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.

Fig. 1.

Dicarboxylate carrier (DIC) and oxoglutarate carrier (OGC) protein expression patterns. Protein samples from rat cortical mitochondria (A) and mouse neurons and glial cells (B) were assayed for DIC and OGC expression. The neuronal nuclear antigen (NeuN) was used as a neuronal marker, and the glial fibrillary acidic protein (GFAP) was used as a glial marker. Shown are representative Western blots.

Fig. 2.

Glutathione (GSH) transport through DIC is essential for the maintenance of the mitochondrial (m)GSH pool. A: standard curve for naphthalene-2,3-dicarboxaldehyde (NDA)-GSH fluorescence assay, showing linearity and consistency. Data are means ± SE; n = 11. B: rat cortical mitochondria were incubated at 20°C for 30 min in 4 different conditions: M, buffer alone; M/GSH, buffer containing 2.5 mM GSH; M/GSH/BM, buffer containing 2.5 mM GSH and 7 mM butylmalonate; M/GSH/PS, buffer containing 2.5 mM GSH and 7 mM phenylsuccinate. M₀ is freshly isolated mitochondria. mGSH content decreased in M in comparison with M₀, indicating the importance of GSH transport across the inner mitochondrial membrane (IMM) even under totally unchallenged conditions. Furthermore, mGSH content decreased in M/GSH/BM but not in M/GSH/PS in comparison with M/GSH, indicating that DIC was responsible for GSH transport across the IMM in these mitochondria. Data are means ± SE.

Fig. 3.

Inhibition of DIC-mediated GSH transport increases oxidative stress in rat cortical mitochondria. A: FACS analysis of reactive oxygen species (ROS) content in cortical mitochondria using glutamate/malate as the respiration substrate. DHR-123, dihydrorhodamine-123; MFI, median fluorescence intensity. B: FACS analysis of ROS content in cortical mitochondria using succinate as the respiration substrate. C: summary of intramitochondrial ROS levels in 4 individual experiments. Increased ROS levels were evident in the M and M/GSH/BM groups only when complex I substrates glutamate/malate were used to support respiration. Data are means ± SE.

Fig. 4.

Inhibition of DIC-mediated GSH transport increases H₂O₂ release by rat cortical mitochondria. A: kinetic release of H₂O₂ by cortical mitochondria using glutamate/malate as the respiration substrate. B: kinetic release of H₂O₂ by cortical mitochondria using succinate as the respiration substrate. C: summary of H₂O₂ release in 3 individual experiments. Rates of H₂O₂ release were significantly higher in the M and M/GSH/BM groups only when complex I substrates glutamate/malate were used to support respiration. With succinate, H₂O₂ release was similar in all groups. Data are means ± SE.

Fig. 5.

Inhibition of DIC-mediated GSH transport alters IMM functions. A: membrane potential was drastically reduced in M/GSH/BM in comparison with either M₀ or M/GSH, indicating severe impairment with DIC inhibition. Shown is a representative experiment. TMRM, tetramethylrhodamine methyl ester. B: state 4 respiration rates increased in the M/GSH/BM group, especially when complex I substrates were used, indicating increased oxygen consumption under basal conditions with DIC inhibition. Data are means ± SE. C: phosphorus-to-oxygen (P/O) ratio values decreased in the M/GSH/BM group regardless of the respiration substrate used, indicating a reduced efficiency in oxidative-phosphorylation in these mitochondria with DIC inhibition. Data are means ± SE.

Fig. 6.

Inhibition of DIC-mediated GSH transport impairs complex I activity A–C: representative activity assays for complexes I, II, and III, respectively. A: decreased consumption of NADH in M/GSH/BM, indicating a reduced complex I enzymatic activity with DIC inhibition. B: similar rates in 2,6-dichlorophenol indophenol (DCIP) consumption in all three samples, indicating no difference in complex II activity. C: similar rates in accumulation of reduced cytochrome c, indicating no difference in complex III activity. D–F: summaries of complexes I, II, and III activities in multiple experiments. Only complex I activity was significantly impaired in the M/GSH/BM group in which DIC was inhibited. Data are means ± SE; n = 8 for complexes I and II and n = 5 for complex III.

Fig. 7.

Inhibition of DIC-mediated GSH transport does not alter complexes I, II, and III integrity. Protein levels of representative subunits of complexes I, II, and III and cytochrome c (Cyto c) were similar in all three groups, indicating a lack of protein dislodgement from the IMM with DIC inhibition. Equal protein loading in each lane was verified by Ponceau staining. Shown are representative Western blots from 4 experiments.

Fig. 8.

DIC gene silencing decreases mGSH in PC12 cells. A: decreased DIC and OGC protein expression in PC12 cells following gene silencing with short hairpin (sh)RNA. Shown are representative Western blots from 4–5 experiments. Cytochrome c was used as a loading control. B: mitochondria from PC12 cells were incubated at 20°C for 30 min in buffer containing 2.5 mM GSH. mGSH content decreased in the DIC-shRNA group but was unaffected in the OGC-shRNA group, indicating that DIC was responsible for GSH transport in these mitochondria. These results are in line with those obtained from experiments using pharmacological agents. Data are means ± SE.