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Ryanodine Receptor Channel-Dependent Glutathione Transport in the Sarcoplasmic Reticulum of Skeletal Muscle
Biochemical and Biophysical Research Communications 287, 696 –700 (2001) doi:10.1006/bbrc.2001.5648, available online at http://www.idealibrary.com on
Ryanodine Receptor Channel-Dependent Glutathione Transport in the Sarcoplasmic Reticulum of Skeletal Muscle Miklo´s Csala,* Rosella Fulceri,† Jo´zsef Mandl,* Angelo Benedetti,† ,1 and Ga´bor Ba´nhegyi* *Department of Medical Chemistry, Pathobiochemistry and Molecular Biology, Semmelweis University, P.O. Box 260, H-1444, Budapest, Hungary; and †Dipartimento di Fisiopatologia e Medicina Sperimentale, Universita` di Siena, 53100 Siena, Italy
Received August 21, 2001
We found that glutathione transport across endo/ sarcoplasmic reticulum membranes correlates with the abundance of ryanodine receptor type 1 (RyR1). The transport was the fastest in muscle terminal cisternae, fast in muscle microsomes and slow in liver, heart, and brain microsomes. Glutathione influx could be inhibited by RyR1 blockers and the inhibitory effect was counteracted by RyR1 agonists. The effect of blockers was specific to glutathione, as the transport of other small molecules was not hindered. Therefore, the glutathione transport activity seems to be associated with RyR1 in sarcoplasmic reticulum. © 2001 Academic Press
Variations in intracellular Ca 2⫹ concentrations represent one of the most important second messenger pathways in living organisms. A major mechanism for increasing cytosolic Ca 2⫹ is the release of Ca 2⫹ from internal stores (endoplasmic or sarcoplasmic reticulum, ER or SR) via the members of a superfamily of intracellular calcium-release channels including ryanodine receptors (RyR) (1). Hypersensitive thiols of RyRs are subjects of oxidoreduction, which cause the activation or inhibition of Ca 2⫹ release (2–5). Generally speaking, thiol oxidation by reactive oxygen species, glutathione disulfide (GSSG), and other thiol reagents activate, while reducing agents, such as glutathione (GSH), dithiothreitol and mercaptoethanol, inhibit the channel. RyR type 1 (RyR1) from skeletal muscle can Abbreviations used: RyR, ryanodine receptor; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; GSH, reduced glutathione; GSSG, glutathione disulfide; Mops, 4-morpholinepropanesulfonic acid. 1 To whom correspondence should be addressed at Dipartimento di Fisiopatologia e Medicina Sperimentale, Viale A. Moro n°1, 53100 Siena, Italy. Fax: ⫹0577 234009. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
function as a transmembrane redox sensor (6). A large transmembrane redox potential inhibits, while dissipation of this potential activates the channel. Therefore, it is logical to consider that GSH/GSSG transport across the SR membrane is involved in regulating the local redox potential gradient necessary for the redox regulation of RyR1. GSH and GSSG constitute the most important redox buffer of animal cells both in the cytosol and in organelles. The transport of glutathione through endomembranes is a less explored field of cell biology. Its importance is especially evident in the regulation of particular redox potentials found in each intracellular compartment. In a typical mammalian cell, the ratio of [GSH]/[GSSG] in the cytosol is 30 –100:1 resulting in a redox potential of about ⫺230 mV. The lumen of ER is more oxidized (⫺180 mV) with a 1–3:1 ratio of [GSH]/ [GSSG] (7). The tightly controlled redox potential is essential, not only for the regulation of the RyR1 calcium channel in the SR of skeletal muscle, but also in the oxidative folding of secretory and membrane proteins in the ER lumen in liver and other secretory organs (8 –10). We have reported that GSH is transported through the membrane of hepatic ER at a relatively slow rate, while the membrane is practically impermeable toward GSSG (11). Recent data showed that both compounds could permeate the membrane of SR vesicles from skeletal muscle, although with different velocity (6). Here we report evidence for the involvement of RyR1 in GSH/GSSG transport across the SR membrane of skeletal muscle. We observed that the initial rate of GSH and GSSG transport is higher in terminal cysternae vesicles, which have a higher relative abundance of RyR1. As well, activators and inhibitors of the RyR1 calcium channel increase or decrease, respectively, the rate of transport. We suggest that RyR1 may behave as a glutathione transporter on its own, or alternatively
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directly interact with a putative GSH/GSSG transporter(s). MATERIALS AND METHODS Preparation of SR and ER vesicles. Microsomal vesicles were prepared from skeletal muscle, liver, brain, and heart of New Zealand White rabbits (Mu¨llner & Sons, Szilasliget, Hungary). SR membrane vesicles (total microsomal and purified terminal cisternae fractions) were prepared from the dominantly white hind limb skeletal muscles according to the method of Saito et al. (12). Cardiac SR vesicles were isolated by the same procedure. Liver and brain ER vesicles were prepared as described earlier for rat liver (11). Intactness of the vesicles was assessed by light scattering method (see below) using nonpermeant compounds (i.e., sucrose, maltose, UDPglucuronate). In the case of liver microsomes, membrane permeability was also confirmed by estimating the latency of the intravesicular enzyme UDP-glucuronosyltransferase, which was higher than 