Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity

The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.     

alpha-Phenyl-n-tert-butyl-nitrone reduces lipopolysaccharide-induced white matter injury in the neonatal rat brain

Lipopolysaccharide (LPS)-induced white matter injury in the neonatal rat brain is at least partially associated with oxidative stress. alpha-Phenyl-n-tert-butyl-nitrone (PBN) (100 mg/kg) significantly attenuated LPS (1 mg/kg)-induced brain injury, as indicated by the reduction in bilateral ventricular enlargement, apoptotic cell death of oligodendrocytes (OLs), and the loss of OL immunoreactivity in the neonatal rat brain. Protection of PBN was linked with the attenuated oxidative stress induced by LPS, as indicated by the decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde positive OLs following LPS exposure. Interestingly, while LPS exposure elevated, rather than depleted, levels of the reduced glutathione (GSH) and the GSH/GSSG (oxidized form) ratio, LPS exposure significantly suppressed glutathione peroxidase activity in the rat brain. PBN attenuated LPS-induced alterations in glutathione homeostasis in the rat brain. Additionally, the inflammatory responses were also reduced in the PBN-treated brain, as indicated by the decreased number of activated microglia following LPS exposure and by the consequently decreased elevation of interleukin1-beta and tumor necrosis factor-alpha contents in the rat brain. The overall results suggest that antioxidant PBN, more than a straightforward free radical scavenger, may also involve anti-inflammatory and anti-apoptotic properties in protection of the neonatal rat brain from LPS-induced injury.     

Some observations on mitochondrial-bound hexokinase and creatine kinase of the heart

A large part of the hexokinase activity of the rat brain 20,000g supernatant became mitochondrial bound when incubated with rat heart mitochondria which had been pretreated with glucose-6-phosphate. This binding was dependent on small-molecular compounds (as yet unidentified) of the brain supernatant. Divalent cations, spermine, and pentalysine strongly stimulated the binding of brain supernatant hexokinase to heart mitochondria. Inorganic phosphate, alpha-glycerophosphate, and fructose-1,6-diphosphate showed some stimulatory effect. No effect was observed with insulin or glucose. Mitochondria isolated from hearts of fasted rats had less specific hexokinase activity than mitochondria from fasted and then carbohydrate refed rats. This dietary treatment had no significant effect on the total heart hexokinase activity. Oligomycin did not inhibit the formation of creatine phosphate or glucose-6-phosphate by isolated rabbit heart mitochondria incubated in the presence of phosphoenolpyruvate and pyruvate kinase. However, the presence of creatine inhibited the formation of glucose-6-phosphate when the ATP/ADP ratio was low, indicating that creatine kinase has a greater access to ATP/ADP translocation than has hexokinase.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

Cholic acid binding by glutathione S-transferases from rat liver cytosol.

Cholic acid-binding activity in cytosol from rat livers appears to be mainly associated with enzymes having glutathione S-transferase activity; at least four of the enzymes in this group can bind the bile acid. Examination of the subunit compositions of different glutathione S-transferases indicated that cholic acid binding and the ability to conjugate reduced glutathione with 1,2-dichloro-4-nitrobenzene may be ascribed to different subunits.     

Coupling of Dopamine Oxidation (Monoamine Oxidase Activity) to Glutathione Oxidation Via the Generation of Hydrogen Peroxide in Rat Brain Homogenates

Abstract: Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline. a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized: this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons.     

Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.     

Deoletion of brain glutathione is accompanied by impaired micochondrial function and decreased N-acetyl aspartate concentration

The effect of depletion of reduced glutathione (GSH) on brain mitochondrial function and N-acetyl aspartate concentration has been investigated. Using pre-weanling rats, GSH was depleted by L-buthionine sulfoximine administration for up to 10 days. In both whole brain homogenates and purified mitochondrial preparations complex IV (cytochrome c oxidase) activity was decreased, by up to 27%, as a result of this treatment. In addition, after 10 days of GSH depletion, citrate synthase activity was significantly reduced, by 18%, in the purified mitochondrial preparations, but not in whole brain homogenates, suggesting increased leakiness of the mitochondrial membrane. The whole brain N-acetyl aspartate concentration was also significantly depleted at this time point, by 11%. It is concluded that brain GSH is important for the maintenance of optimum mitochondrial function and that prolonged depletion leads also to loss of neuronal integrity. The relevance of these findings to Parkinson's disease and the inborn errors of glutathione mtabolism are also discussed.     

Effect of clofibrate treatment on glutathione content and the activity of the enzymes related to peroxide metabolism in rat liver and heart

Clofibrate treatment was shown to increase the content of reduced glutathione in rat liver and kidney, but did not alter the glutathione level in heart, brain, spleen and small intestine. Clofibrate did not affect the activity of superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase in rat liver and heart. The drug decreased the activity of glutathione-S-transferase in the cytosolic fraction of liver homogenate. Glutathione-S-transferase activity in small intestine was also reduced. The administration of clofibrate decreased the content of polypeptides with mol. wt of 22,000 and 24,000 (possible monomers of glutathione-S-transferase) in the cytosolic fraction of liver cells.     

