3-nitrotyrosine and Hypercapnia

Hypercapnia is regularly observed in chronic lung disease, such as bronchopulmonary dysplasia in preterm infants. Hypercapnia results in increased nitric oxide synthase activity and in vitro formation of nitrates. Neural vasculature of the immature subject is particularly sensitive to nitrative stress. We investigated whether exposure to clinically relevant sustained high CO(2) causes microvascular degeneration in the newborn brain by inducing nitrative stress, and whether this microvascular degeneration has an impact on brain growth. Newborn rat pups were exposed to 10% CO(2) as inspired gas (Pa(CO(2)) = 60-70 mmHg) starting within 24 h of birth until postnatal day 7 (P7). Brains were notably collected at different time points to measure vascular density, determine brain cortical nitrite/nitrate, and trans-arachidonic acids (TAAs; products of nitration) levels as effectors of vessel damage. Chronic exposure of rat pups to high CO(2) (Pa(CO(2)) approximately 65 mmHg) induced a 20% loss in cerebrovascular density at P3 and a 15% decrease in brain mass at P7; at P30, brain mass remained lower in CO(2)-exposed animals. Within 24 h of exposure to CO(2), brain eNOS expression and production of nitrite/nitrate doubled, lipid nitration products (TAAs) increased, and protein nitration (3-nitrotyrosine immunoreactivity) was also coincidently augmented on brain microvessels (lectin positive). Intracerebroventricular injection of TAAs (10 microM) replicated cerebrovascular degeneration. Treatment of rat pups with NOS inhibitor (L-N(omega)-nitroarginine methyl ester) or a peroxynitrite decomposition catalyst (FeTPPS) prevented hypercapnia-induced microvascular degeneration and preserved brain mass. Cytotoxic effects of high CO(2) were reproduced in vitro/ex vivo on cultured endothelial cells and sprouting microvessels. In summary, hypercapnia at values frequently observed in preterm infants with chronic lung disease results in increased nitrative stress, which leads to cerebral cortical microvascular degeneration and curtails brain growth.

Topics: Animals; Animals, Newborn; Brain; Hypercapnia; Neurodegenerative Diseases; Nitrates; Nitric Oxide Synthase Type III; Nitrites; Nitroarginine; Oxygen; Rats; Rats, Sprague-Dawley; Tyrosine

Permissive hypercapnia, achieved using low tidal volume ventilation, has been an effective protective strategy in patients with acute respiratory distress syndrome. To date, no such protective effect has been demonstrated for the chronic neonatal lung injury, bronchopulmonary dysplasia. The objective of our study was to determine whether evolving chronic neonatal lung injury, using a rat model, is resistant to the beneficial effects of hypercapnia or simply requires a less conservative approach to hypercapnia than that applied clinically to date. Neonatal rats inhaled air or 60% O2 for 14 days with or without 5.5% CO2. Lung parenchymal neutrophil and macrophage numbers were significantly increased by hyperoxia alone, which was associated with interstitial thickening and reduced secondary crest formation. The phagocyte influx, interstitial thickening, and impaired alveolar formation were significantly attenuated by concurrent hypercapnia. Hyperoxic pups that received 5.5% CO2 had a significant increase in alveolar number relative to air-exposed pups. Increased tyrosine nitration, a footprint for peroxynitrite-mediated reactions, arteriolar medial wall thickening, and both reduced small peripheral pulmonary vessel number and VEGF and angiopoietin-1 (Ang-1) expression, which were observed with hyperoxia, was attenuated by concurrent hypercapnia. We conclude that evolving chronic neonatal lung injury in a rat model is responsive to the beneficial effects of hypercapnia. Inhaled 5.5% CO2 provided a significant degree of protection against parenchymal and vascular injury in an animal model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a phagocyte influx.

Topics: Actins; Air; Animals; Animals, Newborn; Blood Gas Analysis; Blood Vessels; Carbon Dioxide; Elastin; Fluorescent Antibody Technique; Heart Rate; Hypercapnia; Lung; Lung Injury; Neutrophils; Organ Size; Peroxidase; Rats; Subcellular Fractions; Tyrosine; von Willebrand Factor

Chronic lung hypoxia results in hypoxic pulmonary hypertension. Concomitant chronic hypercapnia partly inhibits the effect of hypoxia on pulmonary vasculature. Adult male rats exposed to 3 weeks hypoxia (Fi(02)=0.1) combined with hypercapnia (Fi(C02)=0.04-0.05) had lower pulmonary arterial blood pressure, increased weight of the right heart ventricle, and less pronounced structural remodeling of the peripheral pulmonary arteries compared with rats exposed only to chronic hypoxia (Fi(02)=0.1). According to our hypothesis, hypoxic pulmonary hypertension is triggered by hypoxic injury to the walls of the peripheral pulmonary arteries. Hypercapnia inhibits release of both oxygen radicals and nitric oxide at the beginning of exposure to the hypoxic environment. The plasma concentration of nitrotyrosine, the marker of peroxynitrite activity, is lower in hypoxic rats exposed to hypercapnia than in those exposed to hypoxia alone. Hypercapnia blunts hypoxia-induced collagenolysis in the walls of prealveolar pulmonary arteries. We conclude that hypercapnia inhibits the development of hypoxic pulmonary hypertension by the inhibition of radical injury to the walls of peripheral pulmonary arteries.

Topics: Animals; Blood Pressure; Chronic Disease; Disease Models, Animal; Hypercapnia; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung Injury; Male; Oxidative Stress; Pulmonary Artery; Rats; Rats, Wistar; Time Factors; Tyrosine

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO(2). In the presence of CO(2), lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion of L-[U-(14)C]arginine to L-[U-(14)C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 +/- 3 vs. 6 +/- 1 (SE) nmol. 6.5 h(-1). 10(6) AMs(-1)]. Western blotting studies of immunoprecipitated SP-A indicated that CO(2) enhanced SP-A nitration by AMs and decreased carbonyl formation. CO(2) (0-1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO(2). Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr(164) and Tyr(166)) in the absence of CO(2) and three tyrosines (Tyr(164), Tyr(166), and Tyr(161)) in the presence of 1.2 mM CO(2). These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO(2) increased nitration, at least partially, by enhancing enzymatic nitric oxide production.

Topics: Carbon Dioxide; Cells, Cultured; Enzyme Activation; Epithelial Cells; Humans; Hypercapnia; Lipid Metabolism; Lipopolysaccharides; Macrophages, Alveolar; Mass Spectrometry; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrites; Oxidation-Reduction; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Respiratory Distress Syndrome; Tyrosine

Ventilator strategies allowing for increases in carbon dioxide (CO(2)) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O(2)(-)), nitric oxide (.NO), and their product peroxynitrite (ONOO(-)). The reaction of CO(2) and ONOO(-) can yield the nitrosoperoxocarbonate adduct ONOOCO(2)(-), a more potent nitrating species than ONOO(-). Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of.NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO(2) (hypercapnia) revealed a significant increase in.NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-L-arginine. Our studies reveal that hypercapnia modifies.NO-dependent pathways to amplify cell injury. These results affirm the underlying role of.NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.

Topics: Animals; Apoptosis; Carbon Dioxide; Cells, Cultured; Cytokines; Fetus; Hypercapnia; Interferon-gamma; Interleukin-1; Lipopolysaccharides; Nitrates; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Pulmonary Alveoli; Rats; Respiratory Mucosa; Tumor Necrosis Factor-alpha; Tyrosine