Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.     

Similarities between rat liver mitochondrial and cytosolic glutathione reductases and their apoenzyme accumulation in riboflavin deficiency

Glutathione reductase has been purified 5,500-fold from rat liver mitochondrial matrix in a yield of 30%. The mitochondrial enzyme was immunochemically indistinguishable from that of the cytosol and the subunit molecular weight was apparently similar to that of the cytosolic enzyme, that is, 50,000 daltons. The optimum pH and kinetic properties investigated were not significantly different from those of the cytosolic enzyme. When rats were fed a riboflavin-deficient diet, the enzyme activity in the mitochondria decreased to a greater extent than that in the cytosol, and greater accumulation of apo-enzyme in the former than that in the latter was confirmed by the amount of immunoprecipitable protein, activation by FAD addition in vitro, and the enzyme activity recovery after injection of riboflavin, into riboflavin-deficient rats.     

Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper

H(2)O(2) inactivation of particular GST isoforms has been reported, with no information regarding the overall effect of other ROS on cytosolic GST activity. The present work describes the inactivation of total cytosolic GST activity from liver rats by the oxygen radical-generating system Cu(2+)/ascorbate. We have previously shown that this system may change some enzymatic activities of thiol proteins through two mechanisms: ROS-induced oxidation and non-specific Cu(2+) binding to protein thiol groups. In the present study, we show that nanomolar Cu(2+) in the absence of ascorbate did not modify total cytosolic GST activity; the same concentrations of Cu(2+) in the presence of ascorbate, however, inhibited this activity. Micromolar Cu(2+) in either the absence or presence of ascorbate inhibited cytosolic GST activity. Kinetic studies show that GSH but no 1-chloro-2,4-dinitrobenzene prevent the inhibition on cytosolic GST induced by micromolar Cu(2+) either in the absence or presence of ascorbate. On the other hand, NEM and mersalyl acid, both thiol-alkylating agents, inhibited GST activity with differential reactivity in a dose-dependent manner. Taken together, these results suggest that an inhibitory Cu(2+)-binding effect is likely to be negligible on the overall inhibition of cytosolic GST activity observed by the Cu(2+)/ascorbate system. We discuss how modification of GST-thiol groups is related to the inhibition of cytosolic GST activity.     

Purification and properties of rat liver mitochondrial glutathione peroxidase.

Glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) was purified from rat liver mitochondria. The enzyme was shown to be pure by polyacrylamide-gel electrophoresis and to contain multiple forms that differed in charge. Selenium was specifically associated with the enzyme. The enzyme was inhibited by iodoacetic acid and iodoacetamide in an unusual pattern of reduction by sulfhydryl compounds and pH dependency. The mitochondrial and cytoplasmic forms of the enzyme were compared, and an explanation of the inhibition patterns is offered.     

Role of calcium and cyclophilin D in the regulation of mitochondrial permeabilization induced by glutathione depletion

The mitochondrial permeability transition (MPT) is a calcium and oxidative stress sensitive transition in the permeability of the mitochondrial inner membrane that plays a crucial role in cell death. However, the mechanism regulating the MPT remains controversial. To study the role of oxidative stress in the regulation of the MPT, we used diethyl maleate (DEM) to deplete glutathione (GSH) in human leukemic CEM cells. GSH depletion increased mitochondrial calcium and reactive oxygen species (ROS) levels in a co-dependent manner causing loss of mitochondrial membrane potential (deltapsi(m)) and cell death. These events were inhibited by the calcium chelator BAPTA-AM and the antioxidants N-acetylcysteine (NAC) and the triphenyl phosphonium-linked ubiquinone derivative MitoQ. In contrast, the MPT inhibitor cyclosporine A (CsA) and small interference RNA (siRNA) knockdown of cyclophilin D (Cyp-D) were not protective. These results indicate that mitochondrial permeabilization induced by GSH depletion is not regulated by the classical MPT.     

The kinetics of rat liver and heart mitochondrial beta-hydroxybutyrate dehydrogenase.

The kinetic mechanisms of the beta-hydroxybutyrate dehydrogenase from rat heart and liver mitochondria were investigated. Both enzymes, show an Ordered Bi Bi mechanism and there are no major differences in the kinetic constants. In both cases, the solubilized enzyme, re-activated with phosphatidylcholine, shows kinetic properties very similar to those of the enzyme bound to the mitochondrial membrane.     

