Cytochrome P450 is responsible for nitric oxide generation from NO-aspirin and other organic nitrates

Nitric oxide (NO) biotransformation from NO-aspirin (NCX-4016) is not clearly understood. We have previously reported that cytochrome P450 (P450) plays important role in NO generation from other organic nitrates such as nitroglycerin (NTG) and isosorbide dinitrate (ISDN). The present study was designed to elucidate the role of human cytochrome P450 isoforms in NO formation from NCX-4016, using lymphoblast microsomes transfected with cDNA of human P450 or yeast-expressed, purified P450 isoforms. CYP1A2 and CYP2J2, among other isoforms, were strongly related to NO production from NCX-4016. In fact, these isoforms were detected in human coronary endothelial cells. These results suggest that NADPH-cytochrome P450 reductase and the P450 system participate in NO formation from NCX-4016, as well as other organic nitrates.     

Nitrate tolerance induced by nicorandil or nitroglycerin is associated with minimal loss of nicorandil vasodilator activity.

In the current study, the vasodilator and tolerance-inducing actions of a recently developed organic nitrate vasodilator, nicorandil, were compared to nitroglycerin (NTG) in an isolated coronary artery preparation. The order of potency for relaxing U46619-constricted bovine-isolated coronary artery rings was NTG greater than isosorbide dinitrate (ISDN) greater than nicorandil. NTG was approximately 250-fold more potent than nicorandil (mean EC50 values for relaxation; 0.044 and 11.2 microM, respectively; n = 6-8). Coronary artery rings preexposed for 60 min to NTG (30 microM) were subsequently markedly less responsive to the relaxant effects of NTG (7.5-fold increase in mean EC50 value, 68.4% decrease in Emax; p less than 0.001) and ISDN (14.1-fold increase in mean EC50 value; p less than 0.001), although only marginally less responsive to nicorandil (1.75-fold increase in mean EC50 value; p less than 0.05). Thus, the coronary artery relaxant actions of nicorandil were significantly less affected by NTG-induced tolerance than were the relaxant actions of the related organic nitrate compounds, NTG and ISDN. To compare the tolerance-inducing actions of NTG and nicorandil, the relaxant actions of a series of nitric oxide (NO)-containing vasodilators were determined in control coronary artery rings and in rings preexposed for 60 min to either 30 microM NTG or 5,000 microM nicorandil. Quantitatively, similar changes in coronary artery ring responsiveness were produced by tolerance induced by NTG and nicorandil; marked attenuation of responsiveness to NTG and to the nonnitrate compound 3-morpholinosydnonimine (SIN-1), but only marginal attenuation of responsiveness to nicorandil and NO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevention of vascular nitroglycerin tolerance by inhibition of protein kinase C.

We investigated whether in vivo inhibition of protein kinase C (PKC) can prevent the development of vascular tolerance and restore the sensitivity of isolated vessels to nitroglycerin (NTG). Tolerance was induced in male Wistar rats by a constant i.v. infusion of NTG 1 mg kg-1 h-1, a dose which did not alter blood pressure. After 72 h, the aorta was removed and the sensitivity of aortic rings to NTG tested. Chronic NTG infusion resulted in a 5.5 fold decrease in NTG-sensitivity as compared with controls (vehicle), indicating the development of vascular tolerance. The simultaneous in vivo administration of the specific PKC inhibitor N-benzoyl-staurosporine (30 mg kg-1 day-1) prevented this decrease in NTG sensitivity. These results suggest a role for PKC activation in the development of vascular NTG tolerance.     

