Renal mitochondrial glutathione transport

Life Sciences, Vol. 49, pp. 393-398 Printed in the U.S.A.

Pergamon Press

RENAL MITOCHONDRIALGLUTATHIONETRANSPORT Rick G. Schnellmann Department of Physiology and Pharmacology, College of Veterinary Medicine University of Georgia, Athens, GA 30602 (Received in final form May 29, 1991)

Summary Freshly isolated t i g h t l y coupled rabbit renal cortical mitochondria rapidly accumulated glutathione (GSH) against an e l e c t r i cal and concentration gradient, and in the presence and absence of pyruvate/malate, succinate, antimycin A, or FCCP. Mitochondrial GSH uptake was dependent on medium GSH concentration, was not saturable, and reached equilibrium within 1 min of addition. Mitochondrial GSH uptake was p a r t i a l l y inhibited by glycine, ophthalmic acid, and serine but not glutamate, cysteine, ?-glutamyl-glutamate, or proline. These results show that 1) mitochondrial GSH uptake is by both a carrier-mediated process and by diffusion, and 2) the GSH carrier system has structural specificity with the glycine residue being a recognition site. Glutathione (GSH) is found in high concentrations (mM) in many organs of the body including the kidney, heart and l i v e r (1,2). Two important cellular functions of GSH are the detoxification of reactive oxygen species under normal and abnormal conditions and the detoxification of xenobiotics ( I ) . GSH is located in two subcellular compartments, cytosol and mitochondria (3). iFor example, rabbit renal cortex contains I~-14 nmol GSH'mg cortical protein- i of which 1.9 nmol GSH'mg tubular protein-lis located in the mitochondria (4,5). Rabbit renal proximal tubule suspensions contain approximately 18 nmol GSH'mg tubular protein -I (6). Thus, the mitochondrial and cytosolic compartments of rabbit renal proximal tubules contain I0-]5% and 85-90%, respectively, of the cellular GSH. The source of mitochondrial GSH was unknown until G r i f f i t h and Meister (7) demonstrated that rat l i v e r mitochondria did not have the enzymes required for the synthesis of GSH and thereby suggested that GSH must be transported into mitochondria. The goal of this study was to examine the transport of GSH into rabbit renal cortical mitochondria (RCM) and compare these results with our previous studies using rabbit renal proximal tubules (4). Methods Tightly coupled washed RCM w e r e isolated from New Zealand White rabbits as previously described (8) and resuspended in 0.27 M sucrose. Mitochondria (2 mg protein'ml -I) were suspended in a buffer consisting of 130 mM KCI, 9 mM Tris-PO4, 4 mM Tris-HCl, I mM EGTA (pH 7.4). Respiratory substrates were either pyruvate/malate (5/5 mM) or succinate/rotenone o (10 mM~lO pM). Mitochondrial suspensions were oxygenated and warmed to 22 or 37 C for two min prior to any additions. WhenGSH was added to mitochondrial suspensions, i t was dissolved in incubation medium immediately prior to 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc

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Concentrations of GSH greater than I mMwere adjusted to pH 7.4.

