Characterization of L-arginine uptake by plasma membrane vesicles isolated from cultured pulmonary artery endothelial cells.

We investigated the mechanisms of [3H]-L-arginine transport via System Y+ using plasma membrane vesicles derived from cultured pulmonary artery endothelial cells. [3H]-L-arginine uptake into plasma membrane vesicles was Na-independent, sensitive to trans-stimulation, unaffected by proton-conducting ionophores, and selectively inhibited by cationic amino acids. Kinetic experiments performed over a wide range of substrate concentrations revealed only one population of L-arginine transporters with Km = 130 microM. To elucidate the driving force for L-arginine transport, we measured [3H]-L-arginine uptake by plasma membrane vesicles at different transmembrane ion gradients. Plasma membrane vesicles accumulated [3H]-L-arginine only when a membrane potential was imposed across the vesicles, and the velocity of uptake was linearly related to the magnitude of the created membrane potential. The presence of potassium ions inside the vesicles was not essential for uptake of L-arginine into vesicles, but it was essential for trans-stimulation of L-arginine transport. [3H]-L-arginine accumulated in plasma membrane vesicles can be released by agents that dissipate transmembrane potassium gradients (e.g. saponin, gramicidin, and nigericin). Diazoxide and pinacidil, activators of K(+)-channels, had no significant effect on [3H]-L-arginine uptake, whereas tetraethylammonium chloride, 4-aminopyridine, and glibenclamide, inhibitors of K(+)-channels, caused decreases in [3H]-L-arginine transport by plasma membrane vesicles. This study demonstrates for the first time a specific role for potassium ions in the mechanism of L-arginine transport, particularly in the phenomenon of trans-stimulation.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.

The effect of oxidant gases on membrane fluidity and function in pulmonary endothelial cells.

Free radicals and oxidant gases, such as oxygen (O2) and nitrogen dioxide (NO2), are injurious to mammalian lung cells. One of the postulated mechanisms for the cellular injury associated with these gases and free radicals involves peroxidative cleavage of membrane lipids. We have hypothesized that oxidant-related alterations in membrane lipids may result in disordering of the plasma membrane lipid bilayer, leading to derangements in membrane-dependent functions. To test this hypothesis, we examined the effect of exposure to high partial pressures of O2 or NO2 on the physical state and function of pulmonary endothelial cell plasma membranes. Both hyperoxia (95% O2 at 1 ATA) and NO2 exposure (5 ppm) caused early and significant decreases in fluidity in the hydrophobic interior of the plasma membrane lipid bilayer and subsequent depressions in plasma membrane-dependent transport of 5-hydroxytryptamine. Lipid domains at the surface of pulmonary endothelial cell plasma membranes are more susceptible to NO2-induced injury than to hyperoxic injury. Alterations in the fluidity of these more superficial domains are associated with derangements in surface dependent functions, such as receptor-ligand interaction. These results support our hypothesis and advance our understanding of how the chemical events of free radical injury associated with high O2 and NO2 tensions are translated into functional manifestations of O2 and NO2-induced cellular injury.

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells.

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.

Overexpression of plasma membrane annexin II in NO2-exposed pulmonary artery endothelial cells.

Because exposure to nitrogen dioxide (NO2) alters plasma membrane structure and function in pulmonary artery endothelial cells (PAEC), we examined whether NO2 exposure is associated with upregulation of plasma membrane-specific proteins in PAEC. Exposure to 5 ppm NO2 for 24 h had no significant effect on total protein synthesis. However, two-dimensional gel electrophoresis of isolated plasma membranes from [35S]-methionine pulse-labeled PAEC exposed to NO2 for 24 h demonstrated 3- to 9-fold increases in the synthesis of several proteins with molecular masses of 36, 39, and 40 kDa compared with controls. N-terminal amino acid sequencing and immunodetection analysis identified the 36kDa plasma membrane protein as annexin II (lipocortin II). Northern blotting analysis demonstrated that the mRNA expression for annexin II in NO2-exposed cells was also increased. These results suggest that exposure to NO2 results in induction of plasma membrane annexin II, an important multifunctional calcium- and phospholipid-binding protein in PAEC.

