Robert M. Jackson

Spatial mapping of pulmonary and vascular nitrotyrosine reveals the pivotal role of myeloperoxidase as a catalyst for tyrosine nitration in inflammatory diseases

Nitrotyrosine (NO(2)Tyr) formation is a hallmark of acute and chronic inflammation and has been detected in a wide variety of human pathologies. However, the mechanisms responsible for this posttranslational protein modification remain elusive. While NO(2)Tyr has been considered a marker of peroxynitrite (ONOO(-)) formation previously, there is growing evidence that heme-protein peroxidase activity, in particular neutrophil-derived myeloperoxidase (MPO), significantly contributes to NO(2)Tyr formation in vivo via the oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (.NO(2)). Coronary arteries from a patient with coronary artery disease, liver and lung tissues from a sickle cell disease patient, and an open lung biopsy from a lung transplant patient undergoing rejection were analyzed immunohistochemically to map relative tissue distributions of MPO and NO(2)Tyr. MPO immunodistribution was concentrated along the subendothelium in coronary tissue and hepatic veins as well as in the alveolar epithelial compartment of lung tissue from patients with sickle cell disease or acute rejection. MPO immunoreactivity strongly colocalized with NO(2)Tyr formation, which was similarly distributed in the subendothelial and epithelial regions of these tissues. The extracellular matrix protein fibronectin (FN), previously identified as a primary site of MPO association in vascular inflammatory reactions, proved to be a major target protein for tyrosine nitration, with a strong colocalization of MPO, NO(2)Tyr, and tissue FN occurring. Finally, lung tissue from MPO(-/-) mice, having tissue inflammatory responses stimulated by intraperitoneal zymosan administration, revealed less subendothelial NO(2)Tyr immunoreactivity than tissue from wild-type mice, confirming the significant role that MPO plays in catalyzing tissue nitration reactions. These observations reveal that (i) sequestration of neutrophil-derived MPO in vascular endothelial and alveolar epithelial compartments is an important aspect of MPO distribution and action in vivo, (ii) MPO-catalyzed NO(2)Tyr formation occurs in diverse vascular and pulmonary inflammatory pathologies, and (iii) extracellular matrix FN is an important target of tyrosine nitration in these inflammatory processes.

REACTIVE SPECIES MEDIATED INJURY OF HUMAN LUNG EPITHELIAL CELLS AFTER HYPOXIA-REOXYGENATION

This study tested the hypothesis that hypoxia exposure predisposed lung epithelial cells to reactive oxygen species-(ROS) mediated cellular injury. Human lung carcinoma cells (ATCC line H441) having epithelial characteristics (including lamellar bodies, surfactant protein [SP]-A, and SP-B) were cultured in air (air/5% CO 2) or hypoxia (< 1% O 2 /5% CO 2) for 0 to 24 hours before imposition of oxidant stress. Cellular manganese superoxide dismutase (MnSOD) activity (units/mg protein) decreased significantly after 24 hours of hypoxia. In normoxic culture after hypoxia, the cells produced increased ROS, detected as dichlorofluorescein (DCF) fluorescence and H 2 O 2 accumulation in medium. Antioxidants N-acetylcysteine (N-Ac) and ebselen inhibited increased DCF fluorescence after hypoxia. To test their ability to tolerate oxidant stress, some cells were incubated with antimycin A (100 μ M) and trifluorocarbonylcyanide phenylhydrazone (10 μ M) (anti A + FCCP), a mitochondrial complex III inhibitor and respiratory chain uncoupler, which together increase mitochondrial superoxide (O 2 -) and H 2 O 2 production. Lung epithelial cells preexposed to hypoxia released more lactate dehydrogenase (LDH) than normoxic controls in response to increased O 2 - production. Increased LDH release from hypoxia-preexposed cells treated with anti A + FCCP was inhibited by 1 mM N-Ac. Rotenone and myxothiazole increased DCF oxidation more in hypoxic than in normoxic cells, suggesting that mitochondrial electron transport complex I may have been altered by hypoxia preexposure.

Atrial natriuretic peptide attenuates the development of pulmonary hypertension in rats adapted to chronic hypoxia.

