High O2 Tensions Research Articles

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.

The role of nutritional factors in the prevention of peroxidative damage to tissues

The multi-level defence system present in vertebrate cells to protect against chain reactions initiated by free radicals (mainly toxic metabolites of oxygen) is outlined. It comprises superoxide dismutases (Cu-Zn and Mn dependent), glutathione peroxidase (Se dependent), vitamin E and glutathione S-transferase.The protective role of superoxide dismutase (SOD) was demonstrated by studies on trout (Salmo gairdneri) retina (Desrochers and Hoffert 1983). This tissue is subject to very high O2 tensions but has a high resistance to O2 toxicity that is dependent on a high dismutating capacity. The activities of both the cytosolic Cu-Zn SOD and the mitochondrial Mn SOD in the liver of rainbow trout were significantly altered in response to changes in dietary intake of these minerals.Deficiency of Se did not affect the growth rate of rainbow trout but led to greatly reduced activities of hepatic and plasma glutathione (GSH) peroxidase. There was no evidence of a Se-independent GSH peroxidase activity. In rainbow trout depleted of Se there was a compensatory increase in hepatic GSH S-transferase activity. This enzyme in conjunction with GSH has been shown to inhibit lipid peroxidation in the Festimulated microsomal systemin vitro. A dietary synergism between vitamin E and Se in rainbow trout has been demonstrated.The vitamin E requirement of rainbow trout is related to the polyunsaturated fatty acid (PUFA) content of the diet. The molar ratio PUFA: vitamin E in rainbow trout tissues is lower than that in rats. Time course studies on the uptake of orally administered vitamin E showed rapid incorporation into biomembranes; few differences between normal and vitamin E deficient fish were evident. The ester form of vitamin E was more slowly assimilated than the alcohol.

Stimulation of respiration and nitrogenase in bacteroids of Siratro (Macroptilium atropurpureum) by plant nodule cytosol

Nitrogenase activity (acetylene reduction) of isolated Siratro (Macroptilium atropurpureum) bacteroids was stimulated by addition of plant cytosol fractions which also preserved activity at high (up to 3%) O2 tensions. These effects were not due to leghaemoglobin. Boiling removed some, but not all, of the protective capacity of the cytosol. Heat treated cytosol substantially stimulated the respiration of siratro bacteroids. Of a wide variety of compounds tested, only ascorbate could mimic the cytosol. Ascorbate was present in the cytosol fraction, in significant quantities. The effect of ascorbate was evident at low O2 concentrations and in purified bacteroids, and was inhibited by cyanide. Siratro bacteroids appear to possess an oxidase which could serve a protective role in vivo.

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.

Relationship of oxygen and glutathione in protection against carbon tetrachloride-induced hepatic microsomal lipid peroxidation and covalent binding in the rat. Rationale for the use of hyperbaric oxygen to treat carbon tetrachloride ingestion.

CCl4 exerts its toxicity through its metabolites, including the free radicals CCl3. and CCl(3)00.. Oxygen strongly inhibits the hepatic cytochrome P-450-mediated formation of CCl3. from CCl4 and promotes the conversion of CCl3. to CCl(3)00.. Both these free radicals injure the hepatocyte by causing lipid peroxidation and binding covalently to cell structures. A reduced glutathione (GSH)-dependent mechanism can protect the liver microsomal membrane against CCl4-induced damage under aerobic conditions but not under anaerobic conditions (Burk, R.F., K. Patel, and J.M. Lane, 1983, Biochem. J., 215:441-445). Experiments were carried out using rat liver microsomes to examine the effect of O2 tensions found in the liver and of GSH on CCl4-induced covalent binding and lipid peroxidation. An NADPH-supplemented microsomal system was used. CCl4 or 14CCl4 was added to the sealed flask that contained the system, and after 20 min CHCl3 production, thiobarbituric acid-reactive substances (an index of lipid peroxidation), and covalent binding of 14C were measured. O2 tensions of 0, 1, 3, 5, and 21% were studied. Increases in O2 tension caused a fall in CHCl3 production, which indicated that it decreased CCl3.. GSH had no significant effect on CHCl3 production at any O2 tension. Lipid peroxidation and covalent binding of 14C fell progressively as O2 tension was increased from 1 to 21%. The addition of GSH decreased both lipid peroxidation and covalent binding, but did so better at the higher O2 tensions than at the lower ones. These results indicate that low O2 tensions such as are found in the centrilobular areas of the liver favor conversion of CCl4 to free radical products which cannot be detoxified by the GSH-dependent mechanism. They suggest that hyperbaric O2 might decrease free radical formation in the liver in vivo and promote formation of CCl(3)00. from CCl3.. This should result in diminished CCl4-induced lipid peroxidation and liver damage. Rats given CCl4 (2.5 ml/kg) were studied in metabolic chambers. Production of CHCl3 and ethane, the latter an index of lipid peroxidation, were measured. Rats in two atmospheres of 100% O2 produced much less CHCl3 and ethane than rats in air. This strongly suggests that hyperbaric O2 is decreasing free radical formation from CCl4 and/or promoting the formation of CCl(3)00. from CCl3.. These results provide the rationale for the use of hyperbaric O2 in the treatment of CCl4 ingestion.

