For many diseases, mediation of pathogenesis by nitric oxide (NO) has been suggested. In this study, we explored NO-induced viral pathogenesis with a focus on nucleic acid damage as evidenced by 8-nitroguanosine formation in vivo. Wild-type mice and littermate mice deficient in inducible NO synthase (iNOS) were infected with influenza or Sendai virus. Formation of 8-nitroguanosine in virusinfected lungs was assessed immunohistochemically with an antibody specific for 8-nitroguanosine. Extensive nitration of RNA either treated with peroxynitrite or obtained from cultured RAW 264 cells expressing iNOS was readily detected by this antibody. Strong 8-nitroguanosine immunostaining was evident primarily in the cytosol of bronchial and bronchiolar epithelial cells of virus-infected wild-type mice but not iNOS-deficient mice. This staining colocalized with iNOS immunostaining in the lung. 8-Nitroguanosine staining disappeared after addition of exogenous authentic 8-nitroguanosine during the antibody reaction and after pretreatment of tissues with sodium hydrosulfite, which reduces 8-nitroguanosine to 8-aminoguanosine. NO was generated in excess in lungs of wild-type mice but was eliminated in iNOSdeficient mice after virus infection; this result also correlated well with formation of 8-nitroguanosine and 3-nitrotyrosine. One consequence of the lack of iNOS expression was marked improvement in histopathological changes in the lung and the lethality of the infection without effects on cytokine responses and viral clearance. It is intriguing that 8-nitroguanosine markedly stimulated superoxide generation from cytochrome P450 reductase and iNOS in vitro. The present data constitute a demonstration of 8-nitroguanosine formation in vivo and suggest a potential role for NO-induced nitrative stress in viral pathogenesis.

Host defense functions of nitric oxide (NO) are known for many bacterial infections. In this study, we investigated the antimicrobial effect of NO in murine salmonellosis by using inducible NO synthase (iNOS)-deficient mice infected with an avirulent or virulent Salmonella enterica serovar Typhimurium strain. All iNOS-deficient mice died of severe septicemia within 6 days after intraperitoneal injection with an avirulent strain (LT2) to which wild-type mice were highly resistant; 50% lethal doses (LD 50 s) of the LT2 strain for iNOS-deficient and wild-type mice were 30 CFU and 7 ؋ 10 4 CFU, respectively. Lack of NO production in iNOS-deficient mice was verified directly by electron spin resonance spectroscopy. Bacterial yields in liver and blood were much higher in iNOS-deficient mice than in wild-type mice throughout the course of infection. Very small amounts of a virulent strain of serovar Typhimurium (a clinical isolate, strain Gifu 12142; LD 50 , 50 CFU) given orally caused severe septicemia in iNOS-deficient animals; wild-type mice tolerated higher doses (LD 50 , 6 ؋ 10 2 CFU). Histopathology of livers from infected iNOS-deficient mice revealed extensive damage, such as diffuse hepatocellular apoptosis and increased neutrophil infiltration, but livers from infected wild-type mice showed a limited number of microabscesses, consisting of polymorphonuclear cells and macrophages and low levels of apoptotic change. The LT2 strain was much more susceptible to the bactericidal effect of peroxynitrite than the Gifu strain, suggesting that peroxynitrite resistance may contribute to Salmonella pathogenicity. These results indicate that NO has significant host defense functions in Salmonella infections not only because of its direct antimicrobial effect but also via cytoprotective actions for infected host cells, possibly through its antiapoptotic effect.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 39,59-cyclic monophosphate (8-bromocGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-59-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells.

Cell-surface Toll-like receptors (TLRs) initiate innate immune responses, such as inducible nitric oxide synthase (iNOS) induction, to microorganisms' surface pathogens. TLR2 and TLR4 play important roles in gastric mucosa infected with Helicobacter pylori (H. pylori), which contains lipopolysaccharide (LPS) as a pathogen. The present study investigates their physiological roles in the innate immune response of gastric epithelial cells to H. pylori-LPS. Changes in the expression of iNOS, TLR2, and TLR4, as well as downstream activation of mitogen-activated protein kinases and nuclear factor-kappaB (NF-kappaB), were analyzed in normal mouse gastric mucosal GSM06 cells following stimulation with H. pylori-LPS and interferon-gamma. Specific inhibitors for mitogen-activated protein kinases, NF-kappaB, and small interfering RNA for TLR2 or TLR4 were employed. The immunohistochemistry of TLR2 was examined in human gastric mucosa. H. pylori-LPS stimulation induced TLR2 in GSM06 cells, but TLR4 was unchanged. TLR2 induction resulted from TLR4 signaling that propagated through extracellular signal-related kinase and NF-kappaB activation, as corroborated by the decline in TLR4 expression on small interfering RNA treatment and pretreatment with inhibitors. The induction of iNOS and the associated nitric oxide production in response to H. pylori-LPS stimulation were inhibited by declines in not only TLR4 but also TLR2. Increased expression of TLR2 was identified in H. pylori-infected human gastric mucosa. TLR4 signaling initiated by H. pylori-LPS and propagated via extracellular signal-regulated kinase and NF-kappaB activation induced TLR2 expression in gastric epithelial cells. Induced TLR2 cooperated with TLR4 to amplify iNOS induction. This positive correlation may constitute a mechanism for stimulating the innate immune response against various bacterial pathogens, including H. pylori-LPS.