Allen M. Miles

Superoxide Modulates the Oxidation and Nitrosation of Thiols by Nitric Oxide-derived Reactive Intermediates CHEMICAL ASPECTS INVOLVED IN THE BALANCE BETWEEN OXIDATIVE AND NITROSATIVE STRESS

Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O2 were nearly equivalent. The presence of superoxide dismutase reversed O2-mediated inhibition, while catalase had no effect. Increasing the flux of O2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and O2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.

Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes

It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG‐monomethyl‐L‐arginine (L‐NMMA)‐inhibitable conversion of 14C‐labeled L‐arginine to L‐[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L‐NMMA‐inhibitable nitrite (NO2−) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 × 106 cells/well) were unable to generate significant amounts of NO2− when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4‐ and 6‐h elicited rat PMNs increased 21‐ and 42‐fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4‐ and 6‐h elicited rat PMNs, respectively. Approximately 20–30% of the total iNOS activity was Ca2+‐dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 μM/106 cells for the 4‐ and 6‐h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation‐induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time‐dependent fashion and that 20–30% of the total inducible NOS is Ca2+‐dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

Reactive Oxygen Metabolites

There is a large body of circumstantial evidence to suggest that reactive oxygen metabolites are important mediators of the pathophysiology observed in a variety of inflammatory disorders. Intravenous administration of certain anti-oxidants with well-defined mechanisms of action, for example Superoxide dismutase and catalase, has been suggested as a possible mode of therapy for some inflammatory disorders. Unfortunately, the circulating half-lives of these enzymes are very short. This limits the use of these compounds to only very acute inflammatory conditions, or requires multiple injections or continuous infusion over long periods of time in more chronic conditions. The development and use of antioxidants with well-characterised mechanisms of action that can be administered over many days or weeks should prove useful in defining a role for reactive oxygen metabolites in inflammatory tissue injury. These agents may also be valuable as pharmacological agents in treating these disease processes.

ANTIOXIDANT PROPERTIES OF AMINOSALICYLATES

Publisher Summary This chapter discusses the techniques that may be used to assess the antioxidant properties of several different anti-inflammatory drugs including the aminosalicylates. The most popular method for measuring the superoxide (O 2 − ) scavenging properties of various compounds is the inhibition of O 2 − -mediated reduction of cytochrome. In most cases, this assay is entirely appropriate; however, it should be noted that some low molecular weight, easily oxidizable compounds reduce hemoproteins, such as cytochrome c in a superoxide-independent mechanism, thereby introducing an artifactually high background rate of reduction. There are a variety of methods available to measure the H 2 O 2 decomposing activity of various substances. All these methods are based on the ability of a hemoprotein peroxidase (usually horseradish peroxidase) to catalyze the H 2 O 2 -dependent oxidation of an electron-donating detector molecule. Hydroxyl radicals interact with certain carbohydrates, proteins, nucleotide bases, and lipids to produce peroxyl radicals (ROO) as intermediates. Peroxyl radicals are slightly less reactive than .OH and thus possess extended half-lives of seconds instead of nanoseconds.

Effects of aminosalicylates and immunosuppressive agents on nitric oxide-dependent N-nitrosation reactions

Recent studies have demonstrated that nitric oxide (NO) rapidly and spontaneously decomposes in oxygenated solutions to generate potent N-nitrosating agents. These electrophilic substances have been shown to mediate mutagenesis and carcinogenesis via the formation of aliphatic and aromatic nitrosamines. We have also demonstrated that extravasated neutrophils and macrophages produce significant amounts of N-nitrosating agents derived exclusively from NO. During the course of these studies, we found that certain antioxidants, including 5-aminosalicylic acid (5-ASA), inhibited the leukocyte-mediated N-nitrosation reaction. Because 5-ASA and other anti-inflammatory and immunosuppressive drugs are used to treat inflammatory bowel disease, we wondered if any of these other compounds might also modulate N-nitrosation reactions in vitro. Therefore, the objectives of this study were to assess the ability of aminosalicylates and certain immunosuppressive agents to inhibit NO-dependent N-nitrosation of a model aromatic amine (2,3-diaminonaphthalene) and to determine whether this inhibitory activity correlated with their oxidation potential. We found that the concentrations necessary to inhibit the N-nitrosation reaction by 50% (IC50) were 25, 50 and 100 microM for 5-ASA, olsalazine (dimeric 5-ASA) and sulfasalazine, respectively. In contrast, sulfapyridine, 4-ASA, N-acetyl-5-ASA, 6-mercaptopurine, azathioprine, and methotrexate were either much less effective or inactive at inhibiting the N-nitrosation reaction. Although 5-ASA was able to fully scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl, neither olsalazine nor sulfasalazine was found to be effective at scavenging this weak oxidant. We did find that olsalazine possessed an oxidation potential substantially less than that of sulfasalazine, suggesting that it may, in fact, scavenge more potent oxidizing agents such as the N-nitrosating agent. We conclude that 5-ASA and olsalazine inhibit NO-dependent N-nitrosation reactions by scavenging or decomposing the nitrosating agent(s). We propose that the secondary nitrogen unique to sulfasalazine interacts with the nitrosating agent to yield a secondary nitrosamine, thereby competing for N-nitrosation of our detector.

Pathophysiology and Reactive Oxygen Metabolites

Aerobic metabolism provides an organism with a distinct metabolic advantage in that it allows for the complete combustion of glucose to carbon dioxide and water, and in the process produces 36 moles of ATP. However, all aerobic organisms pay a price for this metabolic advantage. It is known that during normal metabolism a small but significant flux of reactive oxygen metabolites (ROMs) are produced by the mitochondrial electron transport chain and by a variety of different oxidases. More specialized cells such as erythrocytes may generate even greater amounts of ROMs via the spontaneous autooxidation of important biological substrates such as hemoglobin. Thus, virtually all aerobic organisms possess several different enzymatic and nonenzymatic antioxidants including superoxide dismutase (SOD) or SOD-like chelates, catalase and/or peroxidases, as well as a variety of metal binding proteins and low molecular weight antioxidants.

Stability of S-nitrosothiols in presence of copper, zinc-superoxide dismutase.

Publisher Summary This chapter presents a spectrofluorometric method used to quantify the effect of copper-containing proteins such as Cu, Zn- OD on the stability of nitrosothiol such as S-nitrosoglutathion (GSNO). GSNO regulate the hexose-monophosphate pathway in immune cells such as neutrophils. The procedure described in the chapter allows for the determination of concentrations of GSNO as low as 500 n M . This method can be applied for the quantification of a number of S-nitrosothiols, including S-nitroso cysteine, S nitrosocysteinylglycine, S-nitrosoalbumin, and S-nitrosohemoglobin. The effect of different enzymatic activities on the stability of S-nitrosothiols has been limited because of the low detection level of the methodology used. This simple, rapid, reliable, and sensitive method provides laboratories equipped with a spectrofluorometer the ability to evaluate certain aspects of the metabolism of S-nitrosothiols in the presence of physiologically relevant concentrations of glutathione (GSH).