Preservation of platelet responsiveness to nitroglycerine despite development of vascular nitrate tolerance

AIMS: Organic nitrates, via nitric oxide (NO) release, induce vasodilatation and inhibit platelet aggregation. Development of nitrate tolerance in some vascular preparations may be associated with diminished responsiveness to NO. To date it is not known to what extent vascular tolerance to organic nitrates is associated with acquired platelet hypo-responsiveness to NO. In the current study we compared the acute and chronic effects of sustained release (SR) isosorbide 5' mono-nitrate (ISMN) and transdermal nitroglycerine (TD-NTG) on blood vessels (effects on apparent arterial stiffness) and platelets (effects on responsiveness to NO donors) in patients with stable angina pectoris (SAP). METHODS: Patients (n = 34) with SAP entered a blinded randomized crossover study of ISMN (120 mg) vs. intermittent TD-NTG (15 mg 24 h(-1)). Effects of each nitrate on pulse wave reflection (augmentation index (AIx)), platelet response to adenosine di-phosphate (ADP 1 micromol l(-1)), nitroglycerine (NTG 100 micromol l(-1)) and the non-nitrate NO donor sodium nitroprusside (SNP 10 micromol l(-1)), were measured pre-dose, 4 and 8 h post dose, on three occasions: 1) at the end of a pre-nitrate phase, 2) after dosing for 7 days and 3) following 14 days of full dose therapy with either nitrate. RESULTS: Acutely, both ISMN and TD-NTG markedly reduced AIx. After 14 days, these effects were significantly attenuated (ANOVA, P = 0.018) but not abolished, indicating development of nitrate tolerance. Neither nitrate preparation affected ADP (1 micromol l(-1))-induced platelet aggregation. Platelet responsiveness to NTG (100 micromol l(-1)) and SNP (10 micromol l(-1)) was not diminished during chronic nitrate therapy, and there was no evidence of 'rebound' hyper-aggregability during 'nitrate-free' periods. CONCLUSIONS: Chronic therapy with either ISMN or TD-NTG is associated with development of vascular tolerance. Despite the induction of vascular tolerance, platelet responsiveness to NTG and SNP remains unaffected. Therefore, development of vascular tolerance is unlikely to compromise the anti-aggregatory effects of organic nitrates, or those of endogenous NO.     

Antiplatelet activities of FK409, a new spontaneous NO releaser.

1. We reported that (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409) released nitric oxide (NO) spontaneously with a chemiluminescence analyzer. The aim of this study was to compare antiplatelet activities of FK409, a new NO releaser, with those of isosorbide dinitrate (ISDN) in vivo and in vitro. In order to elucidate the differences in antiplatelet activities between FK409 and ISDN, we compared their modes of action. 2. In in vitro experiments, FK409 had a more potent inhibitory effect on rat platelet aggregation induced by adenosine 5'-diphosphate (2.0 microM) than ISDN (IC50 = 4.32 +/- 0.95 microM and > 100 microM respectively). 3. In the rat extracorporeal shunt model (in vivo experiments), FK409 suppressed thrombus formation dose-dependently from 0.32 mg kg-1, p.o. and showed the maximum inhibition (52% inhibition vs. vehicle treatment) at 10 mg kg-1, p.o., while ISDN showed no inhibition at 10 mg kg-1 and only 17% inhibition at 32 mg kg-1, p.o. 4. FK409 could generate nitrite, which is an oxidative product of NO, much faster than ISDN in phosphate buffer solution and rat plasma during 60-min incubation at 37 degrees C. 5. These data show that FK409 has more potent antiplatelet effects than ISDN, by acting through spontaneously released NO.     

Calcitonin gene-related peptide-dependent vascular relaxation of rat aorta. An additional mechanism for nitroglycerin