Nitric oxide exposure and sulfhydryl modulation alter L-arginine transport in cultured pulmonary artery endothelial cells.

The effect of nitric oxide (NO) exposure and sulfhydryl-reactive chemicals on L-arginine transport in pulmonary artery endothelial cells was evaluated. Exposure of pulmonary artery endothelial cells to 7.5 ppm (0.4 microM) NO for 4 h resulted in a significant (p < 0.05) reduction of Na(+)-dependent but not Na(+)-independent L-arginine transport. More prolonged exposure for 12-24 h reduced both Na(+)-dependent and Na(+)-independent transport of L-arginine with maximal loss of transport after 18 h of exposure (p < 0.02 for both). Similarly, incubation of cells in the presence of 50-200 microM S-nitroso-acetyl-penicillamine (SNAP) (but not 500 microM each of nitrate or nitrite) for 2 h also reduced both the Na(+)-dependent and Na(+)-independent transport of L-arginine (p < 0.05 for all concentrations). The SNAP-induced reduction of L-arginine transport was blocked by the NO scavenger oxyhemoglobin. When cell monolayers were exposed to varying concentrations of the sulfhydryl reactive chemicals N-ethylmaleimide (NEM) and acrolein, a dose-dependent reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes was observed. Na(+)-dependent L-arginine transport was more susceptible to inhibition by exposure to NO and to sulfhydryl reactive chemicals. Incubation of cells with 0.5 mM of the thiol-containing agent N-acetyl-L-cysteine prior to and during NEM or acrolein exposure blocked NEM and acrolein-induced reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes. Similarly, NO-induced reductions of Na(+)-dependent and Na(+)-independent L-arginine transport were reversed to control levels 24 h after termination of NO exposure. Treatment with the disulfide reducing agent dithiothreitol after exposure to NO resulted in partial reversal of the decreases in L-arginine transport. These results demonstrate that exposure to exogenous NO is responsible for reversible reductions of plasma membrane-dependent L-arginine transport mediated by both the Na(+)-dependent (system Bo,+) and the Na(+)-independent (system y+) transport processes. Modulation of the sulfhydryl status of plasma membrane proteins involved in L-arginine transport, such as L-arginine transporters and/or Na+/K(+)-ATPase, may be responsible, at least in part, for reductions in overall L-arginine transport in pulmonary artery endothelial cells.

Nitrogen dioxide-induced expression of a 78 kDa protein in pulmonary artery endothelial cells.

Exposure to nitrogen dioxide (NO2) activates signal transduction in cultured pulmonary artery endothelial cells (PAEC). We examined whether NO2-induced activation of signal transduction results in increased expression of proteins in PAEC. Exposure to 5 ppm NO2 for 4, 12, and 24 h had no significant effect on total protein synthesis. However, two-dimensional gel electrophoresis of [35S]-methionine-labeled PAEC exposed to NO2 for 24 h, but not 4 and 12 h, demonstrated increased synthesis of several proteins including a two- to five-fold increase of some proteins with molecular masses of 47, 64, 78, and 105 kDa compared to controls. N-terminal amino acid sequencing and immunodetection analysis identified the 78 kDa protein as 78 kDa glucose-regulated protein (GRP-78). Induction of GRP-78 by NO2 exposure was regulated at the transcriptional level, and the induction required de novo protein synthesis. Exposure to NO2 for 24 h also significantly (p < .05) decreased glycosylation of proteins in PAEC. Exposure of cell monolayers to tunicamycin, an inhibitor of protein glycosylation, mimicked the effect of NO2 exposure on expression of GRP-78. Increased expression of GRP-78 was also detected when cell monolayers were exposed to the calcium ionophore A 23187, to 2-deoxyglucose, or to glucose-free medium, which are also known to cause perturbations in protein glycosylation. These results demonstrate that exposure to NO2 increases expression of a number of proteins including GRP-78 in PAEC. Increased expression of GRP-78 in NO2-exposed cells appears to be associated with inhibition of glycosylation or through coordinated alterations in metabolic events that lead to inhibition of protein glycosylation.

