Mitochondrial glutathione transport: physiological, pathological and toxicological implications

Abstract

Although most cellular glutathione (GSH) is in the cytoplasm, a distinctly regulated pool is present in mitochondria. Inasmuch as GSH synthesis is primarily restricted to the cytoplasm, the mitochondrial pool must derive from transport of cytoplasmic GSH across the mitochondrial inner membrane. Early studies in liver mitochondria primarily focused on the relationship between GSH status and membrane permeability and energetics. Because GSH is an anion at physiological pH, this suggested that some of the organic anion carriers present in the inner membrane could function in GSH transport. Indeed, studies by Lash and colleagues in isolated mitochondria from rat kidney showed that most of the transport (>80%) in that tissue could be accounted for by function of the dicarboxylate carrier (DIC, Slc25a10) and the oxoglutarate carrier (OGC, Slc25a11), which mediate electroneutral exchange of dicarboxylates for inorganic phosphate and 2-oxoglutarate for other dicarboxylates, respectively. The identity and function of specific carrier proteins in other tissues is less certain, although the OGC is expressed in heart, liver, and brain and the DIC is expressed in liver and kidney. An additional carrier that transports 2-oxoglutarate, the oxodicarboxylate or oxoadipate carrier (ODC; Slc25a21), has been described in rat and human liver and its expression has a wide tissue distribution, although its potential function in GSH transport has not been investigated. Overexpression of the cDNA for the DIC and OGC in a renal proximal tubule-derived cell line, NRK-52E cells, showed that enhanced carrier expression and activity protects against oxidative stress and chemically induced apoptosis. This has implications for development of novel therapeutic approaches for treatment of human diseases and pathological states. Several conditions, such as alcoholic liver disease, cirrhosis or other chronic biliary obstructive diseases, and diabetic nephropathy, are associated with depletion or oxidation of the mitochondrial GSH pool in liver or kidney.

Fig. 1

Structure of GSH.

Fig. 2

Reactions by which GSH reacts with NO to form GSNO. GSH, as the thiolate, reacts with NO in the presence of O₂ and forms GSNO. GSNO can release NO (function of GSNO as an NO donor) or it may react in the presence of the thiolate to form a species that can glutathionylate protein sulfhydryl groups. SOD, superoxide dismutase. Adapted from [43].

Fig. 3

NO formation from GSNO. GSNO acts as an NO donor in two reactions, the first of which is mediated by a flavoprotein containing FMN. The HNO generated from the first reaction can react with another molecule of GSNO to form an intermediate that decomposes to GSH and NO.

Fig. 4

Mitochondrial transport of GSH. Generalized summary scheme, simplified from [56], illustrating the basic function of the DIC and OGC in GSH transport and their relationships with the citric acid and GSH redox cycles.

Fig. 5

Genetic modulation of mitochondrial GSH transport in NRK-52E cells and susceptibility to chemically induced apoptosis. The cDNA for the DIC was transiently overexpressed in NRK-52E cells (NRK-DIC) and the cDNA for either the OGC or a double-cysteine mutant of the OGC (NRK-OGC or NRK-OGC-M, respectively) was stably overexpressed in NRK-52E cells. A. Uptake rates for GSH into mitochondria from different genetically modified NRK-52E cell populations. Mitochondria from each cell population were incubated with [³H]-GSH (final concentration = 5 mM). Data are expressed as uptake rates and are means ± SEM of measurements from 3–5 separate experiments. B. Fraction of apoptotic cells. Each cell population was incubated for 4 hr with either medium (= Control), 10 μM tert-butyl hydroperoxide (tBH), or 50 μM S-(1,2-dichlorovinyl)-L-cysteine (DCVC). The fraction of cells undergoing apoptosis was estimated by propidium iodide staining, flow cytometry and FACS analysis. Results are means ± SEM of 4–5 separate experiments. These data were derived from studies originally presented in refs. and , and were combined to illustrate toxicologic effects of genetic manipulation of mitochondrial GSH carriers.