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Role of Glutathione and Nitric Oxide in the Energy Metabolism of Rat Liver Mitochondria Manabu Nishikawaa, Eisuke F

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Role of Glutathione and Nitric Oxide in the Energy Metabolism of Rat Liver Mitochondria Manabu Nishikawaa, Eisuke F FEBS 19304 FEBS Letters 415 (1997) 341-345 Role of glutathione and nitric oxide in the energy metabolism of rat liver mitochondria Manabu Nishikawaa, Eisuke F. Satoa, Tetsuo Kurokib, Masayasu Inouea* ^Department of Biochemistry, Osaka City University Medical School, 1-4-54 Asahimachi, Abenoku, Osaka 545, Japan hDepartment of Internal Medicine, Osaka City University Medical School, 1-4-54 Asahimachi, Abenoku, Osaka 545, Japan Received 1 August 1997; revised version received 3 September 1997 ble NO donor [16], cellular GSH might play important roles Abstract Previous studies have suggested that nitric oxide (NO) inhibited mitochondrial respiration. NO and/or its inter- in regulating the fates of NO functions. mediate^) react with various molecules, such as hemeproteins During the experiments with ascites hepatoma cells [10], we and free SH groups. The inhibitory effect of NO on mitochon- found that the activity of NO to inhibit mitochondrial respi- drial respiration was decreased by exogenously added glutathione ration was increased by treating cells with digitonin, a mem- (GSH). However, a decrease of intramitochondrial GSH by brane permeabilizing agent. The enhancing effect of digitonin pretreating animals with L-buthionine sulfoximine had no was inhibited by adding thiol compounds in the incubation appreciable effect on the inhibitory effect of isolated mitochon- medium. Because GSH is the major low molecular weight dria. Furthermore, the effect of NO was not affected by depleting thiol, cellular GSH might be an important factor that affects free SH residues in mitochondria by /V-ethyhnaleimide. These the NO-dependent inhibition of mitochondrial energy metab- results suggest that cytosolic but not intramitochondrial GSH olism. The present work describes the effects of GSH and might be an important factor that determines the NO-dependent regulation of mitochondrial energy metabolism. related thiols in and around mitochondria on the inhibitory action of NO. © 1997 Federation of European Biochemical Societies. Key words: Mitochondria; Glutathione; Nitric oxide; 2. Materials and methods Energy metabolism 2.1. Chemicals NO and argon gases were obtained from Kinkisanki Co. (Osaka, Japan). All other reagents used were of the highest purity and ob- 1. Introduction tained from Sigma Chemical Co. (St. Louis, MO, USA). Nitric oxide (NO) is a reactive gaseous free radical which 2.2. Preparation of NO solution regulates the activity of various enzymes through posttransla- NO solution was prepared after bubbling NO gas through 50 mM tional modification, such as nitrosylation of metal complexes HEPES-NaOH buifer, pH 7.4, as described previously [17]. Briefly, two small tubes were fitted with an air-tight septum with glass tubes and free cysteinyl residues [1]. Potential sites of NO-iron com- inserted for delivery and escape of gases with the first tube containing plex in cells include heme and non-heme iron groups in en- 5 M KOH and the second containing the HEPES-NaOH buffer. Ar- zymes, such as cytochrome c oxidase [2-4] and other proteins gon was delivered into two tubes at a flow rate of 100 ml/min. After in the mitochondrial electron transport system [5,6]. Some 15 min, argon was replaced by NO (100 ml/min). After 15 min, the metabolites of NO, such as peroxynitrite, also inhibit various saturated NO solution (1.9 mM) was stored at 4°C and used for experiments within 3 h; NO concentration remained unchanged dur- enzymes including aconitase [7,8]. ing the experiments. NO concentration was determined using electron Previous studies in this laboratory had suggested that low spin resonance and the NO trapping agent, 2-(4-carboxyphenyl)- levels of NO reversibly inhibited the respiration and ATP syn- 4,4,5,5-tetramethylimidazoline-l-oxyl 3-oxide (carboxy-PTIO) [18,19]. thesis of isolated mitochondria [9] and ascites tumor cells [10] particularly under physiologically low oxygen tensions. Ki- 2.3. Preparation of S-nitrosoglutathione (GSNO) netic analysis revealed that cytochrome c oxidase was the GSNO was prepared according to the method of Hart [20] by in- cubating equimolar GSH and sodium nitrite in acidified water at 0°C. primary site for the inhibition by NO [10]. GSNO solutions were freshly prepared from frozen stored GSNO NO and/or its intermediary metabolite(s) also react with powder before each experiment. The concentration of GSNO was determined spectrophotometrically at 335 nm (e = 992 M_1 cm-1) free sulfhydryl groups of various compounds to form S-nitro- 1 -1 sothiols [11]. In mammalian cells, reduced glutatione (GSH), a and at 545 nm (e= 15.9 M" cm ). major non-protein thiol, is synthesized exclusively in a cyto- 2.4. Isolation of mitochondria solic compartment