NO2-induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine-derived diacylglycerol in cultured pulmonary artery endothelial cells.

The present study examines whether nitrogen dioxide (NO2)-induced activation of protein kinase C (PKC) is associated with increased expression of specific PKC isoforms and/or with enhanced generation of phosphatidylcholine(PC)-derived diacylglycerol (DAG) in pulmonary artery endothelial cells (PAEC). Western blot analysis revealed that exposure to 5 ppm NO2 resulted in increased expression of PKC alpha and epsilon isoforms in both cytosol and membrane fractions in a time-dependent fashion compared with controls. A time-dependent elevated expression of PKC isoform beta was observed in the cytosol fraction only of N02-exposed cells. PKC isoform gamma was not detectable in either the cytosolic or membrane fractions from control or N02-exposed cells. Scatchard analysis of [3h]phorbol 12,13-dibutyrate (PDBu) binding showed that exposure to N02 for 24 h increased the maximal number of binding sites (Bmax) from 15.2 +/- 2.3 pmol/mg (control) to 42.3 +/- 5.3 pmol/mg (p < 0.01, n = 4) (NO2-exposed). Exposure to NO2 significantly increased PC specific-phospholipase C and phospholipase D activities in the plasma membrane of PAEC (p < 0.05 and p < 0.001, respectively). When [3H]-myristic acid-labeled cells were exposed to NO2, significantly increased radioactivity was associated with cellular DAG. These results show for the first time that exposure of PAEC to NO2 results in elevated expression of specific PKC isoforms and in enhanced generation of cellular DAG, and the latter appears to arise largely from the hydrolysis of plasma membrane PC.

Results of a prospective study evaluating the effects of mantle irradiation on pulmonary function.

Thirty patients with Stages I-III Hodgkin's disease receiving mantle irradiation were prospectively evaluated prior to therapy with spirometry, lung volumes, and tests of diffusing capacity (DLCO). Follow-up examinations were performed at 3, 6, and 12 months and then yearly. Sixteen patients had Hodgkin's disease involving the mediastinum at presentation, 10 were smokers, and 16 received either preirradiation or postirradiation chemotherapy. Mantle doses ranged between 2300 cGy and 4000 cGy (mode of 3750 cGy) given at 150 cGy to 170 cGy tumor dose per day with split-course technique. Pulmonary function test results were translated to percent change from predicted values obtained from normal standards for each age, sex, race, and height. These percent changes were then analyzed as a linear function of time. Twenty patients have been tested greater than or equal to 4 years after treatment with a median time from treatment to last pulmonary function test of 8 years. Changes over time in spirometry included an early, mild decrease in both forced vital capacity (FVC) and forced expiratory volume at 1 second (FEV1), which returned to baseline by 2 years and then gradually decreased to a 10-15% deficit as compared with predicted values at 6-10 years. Additionally, there was a very slight decrease in FEV1/FVC beginning at 1 year and gradually increasing to an 8% deficit at 6-10 years. Changes over time in lung volumes included a mild nadir of total lung capacity (TLC) and functional residual capacity (FRC) at 6 months to a year, which returned to baseline at 2-4 years and then gradually dropped to a 5-10% deficit at 6-10 years. Mean DLCO for the study group was 20% below predicted values prior to treatment and dropped to a low of 30% below predicted at 6 months following treatment, then gradually returned to baseline by 4 years and showed continued improvement to an overall deficit of approximately 10% at 6-10 years. With the exception of FEV1/FVC, the changes noted in spirometry and lung volumes were of insufficient degree to be classified as abnormal. The decrease in FEV1/FVC is indicative of a significant and progressive obstructive ventilatory defect. The effects on pulmonary function tests of smoking, the presence of mediastinal involvement by Hodgkin's disease, and exposure to chemotherapy were assessed by statistical analysis. No subsets of patients demonstrated consistent evidence of a restrictive ventilatory defect expected after irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of nitrogen dioxide on surface membrane fluidity and insulin receptor binding of pulmonary endothelial cells.

