Superoxide dictates the mode of U937 cell ascorbic acid uptake and prevents the enhancing effects of the vitamin to otherwise nontoxic levels of reactive oxygen/nitrogen species

Exposure of U937 cells to low micromolar levels of ascorbic acid or dehydroascorbic acid, while resulting in identical ascorbic acid accumulation, is unexpectedly associated with remarkably different responses to exogenous oxidants. We observed that otherwise nontoxic levels of hydrogen peroxide, tert-butylhydroperoxide or peroxynitrite promote toxicity in cells preloaded with ascorbic acid, whereas hardly any effect was detected in cells pretreated with dehydroascorbic acid. Further experiments performed with peroxynitrite in cells preloaded with ascorbic acid provided evidence for a very rapid nonapoptotic death, preceded by early Bax mitochondrial translocation and by mitochondrial permeability transition. The notion that conversion of extracellular ascorbic acid to dehydroascorbic acid prevents the enhancing effects on oxidant toxicity and nevertheless preserves the net amount of vitamin C accumulated was also established using ascorbate oxidase as well as various sources of superoxide, namely, xanthine/xanthine oxidase or ATP-driven NADPH oxidase activation. These findings suggest that superoxide-dependent conversion of extracellular ascorbic acid to dehydroascorbic acid represents an important component of the overall survival strategy of some cell types to reactive oxygen/nitrogen species.     

Studies on the NADPH oxidizing activity in polymorphonuclear leucocytes: The mode of association with the granule membrane the relationship to myeloperoxidase and the interference of hemoglobin with NADPH oxidase determination

Phospholipase C-treated polymorphonuclear leucocytes were used to study the properties of NADPH oxidase activity of stimulated polymorphonuclear leucocytes.A comparison of the effects of phospholipase C treatment of whole leucocytes on the NADPH oxidase activity with other granule enzymes showed that the activities of β-glucuronidase and acid phosphatase were un-affected, whereas the NADPH oxidase activity was stimulated 4-fold and myeloperoxidase was inhibited about 30%.The distribution of NADPH oxidase activity among subcellular fractions of polymorphonuclear leucocyte homogenates was unaffected by phospholipase C whereas the other enzymes were released into the medium in soluble form; β-glucuronidase > acid phosphatase and myeloperoxidase.A number of solubilizing agents and procedures were tested for their ability to release NADPH oxidase activity from granules of phospholipase C-stimulated polymorphonuclear leucocytes. All procedures used caused appreciable release of granule protein but no release of NADPH oxidase activity. Most of the procedures used strongly inhibited the oxidase activity. These results indicate that the enzyme is tightly bound to granule structures and that the integrity of these structures is required for activity.Some of the solubilizing agents used (KCI, guanidium chloride) were very effective in solubilizing myeloperoxidase.The differential response of myeloperoxidase and NADPH oxidase to treatment with phospholipase C or solubilizing procedures suggests that the two activities are not due to the same enzyme. However, definite conclusion cannot be drawn because of the complex nature of myeloperoxidase.It was found necessary to lyse any erythrocytes present as contaminants of polymorphonuclear leucocytes preparations, since hemoglobin was converted to methemoglobin during the NADPH oxidase assay and methemoglobin exhibits appreciable NADPH oxidase activity.     

The determination and localization of sialic acid in guinea-pig granulocytes.

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1--3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5--2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5'-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.     

Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium.     

The effect of phagocytosis of Candida albicans blastospores on alkaline phosphatase levels in rat polymorphonuclear leucocytes

Summary The phagocytic function of young polymorphonuclear leucocytes with high levels of leucocyte alkaline phosphatase, which are present in the peripheral blood during an inflammatory response, was compared with that of normal polymorphonuclear leucocytes.Candida albicans blastospores were used as phagocytic targets. The phagocytic index of polymorphonuclear leucocytes with low levels of leucocyte alkaline phosphatase was higher than that of cells with high levels of enzyme.Monitoring of leucocyte alkaline phosphatase levels with increasing times of incubation of leucocytes with the blastospores showed a progressive decline in the level of the enzyme. Thus loss of the enzyme is linked to the phagocytic function, although the mechanism of this dynamic process is unclear.     

