Introduction

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Cellular thiol-disulfide status, which is predominantly regulated by GSH, is critical to numerous mitochondrial functions, including membrane structure and integrity (Lê-Quôc and Lê-Quôc, 1985, 1989), ion homeostasis (Beatrice et al., 1984), intramitochondrial redox status (Yagi and Hatefi, 1984) and activities of numerous sulfhydryl-dependent enzymes (Lê-Quôc, 1985). Distinct pools of GSH are found in cytosolic and mitochondrial fractions of cells (Meredith and Reed, 1982; Schnellmann et al., 1988). The importance of mitochondrial GSH is highlighted by several studies in both liver and kidney, that have concluded that most chemical exposures that are associated with GSH depletion require depletion of mitochondrial, rather than cytosolic, GSH to elicit cellular injury (Lash and Anders, 1987; Olafsdottir et al., 1988; Reed, 1990, 1993; Shan et al., 1993). Available data indicate that little or no de novo synthesis of GSH occurs in mitochondria (Griffith and Meister, 1985; Martensson et al., 1990). Hence, the mitochondrial pool of GSH must derive from transport of cytosolic GSH across the mitochondrial inner membrane (Smith et al., 1996).

In renal cortex, mitochondria contain about 15 to 30% of total cellular GSH (Schnellmann et al., 1988), which is approximately the volume fraction of mitochondria in renal proximal tubular cells. Therefore, there should be little concentration gradient of GSH across the mitochondrial inner membrane between the cytosolic and mitochondrial matrix compartments. Transport of GSH into mitochondria, however, is energetically unfavored because GSH is a negatively charged molecule at physiological pH and the mitochondrial matrix is negatively charged relative to the cytosol. GSH molecules have one free amino group, approximately 98% of which are protonated at pH 7.0, two free carboxyl groups, of which >99.9% are deprotonated at pH 7.0, and one thiol group, of which approximately .6% are deprotonated at pH 7.0. This makes a net charge of at least -1 for GSH at pH 7.0. At the slightly alkaline pH of the mitochondrial matrix (pH = 7.8), the fraction of protonated amino groups on GSH molecules is approximately 86% and the fraction of deprotonated thiol groups is approximately 4%. At the higher pH of the matrix, therefore, a larger proportion of the GSH molecules will have a net charge of -2. The existence of these charges on the GSH molecule indicates that the transport mechanism for GSH into mitochondria cannot be simple diffusion, but must be either energy-dependent or linked to the transport of other chemicals (Lash, 1995).

Because GSH has a net negative charge, it is conceivable that one or more of the known anion transporters in the mitochondrial inner membrane could be involved in the uptake of GSH in renal mitochondria. There are eight known carriers in the mitochondrial inner membrane that can catalyze transport of organic anions (table 1). Many of the organic anion substrates for these carriers are citric acid cycle intermediates. High activity of these transporters is expected in tissues such as the kidney because of high rates of mitochondrial respiration. Because the adenine nucleotide translocase and the phosphate transporter have fairly restricted substrate specificity, these two carriers are not likely to be directly involved in GSH uptake. In contrast, each of the other six transporters could conceivably mediate mitochondrial GSH uptake. Although the dicarboxylate, 2-oxoglutarate, tricarboxylate and the monocarboxylate transporters each differ with respect to substrate specificity and inhibitor sensitivity, they share a common 30 kDa molecular weight subunit, suggesting that they belong to a carrier "superfamily" (Palmieri et al., 1996). Each of these transporters are electroneutral, meaning that there is no net transfer of charge across the membrane. The glutamate carrier, which exchanges glutamate for hydroxide ions, is similarly electroneutral. In contrast, the glutamate-aspartate carrier is electrogenic, mediating net transfer of one positive charge into the mitochondrial matrix.

Previous work on renal mitochondrial GSH uptake by Schnellmann (1991) focused on the energetics of the transport process. Uptake of GSH against a concentration and electrical gradient was observed and it was concluded that at low concentrations, GSH exchanges with other substrates whereas at higher concentrations, a non-saturable mechanism may predominate. Neither energy dependence nor a proton gradient were required for GSH uptake. In an earlier study (McKernan et al., 1991), we showed similarly that GSH uptake by renal cortical mitochondria exhibited neither energy nor pH dependence and was cis-inhibited and trans-stimulated by dicarboxylates such as malate and succinate but was unaffected by mono- or tricarboxylates.

The goal of our study was to define unambiguously the role of the various organic anion transporters in the uptake of GSH in suspensions of isolated mitochondria from rat renal cortex. This was accomplished by use of carrier substrates and specific inhibitors of the various mitochondrial anion transporters and alteration of mitochondrial energetics and extramitochondrial phosphate and pH status. The results showed conclusively that the dicarboxylate and 2-oxoglutarate carriers are involved whereas the mono- and tricarboxylate carriers and both the glutamate-aspartate and glutamate carriers are not involved in mitochondrial GSH uptake. Preliminary reports of some of this work have been presented (Chen and Lash, 1996, 1997).

