Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

Lash, Lawrence H.; Jones, Dean P.

doi:10.1016/0003-9861(85)90065-7

Cited by 186 publications

(76 citation statements)

References 44 publications

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“…Both wild-type cells (NRK-52E-WT) and transfectants overexpressing rDCC-His 6 (NRK-52E-rDCCϩ) were incubated with 1 mM GSH for 24 h, followed by incubation for 1, 2, or 4 h without treatment, or with tBH (10 or 50 M) or DCVC (50 or 200 M), in the presence of 20 M GSH. The 20 M GSH was chosen to mimic the concentration of GSH in the extracellular or periplasmic space and is that to which renal cells are normally exposed (Lash and Jones, 1985).…”

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confidence: 99%

Protection of NRK-52E Cells, a Rat Renal Proximal Tubular Cell Line, from Chemical-Induced Apoptosis by Overexpression of a Mitochondrial Glutathione Transporter

Lash¹,

Putt²,

Matherly³

2002

J Pharmacol Exp Ther

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The dicarboxylate carrier (DCC) is one of two carriers responsible for glutathione (GSH) transport into rat kidney mitochondria. The central hypothesis of the present study was that overexpression of this carrier in renal proximal tubular cells increases content of mitochondrial GSH, which in turn can protect these cells from chemical-induced injury. We first cloned the carrier protein and verified its properties. This was accomplished by reverse transcribing total rat kidney RNA and polymerase chain reaction amplification with primers based on the complete cDNA sequence for the mitochondrial DCC protein. DCC was expressed as a His 6 -tagged protein, purified from Escherichia coli inclusion bodies, and reconstituted into proteoliposomes for transport assays. Time-and concentration-dependent uptake of both L-[ 3 H-glycyl]GSH and [2-14 C]malonate was observed with kinetics, substrate specificity, and inhibitor sensitivities similar to those observed in rat kidney proximal tubules. We next transiently transfected NRK-52E cells with the cDNA for rat kidney DCC to overexpress the protein. The presence of the recombinant DCCHis 6 protein was confirmed by immunoblots. Transport of both GSH and malonate into the mitochondrial fraction of transfected cells was enhanced 2.45-to 11.3-fold, compared with that in wild-type cells. Transfected cells exhibited markedly less apoptosis from tert-butyl hydroperoxide or S-(1,2-dichlorovinyl)-L-cysteine than did wild-type cells, validating the central hypothesis and providing us with a valuable and novel tool with which to further study GSH and thiol redox status in renal mitochondria, and the function of GSH transport in regulation of processes such as apoptosis and oxidative phosphorylation.

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confidence: 99%

Protection of NRK-52E Cells, a Rat Renal Proximal Tubular Cell Line, from Chemical-Induced Apoptosis by Overexpression of a Mitochondrial Glutathione Transporter

Lash¹,

Putt²,

Matherly³

2002

J Pharmacol Exp Ther

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“…Normal plasma levels of Hcy are generally similar to those of Cys and GSH (Lash and Jones, 1985). However, under certain pathophysiological conditions, the plasma levels of Hcy can increase from approximately 5 to 10 M to as much as 200 M (Stabler et al, 1988;Malinow, 1990;Selhub, 1999).…”

Section: Introductionmentioning

confidence: 99%

Handling of the HomocysteineS-Conjugate of Methylmercury by Renal Epithelial Cells: Role of Organic Anion Transporter 1 and Amino Acid Transporters

Zalups¹,

Ahmad²

2005

J Pharmacol Exp Ther

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Recently, the activity of the organic anion transporter 1 (OAT1) protein has been implicated in the basolateral uptake of inorganic mercuric species in renal proximal tubular cells. Unfortunately, very little is known about the role of OAT1 in the renal epithelial transport of organic forms of mercury, such as methylmercury (CH 3 Hg ϩ ). Homocysteine (Hcy) S-conjugates of methylmercury [(S)-(3-amino-3-carboxypropylthio)(methyl)mercury (CH 3 Hg-Hcy)] have been identified recently as being potentially important biologically relevant forms of mercury. Thus, the present study was designed to characterize the transport of CH 3 Hg-Hcy in Madin-Darby canine kidney (MDCK) cells (which are derived from the distal nephron) that were transfected stably with the human isoform of OAT1 (hOAT1). Data on saturation kinetics, time dependence, substrate specificity, and temperature dependence demonstrated that CH 3 Hg-Hcy is a transportable substrate of hOAT1. However, substrate-specificity data from the control MDCK cells also showed that CH 3 Hg-Hcy is a substrate of one or more transporter(s) that is/are not hOAT1. Additional findings indicated that at least one amino acid transport system was probably responsible for this transport. It is noteworthy that the activity of amino acid transporters accounted for the greatest level of uptake of CH 3 HgHcy in the hOAT1-expressing cells. Furthermore, rates of survival of the hOAT1-transfected MDCK cells were significantly lower than those of corresponding control MDCK cells when they were exposed to cytotoxic concentrations of CH 3 Hg-Hcy. Collectively, the present data indicate that CH 3 Hg-Hcy is a transportable substrate of OAT1 and amino acid transporters and, thus, is probably a transportable mercuric species taken up in vivo by proximal tubular epithelial cells.Homocysteine (Hcy) is a sulfhydryl-containing amino acid formed from methionine after it undergoes demethylation. After its formation, Hcy is broken down by one of two mechanisms: remethylation or transsulfuration. In the remethylation pathway, a methyl group (from N 5 -methyltetrahydrofolate or betaine) is transferred to Hcy to re-form Met. In the alternative pathway, Hcy is converted to cysteine and ␣-ketobutyrate by trans-sulfuration, which requires the sequential actions of cystathionine-␤-synthase and ␥-cystathionase (Selhub, 1999).In blood, Hcy, Cys, and glutathione (GSH) make up a very important pool of nonprotein thiols to which various electrophiles, including metals such as mercury (Hg) and cadmium (Cd), can interact. Normal plasma levels of Hcy are generally similar to those of Cys and GSH (Lash and Jones, 1985). However, under certain pathophysiological conditions, the plasma levels of Hcy can increase from approximately 5 to 10 M to as much as 200 M (Stabler et al., 1988;Malinow, 1990;Selhub, 1999). Hyperhomocysteinemia is a significant clinical problem (resulting from altered intracellular metabolism of Hcy or Met) that has been implicated as a risk factor in the development of cardiovascular diseas...