Carbon monoxide exposure in rat heart: glutathione depletion is prevented by antioxidants

Rat hearts were perfused for 15min with buffer equilibrated with 0.01% or 0.05% CO. The buffer was equilibrated with 21% O(2) throughout. The ventricular glutathione content decreased by 76% and 84%, 90min post-exposure to 0.01% and 0.05% CO, respectively, compared with 0% CO controls (0.45+/-0.01 micromol/g wet tissue; +/-SEM, n=3). Both reduced and oxidised glutathione contributed to this decline. When ascorbate and Trolox C were included during exposure to 0.05% CO the glutathione pool was partly protected; here the glutathione decrease was 46%. In most hearts additional creatine kinase activity in the perfusate indicated minor tissue injury occurring immediately after the start and/or about 10min after the end of exposure to 0.01% CO or 0.05% CO. Ventricle lactate levels were unaffected by exposure to 0.01% CO. This evidence supports a role for oxidative stress in CO cardiotoxicity.     

The effects of selenium and copper deficiencies on glutathione S-transferase and glutathione peroxidase in rat liver.

Selenium (Se) deficiency in rats produced significant increases in the activity of hepatic glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene as substrate and in various GST isoenzymes when determined by radioimmunoassay. These changes is GST activity and concentration were associated with Se deficiency that was severe enough to provoke decreases of over 98% in hepatic Se-containing glutathione peroxidase activity (Se-GSHpx). However, decreases in hepatic Se-GSHpx of 60% induced by copper (Cu) deficiency had no effect on GST activity or concentration. Increased GST activity in Se deficiency has previously been postulated to be a compensatory response to loss of Se-GSHpx, since some GSTs have a non-Se-glutathione peroxidase (non-Se-GSHpx) activity. However, the GST isoenzymes determined in this study, GST Yb1Yb1, GST YcYc and GST YaYa, are known to have up to 30-fold differences in non-Se-GSHpx activity, but they were all significantly increased to a similar extent in the Se-deficient rats.     

Synthesis of N-acetyl-l-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport

1. The synthesis and efflux of N-acetyl-l-aspartate from brain mitochondria of rats of different ages has been studied. 2. Brain mitochondrial State 3 (+ADP) respiration rate, using 10mm-glutamate and 2.5mm-malate as substrates, increases during the suckling period and reaches approx. 50% of the adult value at 17 days after birth [adult State 3 respiration rate=160+/-7ng-atoms of O/min per mg of mitochondrial protein(mean+/-s.d.; n=3)]. 3. The influence of 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate on aspartate efflux from brain mitochondira from rats of different ages oxidizing glutamate and malate was studied. In all cases the aspartate efflux in State 3 was greater than in State 4, but, whereas the aspartate efflux in State 3 increased as the animals developed, that of State 4 showed only a small increase. However, the rate of aspartate efflux in the presence of pyruvate or 3-hydroxybutyrate as well as glutamate and malate was approx. 60-65% of that in the presence of glutamate and malate alone. 4. An inverse relationship between aspartate efflux and N-acetylaspartate efflux was observed with adult rat brain mitochondria oxidizing 10mm-glutamate and 2.5mm-malate in the presence of various pyruvate concentrations (0-5mm). 5. N-Acetylaspartate efflux by brain mitochondria of rats of different ages was studied in States 3 and 4, utilizing 5mm-pyruvate or 10mm-dl-3-hydroxybutyrate as acetyl-CoA sources. A similar pattern of increase during development was seen in State 3 for N-acetylaspartate efflux as for aspartate efflux (see point 3 above). Also only very small increases in N-acetylaspartate efflux occurred during development in State 4.6. Rat brain mitochondria in the presence of iso-osmotic N-acetylaspartate showed some swelling which was markedly increased in the presence of malate. 7. It is concluded that N-acetylaspartate may be synthesized and exported from both neonatal and adult rat brain mitochondria. It is proposed that the N-acetylaspartate is transported by the dicarboxylic acid translocase and may be an additional mechanism for mitochondrial/cytosolic carbon transport to that of citrate.     

Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption.

