The effect of oxidant stress on diamide-treated human granulocytes

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.     

The effect of oxidant stress on diamide-treated human granulocytes.

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xamthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.     

The effects of arachidonic and other fatty acids on canine neutrophil metabolism

The effect of arachidonic acid on the metabolic activity and chemiluminesence of canine neutrophils was investigated to gain further insight into its role in the neutrophil metabolic burst. Arachidonic acid was found to stimulate metabolic activity and luminol-augmented chemiluminescence. The increased metabolic activity was detected by both oxygen uptake measurements and assays of hexose monophosphate shunt activity. An inhibitor of lipoxygenase and cyclooxygenase,5, 8, 11, 14-eicosatetraynoic acid prevented the hexose monophosphate shunt response to arachidonic acid. Aspirin or indomethacin, blockers of cyclooxygenase, inhibited chemiluminescence but failed to block the metabolic response to arachidonic acid. Since superoxide dismutase and 2-deoxyglucose, a blocker of glucose metabolism, inhibited the chemiluminescent response of neutrophils to arachidonic acid, it is likely that oxygen radicals produced via the hexose monophosphate shunt are required for the chemiluminescent reaction. In addition it was found that inhibition of cyclooxygenase activity blocked chemiluminescence but not the metabolic stimulation induced by sodium fluoride, suggesting that the chemiluminescence stimulated by sodium fluoride is associated with endogenous fatty acid stores. From these studies it can be concluded that arachidonic acid products of the cyclooxygenase pathway do not play a significant role in the metabolic response of neutrophils when arachidonic acid or sodium fluoride is the stimulant while the lipoxygenase pathway appears to be involved. The metabolic response is not linked to the chemical reaction that causes neutrophil, chemiluminesence, although the chemiluminescent response depends on hexose monophosphate shunt activity and presumably the oxygen radicals that ultimately result from that process.     

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. II. Presence of the NADPH2 oxidizing activity in a myeloperoxidase-deficient subject.

1. The biochemical properties of leucocytes from a myeloperoxidase-deficient subject were compared with those of leucocytes from healthy subjects. 2. Myleoperoxidase-deficient leucocytes responded to phagocytosis of heat-killed bacteria with increased respiration, increased oxidation of glucose through the hexose monophosphate shunt and increased production of H2O2 as normal leucocytes do. 3. The ability of granules isolated from myeloperoxidase-deficient leucocytes to oxidize nicotinamide coenzymes was comparable to that of granules isolated from normal leucocytes. 4. The results argue against the hypothesis that oxidation of NADPH2 in leucocytes is performed by myeloperoxidase.     

The use of acetylated cytochrome c in detecting superoxide anion production in rabbit alveolar macrophages

Polymorphonuclear phagocytes have been shown to undergo marked alteration in oxidative metabolism during phagocytosis. These alterations, collectively known as the "respiratory burst", include increased glucose oxidation through the hexose monophosphate shunt (1), increased oxygen consumption (1), and increased superoxide (O-2)3 (2) and H2O2 production (3). Similar metabolic events have also been shown to occur in the rabbit alveolar macrophage (AM). There is consistent evidence that the macrophage undergoes increased oxygen consumption (4-6) and hexose monophosphate shunt activity (4-9) upon phagocytosis. There are conflicting data, however, concerning the ability of the macrophage to produce O-2. Some studies suggest that macrophages are incapable of producing measurable amounts of O-2 upon phagocytosis (7, 10-12). Other studies, however, suggest that macrophages are indeed capable of producing substantial amounts of O-2 during phagocytosis (8, 13-15). This study was designed to resolve the discrepancies in the literature concerning O-2 production in macrophages.     

