Cyclosporin A Inhibits Creatine Uptake by Altering Surface Expression of the Creatine Transporter

Abstract

The immunosuppressive drug cyclosporin A (CsA) inhibited the hCRT-1 cDNA-induced creatine uptake inXenopus oocytes and the endogenous creatine uptake in cultured C2C12 muscle cells in a dose- and time-dependent manner. FK506, another potent immunosuppressant, was unable to mimic the effect of CsA suggesting that the inhibitory effect of CsA was specific. To delineate the mechanism underlying, we investigated the effect of CsA on theK m and V max of creatine transport and also on the cell surface distribution of the creatine transporter. Although CsA treatment did not affect theK m (20–24 μm) for creatine, it significantly decreased the V max of creatine uptake in both oocytes and muscle cells. CsA treatment reduced the cell surface expression level of the creatine transporter in the muscle cells by ∼60% without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in creatine uptake. Taken together, our results suggest that CsA inhibited creatine uptake by altering the surface abundance of the creatine transporter. We propose that CsA impairs the targeting of the creatine transporter by inhibiting the function of an associated cyclophilin, resulting in an apparent loss in surface expression of the creatine transporter. Our results also suggest that prolonged exposure to CsA may result in chronically creatine-depleted muscle, which may be a cause for the development of CsA-associated clinical myopathies in organ transplant patients. The immunosuppressive drug cyclosporin A (CsA) inhibited the hCRT-1 cDNA-induced creatine uptake inXenopus oocytes and the endogenous creatine uptake in cultured C2C12 muscle cells in a dose- and time-dependent manner. FK506, another potent immunosuppressant, was unable to mimic the effect of CsA suggesting that the inhibitory effect of CsA was specific. To delineate the mechanism underlying, we investigated the effect of CsA on theK m and V max of creatine transport and also on the cell surface distribution of the creatine transporter. Although CsA treatment did not affect theK m (20–24 μm) for creatine, it significantly decreased the V max of creatine uptake in both oocytes and muscle cells. CsA treatment reduced the cell surface expression level of the creatine transporter in the muscle cells by ∼60% without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in creatine uptake. Taken together, our results suggest that CsA inhibited creatine uptake by altering the surface abundance of the creatine transporter. We propose that CsA impairs the targeting of the creatine transporter by inhibiting the function of an associated cyclophilin, resulting in an apparent loss in surface expression of the creatine transporter. Our results also suggest that prolonged exposure to CsA may result in chronically creatine-depleted muscle, which may be a cause for the development of CsA-associated clinical myopathies in organ transplant patients. cyclosporin A cyclosporin C FKBP, FK506-binding protein synthetic RNA peptidyl-prolyl isomerase creatine transporter-1 human creatine transporter-1 anti-C creatine transporter choline chloride Cyclosporin A (CsA)1 is a potent immunosuppressive agent, which is used widely in organ transplants to prevent graft rejection and also to treat autoimmune disorders (1Borel J.F. Pharmacol. Rev. 1989; 41: 259-371Google Scholar). A large body of evidence suggests that prolonged administration of cyclosporin induces a number of toxic side effects (2Bennett W.M. Burdmann E. Andoh T. Elzinga L. Franceschini N. Miner. Electrolyte Metab. 1994; 20: 214-220PubMed Google Scholar, 3Garcia-Escrig M. Matinez J. Fernandez-Ponsati J. Soto O. Clin. Neuropharmacol. 1994; 17: 298-302Crossref PubMed Scopus (14) Google Scholar, 4Hoyer P.F. Contrib. Nephrol. 1995; 114: 111-123Crossref PubMed Google Scholar), including adverse effects of cyclosporin on skeletal muscle (5Arellano F. Krupp P. Lancet. 1991; 337: 915Abstract PubMed Scopus (33) Google Scholar, 6Biring M.S. Fournier M. Ross D.J. Lewis M.I. J. Appl. Physiol. 1998; 84: 1967-1975Crossref PubMed Scopus (69) Google Scholar, 7Budak-Alpdogan T. Kalayoglu-Besisik S. Sargin D. Tangun Y. Bone Marrow Transplant. 1998; 22: 115-116Crossref PubMed Scopus (3) Google Scholar, 8Fernandez-Sola J. Campsitol J. Casademont J. Grau J.M. Urbano-Marquez A. Lancet. 1990; 335: 362-363Abstract PubMed Scopus (43) Google Scholar, 9Hardiman O. Sklar R.M. Brown Jr., R.H. Neurology. 1993; 43: 1432-1434Crossref PubMed Google Scholar). The CsA-induced clinical myopathies were, however, reversed after cessation of cyclosporin and the muscle weakness returned with exacerbation upon reinstitution of cyclosporin therapy (5Arellano F. Krupp P. Lancet. 