Hypoxia increases glutathione redox cycle and protects rat lungs against oxidants

Preexposure to hypoxia increased survival and lung reduced glutathione-to-oxidized glutathione ratios (GSH/GSSG) and decreased pleural effusions in rats subsequently exposed to continuous hyperoxia. In addition, lungs from hypoxia-preexposed rats developed less acute edematous injury (decreased lung weight gains and lung lavage albumin concentrations) than lungs from normoxia-preexposed rats when isolated and perfused with hydrogen peroxide (H2O2) generated by xanthine oxidase (XO) or glucose oxidase (GO). In contrast, when perfused with elastase or exposed to a hydrostatic left atrial pressure challenge, lungs isolated from hypoxia-preexposed rats developed the same acute edematous injury as lungs from normoxia-preexposed rats. The mechanism by which hypoxia preexposure conferred protection against H2O2 appeared to depend on hexose monophosphate shunt (HMPS)-dependent increases in lung glutathione redox cycle activity. First, before perfusion with GO, lungs from hypoxia-preexposed rats had increased glutathione peroxidase and glucose 6-phosphate dehydrogenase (but not catalase or glutathione reductase) activities compared with lungs from normoxia-preexposed rats. Second, after perfusion with GO, lungs from hypoxia-preexposed rats had increased H2O2 reducing equivalents, as reflected by increased GSH/GSSG and NADPH/NADPH+, compared with lungs from normoxia-preexposed rats. Third, pretreatment of rats with an HMPS inhibitor, (6-aminonicotinamide) or a glutathione reductase inhibitor, [1,3-bis(2-chloroethyl)-1-nitrosourea] prevented hypoxia-conferred protection against H2O2-mediated acute edematous injury in isolated lungs. These findings suggest that increased detoxification of H2O2 by glutathione redox cycle and HMPS-dependent mechanisms contributes to tolerance to hyperoxia and resistance to H2O2 of lungs from hypoxia-preexposed rats.     

Xanthine oxidase is increased and contributes to paraquat-induced acute lung injury

Two lines of investigation suggested that xanthine oxidase- (XO) derived O2 metabolites contribute to paraquat- (PQ) induced acute lung injury. First, PQ treatment increased lung XO activity and decreased lung xanthine dehydrogenase activity. Second, lung albumin uptake increased compared with control values in untreated XO-replete but not tungsten-treated XO-depleted lungs in rats treated with PQ.     

Hyperoxia and self- or neutrophil-generated O2 metabolites inactivate xanthine oxidase

Xanthine oxidase (XO) and xanthine dehydrogenase (XD) activities decreased in lungs isolated from rats and cultured lung endothelial cells that had been exposed to hyperoxia. Purified XO activity also decreased after addition of a variety of chemically generated O2 metabolite species (superoxide anion, hydrogen peroxide, hydroxyl radical, or hypochlorous acid), hypoxanthine, or stimulated neutrophils in vitro. XO inactivation by chemically, self-, or neutrophil-generated O2 metabolites was decreased by simultaneous addition of various O2 metabolite scavengers but not their inactive analogues. Since XO appears to contribute to a variety of biological processes and diseases, hyperoxia- or O2 metabolite-mediated decreases in XO activity may be an important cellular control mechanism.     

