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Biomedical subjects

H J Forman

Publications and source records attributed to H J Forman.

At least 73 records · Page 4Linked to original sources
PubMed1990

Protection of alveolar macrophages from hyperoxia by gamma-glutamyl transpeptidase.

Exposure to hyperoxia causes loss of alveolar macrophage cell function. Toxicity was measured as suppression of the respiratory burst stimulated by phorbol myristate acetate subsequent to exposure (43.5% depression by 2-h exposure to 5 atm absolute O2 vs. controls). The presence of extracellular glutathione significantly protected these cells (7% loss). gamma-Glutamyl transpeptidase, a membrane enzyme with its active site directed outward, was necessary for use of extracellular glutathione. This was demonstrated using the gamma-glutamyl transpeptidase inhibitor, serine-borate complex, which significantly blocked both protection of cells by extracellular glutathione and extracellular glutathione-dependent synthesis of glutathione. The principal use of glutathione in antioxidant defense is as a substrate for glutathione peroxidase. The apparent Km for glutathione of glutathione peroxidase of rat alveolar macrophages was determined to be 2 mM; however, rat alveolar macrophages have approximately 1.3 mM intracellular glutathione, which is insufficient for maximal enzymatic activity. During hyperoxic exposure, this deficit would probably be more significant. Thus the ability of extracellular glutathione along with gamma-glutamyl transpeptidase activity to provide amino acids for de novo glutathione synthesis appears to be a potentially important component of antioxidant defense.

Animals↗
PubMed1990

Chemoattractant and leukotriene B4 production from rat alveolar macrophages exposed to nitrogen dioxide.

The present study examined the hypothesis that exposure of alveolar macrophages to nitrogen dioxide (NO2) resulted in enhanced production of a lipophilic chemotactic agent for neutrophils, possibly leukotriene B4 (LTB4). Neutrophil migration was significantly increased in response to the reconstituted ethyl acetate extract of the medium surrounding macrophages exposed for 1 h to 5 or 20 ppm NO2. Compared with air-treated macrophages, production of LTB4 was found to be significantly increased by exposure to 5 ppm NO2, but unchanged by exposure to 20 ppm NO2. Treatment of macrophages with the calcium ionophore A23187 at a concentration of 2 microM for 15 min following a 1-h exposure to 5 ppm NO2 led to a significant increase in the production of LTB4 compared with A23187-treated air controls; however, LTB4 production in response to the calcium ionophore was unchanged following exposure to 20 ppm NO2. Thus, while increased neutrophil migration in response to products from macrophages exposed to 5 ppm NO2 correlated with the increased production of LTB4, increased migration in response to products from macrophages exposed to 20 ppm NO2 suggested the presence of another chemotactic lipid. Lipid peroxidation processes induced by NO2 at 5 ppm may lead to the formation of hydroperoxides that enhance the formation of LTB4; yet at 20 ppm, significantly higher concentrations of hydroperoxides may be responsible for impaired LTB4 formation. Phorbol ester-stimulated macrophage superoxide production was significantly inhibited in a dose-dependent manner following exposure to NO2 concentrations of 1, 5, or 20 ppm.

Animals↗
PubMed1989

Inhibition by linoleic acid hydroperoxide of alveolar macrophage superoxide production: effects upon mitochondrial and plasma membrane potentials.