1991; 337: 915Abstract PubMed Scopus (33) Google Scholar, 8Fernandez-Sola J. Campsitol J. Casademont J. Grau J.M. Urbano-Marquez A. Lancet. 1990; 335: 362-363Abstract PubMed Scopus (43) Google Scholar). In vitro, CsA inhibited biochemical differentiation of cultured human myoblasts in a dose-dependent manner without significantly altering their proliferation (10Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Palvath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar). Moreover, the muscles of CsA-treated mice were found to be deficient in regenerated muscle fibers (10Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Palvath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar). Together, these studies suggest that CsA have profound biochemical and morphological effects on the skeletal muscle. Currently, the cellular mechanism(s) involved in the development of the CsA-induced toxicity, including the possibility of altered energy state of the muscle, is not fully understood. Muscles store millimolar concentration of a high-energy phosphate intermediate, phosphocreatine. During high muscle activity, the enzyme creatine kinase replenishes the consumed ATP quickly by catalytic transfer of the phosphocreatine phosphate group to the ADP (11Walker J.B. Adv. Enzymol. 1979; 50: 177-242PubMed Google Scholar, 12Bessman S.P. Geiger P.J. Science. 1981; 211: 448-451Crossref PubMed Scopus (578) Google Scholar). Although phosphocreatine is synthesized in the muscle from creatine, the de novo synthesis of creatine occurs mainly in the human liver, kidney, and pancreas (11Walker J.B. Adv. Enzymol. 1979; 50: 177-242PubMed Google Scholar). Thus, to meet their energy demands and to maintain equilibrium between the intracellular concentration of creatine and phosphocreatine, muscles possess a mechanism to actively accumulate creatine from the circulating plasma (see Ref. 13Wyss M. Wallimann T. Mol. Cell. Biochem. 1994; 133/134: 51-66Crossref Scopus (98) Google Scholar for a review). Normally, muscles maintain a steep creatine concentration gradient, which is ∼500–1000-fold higher inside the muscle than the plasma creatine concentration (14Beis I. Newsholme E.A. Biochem. J. 1975; 152: 23-32Crossref PubMed Scopus (363) Google Scholar), primarily with the help of a high affinity creatine transporter (13Wyss M. Wallimann T. Mol. Cell. Biochem. 1994; 133/134: 51-66Crossref Scopus (98) Google Scholar, 15Daly M.M. Seifter S. Arch. Biochem. Biophys. 1980; 203: 317-324Crossref PubMed Scopus (54) Google Scholar). This creatine concentration gradient (higher inside) is tightly regulated to maintain normal muscle function (12Bessman S.P. Geiger P.J. Science. 1981; 211: 448-451Crossref PubMed Scopus (578) Google Scholar, 13Wyss M. Wallimann T. Mol. Cell. Biochem. 1994; 133/134: 51-66Crossref Scopus (98) Google Scholar). Conceivably, chronic dysfunction of the creatine transporter resulting in little or no creatine transport could adversely affect the energy metabolism in the muscle as a result of creatine depletion, which in turn might lead to muscle myopathy. Commensurate with the above idea, a number of studies documented both ultrastructural and functional abnormalities in the muscles of chronically creatine-depleted animals (16Fitch C.D. Jellinek M. Mueller E.J. J. Biol. Chem. 1974; 249: 1060-1063Abstract Full Text PDF PubMed Google Scholar, 17Mekhfi H. Heorter J. Lauer C. Wisnewsky C. Schwartz K. Ventura-Clapier R. Am. J. Physiol. 1990; 258: H1151-H1158PubMed Google Scholar). Recently, we (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar) and others (19Guimbal C. Kilimann M.W. J. Biol. Chem. 1993; 268: 8418-8421Abstract Full Text PDF PubMed Google Scholar, 20Nash S.R. Giros B. Kingsmore S.F. Rochelle J.M. Suter S.T. Gregor P. Seldin M.F. Caron M.G. Receptors and Channels. 1994; 2: 165-174PubMed Google Scholar, 21Sora I. Richman J. Santoro G. Wei H. Wang Y. Vanderah T. Horvath R. Nguyen M. Waite S. Roeske W.R. Yamamura H.I. Biochem. Cell Biol. 1994; 204: 419-427Google Scholar, 22Mayser W. Schloss P. Betz H. FEBS Lett. 1992; 305: 31-36Crossref PubMed Scopus (81) Google Scholar, 23Saltarelli M.D. Bauman A.L. Moore K.R. Bradley C.C. Blakely R.D. Dev. Neurosci. 1996; 18: 524-534Crossref PubMed Scopus (62) Google Scholar) reported cloning of a high affinity creatine transporter (CRT-1) from a variety of mammalian tissues. The CRT-1 mRNA is most abundantly expressed in the skeletal muscle among all tissues examined (19Guimbal C. Kilimann M.W. J. Biol. Chem. 1993; 268: 8418-8421Abstract Full Text PDF PubMed Google Scholar, 20Nash S.R. Giros B. Kingsmore S.F. Rochelle J.M. Suter S.T. Gregor P. Seldin M.F. Caron M.G. Receptors and Channels. 