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H J Forman

Publications and source records attributed to H J Forman.

At least 91 records · Page 5Linked to original sources
PubMed1983

Depolarization and increased conductance precede superoxide release by concanavalin A-stimulated rat alveolar macrophages.

Rat alveolar macrophages release superoxide into the extracellular medium when stimulated by concanavalin A. This process, the respiratory burst, is characterized by a delay between binding of the stimulus and release of superoxide. It has been proposed that a key event that occurs during this delay period is the alteration of membrane electrical potential. Microelectrode impalement was used to directly measure electrical properties of the plasma membrane. Upon addition of concanavalin A, the membrane potential depolarized 21%, and membrane electrical resistance decreased 16%. Parallel chemical measurement of superoxide release indicated that these changes in electrical properties precede the release of superoxide.

Animals↗
PubMed1983

Paraquat inhibits mixed-function oxidation by rat lung.

The effect of paraquat on 7-ethoxycoumarin (7-EC)deethylation, an NADPH-dependent cytochrome P-450-linked reaction, was studied in isolated rat lungs. Control lungs oxidatively deethylated 7-EC at a constant rate, metabolizing 178 +/- 12 nmol/g dry wt in 50 min (n=3, mean +/- SE). When glucose was omitted from the perfusate, 7-EC deethylation was lower (85 +/- 17 nmol/g, P less than 0.05), and the rate progressively decreased, suggesting inadequate pentose shunt regeneration of NADPH. During perfusion with paraquat (10(-3) M), 7-EC metabolism was still lower (75 +/- 5 nmol/g in the presence of glucose and 18 +/- 4 nmol/g in the absence of glucose, P less than 0.05). Addition of lactate plus pyruvate partially restored 7-EC deethylase activity in control lungs but had no effect in lungs perfused with paraquat. Varying perfusate paraquat concentrations (10(-6) to 10(-3) M) inhibited 7-EC deethylation in a dose-dependent fashion (r = 0.79, P less than 0.02). Oxidative demethylation of p-nitroanisole, another cytochrome P-450-linked reaction, was similarly inhibited by paraquat. In contrast, microsomes isolated from paraquat-perfused lungs showed no inhibition of 7-EC metabolism (measured in the presence of an NADPH-regenerating system). We conclude that paraquat depletes NADPH in the isolated lung to a sufficient extent to impair mixed-function oxidation. Such NADPH depletion may play a role in the toxicity of paraquat.

Animals↗
PubMed1983

Effect of extracellular calcium on superoxide release by rat alveolar macrophages.

Alveolar macrophages can be stimulated to release superoxide into the extracellular medium. The mechanism of activation of superoxide release has been reported to be mediated by the movement of Ca2+ into the cytosol, but the involvement of extracellular Ca2+ in this process has remained uncertain. Extracellular Ca2+ was not an absolute requirement for activation of superoxide release; however, 1.3 mM extracellular Ca2+ caused an approximate twofold enhancement of superoxide release stimulated by either concanavalin A or formyl-methionyl-leucyl-phenylalanine but had no effect on digitonin-stimulated superoxide release. Concanavalin A-stimulated superoxide was inhibited by the "Ca2+ entry blockers," Mn2+ (1 microM) or methoxyverapamil (D-600) (100 microM). Inhibition by Mn2+ was competitive with extracellular Ca2+, whereas inhibition by D-600 was noncompetitive. Neither inhibitor, however, affected superoxide release after initiation. Thus activation of superoxide release by alveolar macrophages can be accomplished, in part, through the entrance of Ca2+ into the cell.

Animals↗
PubMed1983

Roles of selenium and sulfur-containing amino acids in protection against oxygen toxicity.

Tolerance and adaptation to hyperoxia have been correlated with increases in antioxidant enzymes. This study evaluated whether selenium deficiency would prevent an increase in glutathione peroxidase (GSHPX), a selenium-containing enzyme, during oxygen exposure, and, thus, inhibit adaptation. Because the Torula yeast-based diet, which was used to produce selenium deficiency, was also deficient in cysteine and methionine, the effects of these deficiencies were also evaluated. When rats were exposed to 80% oxygen for 1 week, mortality was 80% for rats deficient in both selenium and the sulfur-containing amino acids, 40% for selenium-deficient rats, 35% for cysteine- and methionine-deficient rats, and 0% for rats fed either a standard laboratory diet or a selenium, cysteine-, and methionine-supplemented Torula yeast diet. However, only one of the six surviving rats with low selenium and none of the rats from any other dietary group died during a subsequent 96 hours of 98% oxygen, indicating adaptation to hyperoxia (LD50 for unadapted rats is 72 hours.) GSHPX activity (per gram of dry weight) was decreased 85% in lungs from unexposed rats fed the low selenium diets. After oxygen exposure, lung GSHPX activity was elevated in all dietary groups. Rats fed the high selenium diets had a 47% increase in enzyme activity, whereas rats with high selenium had a 214% increase. Although hyperoxia caused a relatively high percentage increase in the low Se rats, the resulting absolute GSHPX activity was only 34 to 70% of that of unexposed high selenium rats. The results indicate that both selenium and sulfur-containing amino acids contribute to antioxidant defense. However, although the stress of hyperoxic exposure produces an increase in glutathione peroxidase activity, the absolute lung GSHPX activity is better correlated with tolerance than with adaptation to hyperoxia.