95% (13). The integrity of SR vesicles was also assessed on the basis of their ATP-dependent Ca 2⫹ accumulation according to (14). An almost complete calcium release was observed in terminal cisternae vesicles upon caffeine addition, indicating the purity of the fraction (14). Microsomal preparations were frozen and maintained in liquid N 2 until used. Transport measurements by rapid filtration method. Rapid filtration experiments were executed as described in detail earlier (15, 16). Briefly, microsomal vesicles (1 mg protein/ml) were incubated in a buffer containing 100 mM KCl, 20 mM NaCl, 1 mM MgCl 2, 20 mM Mops, 1 mM GSH, and its radiolabeled analogue [ 3H]GSH (10 Ci/ ml) at 37°C. At the indicated times, vesicles were filtered through cellulose acetate-nitrate filter membranes (pore size 0.22 m) and washed quickly on the filter with the same buffer containing 1 mM flufenamic acid, the inhibitor of GSH transport in ER (11) and SR (6) vesicles. The radioactivity retained on the filter was measured by liquid scintillation. Alamethicin (50 g/mg protein) was included in parallel incubates to distinguish the intravesicular and the bound radioactivity. Alamethicin, a pore-forming antibiotic, makes the microsomal vesicles permeable toward various hydrophilic compounds such as UDP-glucuronate (13), sucrose, glucose-6-phosphate (15), GSH, and GSSG (11). The alamethicin-treated vesicles were recovered on filters and washed as above. More than 95% of the microsomal proteins was retained by the filters, indicating that alamethicin treatment did not affect the vesicular structure of microsomes as reported in (13). The alamethicin-releasable portion of radioactivity (assumed as intravesicular) was calculated by subtraction. Transport measurements by the light scattering technique. Osmotically-induced changes in microsomal vesicle size and shape (17) were monitored at 400 nm at right angles to the incoming light beam, using a fluorimeter (Hitachi F-4500) equipped with a temperature-controlled cuvette holder (37°C) and magnetic stirrer. SR or ER vesicles (50 g/ml protein) were equilibrated for 2 h in a hypotonic medium (5 mM K-Pipes, pH 7.0). The osmotically-induced changes in light scattering were measured after the addition of a small volume (⬍5%) of the total incubation volume of concentrated and neutralized solutions of the compounds to be tested as described in detail elsewhere (16). Materials. GSH, GSSG, ruthenium red, ryanodine, ATP, ADP, AMP, oleoyl-CoA were from Sigma (St. Louis, MO). [ 3H]GSH was from NEN Life Science Products, Inc. (Boston, MA). All other chemicals were of analytical grade.
RESULTS In the first set of experiments, the transport of radiolabeled GSH was measured in liver and skeletal
FIG. 1. Transport of GSH into liver and muscle microsomes detected by the rapid filtration method. Microsomal vesicles (1 mg protein/ml) were incubated in the presence of 3 mM GSH and tracer amounts of [ 3H]glutathione (10 Ci/ml) as described under Materials and Methods. Microsomal vesicles permeabilized with alamethicin (50 g/mg protein) were incubated in parallel experiments to evaluate radioactivity bound to microsomal membranes. At the indicated time points aliquots were withdrawn to measure 3H associated with the microsomes. The alamethicin-releasable portions of radioactivity (regarded as intravesicular) calculated by subtraction are shown. Data are means ⫾ S.E.M. of 4 – 6 measurements. (Œ) liver microsomes; (■) muscle microsomes.
muscle microsomes by a rapid filtration technique. The radioactivity associated with microsomes was measured in vesicles incubated both in the presence and absence of the pore-forming antibiotic, alamethicin to determine net intravesicular accumulation. The radioactivity associated to alamethicin-treated vesicles can be attributed to the binding of glutathione to the membrane or proteins. Alamethicinpermeabilized microsomes retained amounts of radioactivity less than 20% of that associated to untreated microsomes. Intravesicular GSH content was calculated as the alamethicin-releasable portion of the total radioactivity associated with the vesicles. The initial rates, time course and steady-state level of GSH uptake in liver microsomes were similar to our previous observations gained by an alternative method (11). Both the extent and the rate of GSH uptake were significantly higher in muscle microsomes than in hepatic microsomes (Fig. 1). The results gained by rapid filtration experiments might underestimate the rate of transport due to the unavoidable efflux of the investigated compound during the washing procedure. This discrepancy is obviously larger in case of a faster transport process. Therefore, the results were confirmed by permeability measurements using the light scattering method,
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FIG. 2. Transport of GSH and GSSG into liver, heart, brain, and muscle microsomes, and the terminal cisternae fraction detected by the light scattering method. Vesicles (50 g protein/ml) were preequilibrated in a hyposmotic buffer. Osmotically-induced changes of light scattering following the addition of GSH (25 mM, a) or of GSSG (25 mM, b) were measured as described under Materials and Methods. The osmolytes were added (arrow) to liver microsomes (L), heart microsomes (H), brain microsomes (B), to total muscle microsomal fraction (M), or to purified terminal cisternae fraction (TC). Representative traces are shown of 6 –10 similar experiments on three different microsomal preparations.