	Materials and Methods

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Materials

Acivicin, GSH, DTT, dibutylphthalate, L--glutamyl-L-glutamate, iodoacetic acid, 1-fluoro-2,4-dinitrobenzene, AA, PEP, CsA, oligomycin, atractyloside and Lubrol PX were purchased from Sigma Chemical Co. (St. Louis, MO). Phenylsuccinate, butylmalonate and TEC were purchased from Aldrich Chemical Co. (Milwaukee, WI). [U-¹⁴C]Sucrose (specific activity = 1.74 mCi/mg), ³H₂O (1 mCi/ml) and L-[G-³H]glutamic acid (specific activity = 327 mCi/mg) were purchased from Amersham (Arlington Heights, IL). Other chemicals were of the highest purity available and were obtained from commercial sources.

Preparation of Mitochondria

Male Fisher 344 rats (175-225 g) were purchased from Charles River Laboratories (Wilmington, MA). Rats were housed in cages in the Wayne State University vivarium with a 12-hr light-dark cycle and were given food and water ad libitum. Rats were anesthetized with an injection of sodium pentobarbital (50 mg/kg body weight i.p.). After removal from the abdomen, rat kidneys were immediately placed in ice-cold buffer and rats were killed by pneumothorax and exsanguination. The mitochondrial isolation buffer contained 20 mM triethanolamine/HCl, pH 7.4, 225 mM sucrose, 3 mM potassium phosphate (pH 7.4), 5 mM MgCl₂, 20 mM KCl and .1 mM phenylmethylsulfonyl fluoride to inhibit proteolysis. EGTA (2 mM) was included in the buffer at all preparatory stages, except the final resuspension, to remove calcium ions. After decapsulation, the cortex was sliced into small pieces and was homogenized with a hand-held Dounce homogenizer. Mitochondria from homogenates of rat renal cortex were isolated by the differential centrifugation method of Johnson and Lardy (1967), as described previously (Lash and Sall, 1993). In those experiments in which the concentration of extramitochondrial phosphate was varied between 0 and 10 mM, mitochondria were prepared in phosphate-free isolation buffer. The specified amount of phosphate was then added to the mitochondria at the time of the experiments.

Determination of Mitochondrial Respiratory Function

After incubating mitochondria in buffer at 20°C for various times (ranging from 0 to 15 min), oxygen consumption in mitochondrial suspensions was measured with a Gilson 5/6H oxygraph in a thermostated, air-tight, 1.6 ml chamber at 20°C using a Clark-type oxygen electrode. State 3 rates were measured by addition of 3.3 mM succinate and 0.3 mM ADP in the presence of 5 µM rotenone in ethanol (final concentration = 0.3%, v/v) to 1.5 to 3.0 mg mitochondrial protein; state 4 rates were measured as the rate of oxygen consumption after exhaustion of ADP. Respiratory control ratios (RCR = state 3 rate/state 4 rate) were used to assess functional integrity (Estabrook, 1967).

Measurement of GSH Uptake

Before incubation with GSH, mitochondria were pretreated with 0.25 mM acivicin in the presence of 5 mM DTT for 15 min at 20°C to inhibit the activity of GGT (EC 2.3.2.2) that derived from contaminating brush-border membranes. Pretreatment with 0.25 mM acivicin produced greater than 98% inhibition of GGT activity, both in the presence or absence of DTT. Uptake of GSH into mitochondria was determined by one of three different methods (see Lash, 1995 for a discussion of these methods). The isolation buffer (without EGTA), with additions as indicated, was used in all incubations for measurement of GSH uptake.

Single-step centrifugation method. This method was used solely for evaluation of matrix volume during incubations with GSH (see below). Aliquots (0.5 ml) of samples at each time point during incubations with GSH were placed in 1.5 ml-polyethylene microcentrifuge tubes and were centrifuged at 13,000 × g for 30 sec to separate extramitochondrial space (supernatants) from mitochondrial matrix space (pellets).

Centrifugation-resuspension method. Aliquots (0.5 ml) of samples at each time point during incubations with GSH were placed in 1.5 ml-polyethylene microcentrifuge tubes and were centrifuged at 13,000 × g for 30 sec. After removal of supernatants, pellets were resuspended in 0.5 ml ice-cold buffer and were centrifuged again. Pellets were resuspended and centrifuged one more time to wash out the incubation medium, and were finally resuspended in 10% (v/v) perchloric acid to release mitochondrial matrix contents.