Reversed-phase chromatography separates lysyl-tRNA from mammalian tissues into five isoaccepting species. The major peaks are designated lysyl-tRNA₂ and lysyl-tRNA₅. In ribosomal binding experiments lysyl-tRNA₂ binds exclusively for AAG, whereas lysyl-tRNA₅ codes preferentially for AAA. The results presented here show that the lysyl-tRNA₅ peak in liver tissue is a mixture of two different lysyl-tRNAs which we have designated as lysyl-tRNA₅ and lysyl-tRNA_5B. These two lysyl-tRNAs were distinguished by the fact that lysyl-tRNA_5B could still accept lysine after iodine oxidation, whereas lysyl-tRNA₅ could not. Lysyl-tRNA_5B, however, was modified by the iodine treatment because it eluted at a different position during RPC-5 chromatography. Similar results were obtained when cyanogen bromide was used in place of iodine as the modifying reagent. It was also shown that iodine oxidation of lysyl-tRNA_5B caused a loss of the ability of this tRNA to bind to ribosomes in response to both ApApA and ApApG. Reversal of the effects of iodine by incubation with sodium thiosulfate restored some of the ribosomal binding activity. Under these conditions lysyl-tRNA_5B bound in response to ApApG, but not to ApApA. Based upon these data, lysyl-tRNA_5B appears to be a sulfur-containing lysyl-tRNA which codes for AAG exclusively. It is postulated that this tRNA may contain a 2-thiocytidine in the anticodon loop.     

Changes induced in rat liver polysomal polyadenylated RNAs by depletion of circulating glucocorticoids.

The effects of adrenalectomy on the complexity and the relative abundances of rat liver polyadenylated mRNAs have been investigated. The qualitative and quantitative changes induced by adrenalectomy have been measured by hybridisation of polysomal polyadenylated RNAs from the livers of normal and adrenalectomised rats with total cDNAs, fractionated cDNAs, cDNA representing RNAs specific to normal liver, total unique-sequence DNA and unique-sequence DNA complementary to normal liver polysomal RNA. These analyses indicated that, by 14 days after adrenalectomy, the equivalent of about 7000 sequences of average length 2000 nucleotides can no longer be detected in liver polysomes. Many other sequences are decreased in abundance as compared to normal liver, but some abundant sequences become more abundant. Administration of a glucocorticoid hormone (dexamethasone) very rapidly reverses these changes.     

Different involvement of the mitochondrial, plastidial and cytosolic ascorbate–glutathione redox enzymes in heat shock responses

Plant survival under heat stress requires the activation of proper defence mechanisms to avoid the impairment of metabolic functions. Heat stress leads to the overproduction of reactive oxygen species (ROS) in the cell. In plants, the ascorbate (ASC)-GSH cycle plays a pivotal role in controlling ROS levels and cellular redox homeostasis. Ascorbate peroxidase (APX) is the enzyme of this cycle mainly involved in ROS detoxification. In this study, the ASC-GSH cycle enzymes were analysed in the cytosol, mitochondria and plastids of tobacco Bright Yellow-2 cultured cells. The cells were also subjected to two different heat shocks (HSs; 35 or 55°C for 10 min) and the cell compartments were isolated in both conditions. The results reported here indicate that moderate HS (35°C) does not affect cell viability, whereas cell exposure to 55°C HS induces programmed cell death (PCD). In relation to ASC-GSH cycle, the three analysed compartments have specific enzymatic profiles that are diversely altered by the HS treatments. The cytosol contains the highest activity of all ASC-GSH cycle enzymes and the data reported here suggest that it acts as a redox buffer for the whole cells. In particular, the cytosolic APX seems to be the most versatile enzyme, being its activity enhanced after moderate HS and reduced during PCD induction, whereas the other APX isoenzymes are only affected in the cells undergoing PCD. The relevance of the changes in the different ASC-GSH cycle isoenzymes in allowing cell survival or promoting PCD is discussed.     

End group analysis of the cytosolic and mitochondrial fumarases from rat liver

Some molecular properties of the cytosolic and mitochondrial fumarases were compared. The carboxyl(C)-terminal amino acid of both the cytosolic and mitochondrial fumarases of rat liver cell was identified as leucine by using carboxypeptidase (CPase) A. As the amino(N)-terminal amino acid of both the cytosolic and mitochondrial fumarases could not be identified by the dansyl chloride method or by the cyanate method, the N-termini of these two fumarases seems to be masked. Both fumarases, after S-carboxymethylation, were completely digested with pronase E and CPase A and B, and the amino acids with blocked amino group were analyzed by high voltage paper electrophoresis and amino acid analysis after acid hydrolysis of these amino acid derivatives. The N-termini of the mitochondrial and cytosolic fumarases were identified as pyroglutamic acid and N-acetylalanine, respectively. To compare the primary structures of the two fumarases in detail, each fumarase was digested with an arginine-specific protease or cleaved with cyanogen bromide. The electrophoretic profiles of the digests of these fumarases were indistinguishable from each other.     

