Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2(-). The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2(-) generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2(-)-dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, NO2(-) is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrate reduction: evaluation of its role in nitrite and nitric oxide generation in anoxic tissues

In addition to nitric oxide (NO) generation from specific NO synthases, NO is also formed during anoxia from nitrite reduction, and xanthine oxidase (XO) catalyzes this process. While in tissues and blood high nitrate levels are present, questions remain regarding whether nitrate is also a source of NO and if XO-mediated nitrate reduction can be an important source of NO in biological systems. To characterize the kinetics, magnitude, and mechanism of XO-mediated nitrate reduction under anaerobic conditions, EPR, chemiluminescence NO-analyzer, and NO-electrode studies were performed. Typical XO reducing substrates, xanthine, NADH, and 2,3-dihydroxybenz-aldehyde, triggered nitrate reduction to nitrite and NO. The rate of nitrite production followed Michaelis-Menten kinetics, while NO generation rates increased linearly following the accumulation of nitrite, suggesting stepwise-reduction of nitrate to nitrite then to NO. The molybdenum-binding XO inhibitor, oxypurinol, inhibited both nitrite and NO production, indicating that nitrate reduction occurs at the molybdenum site. At higher xanthine concentrations, partial inhibition was seen, suggesting formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. The pH dependence of nitrite and NO formation indicate that XO-mediated nitrate reduction occurs via an acid-catalyzed mechanism. With conditions occurring during ischemia, myocardial xanthine oxidoreductase and nitrate levels were determined to generate up to 20 microM nitrite within 10-20 min that can be further reduced to NO with rates comparable to those of maximally activated NOS. Thus, XOR catalyzed nitrate reduction to nitrite and NO occurs and can be an important source of NO production in ischemic tissues.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. Evaluation of its role in nitric oxide generation in anoxic tissues

Xanthine oxidase (XO)-catalyzed nitrite reduction with nitric oxide (NO) production has been reported to occur under anaerobic conditions, but questions remain regarding the magnitude, kinetics, and biological importance of this process. To characterize this mechanism and its quantitative importance in biological systems, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism. Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. The substrate dependence of anaerobic XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems. It was determined that under conditions occurring during no-flow ischemia, myocardial XO and nitrite levels are sufficient to generate NO levels comparable to those produced from nitric oxide synthase. Thus, XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions.     

Characterization of the effects of oxygen on xanthine oxidase-mediated nitric oxide formation

Under anaerobic conditions, xanthine oxidase (XO)-catalyzed nitrite reduction can be an important source of nitric oxide (NO). However, questions remain regarding whether significant XO-mediated NO generation also occurs under aerobic conditions. Therefore, electron paramagnetic resonance, chemiluminescence NO-analyzer, and NO-electrode studies were performed to characterize the kinetics and magnitude of XO-mediated nitrite reduction as a function of oxygen tension. With substrates xanthine or 2,3-dihydroxybenz-aldehyde that provide electrons to XO at the molybdenum site, the rate of NO production followed Michaelis-Menten kinetics, and oxygen functioned as a competitive inhibitor of nitrite reduction. However, with flavin-adenine dinucleotide site-binding substrate NADH as electron donor, aerobic NO production was maintained at more than 70% of anaerobic levels, and binding of NADH to the flavin-adenine dinucleotide site seemed to prevent oxygen binding. Therefore, under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in tissues. Studies of the pH dependence of NO formation indicated that lower pH values decrease oxygen reduction but greatly increase nitrite reduction, facilitating NO generation. Isotope tracer studies demonstrated that XO-mediated NO formation occurs in normoxic and hypoxic heart tissue. Thus, XO-mediated NO generation occurs under aerobic conditions and is regulated by oxygen tension, pH, nitrite, and reducing substrate concentrations.     