Nitrogen dioxide (NO2), an environmental oxidant pollutant, is known to peroxidize membrane lipids of lung cells. We evaluated the ability of NO2 to alter the surface membrane fluidity, lipid composition, and insulin receptor binding of porcine pulmonary artery endothelial cells in culture. After 3- to 24-hr exposure to 5 ppm NO2, cells were labeled with either 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a cationic fluorescent aromatic hydrocarbon that anchors at the lipid-water interface, or fluorescamine, a fluorescent molecular probe that covalently binds with amino groups of surface phospholipids and proteins. Membrane fluidity was measured by monitoring changes in the steady-state fluorescence anisotropies (rs) for TMA-DPH and fluorescamine. Insulin specific receptor binding was monitored by measuring time-dependent binding of 125I-insulin. Following NO2 exposure, rs values for TMA-DPH and fluorescamine were increased significantly in a time-dependent fashion, with maximum increases at 24 hr (P less than 0.001). Similar increases in rs values were observed in isolated plasma membranes as well as in lipid vesicles prepared from total lipid extracts of endothelial cells or their plasma membranes. Phosphatidylethanolamine plus phosphatidylserine content in lipid extracts from 24-hr but not 3- to 12-hr NO2-exposed cells was increased significantly (P less than 0.01) compared to control cells. Specific binding of 125I-insulin to cells exposed to NO2 for 12 and 24 hr (but not 3 and 6 hr) was reduced significantly (P less than 0.05) compared to binding in control cells. Scatchard analysis of the binding data indicated that NO2 exposure caused a 5-fold reduction in insulin receptor binding sites in endothelial cells. Recovery was achieved 24 hr after NO2 exposure with, but not without, changing culture medium. These results indicate that NO2 exposure causes reversible changes in the physical state of lipids in the superficial lipid domains of the pulmonary endothelial cell plasma membrane, and these alterations may interfere with plasma membrane-dependent functions such as receptor-ligand interaction.

Environmental influences on uptake of serotonin and other amines.

Lungs accumulate 5-hydroxytryptamine (serotonin, 5-HT) from the perfusate by a sodium-dependent, energy-requiring, saturable process. The rate-limiting step for uptake is the transport of 5-HT and not its subsequent metabolism to 5-hydroxyindoleacetic acid. Autoradiographic studies indicate that the pulmonary endothelium is the cellular site of uptake. The effect of hyperoxia on lung clearance of 5-HT was studied with isolated perfused and ventilated lungs from rats that were previously exposed to hyperoxia. Lungs were perfused with recirculating electrolyte solution and initial [5-HT] of 0.24 microM. The calculated fractional 5-HT clearance (fracion of 5-HT removed in a single pass) ws 0.77 +/- 0.02 (mean +/- SE: n = 44) for control rats. Mean fractional clearance decreased by 20% in rats exposed to 1 atm O2 for 18 hr and 30% after 4 atmospheres absolute (ata) O2 for 1 hr (p < 0.05). The effects of O2 at 4 ata were in part reversed by exposure to air for 3.5 hr and in part prevented by injection of superoxide dismutase (60 nmole/kg body weight). This degree of O2 exposure at either 1 or 4 ata had no effect on lung content of adenine nucleotides or the distribution of 3H-5HT on autoradiography. Rats maintained for 6 weeks on a vitamin E-deficient diet showed an increased effect of hyperoxia on 5-HT clearance and did not show reversal of changes after 24 hr of air breathing. The results indicate that exposure to elevatd po2 results in reversible depression of pulmonary 5-HT clearance that is potentiated by vitamin E deficiency. This suggests alteration of pulmonary endothelial membrane transport properties due to O2 toxicity.

Carbonic anhydrase inhibition in glaucoma: hazard or benefit for the chronic lunger?