Nitrous oxide production from nitrification and denitrification in marine sediment at low oxygen concentrations

Measurements of N2O production (release of free N2O), nitrification, and denitrification were made simultaneously in NH4Cl- and KNO3-amended suspensions of marine sediment. An open flow system was designed for the application of low partial pressures of O2 (0–10 kPa) to the sediment. The overall rate of N2O production increased dramatically at the lowest O2 tensions (0–0.2 kPa) and had a maximum at complete anoxia. The specific rates of N2O production from nitrification (N2On) and from denitrification (N2Od) were determined after separation of the processes with inhibitors. Within the range of 0–0.2 kPa O2, the rate of N2On production showed an apparent maximum of 0.1 kPa O2 where the production accounted for 25% of the total activity of nitrification ([Formula: see text] oxidation). The rate of N2Od production, however, continued to increase as the O2 fell to zero. The proportion of N2Od to the total N2Od plus N2 produced from denitrification increased at the higher O2 tensions and reached the maximum of about 50% at 5 kPa O2. Except for a narrow range between 0.1 and 0.2 kPa O2, denitrification was the main source of N2O at 0–10 kPa O2.

Geotaxis in the Ciliated Protozoon Loxodes

ABSTRACT Geotaxis is demonstrated in the ciliated protozoon Loxodes. This behaviour is mediated by a mechanoreceptor which is probably the Müller body, an organelle characteristic of loxodid ciliates. The geotactic response is sensitive to dissolved oxygen tension: in anoxia or at very low O2 tensions the ciliates tend to swim up and at higher O2 tensions they tend to swim down. This behaviour, in conjunction with a kinetic response allows the ciliates to orientate themselves in vertical O2 gradients and to congregate in their optimum environment. In two appendices, models of the behaviour predicting vertical distribution patterns and considerations of the minimum size of a functional statocyst are offered.

Spontaneous lipid peroxidation in rabbit spermatozoa: a useful model for the reaction of O2 metabolites with single cells.

Mature mammalian spermatozoa comprise a relatively homogeneous population of cells. In suspension, their motility provides a readily visualized index of cell viability. Suspensions of spermatozoa are, therefore, convenient and useful systems for studying the reactions of O2 and its partially reduced metabolites which cause cell damage. It has been known for nearly 40 years that high O2 tensions are deleterious to the motility of human sperm (McLeod, 1943). Bull and ram spermatozoa were shown to lose motility on storage at 4°C in concert with loss of phospholipid, particularly plasmalogen, suggesting that this loss was due to lipid peroxidation leading to plasma membrane damage (Jones and Mann, 1973; Mann and Lutwak-Mann, 1981). Since most mammalian sperm contain little or no catalase (Mann, 1964), O2-induced damage to spermatozoa has been attributed to H2O2 (Wales et al., 1959). Another source of O2-induced damage could be the superoxide anion O2: Menella and Jones (1980) demonstrated superoxide dismutase (SOD) activity in spermatozoa from a variety of mammalian species, suggesting that O2 might be produced in these cells. But no documentation of O2 production was provided.KeywordsLipid PeroxidationSperm CellSperm Cell ConcentrationOrder Rate ConstantDouble Reciprocal PlotThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Short-term modulation of glycogen metabolism, glycolysis and gluconeogenesis by physiological oxygen concentrations in hepatocyte cultures.