Ascorbic acid analysis using high-performance liquid chromatography with coulometric electrochemical detection

A method for the detection of ascorbic acid using high-performance liquid chromatography with coulometric electrochemical detection and a technique for stabilization of the vitamin are described. Since less than 1 pmol of ascorbic acid can be detected, this assay provides significantly greater sensitivity than nearly all of the currently available procedures. Stabilization of 10 pmol or less of ascorbic acid at room temperature for up to 4 h and for several weeks at -70 degrees C facilitates storage of a large number of samples and measurement of ascorbic acid using an automated sampling device. This method was used to quantitate the amounts of ascorbic acid in human polymorphonuclear leukocytes and bovine adrenomedullary chromaffin granules. The calculated concentrations found for human neutrophils (1.35 mM) and bovine chromaffin granules (10.0 mM) are in agreement with previously published data. The assay is suitable for the determination of ascorbic acid in biological samples where only a small amount of tissue is available or very low amounts of ascorbic acid are found. This method is the first application of coulometric electrochemical detection to ascorbic acid HPLC analysis.     

Inhibition of phagocytosis of polymorphonuclear leucocytes by adenosine and HoCl3 in vitro

Phagocytosis of polymorphonuclear leucocytes treated with NaF, HoCl3 and adenosine were studied. The highest concentration used was 25 mM of NaF, 25 mM of adenosine and 5 mM of HoCl3. It was ascertained that these substances, inhibitors of erythrocyte contractile protein, inhibit both phagocytosis and ability of polymorphonuclear leucocytes to change their shape. These unfavourable effects may be induced by the chemicals interfering with polymorphonuclear leucocytes contractile protein. NaF, HoCl3 and adenosine are also responsible for morphological changes in the cell nucleus.     

Effect of Temperature on the Morphometrical and Physical Parameters of Erythrocytes and Polymorphonuclear Leucocytes in <Emphasis Type="Italic">Carassius gibelio</Emphasis> (Bloch)

Dynamics of the morphometric and physical properties of hemocytes of the Prussian carp Carassius gibelio (Bloch) under the influence of a temperature factor has been studied with atomic force microscopy in experiments in vitro. It is found that, at a low incubation temperature (5°C), as opposed to room temperature (20°C), morphometric parameters change in erythrocytes; at a high temperature (40°C) they change in polymorphonuclear leucocytes. The low incubation temperature reduces the adhesion and elasticity of polymorphonuclear leucocytes and erythrocytes of C. gibelio, whereas a high incubation temperature leads to a decrease in adhesion in polymorphonuclear white blood cells.     

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis

Summary The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes.The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O₂ ^– and H₂O₂, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O₂ ^– and H₂O₂ by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H₂O₂ is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H₂O₂ degrading enzymes in guinea pig cells.The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported.It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H₂O₂ degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.     

Ascorbic acid metabolism and glycolysis in the polymorphonuclear leucocyte of the guinea pig

Exudate leucocytes lost approximately 30% of their original intracellular ascorbic acid content during two hour incubation in glucose medium. The same loss was observed for cells containing initially both high and low levels of ascorbic acid. High concentrations of ascorbic acid in the incubation medium depressed lactic acid production and increased oxygen uptake by the cells. Iodoacetate and fluoride at low concentrations decreased ascorbic acid loss from cells during incubation; at high concentrations they increased loss. Ascorbic acid uptake from the medium was inhibited by iodoacetate but stimulated by fluoride.     

The unsuitability of the uridine incorporation assay for the measurement of phagocytosis of Escherichia coli

The uridine incorporation technique for assaying phagocytosis is based on the fact that polymorphonuclear leucocytes are impermeable to labelled uridine, and therefore ingested bacteria inside phagocytic vacuoles will be unable to take it up. Extracellular bacteria, including those adherent to the phagocytic cell surface, can do so however. Differences in uptake between bacteria alone and in the presence of phagocytic cells can be used to measure ingestion. The present paper describes the application of this technique to Escherichia coli O-86 as the test organism. It appears that with this test species, the method is unsuccessful, because exposure of the non-ingested bacteria to some soluble product of the triggered polymorphonuclear leucocytes causes a large increase in their uridine uptake rates, over that of the control bacteria. The nature of the product responsible is unknown. It is unconnected with change in the pH of the medium, is heat stable, and is only produced by polymorphonuclear leucocytes which are actively phagocytosing. It may be that a release of phagolysosome contents is responsible.     

Enhancement of chemotactic response and microtubule assembly in human leukocytes by ascorbic acid.