Carbonic anhydrase inhibitors, of proven value in the longterm management of glaucoma, have a number of troublesome side effects, most of which are well-known. However, their potential hazards to patients with chronic obstructive lung disease have received little attention. The author reviews the physiological effects of carbonic anhydrase inhibition on carbon dioxide metabolism and the implications for the chronic lung patient. The untoward pulmonary complications associated with the use of carbonic anhydrase inhibitors are of particular importance to ophthalmologists, since chronic lung disease and glaucoma, both common disorders in the elderly, frequently coexist in the same patient.

Hyperoxia reduces plasma membrane fluidity: a mechanism for endothelial cell dysfunction.

To evaluate the relative contributions of three possible mechanisms that can be advanced to explain the observation that hyperoxia decreases serotonin uptake by endothelial cells, we examined the effect of high O2 tensions on Na+-K+-ATPase activity, ATP content, and plasma membrane fluidity in cultured endothelial cells. Confluent monolayers of pulmonary artery and aortic endothelial cells were exposed to 95% O2 (hyperoxia) or 20% O2 (controls) in 5% CO2 at 1 ATA for 4-42 h. Exposure to high O2 tensions had no effect on Na+-K+-ATPase activity or ATP content in pulmonary artery or aortic endothelial cells in culture. However, hyperoxia decreased the fluidity of the plasma membrane of pulmonary artery and aortic endothelial cells in culture, and the time course for the decrease in fluidity parallels that of the hyperoxic inhibition of serotonin transport. These results indicate that hyperoxia decreases fluidity in the hydrophobic core of the plasma membranes of cultured endothelial cells. Such decreases in plasma membrane fluidity may be responsible for hyperoxia-induced alterations in membrane function including decreases in transmembrane transport of amines.

Lung serotonin metabolism.

The pulmonary vascular endothelium, a metabolically active tissue, serves as an important site of injury in many types of clinical and experimental lung disease. Removal of 5-HT from the circulation constitutes one of the endothelial metabolic functions that is depressed early in the course of lung injury. Experimental evidence confirms that measuring 5-HT uptake detects alterations in endothelial cell function that precede the abnormalities detected by more conventional diagnostic tests such as radiographs, pulmonary function tests, and arterial blood gases. In addition, depression of 5-HT uptake can lead to increased concentrations of 5-HT in the pulmonary vasculature, which may contribute to the pathogenesis of lung injury. The development of an ideal method for measuring 5-HT uptake accurately in the lungs of critically ill patients has just begun. As yet, numerous variables reviewed in this article confound clinical measurements of 5-HT uptake. However, if investigators can continue to refine and develop the techniques for measuring 5-HT uptake in human patients, clinicians can look forward to the addition of a sensitive tool to their diagnostic armamentarium. Hopefully, the ability to detect diffuse lung injury earlier in its course will enable future clinicians to institute therapy that will prevent the pathologic progression to morbidity and death seen all too frequently in current medical practice.

Effect of NO2 exposure on antioxidant defense of endothelial cells.

Nitrogen dioxide (NO2), an environmental oxidant pollutant, is toxic to lung cells. We evaluated the changes in antioxidant enzyme activities in porcine pulmonary artery (PA) and aortic (AO) endothelial cells in monolayer cultures. Confluent PA or AO endothelial cells were exposed to 3 or 5 ppm NO2 or air (control) for 3-24 h and assayed for GSH-reductase (GSH-red), GSH-peroxidase (GSH-per), and glucose-6-phosphate dehydrogenase (G6PDH) activities as well as for intracellular GSH content. After 3, 6, or 12 h exposure to 3 or 5 ppm, GSH-red and G6PDH activities were not different from those of controls in both PA and AO endothelial cells. Exposure to 3 or 5 ppm NO2 for 24 h resulted in significant increases in GSH-red (P less than 0.05) and G6PDH (P less than 0.001) activities in both cell types. GSH-per activity and GSH content in NO2-exposed PA and AO endothelial cells were not different from those of controls, irrespective of NO2 concentration and exposure time. These results indicate that enzyme activities of G6PDH and GSH-red are increased in PA and AO endothelial cells exposed to NO2, and this response is comparable, in part, to that in the lungs from animals exposed to NO2.