Linoleic acid hydroperoxide (LOOH) is a naturally occurring product of lipid peroxidation. Incubation of rat alveolar macrophages with LOOH produced alterations of membrane properties and function at concentrations of LOOH as low as 0.1 microM. These included phorbol myristate acetate (PMA)-stimulated superoxide production, mitochondrial membrane potential, and plasma membrane potentials. These effects were clearly separated from gross loss of structural integrity as measured by lactate dehydrogenase release, in terms of both time of incubation and concentration of LOOH. PMA-stimulated superoxide production measured 15 min after addition of 10 microM LOOH was inhibited approximately 50%; however, addition of this concentration of the hydroperoxide after PMA stimulation was without effect. Superoxide production was also measured in a cell-free system produced by incubation of alveolar macrophages with sodium dodecyl sulfate. Prior incubation of alveolar macrophages with LOOH, H2O2, or t-butyl hydroperoxide, under conditions that significantly inhibited superoxide production by the intact cells, did not produce inhibition of the NADPH-dependent superoxide generating system in the cell-free preparation. These results suggest that the effect of LOOH was upon signal transduction involved in the stimulation of superoxide production rather than on the NADPH oxidase itself. Measurements of membrane potential changes were made using the lipophilic ions, 3,3'-dipentyloxacarbocyanine (DiOC5(3] and bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V). On the basis of their charge, DiOC5(3) fluorescence primarily reports mitochondrial potential and oxonol V absorbance reports plasma membrane potential. With 10 microM LOOH, depolarization of the plasma and mitochondrial membranes appeared to occur within seconds. As prior depolarization depresses superoxide production, these hydroperoxide-induced changes in membrane potential may be responsible for decreased PMA-stimulated superoxide production.

Animals↗
PubMed1989

Adhering lung macrophages produce superoxide demonstrated with desferal-Mn(IV).

It has been shown that H2O2, the dismutation product of O2., is produced at cell-surface interfaces. Nevertheless, the relationships between the degree of attachment itself, type of surface, and O2. production are not clear. Superoxide production can be measured by the O2.-dependent reduction of nitroblue tetrazolium to an insoluble formazan. Superoxide dismutase (SOD) may be unable to scavenge O2. produced between alveolar macrophages (AM) and a surface. Desferal-Mn(IV) (Des-Mn), a low molecular weight mimic of SOD, is protective against paraquat toxicity in vivo, presumably because of specificity for O2-. Using that assumption, Des-Mn was used to measure O2. production that occurred during adherence of AM. AM suspensions were placed on fibronectin-coated glass coverslips or uncoated glass coverslips or non-stick tissue culture plates. Adherence to the surfaces varied with fibronectin greater than glass greater than non-stick and the percent formazan positive cells was 60, 24, and 4, respectively. With SOD present, the percentage of formazan positive cells were 40, 17, and 2; however, in the presence of Des-Mn the percent stained cells was 4, 4, and 0. When phorbol myristate acetate (PMA) was added during adherence, the percent of formazan positive cells was 82, 57, and 44, respectively. With PMA, Des-Mn was able to inhibit 88-100% of formazan staining whereas SOD inhibition decreased more markedly with increasing adherence. These results indicated that the degree of attachment correlated with both the degree of NBT reduction and the relative effectiveness of Des-Mn versus SOD to scavenge O2..

Animals↗
PubMed1989

Sublethal oxidant injury inhibits signal transduction in rat type II pneumocytes.

The production of reduced forms of O2 in oxidant injury can lead to lipid peroxidation, which would pose a threat to cell membrane integrity and therefore to signal transduction pathways located in the membrane. We studied the effects of oxidant injury with t-butyl hydroperoxide (tBOOH) on signal transduction in rat type II pneumocytes in culture. Exposure of 1 x 10(6) type II pneumocytes to tBOOH concentrations less than or equal to 100 microM did not cause significant release of lactate dehydrogenase and was therefore termed sublethal. Exposure to sublethal concentrations of tBOOH caused a dose-dependent inhibition of surfactant secretion stimulated both by terbutaline (10(-4)M) and by extracellular ATP (10(-5)M). Adenosine 3',5'-cyclic monophosphate production in response both to terbutaline and to extracellular ATP was also inhibited by exposure to 100 microM tBOOH. In addition, exposure to 100 microM tBOOH inhibited inositol phosphate formation in response to extracellular ATP. We conclude that sublethal oxidant injury inhibits not only receptor-mediated agonist stimulation of surfactant secretion but also receptor-mediated agonist stimulation of second messenger formation in type II pneumocytes in culture. Such inhibition may be important in the pathogenesis of both the adult and neonatal forms of respiratory distress syndrome, in which alveolar surfactant deficiency may be exacerbated by oxidant injury.

