Nitrite reduces acute lung injury and improves survival in a rat lung transplantation model.

Ischemia/reperfusion injury (IRI) is the most common cause of early mortality following lung transplantation (LTx). We hypothesized that nitrite, an endogenous source of nitric oxide (NO), may protect lung grafts from IRI. Rat lung grafts were stored in preservation solution at 4°C for 6 hours. Both grafts and recipients were treated with nitrite. Nitrite treatment was associated with significantly higher levels of tissue oxygenation, lower levels of cytokines and neutrophil/macrophage infiltration, lower myeloperoxidase activity, reduced oxidative injury and increased cGMP levels in grafts than in the controls. Treatment with either a nitric oxide scavenger or a soluble guanylyl cyclase (sGC) inhibitor diminished the beneficial effects of nitrite and decreased cGMP concentrations. These results suggest that nitric oxide, generated from nitrite, is the molecule responsible for the effects of nitrite via the nitric oxide/sGC/cGMP pathway. Allopurinol, a xanthine oxidoreductase (XOR) inhibitor, abrogated the protective effects of nitrite, suggesting that XOR is a key enzyme in the conversion of nitrite to nitric oxide. In vitro experiments demonstrated that nitrite prevented apoptosis in pulmonary endothelial cells. Nitrite also exhibits longer survival rate in recipients than control. In conclusion, nitrite inhibits lung IRI following cold preservation and had higher survival rate in LTx model.

Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase.

Mutations in copper, zinc superoxide dismutase (SOD) have been implicated in the selective death of motor neurons in 2 percent of amyotrophic lateral sclerosis (ALS) patients. The loss of zinc from either wild-type or ALS-mutant SODs was sufficient to induce apoptosis in cultured motor neurons. Toxicity required that copper be bound to SOD and depended on endogenous production of nitric oxide. When replete with zinc, neither ALS-mutant nor wild-type copper, zinc SODs were toxic, and both protected motor neurons from trophic factor withdrawal. Thus, zinc-deficient SOD may participate in both sporadic and familial ALS by an oxidative mechanism involving nitric oxide.

Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance.

We sought to examine mechanisms underlying nitroglycerin (NTG) tolerance and "cross-tolerance" to other nitrovasodilators. Rabbits were treated for 3 d with NTG patches (0.4 mg/h) and their aortic segments studied in organ chambers. Relaxations were examined after preconstriction with phenylephrine. In NTG tolerant rabbit aorta, relaxations to cGMP-dependent vasodilators such as NTG (45 +/- 6%), SIN-1 (69 +/- 7%), and acetylcholine (ACh, 64 +/- 5%) were attenuated vs. controls, (90 +/- 2, 94 +/- 3, and 89 +/- 2% respectively, P < 0.05 for all), while responses to the cAMP-dependent vasodilator forskolin remained unchanged. In tolerant aorta, endothelial removal markedly enhanced relaxations to NTG and SIN-1 (82 +/- 4 and 95 +/- 3%, respectively). Other studies were performed to determine how the endothelium enhances tolerance. Vascular steady state .-O2 levels (assessed by lucigenin chemiluminescence) was increased twofold in tolerant vs. control vessels with endothelium (0.31 +/- 0.01 vs. 0.61 +/- 0.01 nmol/mg per minute). This difference was less in vessels after denudation of the endothelium. Diphenylene iodonium, an inhibitor of flavoprotein containing oxidases, and Tiron a direct .-O2 scavenger normalized .-O2 levels. In contrast, oxypurinol (1 mM) an inhibitor of xanthine oxidase, rotenone (50 microM) an inhibitor of mitochondrial electron transport and NG-nitro-L-arginine (100 microM) an inhibitor of nitric oxide synthase did not affect the chemiluminescence signals from NTG-tolerant aortas. Pretreatment of tolerant aorta with liposome-entrapped, pH sensitive superoxide dismutase (600 U/ml) significantly enhanced maximal relaxation in response to NTG, SIN-1, and ACh, and effectively reduced chemiluminescence signals. These studies show that continuous NTG treatment is associated with increased vascular .-O2-production and consequent inhibition of NO. mediated vasorelaxation produced by both exogenous and endogenous nitrovasodilators.

Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite.

The evanescent nature of reactive oxygen and nitrogen species, the multiple cellular mechanisms evolved to maintain these substances at low (submicromolar) concentrations within the vascular system, and the often multifaceted nature of their reactivities have made measurement of these compounds within the vasculature problematic. This review attempts to provide a critical description of some of the most common approaches to quantification of nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite, with attention to key issues that may influence the utility of a particular assay when adapted for use in vascular cells and tissues.