Pulmonary endothelial cell pathobiology: implications for acute lung injury.

Pulmonary endothelial cells form a continuous monolayer on the luminal surface of the lung vasculature. Until the mid-1970s, the pulmonary endothelium was felt to provide little more than a passive surface for the exchange of gases, water, macromolecules, and some cell traffic. Recent evidence indicates that the pulmonary endothelium is a metabolically active surface, which provides a regulatory interface for the continual processing of blood-borne vasoactive molecules, plays an active role in hemostasis and immunologic and inflammatory events, regulates vascular tone, and interacts with inflammatory cells and neighboring vascular cell types. These metabolic properties are both constitutive and capable of being induced in response to stimuli or injury. Virtually any agent that causes pulmonary endothelial cell injury will lead to impairments in the functional metabolic properties of these cells, resulting in alterations in hemodynamics, hemofluidity, permeability, gas exchange, and intercellular signaling. The net result in the lung is often the clinical picture of acute lung injury with respiratory distress, refractory hypoxemia, diffuse alveolar infiltrates, and respiratory failure.

Molecular cloning, characterization and expression of a nitric oxide synthase from porcine pulmonary artery endothelial cells.

The lack of sequence information and clones of porcine pulmonary artery endothelial cell (PAEC) constitutive nitric oxide synthase (ecNOS) cDNA limits comparative analysis between porcine and human PAEC. Therefore, we cloned, characterized and expressed the ecNOS cDNA from porcine PAEC. Two oligonucleotide primers were designed based on the published human ecNOS cDNA sequence and used to clone porcine PAEC ecNOS using 5' and 3' rapid amplification of cDNA ends reverse transcriptase polymerase chain reaction technique. A full-length ecNOS cDNA was cloned and sequenced, representing a protein of 1205 amino acids with a molecular mass of 134 kDa. A mammalian expression vector (pcDNA3) containing this cDNA was transfected into COS-7 cells, and ecNOS activity was detected by monitoring the formation of [3H]-citrulline from [3H]-L-arginine. Expression of ecNOS activity was predominantly associated (> 90%) with the total membrane fraction of these transfected cells. The deduced amino acid sequence of porcine ecNOS cDNA, containing binding sites for NADPH, flavin adenine dinucleotide and bound flavin mononucleotide, shows 94% identity to human ecNOS. The molecular weight of porcine ecNOS mRNA was estimated to be 4.7 kb by Northern blot analysis, similar to human ecNOS mRNA. This suggests that porcine ecNOS is similar to human ecNOS in deduced amino acid sequence and structure.

Biochemical and metabolic response to nitrogen dioxide-induced endothelial injury.