Dibutylphthalate method. Aliquots (0.5 ml) of samples at each time point during incubations with GSH were layered on the top of a 0.5 ml-dibutylphthalate layer and a 0.55 ml-10% (v/v) perchloric acid layer in 1.5 ml-polyethylene microcentrifuge tubes. Mitochondria were immediately centrifuged at 13,000 × g for 30 sec through the dibutylphthalate layer into the acid layer

Measurement of GSH Efflux

After 15-min preincubation with acivicin, mitochondria were incubated with various concentrations of GSH for 10 min to reach their equilibrium GSH content. After mitochondria were centrifuged at 15,000 × g for 3 min, pellets were resuspended in a large volume of buffer (50 ml) and recentrifuged to wash out incubation medium that was trapped in the pellets. The final pellets were resuspended in 5 ml buffer, which was the same volume for GSH preloading. The mitochondrial suspension was incubated either at 20 or 4°C for various periods of time. GSH content in mitochondria and in the medium was measured at each time point by high-performance liquid chromatography as above.

Measurement of Glutamate Uptake

Uptake of 5 or 10 mM glutamate by renal cortical mitochondria was measured by the centrifugation-resuspension method and intramitochondrial content of glutamate was measured by derivatization with 1-fluoro-2,4-dinitrobenzene and high-performance liquid chromatography as described above for GSH. Uptake of 0.1 mM glutamate was measured with L-[G-³H]glutamic acid. Samples were processed by the centrifugation-resuspension method and intramitochondrial content of glutamate was determined by scintillation counting.

Determination of Mitochondrial Matrix Volume

Mitochondria were incubated with 5 mM GSH in buffer containing 0.1 µCi [¹⁴C]-sucrose and 1 µCi ³H₂O. At each time point of incubation, mitochondrial suspensions were either centrifuged through buffer (single-step centrifugation method) or were centrifuged through a layer of dibutylphthalate into perchloric acid, and radioactivity in both pellet and supernatant fractions was counted separately in the ¹⁴C and ³H channels. The mitochondrial matrix volume was then calculated with the following equation: The matrix volume (Vm) is the volume of ³H₂O in the pellet (VH₂O), which is total pellet volume calculated from net ³H counts over specific ³H counts (A³H), minus the volume of [¹⁴C]-sucrose in the pellet (Vsucrose), which is the extramitochondrial medium volume calculated from net ¹⁴C counts over specific ¹⁴C counts (A¹⁴C). Specific ¹⁴C and ³H counts were determined by standard samples that only contained a known amount of either ³H₂O or [¹⁴C]-sucrose.

Measurement of GGT Activity

GGT activity was measured as formation of p-nitroanilide by following the increase in absorbance at 410 nm with -glutamyl-p-nitroanilide and glycylglycine as substrates according to the method of Orlowski and Meister (1963).

Protein Assay

Mitochondrial protein content was determined spectrophotometrically by a Coomassie Blue G dye-binding assay (Read and Northcote, 1981).

Calculation of Initial Rate of GSH Uptake

GSH uptake appeared to be linear for only 1 to 2 min (see fig. 1). For this reason, initial rates of uptake were calculated from uptake time courses (0, 0.33, 1, 2, 3, 5 min) by curve-fitting and the following method. To calculate the first-order rate constant k of GSH uptake, linear curve-fitting was performed on the plot of ln[P_total/(P_total - P_t)] vs. time according to Halestrap (1975). P_total represents the total uptake of GSH at equilibrium, which was estimated by performing an exponential decay curve-fitting on the time course data of GSH uptake. P_t represents the GSH uptake at time t (0, 1, 3 or 5 min). Thus, the initial rate of GSH uptake was determined from the first-order rate equation v = k (P_total).

Data Analysis

All values are expressed as the means ± S.E. of measurements from the indicated number of individual experiments. Significant differences between means were first assessed by a one-way or two-way analysis of variance. When significant "F-values" were obtained, the Fisher's protected least significant difference t test was performed to determine which means were significantly different from one another, with two-tail P < .05 considered significant.

	Results

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Mitochondrial Respiratory Function

Freshly isolated renal cortical mitochondria exhibited RCR values above 4.0, and mitochondria incubated at 20°C for 15 min with or without pretreatment with a thiol (e.g., DTT, GSH) exhibited RCR values of 3.2 to 3.7 or 2.4 to 2.7, respectively (table 2). We concluded, therefore, that pretreatment of mitochondria with DTT was beneficial in maintaining functional integrity. All mitochondria were pretreated with acivicin to inhibit degradation of GSH by contaminating GGT, and this pretreatment had no effect on RCR values, indicating no adverse effect on mitochondrial integrity.