Peroxynitrite stimulates vascular smooth muscle cell cyclic GMP synthesis.

Peroxynitrite stimulated the synthesis of cyclic GMP by rat aortic smooth muscle in a time- and dose-dependent manner. Peak formation of cyclic GMP occurred at 1 min with 100 microM peroxynitrite and was inhibited by oxyhemoglobin. Peroxynitrite was less potent than nitric oxide in stimulating cyclic GMP synthesis. Peroxynitrite also enhanced endothelial-dependent cyclic GMP synthesis, via generation of a long-lived substance, which was prevented by inhibition of glutathione synthesis. These data show that peroxynitrite stimulates cyclic GMP synthesis, inferring production of low yields of nitric oxide or associated derivatives. Additionally, vascular exposure to peroxynitrite potentiates endothelial-dependent activation of guanylate cyclase.

Nitronyl nitroxides as probes to study the mechanism of vasodilatory action of nitrovasodilators, nitrone spin traps, and nitroxides: role of nitric oxide.

Nitronyl nitroxides have been used to trap nitric oxide (.NO) produced during visible irradiation of nitrovasodilators such as sodium nitroprusside (Joseph et al., Biochem. Biophys. Res. Commun. 192:926-934; 1993). We have also shown that nitrone and nitroso spin traps exert a potent vasodilatory effect in the isolated perfused rat heart (Konorev et al., Free Radic. Biol. Med. 14:127-137, 1993). The objective of this study was to investigate the effect of nitronyl nitroxides on the vasodilatory action of sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy free radical (TEMPOL) in the isolated perfused rat heart model. In this study, we have used the following nitronyl nitroxides as nitric oxide traps: 2-(p-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-3-oxide 1-oxyl (SLI) and 2(1',1'-dimethyl-2'-hydroxyethyl)-4,4,5,5-tetramethyl imidazoline-3-oxide 1-oxyl (SLII). Under in vitro conditions, both SLI and SLII trapped .NO released from SNP/light treatment and from spontaneous decomposition of SNAP, forming the corresponding imino nitroxides, which were characterized by electron spin resonance (ESR) technique. In isolated hearts, SNP (2 mumol/l) and SNAP (20 mumol/l) increased coronary flow rate to a maximum of 185% and 190%, respectively. SNP-induced vasodilation was inhibited by SLI (0.05-3 mmol/l) from 162% to 131% of baseline, and SNAP-induced vasodilation was inhibited by SLII (0.05-1.2 mmol/l) from 190% to 136% of baseline. In contrast, neither SLI nor SLII inhibited the vasodilatory action elicited by POBN or TEMPOL.(ABSTRACT TRUNCATED AT 250 WORDS)

Liposome-delivered superoxide dismutase prevents nitric oxide-dependent motor neuron death induced by trophic factor withdrawal.

Inhibition of nitric oxide synthesis prevents rat embryonic motor neurons from undergoing apoptosis when initially cultured without brain-derived neurotrophic factor. Using an improved cell culture medium, we found that the partial withdrawal of trophic support even weeks after motor neurons had differentiated into a mature phenotype still induced apoptosis through a process dependent upon nitric oxide. However, nitric oxide itself was not directly toxic to motor neurons. To investigate whether intracellular superoxide contributed to nitric oxide-dependent apoptosis, we developed a novel method using pH-sensitive liposomes to deliver Cu, Zn superoxide dismutase intracellularly into motor neurons. Intracellular superoxide dismutase prevented motor neuron apoptosis from trophic factor withdrawal, whereas empty liposomes, inactivated superoxide dismutase in liposomes or extracellular superoxide dismutase did not. Neither hydrogen peroxide nor nitrite added separately or in combination affected motor neuron survival. Our results suggest that a partial reduction in trophic support induced motor neuron apoptosis by a process requiring the endogenous production of both nitric oxide and superoxide, irrespective of the extent of motor neuron maturation in culture.

The mechanism of cardioprotection by S-nitrosoglutathione monoethyl ester in rat isolated heart during cardioplegic ischaemic arrest.

