The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon y. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by Nwmonomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with 02-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with 02-which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.

The purpose of this study was to investigate the induction of inducible nitric oxide synthase (iNOS) mRNA in the brain tissue of rats and mice under the following experimental conditions: in rats infected with borna disease virus and rabies virus, in mice infected with herpes simplex virus, and in rats after the induction of experimental allergic encephalitis. The results showed that iNOS mRNA, normally nondetectable in the brain, was present in animals after viral infection or after induction of experimental allergic encephalitis. The induction of iNOS mRNA coincided with the severity of clinical signs and in some cases with the presence of inflammatory cells in the brain. The results indicate that nitric oxide produced by cells induced by iNOS may be the toxic factor accounting for cell damage and this may open the door to approaches to the study of the pathogenesis of neurological diseases.The mechanisms involved in the development of central nervous system (CNS) lesions are readily understood only in those pathological conditions in which there is evidence that a virus destroys its target cell as a direct cytopathic consequence of viral replication (e.g., polio virus or other neurotrophic viruses; refs. 1 and 2). However, the effector mechanisms involved in tissue damage associated with a far wider variety of viral infections of the CNS, involving such viruses as the measles and rubella viruses as well as human immunodeficiency virus 1, are unclear. Likewise, the mediators responsible for the CNS damage associated with chronic neurologic diseases such as multiple sclerosis remain the subject of speculation. Interestingly, morphologic analyses have revealed that lesions in affected brain tissues are frequently surrounded by infiltrating inflammatory cell populations. Although the precise role that these cells play in CNS pathology is the subject of ongoing investigation, previous studies have focused on the ability of leukocyte populations to generate proinflammatory cytokines (e.g., interleukin 1, tumor necrosis factor, etc.), neurotoxins (e.g., quinolinic acid), or reactive oxygen intermediates (3). Recently, increased attention has focused on the possibility that reactive nitrogen intermediates (NOI) generated by a family of cytochrome P-450 reductase-like enzymes, the nitric oxide synthases (NOS), directly damage host tissues in a diverse array of pathogenic states (4).To date, at least three NOS genes have been cloned and characterized, and these have been provisionally categorized on the basis of their sensitivity to regulation by Ca2+ transients (4). In this schema, NOS forms that bind calmodulin in a reversible Ca2+-dependent manner are termed the constitutive forms of NOS, and those forms of the enzyme that bind calmodulin tightly at resting [Ca2+] are termed inducible NOSs (iNOSs). After the addition of rapid-acting agonists, the constitutive NOS system generates only low levels of the NOI, nitric oxide (NO), whereas the iNOS system begins to generate NO several hours after exposure to cyto...

Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) We have reported (1) that Borna disease virus, rabies virus, and herpes simplex virus induce the increased expression of inducible nitric oxide synthase (iNOS) mRNA in the brains of intrathecally infected mice and rats. We and others (2) also found that the induction of experimental allergic encephalomyelitis resulted in a similar increase in iNOS mRNA expression, suggesting that a similar phenomenon may occur in the brains of patients with multiple sclerosis (MS). We investigated this hypothesis by detecting iNOS mRNA in extracts of brains from patients who died with MS and from extracts of brain tissues from patients who died from nonneurological diseases. We have also colocalized the iNOS expressing cells by using immunocytochemical techniques combined with reverse transcriptase (RT) driven-in situ PCR and demonstrated the presence of iNOS protein by the same double labeling procedures. Finally, as a surrogate marker for nitric oxide (NO) presence, we have immunocytochemically localized nitroty-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.rosine adducts in histological sections of brain from MS patients. MATERIALS AND METHODSSources and Selection of Specimens. The samples used in these studies were collected from patients with an antemortem diagnosis of MS based on clinical presentation and reconfirmed by histopathology after death. All MS samples were found to contain pathognomonic MS plaques; control brains were examined and found to be pathologically unremarkable. In most cases, autopsy samples were of frontal lobes, which provided mixed white and gray matter.In two of the MS cases, special efforts were made to preserve the tissue morphology. Small portions of the brain specimens were frozen in liquid nitrogen immediately after sectioning and then stored at -70°C. This procedure preserved the architecture of the brain significantly better than direct freezing of fresh brain specimens at -80°C, which results in ice crystal formation in the tissue and distorts its architecture.Cell Culture and Cytokine Activation. A549 human pulmonary epithelial cells were purchased from the American Type Culture Collection. Cells were grown in Ham's F-12 medium supplemented with 10% (vol/vol) fetal calf serum, penicillin (50 units/ml), streptomycin (50 ,ug/ml), and 5 ,tM L-glutamine. Cells were seeded into 6-well tissue culture clusters (Falcon) for Northern blot analysis or seeded into 8-well Lab-Tek culture slides (Nunc) for RT-in situ PCR. For those cultures stimulated for the expression of iNOS, interleukin 1(3 (IL-1,B at 100 units/ml), tumor necrosis factor a (TNF-a at 10 ng/ml), and interferon y (IFN-,y at 500 units/ml) were added for 8 h.For use in Northern blot analysis, the cells were scraped from the culture substrate, washed three ti...

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl− currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3−/−) mice, hypoxia-induced, but not submaximal noradrenaline–induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3−/− mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline–induced Ca2+ and contractile responses in PASMCs.

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCɛ activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCɛ protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression, prevented hypoxia-induced increases in total PKC and PKCɛ activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCɛ translocation peptide inhibitor or PKCɛ gene deletion decreased hypoxic increase in [Ca 2+ ] i in PASMCs, whereas the conventional PKC inhibitor GÖ6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCɛ, contributing to hypoxia-induced increase in [Ca 2+ ] i and contraction in PASMCs. KeywordsHypoxia; protein kinase C; reactive oxygen species; mitochondria; intracellular calcium; pulmonary artery smooth muscle cells Hypoxic pulmonary vasoconstriction (HPV) is observed in isolated lungs, pulmonary arteries, and pulmonary artery smooth muscle cells (PASMCs). The pulmonary circulation differs from the systemic circulation in response to oxygen tension; pulmonary arteries constrict to physiological hypoxia (~ 20-60 mmHg PO 2 ), whereas systemic arteries vasodilate. The mechanisms for these opposing responses to hypoxia appear to lie within the vascular SMCs. Hypoxia increases intracellular Ca 2+ concentration ([Ca 2+ ] i ) and contracts PASMCs. In contrast, SMCs from systemic arteries display decreased [Ca 2+ ] i and relax in response to hypoxia. The response of PASMCs to acute hypoxia involves calcium entry through voltagedependent and store-operated Ca 2+ channels, as well as Ca 2+ release from the sarcoplasmic reticulum [1][2][3][4][5][6][7][8]. Hypoxia-dependent changes in reactive oxygen species (ROS) concentration have been proposed to mediate HPV by several laboratories, although the details of this hypothesis differ greatly [9; 10]. However, the signaling pathways underlying artery-specific, acute hypoxic vasoconstriction remain to be fully elucidated. AnimalsPKCɛ −/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME); Swiss-Webster mice from Taconic (Germantown, NY). Glutathione peroxidase-1 (Gpx1) overexpression mice were generated and maintained as described previously [18]. All animal experiments were approved by the Institutional Animal Care and Use Committee of Albany Medical College. To examine the effects of pharmacological reagents, control experiments were carried out in cells or tissues from the same mice. For experimen...