Nitric oxide produced in endothelial cells affects vascular tone. To investigate the role of endothelial nitric oxide synthase (eNOS) in blood pressure regulation, we have generated mice heterozygous (؉͞؊) or homozygous (؊͞؊) for disruption of the eNOS gene. Immunohistochemical staining with anti-eNOS antibodies showed reduced amounts of eNOS protein in ؉͞؊ mice and absence of eNOS protein in ؊͞؊ mutant mice. Male or female mice of all three eNOS genotypes were indistinguishable in general appearance and histology, except that ؊͞؊ mice had lower body weights than ؉͞؉ or ؉͞؊ mice. Blood pressures tended to be increased (by approximately 4 mmHg) in ؉͞؊ mice compared with ؉͞؉, while ؊͞؊ mice had a significant increase in pressure compared with ؉͞؉ mice (Ϸ18 mmHg) or ؉͞؊ mice (Ϸ14 mmHg). Plasma renin concentration in the ؊͞؊ mice was nearly twice that of ؉͞؉ mice, although kidney renin mRNA was modestly decreased in the ؊͞؊ mice. Heart rates in the ؊͞؊ mice were significantly lower than in ؉͞؊ or ؉͞؉ mice. Appropriate genetic controls show that these phenotypes in F 2 mice are due to the eNOS mutation and are not due to sequences that might differ between the two parental strains (129 and C57BL͞6J) and are linked either to the eNOS locus or to an unlinked chromosomal region containing the renin locus. Thus eNOS is essential for maintenance of normal blood pressures and heart rates. Comparisons between the current eNOS mutant mice and previously generated inducible nitric oxide synthase mutants showed that homozygous mutants for the latter differ in having unaltered blood pressures and heart rates; both are susceptible to lipopolysaccharide-induced death.

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion͞4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calciumindependent L-citrulline formation), and nitrite ؉ nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion͞4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.

Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon y, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shockIn biological systems, nitric oxide (NO) is produced via the oxidation of L-arginine by enzymes known as nitric oxide synthases (NOSs). Three NOS isozymes have been described (for recent reviews see refs. 1 and 2). These include constitutively expressed neuronal (3, 4) and endothelial (5, 6) enzymes and a cytokine-induced enzyme found in macrophages (7), hepatocytes (8), and a variety of other cells (9, 10). Although the biological functions of these enzymes are not completely understood, neuronal NOS is thought to play a role in neurotransmission (11), endothelial NOS is involved in regulation ofvascular tone (12, 13), and inducible NOS (iNOS) is involved in immune defense (14,15). The constitutively synthesized neuronal and endothelial enzymes produce small amounts of NO in response to increases in intracellular calcium levels. In contrast, iNOS is synthesized de novo in response to a variety of inflammatory stimuli and produces large amounts of NO over prolonged periods of time (16). NO produced by iNOS has been shown to be beneficial through its antitumor (17, 18) and antimicrobial (15) activities, but it is also thought to cause tissue damage and contribute to pathology in a variety of inflammatory conditions including rheumatoid arthritis (19,20), inflammatory bowel disease (21), and septic shock (22,23).Septic shock is usually the result of a systemic Gram-negative bacterial infection and is characterized by hypotension and the failure of a number of organ systems, especially the liver, kidney, and heart (24). The bacterial membrane component, lipopolysaccharide (LPS), induces the production of host inflammatory mediators such as tumor necrosis factor a, interferon y (IFN-,y), and interleukin 1,B, which in turn cause an increase in the expression of iNOS. The large amount of NO produced by iNOS has been hypothesized to contribute to LPS-induced hypotension and mortality.To be...

Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3(-/-)) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.

The role of nitric oxide (NO) in lung injury remains unclear. Both beneficial and detrimental roles have been proposed. In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury. Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later. Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation. In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells. This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity. By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining. These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice.