Sequential enzyme-catalyzed metabolism of 4-nitrotoluene to S-(4-nitrobenzyl)glutathione

[14C]-4-Nitrotoluene was metabolized by rat liver postmitochondrial supernatant containing NADPH, reduced glutathione and a sulfate activating system to 4-nitrobenzyl alcohol, 4-nitrobenzyl sulfate, and S-(4-nitrobenzyl) glutathione. Formation of both sulfur-containing metabolites was dependent on the presence of a sulfate activating system. These results suggest that the glutathione conjugate was derived from 4-nitrobenzyl sulfate. Reaction of 4-nitrobenzyl sulfate with glutathione was not detected in pH 7.4 buffer, but rat liver cytosol catalyzed the formation of the glutathione conjugate from 4-nitrobenzyl sulfate. These results show that 4-nitrotoluene is metabolized in rat liver by sequential side chain oxidation, sulfation, and glutathione conjugation. Furthermore, they indicate that, unlike certain other arylmethyl sulfates, 4-nitrobenzyl sulfate is not highly reactive.     

A particle concentration fluorescence immunoassay for prostaglandin D synthase in the rat central nervous system

A solid phase, particle concentration fluorescence immunoassay (PCFIA) was developed for the measurement of prostaglandin (PG) D synthase in the 100,000g supernatant of various regions of the rat central nervous system. In this assay, the enzyme (in the range of 1-25 micrograms protein of brain supernatant or 1-100 ng of the purified enzyme) is attached to submicrometer carboxypolystyrene beads coated with polyclonal anti-rat brain PGD synthase IgG. The total particle-bound enzyme is assayed with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-PGD synthase IgG after incubation for 1 h. The optimum assay condition was obtained when carboxyl particles coated with ca. 500 micrograms/ml of polyclonal IgG at pH 5.0 and 5 micrograms/ml of FITC-IgG were used. No significant fluorescence was observed when FITC conjugates or carboxyl particles were prepared using IgG from nonimmunized rabbits. Heat treatment of the brain supernatant decreased the specific binding of the enzyme in parallel with the loss of enzyme activity, indicating that the denatured enzyme is not recognized by this assay method. The PGD synthase immunoreactivity was widely distributed in the brain regions and was highest in the paraflocculus. Although slight discrepancy was observed between the concentration by PCFIA and the enzyme activity measured by using [14C]PGH2 in some brain regions, there is a considerable correlation (0.727) between the values by both methods in the same brain regions. The PCFIA now developed showed higher sensitivity (around 10 times), greater reliability, and larger number of samples measurable at once than the radio-TLC assay using [14C]PGH2. This method could provide valuable information concerning the regulatory mechanisms of PGD synthase.     

Nature of the Cytoplasmic Factor(s) Modulating Adenylate Cyclase in the Developing Rat Brain

Abstract: Adenylate cyclase activity in the particulate fraction from rat brain was markedly enhanced by the cytoplasmic fraction, which itself contained negligible enzyme activity, indicating the presence of some stimulatory factor(s) in the supernatant. Activation of adenylate cyclase was dependent on the supernatant concentration up to 1 mgiml, but higher concentration of the supernatant did not produce further activation of the enzyme. The supernatant retained its stimulatory activity after boiling for 5 min, extensive dialysis, and phospholipase A and DNAase treatments, but was completely inactivated by digestion with trypsin. Ability of the supernatant to activate adenylate cyclase was low during fetal life, increased severalfold neonatally, and declined somewhat thereafter to an adult level. Adenylate cyclase in the particulate fraction from 2-day-old rat brain was also activated by GTP, calcium-dependent regulator (CDR) of cyclic AMP phosphodiesterase in the presence of 100 pM-Ca¹, and by NaF. The supernatant produced additive activation of the enzyme with NaF but not with GTP or CDR, suggesting a common site of action of the supernatant factor(s) and the latter two agents. DEAE-cellulose chromatography of the boiled supernatant resolved the heat-stable proteins into several peaks. Adenylate cyclase activator eluted in two distinct peaks, one of which also contained CDR activity. It is concluded that rat brain supernatant contains some factor in addition to CDR which activates particulate adenylate cyclase.     

Promotion of Lipid and Protein Oxidative Damage in Rat Brain by Ethylmalonic Acid

High concentrations of ethylmalonic acid are found in tissues and biological fluids of patients affected by ethylmalonic encephalopathy, deficiency of short-chain acyl-CoA dehydrogenase activity and other illnesses characterized by developmental delay and neuromuscular symptoms. The pathophysiological mechanisms responsible for the brain damage in these patients are virtually unknown. Therefore, in the present work we investigated the in vitro effect of EMA on oxidative stress parameters in rat cerebral cortex. EMA significantly increased chemiluminescence and thiobarbituric acid-reactive species levels (lipoperoxidation), as well as carbonyl content and oxidation of sulfhydryl groups (protein oxidative damage) and DCFH. EMA also significantly decreased the levels of reduced glutathione (non-enzymatic antioxidant defenses). In contrast, nitrate and nitrite levels were not affected by this short organic acid. It is therefore presumed that oxidative stress may represent a pathomechanism involved in the pathophysiology of the neurologic symptoms manifested by patients affected by disorders in which EMA accumulates.     