Mechanism of priming of human neutrophils by a soluble lymphoblastoid cell factor

Treatment of human neutrophils (PMN) with a cytokine-like factor in the supernatants of human lymphoblastoid cells (Raji) increased the random mobility and enhanced the migration of treated cells in response to other chemoattractants nearly 21/2-fold, although the supernatant itself was not a chemoattractant. Supernatant treatment also increased the adherence of bacteria threefold and the bacterial killing fourfold compared with PMN treated with control media. In examining the metabolic basis for the enhanced bactericidal ability, we observed a significant increase in spontaneous hexose monophosphate shunt activity of Raji cell supernatant (RS)-treated neutrophils even in the absence of additional stimuli. RS-treated PMN also had significantly enhanced production of superoxide anion and chemiluminescence response upon subsequent stimulation with a variety of soluble and particulate stimuli. Unlike other agents that prime neutrophil activation, however, the factor(s) in RS did not cause degranulation. It also differed in its ability to progressively enhance PMN functions with a longer period of preincubation (up to 3 hr). These data suggest that the RS factor(s) primes neutrophils by a unique mechanism. The neutrophil-enhancing activities of RS, which are the opposite of those activities described for leukocyte inhibitory factor, eluted off a Sephacryl S-200 column at approximately 30,000 m.w. This factor expands the relationship between neutrophils and lymphocytes, and may be a useful agent to provide valuable insights into the mechanism of respiratory burst activation and regulation.     

Hexose monophosphate pathway in synapses

Abstract— Synaptosomes isolated from rat cerebral cortex converted [l-¹⁴C]glucose more rapidly than [6-²⁴C]glucose to ^,14CO₂. The ratio of C-l: C-6 in ¹⁴CO₂ was 3-9, thus suggesting that the hexose monophosphate shunt (HMP) pathway was functional in synapses in vitro. When changes in the ratio of C-l: C-6 in ¹⁴CO₂ were used as an index of shunt activity, glucose oxidation by this route was stimulated by electron acceptors as well as by neurohormones, including norepinephrine, acetylcholine and serotonin. Brain mince also exhibited a C-l: C-6 ratio of 3-2 when short (15 min) incubations were employed. Negative results previously reported are attributable to prolonged incubation during which depletion of NADP or randomization of the labelled carbons in radioactive glucose could have occurred. Our experiments excluded the incorporation of glucose into macromolecules as a specific role for the hexose monophosphate pathway. The generation of NADPH for numerous metabolic reactions including the maintenance of membrane SH groups and the oxidation and hydroxylation reactions may represent the functions of the hexose monophosphate in synaptosomes and account for its stimulation by neurohormones.     

Effects of cyclooxygenase inhibitors and prostaglandin E2 on macrophage activation in vitro

The effect of the cyclooxygenase inhibitors, indomethacin and diclofenac, and of PGE₂ on either resting or stimulated macrophages was investigated. Peritoneal macrophages were obtained from untreated mice and cultured for 10 days. Macrophage activation was induced by zymosan phagocytosis and was monitored by testing for plasminogen activator secretion and the cellular levels of lactate dehydrogenase, β-glucoronidase and alkaline phosphodiesterase I.It was found that cyclooxygenase inhibitors activate resting macrophages and enhance the degree of activation obtained after zymosan phagocytosis. Addition of exogenous PGE₂, on the other hand, had the opposite effect, it suppressed activation induced either by cyclooxygenase inhibitors, phagocytosis or a combination of both. Cyclooxygenase inhibitors and PGE₂ did not affect the hexose monophosphate shunt activity of resting macrophages and had only a minor effect on the respiratory on the respiratory macrophages and had only a minor effect on the respiratory burst occuring during zymosan phagocytosis. It appears, therefore, that the observed changes in the state of activation of the machrophages are not related to hexose monophosphate shunt activity.The described effects suggest that PGE₂ and possibly other cyclooxygenase products may function as inhibitory feed-back regulators of macrophage activation.     