We investigated the involvement of calcitonin gene-related peptide (CGRP) in the vasodilatory mechanism of action of nitric oxide (NO) donors. The functional role of CGRP in NO donor-induced vasodilation of isolated rat aortic rings was determined by incubating these drugs with and without CGRP(8-37), a selective CGRP receptor antagonist. CGRP(8-37) (0.63 microM) induced rightward shifts in the vasodilatory concentration-response curves for nitroglycerin (NTG), Piloty's acid (PA), and SIN-1 (linsidomine). The EC(50) values for NTG, PA, and SIN-1 were increased by 8.3-, 5.2-, and 2.3-fold, respectively (P < 0.05). The release of CGRP from rat aorta in response to NTG and PA was measured specifically by radioimmunoassay. Thirty-minute incubations of NTG or PA with rat aorta induced 189.5 and 214.6% increases, respectively, in CGRP release when compared with the control (P < 0.05). The concentration-response curves of sodium nitroprusside (SNP), S-nitroso-acetylpenicillamine (SNAP), tetranitromethane (TNM), diethylamine NO complex (DEA-NO), and diethylenetriamine/nitric oxide adduct (DETA NONOate) were not inhibited significantly by CGRP(8-37) co-incubation (P 0.05). NO donors also were incubated with aortic strips, and NTG and PA alone induced significant formation of hydroxylamine, a NO(-) metabolite (232.4 and 364.9%, respectively, P < 0.05). These results indicate that only NTG and PA, and to a lesser extent SIN-1, stimulate the release of CGRP from the rat aorta, which subsequently contributes to the vasodilatory activity of these agents. The hydroxylamine formation suggests a possible link between NO(-) generation and CGRP release from the vascular wall.     

Differential Metabolism of Organic Nitrates by Aldehyde Dehydrogenase 1a1 and 2: Substrate Selectivity, Enzyme Inactivation, and Active Cysteine Sites

Organic nitrate vasodilators (ORN) exert their pharmacologic effects through the metabolic release of nitric oxide (NO). Mitochondrial aldehyde dehydrogenase (ALDH2) is the principal enzyme responsible for NO liberation from nitroglycerin (NTG), but lacks activity towards other ORN. Cytosolic aldehyde dehydrogenase (ALDH1a1) can produce NO from NTG, but its activity towards other ORN is unknown. Using purified enzymes, we showed that both isoforms could liberate NO from NTG, isosorbide dinitrate (ISDN), and nicrorandil, while only ALDH1a1 metabolized isosorbide-2-mononitrate and isosorbide-5-mononitrate (IS-5-MN). Following a 10-min incubation with purified enzyme, 0.1 mM NTG and 1 mM ISDN potently inactivated ALDH1a1 (to 21.9% ± 11.1% and 0.44% ± 1.04% of control activity, respectively) and ALDH2 (no activity remaining and 4.57% ± 7.92% of control activity, respectively), while 1 mM IS-5-MN exerted only modest inactivation of ALDH1a1 (reduced to 89% ± 4.3% of control). Cytosolic ALDH in hepatic homogenates incubated at the vascular EC₅₀ concentrations of ORN was inactivated by NTG (to 45.1% ± 8.1% of control activity) while mitochondrial ALDH was inactivated by NTG and nicorandil (to 68.2% ± 10.0% and 78.7% ± 19.8% of control, respectively). Via site-directed mutagenesis, the active sites of ORN metabolism of ALDH2 (Cys-319) and ALDH1a1 (Cys-303) were found to be identical to those responsible for their dehydrogenase activity. Cysteine-302 of ALDH1a1 and glutamate-504 of ALDH2 were found to modulate the rate of ORN metabolism. These studies provide further characterization of the substrate selectivity, inactivation, and active sites of ALDH2 and ALDH1a1 toward ORN.Key words: aldehyde dehydrogenase, nitric oxide, organic nitrate, site-directed mutagenesis     

cDNA microarray analysis of vascular gene expression after nitric oxide donor infusions in rats: implications for nitrate tolerance mechanisms