Kinetics of GSH Uptake and Efflux

The time course for uptake of 5 mM GSH in renal cortical mitochondria was measured at 20°C by the centrifugation-resuspension method (fig. 1A). Initial mitochondrial contents of GSH were between 2.5 and 4.0 nmol/mg protein. In the presence of 5 mM GSH, these contents increased rapidly to equilibrium levels of approximately 7.0 nmol/mg protein within 5 min. Incubation with 2.5 mM extramitochondrial GSH for 5 min resulted in little net uptake of GSH (fig. 1B). In contrast, intramitochondrial contents of GSH after 5-min incubations with extramitochondrial concentrations of GSH of 5 mM or higher were 7.5 to 8.5 nmol/mg protein, with apparent saturation between 5 and 10 mM GSH.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   GSH uptake into renal cortical mitochondria as measured by the centrifugation- resuspension method. After preincubation with 0.25 mM acivicin, mitochondria (4-6 mg/ml) were incubated with various concentrations of GSH (2.5, 5, 10 or 15 mM) at 20°C. At each time point of incubation (0, 1, 3, 5, 10 or 30 min), mitochondrial GSH content was determined by the centrifugation-resuspension method described in "Materials and Methods." A, Time course of GSH uptake into mitochondria with 5 mM extramitochondrial GSH. Results are the means ± S.E. of six experiments. B, Concentration dependence of GSH uptake. The mitochondrial GSH content at 5 min of incubation was plotted vs. concentration of exogenous GSH.

Concentration dependence of GSH efflux from mitochondria was determined after preloading mitochondria with 0, 5, 7.5 or 10 mM GSH (fig. 2). The efflux curves were nearly linear, suggesting that efflux rates were constant over the incubation period. The concentration of GSH with which mitochondria were preloaded did not markedly affect the rate of GSH efflux above a preloading concentration of 5 mM. Rates of GSH efflux were much slower than uptake. In mitochondria preloaded with 5 mM GSH and incubated at 20°C, more than 50% of intramitochondrial GSH was retained after 30 min of incubation. Efflux of GSH was also assessed at 4°C as a control experiment to show that minimal loss of intramitochondrial GSH occurs during sample processing in mitochondrial GSH uptake determinations, which were performed at 0 to 4°C (data not shown). Mitochondria only lost 10% of their GSH content in the first 10 min and 25% in 30 min. Because sample processing takes about 2 min, these results indicate that minimal GSH is lost from mitochondria during the uptake determinations.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Efflux of GSH from renal cortical mitochondria. Freshly isolated mitochondria (4 to 6 mg/ml) were preincubated at 20°C with 0.25 mM acivicin for 15 min followed by incubation at 20°C with various concentrations of GSH (0-10 mM) for 10 min. After removing the incubation medium by the centrifugation and resuspension method, the GSH-loaded mitochondria were resuspended in GSH-free buffer and incubated for 0, 5, 10, 15 or 30 min at 20°C. The mitochondrial GSH content was then determined at each time point. Results are means ± S.E. of five experiments. For clarity, error bars were not shown, but were approximately 15 to 20% of means.

Measurement of Matrix Volume and Membrane Binding

To validate that the centrifugation-resuspension method gave accurate values for mitochondrial GSH uptake, two alternative methods were used so that precise corrections for contamination of mitochondrial matrix space with extramitochondrial volume and nonspecific binding or association of GSH to mitochondrial membranes could be made. The single-step centrifugation method involves centrifugation of the mitochondrial suspension in a microcentrifuge tube and removal of the supernatant. This contrasts with the centrifugation-resuspension method, in which pellets are resuspended in buffer and recentrifuged. In the dibutylphthalate method, mitochondrial suspensions are centrifuged through a layer of the oil dibutylphthalate to effect separation of intra- and extramitochondrial space. Note that it is not possible, by the procedure used, to determine matrix volume with the centrifugation-resuspension method because the extracellular supernatant is removed and discarded during sample processing. However, validity of this method is assessed by comparison of the final, calculated results using the two alternate methods after all the corrections are obtained for extra-matrix volume and nonspecific association of GSH.

To validate further the methodology for transport measurement, matrix volumes after incubation for various times (up to 5 min) with 5 mM GSH were measured using radiolabeled sucrose and water and samples were processed by the single-step centrifugation and the dibutylphthalate methods (table 3). Matrix volumes obtained from the two methods for measurement of uptake were approximately the same, about 1 µl/mg protein, and did not vary significantly over the 5-min incubation time period. In contrast, total pellet volumes, which are the sum of matrix volumes and extramitochondrial medium volumes trapped in the pellets, were significantly smaller when mitochondria were centrifuged through dibutylphthalate than when they were processed by the single-step centrifugation method. However, extramitochondrial space in the pellets still accounted for nearly 80% of total pellet volumes. Although matrix volumes calculated for mitochondria that were processed by the single-step centrifugation and dibutylphthalate methods were the same, a much higher degree of variation was obtained with the former method (23 to 44% vs. 4 to 23%). This is likely due to the much higher total pellet volumes by the former method and the fact that there is often inherently more error involved in subtracting a smaller parameter from a larger one, which is what is done to obtain the matrix volume data.