Hyperoxia and xanthine dehydrogenase/oxidase activities in rat lung and heart

Cell injury from hyperoxia is associated with increased formation of superoxide radicals (O2-). One potential source for O2- radicals is the reduction of molecular O2 catalyzed by xanthine oxidase (XO). Physiologically, this reaction occurs at a relatively low rate, because the native form of the enzyme is xanthine dehydrogenase (XD) which produces NADH instead of O2-. Reports of accelerated conversion of XD to XO, and increased formation of O2- formation in ischemia-reperfusion injury, led us to examine whether hyperoxia, which is known to increase O2- radical formation, is associated with increased lung XO activity, and accelerated conversion of XD to XO. We exposed 3-month-old rats either to greater than 98% O2 or room air. After 48 h, we sacrificed the rats and measured XD and XO activities and uric acid contents of the lungs. We also measured the activities of the two enzymes in the heart as a control organ. We found that the activity of XD was not altered significantly by hyperoxia in rat lungs or hearts, but XO activity was markedly lower in the lung, whether expressed per whole organ or per milligram protein, and remained unchanged in the heart. Lung uric acid content was also significantly lower with hyperoxia. The decrease in lung XO activity may reflect inactivation of the enzyme by reactive O2 metabolites, possibly as a negative feedback mechanism. The concomitant decrease in uric acid content suggests either decreased production mediated by XO due to its inactivation or greater utilization of uric acid as an antioxidant. We examined these postulates in vitro using a xanthine/xanthine oxidase system and found that H2O2, but not uric acid, has an inhibitory effect on O2- formation in the system. We therefore conclude that hyperoxia is not associated with increased conversion of XD to XO, and that the exact contribution of XO to hyperoxic lung injury in vivo remains unclear.     

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.     

Polyethylene glycol-attached antioxidant enzymes decrease pulmonary oxygen toxicity in rats

When exposed continuously to hyperoxia (100% O2, 760 Torr barometric pressure), rats pretreated with polyethylene glycol (PEG)-attached superoxide dismutase and catalase (PEG-SOD + PEG-CAT) lived longer (79.1 + 7.6 h) than rats pretreated with saline (60.7 +/- 2.1 h) or PEG-inactivated-SOD + PEG-inactivated-CAT (62.3 +/- 1.6 h). Rats pretreated with PEG-SOD + PEG-CAT also had less hyperoxia-induced acute oxidative edematous lung injury, as assessed by increases in lung oxidized glutathione (GSSG) contents, pleural effusions, and lung lavage albumin concentrations than saline-pretreated rats. Rats pretreated with the long-lived conjugates PEG-inactivated-SOD + PEG-inactivated-CAT or PEG-albumin also had decreased acute oxidative edematous lung injury compared with rats pretreated with PEG, SOD + CAT + PEG, SOD + CAT, or saline. In vitro studies suggested that PEG itself may have contributed to protection by scavenging hydroxyl radical (.OH) but not superoxide (O2-.) or H2O2. Compared with more effective endogenous (via preexposure to hypoxia) or exogenous (via liposomes) means for increasing lung antioxidant enzymes, PEG enzymes are less protective against lung injury from continuous hyperoxia.     

O2 metabolites and neutrophil elastase synergistically cause edematous injury in isolated rat lungs

Addition of glucose oxidase (GO) increased H2O2 concentrations and decreased antielastolytic activities of beta-D-glucose containing perfusates of isolated rat lungs. Pretreatment with GO also caused acute edematous injury (increased lung weight gains, increased recovery of Ficoll in lung lavages, and increased pulmonary arterial pressures) in isolated lungs perfused with purified human neutrophil elastase (NE). Acute edematous injury in isolated lungs pretreated with GO and then NE exceeded levels found in lungs following addition of GO or NE alone or NE before GO. Simultaneous addition of catalase (an H2O2 scavenger) or methoxy-succinyl-L-alanyl-L-alanyl-prolyl-L-valine-chloromethyl ketone (an NE inhibitor, but not aminotriazole-inactivated catalase, N-tosyl-L-phenyl-alanine chloromethyl ketone (a chymotrypsin inhibitor) or N-alpha-p-tosyl-L-lysine chloromethyl ketone (a trypsin inhibitor), prevented acute edematous injury in isolated lungs perfused with both GO and NE. This observation indicated that injury was dependent on both H2O2 and NE, especially since the relative inactivating specificities of the inhibitors for H2O2 or NE, respectively, were confirmed under similar conditions in vitro. The synergistic nature of the interaction between H2O2 and NE-mediated injury was further clarified when GO- and NE-induced lung injury was prevented by addition of an oxidant-resistant NE inhibitor (Eglin-C), but not an oxidant-sensitive NE inhibitor (human alpha 1-protease inhibitor, alpha 1PI). Moreover, treatment with H2O2 also decreased the ability of alpha 1PI but not Eglin-C to decrease NE activity in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of neutrophils in xanthine/xanthine oxidase-induced oxidant injury in isolated rabbit lungs.