To test the hypothesis that chronic infusion of atrial natriuretic peptide (ANP) instituted before hypoxic exposure attenuates the development of pulmonary hypertension in hypoxia adapted rats, ANP (0.2 and 1.0 microgram/h) or vehicle was administered intravenously via osmotic minipump for 4 wk beginning before exposure to 10% O2 or to room air. Low dose ANP increased plasma ANP levels by only 60% of vehicle controls after 4 wk and significantly decreased mean pulmonary arterial pressure (MPAP) (P less than 0.01), the ratio of right ventricular weight to body weight (RV/BW) (P less than 0.01), and the wall thickness of small (50-100 microns) pulmonary arteries (P = 0.01) in hypoxia-adapted rats. ANP did not alter any of these parameters in air-control rats. High dose ANP increased plasma ANP levels by 230% of control and produced greater reductions in MPAP (P less than 0.001) and RV/BW) (P less than 0.05), but not in pulmonary arterial wall thickness, than the low dose. Neither dose of ANP altered mean systemic arterial pressure in either hypoxic or normoxic rats. The data demonstrate that chronic infusion of exogenous ANP at a dose that does not affect MPAP or RV weight in air-control rats attenuates the development of pulmonary hypertension and RV enlargement in rats adapted to chronic hypoxia.

Vasopressin Lowers Pulmonary Artery Pressure in Hypoxic Rats by Releasing Atrial Natriuretic Peptide

The authors previously demonstrated that arginine vasopressin (AVP) lowers pulmonary artery pressure in rats with hypoxic pulmonary hypertension by activation of the V1 receptor. The pulmonary depressor effect of AVP in hypoxia-adapted rats is not due to its effect on cardiac output. The current study tested two alternative hypotheses: that AVP lowers pulmonary artery pressure in the hypoxia-adapted lung by (1) dilating pulmonary vasculature directly, or (2) releasing atrial natriuretic peptide (ANP) from the heart. The first hypothesis was tested by injecting AVP into the pulmonary arteries of isolated, buffer perfused lungs and monitoring pulmonary artery pressure, and by exposing preconstricted pulmonary artery rings to graded doses of AVP and monitoring the tension generated. AVP caused minimal vasodilation in perfused lungs and only a small vasodilator effect in pulmonary artery rings. The second hypothesis was tested by injecting AVP (160 ng/kg) or vehicle intravenously in conscious hypoxia-adapted (4 weeks) or air control rats and measuring ANP in arterial blood and atria, and by testing pretreatment with the V1 receptor antagonist d(CH2)5 Tyr(Me)AVP (130 micrograms/kg) on the AVP-induced increase in plasma ANP. AVP produced a 7-fold increase in plasma ANP (209 +/- 33 to 1346 +/- 233 pg/ml; p less than 0.05) in hypoxia-adapted rats and a 5-fold increase in ANP (122 +/- 22 to 573 +/- 174 pg/ml; p less than 0.05) in air controls. ANP release was abolished by pretreatment of both groups with d(CH2)5 Tyr(Me)AVP. The AVP-induced ANP release came mainly from left atrium. These data strongly suggest that the pulmonary depressor effects of AVP in hypoxia-adapted rats is due to augmented V1 receptor-induced release of ANP from left atrium.

Review: Re-Expansion, Re-Oxygenation, and Rethinking

ABSTRACT In a 1902 American Journal of the Medical Sciences case report, Riesman described “albuminous expectoration” following thoracentesis, a phenomenon that is now recognized as re-expansion pulmonary edema (RPE). Both cellular and biochemical mechanisms that produce lung injury in RPE have been described recently. Pathophysiologically, this unilateral edematous lung injury resembles the adult respiratory distress syndrome (ARDS) because both are characterized by intra-alveolar-activated neutrophils and markedly increased lung capillary permeability. Biochemical mechanisms that operate in RPE are analogous to those in diverse re-oxygenation (reperfusion) injuries that have been described recently in the heart, kidney, brain, and intestine. Re-oxygenated lung tissue appears to produce excess superoxide and other cytotoxic oxygen metabolites, although lung xanthine oxidase, the commonly recognized source of these oxidants, is exceedingly low. Riesman’s critical analyses of the re-expansion edema fluid in his case provided an impetus for others to hypothesize that increased permeability pulmonary edema in RPE represented re-oxygenation injury of the lung microvasculature.