1. This study was designed (i) to assess the effect of S-nitrosoglutathione monoethyl ester (GSNO-MEE), a membrane-permeable analogue of S-nitrosoglutathione (GSNO), on rat isolated heart during cardioplegic ischaemia, and (ii) to monitor the release of nitric oxide (.NO) from GSNO-MEE in intact hearts using endogenous myoglobin as an intracellular .NO trap and the hydrophilic N-methyl glucamine dithiocarbamate-iron (MGD-Fe2+) complex as an extracellular .NO trap. 2. During aerobic perfusion of rat isolated heart with GSNO-MEE (20 mumol 1(-1), there was an increase in cyclic GMP from 105 +/- 11 to 955 +/- 193 pmol g-1 dry wt. (P < 0.05), and a decrease in glycogen content from 119 +/- 3 to 96 +/- 2 mumol g-1 dry wt. (P < 0.05), and glucose-6-phosphate concentration from 258 +/- 22 in control to 185 +/- 17 nmol g-1 dry wt. (P < 0.05). During induction of cardioplegia, GSNO-MEE caused the accumulation of cyclic GMP (100 +/- 6 in control vs. 929 +/- 168 pmol g-1 dry wt. in GSNO-MEE-treated group, P < 0.05), and depletion of glycogen from 117 +/- 3 to 103 +/- 2 mumol g-1 dry wt. (P < 0.05) in myocardial tissue. 3. Inclusion of GSNO-MEE (20 mumol l-1) in the cardioplegic solution improved the recovery of developed pressure (46 +/- 8 vs. 71 +/- 3% of baseline, P < 0.05), and rate-pressure product from 34 +/- 6 to 63 +/- 5% of baseline (P < 0.05), and reduced the diastolic pressure during reperfusion from 61 +/- 7 in control to 35 +/- 5 mmHg (P < 0.05) after 35 min ischaemic arrest. GSH-MEE (20 mumol l-1) in the cardioplegic solution did not elicit the protective effect. 4. During cardioplegic ischaemia, GSNO-MEE (20-200 mumol l-1) induced the formation of nitrosylmyoglobin (MbNO), which was detected by electron spin resonance (ESR) spectroscopy. Inclusion of MGD-Fe2+ (50 mumol l-1 Fe2+ and 500 mumol l-1 MGD) in the cardioplegic solution along with GSNO-MEE yielded an ESR signal characteristic of the MGD-Fe2+ -NO adduct. However, the MGD-Fe2+ trap did not prevent the formation of the intracellular MbNO complex in myocardial tissue. During aerobic reperfusion, denitrosylation of the MbNO complex slowly occurred as shown by the decrease in ESR spectral intensity. GSNO-MEE treatment did not affect ubisemiquinone radical formation during reperfusion. 5. GSNO-MEE (20 microliters l-1) treatment elevated the myocardial cyclic GMP during ischaemia (47 +/- 3 in control vs. 153 +/- 34 pmol g-1 dry wt. after 35 min ischaemia, P < 0.05). The cyclic GMP levels decreased in the control group during ischaemia from 100 +/- 6 after induction of cardioplegia to 47 +/- 3 pmol g-1 dry wt. at the end of ischaemic duration. 6. Glycogen levels were lower in GSNO-MEE (20 mumol l-1)-treated hearts throughout the ischaemic duration (26.7 +/- 3.1 in control vs. 19.7 +/- 2.4 mumol g dry-t wt. in GSNO-MEE-treated group at the end of ischaemic duration), because of rapid depletion of glycogen during induction of cardioplegia. During ischaemia, the amounts of glycogen consumed in both groups were similar. Equivalent amounts of lactate were produced in both groups (148 +/- 4 in control vs. 141 +/- 4 mumol g-1 dry wt. in GSNO-MEE-treated group after 35 min in ischaemia). 7. The mechanism(s) of myocardial protection by GSNO-MEE against ischaemic injury may involve preischaemic glycogen reduction and/or elevated cyclic GMP levels in myocardial tissue during ischaemia.

S-nitrosoglutathione improves functional recovery in the isolated rat heart after cardioplegic ischemic arrest-evidence for a cardioprotective effect of nitric oxide.

The objective of this study was to assess the cardioprotective effect of the nitric oxide (.NO) donor, S-nitrosoglutathione (GSNO) and to investigate the mechanism of cardioprotection in a model of ischemia and reperfusion in isolated rat hearts. The role of .NO in myocardial protection was investigated by using nitronyl nitroxide as the .NO trap. Electron spin resonance spectroscopy was used to demonstrate that nitronyl nitroxide can trap .NO released from GSNO in a cardioplegic solution. .NO traps, oxyhemoglobin (4 mumol/l, n = 4) and nitronyl nitroxide (400 mumol/l, n = 5), inhibited the (2 mumol/l) GSNO-induced coronary vasodilation from the control value of 122% (n = 6) above base-line value to 73 and 60%, respectively. In the ischemia-reperfusion protocol, GSNO (20 mumol/l) was added to the cardioplegic solution during a 35-min ischemic arrest (n = 8). GSNO improved the functional recovery of ischemic hearts as compared to control (n = 6) as measured by the developed pressure (76 +/- 3 to 95 +/- 5% of base-line), rate pressure product (68 +/- 3 to 83 +/- 4% of base-line) and diastolic pressure (31 +/- 2 to 19 +/- 3 mm Hg). Reduction of coronary flow rate during reperfusion to control values in GSNO-treated hearts did not eliminate the improvement of functional recovery induced by GSNO. GSNO increased cyclic GMP production and slowed the accumulation of lactate (154 +/- 7 in control to 114 +/- 4 mumol/g dry wt.) and glucose-6-phosphate (3.66 +/- 0.19 in control to 2.18 +/- 0.10 mumol/g dry wt.) in myocardial tissue during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of lymphatics in removal of sheep lung surfactant lipid.