Reactive oxygen species have been shown to play an important role in the pathogenesis of lung injury. This study was designed to clarify the role of intrapulmonary neutrophils in the development of xanthine/xanthine oxidase (X/XO)-induced lung injury in isolated buffer-perfused rabbit lungs. We measured microvascular fluid filtration coefficient (K(f)) and wet-to-dry weight ratio to assess lung injury. X/XO induced a significant increase in K(f) and wet-to-dry weight ratio in neutrophil-replete lungs, whereas the lung injury was attenuated in neutrophil-depleted lungs. A neutrophil elastase inhibitor, ONO-5046, also attenuated the lung injury. In addition, X/XO induced a transient pulmonary arterial pressure (P(pa)) increase. The thromboxane inhibitor OKY-046 attenuated the P(pa) increase but did not alter the increase in permeability. Neutrophil depletion reduced the K(f) increase but had no effect on the P(pa) increase. These results suggest that intrapulmonary neutrophils activated by X/XO play a major role in development of the lung injury, that neutrophil elastase is involved in the injury, and that the X/XO-induced vasoconstriction is independent of intrapulmonary neutrophils.     

Responses of baboons to prolonged hyperoxia: physiology and qualitative pathology.

Cardiopulmonary responses to prolonged hyperoxia and their relationships to the development of lung pathology have not been fully characterized in primates. In this study, circulatory hemodynamics and pulmonary function, vascular permeability, and leukocyte sequestration were measured in male baboons after 100% O2 exposure and related to ultrastructural changes of lung injury by electron microscopy. Three groups of animals were exposed to 100% O2 in an exposure cage for 40, 66, and 80 h, respectively. A fourth group of animals was exposed in a cage for 80 h and then anesthetized and ventilated with 100% O2 for additional time. These animals were exposed for a total duration of 110 h or until death from the injury. Physiological responses to hyperoxia were characterized by decreases in total lung capacity and inspiratory capacity at 80 and 110 h. A significant increase in pulmonary leukocyte accumulation was noted by 80 h. Extravascular lung water and permeability surface-area product increased at 80 and 110 h. Cardiac output and stroke volume also decreased, and systemic vascular resistance increased after 80 and 110 h of hyperoxia. Histopathological changes were present in the lungs of all but the 40-h exposure group. Animals exposed for 66 h showed endothelial injury and neutrophil accumulation. By 80 h, animals showed endothelial cell destruction, interstitial edema, and type I cell injury. At 110 h, animals showed substantial destruction of endothelial and type I epithelial cells, exposure of alveolar basement membrane, congestion of capillaries, and substantial interstitial edema. The data indicate that histological changes by electron microscopy precede physiological responses to hyperoxic pulmonary injury in baboons by as much as 14 h and that the physiological responses to early hyperoxic injury are relatively insensitive to the pathological injury.     

Dimethylthiourea consumption reflects H2O2 concentrations and severity of acute lung injury

Even though dimethylthiourea (DMTU) effectively scavenges O2 metabolites in vitro, it is often unclear if scavenging of O2 metabolites is the mechanism by which DMTU decreases tissue injury in biological models. Since DMTU not only scavenges O2 metabolites but is also consumed in a dose-response manner following reaction with hydrogen peroxide (H2O2) in vitro, we wondered whether DMTU would also be consumed by O2 metabolites in biological systems and if DMTU consumption would then reflect O2 metabolite concentrations and O2 metabolite-mediated injury. Our results supported this possibility. We found that selected nonprotecting concentrations of DMTU were consumed in isolated rat lungs perfused with H2O2 and that the amounts of DMTU consumed reflected both the added amounts of H2O2 and the corresponding degrees of H2O2-induced acute edematous injury. DMTU consumption was relatively specific for reaction with H2O2 occurring in isolated lungs that were injured by H2O2 but not lungs injured by elastase, oleic acid, histamine, or a venous pressure challenge. Our results suggest that measurement of DMTU consumption may be useful for assessing the presence and toxicity of O2 metabolites and the specificity of the protective effects of DMTU in biological systems.     

Effects of neutrophil elastase inhibitor (ONO-5046) on lung injury after intestinal ischemia-reperfusion.