The incubation of human leukocytes with ascorbic acid increased chemotaxis of the cells. In addition, ascorbic acid promoted the assembly of intracellular polymorphonuclear leukocyte (PMN) with colchicine blocked the effect of ascorbic acid on promoting microtubule assembly. Not only did ascorbic acid promote the assembly of microtubules in vivo, but it enhanced the assembly of bovine brain tubulin into microtubules in vitro as quantitated by a glass-fiber filtration assay and by promotion of viscosity changes. The enhancement in leukocyte mobility by ascorbate at concentrations achievable in normal tissues correlates with its ability to assemble microtubule organelles.     

A comparison of the effects of phytohaemagglutinin and of calcium ionophore A23187 on the metabolism of glycerolipids in small lymphocytes.

1. The effects of phytohaemagglutinin and of a Ca2+ ionophore (A23187) on glycerolipid metabolism in lymphocytes from pig lymph nodes were compared (a) by studying the incorporation of [32P]Pi and [3H]glycerol, and (b) by following the redistribution of [3H]glycerol among the lipids caused by these agents in pulse-chase experiments. 2. Phytohaemagglutinin only stimulated 32P incorporation into phosphatidylinositol and, to a slight extent, phosphatidate. Removal of most of the extracellular Ca2+ somewhat decreased this response. 3. Ionophore A23187 stimulated the labelling of phosphatidate and phosphatidylinositol with 32P to a much greater extent than did phytohaemagglutinin: the increase in phosphatidate labelling, but not that of phosphatidylinositol, was almost abolished by the removal of extracellular Ca2+. 4. The combined effects of phytohaemagglutinin and ionophore appeared to be additive, rather than synergistic. 5. Treatment with ionophore A23187 somewhat decreased the total incorporation of [3H]glycerol into glycerolipids, possibly because it lowered cell ATP content. In these experiments di- and tri-acylglycerol behaved anomalously, triacylglycerol labelling being suppressed completely, whereas that of diacylglycerol was enhanced. The pulse-chase results revealed that triacylglycerol was converted into diacylglycerol in the ionophore-treated cells, and the availability of this diacylglycerol probably led to the enhanced labelling of phosphatidate and phosphatidylinositol in the these cells. 6. Thus an increase in intracellular Ca2+ concentration appeared to have three effects on glycerolipid metabolism: (a) slight inhibition of some metabolic step preceding phosphatidate synthesis, (b) inhibition of diacylglycerol acyltransferase and (c) activation of a triacylglycerol lipase. 7. In contrast, it seems likely that the only effect of phytohaemagglutinin is to stimulate phosphatidylinositol breakdown. 8. Pig polymorphonuclear leucocytes treated with ionophore A23187 showed metabolic changes that were similar to those demonstrated with lymphocytes. 9. A possible similarity is suggested between Ca2+-stimulated triacylglycerol lipase in lymphocytes and polymorphonuclear leucocytes and previous observations of enhanced triacylglycerol metabolism in stimulated cells whose metabolic functions involve membrane fusion.     

Decreased levels of ascorbic acid in lung following exposure to ozone

Mice were exposed to concentrations of 20, 40 and 200 ppm ozone in air for 30 min. Ozone exposure decreased lung ascorbic acid levels and increased lung weight by up to 50% in a dose related manner. On incubation in Krebsphosphate solution, lung slices from mice exposed to 200 ppm ozone released a smaller fraction of their content of ascorbic acid into the medium than did lung slices from control mice, suggesting that there was a preferential loss of extracellular ascorbic acid during ozone exposure. These results are consistent with the proposed function of ascorbic acid as an extracellular antioxidant in lungs.     