The effects of disulfiram on rat liver mitochondrial monoamine oxidase.

Monoamine oxidase (MAO) of rat liver mitochondria was found to be inhibited by disulfiram. The inhibition is pH and time dependent: 50% inhibition was observed by 16.5 μM of disulfiram at pH 9.1 after 30 min of preincubation. At pH 7.4 only slight inhibition was produced despite the high concentration of disulfiram (330 μM) and the preincubation period. The inhibition is irreversible and appears to be of mixed type: noncompetitive at low concentration range of the substrate and uncompetitive at high concentration range. Glutathione at twice the concentration of disulfiram abolished the inhibitory effect of the drug. Ethanol, while by itself has only slight effect on MAO activity, enhanced the inhibitory effect of disulfiram at pH 7.4. At pH 9.1, ethanol alone has no effect on MAO; however, it was found to weaken the inhibitory effect of disulfiram.     

On the regulation of cold-labile cytosolic and of mitochondrial acetyl-CoA hydrolase in rat liver

The discovery of a cold-labile cytosolic acetyl-CoA hydrolase of high activity in rat liver by Prass et al. [(1980) J. Biol. Chem. 255, 5215-5223] has questioned the importance of mitochondrial acetyl-CoA hydrolase for the formation of free acetate [Grigat et al. (1979) Biochem. J. 177, 71-79] under physiological conditions. Therefore this problem has been reevaluated by comparing various properties of the two enzymes. Cold-labile cytosolic acetyl-CoA hydrolase bands with an apparent Mr of 68000 during SDS/polyacrylamide gel electrophoresis, while the native enzyme elutes in two peaks with apparent Mr of 136000 and 245000 during gel chromatography in the presence of 2 mM ATP. The mitochondrial enzyme elutes under the same conditions with an apparent Mr of 157000. Under conditions where the cold-labile enzyme binds strongly to DEAE-Bio-Gel and ATP-agarose, the mitochondrial enzyme remains unbound. The cold-labile enzyme can be activated 14-fold by ATP, half-maximal activation occurring already at 40 microM ATP. AdoPP[NH]P, AdoPP[CH2]P and GTP have a similar though weaker effect. ADP as well as GDP can completely inhibit the cold-labile enzyme with 50% inhibition occurring for both nucleotides at about 1.45 microM. The binding of ATP and ADP is competitive. Acetyl phosphate and pyrophosphate have no effect on the activity of the cold-labile enzyme. The mitochondrial acetyl-CoA hydrolase is not affected by these nucleotides. CoASH is a strong product inhibitor (approximately equal to 80% inhibition at 40 microM CoASH) of the cold-labile enzyme, but only a weak inhibitor of the mitochondrial enzyme. Under in vivo conditions the activity of the cold-labile cytosolic acetyl-CoA hydrolase can be no more than 7% of the activity calculated for mitochondrial acetyl-CoA hydrolase under the same conditions. Accordingly the mitochondrial enzyme seems to be mainly responsible for the formation of free acetate by the intact liver, especially in view of the fact that the substrate specificity of the mitochondrial enzyme is much higher (activity ratios acetyl-CoA/butyryl-CoA 4.99 and 1.16 for the mitochondrial and the cold-labile enzyme respectively). Alloxan diabetes neither increased the activity of the cold-labile enzyme nor that of the mitochondrial enzyme. No experimental support has been found yet for the hypothesis that the acetyl-CoA hydrolase activity of the cold-labile enzyme represents the side-activity of an acetyl-transferase.     

The distribution of glutathione in the rat liver lobule.

A quantitative cytochemical method was developed for measuring the GSH (reduced glutathione) content of hepatocytes in different regions of the rat liver lobule. Use of this method enabled us to show that GSH is not evenly distributed within the rat liver lobule. The hepatocytes located within 100 micrometer of the central vein contain much less GSH than do those in other regions of the rat liver lobule. We suggest that this partially explains the peculiar susceptibility of these cells to electrophilic attack by toxic metabolites formed via the microsomal cytochrome P-450 system.     