Adaptation, Physiological↗
PubMed1982

Hyperoxia inhibits stimulated superoxide release by rat alveolar macrophages.

Factors responsible for the loss of respiratory burst capacity (stimulated extracellular O2-. release) of alveolar macrophages (AM) exposed to prolonged hyperoxia were assessed. Specific pathogen-free rats were exposed to 1 ATA O2 for 24-72 h, and lungs of survivors lavaged. Release of O2-. by cells after addition of concanavalin A, which stimulated AM but not polymorphonuclear leukocytes (PMN), or digitonin, which stimulated both cell types, was measured using cytochrome c reduction +/- superoxide dismutase. O2-. release by AM declined 47.2% (P less than 0.05) after 24 h of hyperoxia and 100% after 60 h. Percent PMN in the lavage was less than 3% at 0-36 h but increased to 16% at 48 h and to 44% at 72 h. Although addition of PMN to AM in vitro caused inhibition of AM O2-. release, the percent PMN required for inhibition was not reached in vivo until after a significant decline in AM O2-.-releasing capacity had already occurred. Cell-free lavage fluid from either control or hyperoxic rats did not affect AM O2-. release. AM in culture for 24 h in hyperoxia lost 76.7% (P less than 0.005) of O2-.-releasing capacity vs. cells incubated in 20% O2, although dye exclusion was unaffected. The results indicate that the major cause of loss of AM O2-. release by hyperoxia is a direct effect of O2 on the cells.

Animals↗
PubMed1982

Differential paraquat uptake and redox kinetics of rat granular pneumocytes and alveolar macrophages.

Possible causes for differences in cellular susceptibility to paraquat (PQ) toxicity were investigated with isolated lung cells and perfused lungs. Uptake of radiolabeled PQ was measured as a function of both time and concentration (1 x 10(-6)-1 x 10(-3) M). With alveolar macrophages, uptake was linear with concentration but with time reached a plateau as internal approached external concentration. With granular pneumocytes, uptake was linear with time but nonlinear with external concentration. Accumulation above external concentration occurred and was prevented by inhibitors of ATP synthesis. Apparently, PQ can enter both cell types by diffusion, but granular pneumocytes can also accumulate PQ by an energy-dependent process. With lungs, uptake was nonlinear with time; in the 1st hr a plateau occurred when the intracellular approached extracellular (1 x 10(-5) M) concentration, but a slower second process resulted in a 6-fold accumulation by 4 hr. The effect of PQ on NADPH oxidation was measured with the supernatant fraction (12000 x g. 10 min) from cells and lungs. The maximal rate was about 2 times higher (6.8 +/- 0.8 nmol/mg of protein . min) in granular pneumocytes than in alveolar macrophages, but the estimated kinetic constants were similar (Km-NADPH approximately 5 x 10(-6) M; Km-PQ approximately 2 x 10(-4) M). The results suggest that PQ-dependent NADPH oxidation rates would differ markedly between cell types at low PQ concentration because of energy-dependent transport into granular pneumocytes but would be fairly close at high PQ concentration where diffusion predominates. These conclusions suggest a mechanistic explanation for how differences in the toxic effects of PQ exposure might occur over a range of PQ concentrations.

Animals↗
PubMed1980

Rat alveolar macrophages require NADPH for superoxide production in the respiratory burst. Effect of NADPH depletion by paraquat.

Alveolar macrophages can be stimulated by concanavalin A to produce extracellular superoxide. Conflicting opinions exist, however, concerning the relative importance of the oxidation of either NADPH or NADH in the generation of (Formula: see text) by surface membrane-stimulated phagocytic cells. Alveolar macrophages were obtained from adult male rats by lavage with phosphate-buffered saline. Cells (approximately 10(6)/ml) were incubated in Krebs-Ringer phosphate 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and ferricytochrome c for 15 min at 37 degrees C before addition of concanavalin A. Release of (Formula: see text) was detected as the difference in cytochrome c reduction, followed at 550 nm, in the absence and presence of superoxide dismutase. Superoxide production by concanavalin A-stimulated alveolar macrophages was markedly increased in the presence of glucose but fructose, lactate, and pyruvate were without effect. Paraquat (methylviologen), an oxidation-reduction dye, significantly reduced concanavalin A-stimulated (Formula: see text) production when incubated at 1 mM with alveolar macrophages in the absence of glucose. The effect of paraquat was reversed by glucose, but fructose, lactate, and pyruvate could not reverse paraquat inhibition. Paraquat enhanced oxidation of NADPH (but not NADH) by cell supernatant and increased pentose phosphate shunt activity in resting macrophages, but did not affect mitochondrial respiration or ATP content of alveolar macrophages. These results suggest that paraquat is able to specifically deplete NADPH in alveolar macrophages while not affecting NADH or ATP. Our conclusion is that NADPH is essential for the production of (Formula: see text) by concanavalin A-stimulated alveolar macrophages.