which permits the real-time detection of the transport. The method is based on the detection of osmotic shrinkage and swelling of microsomal vesicles (17, 13–16). Addition of nonpermeable osmolytes causes a permanent shrinking of vesicles leading to a sustained increase in the light scattering signal. Permeant compounds cause a transient shrinking followed by a swelling phase as reflected by a gradual decrease in the light scattering signal. With highly permeant compounds, the transient shrinking phase may be small or even absent because of the very rapid equilibration of the compounds. In accordance with earlier observations (13–17), neither liver nor muscle microsomes nor isolated SR terminal cisternae vesicles were permeable to sucrose, maltose or UDP-glucuronic acid while lower molecular weight compounds (glucose or KCl) rapidly entered the vesicles (data not shown). These observations as well, confirm the integrity of the vesicle membranes. Addition of GSH or GSSG (6.25–25 mM) to liver microsomal vesicles leads to sustained increases in light scattering—the membrane is poorly permeable to them (Fig. 2, traces L). Similar results were obtained with heart (Fig. 2, traces H) and brain microsomes (Fig. 2, traces B). In contrast, the addition of GSH or GSSG to SR vesicles hardly caused a shrinking phase indicating that both compounds crossed the membrane of SR vesicles rapidly (Fig. 2, traces M), in agreement with the results of Feng et al. (6). Since the intra- and extravesicular concentrations of other components of the incubation medium had been equilibrated during a 2-h preincubation, the osmotically induced changes must
be attributed to the movement of GSH or GSSG. Even higher permeability was observed in purified terminal cisternae—GSH and GSSG entered these vesicles instantly (Fig. 2, traces TC). The permeability of the SR membranes was specific to GSH and GSSG: hydrophilic molecules of similar size to GSH/GSSG, such as sucrose, maltose, and maltotetraose, did not enter muscle microsomal or terminal cisternae vesicles (data not shown). The permeability of the SR membrane toward GSH or GSSG was independent of the redox conditions: the addition of their various mixtures (GSH/GSSG from 50:1 to 1:1, 6.25 mM total concentration) resulted in similar light scattering traces (data not shown). Skeletal muscle microsomes and especially the subfraction enriched in terminal cisternae contain the RyR type 1. On the other hand, liver, heart, and brain microsomes do not express this RyR isoform (1). The above results indicate therefore a correlation between a high rate of GSH/GSSG transport and the presence of RyR1. In further experiments we studied the effect of RyR inhibitors (18, 19) on SR permeability to GSH and GSSG. When muscle microsomes or terminal cisternae vesicles were incubated in the presence of 1–5 mM MgCl 2, glutathione (GSH or GSSG) influx was slower and its time course became similar to the transport observed in hepatic microsomes (Fig. 3). The possible role of Cl ⫺ ions was ruled out by the addition of 2–10 mM KCl, which did not influence the permeability of the membranes to glutathione (data not shown). Similarly to Mg 2⫹, addition of 2 M ruthenium red to the SR vesicles caused a dramatic inhibition of glutathione transport (Fig. 3). Ryanodine was also inhibitory in
FIG. 3. Effect of RyR antagonists on GSH and GSSG transport. The effect of Mg 2⫹ ions (Mg) ryanodine (Ry) and ruthenium red (RR) on GSH (a and c) or GSSG (b and d) transport was studied by the light scattering technique in total muscle microsomal fraction (a and b) and in a purified terminal cisternae fraction (c and d). MgCl 2 (1 mM) was present in the hyposmotic buffer during the equilibration. Ryanodine (200 M) and ruthenium red (2 M) were added 2 min before GSH or GSSG. Representative traces are shown of 6 –10 similar experiments on three different microsomal preparations.
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DISCUSSION
FIG. 4. Effect of RyR agonists on GSH and GSSG transport. Total muscle microsomal vesicles were preincubated in hyposmotic buffer containing 1 mM MgCl 2. GSH (a) or GSSG (b) transport was studied by the light-scattering technique. AMP, ADP, or ATP (2 mM of each) or oleoyl-CoA (5 M) were added 2 min before GSH or GSSG. Representative traces are shown of 6 –10 similar experiments on three different microsomal preparations.
micromolar concentrations; maximal effect was reached at 200 M (Fig. 3). None of these agonists influenced the permeability of the membranes to glutamate, cysteine, or to other small permeant compounds (i.e., glucose or phosphate) (data not shown). In a final set of experiments the effect of RyR channel activators on the glutathione transport in muscle microsomes was investigated. RyR1 calcium channels can be activated by a variety of compounds including caffeine, adenine nucleotides, and fatty acyl CoA esters (see 20 and 21 and references therein). Moreover, fatty acyl CoA esters have been shown to counter the inhibitory effect of Mg 2⫹ ions on RyR activity (21). Under the present experimental conditions, transport of GSH or GSSG in SR vesicles is already so rapid that its increase would be hardly measurable. Therefore, muscle microsomes were preincubated in a buffer containing MgCl 2 (1 mM) to maintain the transport of GSH or GSSG at slow rates (see Fig. 3). Administration of adenine nucleotides (AMP, ADP, or ATP) or oleoyl-CoA increased the influx rate of both compounds (Fig. 4) but did not affect the permeability of the membranes to the other small compounds mentioned above (data not shown). Maximal stimulatory effect was observed at 2 mM in the case of adenine nucleotides and at 5 M in case of oleoyl-CoA (Fig. 4). Addition of sucrose or maltose after these agents resulted in a sustained light scattering signal indicating that they did not permeabilize the membrane (data not shown). The effect of caffeine cannot be clearly evaluated by the light scattering assay. Addition of caffeine (10 mM) caused, in fact, increases of both the signal and the background noise, possibly because the drug caused shrinking and/or aggregation of microsomal vesicles.