Vascular nitrate tolerance is often accompanied by changes in the activity and/or expression of a number of proteins. However, it is not known whether these changes are associated with the vasodilatory properties of nitrates, or with their tolerance mechanisms. We examined the hemodynamic effects and vascular gene expressions of 2 nitric oxide (NO) donors: nitroglycerin (NTG) and S-nitroso-N-acetylpenicillamine (SNAP). Rats received 10 microg/min NTG, SNAP, or vehicle infusion for 8 hours. Hemodynamic tolerance was monitored by the maximal mean arterial pressure (MAP) response to a 30-microg NTG or SNAP bolus challenge dose (CD) at various times during infusion. Gene expression in rat aorta after NTG or SNAP treatment was determined using cDNA microarrays, and the relative differences in expression after drug treatment were evaluated using several statistical techniques. MAP response of the NTG CD was attenuated from the first hour of NTG infusion (P <.001, analysis of variance [ANOVA]), but not after SNAP (P >.05, ANOVA) or control infusion (P >.05, ANOVA). Student t-statistics revealed that 447 rat genes in the aorta were significantly altered by NTG treatment (P <.05). An adjusted t-statistic approach using resampling techniques identified a subset of 290 genes that remained significantly different between NTG treatment vs control. In contrast, SNAP treatment resulted in the up-regulation of only 7 genes and the down-regulation of 34 genes. These results indicate that continuous NTG infusion induced widespread changes in vascular gene expression, many of which are consistent with the multifactorial and complex mechanisms reported for nitrate tolerance.     

Parallel tolerance between platelet cyclic GMP and preload effects of nitroglycerin in anaesthetized mini-pigs.

The effects of acute intravenous nitroglycerin (NTG) administration on platelet cyclic GMP in relation to changes in indices of preload (end-diastolic volume) and afterload (effective arterial elastance) were evaluated in the anaesthetized mini-pig, using pressure-volume analysis. NTG (1-30 micrograms kg-1 min-1, i.v.) elicited a dose-dependent fall in preload and afterload, and an increase in arterial blood platelet cyclic GMP. Repeated doses of NTG (30 micrograms kg-1 min-1) resulted in tolerance to the preload but not afterload effects. The increases in platelet cyclic GMP were also attenuated, being highly correlated with the preload changes. Therefore, platelet cyclic GMP appears to reflect NTG-induced venous tolerance, rather than arterial responsiveness. The measurement of platelet cyclic GMP may represent a simple approach for monitoring the degree of venous tolerance to NTG in animals or patients, facilitating further mechanistic investigations.     

Comparative vasorelaxing profiles of nicorandil, isosorbide dinitrate and nitroglycerin in isolated coronary arteries of the dog

The vasorelaxing effects of nicorandil (NCR), isosorbide dinitrate (ISDN) and nitroglycerin (NTG) were studied in isolated canine coronary arteries. In rings of coronary arteries precontracted with prostaglandin F2 alpha (3 x 10(-6) M) or KCl (30 mM), removal of the endothelium significantly augmented the relaxing effects of NCR, while it did not affect those of ISDN and NTG. In unrubbed rings precontracted with KCl (30 mM), methylene blue (5 x 10(-6) M) significantly inhibited vasorelaxing responses to the three drugs. The order of the inhibition was as follows: NTG greater than ISDN greater than NCR. When the unrubbed tissue was incubated with NTG (10(-5) M) or ISDN (10(-4) M) for 10 min, it developed acute tolerance in relaxing response to NTG or ISDN. Unlike NTG and ISDN, NCR did not develop any tolerance. The treatment with N-acetylcysteine (5 x 10(-5) M) tended to potentiate relaxant effects of NTG and to reduce the degree of acute tolerance to NTG. The results suggest that cGMP plays a role in the relaxation of the coronary artery induced by the drugs and furthermore that the mode of the vasorelaxing action of NCR may be somewhat different from that of NTG or ISDN.     

Detection of nitric oxide in exhaled air during administration of nitroglycerin in vivo.