1994; 2: 165-174PubMed Google Scholar). Analysis of the encoded protein sequence predicts that the creatine transporter contains 12 transmembrane helical domains interconnected by extracellular and intracellular (19Guimbal C. Kilimann M.W. J. Biol. Chem. 1993; 268: 8418-8421Abstract Full Text PDF PubMed Google Scholar, 20Nash S.R. Giros B. Kingsmore S.F. Rochelle J.M. Suter S.T. Gregor P. Seldin M.F. Caron M.G. Receptors and Channels. 1994; 2: 165-174PubMed Google Scholar). of the CRT-1 in expression a and creatine uptake that was both and to cultured cells W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar, C. Kilimann M.W. J. Biol. Chem. 1993; 268: 8418-8421Abstract Full Text PDF PubMed Google Scholar, 20Nash S.R. Giros B. Kingsmore S.F. Rochelle J.M. Suter S.T. Gregor P. Seldin M.F. Caron M.G. Receptors and Channels. 1994; 2: 165-174PubMed Google Scholar). In the we the effects of CsA on creatine uptake in oocytes and cultured muscle cells and endogenous creatine We that CsA inhibited the of both the and endogenous creatine Our results suggest that the in the creatine uptake is to an of the surface abundance of the creatine transporter. Cyclosporin A (CsA) and were from J. and of and were from and was from the were or and were from C2C12 myoblasts were in with and in a uptake cells were in a of and were for of myoblasts was by the of myoblasts with the differentiation and by an of were to the differentiation after the cells were to the differentiation which cells were for were used for uptake The of the human CRT-1 was (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar). RNA was from the RNA and was used to and oocytes M. H. Qian X. Sarkar H.K. 1994; Google Scholar). The oocytes were used for the functional studies as (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar, S. Qian X. H. Sarkar H.K. J. PubMed Scopus Google Scholar). uptake were the creatine uptake studies in oocytes were as (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar) concentration of activity, as a The creatine uptake in cultured C2C12 cells were as were with the choline chloride choline The uptake was by the with the as the the choline chloride was with an of for a of the uptake was by the from by with were in of and the of in the was a concentration in a of the was the To the effect of CsA and on creatine C2C12 cells or oocytes were in the of a concentration of the drug for The drug was to the cell or to the with and cells or oocytes were in with an of the cells and oocytes were used for uptake m max for creatine uptake were as (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar) by creatine uptake of A a synthetic to an of the creatine transporter was in the by from of the high the synthetic in an This anti-C creatine transporter was used for H. T. J. S. A. 1979; Scopus Google Scholar) without Cell surface expression of the creatine transporter in and CsA-treated C2C12 cells was the as Enzymol. 1998; PubMed Scopus Google Scholar, M.M. G. PubMed Scopus Google Scholar). and CsA-treated were as which cells were with and was in for with cells were with in to the cells were in by on for The were by for and a of the of of was with of with were with with high and with The were from the with of and a of it was by cell and were on a and a The was with and in and for and with of the for with the was with a of the for The were the and by of the To the effect of cyclosporin on creatine uptake CsA or was to the after the oocytes were with the hCRT-1 The oocytes were in the and of CsA for an of to for uptake in the creatine uptake in oocytes with or CsA for from that of the uptake higher however, CsA inhibited the creatine uptake Thus, in treatment with CsA for inhibited the creatine uptake in oocytes by results not that the uptake level in oocytes with CsA for from that of the uptake. the uptake was inhibited by in oocytes with CsA for and not in of CsA to the uptake of the oocytes also no effect on the creatine uptake not To the of we examined the effects of cyclosporin C a cyclosporin A and FK506, a immunosuppressive drug T. K. M. Google Scholar), on creatine uptake. in inhibited the creatine uptake. We used concentration of concentration is to the of FKBP, and higher concentration is toxic for the oocytes D. S. 1994; Full Text PDF PubMed Scopus Google Scholar). In our CsA was to the and the inhibitory effect of CsA was not apparent for another To the mechanism of of creatine we examined the expression level of the creatine transporter in oocytes by the of the creatine uptake as a function of The creatine uptake and after were and not the creatine uptake to the of the it is to that the were and to the cell surface Thus, these results the possibility in CsA might affect the targeting of the creatine transporter to the cell To the