At the indicated sample times, 0.5 ml of the mitochondrial suspension was quickly centrifuged (16,000 x g) through 0.4 ml of butyl-dioctyl phthalate (2:1) into 0.2 ml of 5 percent meta-phosphoric acid in 100 mM phosphate - 5 mM EDTA buffer. The samples were frozen at -800 C. until analyzed. Extraparticulate water or that water carried down with the mitochondria and matrix water were determined as previously.described (9), and were found to be 1.9 and 1.1 ~l'mg mitochondrial protein -I , respectively. Mitochondrial GSH content was corrected for extraparticulate GSH. GSH was determined by the method of Hissin and H i l l (10) and periodically validated using the method of Griffith (11). Protein content was determined as previously described (12). The data are presented as the mean ± SE. Each mitochondrial isolation represented an N of I. Data were analyzed by ANOVA or Student's t - t e s t . Multiple means were tested for significance using Fisher's protected LSD test. A P value of less than 0.05 was considered significant. ?-Glutamyl-glutamate and ophthalmic acid (?-L-glutamyl-L-e-aminobutyrylglycine) were obtained from Aldrich Chemical Co. (Milwaukee, WI) and Bachem Fine Chemicals (Torrance, CA), respectively. The remaining biochemicals used in this study were purchased from Sigma Chemical Co. (St. Lguis, MO). Radiola~ l e d 3H20 (I mCi'gram-I). [U-T4C]sucrose (671 mCi'mmol-~), and [carboxylC]dextran (0.58 mC1"gram ) were obtalned from DuPont Co. (Wilmington, DE). Results Freshly isolated t i g h t l y coupled rabbit RCM have a GSH concentration of 23 nmol'mg protein -~ or approximately 2-3 mM (Figure I ) . Mitochondria respiring on pyruvate/malate rapidly accumulated GSH from the buffer against an e l e c t r i cal gradient (Figure I ) . The shortest time point that could be examined in this study was 15 sec; thus the rate of mitochondrial uptake of GSH from a Ted!um.containing 5 mM GSH was greater than 30 ± 3 and 38 ± 3 nmol'mg protein•mln- l at 220 and 370 C., respectively. These rates are not s i g n i f i c a n t l y different from one another. Mitochondria incubated at 570 C. exhibited maximum uptake of GSH after 0.25 min of e,xposure (9.5 nmol'mg-1) then decreased after 0.5 min of exposure (8.1 nmol'mg-~) and remained at this level for the remaining 4.5 m i n . The 'overshoot' phenomenonwas not observed when mitochondria were incubated with GSH at 220 C. Using medium GSH concentrations similar to those found in tubules under various conditions (4,6), mitochondria exhibited a non-saturable increase in GSH content (Figure I ) . Mitochondrial GSH uptake was dependent on medium GSH concentrations with medium GSH concentrations of 1,2,5, and 10 mM resulting in mitochondrial GSH concentrations of 3.8, 4.8, 9, and 19.9 mM. MediumGSH concentrations less than 0.5 mM had minimal effects on mitochondrial GSH content (data not shown). To determine i f electron flow through the electron transport chain or a proton gradient was necessary for mitochondrial GSH uptake, mitochondrial GSH uptake was examined in the absence or presence of pyruvate/malate, succinate, the site 2 electron transport inhibitor antimycin A, or the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). Mitochondrial GSH uptake was unaffected by the metabolic substrate present or absent, electron flow or a proton gradient (Figure 2). To examine any specificity in the uptake of GSH by mitochondria, mitochondrial GSH uptake was determined in the presence of varying amino acids and other ?-glutamyl compounds. Glycine, ophthalmic acid, and serine decreased mitochondrial GSH uptake 27, 18, and 10 percent, respectively (Figure 2). Glutamate, ?-glutamyl-glutamate, cysteine, or proline had no effect on mito-

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FIG. l The effect of time, temperature and medium GSH concentration on mitochondrial GSH content. MediumGSH concentration was 5 mM (upper panel) and time of exposure was 0.25 min (lower panel). Datum points (N=3-5) with different letters are significantly different from one another (P ~ 0.05). chondrial GSH uptake. Discussion Assuming an i n t r a c e l l u l a r water content of 2.4 #l'mg tubular protein -1 (13) and a tubular GSH content of 13-18 nmol'mg tubular p r o t e i n " (5,6), the concentration of GSH in rabbit renal proximal tubules is approximately 5.5 7.5 mM._ Since the mitochondrial GSH pool in tubules is 1.9 nmol'mg tubular protein -I (4) and approximately 24 percent tubular protein is mitochondrial prote~n (14), the mitochondrial GSH content is 7.9 nmol-mg mitochondrial prot e i n - ' . Using the experimentally determined mitochondrial matrix volume of 1.1 #l'mg mitochondrial protein" , the concentration of GSH in mitochondria is 7.2 mM. Thus, there is not a significant difference in the concentration of GSH between the cytosol and the mitochondria in rabbit renal proximal tubules. The mitochondrial

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FIG. 2 The effect of no substrate (control), pyruvate/malate, succinate, I #M antimycin A, and 1 #M FCCP on mitochondrial GSH uptake after a 2 min exposure (upper panel). The presence of GSH was the only parameter that significantly (P < 0.05) altered mitochondrial GSH content (none < I < 5 mM GSH). The effect of 5 mM glycine (GLY), glutamate (GLU), cysteine (CYS), ?-glutamyl-glutamate (GGG), ophthalmic a c i d (OPH), serine (SER), or proline (PRO) on the mitochondrial uptake of I mM GSH after a 2 min exposure (lower panel). The compounds were added 0.3 min prior to the GSH. *, Significantly different from GSH alone (N=3-5, P ~ 0.05). gradient suggests a GSH transport/exchange system predominates when medium GSH concentrations are low, while the non-saturability of GSH uptake suggests a diffusional process may predominate when medium GSH concentrations are high. The fact that mitochondrial GSH uptake is not an energy dependent process nor requires a proton gradient is consistent with the presence of a mitochondrial GSH exchange system. The mitochondrial GSH uptake system exhibited some structural specificity. Glycine, ophthalmic acid, and serine decreased mitochondrial GSH uptake while glutamate, ?-glutamyl-glutamate, cysteine, or proline had no effect. These results suggest that the glycine moiety is an important recognition site of

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mitochondrial GSH transport system.