1. Direct evidence for nitric oxide (NO) formation from nitroglycerin (GTN) was obtained by measurements of NO concentrations in exhaled air in artificially-ventilated, pentobarbitone-anaesthetized rabbits. 2. The concentration of endogenously formed NO was 23 +/- 5 parts per billion (p.p.b.). Infusions of GTN (1-100 micrograms kg-1 min-1, i.v.) induced dose-dependent and biphasic increments in exhaled NO and concomitant reductions in systemic blood pressure. 3. Tolerance to the blood pressure reduction developed in parallel with a decrease in GTN-induced exhaled NO, a pattern which was unaffected by administration of N omega-nitro-L-arginine methyl ester (L-NAME, 30 mg kg-1), L-cysteine (200 mg kg-1), N-acetylcysteine (200 mg kg-1) or glutathione (200 mg kg-1). 4. Intravenous infusions of adenosine (0.7 mg ml-1, 250 microliters kg-1 min-1) and GTN (1 mg ml-1, 250 microliters kg-1 min-1) elicited similar decrements in pulmonary vascular resistance. GTN elicited a substantial increase in exhaled NO (50 +/- 10 p.p.b.) whereas adenosine evoked a markedly smaller increase (7 +/- 1 p.p.b.). L-NAME (30 mg kg-1, i.v.) abolished NO in exhaled air, and evoked an increase in pulmonary vascular resistance from 116 +/- 19 to 147 +/- 9 pulmonary vascular resistance units. After L-NAME the change in pulmonary vascular resistance induced by adenosine or GTN was increased to a similar degree. However, while the increase in exhaled NO induced by nitroglycerin was unaffected, the response to adenosine was abolished. 5. The present data demonstrate that NO is formed from GTN in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

大气细颗粒物有机成分在体染毒对大鼠胸主动脉血管反应的影响及其与NO/NOS的关系

目的探究大气细颗粒物PM2.5有机成分在体染毒对大鼠胸主动脉血管反应性的影响及其与血管内皮细胞NO/NOS系统的关系。方法 Wistar大鼠随机分为两组:①染毒组:PM2.5染毒溶液1 ml.kg-1.d-1,iv;②对照组:生理盐水1 ml.kg-1.d-1,iv。染毒10 d后,击晕大鼠,主动脉采血,用于检测血清一氧化氮(NO)和内皮型一氧化氮合酶(eNOS)含量、诱导型一氧化氮合酶(iNOS)活性。分离主动脉,用于离体主动脉实验和免疫组织化学染色分析,观察主动脉血管反应性和内皮细胞eNOS和iNOS蛋白表达。结果 PM2.5有机成分在体染毒后,可增强大鼠离体胸主动脉对60 mmol·L-1 KCl及苯肾上腺素(PE,10-5 mol·L-1)的收缩效应,减弱其对乙酰胆碱(ACh,10-9～10-5 mol·L-1)的舒张效应,但主动脉对硝普钠(SNP,10-9～10-5 mol·L-1)的舒张效应则不受影响。PM2.5有机成分在体染毒后,大鼠血清NO含量及iNOS活性增加,但eNOS含量则无明显变化;主动脉血管内皮细胞iNOS表达增加,eNOS表达减少。结论 PM2.5有机成分可影响大鼠胸主动脉环血管反应性,NO/NOS系统功能紊乱可能是其机制之一。     

Hydroxyimine NO-donors; FK409 and derivatives

FK409 was discovered during a screening program for prostaglandin like compounds from microbial products by measuring inhibition of platelet aggregation and vasodilation. FK409, a semisynthetic compound derived from acidic nitrosation of microbial broth, was shown to be a potent vasodilator with a similar pharmacological profile to organic nitrates such as nitroglycerin (NTG). FK409 dilated the larger diameter coronary vascular vessels more potently than those with a smaller diameter in vitro, and was more potent than NTG in a dog angina pectoris model. Clinical development of FK409 for angina pectoris included a preliminary open efficacy study in about twenty patients with angina pectoris showing an improvement in 60-70 % of patients. Anemia proved a drug related adverse event. A new study was carried out on around 20 patients with ischemic heart disease, but in the longer term the anemia remained. It was concluded that FK409 had a comparable efficacy to organic nitrates, but an undesirable adverse effect, and development was terminated. Back-up compounds for FK409, explored improvement of the pharmacokinetic profile: an increase in duration of action and a reduction of the risk of anemia. The pharmacological action of FK409 was associated with increased cGMP levels in aortic smooth muscle; and release of NO was observed by physicochemical methods. Synthesis of chemically more stable derivatives of FK409 with slower NO release was aimed at longer pharmacokinetic profiles and a lower incidence of anemia. FR144220 and FR146881 were identified as chemically more stable compounds with a longer duration of pharmacological action.     