The underlying mechanisms of lung endothelial injury after intestinal ischemia-reperfusion (I/R) injury are not fully known. Here we investigated the effects of posttreatment with a neutrophil elastase inhibitor (NEI; ONO-5046) on lung injury after intestinal I/R injury in a rat model. Intestinal I/R was produced by 90 min of ischemia followed by either 60 or 240 min of reperfusion. For all experimental groups, the endothelial permeability index increased, neutrophil H(2)O(2) production increased in the pulmonary vasculature blood, neutrophil counts increased in bronchoalveolar lavage fluid (BALF), and the cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels were increased in BALF after 240 min (P < 0.01). In rats treated with NEI from 60 min after reperfusion, the lung endothelial permeability index was significantly reduced (P < 0.05), whereas neutrophil H(2)O(2) production in pulmonary vasculature blood and neutrophil count in BALF were significantly suppressed by NEI (P < 0.05 and P < 0.01, respectively). In addition, NEI significantly suppressed the increase of CINC-1 and CINC-3 levels in BALF (P < 0.05). Our study clearly indicates that posttreatment with NEI reduces neutrophil activation in the pulmonary vessels and neutrophil accumulation in the lungs and suggests that ONO-5046, even when administered after the primary intestinal insult, can prevent the progression of lung injury associated with intestinal I/R.     

IL-4 increases surfactant and regulates metabolism in vivo

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.     

Production of arachidonic acid metabolites by endothelial cells in hyperoxia

This study investigated the response of bovine pulmonary artery endothelial cells to incubation in hyperoxia (95% O2-5% CO2). Changes in cell number and morphology, release of lactate dehydrogenase, and production of arachidonic acid metabolites were assessed during continuous exposure of confluent endothelial monolayers to air (air-5% CO2, "controls") or O2 (95% O2-5% CO2, "O2-exposed") for periods of 12-72 h. Control monolayer cell numbers remained constant (approximately 2,000,000 cells/flask), whereas the number of cells in O2-exposed monolayers decreased progressively to 30% of controls (P less than 0.01) by 72 h. As assessed by radioimmunoassay, both control and O2-exposed cells produced the prostacyclin metabolite, 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin F2 alpha (PGF2 alpha), but no thromboxane metabolite (TxB2) was detected. The O2-exposed cells released significantly more 6-keto-PGF1 alpha and PGF2 alpha than control cells when apparent net production rates over the entire 72-h period were compared. In addition, both control and O2-exposed (48 h) endothelial monolayers released immunoreactive leukotriene B4 (LTB4) on stimulation with calcium ionophore (10 microM A23187). As with the cyclooxygenase products, O2-exposed cells released more immunoreactive LTB4 than did controls. Both cyclooxygenase and lipoxygenase metabolites of arachidonic acid are released by cultured endothelial cells during the development of O2 toxicity.     

Perflubron attenuates neutrophil adhesion to activated endothelial cells in vitro

Infiltration of activated neutrophils into the lung appears to be a key element in the severe lung injury that develops in animal models of acute lung injury. Partial liquid ventilation with perflubron has been shown to ameliorate tissue damage compared with conventional mechanical ventilation in acute lung injury models. Pilot experiments indicated that indirect exposure to perflubron could modulate the degree to which subsequent neutrophil binding to endothelial cell monolayers was upregulated after lipopolysaccharide activation. Endothelial cell monolayers preexposed to perflubron showed >40% reductions in the surface steady-state levels of E-selectin and intercellular adhesion molecule-1 achieved after proinflammatory activation (P < 0.05), which correlated with a reduction in the real-time association constants measured by biosensor techniques. These results indicate that direct contact with the perflubron liquid phase is not necessary to attenuate inflammatory responses. Rather, diffusion of perflubron from the alveolar space into the adjacent pulmonary vascular endothelial layer may modulate neutrophil adhesion and thereby reduce the rate of infiltration of activated neutrophils into the injured lung.     

