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.     

Effect of concanavalin A and its succinylated derivative on the oxidative metabolism of guinea pig peritoneal macrophages

The effect of concanavalin A and its succinylated derivative on the metabolic regulation of guinea pig peritoneal macrophages was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of macrophages caused a marked increase in the rate of oxygen consumption due to the activation of the hexose monophosphate shunt. Divalent succinylated concanavalin A, also induced a similar change in the rate of oxygen metabolism. The metabolic change induced by these two types of lectin was reversibly inhibited by alpha-methyl-D-glucoside, a haptenic inhibitor of these lectins, and was temperature dependent (observed at above 15 degrees C). It is suggested that the binding of these lectins to the surface glycoproteins, and not their cross-linking into caps, is required for the activation of oxygen metabolism of macrophages, and that highly fluid state of the plasma membrane seems to be an essential requirement for the transduction of glycoprotein perturbation on the macrophage surface into cellular interior via transmembrane control mechanism.     

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.     

D-Penicillamine: analysis of the mechanism of copper-catalyzed hydrogen peroxide generation

Recent studies have suggested that the inhibition of lymphocyte mitogenesis by D-penicillamine in the presence of copper could be mediated by the formation and action of hydrogen peroxide. To explore this possibility further, we first sought evidence of H2O2 generation by D-penicillamine in a cell-free system by a) measurement of copper-catalyzed D-penicillamine oxidation and the requirement for oxygen in this process; b) direct measurement of H2O2 formation during D-penicillamine oxidation by the peroxidase-mediated oxidation of fluorescent scopoletin; and c) evaluation of the possible synthesis of O2- during D-penicillamine oxidation. The addition of copper to D-penicillamine in physiologic buffer catalyzed D-penicillamine oxidation in a dose-dependent fashion. D-penicillamine oxidation was accompanied by O2 consumption with a molar ratio of approximately 2:1, but did not occur under anaerobic conditions. Furthermore, D-penicillamine oxidation resulted in the formation of amounts of H2O2 stoichiometrically equivalent to oxygen consumption (i.e., 1:1). Copper-catalyzed D-penicillamine oxidation caused reduction of nitroblue tetrazolium in a reaction blocked by superoxide dismutase, suggesting the formation of O2-. Additional studies confirmed that D-penicillamine inhibited PHA-induced mitogenesis of lymphocytes in the presence of copper, and that catalase protected the cells from this action. Furthermore, when polymorphonuclear leukocytes were incubated with D-penicillamine plus copper, hexose monophosphate shunt activity increased up to threefold with abrogation of this stimulation by catalase. None of the effects of D-penicillamine plus copper on cells were diminished by hydroxyl radical scavengers mannitol or benzoate. These results are consistent with oxygen-dependent copper-catalyzed oxidation of D-penicillamine in aqueous solutions leading to the formation of O2- and H2O2. H2O2 produced by this reaction can inhibit lymphocyte mitogenesis and stimulate neutrophil hexose monophosphate shunt activity in vitro and may be relevant to the therapeutic effects of D-penicillamine in vivo.     

Effect of cytochalasin B on the oxidative metabolism of human peripheral blood granulocytes.

Pretreatment of human granulocytes with cytochalasin B before addition of opsonized zymosan particles resulted in strong inhibition of the oxygen consumption, the hydrogen peroxide production, and the hexose monophosphate shunt activity as compared to normal phagocytosing cells. In contrast, however, no effect of cytochalasin B was found on the generation of superoxide anions. These seemingly controversial results can be explained by the action of cytochalasin B on the cell membrane.     

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.     

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.     

Carbon balance and energetics of cyooprotectant synthesis in a freeze-tolerant insect: responses to perturbation by anoxia

Summary The capacity for polyol synthesis by larvae of Eurosta solidaginis was evaluated under aerobic versus anoxic (N₂ gas atmosphere) conditions. Glycerol production occurred readily in aerobic larvae at 13°C. Under anoxic conditions, however, net glycerol accumulation was only 57% of the aerobic value after 18 d, but the total hydroxyl equivalents available for cryoprotection were balanced by the additional synthesis of sorbitol. The efficiency of carbon conversion to polyols was much lower in anaerobic larvae. The ATP requirement of glycerol biosynthesis necessitated a 22% greater consumption of carbohydrate, when anaerobic and resulted in the accumulation of equimolar amounts of l-lactate and l-alanine as fermentative end products. The ratio of polyols produced to glycolytic end products formed was consistent with the use of the hexose monophosphate shunt to generate the reducing equivalents needed for cryoprotectant synthesis. A comparable experiment analyzed sorbitol synthesis at 3°C under aerobic versus anoxic conditions. Sorbitol synthesis was initiated more rapidly in anaerobic larvae, and the final sorbitol levels attained after 18 d were 60% higher than in aerobic larvae. The enhanced sorbitol output under anoxia may be due to an obligate channeling of a high percentage of total carbon flow through the hexose monophosphate shunt at 3°C. Carbon processed in this way generates NADPH which, along with the NADH output of glycolysis, must be reoxidized if anaerobic ATP synthesis is to continue. Redox balance within the hexose monophosphate shunt is maintained through NADPH consumption in the synthesis of sorbitol.     

Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes

To compare the kinetics of the O-2-generating enzyme in nonactivated and activated macrophages and granulocytes from the mouse peritoneal cavity, we sought conditions in which the activity of this enzyme in cell lysates was comparable to that in intact cells. Pretreatment of macrophages with 10 mM diethyldithiocarbamate inhibited endogenous superoxide dismutase by 70% and enhanced O-2 secretion up to 15-fold, so that it was comparable to H2O2 secretion. O-2 secretion was terminated by detergent lysis and reconstituted by addition of NAD(P)H to the lysates. Optimal detection of O-2 production in lysates depended on prior stimulation of the respiratory burst, lysis with 0.05% deoxycholate rather than any of 4 other detergents or sonication, acetylation of the cytochrome c used as an indicator, and addition of NADPH rather than NADH. Kinetic analysis using NADPH-reconstituted deoxycholate lysates, together with spectra of oxidized and reduced cells, failed to reveal either marked differences in the Vmax of the O-2-generating enzyme or correlations between O-2 secretion and cytochrome b559 content among 5 macrophage populations whose H2O2 secretion ranged from 0 to 365 nmol/90 min/mg of protein. In contrast, the Km of the oxidase for NADPH varied markedly and inversely with the capacity of the intact cells to secrete O-2 or H2O2: J774G8 histiocytoma cells, 1.43 mM; resident macrophages, 0.41 mM; proteose peptone-elicited macrophages, 0.20 mM; casein-activated macrophages, 0.05 mM; NaIO4-activated macrophages, 0.05 mM; and granulocytes, 0.04 mM. These results suggest that macrophage activation, a process that enhances oxygen-dependent antitumor and antimicrobial functions, may equip the cell to secrete increased amounts of reactive oxygen intermediates largely by increasing the affinity of the oxidase for NADPH.     

Glycolysis in the eye tissues of the rabbit in ontogeny. I. The enzymes of glycolysis and hexosemonophosphate shunt]

The activity of the enzymes of glycolysis (phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase) and hexose monophosphate shunt (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) was determined in the eye tissues of the rabbit at different stages of ontogenesis. The activity of these enzymes in the retina was shown to be higher than in other eye tissues. In the uveal tract (iris, ciliary bodies, uvea) the activity of glycolytic enzymes changes with the age. The greatest changes in the activity of enzymes were found during the period of the opening of eyelids. The activity of the enzymes of hexose monophosphate shunt in the eye tissues increases with the age. The relative activity of dehydrogenases of the hexose monophosphate shunt after the establishment of visual function is, however, not high and does not exceed that of phosphofructokinase and pyruvate kinase in the eye tissues of the rabbit.     

IFN-gamma primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-gamma. We hypothesize that synergistic cellular responses to IFN-gamma and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of approximately 20 s. IFN-gamma increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-gamma. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-gamma priming for oxidant production. MPO inhibitors blocked cell responses to IFN-gamma and CpG DNA. In the absence of IFN-gamma exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-gamma and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-gamma plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-gamma treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.     

Stoichiometric conversion of oxygen to superoxide anion during the respiratory burst in neutrophils. Direct evidence by a new method for measurement of superoxide anion with diacetyldeuteroheme-substituted horseradish peroxidase

Extracellular release of superoxide anion (O-2) and hydrogen peroxide (H2O2) during the respiratory burst of porcine and human neutrophils was studied by using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for these oxygen derivatives. The method permitted simultaneous measurement of oxygen consumption and formation of both O-2 and H2O2 in a single reaction mixture. When neutrophils were stimulated with phorbol myristate acetate in the presence of the heme-substituted peroxidase, a rapid accumulation of compound III, a complex of the enzyme with O-2, was observed accompanying an increase in oxygen consumption. During the process, amounts of compound III formed and oxygen consumed were stoichiometric, and no compound II, an indicator of H2O2 formation, was observed. These results establish that neutrophils stimulated with the phorbol ester produce exclusively O-2 as the primary oxygen metabolite and release it into the extracellular medium. When a limited amount of opsonized zymosan was used as the stimulus, compound III formation was also observed but it ceased at an early stage of oxygen consumption. When a sufficient amount of azide was included in the system, however, formation of compound II was noted in the later stage of oxygen consumption. The findings suggest that O-2, formed during phagocytosis, is converted to H2O2 within phagosomes and then diffuses out into the extracellular medium when its decomposition by catalase and/or peroxidases is blocked by azide.     