Since the lung is repeatedly subjected to injury by pathogens and toxicants, maintenance of pulmonary homeostasis requires rapid repair of its epithelial surfaces. Ciliated bronchiolar epithelial cells, previously considered as terminally differentiated, underwent squamous cell metaplasia within hours after bronchiolar injury with naphthalene. Expression of transcription factors active in morphogenesis and differentiation of the embryonic lung, including ␤-catenin, Foxa2, Foxj1, and Sox family members (Sox17 and Sox2), was dynamically regulated during repair and redifferentiation of the bronchiolar epithelium after naphthalene injury. Squamous cells derived from ciliated cells spread beneath injured Clara cells within 6-12 h after injury, maintaining the integrity of the epithelium. Dynamic changes in cell shape and gene expression, indicating cell plasticity, accompanied the transition from squamous to cuboidal to columnar cell types as differentiation-specific cell markers typical of the mature airway were restored. Similar dynamic changes in the expression of these transcription factors occurred in ciliated and Clara cells during regeneration of the lung after unilateral pneumonectomy. Taken together, these findings demonstrate that ciliated epithelial cells spread and transdifferentiate into distinct epithelial cell types to repair the airway epithelium. Keywords: naphthalene; lung injury; transcription; pneumonectomy; bronchioleThe respiratory tract has an extensive cell surface that is directly exposed to inhaled gases, particles, and pathogens. A complex epithelium lines the airways, mediating gas exchange, mucociliary clearance, host defense, and surfactant homeostasis to maintain lung sterility and stability. While the adult lung is not mitotically active, respiratory epithelial cells can proliferate rapidly after injury to maintain lung structure and function.Models in which relatively rare subsets of nonciliated respiratory epithelial cells located in unique environments play critical roles in lung repair have been proposed (1-5). Krause and coworkers have provided evidence that extrapulmonary, bone marrow-derived cells migrate to the lung, contributing to the repair of the respiratory epithelium after injury (6). From a stochastic view, however, models in which rare progenitor cells account for the rapid and extensive repair of the lung are not compatible with the observed short period of proliferation and rapid restoration of epithelial surfaces that is observed after catastrophic injury caused by infection or toxicants. Rather, the remarkable repair capacity of the lung is more consistent with a model in (Received in original form August 30, 2005 and in final form September 30, 2005) This study was supported by NIH HL56387 (J.A.W.) and HL61646 (J

Purpose of review Lungs are extremely susceptible to injury, and despite advances in surgical management and immunosuppression, outcomes for lung transplantation are the worst of any solid organ transplant. The success of lung transplantation is limited by high rates of primary graft dysfunction (PGD) due to ischemia-reperfusion (IR) injury characterized by robust inflammation, alveolar damage and vascular permeability. This review will summarize major mechanisms of lung IR injury with a focus on the most recent findings in this area. Recent findings Over the past 18 months numerous studies have described strategies to limit lung IR injury in experimental settings, which often reveal mechanistic insight. Many of these strategies involved the use of various anti-oxidants, anti-inflammatory agents, mesenchymal stem cells, and ventilation with gaseous molecules. Further advancements have been achieved in understanding mechanisms of innate immune cell activation, neutrophil infiltration, endothelial barrier dysfunction, and oxidative stress responses. Summary Methods for prevention of PGD after lung transplant are urgently needed, and understanding mechanisms of IR injury is critical for the development of novel and effective therapeutic approaches. In doing so, both acute and chronic outcomes of lung transplant recipients will be significantly improved.

NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during wholeblood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.Introduction NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes (1, 2). For example, NO transfer from deoxygenated erythrocytes to glutathione ex vivo forms S-nitrosoglutathione (GSNO) (3). GSNO can signal acute vascular and central ventilatory effects characteristic of oxyhemoglobin desaturation (3-4) that are regulated by γ-glutamyl transpeptidase (GGT), GSNO reductase (GSNOR), and other enzymes (1, 3-6). However, direct measurement of S-nitrosothiol signaling in vivo has proven challenging because of the metabolism and tissue-specific localization of endogenous S-nitrosothiol species (1, 3, 4, 6). We have addressed these challenges by using N-acetylcysteine (NAC) as a bait reactant, allowing the stable NO transfer product, S-nitroso-N-acetylcysteine (SNOAC), to be distinguished by mass spectrometry (MS) from endogenous S-nitrosothiols. We report that NAC is converted to SNOAC in mice in vivo. Furthermore, chronic, systemic administration of either NAC or SNOAC to mice causes hypoxia-mimetic pulmonary arterial hypertension (PAH). These data reveal a previously unappreciated vascular toxicity of NAC and of S-nitrosothiols. Moreover, they suggest that S-nitrosothiol transfer reactions can signal hypoxia in vivo.PAH is characterized by increased pressure in the pulmonary arteries (PAs), increased RV weight, and thickening and remodeling of small PAs. Untreated human PAH can progress to right