Identity of mitochondrial and cytosolic glycerate kinases in rat liver and regulation of their intracellular localization by dietary protein.

Glycerate kinase (ATP: D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of glyconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serin. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase.     

The reduction of diamide by rat liver mitochondria and the role of glutathione.

Diamide is reduced by mitochondria utilizing endogenous substrates with Vmax. 20nmol/min per mg of protein and Km 75micrometer. The reaction is inhibited by: (a) thiol-blocking reagents (N-ethylmaleimide, p-hydroxymercuribenzoate, mersalyl and 2,6-dichlorophenol-indophenol);(b) respiratory inhibitors (arsenicals, malonate and antimycin, but not cyanide or oligomycin; inhibition by antimycin is reversed by ATP); (c) uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol and valinomycin with K+; inhibition by the first of these uncouplers is not reversed by cyanide); (d) reagents affecting energy conservation (Ca2+, increasing pH, phosphate; phosphate inhibition is augmented by catalytic ADP or ATP and augmentation is abolished by respiratory inhibitors). Concentrations of mitochondrial glutathione are high when diamide reduction is uninhibited, but low after adding one of the above inhibitors such that the reduction rate is roughly proportional to the glutathione concentration. Endogenous ATP concentrations are lower in the presence of diamide than without, but the difference is abolished by respiratory inhibitors. With oligomycin added, however, ATP concentrations are higher in the presence of diamide and this positive increment is decreased by antimycin, N-ethylmaleimide and malonate. In the presence of diamide and an uncoupler, the mitochondrial glutathione content does not fall if various reducible substrates are present, although the inhibition of diamide reduction is not relieved. Some of these substrates prevent the fall in reduced glutathione concentration found with diamide and phosphate. They also relieve the inhibition of diamide reduction and the relief is sensitive to butylmalonate. The inhibition of diamide reduction by N-ethylmaleimide, mersalyl or p-hydroxymercuribenzoate is not relieved by reducible substrates, but the latter mitigate the fall in the concentration of glutathione. Inhibitors of carriers of tricarboxylic acid-cycle intermediates also inhibit reduction of diamide. The reduced glutathione concentration remains high when they are added singly, but falls when two of them are combined. It is proposed that diamide may enter the matrix as a protonated adduct formed with the thiol groups of mitochondrial carriers and then be reduced in the matrix by glutathione, which is regenerated via NADH, energy-dependent transhydrogenase and NADP+-specific glutathione reductase. Some of the high-energy equivalents required for the transhydrogeneration may be generated by the substrate phosphorylation step of the tricarboxylic acid cycle.     

Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver

Oxidative stress in the liver is sometimes accompanied by cholestasis. We investigated the localization and role of multidrug-resistance-associated protein (Mrp) 2, a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress. Normal Sprague-Dawley rat (SDR) and Mrp2-deficient Eisai hyperbilirubinemic rat (EHBR) livers were perfused with 500 microM EA. The release of glutamic pyruvic transaminase (GPT) and thiobarbituric-acid-reactive substances (TBARS) from EHBR liver was markedly delayed compared with that from SDR liver. This is mainly due to the higher basal level of glutathione (GSH) in EHBR liver (59.1 +/- 0.3 nmol/mg protein) compared with SDR liver (39.7 +/- 1.5 nmol/mg protein). EA similarly induced a rapid reduction in GSH followed by mitochondrial permeability transition in the isolated mitochondria from both SDR and EHBR. Internalization of Mrp2 was detected before nonspecific disruption of the canalicular membrane and GPT release in SDR liver perfused with 100 microM EA. SDR liver preperfused with hyperosmolar buffer (405 mosmol/L) for 30 min induced internalization of Mrp2 without changing the basal GSH level, while elimination of hepatic GSH by 300 microM EA perfusion was significantly delayed thereafter. Concomitantly, hepatotoxicity assessed by the release of GPT and TBARS was also significantly attenuated under hyperosmolar conditions. In conclusion, preserved cytosolic and intramitochondrial GSH is the key factor involved in the acute hepatotoxicity induced by EA and its susceptibility could be altered by the presence of Mrp2.