Adenosine Triphosphate↗
PubMed1988

Kinetics of uptake and distribution of arachidonic acid by rat alveolar macrophages.

The time course of uptake and distribution of 3H-arachidonic acid (3H-AA) into rat alveolar macrophage phospholipid pools was examined. Macrophages incubated with exogenous 3H-AA in RPMI-1640 containing 0.1% bovine serum albumin (BSA), incorporated this radiolabel into phosphatidylcholine and phosphatidylinositol (PI) with plateaus reached within 2 to 4 hours, which remained relatively constant for up to 18 hours. Incorporation of 3H-AA into phosphatidylethanolamine was small, but continued to increase for 14 hours. Analysis of phosphate content in phospholipid pools revealed that treatment with exogenous 5 nM arachidonic acid had no effect upon pool sizes, but there was a selective incorporation of 3H-AA into PI. Cells were incubated with 3H-AA in RPMI alone or medium containing either 0.2% lactalbumin, fetal calf serum at variable concentrations, 10% Nu Serum, or 0.1% BSA. Incubation of macrophages with 3H-AA in RPMI alone or containing 0.2% lactalbumin, resulted in approximately 70% of the radiolabel taken up by the cells being incorporated into triglyceride. The addition of BSA to RPMI-1640 medium was found to facilitate selective uptake of 3H-AA into phospholipids. Approximately 70% of incorporated 3H-AA was releasable through the action of exogenous phospholipase A2.

Animals↗
PubMed1988

Role of selenium-dependent glutathione peroxidase in antioxidant defenses in rat alveolar macrophages.

Glutathione peroxidase is a crucial component of cellular antioxidant defenses. Using tertiary butyl hydroperoxide (tBOOH) as a model for oxidant stress in alveolar macrophages, we determined the effectiveness of glutathione peroxidase in preventing both cell "death" (lactate dehydrogenase release) and more subtle alterations in cell function. The KM of glutathione peroxidase for tBOOH was 54 microM, and the Vmax was 26 nmol/min/10(6) cells in alveolar macrophages. Concentrations of tBOOH greater than 100 microM caused lactate dehydrogenase release; however, a lag greater than 30 min was observed when with 10 mM tBOOH. With 200 microM tBOOH, the rate of decrease in membrane potential, measured by 3,3'-dipentyloxacarbocyanine iodide fluorescence, inversely correlated with glutathione peroxidase. Computer-enhanced microscopy showed that this fluorescence predominately was in mitochondria. NADPH fluorescence was altered in selenium-deficient alveolar macrophages; the tBOOH-dependent rate of NADPH oxidation was slowed, and higher concentrations of tBOOH were required to disturb the steady state NADPH/NADP+ ratio. Although alteration in NADPH or glutathione oxidation can reflect oxidant stress and can adversely affect cell function, such a change does not dictate irreversible injury. Nevertheless, irreversible injury by oxidants appears to involve an overwhelming of the glutathione-NADPH antioxidant system.

Animals↗
PubMed1987

A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages.

Superoxide production in alveolar macrophages is stimulated by agonists which act through Ca2+-mediated (concanavalin A) and/or protein kinase C (phorbol ester or diacylglycerol analogues) -mediated events. Simultaneous addition of saturating concentrations of concanavalin A and a protein kinase C activator (either phorbol 12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol) caused a supra-additive enhancement of the initial rate of O2-. production. This synergism closely correlated with the known time-course of Ca2+ mobilization induced by concanavalin A; however, it occurred under conditions in which protein kinase C activation is reportedly not Ca2+ dependent. Phorbol ester-induced O2-. production was partially inhibited by the Ca2+ ionophore, A23187. Although phorbol ester-stimulated O2-. production initially was enhanced by concanavalin A, the duration of this O2-. production was reduced in comparison to that induced by phorbol ester alone. These results suggest a dual role for intracellular Ca2+ in both stimulatory and inhibitory regulation of O2-. production.