Regulation of angiogenesis in vitro by collagen metabolism

Summary The role of collagen in microvascular growth was investigated using the aortic ring model of angiogenesis. Collagen production by vasoformative outgrowths in plasma clot culture of rat aorta was either stimulated with ascorbic acid or inhibited with the proline analogue cis-hydroxyproline. Microvessels proliferating in the absence of ascorbic acid supplements became ectatic and developed large lumina. In contrast, newly formed microvessels in the presence of ascorbic acid remained small and maintained thin lumina throughout the angiogenic process. Biochemical studies demonstrated enhanced collagen production and deposition in cultures treated with ascorbic acid. Ultrastructural studies of these cultures showed a marked increase in newly formed interstitial collagen in the perivascular matrix and in regions of the plasma clot containing nonendothelial mesenchymal cells. Small microvessels with thin lumina similar to the ones observed in ascorbic acid-treated plasma clot cultures were obtained by growing aortic explants in gels of interstitial collagen in the absence of ascorbic acid. Inhibition of collagen production with the proline analogue cis-hydroxyproline had a marked anti-angiogenic effect in both plasma clot and collagen gel cultures. The anti-angiogenic effect of cis-hydroxyproline was abolished by addingl-proline to the culture medium, thereby restoring normal metabolism. These results support the hypothesis that angiogenesis is regulated by collagen production and suggest that the size of newly formed microvessels is influenced by the degree of collagenization of the extracellular matrix.     

Interaction of polymorphonuclear leukocytes, bradykinin and carragenin. Transference reaction in the rat

1. Injected in the paw of the rat, polymorphonuclear leucocytes do not increase the oedematogen action of bradykinin, but increase the action of lambda carrageenan. 2. This potentiation of carrageenan action is not modified when PG biosynthesis in leucocytes is inhibited by indomethacin or aspirin. It does not appear in rats previously treated by indomethacin or aspirin. 3. Our results suggest that rat polymorphonuclear leucocytes increase the inflammatory reaction, when they are stimulated by carrageenan, by the release of a phospholipase A2 activity which induces PG biosynthesis in rat paw tissues.     

Redox Changes in Perfusates Following Intracerebral Penetration of Microdialysis Probes

Microdialysis probe insertion into rat cerebral cortex significantly affects the levels of redox-active substances in brain extracellular fluid. Ascorbic acid levels are high immediately after probe insertion, decline rapidly, and then rise as the rat recovers from anesthesia 5–8 hours after surgery. Uric acid is at a low level for 5 hours and then rapidly increases in parallel with ascorbic acid. High ascorbic acid levels immediately after probe insertion are likely due to a shift from intracellular to extracellular fluids, whereas the delayed increase in uric acid may be due to increased enzymatic formation. After removal from the brain, hydrogen peroxide (H₂O₂) in microdialysis samples produces catalase-sensitive oxidative chemiluminescence. Microdialysis samples also produce high level catalase-resistant chemiluminescence associated with ascorbic acid levels after penetration injury. Although ascorbic acid is likely an antioxidant at concentrations estimated to be in brain extracellular fluid, it may have prooxidant effects when complexed with transition metals released into the neuronal microenvironment during traumatic brain injury.     

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.     

Adenosine production inside rat polymorphonuclear leucocytes.

Adenosine synthesis was studied during 2-deoxyglucose-induced ATP catabolism in intact rat polymorphonuclear leucocytes. When both adenosine kinase (EC 2.7.1.20) and adenosine deaminase (EC 3.5.4.4) were selectively inhibited, adenosine accumulated. Adenosine formation took place inside the intact cells by a metabolic pathway independent of the ecto-5'-nucleotidase (EC 3.1.3.5). Distinct metabolic pathways are proposed for adenosine production from intracellular or extracellular nucleotides.     

The effect of ascorbic acid on the nature and production of collagen and elastin by rat smooth-muscle cells.

1. The effects of various concentrations of ascorbic acid on the quality and quantity of the insoluble extracellular matrices produced by two strains of cultured rat smooth-muscle cells were studied. 2. Ascorbic acid was necessary for the appearance of insoluble collagen in the extracellular matrix. 3. Secretion of soluble collagen continued in the absence of ascorbic acid, but this soluble collagen was markedly underhydroxylated. 4. The amount of insoluble collagen present in the matrix was directly related to the ascorbic acid concentration. 5. The insoluble collagen that appeared in the matrix under conditions where ascorbic acid was limiting was no more than 7% underhydroxylated. 6. In contrast, the amount of insoluble elastin produced was inversely proportional to the ascorbic acid concentration. 7. The elastin produced in the absence of ascorbic acid had the expected amino acid composition, but hydroxyproline was absent. 8. The hydroxyproline content of elastin was also directly dependent on the ascorbic acid concentration. 9. Ascorbic acid had variable effects on the quantity of glycoprotein(s) present in the matrix. 10. The appearance of insoluble collagen in the extracellular matrices produced by cultured human fibroblasts and calf endothelial cells was also completely dependent on the presence of ascorbic acid.