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.     

Hepatic glutathione S-transferases: identification by gel filtration and in vitro inhibition by organic anions.

In the gel filtration of 100,000 g rat liver supernatant, four major glutathione S-transferase activities, S-aryl-, S-epoxide-, S-aralykyl, and S-alkyltransferase, were identified as having an elution volume identical to that of fractions exhibiting either glutathione or sulfobromophthalein sodium binding. The organic anions, sulfobromophthalein sodium, indocyanine green, and bilirubin, were found to be competitive inhibitors of the four glutathione S-transferase activities. These findings indicate that the glutathione S-transferases bind organic anions, and as a group, have a similar molecular weight to a known organic anion-binding protein. It is proposed that these enzymes also serve nonenzymically as a group of binding proteins in the hepatic cytoplasmic transport of organic anions.     

Characterization of pulmonary surfactant protein D: its copurification with lipids

Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II cells. SP-D was purified from the supernatant of rat bronchoalveolar lavage fluids obtained by centrifugation at 33,000 x gav for 16 h. The contents of SP-D and SP-A in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of SP-A in these lavage fluids. 99.1% of SP-A was present in the 33,000g pellet, whereas 71.1% of SP-D was in the 33,000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SP-A, SP-D in the purified and delipidated form failed to compete with 125I-labeled SP-A for phosphatidylcholine binding, and to aggregate phospholipid liposomes. The present study demonstrates that lipids are copurified with SP-D, that SP-D and SP-A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.     

Glutathione monoethylester prevents mitochondrial glutathione depletion during focal cerebral ischemia

Glutathione is a central component in the antioxidant defences of cells. We have recently reported an early and selective loss of total (reduced plus oxidised) glutathione from mitochondria isolated from rat brain following occlusion of the middle cerebral artery. This mitochondrial glutathione depletion showed an apparent association with the tissue damage that developed during subsequent reperfusion, suggesting that it could be an important determinant of susceptibility to cell loss. In the present study, we have investigated whether in vivo treatment with glutathione ethyl ester can modulate mitochondrial glutathione in the brain and whether this treatment can influence the response to focal ischemia. In further support of our previous findings, middle cerebral artery occlusion caused a duration-dependent partial loss of mitochondrial glutathione. Bilateral injections of glutathione ethyl ester immediately prior to induction of unilateral focal ischemia resulted in a substantial increase in glutathione in mitochondria from the striatum of both the non-ischemic hemisphere (190% of saline-treated controls) and the ischemic hemisphere (240% of controls) at 2h after arterial occlusion. Total tissue glutathione was not affected by the ester treatment at this time. A smaller increase in mitochondrial glutathione was observed at 3h of occlusion in the non-ischemic striatum following ester treatment but at this time point glutathione was not significantly altered in mitochondria from the ischemic hemisphere. Pre-ischemic treatment with glutathione ester did not significantly change the volume of tissue infarction assessed at 48 h following ischemia for 2 or 3h. These studies demonstrate that glutathione ethyl ester is a highly effective modulator of the mitochondrial glutathione pool in the intact brain and provides a useful means for further investigating the role of this antioxidant in the development of tissue damage in ischemia and other brain disorders.     

Mutagenicity and irreversible binding of the hepatocarcinogen, 2,4-diaminotoluene.

Mutagenicity of 2,4-diaminotoluene (DAT) in the Salmonella mutagenicity assay was increased with liver fractions from phenobarbital (PB) or beta-naphthoflavone (BNF) treated rats. Substitutions of the hydrogens in the methyl group of 2,4-DAT with deuterium resulted in a decrease in mutagenicity. Incubation of rat liver microsomes with tritiated 2,4-DAT in the presence of NADPH led to the formation of irreversibly bound products to microsomal protein. The rates of binding were not increased using microsomes from PB or BNF-treated rats and was not altered by deuterium substitution in the methyl group. Addition of superoxide dismutase, glutathione (GSH) or rat liver supernatant reduced 2,4-DAT irreversible binding, whereas 2,4-DAT mutagenicity was unaffected by superoxide dismutase addition. Injection of tritiated 2,4-DAT 100 mg/kg to rats lead to its irreversible binding to liver protein and ribosomal RNA and to kidney protein in vivo, again protein binding was not increased after prior treatment with PB or BNF. No irreversible interaction of tritiated 2,4-DAT with DNA either in vitro or in vivo could be demonstrated.