An additional correction factor that is often needed is one for nonspecific binding or association. To test whether a portion of the measured GSH was associated with the mitochondrial inner membrane and not free in the matrix, GSH-loaded mitochondria were treated for 5 min with 0.05% (w/v) Lubrol PX to release matrix contents. Measurement of matrix volume with [¹⁴C]-sucrose and ³H₂O and release of malic dehydrogenase (EC 1.1.1.37), a marker enzyme for the matrix fraction, after Lubrol treatment demonstrated that this concentration of Lubrol completely released the soluble components of the matrix (data not shown). Approximately 65% of total GSH of mitochondria preloaded with 5 mM GSH was Lubrol-sensitive and was released by treatment with the detergent. Hence, 35% of the total GSH measured by the dibutylphthalate method is not free and may be membrane-bound or associated with the membrane in some other manner.

Measurement of GSH uptake in mitochondria incubated for up to 10 min with 5 mM GSH, with samples processed by the dibutylphthalate method, gave apparent uptake at equilibrium of approximately 40 nmol/mg protein (fig. 3). When corrections were made for trapped extramitochondrial medium and for apparent binding or association of GSH to mitochondria, the apparent uptake was reduced to approximately 8 nmol/mg protein. These uptake values are consistent with those found by using the centrifugation-resuspension method (cf. fig. 1A), showing that the centrifugation-resuspension method gives valid measurements of matrix GSH content. Accordingly, we used the centrifugation-resuspension method for processing of samples in all subsequent experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Time course of GSH uptake into mitochondria using the dibutylphthalate method. After acivicin-preincubation, mitochondria (4-6 mg/ml) were incubated with 5 mM GSH for various times (0, 20 sec, or 1, 5 or 10 min) at 20°C. At each time point, mitochondria were centrifuged through the dibutylphthalate layer into perchloric acid. Total GSH amount in the perchloric acid extract was determined by high-performance liquid chromatography analysis. Net matrix GSH content was calculated by subtracting the total GSH amount by the amount of GSH in the extramitochondrial medium and the amount of GSH nonspecifically associated with mitochondrial membranes, as estimated by the Lubrol release experiment (see text). Results are the means ± S.E. of five experiments.

Role of Anion Carriers in GSH Uptake

Substrate specificity. The relationship between the uptake of GSH and organic anions in renal mitochondria was assessed by coincubation of mitochondria with 5 mM GSH and 10 mM of a substrate or specific inhibitor of one of the known mitochondrial anion transporters (table 4). The 5 mM concentration of GSH was chosen because this is the typical cytosolic concentration of GSH to which the mitochondria are exposed. A 2-fold molar excess of competing substrate or inhibitor was chosen to enable significant inhibition to be observed. Higher concentrations were not used because that would significantly alter the osmolarity and composition of the incubation media. Because the mitochondrial matrix volume is sensitive to changes in energetic status and several of the anions tested are respiratory substrates, measurements of GSH uptake were done in both the presence and absence of 2 µM AA. This concentration of AA completely inhibits oxygen consumption and substrate metabolism in renal cortical mitochondria (data not shown). Metabolism of these substrates could lead to rapid changes in matrix volume, which would alter apparent GSH uptake values. Coincubation of mitochondria with malate or succinate, which are both substrates for the dicarboxylate and 2-oxoglutarate transporters, significantly inhibited GSH uptake in either the presence or absence of AA. Similarly, phenylsuccinate, which is a specific inhibitor of the 2-oxoglutarate transporter, also markedly inhibited GSH uptake in either the presence or absence of AA. Butylmalonate, which is a specific inhibitor of the dicarboxylate transporter, had no effect in the absence of AA but inhibited GSH uptake by >80% in the presence of AA. Of the other substrates and inhibitors tested, only glutamate and PEP significantly inhibited GSH uptake. Glutamate is transported by either an electrogenic glutamate-aspartate exchanger or by an electroneutral glutamate-hydroxide exchanger. PEP is transported by the tricarboxylate transporter. Neither aspartate, other substrates of the tricarboxylate transporter, TEC (which inhibits the tricarboxylate transporter), nor substrates of the monocarboxylate transporter inhibited GSH uptake.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Interaction between mitochondrial transport of GSH and glutamate. Time course of glutamate uptake in mitochondria was determined by the same method for GSH uptake with various concentrations of glutamate (0.5, 1, 2.5, 5 and 10 mM) at 20°C. A, The initial rates of GSH uptake (5 mM) were determined by measuring the time course of GSH uptake in the presence of 5 or 10 mM glutamate with or without the addition of 2 µM AA. B, The initial rates of glutamate uptake (5 and 10 mM) were determined by measuring the time course of glutamate uptake in the presence of 2 µM antimycin A (AA) or 5 mM GSH. Results are the means ± S.E. of three to five experiments. *Significant difference (P < .05) from the corresponding control value.