Survival, lung injury, and lung protein nitration in heterozygous MnSOD knockout mice in hyperoxia.

This study tested whether a strain of heterozygous Mn superoxide dismutase (SOD) knockout mice differed from wild types in response to lethal (100 or 85%) or sublethal (50 or 75%) oxygen exposures. Lung MnSOD activity was significantly (-40%) less in the heterozygous mice, and lung catalase activity was also significantly decreased. Total SOD activity, glutathione peroxidase, and glutathione reductase did not differ between heterozygous (+/-) and wild-type (+/+) mice. We exposed both heterozygous and wild-type mice to hyperoxia (50, 75, 85, or 100% oxygen) until death or for 48 hours to assess sublethal lung injury. Survival of the heterozygous and wild-type mice did not differ significantly in 100 or 85% oxygen. No mice of either genotype died in 50 or 75% oxygen (14-day exposures). Hyperoxia exposures significantly increased (by two-way ANOVA) the alveolar lavage protein concentration, percent neutrophils, and lung wet-dry/dry weight ratios. No significant differences occurred between the heterozygous and wild-type mice for any marker of injury at any oxygen level. Lavage fluid total nitrite concentrations did not differ at any oxygen level. Hyperoxia caused a similar degree of nitration of lung structural proteins detected by immunohistochemistry in both groups.

Modulation of rat lung Na+, K+-ATPase gene expression by hyperoxia

Rats exposed to 85% O2 for 5-7 days develop tolerance to otherwise lethal hyperoxia (100% O2). The rate of alveolar fluid clearance increases during adaptation to hyperoxia, due in part to increased alveolar epithelial sodium channel activity. In these studies, we have investigated molecular mechanisms leading to increased lung Na+,K(+)-ATPase activity in hyperoxia. We exposed adult rats to 85% O2 (sublethal hyperoxia) for 7 days, followed by 2, 3, or 4 days in 100% O2. Steady-state levels of the Na+,K(+)-ATPase alpha 1 and beta 1 subunit mRNAs increased in whole lung tissue during hyperoxia exposures. Stability of the Na+,K(+)-ATPase alpha 1 and beta 1 subunit mRNA messages in whole lung RNA did not change significantly. Thus, lung Na+,K(+)-ATPase gene expression in sublethal hyperoxia appears to be regulated in part at the transcriptional level. Alveolar epithelial type II (ATII) cell Na+,K(+)-ATPase alpha 1 and beta 1 subunit proteins, measured by quantitative immunofluorescence, increased significantly after sublethal hyperoxia and 100% O2 exposures. Increases in lung fluid clearance after sublethal hyperoxia are associated with increased ATII cell Na+,K(+)-ATPase protein and whole lung Na+,K(+)-ATPase mRNA expression, which correspond to previously described increases in epithelial sodium channel expression under these conditions.

Lodoxamide tromethamine prevents neutrophil accumulation in reexpansion pulmonary edema

Reexpansion pulmonary edema (RPE) is an acute, unilateral lung injury initiated by cytotoxic oxygen metabolites and temporally associated with an influx of polymorphonuclear neutrophils (PMNs); these toxic oxygen products appear to result from reoxygenation of chronically collapsed lung. Lodoxamide tromethamine (U-42585E) reduces infarct size after reperfusion of ischemic myocardium. The possible protective effects of lodoxamide in RPE were examined. Right lungs of rabbits were collapsed for 7 days by injection of air into the pleural space. Reexpansion was accomplished by chest tube with negative pressure in spontaneously ventilating rabbits. Twelve pairs of animals received either lodoxamide (20 mg/kg/h intravenously (i.v.) from 30 min before reexpansion until they were killed) or an equivalent volume of sterile saline. After 2 h, animals were killed by i.v. pentobarbital. Right and left lungs of six pairs of animals were lavaged with 25 ml saline each; the remaining six pairs of animals were studied by measurement of lung wet/dry weight ratio. Albumin concentrations in lavage fluid (BAL) of lodoxamide-treated animals were 243 ± 165 μg/ml in right lung and 29 ± 15 μg/ml in left lung (p < 0.03); albumin concentration in right lung BAL of untreated animals was 1,180 ± 319 μg/ml (p < 0.02 vs. lodoxamide-treated animals). PMN percentages in right BAL (3.8 ± 3.1) and left BAL (2.9 ± 2.2) did not differ in lodoxamide-treated animals (p > 0.65); PMN percentage in right BAL of untreated animals was 18.7 ± 2.9 (p < 0.001 vs. lodoxamide-treated animals). Incubation of PMNs with lodoxamide (1, 10, and 100 μg/ml) resulted in a dose-dependent decrease in directed migration (p < 0.005) but did not affect PMN adherence. Wet/dry weight ratio was not affected by lodoxamide administration. Thus, lodoxamide prevents PMN influx and decreases the alveolar-capillary protein leak in RPE, primarily by inhibiting PMN chemotaxis.