Carbohydrate metabolism in the rat peritoneal macrophages

Rat peritoneal macrophages derive energy differently from other tissues. Resting rat peritoneal macrophages have been taken for the present investigation. Lactate produced by extracellular glycolysis in the peritoneal lavage fluid, is readily converted into pyruvate by resting peritoneal macrophages and is oxidised in mitochondria. Glycolytic enzymes other than phosphoglucoisomerase and lactate dehydrogenase could not be substantially demonstrated. Glucose-6-phosphate dehydrogenase was detected. The presence of glucose-6-phosphate dehydrogenase along with phosphoglucoisomerase indicates the operation of the hexose monophosphate shunt as a pathway supplementary to glycolysis. Resting rat peritoneal macrophages thus appear to utilize extracellular lactate as their main energy source instead of glucose, bypass glycolysis and have active hexose monophosphate shunt.     

Suppression of monocyte and neutrophil function by recombinant IL-2

Little IS known about the influence of IL-2 on phagocytes. We now describe the effects of human recombinant IL-2 on human neutrophil and monocyte functions related to mobility, phagocytosis, glucose uptake, respiration and degranulation. Neutrophil adherence and hexose monophosphate shunt activities were both suppressed after incubation with IL-2. IL-2 had no effect on neutrophil migration, phagocytosis, deoxyglucose uptake or degranulation, ionocytes demonstrated a greater sensitivity to IL-2 with suppression of monocyte adherence, random and stimulated migration, glucose uptake and hexose monophosphate shunt activity, even after addition of phorbol myristate acetate. Monocyte phagocytosis and degranulation were not affected. All of the effects observed were dose-dependent within a biologically active range for IL-2. These studies suggest that IL-2 may have an important down-regulatory role across a broad range of monocyte functions including movement, deoxyglucose uptake and respiration. However, its role in regulation of neutrophil function is limited to adherence and respiration. IL-2 may be a more versatile cytokine than has previously been appreciated.     

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.     

Hexose monophosphate shunt,the role of its metabolites and associated disorders: A review

The hexose monophosphate (HMP) shunt acts as an essential component of cellular metabolism in maintaining carbon homeostasis. The HMP shunt comprises two phases viz. oxidative and nonoxidative, which provide different intermediates for the synthesis of biomolecules like nucleotides, DNA, RNA, amino acids, and so forth; reducing molecules for anabolism and detoxifying the reactive oxygen species during oxidative stress. The HMP shunt is significantly important in the liver, adipose tissue, erythrocytes, adrenal glands, lactating mammary glands and testes. We have researched the articles related to the HMP pathway, its metabolites and disorders related to its metabolic abnormalities. The literature for this paper was taken typically from a personal database, the Cochrane database of systemic reviews, PubMed publications, biochemistry textbooks, and electronic journals uptil date on the hexose monophosphate shunt. The HMP shunt is a tightly controlled metabolic pathway, which is also interconnected with other metabolic pathways in the body like glycolysis, gluconeogenesis, and glucuronic acid depending upon the metabolic needs of the body and depending upon the biochemical demand. The HMP shunt plays a significant role in NADPH₂ formation and in pentose sugars that are biosynthetic precursors of nucleic acids and amino acids. Cells can be protected from highly reactive oxygen species by NADPH ₂. Deficiency in the hexose monophosphate pathway is linked to numerous disorders. Furthermore, it was also reported that this metabolic pathway could act as a therapeutic target to treat different types of cancers, so treatments at the molecular level could be planned by limiting the synthesis of biomolecules required for proliferating cells provided by the HMP shunt, hence, more experiments still could be carried out to find additional discoveries.     

Peroxidase-induced enhancement of chemiluminescence by murine peritoneal macrophages

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.     

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.     

Phagocytosis of Leishmania enhances macrophage activation by IFN-gamma and lipopolysaccharide

This investigation describes the ability of Leishmania promastigotes to enhance activation of bone marrow-derived murine macrophages in vitro if added together with rIFN-gamma in the presence or absence of LPS. Activation was defined as the capacity for arginine-derived NO2- production and the killing of intracellular Leishmania. Enhanced NO2- production was observed for either CBA or C3H/HeJ macrophages undergoing phagocytosis at the time of activation. Other phagocytic stimuli including inert polystyrene latex beads were as effective as Leishmania. No correlation could be demonstrated between the enhanced NO2- release and secretion of products of the respiratory burst or PGE2. However, TNF-alpha secretion was elevated in cultures undergoing phagocytosis and a relationship between hexosemonophosphate shunt activity and NO2- levels was evident. These studies confirm and extend previous reports that phagocytosis plays an important role in the regulation of macrophage physiology.