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_Dr. Eiserich_eiserich-3

_Dr. Eiserich_eiserich-3 - Nitric Oxide in the Lung Jason P...

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Unformatted text preview: Nitric Oxide in the Lung Jason P. Eiserich Department of Internal Medicine, University of California, Davis E-mail: [email protected] Tel.: 752-4010 ¥ N ¥ ¥ NITRIC OXIDE ¥ ¥ O¥ ¥ Three Nitric Oxide Synthase isoforms NOS1 NOS1 nNOS Constitutive; present in e.g. Constitutive; neurons; role in memory, mood, etc. neurons; Inducible; present in e.g. leukocytes; Inducible; role in inflammation, resistance against pathogens. against Constitutive; present in e.g. Constitutive; endothelial cells; role in vasodilation. NOS2 NOS2 iNOS NOS3 NOS3 eNOS How can we measure airway NO production? q NO in expired breath NO metabolites (nitrite/nitrate) q Condensed q exhalates q (Induced) sputum q Bronchoalveolar lavage fluids NO in the lung: where does it come from? Bronchial and alveolar epithelium (NOS2) Neuronal cells (NOS1) Pulmonary endothelium (NOS3) Inflammatory cells (alveolar macrophages) (NOS2) Contribution of type I NOS to expired gas NO and bronchial responsiveness in mice De Sanctis et al. (1997) Am. J. Physiol. 273: L883-L888. nNOS +/+ Expired NO levels _____________________ nNOS +/+ 6.3 ± 0.9 ppb nNOS -/- nNOS -/3.9 ± 0.4 ppb _____________________ Airway production of nitric oxide Steudel et al. (2000) Am. J. Respir. Crit. Care Med. 162, 1262-1267 Nitric oxide Role in host defense NADPH oxidase O2 O2•– gp91 p22 Rap1A gp91 rac2 p22 Rap1A OPO3 OPO3 OPO3 rac2 p47 p67 p67 p47 p40 OH OH OH p40 Resting Activated Role of NADPH oxidase in host defense q Genetic defect in NADPH oxidase, by deficiency of either p47PHOX, p67PHOX, p22PHOX, or gp91PHOX. Results in chronic granulomatous disease (CGD). CGD leukocytes are unable to consume oxygen, and do not produce H2O2 and O2•– upon activation. Patients with CGD typically have clinical symptoms early in life, and recurrent infections that can lead to death during childhood. q q Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells T.J. Kelley and M.L. Drumm, J. Clin. Invest. 102: 1200-1207, 1998 non-CF non-CF CF CF Cystic Fibrosis: A genetic defect in CFTR Effect on salt and fluid balance Mucous Isotonic absorption Hypotonic fluid Abnormal ASL ion concentration Isotonic hyper absorption Mucoid Pseudomonas Salt absorption Isotonic secretion Defective secretion NORMAL CF Nitric oxide and cystic fibrosis q Exhaled NO levels and epithelial NOS2 expression are Exhaled generally subnormal in CF patients. generally Reduced NO levels may explain failure in fighting infections Reduced with Pseudomonas Aeruginosa. Pseudomonas Cystic fibrosis patients commonly have exaggerated activation Cystic of airway inflammation; possibly related to subnormal NO of q q Antimicrobial functions of NOS2 Studies with NOS2-deficient mice Benefit to host Mycobacterium tuberculosis Leishmania major Listeria monocytogenes Toxoplasma gondii Ectromelia virus Staphylcoccus aureus Detriment to host Influenza virus pneumonitis Immunosuppression associated with Mycobacterium avium infection No major contribution Plasmodium chabaudi Trypanosoma cruzi Pseudomonas aeruginosa Legionella pneumophilia Chlamydia trachomatis Deficiency in both gp91phox and NOS2 Shiloh et al. (1999) Immunity 10, 29-38 q NOS2–/– and gp91phox–/– mice do not develop spontaneous infections. In contrast, gp91phox–/–/NOS2–/– mice spontaneously developed massive contrast, abscesses in the GI tract, caused by normal flora. abscesses Compared to individual knockouts, gp91phox–/–/NOS2–/– mice less resistant to infections with S. typhimurium, E. coli, or L. monocytogenes. S. E. or L. Results suggest that phox and NOS2 are compensatory in resistance to Results