and plays important roles in cellular func- Male Wistar rats (200-250 g) were fasted overnight. Liver mito- tions, such as protection of cells and tissues from oxidative chondria were isolated according to the method described previously stress [12-14]. Because NO and/or its metabolite(s) react with [21] using a medium containing 0.25 M sucrose, 10 mM Tris-HCl (pH thiols [15] and the resulting 5-nitrosothiols function as a sta- 7.4), and 0.1 mM EDTA. EDTA was omitted in the final wash and the mitochondrial samples were suspended in 0.25 M sucrose contain- ing 10 mM Tris-HCl, pH 7.4, at 10-20 mg protein/ml and stored at 4°C until use. Mitochondrial protein was determined by the method *Corresponding author. Fax: (81) (6) 645-2025. of Bradford. E-mail : [email protected] 2.5. Analysis of oxygen consumption Abbreviations: NO, nitric oxide; GSH, reduced glutathione; BSO, Oxygen consumption by isolated mitochondria was determined po- L-buthionine sulfoximine; NEM, /V-ethylmaleimide; GSNO, S-nitro- larographically using a Clark type oxygen electrode fitted to a 2 ml soglutathione; NOS, nitric oxide synthase; GSSG, oxidized glu- water-jacketed closed chamber [22]. Isolated mitochondria (1 mg pro- tathione; Cys, cysteine; NAC, 7V-acetylcysteine tein/2 ml) were suspended in the reaction medium consisting of 0.2 M 0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved. P//S0014-5793 (9 7)01156-3 342 M. Nishikawa et al.lFEBS Letters 415 (1997) 341-345 sucrose, 10 mM KC1, 1 mM MgCl2, 2 mM sodium phosphate and 10 mM Tris-HCl (pH 7.4). Oxygen consumption was monitored in the presence of 5 mM succinate and 600 uM ADP. During the experi- ments, GSNO or NO-saturated solutions were added to the reaction mixture at varying oxygen tensions. 2.6. Decrease of mitochondrial GSH L-Buthionine sulfoximine (BSO) (0.25 mmol/kg) was injected intra- peritoneally to rats every 12 h for 3 days. At the indicated times, liver mitochondria were isolated as described above. 2.7. Depletion of free SH residues in mitochondria Liver mitochondria were incubated with varying concentrations of N-ethylmaleimide (NEM) for 10 min at 4°C. Then, mitochondria were washed twice with 0.25 M sucrose buffer by repeated centrifugations and used for experiments. Time (min) 2.8. Measurement of glutathione and free thiols Freshly isolated mitochondria were immediately sonicated in 100 ul Fig. 2. Effect of GSH on mitochondrial respiration inhibited by of ice-cold 5% sulfosalicylic acid solution at a concentration of 100 mg NO. Mitochondrial respiration was monitored in the presence (B) protein/ml. After centrifugation at 12000Xg for 15 min, concentra- or absence (A) of 5 mM GSH. At the indicated times (arrows), tions of free thiols and total glutathione (GSH and 2GSSG) in the NO-saturated solution was added to the reaction mixture. Added acid-soluble fractions were determined. Total glutathione was meas- NO concentrations of numbers 1, 2, 3 and 4 were 0, 0.5, 1 and 2 ured by the method of Tietze [23]. Levels of free thiols were deter- |xM, respectively. Other conditions were the same as described in mined using Ellman's reagent [24]. Fig. 1. Experiments were performed at least five times using differ- ent samples of mitochondria with similar results. 3. Results cytosolic thiols in the energy metabolism of mitochondria, the 3.1. Effect of NO and GSNO on mitochondrial respiration inhibitory effect of NO was also tested with mitochondria in In the presence of succinate and inorganic phosphate, ADP the presence of a physiological concentration (5 mM) of GSH. increased the rate of oxygen consumption by rat liver mito- The inhibitory effect of NO was markedly decreased by exo- chondria. Consistent with previous observations [9], the state genously added GSH. The effect of GSH also depended on its 3 respiration was reversibly inhibited by a small amount of concentration (Fig. 3). NO (Fig. 1). The inhibitory effect of NO was stronger at low oxygen tensions than at high tensions. On the other hand, the 3.2. Effect of various thiols on inhibitory effect of NO same concentration of GSNO exhibited no significant effect To test the specificity of GSH, the effect of related com- on mitochondrial respiration particularly at high oxygen ten- pounds on the NO action was also studied. The inhibitory sions. When oxygen tensions became lower than 50 \iM, effect of NO was also decreased by the presence of other GSNO inhibited the respiration only slightly. Similar inhibi- thiols, such as cysteine and iV-acetylcysteine (NAC) (Fig. 4). tion was also observed in the dark. Among various thiols used, the inhibitory effect of cysteine As shown in Fig. 2, NO inhibited the respiration of mito- was the most apparent. Oxidized glutathione (GSSG) had no chondria in a dose-dependent manner. To study the role of appreciable effect on the inhibition by NO. 3.3. Effect of mitochondrial GSH on NO-dependent respiratory inhibition Under physiological conditions, GSH levels in the mito- chondrial matrix are fairly high (~10 mM).
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