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

Abstract

Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxo-glutarate 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 H 2 O 2 , methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver. KeywordsGlutathione; Dicarboxylates; Transport; Liver; Mitochondria; Hepatoma cells; Oxidative stress Glutathione (GSH) 1 is an important antioxidant and cofactor in numerous drug metabolism reactions. In isolated hepatocytes, approximately 80% of total cellular GSH is in the cytoplasm whereas 10-15% is in the mitochondria [1]. Mitochondrial GSH is critical to defense against reactive oxygen species and maintains sulfhydryl groups of mitochondrial proteins in the reduced state. There are no enzymes to synthesize GSH in mitochondria [2,3]. Rather, GSH must be transported from cytoplasm into mitochondria. Due to the almost equal concentration of GSH in the cytoplasmic and mitochondrial pools and the anionic property of GSH at physiological pH, it has been presumed that a GSH carrier(s) exist(s) in the inner membrane of mitochondria. By using substrates and specific inhibitors for known anion transporters of mitochondria isolated from rat and rabbit kidneys, our previous work demonstrated that the dicarboxylate carrier (DIC; Slc25a10) and the 2-oxoglutarate carrier (OGC; Slc25a11) are responsible for uptake of GSH from the cytoplasm into the mitochondrial matrix [3][4][5]. Overexpression of either of these two carriers in a rat ✩

The TRβ-selective agonist, GC-1, stimulates mitochondrial oxidative processes to a lesser extent than triiodothyronine

“…Mitochondrial Vit E and coenzyme Q levels increased in T 3 -and GC-1-treated rats according to what previously found in hepatic tissue (Venditti et al 2009a), whereas GSH levels were not modified by treatments although the cytosolic levels decreased (Venditti et al 2009a). This result is consistent with the observation that, during oxidative stressinduced tissue GSH depletion, liver mitochondria conserve GSH level (Venditti et al 1999b) by transport systems located on the inner membrane (Zhong et al 2008).…”

Mitochondrial Glutathione, a Key Survival Antioxidant

“…In line with this, a recent study showed that incubation of rat liver mitochondria with butylmalonate (a dicarboxylate carrier inhibitor) and phenylsuccinate (a 2-oxoglutarate carrier inhibitor) inhibited GSH uptake by 45-50%, suggesting that these two transporters are only partially responsible for GSH uptake in rat liver mitochondria (153). Moreover, in H4IIE cells, a rat hepatoma cell line, that were stably transfected with the cDNA for the 2-oxoglutarate carrier, exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by hydrogen peroxide, methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport and mGSH levels in the liver (153). Because 2-oxoglutarate is a dicarboxylate, it could share the dicarboxylate carrier for its mitochondrial entry; thus, the competition seen between 2-oxoglutarate and GSH does not discard the participation of dicarboxylate carrier in the transport of GSH in liver mitochondria (26).…”

Renal Membrane Transport of Glutathione in Toxicology and Disease

“…Thus, we found significant sequence differences between rat kidney and liver OGC, 97 although the functional implications of these differences are not entirely clear. One thing that is clear, however, is that although both carriers are expressed in kidney and liver mitochondria, their relative contribution to mitochondrial GSH uptake differs in the 2 tissues.…”

“…Although the 2 carriers essentially account for virtually all of the detectable GSH uptake in renal cortical mitochondria, 19,66 function of at least 1 additional carrier must be invoked to account for the observable transport in rat liver mitochondria. 97 To directly demonstrate the toxicological role of mitochondrial GSH transport in renal cells, we used an immortalized cell line derived from normal rat proximal tubules (NRK-52E cells) as a convenient experimental model. 58 Overexpression of the cDNA for either the DIC 59 or the OGC 94 markedly increased mitochondrial GSH transport rates and provided significant protection from oxidant-induced cell death by apoptosis.…”