Localization of a mitochondrial type of NADP-dependent isocitrate dehydrogenase in kidney and heart of rat: an immunocytochemical and biochemical study.

We studied the subcellular localization of the mitochondrial type of NADP-dependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.     

Role of a partially purified glutathione S-transferase from rat liver nuclei in the inhibition of nuclear lipid peroxidation

Glutathione protects isolated rat liver nuclei against lipid peroxidation by inducing a lag period prior to the onset of peroxidation. This GSH-dependent protection was abolished by exposing isolated nuclei to the glutathione S-transferase inhibitor S-octylglutathione. In incubations containing 0.2 mM S-octylglutathione, the GSH-induced lag period was reduced from 30 to 5 min. S-Octylglutathione (0.2 mM) also completely inhibited nuclear glutathione S-transferase activity and reduced glutathione peroxidase activity by 85%. About 70% of the glutathione S-transferase activity associated with isolated nuclei was solubilized with 0.3% Triton X-100. This solubilized glutathione S-transferase activity was partially purified by utilizing a S-hexylglutathione affinity column. The partially purified nuclear glutathione S-transferase exhibited glutathione peroxidase activity towards lipid hydroperoxides in solution. The data from the present study indicate that a glutathione S-transferase associated with the nucleus may contribute to glutathione-dependent protection of isolated nuclei against lipid peroxidation. Evidence was obtained which indicates that this enzyme is distinct from the microsomal glutathione S-transferase.     

Microinjected glutathione S-transferase Yb subunits translocate to the cell nucleus.

We have previously shown that a 30 kDa DNA-binding protein isolated from rat cell nuclei exhibits the chemical and immunological properties of glutathione S-transferase Yb subunits [Bennett, Spector & Yeoman (1986) J. Cell Biol. 102, 600-609]. It was of interest, therefore, to determine whether Yb subunits isolated from rat liver nuclei would return to nuclear fractions upon reintroduction to cell cytoplasms via red-blood-cell-mediated fusion. Labelled Yb subunits were associated with nuclear fractions 60 min after cell fusion. The microinjected protein remained associated with the nuclei for 18 h and was not extractable with low-salt washes. In addition, injected Yb subunits were found to equally distribute between extractable (56%) and residual (44%) nuclear fractions. These experiments demonstrate that glutathione S-transferase Yb subunits isolated from nuclei rapidly translocate to nuclei upon reintroduction into cell cytoplasms.     

Effects of glutathione on the alpha-tocopherol-dependent inhibition of nuclear lipid peroxidation

Endogenous alpha-tocopherol levels in isolated rat liver nuclei were determined to be 0.045 mol% (mol alpha-tocopherol per mol phospholipid x 100). This value corresponds to 970 polyunsaturated fatty acid (PUFA) moieties to one molecule of alpha-tocopherol in the nuclear membrane. Isolated nuclei, when incubated with various concentrations of exogenous alpha-tocopherol, took up only a small percent of initial levels of alpha-tocopherol present in the incubation media. Exogenous alpha-tocopherol, when incorporated in isolated nuclei above a threshold value of 0.085 mol%, effectively inhibited NADPH-induced lipid peroxidation. The addition of 1 mM glutathione lowered the threshold levels of alpha-tocopherol needed to inhibit lipid peroxidation to about 0.040 mol%. We suggest the data indicate a glutathione-dependent enhancement of the ability of alpha-tocopherol to inhibit nuclear lipid peroxidation.     

Metabolic cooperation of ascorbic acid and glutathione in normal and vitamin C-deficient ODS rats.