indigenous flora or invading organisms indigenous q q Physiological function Cell signaling NADPH oxidase O2•–/H2O2 Inflammation Bacterial killing Tissue injury Physiological role Vasodilation, neurotransmission NO synthase NO•/NOx? Host defense, inflammation Cell and tissue injury Nitric oxide Role in inflammation Nitric oxide and inflammation: The answer is blowing in the wind Lundberg et al. (1999) Nature Medicine 3:30 q q Measurement of airborne NO to monitor mucosal NO Measurement production in hollow organs. production Luminal levels of NO are dramatically elevated during Luminal inflammation: inflammation: x x x Asthma: 2- to 10-fold increase Inflammatory bowel disease: 20- to 200-fold increase Cystitis: 30- to 100-fold increase NO and inflammation Anti-inflammatory Anti-inflammatory ↓ leukocyte adhesion to endothelium endothelium ↓ P-selectin expression P-selectin ↓ microvascular thrombosis microvascular ↓ lymphocyte proliferation lymphocyte ↓ mast cell degranulation mast ↓ phagocyte oxidant production production ↓ activation of COX activation ↓ activation of NF-kB, AP-1 activation Pro-inflammatory ↑ vasodilation and vascular leakiness vascular ↑ hypotension or vascular collapse collapse ↑ cytotoxicity cytotoxicity ↑ activation of COX activation ↑ formation of RNS formation Biological fates of nitric oxide Fe NO • Fe-NO O2 NO2• N2O3 ONOO– Activation of guanylyl cyclase, cGMP-dependent signaling NO2– NO3– O2•– ONOOH* NO3– Superoxide anion Peroxynitrite anion Peroxynitrous acid Nitrate •– O2 + NO – • k = 6 x 109 M–1 .sec–1 ONOO H+ pKa = 6.8 ONOOH [OH• + NO2• ] – NO3 +H + Nitration of protein tyrosine residues H OH OH N O O Tyrosine pK a = 10 3-Nitrotyrosine p K a = 7.5 Tyrosine nitration in vivo: q q q q q q q q q ..it happens! Atherosclerosis Atherosclerosis Rheumatoid arthritis Inflammatory bowel disease Ulcerative colitis Celiac disease Allograft rejection Heliobacterium Pylori gastritis Heliobacterium gastritis Preeclampsia Septic shock with renal failure q q q q q q q q q Acute lung injury Iodopathic pulmonary fibrosis Perennial nasal allergy Cystic fibrosis Asthma Alzheimer’s disease Amyotrophic lateral sclerosis Multiple sclerosis Cigarette smoking OVA-induced airway inflammation in mice 3-Nitrotyrosine staining in the lung Normal mice Deficient in eosinophil peroxidase Duguet, A. et al. 2001. Am J Respir Crit Care Med 164, 1119-1126 3-Nitrotyrosine staining in BAL cells Duguet, A. et al. (2001) Am J Respir Crit Care Med 164, 1119-1126 Tyrosine nitration q Associated with the presence of inflammatory-immune Associated cells cells Dependent on oxidant mechanisms induced by heme Dependent peroxidases (myeloperoxidase, eosinophil peroxidase) peroxidases Does not require induction of NOS2 or increased NO Does synthesis; basal levels are sufficient synthesis; q q NO• metabolism during inflammation Myeloperoxidase (MPO) iNOS Lysozyme NO• H2O2 Cl– NO2– HOCl NO2– NO2• ClNO2 NO2• MPO O2•– NO• NADPH oxidase O2 ONOO– Tyrosine nitration: functional implications? q Induced by various biological pathways, including Induced enzymatic mechanisms (heme peroxidases) enzymatic Nitration of essential tyrosine residues (i.e. Nitration phosporylation sites, protein tyrosine radicals) can affect protein structure and function affect Potentially reversible, based on enzymatic Potentially “nitrotyrosine dinitrase” or “protein nitratase” activity in some tissues in q q Functional importance of tyrosine nitration during airway inflammation is still unclear van der Vliet et al. (1999) Am. J. Respir. Crit. Care Med. 160, 1-9 Formation of S-nitrosothiols NO O2 ¥ O2¥Ð H+ , Fe(III) N2O3 RS Ð Fe(II)-NO RSÐ + RSÐ ONOOH RSNO Role of S-nitrosation in cell signaling q Activation of guanine nucleotide-binding protein p21ras Modulation of activation and/or DNA