As both reduced and oxidized forms of glutathione are hydrophilic and charged molecules, they require transporter proteins to cross the ER or SR membranes. The nature of these transporters is not yet clear. However, it is clear that the transport activity for glutathione is different in liver ER compared to muscle SR. In liver ER, only the reduced form of glutathione crosses the membrane (11). In muscle, both GSH and GSSG can permeate the SR membrane [6, and the present study]. Moreover, the transport of GSH appears to be lower in liver ER than in muscle SR. The initial rate of radiolabeled GSH uptake was at least fourfold lower in liver microsomes (Fig. 1, earlier incubation time). In addition, the radiolabeled GSH taken up by liver microsomes is likely to be oxidized to GSSG and retained in this form inside the vesicles (11). We have not yet evaluated the possible retention of GSSG by muscle microsomes, since it was not relevant in the present work. In muscle SR, however, the high permeability to GSSG suggests that it does not occur. Consistently, light-scattering measurements reveal that GSH transport is much more rapid in muscle total SR vesicles— and even more in the subfraction enriched in RyR channels—than in liver microsomes. Moreover, lightscattering measurements reveal that little or no GSH/ GSSG transport occurs in brain and heart microsomes; these are known to possess almost exclusively RyR channels other than the skeletal muscle isoform RyR1. It therefore appears that rapid transport of GSH/GSSG is restricted to ER/SR membranes enriched in RyR1. In skeletal muscle SR, it has been suggested that the GSH/GSSG permeability is in a functional relationship with the RyR channel in that it may contribute to the redox control of RyR channel-mediated Ca 2⫹ fluxes (6). Our present results indicate that the RyR activity can in turn directly control GSH and GSSG transport across the SR membrane. Testamental to this is that the transport of GSH and GSSG can be inhibited or activated by well-known inhibitors and activators of RyR1, respectively. This phenomenon appears to be specific for GSH and GSSG since the transport of other permeants in the SR membranes was unaffected by inhibitors/activators of RyR1. The function of the RyR channel in the generation of the Ca 2⫹ signal during muscle contraction is well known. On the basis of our findings it is likely that passive GSH and GSSG fluxes are activated simultaneously with the release of intraluminal Ca 2⫹ from the SR. The glutathione redox gradient between the cytosol and the lumen of the SR is allowed to equilibrate (at least partly). This phenomenon could play a role in altering the redox state of GSH and protein thiols of the skeletal muscle in contraction-induced injury (22). Since the disappearance of the transmembrane redox gradient favors the open state of the calcium channel
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(6), the RyR1-dependent glutathione permeation may promote the mobilization of calcium in skeletal muscle. As to the molecular nature of the skeletal muscle SR transporter of GSH and GSSG, it can be speculated that it is the RyR1 itself. In particular, another channel can be formed by the supramolecular arrangement (23) of open RyR1s in the presence of agonists allowing the permeation of glutathione. Alternatively, RyR1 may be tightly coupled to the GSH/GSSG transport protein, and the activity of RyR1 channel is directly transduced to the transporter resulting in a degree of coregulation. This possibility is not unprecedented, since protein–protein interactions between the RyR1 and the voltage-sensitive dihydropyridine receptor of the T tubule most likely transduce the activation of the latter to the former. Further work is needed to clarify these possibilities. ACKNOWLEDGMENTS This work was supported by OTKA (National Scientific Research Fund) Grant T32873, a Hungarian Academy of Sciences Grant, a NATO linkage grant and a Telethon Grant No. 10602 to R. Fulceri. M. Csala was a recipient of a FEBS Short-Term Fellowship and a NATO Advanced Fellowship to Siena. Thanks are due to Dr. Roberta Giunti for the methodological advice and Mrs. Vale´ria Mile for her skillful technical assistance. We thank Professor Fyfe Bygrave (Australian National University) for assistance in the preparation of the manuscript.
REFERENCES 1. Sorrentino, V., Barone, V., and Rossi, D. (2000) Curr. Opin. Gen. Dev. 10, 662– 667. 2. Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028 – 33034. 3. Zable, A. C., Favero, T. G., and Abramson, J. J. (1997) J. Biol. Chem. 272, 7069 –7077.