Role of membrane permeability transition in GSH uptake. Although the absence of changes in matrix volume during coincubations with GSH and other organic anions is a crucial control experiment, mitochondria can also undergo a permeability transition whereby the inner membrane becomes permeable to ions and small molecules in the presence of calcium ions and an inducing agent (Gunter and Pfeiffer, 1990). A hallmark characteristic of the permeability transition is that it is prevented by CsA (Broekemeier et al., 1989; Crompton et al., 1988). In the substrate specificity experiments (cf. table 4), the two results that were not readily explainable were the marked inhibition of GSH uptake by PEP and the inhibition of GSH uptake by butylmalonate, but only in the presence of AA.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Effect of cyclosporin A (CsA) on mitochondrial GSH uptake. Time course of uptake of 5 mM GSH into mitochondria was determined with addition of 10 mM PEP or butylmalonate in the absence or the presence of 0.5 nmol CsA/mg protein. The initial rates of GSH uptake were then calculated from time course data. Results are the means ± S.E. of three experiments. *Significant difference (P < .05) from the corresponding control value.

Phosphate, pH and ATP dependence of GSH uptake. Phosphate is critical for the mitochondrial transport of dicarboxylates as the transport mechanism of the dicarboxylate carrier involves electroneutral exchange of phosphate and a dicarboxylate. Because of the inhibition of GSH uptake by dicarboxylates, the effect of the extramitochondrial concentration of phosphate was studied (fig. 6). Mitochondria were resuspended in nominally phosphate-free buffer or in buffers containing up to 5 mM phosphate. GSH uptake was markedly inhibited in buffer containing 0 or 0.5 mM phosphate. No differences were observed in rates of GSH uptake at phosphate concentrations of 1 mM and above.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Phosphate dependence of Mitochondrial GSH uptake. Time course of GSH uptake in mitochondria was determined with various concentrations of inorganic phosphate in the buffer (0, 0.5, 1, 1.5, 3, and 5 mM). Mitochondria were prepared in phosphate-free isolation buffer and the specified amount of inorganic phosphate was then added before the incubations. The initial rates of GSH uptake were then calculted from the time course data. Results are the means ± SE of 3 to 5 experiments. *Significant difference (P < 0.05) from The value for standard uptake conditions (3 mM phosphate).

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

We hypothesized that one or more of the several organic anion carriers of the mitochondrial inner membrane could be involved in GSH uptake from the cytosol. As with carriers in other membranes, those in the mitochondrial inner membrane can have fairly broad and sometimes overlapping substrate specificities. Hence, more than one system could contribute to maintenance of intramitochondrial GSH content. The various, known carriers (cf. table 1) have some well-defined substrates, inhibitors and energetics. Our approach to assessing the role of these systems in mediating GSH uptake was to test for inhibition by specific substrates or inhibitors and to examine energetics requirements.

Accuracy of the transport protocol can be compromised by at least five potential artifacts: 1) contamination of matrix space with extramitochondrial fluid; 2) change in matrix volume during the incubation period, possibly as a consequence of metabolism; 3) efflux of transported substrate during sample processing after the transport reaction has been stopped; 4) production of the membrane permeability transition and 5) contamination of the mitochondrial preparation with plasma membranes, which contain a large array of transport proteins. For GSH, a sixth possible artifact involves degradation to the constituent amino acids by contaminating GGT from brush-border membranes.

The potential problems of contamination of matrix space with extramitochondrial fluid and change in matrix volume due to substrate metabolism were addressed by measurement of matrix volume with [¹⁴C]sucrose and ³H₂O, both in the presence and absence of AA. AA was added to inhibit substrate metabolism and the electron transport chain. In all cases and with both the single-step centrifugation and dibutylphthalate methods, no significant changes were observed in mitochondrial matrix volume, indicating that GSH was being transported into a constant volume. The results of these studies showed that matrix volume was approximately 1 µl/mg protein and that it accounted for 11 to 12% and 20 to 26% of total mitochondrial pellet volume by the single-step centrifugation and dibutyphthalate methods, respectively. Further correction of measured GSH obtained with the dibutylphthalate method by subtraction of nonspecifically bound or associated GSH showed that this method gave essentially identical results to those obtained with the centrifugation-resuspension method. Hence, the centrifugation-resuspension method was adopted for all subsequent experiments. Matrix volume could not be measured with the centrifugation-resuspension method using the [¹⁴C]sucrose/³H₂O procedure because of the washing steps. It should also be noted that additional washing steps did not improve GSH measurements and actually reduced recovered GSH after two steps, presumably due to damage to the mitochondria (data not shown).