Although the coordination of various antioxidants is important for the protection of organisms from oxidative stress, dynamic aspects of the interaction of endogenous antioxidants in vivo remain to be elucidated. We studied the metabolic coordination of two naturally occurring water-soluble antioxidants, ascorbic acid (AA) and reduced glutathione (GSH), in liver, kidney and plasma of control and scurvy-prone osteogenic disorder Shionogi (ODS) rats that hereditarily lack the ability to synthesize AA. When supplemented with AA, its levels in liver and kidney of ODS rats increased to similar levels of those in control rats. Hepato-renal levels of glutathione were similar with the two animal groups except for the slight increase in its hepatic levels in AA-supplemented ODS rats. Administration of L-buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, rapidly decreased the hepato-renal levels of glutathione in a biphasic manner, a rapid phase followed by a slower phase. Kinetic analysis revealed that glutathione turnover was enhanced significantly in liver mitochondria and renal cytosol of ODS rats. Administration of BSO significantly increased AA levels in the liver and kidney of control rats but decreased them in AA-supplemented ODS rats. Kinetic analysis revealed that AA is synthesized by control rat liver by some BSO-enhanced mechanism and the de novo synthesized AA is transferred to the kidney. Such a coordination of the metabolism of GSH and AA in liver and kidney is suppressed in AA-deficient ODS rats. These and other results suggest that the metabolism of AA and GSH forms a compensatory network by which oxidative stress can be decreased.     

Ethanol-induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and gamma-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, gamma-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration.     

Isolation and partial characterization of a mercury-binding nonhistone protein component from rat kidney nuclei

A nonhistone protein component (NHP_Ins) firmly bound to DNA of rat kidney nuclei has been isolated and partially characterized. In vivo studies show that this protein specifically incorporates 35 to 45 times more ²⁰³Hg than any other nuclear protein fractions. No difference in the ratio of NHP_Ins to DNA between normal and mercury-poisoned rat kidney nuclei was observed. NHP_Ins protein gives a single major band by sodium dodecyl sulfate gel electrophoresis. Electrophoretic pattern as well as amino acid composition of this protein isolated from both normal and mercurypoisoned rats are also found to be similar. Cysteine content is 1.3 to 1.4 mole per cent.     

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.     

Epoxide hydrase and mixed-function oxidase activities of rat liver nuclear membranes.

Rat liver nuclei have 2 to 12% of the corresponding microsomal aryl hydrocarbon hydroxylase, aminopyrine and benzphetamine N-demethylase, NADPH-cytochrome c reductase, and epoxide hydrase activities. Nuclear membranes were prepared from isolated liver nuclei by a sucrose density centrifugation technique. A 2.5- to 10.2-fold increase in the specific enzyme activities was observed in nuclear membrane as compared to intact nuclei. Several properties of the rat liver nuclear membrane and microsomal epoxide hydrase have been compared. Nuclear epoxide hydrase was similar to the corresponding microsomal enzyme in being induced by phenobarbital whereas 3-methylcholanthrene did not produce any effects. Nuclear membrane and microsomal epoxide hydrase were inhibited to a similar degree by 1,1,1-trichloropropene oxide, cyclohexene oxide, an trans-stilbene oxide. The apparent K_m value of nuclear membrane epoxide hydrase was 20 μm for benzo(a)pyrene 4,5-oxide, which is 5.5-fold lower than the corresponding microsomal K_m value (112 μm). Nuclear membranes were prepared from isolated nuclei of rat kidney, lung, spleen, and heart by the DNase digestion method. Epoxide hydrase activity in intact nuclei was in the following order: kidney > lung ? spleen, or heart. Increases of 2.2- and 2.5-fold in specific epoxide hydrase activity were observed in kidney and lung when nuclear membranes were compared to intact nuclei. DMSO, dimethylsulfoxide     

Amino acid sequence of rat kidney gamma-glutamylcysteine synthetase

gamma-Glutamylcysteine synthetase catalyzes the first step in the synthesis of glutathione. The enzyme isolated from rat kidney has two subunits (heavy, Mr 73,000; and light, Mr 27,700) which may be dissociated by treatment with dithiothreitol. The heavy subunit exhibits all of the catalytic activity of the isolated enzyme and also feedback inhibition by glutathione. The light subunit has no known function and may not be an integral part of the enzyme. cDNA clones encoding rat kidney gamma-glutamylcysteine synthetase were isolated from a lambda gt11 cDNA library by immunoscreening with antibody against the isolated enzyme and further screening with oligonucleotide probes derived from several peptides whose sequences were determined by the Edman method. The nucleotide sequence of the mRNA for the heavy subunit was deduced from the sequences of the cDNA of three such clones. The sequence, which codes for 637 residues (Mr 72,614), contains all four of the independently determined peptide sequences (approximately 100 residues). This amino acid sequence shows extremely low overall similarity to that of gamma-glutamylcysteine synthetase isolated from Escherichia coli.     