Reduction of organic nitrites to nitric oxide catalyzed by xanthine oxidase: possible role in metabolism of nitrovasodilators

Xanthine oxidase (XO) was shown to catalyze the reduction of isoamyl and isobutyl nitrites to nitric oxide (NO) in the presence of xanthine under anaerobic conditions. NO was produced at a stoichiometric ratio of 2:1 versus urate generation, steady-state analysis of which showed Michaelis-Menten kinetics with xanthine as varied substrate and substrate inhibition with varied organic nitrite. Under the conditions of NO generation from isoamyl nitrite, XO was progressively inactivated by a mechanism involving conversion of Mo=S to Mo=O, yielding "desulfo" enzyme. It is proposed that XO is involved in the metabolism of organic nitrites to NO in vivo and that the observed inactivation serves to explain the phenomenon of tolerance.     

Nitric oxide production from nitrite occurs primarily in tissues not in the blood: critical role of xanthine oxidase and aldehyde oxidase

Recent studies have shown that nitrite is an important storage form and source of NO in biological systems. Controversy remains, however, regarding whether NO formation from nitrite occurs primarily in tissues or in blood. Questions also remain regarding the mechanism, magnitude, and contributions of several alternative pathways of nitrite-dependent NO generation in biological systems. To characterize the mechanism and magnitude of NO generation from nitrite, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The addition of nitrite triggered a large amount of NO generation in tissues such as heart and liver, but only trace NO production in blood. Carbon monoxide increased NO release from blood, suggesting that hemoglobin acts to scavenge NO not to generate it. Administration of the xanthine oxidase (XO) inhibitor oxypurinol or aldehyde oxidase (AO) inhibitor raloxifene significantly decreased NO generation from nitrite in heart or liver. NO formation rates increased dramatically with decreasing pH or with decreased oxygen tension. Isolated enzyme studies further confirm that XO and AO, but not hemoglobin, are critical nitrite reductases. Overall, NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations.     

In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics

Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.     

Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and 相似文献   

Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism

Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O₂^∙−) as part of the innate host defense system, but exaggerated and sustained O₂^∙− generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O₂^∙− and peroxynitrite (ONOO⁻) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O₂^∙− and ONOO⁻ production in macrophages, which was significantly reduced by nitrite (10 µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O₂^∙− generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response.     

Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall

Nitric oxide (NO) has been shown to be the endothelium-derived relaxing factor (EDRF), and its impairment contributes to a variety of cardiovascular disorders. Recently, it has been recognized that nitrite can be an important source of NO; however, questions remain regarding the activity and mechanisms of nitrite bioactivation in vessels and its physiological importance. Therefore, we investigated the effects of nitrite on in vivo hemodynamics in rats and in vitro vasorelaxation in isolated rat aorta under aerobic conditions. Studies were performed to determine the mechanisms by which nitrite is converted to NO. In anesthetized rats, nitrite dose dependently decreased both systolic and diastolic blood pressure with a threshold dose of 10 microM. Similarly, nitrite (10 microM-2 mM) caused vasorelaxation of aortic rings, and NO was shown to be the intermediate factor responsible for this activity. With the use of electrochemical as well as electron paramagnetic resonance (EPR) spectroscopy techniques NO generation was measured from isolated aortic vessels following nitrite treatment. Reduction of nitrite to NO was blocked by heating the vessel, suggesting that an enzymatic process is involved. Organ chamber experiments demonstrated that aortic relaxation induced by nitrite could be blocked by both hemoglobin and soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). In addition, both electrochemical and EPR spin-trapping measurements showed that ODQ inhibits nitrite-mediated NO production. These findings thus suggest that nitrite can be a precursor of EDRF and that sGC or other heme proteins inhibited by ODQ catalyze the reduction of nitrite to NO.     

A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.     

Reduction of organic nitrates catalysed by xanthine oxidoreductase under anaerobic conditions.