Influence of N-acetylcysteine on bioactivation of nitroglycerin to nitric oxide and S-nitrosothiols in the liver and brain of mice

Three-day nitroglycerin (NTG) administration at progressively increasing doses caused a drop in the liver S-nitrosothiol (SNT) and malonyldialdehyde (MDA) concentrations below the control levels. It suggests that NTG administered in this way, exhibits antioxidant activity due to releasing the biologically active SNT and nitric oxide (NO). On the other hand, in the brain, NTG did not influence SNT concentrations, but slightly elevated NO formation. N-acetylcysteine (NAC) given jointly with NTG substantially stimulated NTG bioactivation to the biologically active NO and SNT as well in the liver as in the brain. It was accompanied by a rise in non-protein sulfhydryl thiol (NPSH) level and additional suppression of lipid peroxidation in hepatocytes. Therefore, is seems that the combined administration of NTG and thiols or other antioxidants is very much justified not only because of their influence on the vascular endothelial cells but also on such organs as the liver and brain.     

Close correlation of the cardioprotective effect of FK409, a spontaneous NO releaser, with an increase in plasma cyclic GMP level.

FK409 ((+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide) , which has been reported by us to be a new spontaneous nitric oxide (NO) releaser, prevented myocardial infarction following occlusion and reperfusion in rat coronary artery and increased plasma cyclic GMP level in rats, dose-dependently and significantly at 32 mg kg-1. Isosorbide dinitrate (ISDN), which is the most popular orally active NO donor used in the treatment of ischaemic cardiovascular diseases, did not show significant effects at 32 mg kg-1 in either experiment. Therefore, it is suggested that FK409 can attenuate myocardial injury during ischaemia and reperfusion in contrast to ISDN and a change in plasma cyclic GMP level may serve as an indicator of the cardioprotective effect of NO-releasing drugs.     

cDNA Microarray analysis of vascular gene expression after nitric oxide donor infusion in rats: Implications for nitrate tolerance mechanisms

Vascular nitrate tolerance is often accompanied by changes in the activity and/or expression of a number of proteins. However, it is not known whether these changes are associated with the vasodilatory properties of nitrates, or with their tolerance mechanisms. We examined the hemodynamic effects and vascular gene expressions of 2 nitric oxide (NO) donors: nitroglycerin (NTG) and S-nitroso-N-acetylpenicillamine (SNAP). Rats received 10 μg/min NTG, SNAP, or vehicle infusion for 8 hours. Hemodynamic tolerance was monitored by the maximal mean arterial pressure (MAP) response to a 30-μg NTG or SNAP bolus challenge dose (CD) at various times during infusion. Gene expression in rat aorta after NTG or SNAP treatment was determined using cDNA microarrays, and the relative differences in expression after drug treatment were evaluated using several statistical techniques. MAP response of the NTG CD was attenuated from the first hour of NTG infusion (P<.001, analysis of variance [ANOVA]), but not after SNAP (P>.05, ANOVA) or control infusion (P> .05, ANOVA). Student t-statistics revealed that 447 rat genes in the aorta were significantly altered by NTG treatment (P <.05). An adjusted t-statistic approach using resampling techniques identified a subset of 290 genes that remained significantly different between NTG treatment vs control. In contrast, SNAP treatment resulted in the up-regulation of only 7 genes and the downregulation of 34 genes. These results indicate that continuous NTG infusion induced widespread changes in vascular gene expression, many of which are consistent with the multifactorial and complex mechanisms reported for nitrate tolerance.     