Animals↗
PubMed1986

Progressive loss of the macrophage respiratory burst in oxygen toxicity.

The respiratory burst of rat alveolar macrophages stimulated by a variety of agents declines as a function of time of exposure to hyperoxia. Previous studies have evaluated this effect in terms of the stimulated O(2) production of a population of cells. The present study was designed to determine whether this decline is due to a "turning off" of the respiratory burst activity of some cells within the alveolar macrophage population or a general suppression of the activity of all cells. The phorbol myristate acetate (PMA) initiated respiratory burst of individual rat alveolar macrophages was monitored using the reaction of nitroblue tetrazolium (NBT), which results in the formation of a precipitate on active cells. The formazan staining was evaluated using black and white photographs of the cells and comparison to a scale constructed from photographed cells of four differing intensities of staining. Frequency distributions indicated that when the respiratory burst capability in the population of alveolar macrophages is impaired approximately 50% by oxygen exposure and/or culture in plastic vessels with artificial media, there is a gradual shift in NBT reduction rather than an "all or nothing" mechanism, in which the distribution would have reflected a shift from darkly stained cells to the very lessened or negligible staining observed at the end stage of oxygen toxicity.

Animals↗
PubMed1986

Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production.

The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity.

Animals↗
PubMed1985

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages.

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.

Animals↗
PubMed1985

Oxygen toxicity: loss of lung macrophage function without metabolite depletion.

Hyperoxia inhibited concanavalin A stimulated O2- release (respiratory burst) of alveolar macrophages obtained by bronchoalveolar lavage from rats. After 36 h of normobaric 100% O2, a partial reversal (48%) of the inhibition was produced by addition of glucose. Since oxidant-induced, reversible NADPH depletion correlates with reversible inhibition of the respiratory burst, intracellular NADPH was assayed to determine whether irreversible inhibition of the respiratory burst was related to persistent changes in this metabolite. The cellular concentrations of ATP, glutathione, and ascorbate were also measured. After 36 h of hyperoxia, NADPH concentration in alveolar macrophages rose slightly while ATP and glutathione content remained at control levels. Ascorbate levels fell significantly but were not responsible for respiratory burst inhibition. Thus, irreversible loss of cellular function in hyperoxia is not due to persistent alterations in these metabolites. Significant amounts of both glutathione and ascorbate were found in extracellular fractions of lung washings, indicating high concentrations in the aqueous subphase in the lung fluid lining. There was no change in total content of these extracellular antioxidants following O2 exposure.

Animals↗
PubMed1985

Selenium deficiency potentiates paraquat-induced lipid peroxidation in isolated perfused rat lung.

Glutathione peroxidase (GSHPx), a seleno-enzyme, reduces lipid hydroperoxides while producing oxidized glutathione (GSSG), which can efflux from cells. To study the role of GSHPx in antioxidant defense, isolated lungs from selenium-deficient rats were perfused for 2 h with or without 1 mM paraquat. Perfusate GSSG was measured as an index of GSHPx activity, and malondialdehyde (MDA) as an index of lipid peroxidation. Selenium deficiency decreased lung GSHPx activity 75-80%. During perfusion control lungs showed GSSG efflux of 8.5 +/- 4.5 nmol/h and with paraquat 49.1 +/- 12.1 nmol/h. Selenium-deficient lungs with or without paraquat showed GSSG efflux of 16.4 +/- 5.3 and 13.7 +/- 8.9 nmol/h, respectively. MDA efflux occurred only in paraquat-perfused selenium-deficient lungs (7.8 +/- 2.7 nmol/h). Lung homogenates from this group had lower GSH + GSSG than the other three groups. These results indicate an inverse correlation between GSSG efflux and MDA accumulation from paraquat-perfused lungs and suggest that increased turnover of the GSHPx reaction protects paraquat-perfused lungs from lipid peroxidation.

Animals↗