Release of mitochondrial matrix content by treatment with the detergent Lubrol indicated that 35% of the GSH measured by the dibutylphthalate method was not free in the matrix but was specifically or nonspecifically associated with the mitochondrial membrane. It is unknown to which components on the membrane GSH molecules associate or what type of interactions are involved in the binding or association. Because GSH is a negatively charged molecule at physiological pH, there may be electrostatic interactions between GSH and some positively charged molecules on the mitochondrial membrane. The similarity between measured contents of matrix GSH obtained by the centrifugation-resuspension method and those obtained by the dibutylphthalate method after correcting for extramitochondrial volume and non-specific association, suggest that the association was an artifact that occurred during processing of the samples by the dibutylphthalate method. Furthermore, complete release of matrix GSH after induction of the membrane permeability transition has been reported, suggesting that a significant portion of matrix GSH is not normally bound to mitochondrial constituents.

The potential problem of efflux of transported GSH during sample processing was assessed by measuring GSH efflux from GSH-preloaded mitochondria at both 4 and 20°C. Rates of GSH efflux at 4°C, which was the temperature at which samples were processed for uptake measurements, was markedly slower than uptake. Only a 10% loss of intramitochondrial GSH occurred in 10 min. Because the samples were processed for measurement of uptake in less than 3 min, minimal loss of transported GSH occurred during this procedure and was, therefore, not a problem in the methodology. It is not surprising that efflux was a slow process because there were no exchangeable substrates in the extramitochondrial medium. Whether or not GSH efflux and uptake occur by the same carriers is unclear, and this requires further work.

The membrane permeability transition can occur in the presence of calcium ions and a variety of inducing agents, including oxidants, phosphate ions and several intermediary metabolites (Gunter and Pfeiffer, 1990). The result of the transition is that the inner membrane becomes permeabilized to low-molecular-weight compounds. The lack of an effect of CsA on the measured accumulation of GSH in mitochondria indicates that a permeability transition was not occurring under the conditions of the transport measurements.

Renal basolateral and brush-border plasma membranes contain a large array of transporters, including those for GSH (Inoue and Morino, 1985; Lash and Jones, 1983, 1984). As explained in our previous study (McKernan et al., 1991), several of the properties of the two plasma membrane transport systems for GSH and those observed for renal mitochondria differ and the contamination of the mitochondrial preparation with plasma membranes is very small, indicating that the observed transport in this study could not be accounted for by contaminating plasma membranes. Some of the properties that differ between GSH transport in mitochondria and plasma membranes include membrane potential and sodium ion dependence, charge transfer and substrate specificity.

Finally, the presence of contaminating brush-border membranes, which contain GGT, in the mitochondrial fraction may complicate measurement of GSH uptake because of degradation of GSH to its constituent amino acids. Even though the extent of contamination is small (<5%) (McKernan et al., 1991), high enough GGT activity is present to cause significant and rapid GSH degradation. This limitation was readily circumvented by the use of acivicin, thus enabling us to detect transport of intact tripeptide. Because the high-performance liquid chromatography method gives a direct measurement of the S-carboxymethyl-N-dinitrophenyl derivative of GSH, along with derivatives of glutamate and cysteine, significant degradation of GSH during the incubation period can be readily detected, and this did not occur.

From the substrate specificity studies, a role for the two previously characterized dicarboxylate transporters, the dicarboxylate carrier, which exchanges dicarboxylates such as malate or succinate with phosphate, and the 2-oxoglutarate carrier, which exchanges 2-oxoglutarate with dicarboxylates such as malate, are supported. The mono- and tricarboxylate transporters do not appear to be involved in GSH uptake because substrates for these transporters did not affect GSH uptake. Experiments using CsA showed that the observed inhibition of GSH uptake by PEP was due to induction of the permeability transition rather than to competition for transport.

The dependence of GSH uptake on the presence of phosphate showed an unusual concentration profile. At less than 1 mM phosphate, rates of GSH uptake were decreased by >80%. It appears, therefore, that the transporters are readily saturated with phosphate above that concentration. A requirement for phosphate is consistent with the dicarboxylate carrier having a role in GSH uptake. The 2-oxoglutarate transporter, which is another member of the organic anion carrier "superfamily" (Palmieri et al., 1996), also appears to mediate GSH uptake based on substrate specificity and inhibition of GSH uptake by phenylsuccinate.