Allyl alcohol toxicity in isolated renal epithelial cells: protective effects of low molecular weight thiols

The toxicity of allyl alcohol was studied in freshly isolated renal epithelial cells prepared from male and female rats. Cells from female rats demonstrated a greater susceptibility to allyl alcohol toxicity as assessed by glutathione depletion and loss of cell viability. The sensitivity of female rat renal cells appears to relate to the higher activity of alcohol dehydrogenase found in the female rat kidney, which metabolizes allyl alcohol to the highly reactive aldehyde, acrolein. Pyrazole, an inhibitor of alcohol dehydrogenase, abolished the cytotoxic effects of allyl alcohol whereas inhibition of aldehyde dehydrogenase by disulfiram treatment was found to increase the sensitivity of renal cells to the effects of allyl alcohol. The toxicity of allyl alcohol was decreased by a number of treatments which resulted in increased levels of glutathione or other low molecular weight thiols. These results indicate that acrolein is the toxic metabolite responsible for the renal cell injury following exposure to allyl alcohol, and unless immediately inactivated acrolein interacts with critical nucleophilic sites of the cell and initiates cell injury. These studies demonstrate that freshly isolated kidney cells represent a convenient model system for studies of thiol-mediated protective mechanisms against toxic renal cell injury.     

Ethanol‐induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty‐two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol‐fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long‐term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose‐6‐phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and γ‐glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, γ‐glutamyltranspeptidase, glutathione reductase, and glucose‐6‐phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose‐6‐phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long‐term ethanol administration. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:386‐395, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20101     

Direct evidence for inter-organ transport of glutathione and that the non-filtration renal mechanism for glutathione utilization involves γ-glutamyl transpeptidase

A direct examination of the inter-organ cycle of glutathione metabolism was made by determining glutathione levels in plasma obtained from various blood vessels of the rat. High levels of GSH were found in hepatic vein plasma, relative to arterial and systemic venous levels, reflecting translocation of GSH from the liver to the plasma. Renal vein plasma has a level that is 20% of arterial plasma indicating that the kidney removes glutathione from plasma not only by glomerular filtration (which can account for 20–30% of the glutathione removed), but also by a non-filtration mechanism. Inhibitors of γ-glutamyl transpeptidase decrease the fraction of glutathione removed by the kidney to a value approaching that filtered, indicating that the non-filtration mechanism involves γ-glutamyl transpeptidase.     

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.     

gamma-Glutamyltranspeptidase activity in newborn rat kidney brush border.

gamma-Glutamyltranspeptidase, known to be localized in the proximal tubule cell brush border in the rat, is a membrane-bound enzyme which transfers the gamma-glutamyl moiety of glutathione or its analogue gamma-glutamyl-p-nitroanilide to an amino acid or dipeptide acceptor. Brush borders were isolated from the kidneys of newborn and adult Sprague-Dawley rats and assayed for gamma-glutamyltranspeptidase activity. There is an increase in specific activity in the brush border with maturation. Newborn and adult brush border preparations exhibit similar pH optima, substrate affinities, apparent Km values, patterns of heat inactivation, inhibition by glutathione, and migration on polyacrylamide gels. Polyacrylamide gel electrophoresis of a deoxycholate extract of brush border proteins and subsequent reaction with substrate within the gel reveal the presence of two bands, suggesting the presence of two forms of gamma-glutamyltranspeptidase in the rat kidney brush border.     

Effect of physiological concentrations of insulin and antidiabetic drugs on RNA release from isolated liver nuclei

The addition of 10(-11) M insulin to a cell-free system from rat liver promotes the release of messengerlike RNA from isolated prelabeled nuclei. The stimulation was similar whether the nuclei were preincubated with insulin, or if insulin was added directly to the cell-free system with or without a protease inhibitor. Dot blot hybridization using cloned cDNA for alpha 2u-globulin mRNA showed that this was one of the messages whose release was enhanced by insulin. Nuclei isolated from rats treated with either of the antidiabetics tolbutamide or tolazamide showed no increase in RNA release in the presence of insulin over the concentration range 10(-5) - 10(-14) M. Furthermore, these nuclei did not release detectable levels of alpha 2u-globulin mRNA.