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“…The liver releases GSH at a substantial rate (8) and contributes to maintenance of circulating GSH in the plasma (9). In principle, transport systems in extrahepatic cells could allow uptake of this GSH to protect against chemical injury.…”

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confidence: 99%

Exogenous glutathione protects intestinal epithelial cells from oxidative injury.

Lash¹,

Hagen²,

Jones³

1986

Proc. Natl. Acad. Sci. U.S.A.

132

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Exogenous GSH provided rat small-intestinal epithelial cells with significant protection against injUry induced by t-butyl hydroperoxide or menadione. This protection was found to be dependent upon uptake of intact GSH. Uptake of GSH occurred by a Na+-dependent electrogenic system found in the basolateral membrane. Thus, rat small-intestinal epithelial cells can utilize plasma GSH to support intracellular detoxication systems that function in protection against chemically induced injury.Drug-metabolizing and oxidation-reduction systems in the epithelium of the small intestine represent a first line of defense against ingested xenobiotics and toxins. Glutathione (GSH) is an important determinant of the protection against chemical injury, by serving as a substrate for glutathione transferases (RX:glutathione R-transferase, EC 2.5.1.18) and glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9). Glutathione transferases catalyze the reaction of GSH with electrophilic compounds to form nontoxic conjugates (1), and glutathione peroxidase utilizes GSH as a reductant to reduce toxic peroxides (2, 3). Depletion of GSH potentiates injury from both types of processes (4, 5), and stimulation of processes that support maintenance of intracellular GSH protects against injury (6, 7). The liver releases GSH at a substantial rate (8) and contributes to maintenance of circulating GSH in the plasma (9). In principle, transport systems in extrahepatic cells could allow uptake of this GSH to protect against chemical injury. Although ofobvious toxicological importance, it has not been determined whether exogenous GSH can protect against toxicity in small-intestinal cells, or whether an uptake system, such as has been found in kidney basolateral membrane (10,11), is also present in small intestine. We therefore addressed these issues with isolated rat intestinal cells and purified plasma membrane vesicles. The results show that exogenous GSH provides substantial protection against oxidative injury by t-butyl hydroperoxide or menadione due to the function of a Na+-dependent GSH uptake system which is present in the basolateral membrane of the epithelial cells.EXPERIMENTAL PROCEDURES GSH, phenylmethylsulfonyl fluoride, valinomycin, 1-fluoro-2,4-dinitrobenzene, Percoll, collagenase (type I), and hyaluronidase were purchased from Sigma. Nitrocellulose filters (0.45-,um pore size) were purchased from Gelman.[glycine-2-3H]GSH (1.1 Ci/mmol, 1 Ci = 37 GBq) was purchased from New England Nuclear. The purity of the radiolabeled GSH was routinely assessed by derivatizing it with iodoacetic acid and 1-fluoro-2,4-dinitrobenzene, followed by HPLC analysis (see below). Greater than 92% ofthe 3H counts were eluted in a single peak that coincided with that of authentic GSH. AT-125 [L-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was a gift from D. J. Reed (Oregon State University). All other chemicals were of reagent grade and were purchased locally.Male white rats (Sprague-Dawley-derived, barrier-re...

show abstract

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

Cited by 186 publications

References 44 publications

Protection of NRK-52E Cells, a Rat Renal Proximal Tubular Cell Line, from Chemical-Induced Apoptosis by Overexpression of a Mitochondrial Glutathione Transporter

Protection of NRK-52E Cells, a Rat Renal Proximal Tubular Cell Line, from Chemical-Induced Apoptosis by Overexpression of a Mitochondrial Glutathione Transporter

Handling of the HomocysteineS-Conjugate of Methylmercury by Renal Epithelial Cells: Role of Organic Anion Transporter 1 and Amino Acid Transporters

Exogenous glutathione protects intestinal epithelial cells from oxidative injury.

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