Xanthine oxidoreductase catalyses the anaerobic reduction of glyceryl trinitrate (GTN), isosorbide dinitrate and isosorbide mononitrate to inorganic nitrite using xanthine or NADH as reducing substrates. Reduction rates are much faster with xanthine as reducing substrate than with NADH. In the presence of xanthine, urate is produced in essentially 1:1 stoichiometric ratio with inorganic nitrite, further reduction of which is relatively slow. Organic nitrates were shown to interact with the FAD site of the enzyme. In the course of reduction of GTN, xanthine oxidoreductase was progressively inactivated by conversion to its desulpho form. It is proposed that xanthine oxidoreductase is one of several flavoenzymes that catalyse the conversion of organic nitrate to inorganic nitrite in vivo. Evidence for its further involvement in reduction of the resulting nitrite to nitric oxide is discussed.     

Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin

Terada, Lance S., John E. Repine, Dale Piermattei, andBrooks M. Hybertson. Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin.J. Appl. Physiol. 82(3): 913-917, 1997.The oxygen radical-producing enzyme xanthine oxidase (XO) canpromote neutrophil adherence to endothelium. Recognizing that a balanceoften exists in inflammatory processes, we sought to determine whetherXO initiates antiadherent pathways. We found that bovine pulmonaryarterial endothelial cells (EC) exposed to XO released increasedamounts of nitrite into the media, reflecting an increased productionof nitric oxide (NO). When EC were subjected to shear stress, treatmentwith XO and/or the NO synthase inhibitorN-nitro-L-arginine(L-NNA) increased neutrophilrolling behavior and firm neutrophil adherence to EC in an additivefashion. Both rolling and adherent interactions were abolished bymonoclonal antibodies directed against P-selectin. In addition,treatment of EC with XO and/orL-NNA increased both surfaceexpression of P-selectin and release of von Willebrand factor intomedia. Finally, treatment of EC with the NO donor sodium nitroprussidedecreased XO-mediated neutrophil rolling and adherence. We concludethat XO stimulates EC to produce NO and that NO decreases theP-selectin-dependent neutrophil adhesion initiated by XO. Suchincreases in endogenous NO may constitute an importantnegative-feedback response to the acute proadhesive effects of XO.

     

Nitrates and NO release: contemporary aspects in biological and medicinal chemistry

Nitroglycerine has been used clinically in the treatment of angina for 130 years, yet important details on the mechanism of action, biotransformation, and the associated phenomenon of nitrate tolerance remain unanswered. The biological activity of organic nitrates can be said to be nitric oxide mimetic, leading to recent, exciting progress in realizing the therapeutic potential of nitrates. Unequivocally, nitroglycerine and most other organic nitrates, including NO-NSAIDs, do not behave as NO donors in the most fundamental action: in vitro activation of sGC to produce cGMP. The question as to whether the biological activity of nitrates results primarily or exclusively from NO donation will not be satisfactorily answered until the location, the apparatus, and the mechanism of reduction of nitrates to NO are defined. Similarly, the therapeutic potential of nitrates will not be unlocked until this knowledge is attained. Aspects of the therapeutic and biological activity of nitrates are reviewed in the context of the chemistry of nitrates and the elusive efficient 3e- reduction required to generate NO.     

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR—the nitrite reduction—and propose a new class of molybdenum-containing nitrite reductases.     

Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates

The aims of this study were to test whether xanthine oxidase, lactoperoxidase, and NO are components of the innate immune system of mammary secretion during active involution in dairy cows, and whether the innate immune system is activated by casein hydrolysates. Our laboratory has shown recently that infusion of CNH into mammary glands induced involution and was associated with earlier increases in the concentrations of components of the innate immune system. Intact casein is inactive and served as control. Half of the glands of 8 Holstein cows scheduled for dry off (approximately 60 days before parturition) were injected for 3 days with a single dose of casein hydrolyzates and the contralateral glands with a single dose of intact casein with the same concentration. Involution elicited marked increases in xanthine oxidase and lactoperoxidase activities, and accumulation of urate and nitrate. NO and H(2)O(2) were constantly produced in the mammary gland secretion. Nitrite formed either by autooxidation of NO or by conversion of nitrate to nitrite by xanthine oxidase was converted into the powerful nitric dioxide radical by lactoperoxidase and H(2)O(2) that is derived from the metabolism of xanthine oxidase. Nitric dioxide is most likely responsible for the formation of nitrosothiols on thiol-bearing groups, which allows an extended NO presence in mammary secretion. Nitrite is effectively converted to nitrate, which accumulated in the range of approximately 25 microM -1 mM from the start of the experiment to the complete involution of glands. The mammary secretion in all glands was bactericidal and bacteriostatic during established involution, and this appeared sooner and more acutely in glands treated with casein hydrolyzates, within 8 to 24 h. It is concluded that xanthine oxidase, lactoperoxidase, and NO are components of the mammary innate immune system that form bactericidal and bacteriostatic activities in mammary secretions. The innate immune system play a major role in preventing intramammary infection during milk stasis and its activation may increase its effectiveness.     