4. Xia, R., Stangler, T., and Abramson, J. J. (2000) J. Biol. Chem. 275, 36556 –36561. 5. Sun, J., Xu, L., Eu, J. P., Stamler, J. S., and Meissner, G. (2001) J. Biol. Chem. 276, 15625–15630. 6. Feng, W., Liu, G., Allen, P. D., and Pessah, I. N. (2000) J. Biol. Chem. 275, 35902–35907. 7. Hwang, C., Sinskey, A. J., and Lodish, H. F. (1992) Science 257, 1496 –1502. 8. Frand, A. R., Cuozzo, J. W., and Kaiser, C. A. (2000) Trends Cell Biol. 10, 203–210. 9. Tu, B. P., Ho-Schleyer, S. C., Travers, K. J., and Weissman, J. S. (2000) Science 290, 1571–1574. 10. Cuozzo, J. W., and Kaiser, C. A. (1999) Nat. Cell Biol. 1, 130 – 135. 11. Ba´nhegyi, G., Lusini, L., Puska´s, F., Rossi, R., Fulceri, R., Braun, L., Mile, V., di Simplicio, P., Mandl, J., and Benedetti, A. (1999) J. Biol. Chem. 274, 12213–12216. 12. Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875– 885. 13. Fulceri, R., Ba´nhegyi, G., Gamberucci, A., Giunti, R., Mandl, J., and Benedetti, A. (1994) Arch. Biochem. Biophys. 309, 43– 46. 14. Fulceri, R., Giunti, R., Knudsen, J., Leuzzi, R., Kardon, T., and Benedetti, A. (1999) Biochem. Biophys. Res. Commun. 264, 409 – 412. 15. Ba´nhegyi, G., Marcolongo, P., Fulceri, R., Hinds, C., Burchell, A., and Benedetti, A. (1997) J. Biol. Chem. 272, 13584 –13590. 16. Ba´nhegyi, G., Marcolongo, P., Puska´s, F., Fulceri, R., Mandl, J., and Benedetti, A. (1998) J. Biol. Chem. 273, 2758 –2762. 17. Meissner, G. (1988) Methods Enzymol. 157, 417– 437. 18. Xu, L, Tripathy, A, Pasek, D. A., and Meissner, G. (1998) Ann. NY Acad. Sci. 853, 130 –148. 19. Xu, L., Tripathy, A., Pasek, A. D., and Meissner, G. (1999) J. Biol. Chem. 274, 32680 –32691. 20. Meissner, G. (1994) Annu. Rev. Physiol. 56, 485–508. 21. Fulceri, R., Knudsen, J., Giunti, R., Volpe, P., Nori, A., and Benedetti, A. (1997) Biochem. J. 325, 423– 428. 22. McArdle, A., van der Meulen, J. H., Catapano, M., Symons, M. C., Faulkner, J. A., and Jackson, M. J. (1999) Free Radic. Biol. Med. 26, 1085–1091. 23. Yin, C. C., and Lai, F. A. (2000) Nat. Cell Biol. 2, 669 – 671.
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Ryanodine Receptor Channel-Dependent Glutathione Transport in the Sarcoplasmic Reticulum of Skeletal Muscle Miklo´s Csala,* Rosella Fulceri,† Jo´zsef Mandl,* Angelo Benedetti,† ,1 and Ga´bor Ba´nhegyi* *Department of Medical Chemistry, Pathobiochemistry and Molecular Biology, Semmelweis University, P.O. Box 260, H-1444, Budapest, Hungary; and †Dipartimento di Fisiopatologia e Medicina Sperimentale, Universita` di Siena, 53100 Siena, Italy
Received August 21, 2001
We found that glutathione transport across endo/ sarcoplasmic reticulum membranes correlates with the abundance of ryanodine receptor type 1 (RyR1). The transport was the fastest in muscle terminal cisternae, fast in muscle microsomes and slow in liver, heart, and brain microsomes. Glutathione influx could be inhibited by RyR1 blockers and the inhibitory effect was counteracted by RyR1 agonists. The effect of blockers was specific to glutathione, as the transport of other small molecules was not hindered. Therefore, the glutathione transport activity seems to be associated with RyR1 in sarcoplasmic reticulum. © 2001 Academic Press
Variations in intracellular Ca 2⫹ concentrations represent one of the most important second messenger pathways in living organisms. A major mechanism for increasing cytosolic Ca 2⫹ is the release of Ca 2⫹ from internal stores (endoplasmic or sarcoplasmic reticulum, ER or SR) via the members of a superfamily of intracellular calcium-release channels including ryanodine receptors (RyR) (1). Hypersensitive thiols of RyRs are subjects of oxidoreduction, which cause the activation or inhibition of Ca 2⫹ release (2–5). Generally speaking, thiol oxidation by reactive oxygen species, glutathione disulfide (GSSG), and other thiol reagents activate, while reducing agents, such as glutathione (GSH), dithiothreitol and mercaptoethanol, inhibit the channel. RyR type 1 (RyR1) from skeletal muscle can Abbreviations used: RyR, ryanodine receptor; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; GSH, reduced glutathione; GSSG, glutathione disulfide; Mops, 4-morpholinepropanesulfonic acid. 1 To whom correspondence should be addressed at Dipartimento di Fisiopatologia e Medicina Sperimentale, Viale A. Moro n°1, 53100 Siena, Italy. Fax: ⫹0577 234009. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
function as a transmembrane redox sensor (6). A large transmembrane redox potential inhibits, while dissipation of this potential activates the channel. Therefore, it is logical to consider that GSH/GSSG transport across the SR membrane is involved in regulating the local redox potential gradient necessary for the redox regulation of RyR1. GSH and GSSG constitute the most important redox buffer of animal cells both in the cytosol and in organelles. The transport of glutathione through endomembranes is a less explored field of cell biology. Its importance is especially evident in the regulation of particular redox potentials found in each intracellular compartment. In a typical mammalian cell, the ratio of [GSH]/[GSSG] in the cytosol is 30 –100:1 resulting in a redox potential of about ⫺230 mV. The lumen of ER is more oxidized (⫺180 mV) with a 1–3:1 ratio of [GSH]/ [GSSG] (7). The tightly controlled redox potential is essential, not only for the regulation of the RyR1 calcium channel in the