Hepatic mitochondrial transport of glutathione: Studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells

doi:10.1016/j.abb.2008.03.008

Archives of Biochemistry and Biophysics

Volume 474, Issue 1, 1 June 2008, Pages 119-127

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Abstract

Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K_m and V_max values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45–50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H₂O₂, methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.

Section snippets

Materials

GSH, iodoacetic acid, 1-fluoro-2, 4-dinitrobenzene, antimycin A (AA), phosphoenolpyruvate (PEP), oligomycin, and atractyloside were purchased from Sigma Chemical Co. (St. Louis, MO). Phenylsuccinate and butylmalonate were purchased from Aldrich Chemical Co. (Milwaukee, WI). l-[³H-Glycyl]-GSH (44.8 Ci/mmol) and [¹⁴C]-2-OG (281 mCi/mmol) was purchased from DuPont NEN (Boston, MA).

Preparation of mitochondria

Male Sprague–Dawley rats (175–225 g) were purchased from Harlan (Indianapolis, IN). Rats were housed in cages in the

Kinetics of GSH uptake

The time course for uptake of 5 mM GSH in liver mitochondria was measured at 25 °C by the centrifugation–resuspension method (Fig. 1A). Initial mitochondrial GSH content was about 0.6 nmol/mg protein. In the presence of 7.5 mM extramitochondrial GSH, the intramitochondrial content of GSH increased rapidly within 5 min to an equilibrium level of 2.2 nmol/mg protein. GSH uptake was concentration dependent (Fig. 1B) and was uniphasic (Fig. 2), exhibiting a K_m of 4.08 mM and a V_max of 3.06 nmol/mg protein

Discussion

In the present study, we demonstrate that both the DIC and OGC contribute to, but are not entirely responsible for, the mitochondrial uptake of GSH in rat liver. Unlike our previous findings in rat kidney mitochondria [3], [4], where we found that function of these two inner membrane carriers could account for nearly all of the observed uptake of GSH, in hepatic mitochondria the DIC and OGC only appear to account for approximately 50% of the observable transport. We further demonstrated the

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The transport of GSH by the reconstituted OGC did not have the typical transport properties of mitochondrial carriers [37–42], as transport ceased after two min [16]. Studies of the DIC and OGC overexpressed within cells led to a number of interesting effects [15,16,18], but these changes are likely to be secondary consequences of metabolite redistribution between the mitochondria and the cytosol, and cannot be taken as evidence for changes in GSH transport. In contrast, the L. lactis system provides a relatively simple and well established system to characterise mitochondrial carriers and other membrane proteins [28,29,32].

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^☆: This research was supported by NIH Grant R01-DK40725 to L.H.L. and by the NIEHS Center for Molecular Toxicology with Human Applications (Grant P30-ES06639) at Wayne State University.

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Hepatic mitochondrial transport of glutathione: Studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells☆

Abstract

Section snippets

Materials

Preparation of mitochondria

Kinetics of GSH uptake

Discussion

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Hepatology

J. Biol. Chem.

J. Biol. Chem.

Hepatology

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Gastroenterology

J. Biol. Chem.

J. Biol. Chem.