Dynamic state of S-nitrosothiols in human plasma and whole blood

In the vasculature, nitrosothiols derived from the nitric oxide (NO)-mediated S-nitrosation of thiols play an important role in the transport, storage, and metabolism of NO. The present study was designed to examine the reactions that promote the decomposition, formation, and distribution of extracellular nitrosothiols in the circulation. The disappearance of these species in plasma and whole blood was examined using a high-performance liquid chromatography method to separate low- and high-molecular weight nitrosothiols. We found that incubation of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO) with human plasma resulted in a rapid decomposition of these nitrosothiols such that <10% of the initial concentration was recovered after 10-15 min. Neither metal chelators (DTPA, neocuproine), nor zinc chloride (glutathione peroxidase inhibitor), acivicin (gamma-glutamyl transpeptidase inhibitor), or allopurinol (xanthine oxidase inhibitor) inhibited the decomposition of GSNO. With both CySNO and GSNO virtually all NO was recovered as S-nitrosoalbumin (AlbSNO), suggesting the involvement of a direct transnitrosation reaction. Electrophilic attack of the albumin-associated thiols by reactive nitrogen oxides formed from the interaction of NO with O(2) was ruled out because one would have expected 50% yield of AlbSNO. Similar results were obtained in whole blood. The amount of S-nitrosohemoglobin recovered in the presence of 10 microM GSNO or CySNO was less than 100 nM taking into consideration the detection limit of the assay used. Our results suggest that serum albumin may act as a sink for low-molecular-weight nitrosothiols and as a modulator of NO(+) transfer between the vascular wall and intraerythrocytic hemoglobin.     

Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation.

Several nitric oxide (NO) effects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important effectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-d,l-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1-1000 microM) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 microM) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 microM) prevented NO effects, confirming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 microM), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 microM) prevented SNAP inhibitory effects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 microM) was ineffective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative effect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1-100 microM), inhibited cell proliferation. In addition, sub-effective concentrations of ODQ (100 nM) and TEA (10 microM) synergized in blocking SNAP antiproliferative effects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation.     

Effect of intravenous nitroglycerin therapy on erythrocyte antioxidant enzymes

Intravenous nitroglycerin (GTN) has been used as an anti-ischemic agent for the therapy of unstable and post-infarction angina. Nitric oxide (NO) and S-nitrosothiols constitute the biologically active species formed via nitroglycerin bioactivation. Increased levels of reactive oxygen species can diminish the therapeutic action of organic nitrates by scavenging donated NO and oxidizing tissue thiols important in nitrate biotransformation. Studies reported here show that the red cell activity of antioxidant enzymes, catalase and glutathione peroxidase, are significantly decreased after intravenous nitroglycerin treatment. Catalase activity (739.6 +/- 92.3 k/gHb) decreased to 440.1 +/- 111.9 and 459.8 +/- 130.7 k/gHb after 1 and 24 hr GTN infusion, respectively. Similarly, glutathione peroxidase activity (5.8 +/- 1.8 U/gHb) decreased to 3.2 +/- 1.7 and 3.8 +/- 1.1 U/g Hb after 1 and 24 hr GTN infusion, respectively. The reported decrease in antioxidant enzyme activities can lead to an oxidant milieu and contribute to the generation of nitrate tolerance.