Oxygen-induced lung microvascular injury in neutropenic rabbits and lambs

We did two studies to see if severe neutropenia might reduce the severity or delay development of O2-induced lung microvascular injury. First, we treated 11 rabbits with nitrogen mustard until their circulating neurophil count decreased to less than 50/microliters of blood, after which the rabbits breathed pure O2 until death; nine other rabbits received no nitrogen mustard and had normal numbers of circulating neutrophils during O2 breathing. All rabbits died of respiratory failure with pulmonary edema, and although chemotherapy decreased the number of neutrophils in the lungs by greater than 90%, it did not influence survival time or extravascular lung water content. To see if severe neutropenia might slow the development of O2-induced lung microvascular injury, we assessed the effects of sustained hyperoxia on lung fluid balance in unanesthetized lambs treated with hydroxyurea, so that their absolute neutrophil count was less than 50/microliters of blood. We measured pulmonary arterial and left atrial pressures, cardiac output, lung lymph flow, and concentrations of protein in lymph and plasma during a 2- to 4-h control period and then daily for 2 to 4 h as the lambs continuously breathed pure O2. After 3 days of hyperoxia, lymph flow doubled and the concentration of protein in lymph increased from 3.3 +/- 0.5 to 4.2 +/- 0.3 g/dl. Tracer studies with 125I-albumin before and 3 days after the start of O2 breathing confirmed the development of increased lung vascular permeability to protein. All lambs died of respiratory failure with pulmonary edema after 3-5 days in O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical factors in pulmonary inflammatory disease

Various biochemical events taking place during pulmonary inflammation were examined in the bronchoalveolar lavage (BAL) fluids from patients with acute respiratory distress syndrome (ARDS) and in experimental animal models. In patients with ARDS, active neutrophil elastase was found in the BAL fluids. In these fluids, inactivation of the major elastase inhibitor alpha 1-protease inhibitor (alpha 1-PI) occurred. This was caused by oxidation of a methionine residue at the active site of the alpha 1-PI, and offered indirect evidence of oxidation occurring in the inflamed pulmonary tissues. Studies with experimental animals have been initiated to gain understanding of the relative roles of proteases, oxidants, arachidonate metabolites, complement and contact system components, and other mediators in the pathogenesis of pulmonary inflammation. Intrabronchial instillation of glucose oxidase/glucose to produce oxidants or formylated norleucylleucylphenylalanine or phorbol myristate acetate as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits and rhesus monkeys. The injury was accompanied by leukocytic protease (acid cathepsins) release in rabbit lungs and oxidant formation, and could be inhibited by neutrophil depletion. Oxidant formation was demonstrated by the inactivation of catalase by 3-amino-1,2,4-triazole in the presence of H2O2, a drop in intracellular glutathione levels, and in the rhesus monkey by inactivation of alpha 1-PI.     

Hyperoxic inhibition of newborn rat lung development: protection by deferoxamine.

Prolonged exposure to hyperoxia markedly inhibits normal lung development (alveolarization and respiratory surface area expansion) in immature animals. Since (a) hyperoxia results in excess hydroxyl radical (OH.) formation, (b) (OH.) is implicated in O2-induced lipid peroxidation and DNA alterations, and (c) both OH. formation and its interaction with DNA are Fe++ dependent; chelation of Fe++ should act to protect against pulmonary O2 toxicity and hyperoxic inhibition of lung development. We therefore treated litters of newborn rats with the iron chelator Deferoxamine mesylate (DES) (150 mg/kg/day) during a 10-day exposure to greater than 95% O2. Morphometric analysis demonstrated that compared to the mean airspace size in air control rat pups (Lm = 44.5 microns), hyperoxic exposure resulted in a 34% larger mean air space diameter in O2-saline rat lungs (59.5 microns) versus only an 11% enlargement in O2-DES lungs (51.1 microns*). Lung internal surface area (cm2) per 100-g body weight were air control = 4480, O2-saline = 3570 (decreases 20.3%), and O2-DES = 4125* (decreases 7.9%) (*p less than 0.05 versus O2-saline group). DES-treated animals also had significantly decreased lung conjugated diene levels during hyperoxic exposure and increased lung elastin content (reflective of preserved lung alveolar formation) compared to O2-saline rats. These results indicate that DES treatment substantially ameliorated the inhibitory effects of neonatal hyperoxic exposure on normal lung development.     