Animals↗
PubMed1980

Mechanism for the potentiation of oxygen toxicity by disulfiram.

Rats given disulfiram (200 mg/kg) or diethyldithiocarbamate (200 mg/kg) by intraperitoneal injection were exposed to 2 atmospheres absolute oxygen in a hyperbaric chamber or kept in normoxia. By 12 hr of hyperoxia exposure, none of the control but 30% of the disulfiram-treated and 87% of the diethyldithiocarbamate-treated rats had died. Both disulfiram and diethyldithiocarbamate administration decreased lung cytosolic superoxide dismutase activity, but the pharmacokinetics were different. At 1 hr postinjection of diethyldithiocarbamate superoxide dismutase activity was 40% decreased but returned to control activity within 13 hr (4 hr, 18% inhibited). In contrast, disulfiram administration produced a greater decrease at 4 hr (31%) than at 1 hr (16%) and was still effective at 13 hr (28% less than control). Although disulfiram did not produce as great a decrease at 1 hr as did diethyldithiocarbamate, it's effect was more persistent. In vitro, diethyldithiocarbamate inactivated superoxide dismutase at 10(-4) M, although 10(-3) M disulfiram did not cause any reduction in enzymatic activity. The contrast between the inhibition by disulfiram of lung superoxide dismutase activity in vivo and its lack of effect in vitro suggests metabolism of disulfiram to diethyldithiocarbamate. It is likely that disulfiram administration potentiates oxygen toxicity via in vivo reduction to diethyldithiocarbamate and subsequent inhibition of superoxide dismutase.

Animals↗
PubMed1980

The oxygen-adapted rat model: Tolerance to oxygen at 1.5 and 2 ATA.

The effect of pre-exposure to graded hyperoxia on subsequent survival under hyperbaric conditions was investigated in 250-300 g male Sprague-Dawley rats. The average survival of normal rats was approximately 4 d in 1 ATA O2, 1 d in 1.5 ATA O2, and 16 h in 2 ATA O2. Rats pre-exposed to O2 at 0.8 ATA for 1 week became O2 tolerant and survived a subsequent 1 week in O2 at 1 ATA with zero mortality. These O2-tolerant rats had a three-fold increase of survival duration in O2 at 1.5 ATA, but showed no significant prolongation of survival in 2 ATA O2. Pre-exposure to 0.8 ATA O2 for 2 weeks had no further effect on tolerance. However, rats pre-exposed to O2 at 0.8 ATA for 1 week followed by exposure to O2 at 1 ATA for a second week demonstrated approximately a three-fold increase in subsequent survival at 2 ATA O2. The results indicate that oxygen-tolerant rats remain susceptible to mortality in 1.5-2 ATA O2 and that graded tolerance to O2 develops as a function of the oxygen exposure regimen.

Adaptation, Physiological↗
PubMed1977

Effects of chaotropic agents versus detergents on dihydroorotate dehydrogenase.

As chaotropic salts are generally believed to affect water structure in a manner which increases lipophilicity of water, they may seem to be capable of substituting for detergents in the solubilization of particulate enzyme. Although solubilization either by detergents or by chaotropic salts has been demonstrated with several membrane proteins, the effects these agents have on the properties and activity of an enzyme may be quite different. This is illustrated by the effects on mammalian mitochondrial dihydroorotate dehydrogenase. Stability of the solubilized enzymic activity is dependnet on the presence of a detergent and maximum enzymic activity is observed at the critical micelle concentration of the detergent. Addition of low concentrations of various anions of the chaotropic series further enhances activity while higher concentrations of these anions, although increasing solubility of the enzyme, irreversibly inhibit catalysis.

Animals↗
PubMed1975

Superoxide production and electron transport in mitochondrial oxidation of dihydroorotic acid.

Production of superoxide radical during oxidation of dihydroorotate in rat liver mitochondria was not affected by antimycin A, thenoyltrifluoroacetone, or added ubiquinone but was inhibited by orotate, a product inhibitor of dihydroorotate dehydrogenase. It appears likely that superoxide is generated at the primary dehydrogenase. Dihydroorotate dehydrogenase differs from succinate dehydrogenase both in its utilization of ubiquinone and in the mechanism of cytochrome b reduction. Thenoyltrifluoroacetone completely inhibits fumarate synthesis and reduction of cytochrome b by succinate. Formation of orotate is only partially inhibited by thenolytrifluoroacetone and the inhibitor does not prevent reduction of cytochrome b by dihydroorotate. It is proposed that several pathways exist for linkage of the primary dihydrorotate dehydrogenase with the electron transport chain. One route involves electron transfer from ubiquinone to cytochrome c and is inhibited by thenoyltrifluoroacetone. A second route bypasses ubiquinone and is inhibited by antimycin A. A third pathway utilizes both ubiquinone and cytochrome b and is partiayly inhibited by either thenoyltrifluoroacetone or antimycin A.

Animals↗