Biomedical subjects

E R Block

Publications and source records attributed to E R Block.

At least 73 records · Page 4Linked to original sources

PubMed1990

Hypoxia directly increases serotonin transport by porcine pulmonary artery endothelial cell plasma membrane vesicles.

To determine whether hypoxia has a direct effect on the plasma membrane transport of serotonin (5-HT), we measured 5-HT transport activity: (1) in plasma membrane vesicles isolated from normoxic and hypoxic endothelial cells, (2) in endothelial cell plasma membrane vesicles that were exposed directly to normoxia or hypoxia, and (3) in endothelial cell monolayers incubated in the presence of 1 x 10(-7) M cycloheximide and exposed to normoxia or hypoxia. A 24-h exposure of endothelial cells to hypoxia resulted in a 40% increase (P less than 0.005) in specific 5-HT transport by plasma membrane vesicles derived from these cells. When plasma membrane vesicles were isolated and then directly exposed to normoxia or hypoxia for 1 h at 37 degrees C, a 31% increase (P less than 0.005) in specific 5-HT transport was observed in hypoxic vesicles. Hypoxia did not alter the Km of 5-HT transport (normoxia = 3.47 microM versus hypoxia = 3.76 microM) but markedly increased the maximal rate of transport (Vmax) (normoxia = 202.4 pmol/min/mg protein versus hypoxia = 317.9 pmol/min/mg protein). Cycloheximide alone had no effect on 5-HT transport by normoxic endothelial cells but did block hypoxia-induced increases in 5-HT uptake in endothelial cell monolayers exposed to 24-h hypoxia. These results indicate that hypoxia increases 5-HT transport in pulmonary artery endothelial cells by a direct effect on the plasma membrane, leading to an increase in the effective number of transporter molecules without alteration in transporter affinity for 5-HT, and possibly by an indirect effect involving de novo protein synthesis.

Animals↗

PubMed1990

Fatty acid supplementation protects pulmonary artery endothelial cells from oxidant injury.

Although supplemental fatty acids have been shown to alter the susceptibility of experimental animals to oxidant gases, the relationship between the degree of tissue fatty acyl unsaturation and resistance to oxidant exposure remains undefined. Because vascular endothelial cells have been demonstrated to be sensitive cellular targets in oxidant-induced lung injury, we evaluated the effects of a supplemental fatty acid on the lipid composition and oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. PAEC were incubated in culture medium supplemented with an ethanolic solution of 0.1 mM cis-vaccenic acid (CVA), an 18-carbon monounsaturated fatty acid, or with the ethanol vehicle alone for 3 h. Cells were then exposed to either control or oxidant (hyperoxia: 95% O2; or hydrogen peroxide: 100 microM) conditions. Oxidant-induced cell injury was assessed by phase-contrast microscopy and by measuring the release of intracellular lactate dehydrogenase. Incubation with CVA increased the CVA content of PAEC lipids and protected cells from oxidant-induced injury for up to 72 h after supplementation. CVA had no effect on nonoxidant-induced cell injury. Although the mechanism by which CVA protects cells against oxidant injury remains undefined, evidence is presented that indicates the mechanism does not involve induction of antioxidant enzyme activity, alterations in the physical state of PAEC membranes, or enhancement of PAEC nucleic acid repair mechanisms. These results define a useful model for exploring the relationship between lipid composition and oxidant susceptibility and suggest that fatty acid modifications may constitute an important strategy for protecting cells against oxidant injury.

Animals↗

PubMed1989

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)

Adolescent↗

PubMed1989

Angiotensin receptors in pulmonary arterial and aortic endothelial cells.