To study the role of lung lymphatics in the removal of surfactant lipid from the sheep lung, we injected [1-14C]palmitate intravenously into six animals previously fitted with a cannula draining the caudal mediastinal lymph node. Lung lymph was collected for 100 h after injection of radiolabel. We obtained alveolar lavage material through a tracheostomy in four other animals after intravenous injection of [9,10-3H]palmitate. We measured radioactivity at several time points in lipid extracts from lymph, lavage fluid, and lung tissue. Alveolar lavage disaturated phosphatidylcholine (DSPC) specific activity peaked at about 40 h and was reduced to 30% of this value by 82 h. About 2% of the injected radiolabel was incorporated into lung tissue lipids. Only 4% of the level of labeling achieved in lung tissue lipids was found in lung lymph lipid during 100 h of lymph collection. Sixty-three percent of radiolabel in lymph lipid was recovered in phospholipids, and 29% of phospholipid radiolabel was found in DSPC. The distribution of phosphorus and palmitate radiolabel in lung lymph phospholipid did not closely resemble that of surfactant lipid. No rise in lung lymph DSPC specific activity was observed following the peak in lavage specific activity. If surfactant lipid is removed from the alveolar compartment without extensive recycling, then we conclude that the lung lymphatics do not play a major role in the clearance of surfactant lipid from the alveolar surface.

Chemiluminescent detection of oxidants in vascular tissue. Lucigenin but not coelenterazine enhances superoxide formation.

Lucigenin-amplified chemiluminescence has frequently been used to assess the formation of superoxide in vascular tissues. However, the ability of lucigenin to undergo redox cycling in purified enzyme-substrate mixtures has raised questions concerning the use of lucigenin as an appropriate probe for the measurement of superoxide production. Addition of lucigenin to reaction mixtures of xanthine oxidase plus NADH resulted in increased oxygen consumption, as well as superoxide dismutase-inhibitable reduction of cytochrome c, indicative of enhanced rates of superoxide formation. Additionally, it was revealed that lucigenin stimulated oxidant formation by both cultured bovine aortic endothelial cells and isolated rings from rat aorta. Lucigenin treatment resulted in enhanced hydrogen peroxide release from endothelial cells, whereas exposure to lucigenin resulted in inhibition of endothelium-dependent relaxation in isolated aortic rings that was superoxide dismutase inhibitable. In contrast, the chemiluminescent probe coelenterazine had no significant effect on xanthine oxidase-dependent oxygen consumption, endothelial cell hydrogen peroxide release, or endothelium-dependent relaxation. Study of enzyme and vascular systems indicated that coelenterazine chemiluminescence is a sensitive marker for detecting both superoxide and peroxynitrite.

Superoxide production, risk factors, and endothelium-dependent relaxations in human internal mammary arteries.

BACKGROUND: In a variety of disease states, endothelium-dependent vasodilation is abnormal. Reduced nitric oxide (NO) production, increased destruction of NO by superoxide, diminished cellular levels of L-arginine or tetrahydrobiopterin, and alterations in membrane signaling have been implicated. We examined these potential mechanisms in human vessels. METHODS AND RESULTS: Relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin, as well as superoxide production and NO synthase expression, were examined in vascular segments from patients with identified cardiovascular risk factors. Endothelium-dependent relaxations were also studied after incubation with L-arginine, L-sepiapterin, and liposome-entrapped superoxide dismutase (SOD) and after organoid culture with cis-vaccenic acid. Relaxations to acetylcholine and to a lesser extent the calcium ionophore A23187 were highly variable and correlated with the number of risk factors present among the subjects studied. Treatment of vessels with L-arginine, L-sepiapterin, liposome-entrapped SOD, or cis-vaccenic acid did not augment endothelium-dependent relaxations. Hypercholesterolemia was the only risk factor associated with high levels of superoxide; however, there was no correlation between superoxide production and the response to either endothelium-dependent vasodilator used. CONCLUSIONS: In human internal mammary arteries, depressed endothelium-dependent relaxations could not be attributed to increases in vascular superoxide production, deficiencies in either L-arginine or tetrahydrobiopterin, or reduced membrane fluidity. Variability in signaling mechanisms may contribute to the differences in responses to acetylcholine and the calcium ionophore A23187.

Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry.

Oxidation of lipoproteins is important for the initiation and propagation of the atherosclerotic lesion and may involve secondary oxidants derived from nitric oxide. Nitric oxide (NO) reacts at near diffusion limited rates with superoxide (O2-.) to form the strong oxidant, peroxynitrite (ONOO-). Nitration on the ortho position of tyrosine is a major product of peroxynitrite attack on proteins. Nitrotyrosine was detected in atherosclerotic lesions of formalin-fixed human coronary arteries with polyclonal and monoclonal antibodies. Binding was pronounced in and around foamy macrophages within the atheroma deposits. Nitration was also observed in early subintimal fatty streaks. Antibody binding was completely blocked by co-incubation with 10mM nitrotyrosine, but not by equivalent concentrations of aminotyrosine or phosphotyrosine. The presence of nitrotyrosine indicates that oxidants derived from nitric oxide such as peroxynitrite are generated in human atherosclerosis and may be involved in its pathogenesis.

Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits.

Reactive oxygen species play a central role in vascular inflammation and atherogenesis, with enhanced superoxide (O2.-) production contributing significantly to impairment of nitric oxide (.NO)-dependent relaxation of vessels from cholesterol-fed rabbits. We investigated potential sources of O2.- production, which contribute to this loss of endothelium-dependent vascular responses. The vasorelaxation elicited by acetylcholine (ACh) in phenylephrine-contracted, aortic ring segments was impaired by cholesterol feeding. Pretreatment of aortic vessels with either heparin, which competes with xanthine oxidase (XO) for binding to sulfated glycosaminoglycans, or the XO inhibitor allopurinol resulted in a partial restoration (36-40% at 1 muM ACh) of ACh-dependent relaxation. Furthermore, O2.(-)-dependent lucigenin chemiluminescence, measured in intact ring segments from hypercholesterolemic rabbits, was decreased by addition of heparin, allopurinol or a chimeric, heparin-binding superoxide dismutase. XO activity was elevated more than two-fold in plasma of hypercholesterolemic rabbits. Incubation of vascular rings from rabbits on a normal diet with purified XO (10 milliunits/ml) also impaired .NO-dependent relaxation but only in the presence of purine substrate. As with vessels from hypercholesterolemic rabbits, this effect was prevented by heparin and allopurinol treatment. We hypothesize that increases in plasma cholesterol induce the release of XO into the circulation, where it binds to endothelial cell glycosaminoglycans. Only in hypercholesterolemic vessels is sufficient substrate available to sustain the production of O2.- and impair NO-dependent vasorelaxation. Chronically, the continued production of peroxynitrite, (ONOO-) which the simultaneous generation of NO and O2.- implies, may irreversibly impair vessel function.

Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease.

Plasma xanthine oxidase (XO) activity was defined as a source of enhanced vascular superoxide (O(2)( *-)) and hydrogen peroxide (H(2)O(2)) production in both sickle cell disease (SCD) patients and knockout-transgenic SCD mice. There was a significant increase in the plasma XO activity of SCD patients that was similarly reflected in the SCD mouse model. Western blot and enzymatic analysis of liver tissue from SCD mice revealed decreased XO content. Hematoxylin and eosin staining of liver tissue of knockout-transgenic SCD mice indicated extensive hepatocellular injury that was accompanied by increased plasma content of the liver enzyme alanine aminotransferase. Immunocytochemical and enzymatic analysis of XO in thoracic aorta and liver tissue of SCD mice showed increased vessel wall and decreased liver XO, with XO concentrated on and in vascular luminal cells. Steady-state rates of vascular O(2)( *-) production, as indicated by coelenterazine chemiluminescence, were significantly increased, and nitric oxide (( *)NO)-dependent vasorelaxation of aortic ring segments was severely impaired in SCD mice, implying oxidative inactivation of ( *)NO. Pretreatment of aortic vessels with the superoxide dismutase mimetic manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin markedly decreased O(2)( small middle dot-) levels and significantly restored acetylcholine-dependent relaxation, whereas catalase had no effect. These data reveal that episodes of intrahepatic hypoxia-reoxygenation associated with SCD can induce the release of XO into the circulation from the liver. This circulating XO can then bind avidly to vessel luminal cells and impair vascular function by creating an oxidative milieu and catalytically consuming (*)NO via O(2)( small middle dot-)-dependent mechanisms.