Neil Hogg

Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver

Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.

Mechanism of Nitric Oxide Release from S-Nitrosothiols

S-Nitrosothiols have many biological activities and have been suggested to be intermediates in signal transduction. The mechanism and products of S-nitrosothiol decomposition are of great significance to the understanding of nitric oxide (·NO) biochemistry. S-Nitrosothiols are stable compounds at 37°C and pH 7.4 in the presence of transition metal ion chelators. The presence of trace transition metal ions (present in all buffers) stimulates the catalytic breakdown of S-nitrosothiols to ·NO and disulfide. Thiyl radicals are not formed as intermediates in this process. Photolysis of S-nitrosothiols results in the formation of ·NO and disulfide via the intermediacy of thiyl radicals. Reduced metal ion (e.g. Cu+) decomposes S-nitrosothiols more rapidly than oxidized metal ion (e.g. Cu2+) indicating that reducing agents such as glutathione and ascorbate can stimulate decomposition of S-nitrosothiol by chemical reduction of contaminating transition metal ions. Transnitrosation can also stimulate S-nitrosothiol decomposition if the product S-nitrosothiol is more susceptible to transition metal ion-catalyzed decomposition than the parent S-nitrosothiol. Equilibrium constants for the transnitrosation reactions of reduced glutathione, either with S-nitroso-N-acetyl-DL-penicillamine or with S-nitroso-L-cysteine indicate that S-nitrosoglutathione formation is favored. The biological relevance of S-nitrosothiol decomposition is discussed.

The Simultaneous Generation of Superoxide and Nitric Oxide Can Initiate Lipid Peroxidation in Human Low Density Lipoprotein

Oxidation of low density lipoprotein (LDL) has been shown to occur in the artery wall of atherosclerotic lesions in both animal models and human arteries. The oxidant(s) responsible for initiating this process are under intensive investigation and 15-lipoxygenase has been suggested in this context. Another possibility is that nitric oxide and superoxide, generated by cells present in the artery wall, react together to form peroxynitrite which decomposes to form the highly reactive hydroxyl radical. In the present study we have modelled the simultaneous generation of superoxide and nitric oxide by using the sydnonimine, SIN-1 and have investigated its effects on LDL. SIN-1 liberates both superoxide and nitric oxide during autooxidation resulting in the formation of hydroxyl radicals. We have demonstrated that superoxide generated by SIN-1 is not available to take part in a dismutation reaction since it reacts preferentially with nitric oxide. It follows, therefore, that during the autooxidation of SIN-1 little or no superoxide, or perhydroxyl radical will be available to initiate lipid peroxidation. We have shown that SIN-1 is capable of initiating the peroxidation of LDL and also converts the lipoprotein to a more negatively charged form. The SIN-1-dependent peroxidation of LDL is completely inhibited by superoxide dismutase which scavenges superoxide. Neither sodium nitroprusside or S-nitroso-N-acetyl penicillamine, which only produce nitric oxide, are able to modify LDL. These results are consistent with the hypothesis that a product of superoxide and nitric oxide could oxidize lipoproteins in the artery wall and so contribute to the pathogenesis of atherosclerosis in vivo.

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer--the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40-60% saturated with oxygen (near the Hb P50), an apparent ideal set point for hypoxia-responsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control.

Inhibition of low-density lipoprotein oxidation by nitric oxide. Potential role in atherogenesis.

The effects of nitric oxide (•NO) and nitrovasodilators on the oxidation of low‐density lipoprotein (LDL) have been studied. S‐Nitroso‐N‐acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) inhibited Cu2+‐and 2,2′‐azobis‐2‐amidinopropane hydrochloride‐dependent oxidation of LDL as monitored by oxygen consumption and the formation of thiobarbituric acid‐reactive substances, conjugated dienes, and lipid hydroperoxides. In the case of SNP, inhibition of LDL oxidation occurred only when the incubation mixture was irradiated with visible light. SNAP, however, exerted a dose‐dependent inhibition of Cu2+‐catalyzed oxidation of LDL even in the dark. Addition of •NO dissolved in deoxygenated buffer also inhibited the progression of LDL oxidation. Mouse peritoneal macrophages were less able to degrade LDL that had been oxidized in the presence of SNAP. Using an •NO electrode, it was estimated that a continuous production of •NO (⩽ 760 nM/min) could retard the progression of LDL oxidation. We propose that •NO can inhibit LDL oxidation by acting as a chain‐breaking antioxidant that is capable of scavenging carbon‐centered and peroxyl radicals. Biological implications of this novel •NO antioxidant property are discussed in relation to atherogenesis and contrasted to the prooxidant property of •NO when generated in the presence of superoxide.

Nitrite as Regulator of Hypoxic Signaling in Mammalian Physiology

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

NITRIC OXIDE AND LIPID PEROXIDATION

Nitric oxide can both promote and inhibit lipid peroxidation. By itself, nitric oxide acts as a potent inhibitor of the lipid peroxidation chain reaction by scavenging propagatory lipid peroxyl radicals. In addition, nitric oxide can also inhibit many potential initiators of lipid peroxidation, such as peroxidase enzymes. However, in the presence of superoxide, nitric oxide forms peroxynitrite, a powerful oxidant capable of initiating lipid peroxidation and oxidizing lipid soluble antioxidants. The role of nitric oxide in vascular pathology is discussed.

Peroxynitrite modification of low‐density lipoprotein leads to recognition by the macrophage scavenger receptor

Peroxynitrite is an oxidant which could be formed in the vasculature by the reaction of superoxide with nitric oxide. It is capable of modifying amino acid residues and of initiating lipid peroxidation. In the present study we have shown that peroxynitrite converts low density lipoprotein to a form recognized by the macrophage scavenger receptor and that this process is associated with modification of the protein and lipid, and with the oxidation of α‐tocopherol to α‐tocopherol quinone.

The role of glutathione in the transport and catabolism of nitric oxide.

Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular environments containing 5–10 mM glutathione. Nitric oxide reacts with glutathione under aerobic conditions generating S‐nitrosoglutathione (GSNO). GSNO reacts with glutathione (k = 8.3 × 10−3 M−1 · s−1) to generate nitrous oxide and glutathione disulfide (GSSG). Anaerobically, glutathione reacts with nitric oxide generating nitrous oxide and GSSG (k = 4.8 × 10−4 s−1 at 5 mM GSH). In both aerobic and anaerobic situations the nitroxyl anion may be an intermediate in the synthesis of nitrous oxide and, under aerobic conditions, nitroxyl anion may generate peroxynitrite. We present a hypothesis for the intracellular interaction between nitric oxide and glutathione.

Biological chemistry and clinical potential of S-nitrosothiols.

S-Nitrosothiols are endogenous metabolites of nitric oxide that have been detected in extra- and intracellular spaces. Many biological functions of S-nitrosothiols have been described that can be categorized as being due to one or more of the following: (i) nitric oxide release, (ii) transnitrosation, (iii) S-thiolation, and (iv) direct action. This emphasizes the fact that S-nitrosothiols are more than simply nitric oxide donors. Many of the biological functions that have been described for S-nitrosothiols have clinical correlates. This review describes the biological chemistry, biological actions, and clinical potential of these compounds.