Angiotensin II (ANG II) is formed from angiotensin I by the action of angiotensin-converting enzyme located on the luminal surface of vascular endothelial cells. We determined whether binding sites specific for ANG II exist on pulmonary artery and aortic endothelial cells. The binding of 125I-ANG II to pulmonary artery and aortic endothelial cells was time dependent, saturable, and reversible. Scatchard analysis indicated a single class of high-affinity binding sites with equilibrium dissociation constants (Kd) of 0.85 and 0.81 nM and total binding capacities of 70 and 73 fmol/mg protein in pulmonary artery and aortic endothelial cells, respectively. Angiotensin analogues [Sar1,Ile8]ANG II and [Sar1,Ala8]ANG II, as well as angiotensin I and angiotensin III, competitively displaced 125I-ANG II in both pulmonary artery and aortic endothelial cells. The degree of inhibition of 125I-ANG II binding by these angiotensin analogues and antagonists was comparable except that [Sar1,Ala8]ANG II was 65% less potent than the other antagonists in both cell types. The binding of 125I-ANG II in pulmonary artery and aortic endothelial cells was not affected by vasopressin, substance P, or insulin, suggesting the presence of specific angiotensin receptors on these cells. These receptors appear to recognize the general configuration of angiotensin peptide rather than being specific to ANG II with no major differences between endothelial cells from pulmonary arterial or aortic vessels.

Angiotensin II↗

PubMed1989

Mechanism of hypoxic injury to pulmonary artery endothelial cell plasma membranes.

We exposed monolayer cultures of pulmonary artery endothelial cells or plasma membranes derived from these cells to hypoxic (0 and 5% O2) and normoxic (20% O2; control) conditions and measured cellular contents of malondialdehyde and conjugated dienes, plasma membrane fluidity and lipid composition, and plasma membrane-dependent transport of 5-hydroxytryptamine (5-HT). Hypoxia caused significant increases in malondialdehyde and conjugated dienes, in fluidity, and in 5-HT transport. Hypoxia also caused a significant decrease in plasma membrane total phospholipids and a marked increase in plasma membrane free fatty acids that appeared to be due to release of fatty acids from the plasma membrane phospholipids. The increases in fluidity and 5-HT transport and the alterations in fatty acids were reversible after return to control conditions. These results indicate that hypoxia alters the physical state, lipid composition, and function of endothelial cell plasma membranes by a combination of stimulation of membrane lipid peroxidation and accelerated degradation of membrane phospholipids, the latter probably secondary to activation of membrane phospholipases.

Aerobiosis↗

PubMed1989

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.

Chemical Phenomena↗

PubMed1988

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.

Amino Acids↗

PubMed1988

Plasma membrane fluidity measurements in intact endothelial cells: effect of hyperoxia on fluorescence anisotropies of 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene.

Fluorescence anisotropy measurements are widely used as sensitive indicators of cell membrane fluidity. 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene (TMA-DPH) is a cationic fluorescent aromatic hydrocarbon that anchors at the lipid-water interface of membrane lipid bilayers. Its uptake into porcine pulmonary artery and aortic endothelial cells was monitored and the probe remained specifically localized on the cell surface for at least 4 h. It can therefore be recommended for use for specific plasma membrane lipid fluidity measurements in these cells. The effect of hyperoxia on plasma membrane fluidity was measured by using TMA-DPH. In both cell types, hyperoxic damage resulted in decreases in plasma membrane fluidity. Recovery was achieved 48 h after a 42-h hyperoxic exposure. These results indicate that TMA-DPH is a sensitive probe of plasma membrane lipid domains of pulmonary artery and aortic endothelial cells and that hyperoxia causes reversible changes in the physical state of superficial lipid domains of the plasma membrane of these cells.

Animals↗

PubMed1988

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.

Endothelium↗

PubMed1988

Serotonin transport and fluidity in plasma membrane vesicles: effect of hyperoxia.

Plasma membrane vesicles were prepared from porcine pulmonary artery endothelial cells by a dextran-polyethylene glycol two-phase system. Specific carrier-mediated transport of 5-hydroxytryptamine (5-HT) into the vesicles was examined. Transport required a Na+ gradient (out greater than in) across the membrane, and accumulated 5-HT rapidly effluxed out of the vesicles when the ionophore gramicidin was added. Transport was inhibited by the antidepressant imipramine. 5-HT transport into plasma membrane vesicles appeared saturable and exhibited Michaelis-Menten kinetics (Km 7.4 microM, maximal velocity 217 pmol.min-1.mg membrane protein-1). A 24-h exposure to 95% O2 at 1 atmosphere absolute resulted in a 21% decrease (P less than 0.05) in specific 5-HT transport by plasma membrane vesicles. Hyperoxia also caused a significant (P less than 0.01) decrease in plasma membrane fluidity, as measured with the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. These results indicate that pulmonary artery endothelial cell plasma membrane vesicles provide a good model for studying 5-HT transport activity in vitro. Hyperoxia affects plasma membrane fluidity and 5-HT transport in pulmonary artery endothelial cells, suggesting a possible cause-and-effect relationship between the two.