The mitochondrial GSH transport system may be related to the mitochondrial glycine transport system reported to be present in spinach leaf mitochondria (15), since both systems are p a r t i a l l y inhibitable by serine but not proline. Interestingly, mitochondrial glycine uptake in spinach and pea leaf mitochondria (15,16), and brain and l i v e r mitochondria (17) also occurs by both a carrier-mediated process and diffusion. The mitochondrial GSH transport system, however, does not appear to be related to the GSH transport system located on the basolateral membrane of the renal proximal tubule since the ?-glutamyl moiety appears to be important in plasma membrane GSH transport (18). Recently, the transport of GSH by rat l i v e r mitochondria was reported (19,20). While the uptake of GSH did not appear to be saturable in both studies, the uptake was much slower (min) in the work of Kurosawa et al. (19) than the work of Martensson et al. (20) (sec) and shown here (sec). Martensson et al. ( 2 0 ) provided evidence of h i g h - a f f i n i t y and low-affinity GSH uptake by mitochondria. The h i g h - a f f i n i t y uptake was consistent with a carrier mediated system and was p a r t i a l l y inhibitable by glutamate and ophthalmic acid. Our results are consistent with a h i g h - a f f i n i t y uptake system except glutamate had no effect on GSH uptake. The reason for the differences between this study and that of Martensson et al. (20) remain to be determined but may include species and/or organ differences. The low-affinity uptake of GSH by mitochondria is more consistent with a diffusional process. Finally, the fact that isolated RCM containing 25 percent of their normal GSH content were t i g h t l y coupled supports our previous observation that the cytosolic GSH pool can be depleted and the mitochondrial GSH p o o l severely decreased in rabbit renal proximal tubules before any adverse effect on tubular mitochondrial function or v i a b i l i t y occurs. ACKNOWLEDGEMENTS This work was supported (in part) by the American Heart Association, Georgia A f f i l i a t e . The author would like to thank Dr. Dean P. Jones for his helpful comments and Theresa J. Cross for her technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

A. MEISTERand M.E. ANDERSON, Ann. Rev. Biochem. 5__22711-760 (1983). A. MEISTER, O.W. GRIFFITH, A NOVOGRODSKY, and S.S. TATE, Excerpta Medica, Ciba Found. Symp. 7__22135-161 (1980). F.J. ROMEROand D. GALARIS, Glutathione Metabolism and Physioloqical Functions, J. Vina (ed.), 29-38, CRC Press, Boca Raton (1990). R.G. SCHNELLMANN, S.M. GILCHRIST, and L.J. MANDEL, Kidney Int. 34 229-233 (1988). R.G. SCHNELLMANN. E.A. LOCK, and L.J. MANDEL, Toxicol. Appl. Pharmacol. 90 513-521 (1987). D.P. RODEHEAVER, M.D. ALEO, and R.G. SCHNELLMANN, In Vitro Cell Dev. Biol. 26898-904 (1990). O.W. GRIFFITH and A. MEISTER, Proc. Natl. Acad. Sci. 8__224668-4672 (1985). R.G. SCHNELLMANN, T.J. CROSS, and E.A. LOCK, Toxicol. Appl. Pharmacol. IO0 498-505 (1989). N.S. COHEN, C.-W. CHEUNGand L. RAIJMAN, Biochem. J. 245375-379 (1987). P.J. HISSIN and R. HILF, Anal. Biochem. _74214-226 (1976). O.W. GRIFFITH, Anal. Biochem. 106207-212 (1980). A. GORNALL, C. BARDNILL and M.M. DAVID, J. Biol. Chem. 177751-766 (1949). S.P. SOLTOFFand L.J. MANDEL, J. Gen. Physiol. 84601-622 (1984).

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14. S.I. HARRIS, R.S. BALABAN, L. BARRETT and L.J. MANDEL, J. Biol. Chem. 256 10319-10328 (1981). 15. C. YU, D.L. CLAYBROOKand A.H.C. HUANG, Arch. Biochem. Biophys. 227 180-187 (1983). 16. M.O. PROUDLOVEand A.L. MOORE, FEBS Lett. ]47 26-30 (1982). 17. J. BENAVIDES, M.L. GARCIA, J. LOPEZ-LAHOYA, M. UGARTE, and F. VALDIVIESO, Biochim. Biophys. Acta 598 588-594 (1980). 18. L.H. LASH and D.P. JONES, J. Biol. Chem. 259 14508-14514 (1984). 19. K. KUROSAWA, N. HAYASHI, N. SATO, T. KAMADA, and K. TAGAWA, Biochem. Bio phys. Res. Commun. 167 367-372 (1990). 20. J. MARTENSSON, J.C.K. LAI, and A. MEISTER, Proc. Natl. Acad. Sci. 8_! 71857189 (1990).