Effects of obesity on the pharmacodynamics of nitroglycerin in conscious rats

Literature reports have suggested that hemodynamic response toward organic nitrates may be reduced in obese patients, but this effect has not been studied. We compared the mean arterial pressure (MAP) responses toward single doses of nitroglycerin (NTG), 0.5–50μg) in conscious Zucker obese (ZOB), Zucker lean (ZL), and Sprague-Dawley (SD) rats. NTG tolerance development in these animal groups was separately examined. Rats received 1 and 10μg/min of NTG or vehicle infusion, and the maximal MAP response to an hourly 30μg NTG IVchallenge dose (CD) was measured. Steady-state NTG plasma concentrations were measured during 10μg/min NTG infusion. The E_max and ED₅₀ values obtained were 33.9± 3.6 and 3.5±1.7μg for SD rats, 33.2±4.1 and 3.0±1.4μg for ZL rats, and 34.8±3.9 and 5.3±2.8μg for ZOB rats, respectively. No difference was found in the dose-response curves among these 3 groups (p>.05, 2-way ANOVA). Neither the dynamics of NTG tolerance development, nor the steady-state NTG plasma concentrations, were found to differ among these 3 animal groups. These results showed that ZOB rats are not more resistant to the hemodynamic effects of organic nitrates compared with their lean controls. Thus, the acute and chronic hemodynamic effects by the presence of obesity in a conscious animal model of genetic obesity.     

Decrease in plasma NOx concentration by isosorbide dinitrate,an organic nitrate ester

It has been suggested that isosorbide dinitrate (ISDN)-induced venodilation could be ascribed to preferential accumulation of the agent in venous tissues, resulting in higher concentrations of nitric oxide (NO). Here, the authors investigated whether the venodilating effect of ISDN is associated with a preferential increase in plasma concentrations of NOx (NO2- and NO3-, stable end-products of NO) in venous blood than arterial blood. Plasma NOx was measured by high-performance liquid chromatography-Griess system with a sensitivity of 0.01 microM for NO2- and 0.1 microM for NO3-. Arterial and venous blood samples were obtained after coronary angiography from the aorta and right atrium of patients with or without ischemic heart disease. Nicardipine, a calcium channel blocker, was used as a non-NO-related arteriovasodilator. At 1 mg i.v., it did not cause any changes in NOx concentration in arterial and venous blood irrespective of hemodynamic changes. However, ISDN (3 mg i.v.) increased NO2- and decreased NO3- in both arterial and venous blood, with concomitant venodilation. Further analysis revealed that plasma NO increased in the pulmonary circulation and this increase was preserved after nicardipine and ISDN, and that ISDN, but not nicardipine, increased plasma NO3- in the pulmonary circulation. The authors did not detect higher concentrations of NOx in venous blood relative to their level in arterial blood. Further studies are necessary to clarify the kinetics of NO and NO-related compounds in the whole body.     

Inhibition of lipid peroxidation in synaptosomes and liposomes by nitrates and nitrites

NO is produced endogenously from arginine by the action of NO synthase, and exogenously by nitrovasodilators, including organic nitrates and nitrites. NO has been proposed as a cytotoxic and cytoprotective agent. There is strong evidence that NO acts as an apparent antioxidant in inhibiting lipid peroxidation, via chain termination, and interestingly lipid nitrates and nitrites have been proposed to be products of this chain termination. Both pro- and antioxidant mechanisms may be drawn for nitrates and nitrites; therefore, their effects on lipid peroxidation were measured in two systems, using tocopherol, thiol, and an NO donor for comparison: (1) rat cerebrocortical synaptosomes with Fe(II)-induced lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS), and (2) phospholipid liposomes with an azo-initiator induction system, quantified by a fluorescent probe of peroxide formation. In contrast to the classical nitrate nitroglycerin, novel nitrates which release NO on reaction with thiols and two novel nitrates which spontaneously generate NO in aqueous solution inhibited lipid peroxidation. i-Amyl nitrite inhibited lipid peroxidation, and its properties were further studied with ESR spectroscopy. The data show that classical nitrites and novel nitrates are not prooxidants, but inhibit lipid peroxidation.     

Oxidative stress and mitochondrial aldehyde dehydrogenase activity: a comparison of pentaerythritol tetranitrate with other organic nitrates

Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN- and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations >1 microM. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.