Analysis of the interaction of GSH with glutamate is complicated because degradation of GSH generates glutamate, which is one of its constituent amino acids, and because rates of glutamate uptake were 3- to 5-fold faster than those of GSH uptake. Although glutamate inhibited GSH uptake, GSH did not produce net inhibition of glutamate uptake after corrections were made for glutamate present in the GSH preparation. Hence, the observed inhibition did not appear to be due to competition between GSH and glutamate for a common transporter. Consideration of other evidence also argues against GSH and glutamate being transported by a common carrier. This evidence includes the absence of pH or membrane potential dependence of GSH uptake and the inability of aspartate to inhibit GSH uptake. The absence of a pH dependence indicates that the glutamate carrier, which catalyzes an electroneutral exchange of glutamate with hydroxide ions, is not likely to transport GSH. Although one would expect an effect of changes in extramitochondrial pH, because of the dependence of GSH uptake on phosphate and the interaction with the dicarboxylate carrier, the apparent saturation of the carrier with phosphate and the energization of the mitochondria probably explain the lack of pH dependence. The absence of a membrane potential dependence (McKernan et al., 1991) as well as the lack of inhibition by aspartate indicate that the electrogenic glutamate-aspartate exchanger is also not involved in GSH transport.

Schnellmann (1991) previously reported the presence of uptake of GSH by mitochondria isolated from rabbit kidney cortex. GSH uptake occurred against concentration and electrical gradients, but was not directly dependent on energy or a proton gradient. It was concluded that GSH is likely to be transported into renal mitochondria by an exchange system. Although a detailed study of substrate specificity was not conducted, including no assessment of the interaction of GSH transport with organic anions, Schnellmann (1991) did find modest inhibition (10-27%) of GSH uptake by glycine, ophthalmic acid and serine and no inhibition by glutamate, -glutamyl-glutamate, cysteine or proline. Although the proposed mechanism generally agrees with our results, the substrate specificity appears to differ from what we report here in that glutamate was a potent inhibitor of GSH uptake in our study. This difference may lie in methodological differences. The overall results, however, are consistent with our previous (McKernan et al., 1991) and current report.

More work on mitochondrial transport of GSH has been done with liver mitochondria. Inasmuch as metabolite transport systems in liver and kidney mitochondria are generally similar to each other (Klingenberg, 1979), one may assume that the mechanism of uptake of GSH in mitochondria will be the same in both tissues. Kurosawa et al. (1990) found uptake and accumulation of GSH in rat liver mitochondria only in state 4 respiration. Furthermore, GSH uptake was decreased by AA and was influenced by a proton gradient. Fasted rats were used, indicating that the mitochondria were energy-depleted. Martensson et al. (1990) identified a two-component transport process for GSH into rat liver mitochondria, one with a high-affinity, low-capacity and one with a low-affinity, high-capacity. The low-affinity component was stimulated by ATP and ADP and both components were inhibited by a protonophore, glutamate and ophthalmic acid. Because these investigators also used fasted rats, mitochondria were de-energized, which may explain the apparent pH dependence and effect of adenine nucleotides. In our study, fed rats and energized mitochondria were used, which likely accounts for the absence of pH dependence and phosphate dependence only at low phosphate concentrations. Our observation of inhibition of GSH uptake by oligomycin in the presence of ATP is consistent with the effect of exogenous adenine nucleotides observed by Martensson et al. (1990). The inhibition by oligomycin in the presence of ATP is consistent with a dependence on protonmotive force that is produced by ATP hydrolysis.

Kaplowitz and colleagues (Fernandez-Checa et al., 1991; Garcia-Ruiz et al., 1995) have also studied GSH transport in rat liver mitochondria. In their work, they have demonstrated that the mitochondrial transporter is distinct from that present in either the canalicular or sinusoidal plasma membranes. The uptake of GSH in mitochondria from Xenopus laevis oocytes, that were microinjected with total liver mRNA to express the GSH transport activity of rat liver mitochondria, was stimulated by ATP and inhibited by glutamate. The possible interaction of GSH transport with that of other anions, however, was not investigated.

Hence, this is the first study to demonstrate a role for dicarboxylate transporters (both the dicarboxylate and 2-oxoglutarate carriers) in the uptake of GSH in mitochondria from any tissue. It may also be possible that an additional, as yet unidentified, carrier may also be involved. When differences in energetic status of mitochondria are considered, the results from this study in mitochondria isolated from rat kidney appear to agree with those from rabbit kidney and rat liver, although the specific role of anion carriers in those models has not been studied. Thus, the transport mechanism for GSH described in kidney mitochondria may also apply to mitochondria from other tissues, including the liver. The function of the dicarboxylate and 2-oxoglutarate transporters in uptake of GSH implies that differences in nutritional or metabolic state may modulate mitochondrial GSH status.