SR of skeletal muscle, but also in the oxidative folding of secretory and membrane proteins in the ER lumen in liver and other secretory organs (8 –10). We have reported that GSH is transported through the membrane of hepatic ER at a relatively slow rate, while the membrane is practically impermeable toward GSSG (11). Recent data showed that both compounds could permeate the membrane of SR vesicles from skeletal muscle, although with different velocity (6). Here we report evidence for the involvement of RyR1 in GSH/GSSG transport across the SR membrane of skeletal muscle. We observed that the initial rate of GSH and GSSG transport is higher in terminal cysternae vesicles, which have a higher relative abundance of RyR1. As well, activators and inhibitors of the RyR1 calcium channel increase or decrease, respectively, the rate of transport. We suggest that RyR1 may behave as a glutathione transporter on its own, or alternatively
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directly interact with a putative GSH/GSSG transporter(s). MATERIALS AND METHODS Preparation of SR and ER vesicles. Microsomal vesicles were prepared from skeletal muscle, liver, brain, and heart of New Zealand White rabbits (Mu¨llner & Sons, Szilasliget, Hungary). SR membrane vesicles (total microsomal and purified terminal cisternae fractions) were prepared from the dominantly white hind limb skeletal muscles according to the method of Saito et al. (12). Cardiac SR vesicles were isolated by the same procedure. Liver and brain ER vesicles were prepared as described earlier for rat liver (11). Intactness of the vesicles was assessed by light scattering method (see below) using nonpermeant compounds (i.e., sucrose, maltose, UDPglucuronate). In the case of liver microsomes, membrane permeability was also confirmed by estimating the latency of the intravesicular enzyme UDP-glucuronosyltransferase, which was higher than 95% (13). The integrity of SR vesicles was also assessed on the basis of their ATP-dependent Ca 2⫹ accumulation according to (14). An almost complete calcium release was observed in terminal cisternae vesicles upon caffeine addition, indicating the purity of the fraction (14). Microsomal preparations were frozen and maintained in liquid N 2 until used. Transport measurements by rapid filtration method. Rapid filtration experiments were executed as described in detail earlier (15, 16). Briefly, microsomal vesicles (1 mg protein/ml) were incubated in a buffer containing 100 mM KCl, 20 mM NaCl, 1 mM MgCl 2, 20 mM Mops, 1 mM GSH, and its radiolabeled analogue [ 3H]GSH (10 Ci/ ml) at 37°C. At the indicated times, vesicles were filtered through cellulose acetate-nitrate filter membranes (pore size 0.22 m) and washed quickly on the filter with the same buffer containing 1 mM flufenamic acid, the inhibitor of GSH transport in ER (11) and SR (6) vesicles. The radioactivity retained on the filter was measured by liquid scintillation. Alamethicin (50 g/mg protein) was included in parallel incubates to distinguish the intravesicular and the bound radioactivity. Alamethicin, a pore-forming antibiotic, makes the microsomal vesicles permeable toward various hydrophilic compounds such as UDP-glucuronate (13), sucrose, glucose-6-phosphate (15), GSH, and GSSG (11). The alamethicin-treated vesicles were recovered on filters and washed as above. More than 95% of the microsomal proteins was retained by the filters, indicating that alamethicin treatment did not affect the vesicular structure of microsomes as reported in (13). The alamethicin-releasable portion of radioactivity (assumed as intravesicular) was calculated by subtraction. Transport measurements by the light scattering technique. Osmotically-induced changes in microsomal vesicle size and shape (17) were monitored at 400 nm at right angles to the incoming light beam, using a fluorimeter (Hitachi F-4500) equipped with a temperature-controlled cuvette holder (37°C) and magnetic stirrer. SR or ER vesicles (50 g/ml protein) were equilibrated for 2 h in a hypotonic medium (5 mM K-Pipes, pH 7.0). The osmotically-induced changes in light scattering were measured after the addition of a small volume (⬍5%) of the total incubation volume of concentrated and neutralized solutions of the compounds to be tested as described in detail elsewhere (16). Materials. GSH, GSSG, ruthenium red, ryanodine, ATP, ADP, AMP, oleoyl-CoA were from Sigma (St. Louis, MO). [ 3H]GSH was from NEN Life Science Products, Inc. (Boston, MA). All other chemicals were of analytical grade.
RESULTS In the first set of experiments, the transport of radiolabeled GSH was measured in liver and skeletal
FIG. 1. Transport of GSH into liver and muscle microsomes detected by the rapid filtration method. Microsomal vesicles (1 mg protein/ml) were incubated in the presence of 3 mM GSH and tracer amounts of [ 3H]glutathione (10 Ci/ml) as described under Materials and Methods. Microsomal vesicles permeabilized with alamethicin (50 g/mg protein) were incubated in parallel experiments to evaluate radioactivity bound to microsomal membranes. At the indicated time points aliquots were withdrawn to measure 3H associated with the microsomes. The alamethicin-releasable portions of radioactivity (regarded as intravesicular) calculated by subtraction are shown. Data are means ⫾ S.E.M. of 4 – 6 measurements. (Œ) liver microsomes; (■) muscle microsomes.