Lipopolysaccharide stimulates monocyte adherence by effects on both the monocyte and the endothelial cell

The effects of the LPS moiety of endotoxin on monocyte adherence to an endothelial cell surface were investigated over times before the development of well described LPS-induced endothelial cell surface adhesive molecules. In an in vitro microtiter adherence assay, LPS in concentrations of 10 ng/ml to 10 micrograms/ml incubated for 20 to 60 min with human monocytes significantly stimulated monocyte adherence to human umbilical vein endothelial cell monolayers (HUVEC) and serum-coated plastic surfaces. The time course and concentration dependence of LPS-stimulated monocyte adherence to glutaraldehyde-fixed HUVEC did not differ significantly from that to unfixed HUVEC or serum-coated plastic surfaces. Pretreatment studies suggested that LPS acted on the monocyte within 25 min to stimulate adherence to untreated endothelial cells but required a minimum of 1.5 to 2 h to render the endothelial cell more adhesive for untreated monocytes. The potential role of TNF-alpha, IL-1 alpha, and IL-1 beta in this system was assessed by determining the ability of these cytokines (+/- cytokine antibodies) to increase monocyte adherence. TNF, but neither IL-1, stimulated early monocyte adherence (1 h). This TNF-stimulated monocyte adherence was abrogated by coincubation with anti-rTNF-alpha polyclonal antibody. However, the anti-rTNF antibody had no effect on LPS-induced monocyte adherence to endothelial cells or serum-coated plastic surfaces. An early action of LPS on the monocyte to induce adherence to endothelial cell surfaces may contribute to the initial localization of peripheral blood monocytes in tissues during endotoxemia. The later effects of LPS on the endothelial cell to stimulate monocyte adherence may then amplify these initial monocyte-endothelial cell interactions to prolong and intensify monocyte adherence prior to migration into tissues.     

Evidence for an apical membrane effect in the regulation of the hexose monophosphate shunt pathway in toad bladder studies with amiloride

Aldosterone stimulates Na⁺ transport in toad bladder and, simultaneously with a coincident dose-response relationship, inhibits the hexose monophosphate shunt pathway. Amiloride, an acylguanidine diuretic, inhibits sodium transport when applied to the apical surface of the bladder. In this study amiloride was found to partially reverse the inhibitory effect of aldosterone on the hexose monophosphate shunt pathway. The amiloride effect upon glucose metabolism was detected when it was applied to both surfaces of the bladder simultaneously, in flask experiments, and when it was applied to the apical surface. No effect of amiloride on the shunt pathway was detected when it was applied to the serosal surface only, even at very high concentrations. It may be, but has not been proven, that the effects of aldosterone and amiloride on the hexose monophosphate shunt pathway are mediated by a common site at the apical membrane.     

Glucose metabolism of thraustochytrium roseum,a nonfilamentous marine phycomycete

Summary In uniformly labeled logarithmic-phase cells of Thraustochytrium roseum grown in isotopic glucose, 85% of the respiratory CO₂ was derived from endogenous reserves and only 15% was contributed by exogenous glucose. Experiments with asymetrically labeled glucose showed that the main portion of metabolic CO₂ came from carbon 1 of the glucose molecule, suggesting that the hexose monophosphate shunt is a major pathway for glucose dissimilation in the fungus. The presence of several enzymes of the hexose monophosphate shunt, the Embden-Meyerhof and glyoxylate pathways, and the tricarboxylic acid cycle were demonstrated.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

Carbon dioxide production by red skeletal muscle of goldfish (Carassius auratus L.) aerobic and anaerobic metabolism of glucose and glutamate

Oxygen uptake and metabolic CO2 production by lateral red muscle of goldfish have been measured in vitro. Added glucose 6-phosphate depresses the rate of oxygen uptake by minced red muscle (Crabtree effect). Total CO2 production is stimulated resulting in a respiratory quotient which is considerably greater than one. 14CO2 release from [U-14C] glucose 6-phosphate and [U-14C] glutamate continues during anoxia. No activity of the hexose monophosphate shunt was observed. The results suggest that both aerobic and anaerobic CO2 production is of mitochondrial origin and, at least partially, derived from TCA cycle reactions.     

