Dicarboxylate carrier-mediated glutathione transport is essential for reactive oxygen species homeostasis and normal respiration in rat brain mitochondria

Abstract

Glutathione transport into mitochondria is mediated by oxoglutarate (OGC) and dicarboxylate carrier (DIC) in the kidney and liver. However, transport mechanisms in brain mitochondria are unknown. We found that both carriers were expressed in the brain. Using cortical mitochondria incubated with physiological levels of glutathione, we found that butylmalonate, a DIC inhibitor, reduced mitochondrial glutathione to levels similar to those seen in mitochondria incubated without extramitochondrial glutathione (59% of control). In contrast, phenylsuccinate, an OGC inhibitor, had no effect (97% of control). Additional experiments with DIC and OGC short hairpin RNA in neuronal-like PC12 cells resulted in similar findings. Significantly, DIC inhibition resulted in increased reactive oxygen species (ROS) content in and H(2)O(2) release from mitochondria. It also led to decreased membrane potential, increased basal respiration rates, and decreased phosphorus-to-oxygen (P/O) ratios, especially when electron transport was initiated from complex I. Accordingly, we found that DIC inhibition impaired complex I activity, but not those for complexes II and III. This impairment was not associated with dislodgment of complex subunits. These results suggest that DIC is the main glutathione transporter in cortical mitochondria and that DIC-mediated glutathione transport is essential for these mitochondria to maintain ROS homeostasis and normal respiratory functions.

Mitochondrial Glutathione Transport Is a Key Determinant of Neuronal Susceptibility to Oxidative and Nitrosative Stress

“…Previous studies have shown that whole brain mitochondria utilize the tricarboxylate carrier (Slc25a1) as a mitochondrial GSH transport protein, whereas mitochondria isolated specifically from cerebral cortex, which express both DIC and OGC, use primarily DIC as a mitochondrial GSH transport protein (12,13). Our study shows that CGNs are dependent on DIC, whereas cerebellar astrocytes use both DIC and OGC as mitochondrial GSH transport proteins.…”

“…A second study showed that cortical neurons and astrocytes express both DIC and OGC. However, in isolated mitochondria from whole cortical tissue, only the activity of DIC appeared to influence mitochondrial GSH content (13). To date, the mechanism of mitochondrial GSH transport utilized by neurons specifically has not been examined.…”

Glutathione and mitochondria

“…Moreover, in mouse brain mitochondria another study showed that OGC and DIC are both expressed in cortical neurons and astrocytes (Kamga et al, 2010). In addition, butylmalonate, an inhibitor of DIC, significantly decreased mGSH, suggesting DIC as the major GSH transporter in mouse cerebral cortical mitochondria (Kamga et al, 2010).…”

Mitochondrial Thiols in Antioxidant Protection and Redox Signaling: Distinct Roles for Glutathionylation and Other Thiol Modifications

“…1) (54,73,101,134,167,181). Uptake may be mediated by two members of the mitochondrial carrier family, the dicarboxylate carrier, and the 2-oxoglutarate carrier (27,54,112,136).…”