Hypoxia and kinase activity regulate lung epithelial cell glutathione

ABSTRACT The authors investigated the mechanisms by which hypoxia regulates glutathione (GSH) in lung epithelial cells, and specifically whether the mitogen-activated protein kinase (MAPK) system is involved in the response to hypoxia. Hypoxia decreased cellular GSH content and appeared to decrease the effect of N-acetylcysteine on repletion of GSH after hypoxia. Hypoxia decreased 2 key enzyme activities that regulate GSH synthesis, glutamate cysteine ligase (GCL) (E.C. 6.3.2.2) and glutathione synthase (GS) (E.C. 6.3.2.3). No hypoxia-dependent change occurred in GCL or GS protein expression on Western blots. When epithelial cells were transfected with an adenoviral vector that caused over expression of human catalase protein (Ad.Cat or Ad.mCat), GCL and GS activities did not decrease in hypoxia. Inhibition of p38MAPK (using SB203580) or extracellular signal-regulated kinase (ERK; PD98059) prevented the hypoxia-dependent decrease in GCL and GS activity. To seek in vivo correlation, the authors assayed total glutathione in lungs and livers from MK2−/− (homozygous knockout) mice. MK2−/− mice are presumably unable to phosphorylate heat shock protein 27 (Hsp27) normally, because of absent kinase (MK2) activity. Liver GSH content (expressed per mg protein) was 20%% less in MK2−/− mice than in nontransgenic Black 6 controls. Down-regulation of lung GSH content in hypoxia depends on peroxide tone of the cell and the p38MAPK system.

Rapid Communication: Arginine Vasopressin Lowers Pulmonary Arterial Pressure in Rats Adapted to Chronic Hypoxia

To determine whether pulmonary and systemic vascular responses to arginine vasopressin (AVP) are altered by hypoxic adaptation, AVP, and, as a control, phenylephrine were administered intravenously in graded doses to pentobarbital anesthetized rats that had been exposed to 10% O2 at ambient pressure or room air for 28 days. After a 30-minute interval, d(CH2)5 Tyr(Me)AVP (130 μg/kg), a specific V1 receptor antagonist of AVP, was injected, and AVP (160 ng/kg) was administered again 5 minutes after injection of d(CH2)5 Tyr(Me)AVP. Mean systemic arterial pressure (MSAP) and mean pulmonary artery pressure (MPAP) were monitored before and after injection of AVP and d(CH2)5 Tyr(Me)AVP. AVP had a significant depressor effect (maximal response = – 6.3 ± 0.5 mm Hg following administration of 160 ng/kg AVP) in the pulmonary vascular bed of animals with hypoxic pulmonary hypertension, but no significant effect in normoxic rats. Rats exposed to chronic hypoxia exhibited a blunted systemic pressor response to AVP compared to normoxic rats. In contrast, there were no significant differences in pulmonary and systemic pressor responses to phenylephrine between the hypoxic and normoxic groups. The effects of AVP on MSAP and MPAP were abolished by the specific AVP V1 receptor antagonist, indicating that these effects are V1 receptor mediated. Further study is needed to determine whether AVP is useful in the pharmacologic treatment of hypoxic pulmonary vasoconstriction.