Metabolic fate of glutamine in lymphocytes, macrophages and neutrophils

Eric Newsholme's laboratory was the first to show glutamine utilization by lymphocytes and macrophages. Recently, we have found that neutrophils also utilize glutamine. This amino acid has been shown to play a role in lymphocyte proliferation, cytokine production by lymphocytes and macrophages and phagocytosis and superoxide production by macrophages and neutrophils. Knowledge of the metabolic fate of glutamine in these cells is important for the understanding of the role and function of this amino acid in the maintenance of the proliferative, phagocytic and secretory capacities of these cells. Glutamine and glucose are poorly oxidized by these cells and might produce important precursors for DNA, RNA, protein and lipid synthesis. The high rate of glutamine utilization and its importance in such cells have raised the question as to the source of this glutamine, which, according to current evidence, appears to be muscle.

Arachidonic acid triggers an oxidative burst in leukocytes

The change in cellular reducing potential, most likely reflecting an oxidative burst, was investigated in arachidonic acid- (AA) stimulated leukocytes. The cells studied included the human leukemia cell lines HL-60 (undifferentiated and differentiated into macrophage-like and polymorphonuclear-like cells), Jurkat and Raji, and thymocytes and macrophages from rat primary cultures. The oxidative burst was assessed by nitroblue tetrazolium reduction. AA increased the oxidative burst until an optimum AA concentration was reached and the burst decreased thereafter. In the leukemia cell lines, optimum concentration ranged from 200 to 400 µM (up to 16-fold), whereas in rat cells it varied from 10 to 20 µM. Initial rates of superoxide generation were high, decreasing steadily and ceasing about 2 h post-treatment. The continuous presence of AA was not needed to stimulate superoxide generation. It seems that the NADPH oxidase system participates in AA-stimulated superoxide production in these cells since the oxidative burst was stimulated by NADPH and inhibited by N-ethylmaleimide, diphenyleneiodonium and superoxide dismutase. Some of the effects of AA on the oxidative burst may be due to its detergent action. There apparently was no contribution of other superoxide-generating systems such as xanthine-xanthine oxidase, cytochromes P-450 and mitochondrial electron transport chain, as assessed by the use of inhibitors. Eicosanoids and nitric oxide also do not seem to interfere with the AA-stimulated oxidative burst since there was no systematic effect of cyclooxygenase, lipoxygenase or nitric oxide synthase inhibitors, but lipid peroxides may play a role, as indicated by the inhibition of nitroblue tetrazolium reduction promoted by tocopherol.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Baroreflex sensitivity and oxidative stress in the LDL receptor knockout mice

This study alms at observing the effect of low-density lipoprotein (LDL) receptor deficiency in cholesterol blood levels, baroreflex sensitivity (BRS), nitric oxide (NO) bioavailability, and oxidative stress. The lack of LDL receptors in mice significantly increased the cholesterol blood levels (179 +/- 35 vs. 109 +/- 13 mg/dL) in the knockout (KO) mice compared to control. There was no difference in basal mean arterial pressure and heart rate between the groups. However, in KO mice the BRS was significantly attenuated and the antioxidant enzyme activities, measured in erythrocytes and heart, were significantly decreased. On the other hand, the oxidative damage measured by chemiluminescence and carbonyls was increased, while total plasma nitrate levels were lower in KO mice, indicating a decrease in NO availability. In conclusion, these results indicate that the lack of LDL receptor increased cholesterol blood levels, induced oxidative stress and decreased BRS. (C) 2008 Elsevier GmbH. All rights reserved.

Modulation of rat neutrophil function in vitro by cis- and trans-MUFA

In the present study, the effects of trans-MUFA, elaidic acid (EA; 18 : 1-9t) and vaccenic acid (VA; 18 : 1-11t) on rat neutrophil functions were compared with those of cis-monounsaturated oleic acid (OA) (18 : 1-9c) and saturated stearic acid (SA; 18 : 0) (10-150 mu M). Trans-fatty acids enhanced neutrophil phagocytic capacity, superoxide (O(2)(center dot-)) and hydrogen peroxide production, and candidacidal activity. The same effects were observed for OA. Cells treated with trans-MUFA showed reduced production of NO(center dot), whereas those treated with OA showed an increase in production. Treatment with SA did not provoke significant effect on the parameters investigated. The increase in O(2)(center dot-) production induced by MUFA was not observed when diphenyleneiodonium, an NADPH oxidase inhibitor, was added to the medium. This finding suggests that MUFA stimulate neutrophil NADPH oxidase activity. The addition of 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-inclol-3-yl)-1H-pyrrole-2,5-dione, a protein kinase C (PKC) inhibitor, and wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor, did not affect O(2)(center dot-) production induced by MUFA. Therefore, the mechanisms by which MUFA stimulate NADPH oxidase are not dependent on PKC and do not seem to involve PI3K. Experiments using Zn(2+), an inhibitor of NADPH oxidase H(+) channel, indicated that MUFA activate the NADPH oxidase complex in rat neutrophil due to opening of H(+) channel.