Microbiological Studies of Coli-aerogenes Bacteria

The presence of glucose-6-phosphate markedly stimulated the anaerobic utilization of glyoxylate by either cell-free extracts or partially purified enzyme preparations of coli-aerogenes bacteria. The enzymic reduction of glyoxylate to glycollate was found to occur in the presence of TPN with the following substrates; glucose-6-phosphate, glucose plus ATP, gluconate plus ATP, glucose-1-phosphate or malate. The data indicated that the reduction of glyoxylate to glycollate was coupled to the oxidation of glucose-6-phosphate via the hexose monophosphate shunt pathway. It was propounded that the operation of the hexose monophosphate oxidative pathway might be controlled by TPN-linked glyoxylic reductase, and the mechanisms of enzymic regulation in microbial respiration were also discussed.     

An opsonised microelectrode. Observation of the respiratory burst of a single human neutrophil

A 10 micron diameter gold microvoltammetric electrode, opsonised with human IgG, was used to study the respiratory burst of a single human neutrophil. The electrode oxidised superoxide produced near its surface by the neutrophil back to dioxygen. It is suggested that the current so detected is proportional to the rate of superoxide production by the NADPH oxidase of a single cell. In all cases the response consisted of a relatively rapid rise in current after cell addition, followed by a 2-phase decay. It is further suggested that this complex decay results from the production of superoxide being rate-limited initially by the NADPH concentration and later by the coupled metabolism of the hexose monophosphate shunt.     

Dynamic assessment of hexose monophosphate shunt activity in the intact rabbit lens by proton NMR spectroscopy

A proton nuclear magnetic resonance technique is demonstrated for ascertaining the real-time contribution of the hexose monophosphate shunt to glucose metabolism in the intact incubated rabbit lens. This measurement requires incubation of the tissue in medium supplemented with [1-13C]glucose, and depends on the presence of the 13C label in the methyl position of lactate which creates satellite resonances by way of 13C - 1H spin-spin scalar coupling. The assumptions required to make the measurement are presented. For lenses maintained under control conditions, a basal level corresponding to 5% hexose monophosphate shunt activity was determined. An eight-fold increase in activity was observed under conditions known to stimulate the shunt.     

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 μM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.     

Stimulation of hexose transport by human polymorphonuclear leukocytes: a possible role for protein kinase C

The protein C kinase activators 1-O-oleoyl, 2-O-acetylglycerol, 12-O-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-O-hexadecyl-2-O-ethylglycerol, 1-O-palmitoyl-2-O-acetyl or 1-O-palmitoyl-3-O-acetyl diesters of propanediol, or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxy-glucose was inhibited by trifluoperazine (10-30 microM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.     

Fatty Acyl-CoAs as feedback regulators of hexose monophosphate shunt in rat adipocytes

Summary The high basal glucose utilization through hexose monophosphate shunt found in our experimental conditions were almost completely inhibited by oleate, octanoate and caproate. However, the inhibition of glucose oxidation due to butyrate was about 50% whereas ketone bodies and acetate did not inhibit. The rate of triacylglycerol formation was not significantly modified with the above organic acids except oleate that presented a 5-fold increase on labeling incorporation into lipids. Oleate inhibition of glucose oxidation was completely prevented by the NADPH oxidant menadione. There was no inhibition by octanoate, caproate, butyrate or ketone bodies of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase or malic enzyme in adipose tissue homogenates. In contrast, specifically glucose-6-phosphate dehydrogenase was inhibited by oleoyl-CoA. The oleoyl-CoA inhibition was prevented by enzyme preincubation with low NADP concentration. The data lend further support for the hypothesis that fatty acids and NADP fulfill an important role in the modulation of the hexose monophosphate shunt activity.