The influence of different oxygen concentrations (0% to 20%, v/v) on the main pathways of carbohydrate metabolism was studied in rat hepatocyte cultures. Cells resembling the periportal or the perivenous cell type were obtained after 48 h culture under different hormonal conditions; they are referred to as 'periportal' or 'perivenous', respectively. Using radiochemical techniques the metabolic rates of the two cell types were measured between 48 h and 50 h under different oxygen tensions. Standard physiological substrates of 5 mM glucose, 2 mM lactate and endogenous glycogen were used. In cells incubated under 4% O2, mimicking hepatovenous oxygen levels, compared to cells assayed under 13% O2, mimicking arterial levels, glycogen degradation to free glucose and to lactate was faster while glycogen synthesis was slower; glycolysis, from glucose to lactate, was faster and gluconeogenesis, from lactate to glucose, was only slightly slower. Under anoxic conditions glycogen breakdown was maximal and glycogen synthesis minimal; gluconeogenesis was also minimal, but glycolysis was not maximal, it reached its peak rate at 4% O2. CO2-formation increased up to 6% and then stayed essentially constant at higher O2 tensions. Net glycogen metabolism: in 'perivenous' cells net glycogen synthesis was observed above, net glycogen degradation below 4% O2. In 'periportal' cells, which had a very low glycogen content, net glycogen metabolism was very small. Net glucose metabolism: in 'perivenous' cells net glucose formation was only seen under anoxic conditions. Net glucose utilization was observed at about the same rate under all physiological O2 tensions. In 'periportal' cells net glucose formation increased clearly up to 6% O2 and then remained almost constant. Net lactate metabolism: in 'perivenous' cells net lactate formation occurred below, and net lactate utilization above, 6% O2. In 'periportal' cells a pronounced net lactate utilization was observed under all physiological O2 tensions. Net flow between glucose-6-phosphate and pyruvate was observed in the glycolytic direction in 'perivenous' and in the gluconeogenic direction in 'periportal' cells except under anoxic conditions. When O2 tensions were lowered, the percentage of 'futile cycling' was decreased in the 'perivenous', glycolytic hepatocytes; conversely, it was increased in the 'periportal', gluconeogenic hepatocytes. It is concluded that physiological oxygen concentrations modulate hepatic carbohydrate metabolism and that they contribute further to the proposed metabolic differences between periportal and perivenous cells in vivo.

C2H2 reduction, oxygen uptake and cytochrome c reduction by bacteroids isolated from French-bean nodules

Low concentration of dissolved O2 (3 μM) induced a significant C2H2 reduction by bacteriods isolated from French-beans (Phaseolus vulgaris) inoculated with Rhizobium phaseoli strain 9-6. This activity, determined with or without glucose, declined with increasing O2 concentrations in spite of the generation of a substantial level of ATP. Under these conditions, cytochrome c studied by a rapid spectrometry method on whole cells, appeared more than 80% oxidized. An active C2H2 reduction always corresponded to a high level of cyt c reduction observed with glucose, at low O2 tensions only, and with succinate for a larger range of pO2 values. Our results indicated that inability of glucose to support C2H2 reduction activity for higher O2 tensions was due to a limiting electron allocation to nitrogenase in relation with the poor level of reduced cyt c observed in these conditions.

Gill ventilation and O2 extraction during graded hypoxia in two ecologically distinct species of flatfish, the flounder (Platichthys flesus) and the plaice (Pleuronectes platessa)

Gill ventilation, breathing frequency, breath volume, oxygen extraction from the ventilatory water current and oxygen uptake through the gills were measured in flounder, Platichthys flesus, and plaice, Pleuronectes platessa, at water O2 tensions ranging from 35 to 155 mm Hg at 10° C. Ventilation volumes were similar in the two species at high water O2 tension. Exposure to hypoxic water elicited a larger increase in ventilation in the flounder. The per cent extraction of O2 from water decreased slightly in both species as water O2 tension was lowered. At comparable levels of ventilation O2 extraction was higher in flounder. At the higher levels of water O2 tension, O2 uptake across the gills of flounder was stable, the critical O2 tension being between 60 and 100 mm Hg. The plaice behaved as an oxygen conformer over the entire range of O2 tensions investigated. The superior ability of the flounder in maintaining OZ uptake across the gills during a reduction in water O2 tension may in part explain why the species, unlike plaice, inhabits very shallow waters with large fluctuations in dissolved oxygen.

Depression of serotonin uptake by cultured endothelial cells exposed to high O2 tension.

