxidase. Thus, inhibitory effects of this beta-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II-induced hypertension.     

Functional role of the soluble guanylyl cyclase alpha(1) subunit in vascular smooth muscle relaxation

OBJECTIVE: Soluble guanylyl cyclase (sGC), the predominant receptor for nitric oxide (NO), exists in 2 active isoforms (alpha(2)beta(1) and alpha(1)beta(1)). In vascular tissue sGCalpha(1)beta(1) is believed to be the most important. The aim of our study was to investigate the functional importance of the sGCalpha(1)-subunit in vasorelaxation. METHODS: Aortic and femoral artery segments from male and/or female sGCalpha(1)(-/-) mice and wild-type littermates were mounted in a small-vessel myograph for isometric tension recording. This was supplemented with biochemical measurements of the cGMP concentration and sGC enzyme activity. RESULTS: The functional importance of sGCalpha(1)beta(1) was demonstrated by the significantly decreased relaxing effects of acetylcholine (ACh), sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), NO gas, YC-1, BAY 41-2272 and T-1032 in the sGCalpha(1)(-/-) mice of both genders. Moreover, the basal and SNP-stimulated cGMP levels and basal sGC activity were significantly lower in the sGCalpha(1)(-/-) mice. However, the relaxing effects of NO, BAY 41-2272 and YC-1 seen in blood vessels from sGCalpha(1)(-/-) mice indicate a role for an sGCalpha(1)beta(1)-independent mechanism. The increase in sGC activity after addition of BAY 41-2272 and the inhibition of the ACh-, SNP-, SNAP- and NO gas-induced response by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the sGCalpha(1)(-/-) mice are observations suggesting that the sGCalpha(2)beta(1) isoform is also functionally active. However, the insignificant increase in cGMP in response to SNP and the non-upregulated sGCalpha(2) expression level in the sGCalpha(1)(-/-) mice suggest rather the involvement of (an) sGC-independent mechanism(s). CONCLUSIONS: We conclude that sGCalpha(1)beta(1) is involved in the vasorelaxation induced by NO-dependent and NO-independent sGC activators in both genders. However, the remaining relaxation seen in the sGCalpha(1)(-/-) mice suggests that besides sGCalpha(1)beta(1) also the minor isoform sGCalpha(2)beta(1) and/or (an) sGC-independent mechanism(s) play(s) a substantial role.     

Vascular consequences of endothelial nitric oxide synthase uncoupling for the activity and expression of the soluble guanylyl cyclase and the cGMP-dependent protein kinase

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolemia, hypertension, diabetes mellitus, chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species (ROS), such as the superoxide radical, and the subsequent decrease in vascular bioavailability of nitric oxide (NO). Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include the NAD(P)H oxidase, the xanthine oxidase, and mitochondrial superoxide-producing enzymes. Superoxide produced by the NADPH oxidase may react with NO released by endothelial nitric oxide synthase (eNOS), thereby generating peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, thereby switching an antiatherosclerotic NO-producing enzyme to an enzyme that may initiate or even accelerate the atherosclerotic process by producing superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and has also been demonstrated to occur within the smooth muscle cell layer in the setting of hypercholesterolemia, diabetes mellitus, hypertension, congestive heart failure, and nitrate tolerance. Increased superoxide production by the endothelial and/or smooth muscle cells has important consequences with respect to signaling by the soluble guanylyl cyclase (sGC) and the cGMP-dependent protein kinase I (cGK-I), the activity and expression of which has been shown to be regulated in a redox-sensitive fashion. The present review summarizes current concepts concerning eNOS uncoupling and also focuses on the consequences for downstream signaling with respect to activity and expression of the sGC and cGK-I in various diseases.     