Animals↗

PubMed1988

Interaction between oxygen and cell membranes: modification of membrane lipids to enhance pulmonary artery endothelial cell tolerance to hypoxia.

Because high partial pressures of oxygen (O2) can cause peroxidative cleavage of membrane lipids, it is plausible to hypothesize that hyperoxia alters the physical state and composition of lipids in the membranes of pulmonary endothelial cells and that manipulation of the lipid profile may modify endothelial cell tolerance to hyperoxic injury. To test this, porcine pulmonary artery endothelial cells were exposed to 95% O2 at 1 atmosphere absolute (ATA) in the presence or absence of cis vaccenic acid (CVA), a monounsaturated fatty acid (C18:1#11). Plasma membrane fluidity was assessed by fluorescence spectroscopy, plasma membrane lipid composition was quantitated using thin layer and gas chromatography, and cytotoxicity was monitored by measuring release of lactate dehydrogenase (LDH). Hyperoxia caused peroxidation of membrane lipids and decreased fluidity in three distinct lipid domains within the plasma membrane. Incubation with CVA was associated with a reduction in the degree of unsaturation of the constituent fatty acids within all plasma membrane lipid subclasses except monoglycerides. CVA-treated cells were also more resistant to hyperoxic injury as judged by LDH release. These results support the hypothesis that cells with membranes in which the fatty acyl chains are more resistant to the disordering effects of high O2 tensions may be more resistant to O2 toxicity.

Animals↗

PubMed1987

Effect of plasma membrane fluidity on serotonin transport by endothelial cells.

To evaluate the effect of plasma membrane fluidity of lung endothelial cells on serotonin transport, porcine pulmonary artery endothelial cells were incubated for 3 h with either 0.1 mM cholesterol hemisuccinate, 0.1 mM cis-vaccenic acid, or vehicle (control), after which plasma membrane fluidity and serotonin transport were measured. Fluorescence spectroscopy was used to measure fluidity in the plasma membrane. Serotonin uptake was calculated from the disappearance of [14C]-serotonin from the culture medium. Cholesterol decreased fluidity in the subpolar head group and central and midacyl side-chain regions of the plasma membrane and decreased serotonin transport, whereas cis-vaccenic acid increased fluidity in the central and midacyl side-chain regions of the plasma membrane and also increased serotonin transport. Cis-vaccenic acid had no effect on fluidity in the subpolar head group region of the plasma membrane. These results provide evidence that the physical state of the central and midacyl chains within the pulmonary artery endothelial cell plasma membrane lipid bilayer modulates transmembrane transport of serotonin by these cells.

Animals↗

PubMed1987

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)

Animals↗

PubMed1986

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.

Antioxidants↗

PubMed1986

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.

Adenosine Triphosphate↗

PubMed1986

Nitrogen dioxide-induced changes in cell membrane fluidity and function.

Nitrogen dioxide (NO2), an environmental oxidant pollutant, is toxic to lung cells. One of the postulated mechanisms of NO2-induced pulmonary injury involves peroxidation of membrane lipids. Therefore, we evaluated the effect of 5 ppm NO2 exposure on membrane lipid fluidity, uptake of 5-hydroxytryptamine (5-HT), lactate dehydrogenase (LDH) release, and formation of lipid peroxides in porcine pulmonary artery and aortic endothelial cells in culture. After 3- to 24-h exposure, cells were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), an aromatic hydrocarbon that partitions into the hydrophobic interior of the lipid bilayer of cell membranes. Membrane fluidity was monitored by measuring changes in rotational relaxation time (rho) for DPH by fluorescence spectroscopy. Reductions in membrane fluidity increase the value of rho. The 5-HT uptake was calculated from the disappearance of 1 X 10(-6) M 14C-5-HT from the medium, and LDH release and lipid peroxide formation were measured by spectrophotometric methods. The NO2 caused a significant increase in rhoDPH in both types of endothelial cells after 3 h and progressed with further exposure to NO2. Exposure to NO2 for 24 h, but not 3 or 12 h, significantly (p less than 0.05) reduced 5-HT uptake, increased (p less than 0.01) LDH release, and increased (p less than 0.05) lipid peroxide formation in both pulmonary artery and aortic endothelial cells. These results suggest that oxidant injury caused by NO changes the physical state of membrane lipids, impairs membrane function, and contributes to the biochemical and metabolic abnormalities in the cells.