Nitrogen dioxide (NO2), a major oxidant constituent of vehicle emissions, is toxic to lung cells including endothelial cells. Since NO2 is a reactive free radical, one of the postulated mechanisms of NO2-induced pulmonary injury involves the peroxidation of membrane lipids. Therefore, this study evaluated the dose- and time-dependent effects of nitrogen dioxide exposure by measuring the biochemical and biophysical parameters, as well as the metabolic function, in porcine pulmonary artery and aortic endothelial cells in monolayer cultures. To evaluate the biochemical changes, the antioxidant enzyme GSH-reductase (GSH-red), GSH-peroxidase (GSH-per), and glucose-6-phosphate dehydrogenase (G6PDH) activities, as well as the lipid peroxide formation, glutathione (GSH) content, and lactate dehydrogenase (LDH) release were measured. Biophysical changes were measured by monitoring lipid fluidity in both the hydrophobic and hydrophilic regions of the plasma membrane. The uptake of 5-hydroxytryptamine (5-HT) was measured as a metabolic function of endothelial cells. Confluent porcine pulmonary artery and aortic endothelial cells were exposed to 3 or 5 ppm NO2 or air (control) for 3-24 hours. After 3-, 6-, or 12-hour exposures to 3 or 5 ppm NO2, the GSH-red and G6PDH activities, as well as the lipid peroxide formation and LDH release, were not different from those of controls in both pulmonary artery and aortic endothelial cells. Exposure of the cells to 3 or 5 ppm NO2 for 24 hours resulted in significant increases in GSH-red (p less than 0.05) and G6PDH (p less than 0.001) activities in both cell types. Exposure to 5 ppm NO2 for 24 hours significantly (p less than 0.05) increased lipid peroxide formation and increased (p less than 0.01) LDH release in both the pulmonary artery and aortic endothelial cells. GSH-per activity and GSH content in NO2-exposed pulmonary artery and aortic endothelial cells were not different from those of controls, irrespective of NO2 concentration and exposure time. Fluorescence spectroscopy was used to measure the membrane lipid fluidity. Membrane fluidity in the hydrophobic region was measured by 1,6-diphenyl-1, 3, 5-hexatriene (DPH), an aromatic hydrocarbon that partitions into the hydrophobic interior of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of superoxide dismutase and succinate on the development of hyperbaric oxygen toxicity.

Prolonged exposure to hyperbaric O2 (HBO) causes seizures and eventual death. The precise molecular basis for O2 toxicity is not known but may be due to increased biological production of superoxide anion (O2-). In the present study, superoxide dismutase (SOD), an enzyme that catalyzes the dismutation of O2- to less toxic forms, was evaluated for its ability to protect against HBO-induced seizures and death, and the results were compared to those concurrently obtained with succinate (SUCC), an agent previously reported to protect against HBO-induced seizures. Preconvulsion time and survival time in normal and vitamin E-deficient rats exposed to 100% O2 at 5 ATA were not significantly prolonged by pretreatment with 2 to 20 mg/kg SOD intraperitoneally (ip) or 0.1 to 1.0 mg/kg SOD intrathecally. In contrast, 12 mmol/kg SUCC ip significantly prolonged preconvulsion time in normal and vitamin E-deficient rats and survival time in normal rats. The ability of SUCC to stimulate ATP production may account for its protective role. Reasons for the failure of SOD to protect against O2 toxicity are discussed.

Prevention of acute paraquat toxicity in rats by superoxide dismutase.

Paraquat is a widely used herbicide which causes lung injury in man characterized by progressive parenchymal damage that may lead to fatal respiratory failure. The precise mechanism of injury is unknown but is related to the cyclic oxidation and reduction of paraquat in cells with resultant production of free radicals of oxygen. In this study, superoxide dismutase (SOD), an enzyme that catalyzes the dismutations of superoxide free radical (O2-) to less toxic forms, plus reduced glutathione (GSH), and d-propranolol (PROP), were evaluated for their ability to protect against acute paraquat toxicity. Rats maintained at room air were given 50 mg paraquat dichloride/kg body weight in a single intraperitoneal (IP) injection 60 min prior to receiving either 0.2, 2, 10, or 20 mg/kg/d SOD, 3.6, 7.2, or 14.4 mmol/kg/d GSH, 2 or 20 mg/kg/d PROP, or an equal volume of normal saline (controls) LP. in divided doses for 3 d. SOD significantly prolonged and increased survival at doses of 2, 10, or 20 mg/kg/d (p less than 0.05). In addition, histologic lung morphology in SOD-treated rats showed only minimal intra-alveolar hemorrhage and hypercellularity 24, 48, and 168 h after paraquat challenge. Treatment with GSH, PROP or 0.2 mg/kg/d SOD was not protective. Duration of survival, percent survival, and lung morphology in these groups were not significantly different from controls. These results indicate that a) SOD protects against the development of acute paraquat toxicity in rats, b) one mechanism of paraquat poisoning is increased biologic production of O2-, and c) SOD may have a role in the therapy of paraquat poisoning in man.