Effect of vitamin E on resistance vessel endothelial dysfunction induced by methionine

We tested if vitamin E, a fat-soluble antioxidant, prevents resistance vessel endothelial dysfunction caused by methionine-induced hyperhomocysteinemia in humans. Moderate elevations in plasma homocysteine concentrations are associated with atherosclerosis and hypertension. Homocysteine causes endothelial dysfunction possibly through several mechanisms. No previous study has tested if a fat-soluble antioxidant can prevent endothelial dysfunction caused by experimental hyperhomocysteinemia. Ten healthy subjects participated in a 2 x 2 factorial, double-blind crossover study, receiving L-methionine (100 mg/kg at -6 hours) or vehicle, with and without vitamin E (1,200 IU at -13 hours). Endothelial function of forearm resistance vessels was assessed using forearm blood flow responses to brachial artery administration of endothelium-dependent and endothelium-independent agents. Forearm resistance vessel dilatation to acetylcholine was significantly impaired 7 hours after methionine (placebo, 583 +/- 87% vs methionine 30 +/- 68%; p <0.05). Dilatation to bradykinin was also impaired (placebo, 509 +/- 54% vs methionine 289 +/- 48%; p <0.05). Methionine did not alter vasodilatation to the endothelium-independent vasodilators, nitroprusside, and verapamil. Methionine-induced impairment of resistance vessel dilatation to acetylcholine and bradykinin (p <0.05 vs placebo) was prevented by administration of vitamin E (acetylcholine, p = 0.004; bradykinin, p = 0.004; both vs methionine alone). Experimentally increasing plasma homocysteine concentrations by oral methionine rapidly impairs resistance vessel endothelial function in healthy humans and this effect is reversed with administration of the fat-soluble antioxidant, vitamin E.     

Effects of vitamin E on chronic and acute endothelial dysfunction in smokers

OBJECTIVES: The aims of this study were to determine whether chronic or acute impairment of flow mediated vasodilation (FMD) in the brachial artery of smokers can be restored or preserved by the antioxidant vitamin E. BACKGROUND: Transient impairment of endothelial function after heavy cigarette smoking and chronic endothelial dysfunction in smokers result at least in part from increased oxidative stress. METHODS: We studied 22 healthy male smokers (mean +/- SD, 23 +/- 9 cigarettes per day) randomly assigned to receive either 600 IU vitamin E per day (n = 11, age 28 +/- 6 years) or placebo (n = 11, age 27 +/- 6 years) for four weeks and 11 age-matched healthy male nonsmokers. Flow mediated vasodilation and endothelium-independent, nitroglycerin-induced dilation were assessed in the brachial artery using high resolution ultrasound (7.5 MHz) at baseline and after therapy. Subjects stopped smoking 2 h before the ultrasound examinations. At the end of the treatment period, a third scan was obtained 20 min after smoking a cigarette (0.6 mg nicotine, 7 mg tar) to estimate transient impairment of FMD. RESULTS: Flow mediated vasodilation at baseline was abnormal in the vitamin E (5.3 +/- 3.8, p < 0.01) and in the placebo group (6.4 +/- 3.5, p < 0.05) compared with nonsmoking controls (11.6 +/- 4.7). Using a two-way repeated measures analysis of variance (ANOVA) to examine the effects of vitamin E on FMD, we found no effect for the grouping factor (p = 0.5834) in the ANOVA over time but a highly significant difference with respect to time (p = 0.0065). The interaction of the time factor and the grouping factor also proved to be significant (p = 0.0318). Flow mediated vasodilation values remained similar after treatment for four weeks in both groups but declined faster after smoking a cigarette in subjects taking placebo compared with those receiving vitamin E (p values from successive differences for the time/group factor: 0.0001/0.0017). The transient attenuation of FMD (calculated as the percent change in FMD) was related to the improvement of the antioxidant status, estimated as percent changes in thiobarbituric acid-reactive substances (r = -0.67, p = 0.0024). Nitroglycerin-induced dilation did not differ between study groups at baseline or after therapy. CONCLUSIONS: These results demonstrate that oral supplementation of vitamin E can attenuate transient impairment of endothelial function after heavy smoking due to an improvement of the oxidative status but cannot restore chronic endothelial dysfunction within four weeks in healthy male smokers.     

cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase.

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (IC50 = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 approximately 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.