Serotonin (5-hydroxytryptamine, 5-HT), a vasoactive amine, is removed from the pulmonary circulation by active transcellular transport into endothelial cells, followed by intracellular metabolism. Pulmonary uptake of 5-HT is depressed by high partial pressures of O2 (Po2). To characterized the cellular basis and mechanism for hyperoxic depression of lung 5-HT uptake, we evaluated the effect of high Po2 on 5-HT uptake by calf aortic endothelial cells in primary confluent monolayer. Cells were exposed to either 95% O2, or 14% O2 (controls) in 5% Co2 at 1 ATA for 20 or 42 h. Following exposure 5-[14C]HT (1 X 10-7 M) was added to the culture medium, and 5-HT uptake in pmol/10(6) cells was measured. Exposure to 95% O2 for 20 h or 42 H resulted in significant depression of 5-HT uptake by cultured endothelial cells, and uptakes remained significantly depressed 48 h after exposure to 95% O2 had ended. Inhibition of intracellular metabolism of 5-HT by iproniazid did not affect 5-HT uptake by control or O2-exposed endothelial cells. These results indicate that 1) high Po2 levels depress 5-HT uptake in endothelial cells by direct inhibition of the transcellular transport of 5-HT and 2) hyperoxic depression of 5-HT uptake in cultured endothelial cells is not readily reversible.

Development of Chick Limb Bud Chondrocytes in Cell Culture

For the purpose of describing early chondrogenic metabolic and structural events, measurements were made of oxidative and other enzymatic activities at various stages in the morphologic development of chondrocytes over a period of 18 to 20 days in continuous cell culture. Comparisons were also made between cells grown in 20% O2 and in 35% O2. These cultures exhibited rapid confluence (within 24 hours), early development of cartilaginous nodules by Day 2 to 3 and metachromatic staining of the chondrocyte matrix by Day 3 to 4. Under 35% O2, cell sheets were thicker and there was increased pleomorphism of chondrocyte and fibroblast cell types, with a relative increase of fibroblast components and reduction in chondroblasts and chondrocyte aggregates. Using the von Kossa staining procedure, calcium salt deposition was observed by Day 9. There was no apparent difference in mineralization of cultures grown under the low and high O2 tensions. Under normoxic conditions cytochrome oxidase and malate dehydrogenase (MDH) activities increased rapidly for the first three to four days and then remained essentially constant. Lactate dehydrogenase (LDH) activity increased continuously over the life of the culture. Acid phosphatase increased rapidly until about Day 13 after which it remained constant, whereas alkaline phosphatase showed a bimodal activity profile. Under hyperoxic conditions, cytochrome oxidase, MDH and alkaline phosphatase activity were significantly inhibited. LDH and acid phosphatase activities were markedly inhibited initially but with time showed a degree of recovery.

Effects of oxygen tension on the lipid composition of Azotobacter chroococcum.

Lipid content and composition were determined in Azotobacter chroococcum grown, under nitrogen-fixing conditions, in continuous culture with intense aeration under atmospheres containing between 5 and 40% O2. Total lipid content remained almost constant at approximately 9% of dry weight. Phospholipid content was maximal at 20% O2 where it accounted for 92% of total lipid, and was minimal at 40% O2. Phosphatidylethanolamine was the only species of phosphatide detected in this fraction. Neutral lipid content was minimal at 20% O2 and maximal at 40% O2 where it represented approximately 30% of the total lipid. Glycolipid remained between 2 and 13% of total lipid throughout. The principal fatty acids of A. chroococcum were hexadecanoic (C16:0), hexadecenoic (C16:1), and octadecenoic acid (C18:1) at all O2 tensions, but C18:1 increased at the expense of C16:1 at higher O2 tensions, particularly in free fatty acid and phospholipid fractions. [U-14C]acetate was readily incorporated into lipid at both 20 and 40% O2, but total incorporation was much greater at 20% O2.

Influence of ethylene and oxygen on respiration and peroxide formation in potato tubers

APPLICATION of ethylene to potato tubers induces a respiratory upsurge1, comparable with that in ripening climacteric fruit2, but which can be studied independently of other ethylene-induced processes occurring in fruit. It has been suggested that ethylene catalyses oxygen-requiring processes, reflecting in part oxygen utilisation in the formation of peroxides3. We report here that high O2 tensions, in combination with ethylene, markedly enhance the respiratory rise, which is accompanied by a corresponding increase in peroxides.

Aerobic Glycolysis and Its Role in Maintenance of High O2 Tensions in the Teleost Retina

SummaryLactic acid production in the presence of high oxygen tensions (aerobic glycolysis) was found to occur in the teleost retina. Forty-two percent more glucose was utilized and 33% more lactic acid was produced under anaerobic than under aerobic conditions. Glycolysis was inhibited 33% by elevated oxygen (Pasteur effect), and it was further demonstrated that glycolysis is dependent upon the integrity of the retinal cells, being almost completely abolished by cellular disruption.The shift in the oxygen dissociation curve to the right by acid metabolites causes release of oxygen from hemoglobin in the choroid region. This O2 release explains in part the high PO2 encountered in the vicinity of the retina. The Pasteur effect may function as a possible component of a negative feedback control loop for the generation of high PO2 in trout eyes.