muscle microsomes by a rapid filtration technique. The radioactivity associated with microsomes was measured in vesicles incubated both in the presence and absence of the pore-forming antibiotic, alamethicin to determine net intravesicular accumulation. The radioactivity associated to alamethicin-treated vesicles can be attributed to the binding of glutathione to the membrane or proteins. Alamethicinpermeabilized microsomes retained amounts of radioactivity less than 20% of that associated to untreated microsomes. Intravesicular GSH content was calculated as the alamethicin-releasable portion of the total radioactivity associated with the vesicles. The initial rates, time course and steady-state level of GSH uptake in liver microsomes were similar to our previous observations gained by an alternative method (11). Both the extent and the rate of GSH uptake were significantly higher in muscle microsomes than in hepatic microsomes (Fig. 1). The results gained by rapid filtration experiments might underestimate the rate of transport due to the unavoidable efflux of the investigated compound during the washing procedure. This discrepancy is obviously larger in case of a faster transport process. Therefore, the results were confirmed by permeability measurements using the light scattering method,
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FIG. 2. Transport of GSH and GSSG into liver, heart, brain, and muscle microsomes, and the terminal cisternae fraction detected by the light scattering method. Vesicles (50 g protein/ml) were preequilibrated in a hyposmotic buffer. Osmotically-induced changes of light scattering following the addition of GSH (25 mM, a) or of GSSG (25 mM, b) were measured as described under Materials and Methods. The osmolytes were added (arrow) to liver microsomes (L), heart microsomes (H), brain microsomes (B), to total muscle microsomal fraction (M), or to purified terminal cisternae fraction (TC). Representative traces are shown of 6 –10 similar experiments on three different microsomal preparations.
which permits the real-time detection of the transport. The method is based on the detection of osmotic shrinkage and swelling of microsomal vesicles (17, 13–16). Addition of nonpermeable osmolytes causes a permanent shrinking of vesicles leading to a sustained increase in the light scattering signal. Permeant compounds cause a transient shrinking followed by a swelling phase as reflected by a gradual decrease in the light scattering signal. With highly permeant compounds, the transient shrinking phase may be small or even absent because of the very rapid equilibration of the compounds. In accordance with earlier observations (13–17), neither liver nor muscle microsomes nor isolated SR terminal cisternae vesicles were permeable to sucrose, maltose or UDP-glucuronic acid while lower molecular weight compounds (glucose or KCl) rapidly entered the vesicles (data not shown). These observations as well, confirm the integrity of the vesicle membranes. Addition of GSH or GSSG (6.25–25 mM) to liver microsomal vesicles leads to sustained increases in light scattering—the membrane is poorly permeable to them (Fig. 2, traces L). Similar results were obtained with heart (Fig. 2, traces H) and brain microsomes (Fig. 2, traces B). In contrast, the addition of GSH or GSSG to SR vesicles hardly caused a shrinking phase indicating that both compounds crossed the membrane of SR vesicles rapidly (Fig. 2, traces M), in agreement with the results of Feng et al. (6). Since the intra- and extravesicular concentrations of other components of the incubation medium had been equilibrated during a 2-h preincubation, the osmotically induced changes must
be attributed to the movement of GSH or GSSG. Even higher permeability was observed in purified terminal cisternae—GSH and GSSG entered these vesicles instantly (Fig. 2, traces TC). The permeability of the SR membranes was specific to GSH and GSSG: hydrophilic molecules of similar size to GSH/GSSG, such as sucrose, maltose, and maltotetraose, did not enter muscle microsomal or terminal cisternae vesicles (data not shown). The permeability of the SR membrane toward GSH or GSSG was independent of the redox conditions: the addition of their various mixtures (GSH/GSSG from 50:1 to 1:1, 6.25 mM total concentration) resulted in similar light scattering traces (data not shown). Skeletal muscle microsomes and especially the subfraction enriched in terminal cisternae contain the RyR type 1. On the other hand, liver, heart, and brain microsomes do not express this RyR isoform (1). The above results indicate therefore a correlation between a high rate of GSH/GSSG transport and the presence of RyR1. In further experiments we studied the effect of RyR inhibitors (18, 19) on SR permeability to GSH and GSSG. When muscle microsomes or terminal cisternae vesicles were incubated in the presence of 1–5 mM MgCl 2, glutathione (GSH or GSSG) influx was slower and its time course became similar to the transport observed in hepatic microsomes (Fig. 3). The possible role of Cl ⫺ ions was ruled out by the addition of 2–10 mM KCl, which did not influence the permeability of the membranes to glutathione (data not shown). Similarly to Mg 2⫹, addition of 2 M ruthenium red to the SR vesicles caused a dramatic inhibition of glutathione transport (Fig. 3). Ryanodine was also inhibitory in
FIG. 3. Effect of RyR antagonists on GSH and GSSG transport. The effect of Mg 2⫹ ions (Mg) ryanodine (Ry) and ruthenium red (RR) on GSH (a and c) or GSSG (b and d) transport was studied by the light scattering technique in total muscle microsomal fraction (a and b) and in a purified terminal cisternae fraction (c and d). MgCl 2 (1 mM) was present in the hyposmotic buffer during the equilibration. Ryanodine (200 M) and ruthenium red (2 M) were added 2 min before GSH or GSSG. Representative traces are shown of 6 –10 similar experiments on three different microsomal preparations.