Adult carotid chemoafferent responses to hypoxia after 1, 2, and 4 wk of postnatal hyperoxia.

Exposing newborn rats to postnatal hyperoxia (60% O2) for 1-4 wk attenuates the ventilatory and phrenic nerve responses to acute hypoxia in adult rats. The goal of this research was to increase our understanding of the carotid chemoreceptor afferent neural input in this depressed response with different durations of postnatal hyperoxic exposure. Rats were exposed from a few days before birth to 1, 2, or 4 wk of 60% O2 and studied after 3-5 mo in normoxia. The rats were anesthetized with urethane. Whole carotid sinus nerve (CSN) responses to NaCN (40 microg/kg iv), 10 s of asphyxia and acute isocapnic hypoxia (arterial Po2 45 Torr) were determined. Mean CSN responses to stimuli after postnatal hyperoxia were reduced compared with controls. Responses in rats exposed to 1 wk of postnatal hyperoxia were less affected than those exposed to 2 and 4 wk of hyperoxia, which were equivalent to each other. These studies illustrate the importance of normoxia during the first 2 wk of life in development of carotid chemoreceptor afferent function.     

Cationic neutrophil proteins increase transendothelial albumin movement

Neutrophils play a role in the development of pulmonary edema in many models of the adult respiratory distress syndrome, but the mechanism of their action is not completely understood. We asked whether two neutrophil secretory products, human neutrophil cationic protein (NCP) and human neutrophil elastase (HNE), would nonenzymatically alter the movement of albumin across a cultured endothelial monolayer. Both enzymes were inactivated by heating before use. HNE was additionally enzymatically inactivated with a chloromethylketone oligopeptide (CMK) inhibitor and with alpha 1-proteinase inhibitor (alpha 1-PI). Heated NCP, heated HNE, and CMK-complexed HNE all increased transendothelial albumin transfer. The cation protamine also increased albumin transfer across the endothelium and this increase was blocked by heparin. Alpha 1-PI and fetal bovine serum also prevented the cationic proteins from increasing albumin transfer. Using the release of lactate dehydrogenase as a marker of cytotoxicity, heated HNE was toxic to endothelial cells, heated NCP had only minimal toxicity, and protamine had no toxicity. Changes in endothelial cell shape with gap formation was seen after exposure to both heated HNE and heated NCP. Both the cytotoxicity associated with heated HNE and the cell shape changes associated with heated NCP and heated HNE could be blocked by heparin. These results suggest that in addition to neutrophil proteases and reactive O2 molecules, neutrophil-derived cationic proteins can directly and nonenzymatically contribute to edema formation during acute inflammation.     

NF-kappaB protects lung epithelium against hyperoxia-induced nonapoptotic cell death-oncosis

Prolonged exposure to hyperoxia induces pulmonary epithelial cell death and acute lung injury. Although both apoptotic and nonapoptotic morphologies are observed in hyperoxic animal lungs, nonapoptotic cell death had only been recorded in transformed lung epithelium cultured in hyperoxia. To test whether the nonapoptotic characteristics in hyperoxic animal lungs are direct effects of hyperoxia, the mode of cell death was determined both morphologically and biochemically in human primary lung epithelium exposed to 95% O(2). In contrast to characteristics observed in apoptotic cells, hyperoxia induced swelling of nuclei and an increase in cell size, with no evidence for any augmentation in the levels of either caspase-3 activity or annexin V incorporation. These data suggest that hyperoxia can directly induce nonapoptotic cell death in primary lung epithelium. Although hyperoxia-induced nonapoptotic cell death was associated with NF-kappaB activation, it is unknown whether NF-kappaB activation plays any causal role in nonapoptotic cell death. This study shows that inhibition of NF-kappaB activation can accelerate hyperoxia-induced epithelial cell death in both primary and transformed lung epithelium. Corresponding to the reduced cell survival in hyperoxia, the levels of MnSOD were also low in NF-kappaB-deficient cells. These results demonstrate that NF-kappaB protects lung epithelial cells from hyperoxia-induced nonapoptotic cell death.