Animals↗

PubMed1986

Endotoxin protects against hyperoxic decrease in membrane fluidity in endothelial cells but not in fibroblasts.

We evaluated the ability of endotoxin to protect against hyperoxic depression of plasma membrane fluidity in endothelial cells and fibroblasts in culture. Second- to-fifth passage porcine aortic endothelial cells and human newborn foreskin fibroblasts with 20 ng/ml of endotoxin or diluent in the culture medium were exposed to 20% O2 (control) or 95% O2 (hyperoxic) in 5% CO2 for 4 hours. After exposure, cells were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), an aromatic hydrocarbon that partitions into the hydrophobic core of lipid bilayer membranes, or transparinaric acid (TPA), a natural, conjugated fatty acid that orients parallel to fatty acyl chains of membrane phospholipids. Membrane fluidity was monitored by measuring changes in the steady state fluorescence anisotropies (rs) for DPH and for TPA by using fluorescence spectroscopy. Reductions in membrane fluidity increase the value of rs. Addition of endotoxin to the culture medium of control endothelial cells and fibroblasts had no effect on rs for DPH or TPA. In hyperoxic endothelial cells, rs for DPH and rs for TPA were increased (p less than 0.001). Addition of endotoxin to the medium of hyperoxic endothelial cells prevented the increases in rs for DPH and TPA. Hyperoxia increased rs for DPH (p less than 0.003) but not rs to TPA in fibroblasts, and endotoxin failed to prevent this increase. These results indicate that hyperoxia decreases plasma membrane fluidity in endothelial cells and fibroblasts and demonstrate that endotoxin prevents the decrease in plasma membrane fluidity in endothelial cells, but not in fibroblasts. These membrane-protective effects may represent an alternative mechanism by which endotoxin protects against hyperoxic cellular injury, and this mechanism may be specific for hyperoxic injury to endothelial cells.

Animals↗

PubMed1985

Effect of oxygen and endotoxin on lactate dehydrogenase release, 5-hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells.

We compared the effects of 95% O2 (hyperoxia) alone, endotoxin (20 ng/ml) alone, and 95% O2 plus endotoxin on the release of lactate dehydrogenase (LDH), uptake of 5-hydroxytryptamine (5-HT), and antioxidant enzyme activities in porcine pulmonary arterial and aortic endothelial cells in monolayer culture. Hyperoxia increased LDH release and decreased 5-HT in both endothelial cell types. Hyperoxia also caused a decrease in catalase (CAT) activity and an increase in total superoxide dismutase (SOD) and glutathione reductase (GSH-Red) activities in both cell types. Endotoxin alone had no effect on LDH release, 5-HT uptake, or antioxidant enzyme activities. However, endotoxin prevented the hyperoxic increase in LDH release and the hyperoxic decrease in 5-HT uptake. Endotoxin plus 95% O2 had no consistent effect on the antioxidant enzyme profile in pulmonary artery or aortic endothelial cells. These results indicate that (1) hyperoxia injures both pulmonary artery and aortic endothelial cells in culture and causes changes in the antioxidant enzyme profile that are similar in the two cell types; (2) hyperoxia-induced decreases in CAT activity and increases in SOD activity may be responsible for increased sensitivity of endothelial cells to O2 toxicity; and (3) endotoxin protects against hyperoxic injury to endothelial cells in vitro, but increases in antioxidant enzyme activities are not the mechanism for this protection.

Animals↗