Recovery of cells in vitro from the effects hypoxia and hyperoxia.

AbstractThe cytotoxic effect of high, as well as low, oxygen tension of proliferation and metabolism of Low line cells in culture is reversible even after several days of exposure provided the cells are returned to 95% air + 5% CO2 environment. This suggests that the activity of certain mechanisms within the cells may have been altered or in other ways inhibited by the abnormal environments but are quite rapidly regenerated once the adverse condition is removed. The cells tolerate a low O2 exposure for at least 20 days while continuous exposure to high O2 atmosphere results in degeneration and death after 7–10 days. Both glucose utilization and lactic acid production are elevated in cultures exposed to either low or high O2 tensions, although they are markedly higher in the latter condition. When cell so exposed are returned to an air + 5% CO2 atmosphere, rate of glucose uptake and lactic acid formation soon approaches that found in control cultures.

Studies on succinate dehydrogenase. 14. Intracellular distribution, catalytic properties and regulation of fumarate reductases in yeast.

The cytoplasm of yeast contains several enzymes for the reduction of fumarate to succinate. One of these is excluded on Sephadex G‐200 and in this respect as in all of its known properties, resembles mitochondrial succinate dehydrogenase in the soluble form. Another type is distinguished by the apparent inability to oxidize succinate with any of the conventional electron acceptors. This enzyme has been subdivided into two types (I and II) which may be readily separated by chromatography on hydroxylapatite. Type I and II fumarate reductases differ from mitochondrial succinate dehydrogenase in containing acid‐extractable (rather than covalently bound) FAD, in their intracellular location, in being more stable and more resistant to inhibition by mercurials. The fumarate reductases also differ from the dehydrogenase in molecular weight, in lack of activation by substrate, and in having lower affinities for succinate and malonate and a higher one for fumarate. Conditions which are optimal for the development of mitochondrial succinate dehydrogenase (high O2 tension, low glucose concentration) repress the development of type I and II fumarate reductases and vice versa.“Petite” mutants, which lack succinate dehydrogenase, have a normal content of these fumarate reductases. Type I and II fumarate reductases, while similar in most respects, differ in molecular weight, kinetic constants and reactivity with electron donors.

Adaptation of the soil microbiota to high CO2 and low O2 tensions.

The ability of the soil microbiota to adapt to atmospheres of high CO2 and low O2 tensions was investigated. Subsamples of soil were incubated (conditioned) in gases containing different concentrations of CO2 and O2, and the tolerance to adverse atmospheric conditions of organisms subsequently isolated was compared with that of organisms present in the soil before conditioning. Although apparent adaptation of the fungal, bacterial, and actinomycete populations occurred, it was not established whether physiological adaptation, rather than enrichment of segments within the populations tolerant to these conditions, occurred. Adaptation appeared to be enhanced by conditioning the soil in gases similar to, but not the same as, that under which the organisms were subsequently isolated. Although adaptation, in general, increased as the CO2 tension of the conditioning atmospheres decreased, the CO2 concentration, rather than reduced O2 tensions, induced adaptation. Aspergillus, Fusarium, Penicillium, and Trichoderma exhibited greater tolerance and adaptability to high CO2 and low O2 tensions than other genera of the mycobiota. Possible implications of the capacity to adapt to adverse atmospheric conditions in the ecology of microorganisms in soil are discussed.

EFFECT OF HIGH INTRA-ALVEOLAR O2 TENSIONS ON PULMONARY CIRCULATION IN PERFUSED LUNGS OF DOGS.

The resistance to blood flow in the pulmonary circulation of dogs (PVR) increased when their lungs were ventilated with 95–100% oxygen and were perfused with blood that recirculated only through the pulmonary circulation; the systemic circulation was perfused independently. This increase in PVR occurred even when nerves were cut or blocked but was abolished by inhaled isopropylarterenol aerosol. Elevation of intra-alveolar Po2 without increase in pulmonary arterial blood Po2 was sufficient to increase pulmonary vascular resistance. The pulmonary venules or veins were thought to be the likely site of the constriction. These reactions were qualitatively similar to those produced by injection of serotonin or histamine into the pulmonary circulation. The time course of the response and failure to obtain it when the blood was perfused through the remainder of the body before it re-entered the pulmonary circulation are compatible with a theory that high intra-alveolar O2 tension activates a vasoconstrictor material in the pulmonary parenchyma.