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DISCUSSION
FIG. 4. Effect of RyR agonists on GSH and GSSG transport. Total muscle microsomal vesicles were preincubated in hyposmotic buffer containing 1 mM MgCl 2. GSH (a) or GSSG (b) transport was studied by the light-scattering technique. AMP, ADP, or ATP (2 mM of each) or oleoyl-CoA (5 M) were added 2 min before GSH or GSSG. Representative traces are shown of 6 –10 similar experiments on three different microsomal preparations.
micromolar concentrations; maximal effect was reached at 200 M (Fig. 3). None of these agonists influenced the permeability of the membranes to glutamate, cysteine, or to other small permeant compounds (i.e., glucose or phosphate) (data not shown). In a final set of experiments the effect of RyR channel activators on the glutathione transport in muscle microsomes was investigated. RyR1 calcium channels can be activated by a variety of compounds including caffeine, adenine nucleotides, and fatty acyl CoA esters (see 20 and 21 and references therein). Moreover, fatty acyl CoA esters have been shown to counter the inhibitory effect of Mg 2⫹ ions on RyR activity (21). Under the present experimental conditions, transport of GSH or GSSG in SR vesicles is already so rapid that its increase would be hardly measurable. Therefore, muscle microsomes were preincubated in a buffer containing MgCl 2 (1 mM) to maintain the transport of GSH or GSSG at slow rates (see Fig. 3). Administration of adenine nucleotides (AMP, ADP, or ATP) or oleoyl-CoA increased the influx rate of both compounds (Fig. 4) but did not affect the permeability of the membranes to the other small compounds mentioned above (data not shown). Maximal stimulatory effect was observed at 2 mM in the case of adenine nucleotides and at 5 M in case of oleoyl-CoA (Fig. 4). Addition of sucrose or maltose after these agents resulted in a sustained light scattering signal indicating that they did not permeabilize the membrane (data not shown). The effect of caffeine cannot be clearly evaluated by the light scattering assay. Addition of caffeine (10 mM) caused, in fact, increases of both the signal and the background noise, possibly because the drug caused shrinking and/or aggregation of microsomal vesicles.
As both reduced and oxidized forms of glutathione are hydrophilic and charged molecules, they require transporter proteins to cross the ER or SR membranes. The nature of these transporters is not yet clear. However, it is clear that the transport activity for glutathione is different in liver ER compared to muscle SR. In liver ER, only the reduced form of glutathione crosses the membrane (11). In muscle, both GSH and GSSG can permeate the SR membrane [6, and the present study]. Moreover, the transport of GSH appears to be lower in liver ER than in muscle SR. The initial rate of radiolabeled GSH uptake was at least fourfold lower in liver microsomes (Fig. 1, earlier incubation time). In addition, the radiolabeled GSH taken up by liver microsomes is likely to be oxidized to GSSG and retained in this form inside the vesicles (11). We have not yet evaluated the possible retention of GSSG by muscle microsomes, since it was not relevant in the present work. In muscle SR, however, the high permeability to GSSG suggests that it does not occur. Consistently, light-scattering measurements reveal that GSH transport is much more rapid in muscle total SR vesicles— and even more in the subfraction enriched in RyR channels—than in liver microsomes. Moreover, lightscattering measurements reveal that little or no GSH/ GSSG transport occurs in brain and heart microsomes; these are known to possess almost exclusively RyR channels other than the skeletal muscle isoform RyR1. It therefore appears that rapid transport of GSH/GSSG is restricted to ER/SR membranes enriched in RyR1. In skeletal muscle SR, it has been suggested that the GSH/GSSG permeability is in a functional relationship with the RyR channel in that it may contribute to the redox control of RyR channel-mediated Ca 2⫹ fluxes (6). Our present results indicate that the RyR activity can in turn directly control GSH and GSSG transport across the SR membrane. Testamental to this is that the transport of GSH and GSSG can be inhibited or activated by well-known inhibitors and activators of RyR1, respectively. This phenomenon appears to be specific for GSH and GSSG since the transport of other permeants in the SR membranes was unaffected by inhibitors/activators of RyR1. The function of the RyR channel in the generation of the Ca 2⫹ signal during muscle contraction is well known. On the basis of our findings it is likely that passive GSH and GSSG fluxes are activated simultaneously with the release of intraluminal Ca 2⫹ from the SR. The glutathione redox gradient between the cytosol and the lumen of the SR is allowed to equilibrate (at least partly). This phenomenon could play a role in altering the redox state of GSH and protein thiols of the skeletal muscle in contraction-induced injury (22). Since the disappearance of the transmembrane redox gradient favors the open state of the calcium channel
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(6), the RyR1-dependent glutathione permeation may promote the mobilization of calcium in skeletal muscle. As to the molecular nature of the skeletal muscle SR transporter of GSH and GSSG, it can be speculated that it is the RyR1 itself. In particular, another channel can be formed by the supramolecular arrangement (23) of open RyR1s in the presence of agonists allowing the permeation of glutathione. Alternatively, RyR1 may be tightly coupled to the GSH/GSSG transport protein, and the activity of RyR1 channel is directly transduced to the transporter resulting in a degree of coregulation. This possibility is not unprecedented, since protein–protein interactions between the RyR1 and the voltage-sensitive dihydropyridine receptor of the T tubule most likely transduce the activation of the latter to the former. Further work is needed to clarify these possibilities. ACKNOWLEDGMENTS This work was supported by OTKA (National Scientific Research Fund) Grant T32873, a Hungarian Academy of Sciences Grant, a NATO linkage grant and a Telethon Grant No. 10602 to R. Fulceri. M. Csala was a recipient of a FEBS Short-Term Fellowship and a NATO Advanced Fellowship to Siena. Thanks are due to Dr. Roberta Giunti for the methodological advice and Mrs. Vale´ria Mile for her skillful technical assistance. We thank Professor Fyfe Bygrave (Australian National University) for assistance in the preparation of the manuscript.
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