During studies of the bactericidal action of nitric oxide (NO), we found that it reversibly inhibited the respiration of Escherichia coli and irreversibly inhibited the respiration of Helicobacter pylori. Peroxynitrite, a reaction product of NO and superoxide, irreversibly inhibited the respiration of both H. pylori and E. coli. H. pylori, but not E. coli, generated substantial amounts of superoxide radicals. These results suggest that NO directly inhibits the respiration of E. coli whereas it rapidly reacts with endogenously generated superoxide radicals in H. pylori. The resulting peroxynitrite inactivates the respiration of H. pylori. Nitric oxide (NO)1 is a multifunctional gaseous free radical produced by NO synthase in various types of cells, such as endothelial cells, neurons, neutrophils, and macrophages (1). Nitric oxide is also generated from nitrite in saliva and from food by microorganisms in the oral cavity as well as by nonenzymatic mechanisms under acidic conditions, such as in gastric juice (12). Because NO synthase is also present in gastric mucosa (13,14), physiological concentrations of NO in gastric juice are fairly high.We have shown that the biological activity of NO is augmented significantly by physiologically low levels of oxygen tension (2-5). Although NO plays important roles in defense mechanisms against enteric bacteria (6, 7), few studies have explored the mechanism of its bactericidal action at physiological levels of oxygen tension.Helicobacter pylori is a Gram-negative and microaerophilic bacterium that resides in the mucus layer overlying the gastric epithelium of the human stomach. This organism is thought to play essential roles in the pathogenesis of gastric inflammation, ulceration, and carcinogenesis (8 -11). Although H. pylori is exposed to fairly high concentrations of NO in gastric juice, which has low oxygen tension, the effect of NO on the metabolism of H. pylori remains to be defined. We therefore studied the effects of NO on the respiration of H. pylori and Escherichia coli under physiologically low levels of oxygen tension. MATERIALS AND METHODSReagents-Peroxynitrite solution and carboxy-2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (cPTIO) were obtained from Dojin Co. (Kumamoto, Japan). Mn-type superoxide dismutase (SOD) from E. coli and 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazol[1,2-␣]pyra zin-3-one (MCLA) were obtained from Sigma and Tokyo Kasei Kogyo Co. (Tokyo, Japan), respectively. Nitric oxide solution was prepared as described previously (5).Bacterial Strains and Their Culture-Two types of enteric bacteria, E. coli K-12 JM109 and H. pylori NCTC-11637, were used in all experiments. E. coli was cultured at 37°C with shaking in nutrient broth (Difco) containing 0.5% NaCl. H. pylori was cultured in Brucella broth containing 5% horse serum under a microaerophilic atmosphere, produced with the use of a gas pack BBL CampyPak (Becton, MD), at 37°C for 20 h as described previously (15). The cultured E. coli and H. pylori were harvested at the logarithmic...

Although S-nitrosoglutathione (GS-NO) and other S-nitrosothiols (RS-NO) exhibit activity attributable to nitric oxide (NO), the dynamic aspects of their metabolism remain to be elucidated. To determine the fates and functions of RS-NO, the stability of GS-NO was analyzed in plasma, and various fractions of liver and kidney. GS-NO was fairly stable under physiological conditions in plasma and buffer solutions. However, GS-NO was rapidly decomposed in the presence of either homogenates of rat liver and kidney or their supernatant fractions. The ability of the supernatants to decompose GS-NO remained unchanged after the removal of proteins and large molecular weight compounds. Physiological levels of reducing agents, such as reduced glutathione (GSH), ascorbic acid (AsA), and cysteine, also enhanced the decomposition of RS-NO; the order of their potency was AsA > cysteine >GSH. Considering their intra-cellular concentrations and potency, AsA might principally be responsible for the enhanced decomposition of GS-NO. NO, GS-NO, and related RS-NO inhibited the respiration of Ehrlich ascites tumor cells. The inhibitory effect of GS-NO was enhanced by the reducing agents (cysteine>AsA>GSH). Intravenously administered GS-NO exhibited a depressor action through some ascorbic acid enhancable mechanism. Thus, the metabolism and biological function of GS-NO and related RS-NO might be affected by AsA and other reducing agents.

To elucidate the role of nitric oxide (NO) in the metabolisms of enteric bacteria, its effect on the respiration and growth of Escherichia coli was examined. Respiration of E. coli was reversibly inhibited by NO particularly under low oxygen tensions. Growth of E. coli was also inhibited by NO more strongly under low oxygen tension than at its high concentration. Because the intestinal lumen is anaerobic, even a small amount of NO might strongly inhibit the energy metabolism and growth of E. coli and other enteric bacteria in vivo than in air atmospheric conditions in which oxygen tension is unphysiologically high.

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Glutathione is one of the most abundant thiols in mammalian tissues and plays important roles in the defense mechanism and detoxification of various metabolites, such as reactive xenobiotics and free radicals. Nitric oxide (NO) readily reacts with thiol compounds, thereby generating chemically stable S-nitrosothiols. Although endotoxin has been known to induce NO synthase in various organs, particularly liver and spleen, and enhances the production of NO, correlation between NO and glutathione metabolism in endotoxemic subjects remains to be elucidated. The present work examines the changes in NO and glutathione metabolism in endotoxemic rats. Administration of lipopolysaccharide (LPS) markedly decreased the glutathione levels in plasma and bile, whereas it decreased the hepatic level only slightly. NG-nitro-L-arginine (L-NNA), a NO synthase inhibitor, inhibited the LPS-induced decrease of glutathione in plasma and bile. Administration of LPS increased the biliary levels of gamma-glutamyl transpeptidase (gamma-GTP) without affecting its thiol levels. Acivicin, a gamma-GTP inhibitor, inhibited the LPS-induced decrease of glutathione in plasma and bile without affecting its hepatic levels. Analysis with the use of L-buthionine sulfoximine revealed that the turnover of hepatic glutathione significantly increased in LPS-treated rats by some L-NNA-inhibitable mechanism. These results suggest that endotoxin might enhance the NO production in the liver and other tissues and significantly modulate the interorgan metabolism of reduced glutathione.