binding of transcription factors Modulation (AP-1, NF-κ B) (AP-1, Stimulation of tyrosine phosphorylation by inhibition of Stimulation phosphotyrosine protein phosphatases (PTPs) phosphotyrosine Regulation of apoptosis by inhibition of caspase activity Inhibition of epidermal growth factor receptor tyrosine kinase q q q q H2O2 Parallels between oxidative and nitrosative stress RSNO Oxidative stress GSH GSSG Nitrosative stress GSH GSNO thiol oxidation thiol nitrosation S-OH oxyR HO S-NO oxyR antioxidative and antinitrosative genes RSNO metabolizing activity Hausladen et al., Cell 86, 719 (1996) 2 2 metabolizing Increased Nitrosothiols in Exhaled Breath Condensate in Inflammatory Airway Diseases Corradi et al. (2001) Am. J. Respir. Crit. Care Med.163, 854-858 NO metabolites in asthmatics before and after allergen challenge Dweik et al.(2001) Proc. Natl. Acad. Sci. USA 98, 2622-2627 NO chemical events in asthma NO metabolism during inflammation q Reaction with superoxide to form peroxynitrite Increased consumption by activated heme proteins Increased (phagocyte peroxidases, cyclooxygenase, etc.) (phagocyte Altered metabolism of NO with increased formation Altered of nitrating RNS. of q q NOS2 in airway inflammation Beneficial effect LPS-induced neutrophil adhesion to endothelium Chronic allograft dysfunction Neutrophil inflammation following hyperoxia or ozone exposure LPS-induced lung injury and inflammation Acute allograft rejection Ozone-induced lung injury Allergic airways eosinophilia Influenza virus-induced pneumonia OVA-induced bronchial hyperreactivity LPS-induced mortality Detrimental effect No contribution A role for NOS1 in airways diseases? q OVA-induced airway hyperreactivity suppressed in OVA-induced NOS1-deficient mice NOS1-deficient NOS1 gene polymorphism linked to exhaled NO NOS1 and/or airway hyperreactivity in asthma, cystic fibrosis, and acute chest syndrome fibrosis, Mechanisms unknown; NOS1 may modulate NOS2 Mechanisms induction induction q q Problems/Limitations q Mice are not men. Strong induction of NOS2 in inflammatory-immune Mice cells in rodents, not in humans cells Animal disease models are imperfect. They don’t always reflect human Animal disease. Unconditional knockout of genes may induce compensatory gene Unconditional expression. This may mask anticipated effects. expression. Technical difficulties in quantitating S-nitrosation and tyrosine nitration. Technical Specific protein targets in vivo still unknown. in q q q Summary q NO production usually enhanced during inflammation, potentially as a NO host mechanism and as a feedback regulator of inflammation. E.g. inhaled NO often suppresses inflammation. inhaled NO metabolism during inflammation gives rise to enhanced production NO of RNS that induce tyrosine nitration and S-nitrosation reactions. -nitrosation Nitrosation may represent redox signaling mechanism and regulate Nitrosation gene expression, apoptosis; significance of tyrosine nitration is questionable. questionable. The overall contribution of NO to airway disease depends on many The factors, including temporal and spacial expression of NOS and activation of oxidant producing enzymes. activation q q q Recommended reading q Pitt, B. and St. Croix, C.M. (2001) Am. J. Respir. Cell Mol. Biol. 26, 6-9. Thomassen, M.J. and Kavuru, M.S. (2001) Int. Immunopharmacol 1, Thomassen, 1479-1490. 1479-1490. Nathan, C., and Shiloh, M.U. (2000) Proc. Natl. Acad. Sci. USA 97, Nathan, 8841-8848. 8841-8848. Van der Vliet, A., Eiserich, J.P., Shigenaga, M.K., and Cross, C.E. Van (1999) Am. J. Respir. Crit. Care Med. 160, 1-9. (1999) Gaston, B., Drazen, J.M., Loscalzo, J. and Stamler, J.S. (1994) Am. J. Gaston, Respir. Crit. Care Med. 149, 538-551. Respir. q q q q ...
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