REACTIVE OXYGEN SPECIES AND THE STRUCTURAL REMODELING OF THE VISUAL SYSTEM AFTER OCULAR ENUCLEATION

Redox processes associated with controlled generation of reactive oxygen species (ROS) by NADPH oxidase (Nox) add an essential level of regulation to signaling pathways underlying physiological processes. We evaluated the ROS generation in the main visual relays of the mammalian brain, namely the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG), after ocular enucleation in adult rats. Dihydroethidium (DHE) oxidation revealed increased ROS generation in SC and DLG between 1 and 30 days postlesion. ROS generation was decreased by the Nox inhibitors diphenyleneiodonium chloride (DPI) and apocynin. Real-time PCR results revealed that Nox 2 was upregulated in both retinorecipient structures after deafferentation, whereas Nox 1 and Nox 4 were upregulated only in the SC. To evaluate the role of ROS in structural remodeling after the lesions, apocynin was given to enucleated rats and immunohistochemistry was conducted for markers of neuronal remodeling into SC and DLG. Immunohistochemical data showed that ocular enucleation produces an increase of neurofilament and microtubule-associated protein-2 immunostaining in both SC and DLG, which was markedly attenuated by apocynin treatment. Taken together, the findings of the present study suggest a novel role for Nox-induced ROS signaling in mediating neuronal remodeling in visual areas after ocular enucleation. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 mu M testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra- and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 mu M testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 mu M testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 mu M. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 mu M testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright (C) 2010 John Wiley & Sons, Ltd.

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-. but not the Noxl- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1- or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O(2)(center dot-)) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O(2)(center dot-) production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent. (C) 2011 Elsevier Inc. All rights reserved.

Extracellular Signal-Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal-regulated kinase (ERK) 1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191 +/- 3 mm Hg) compared with female DOCA rats (172 +/- 7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22 +/- 3 mN; n=6) and small mesenteric arteries (13 +/- 2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16 +/- 3 and 10 +/- 2 mN, respectively; P<0.05) and female DOCA rats (15 +/- 1 and 11 +/- 1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 mu mol/L) abrogated increased contraction to phenylephrine in aorta (14 +/- 2 mN) and small mesenteric arteries (10 +/- 2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process. (Hypertension. 2010; 55: 172-179.)

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.

Saccharomyces cerevisiae coq10 null mutants are responsive to antimycin A

Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q(2). Unlike other coq mutants with hampered coenzyme Q(6) (Q(6)) synthesis, coq10 mutants have near wild-type concentrations of Q(6). In the present study, we used Q-cycle inhibitors of the coenzyme QH(2)-cytochrome c reductase complex to assess the electron transfer properties of coq10 cells. Our results show that coq10 mutants respond to antimycin A, indicating an active Q-cycle in these mutants, even though they are unable to transport electrons through cytochrome c and are not responsive to myxothiazol. EPR spectroscopic analysis also suggests that wild-type and coq10 mitochondria accumulate similar amounts of Q(6) semiquinone, despite a lower steady-state level of coenzyme QH(2)-cytochrome c reductase complex in the coq10 cells. Confirming the reduced respiratory chain state in coq10 cells, we found that the expression of the Aspergillus fumigatus alternative oxidase in these cells leads to a decrease in antimycin-dependent H(2)O(2) release and improves their respiratory growth.

Metabolismo oxidativo de heterófilos de avestruzes jovens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estresse oxidativo e aumento da apoptose em neutrófilos de cães com azotemia pré-renal

O presente trabalho tem como objetivo testar a hipótese de que, à semelhança do que ocorre na uremia, cães com azotemia pré-renal sofrem estresse oxidativo, o qual está relacionado com alterações do metabolismo oxidativo e apoptose dos neutrófilos. Para tal, foi determinada a peroxidação lipídica pela quantificação do malondialdeído (MDA) e o status antioxidante total do plasma de 15 cães normais e 10 com azotemia pré-renal, correlacionando-os com a produção de superóxido e o índice apoptótico dos neutrófilos. As determinações do MDA e do status antioxidante total foram estabelecidas empregando-se um conjunto de reagentes comerciais. Por meio de citometria de fluxo capilar, a produção de superóxido e a apoptose de neutrófilos isolados de sangue periférico foram determinadas utilizando-se a sonda hidroetidina e o sistema anexina V-PE, respectivamente. Cães azotêmicos (26,29±5,32g/L) apresentaram menor concentração (p=0,0264) do antioxidante albumina em relação ao grupo-controle (30,36±3,29g/L) e também uma menor (p=0,0027) capacidade antioxidante total (2,36±0,32 versus 2,73±0,24mmol/L), enquanto não houve alteração da peroxidação lipídica plasmática e da produção de superóxido neutrofílica. Concluiu-se que, à semelhança do que ocorre na uremia, condições azotêmicas pré-renais no cão causam estresse oxidativo e aceleração da apoptose dos neutrófilos.

Efeito inibidor do soro urêmico sobre o metabolismo oxidativo dos neutrófilos de cães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)