possibility that the effect of CsA on creatine uptake was cell we examined the effect of CsA on the endogenous creatine uptake in cultured C2C12 muscle cells. uptake in C2C12 cells after the myoblasts were by not The creatine uptake in is to an in the creatine transporter expression as a result of muscle cell We did not morphological between CsA-treated and C2C12 not which is in with the that CsA C2C12 proliferation (10Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Palvath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar). of C2C12 cells with CsA for in than the an effect that was apparent higher CsA concentration not Moreover, number of were found the cells were with CsA in differentiation not which is also with the by (10Abbott K.L. Friday B.B. Thaloor D. Murphy T.J. Palvath G.K. Mol. Biol. Cell. 1998; 9: 2905-2916Crossref PubMed Scopus (196) Google Scholar). concentration of however, the number and the of C2C12 were to in the not we used C2C12 cells and CsA for most of our The creatine uptake in C2C12 decreased cells were with CsA In CsA treatment also inhibited the uptake significantly the uptake in C2C12 the transport of creatine, and their by the gradient of the our the possibility that the effect of CsA on creatine uptake was to a decrease in the creatine uptake in a dose- and time-dependent manner. a after the oocytes were with hCRT-1 CsA or was to the the and the oocytes were in the or of CsA for an the oocytes were used for a uptake as expressed as creatine uptake oocytes were used as oocytes were with CsA μm) or for and In all CsA was a after the oocytes were The and CsA-treated oocytes were used for a creatine uptake expressed as uptake of CsA treatment on creatine, and uptake in C2C12 after the C2C12 myoblasts were to differentiation by CsA was to the differentiation and the cells were for an and and CsA-treated cells were used for a uptake in and or and as a of expressed as of CsA inhibited the endogenous creatine uptake in C2C12 cells both in a and time-dependent manner in treatment with of CsA for in decreased creatine and the decrease in uptake was higher as the CsA concentration was The inhibitory effect of CsA also with of the in uptake was which to not the uptake was inhibited by and and with the results with oocytes the hCRT-1 a and a were for CsA to the endogenous creatine uptake in the C2C12 muscle cells. This may be to a in the of of CsA these cell FK506, another potent immunosuppressive drug that its FKBP, did not mimic the effect of CsA a concentration of which is significantly higher than its immunosuppressive in to the results the oocytes the hCRT-1 We examined CsA inhibited the creatine uptake by altering the m max of creatine To we the of uptake as a function of extracellular of In both CsA-treated or oocytes the hCRT-1 the of uptake as the creatine concentration was results were also the and the CsA-treated C2C12 cells not results were the and the results in CsA treatment did not affect the m for creatine in both oocytes and C2C12 cells. Moreover, the m (20–24 μm) is to the m we reported for the hCRT-1 (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar). CsA treatment in the V max for creatine uptake in CsA-treated C2C12 cells by and in CsA-treated oocytes by Currently, we not CsA max to in cells and is that the might be a in the cell these results suggest that max effect is to a reduction of the of in the cell surface state the plasma of CsA on and of creatine creatine uptake was in CsA-treated for cells or for and C2C12 cells and oocytes as were to the to the m max in a creatine uptake was in CsA-treated for cells or for and C2C12 cells and oocytes as were to the to the m max The effect of CsA on the state of the transporter the plasma might be to an apparent decrease in the synthesis of the creatine transporter or to a decrease in the number of the surface expressed To between these we the expression level of the creatine transporter in and CsA-treated C2C12 by a creatine transporter We the surface abundance of the creatine in and CsA-treated C2C12 a surface Our protein and in the of both and CsA-treated and the of these were in both and with for in the loss of these with a of and We also the in from cells a human CRT-1 creatine transporter the as a not Our in with the of and Wallimann Wallimann T. Mol. Cell. Biochem. 1998; PubMed Google Scholar) and S. H. M. M. F. J. B. G. G. Wallimann T. 1999; PubMed Scopus Google Scholar), and in the of and human a creatine transporter Thus, we that both the and with the expression of the creatine and most the and of the creatine transporter. The results of the surface also in and of the results that the to the Moreover, of the from by that the of the in the CsA-treated cells was of that of the cells. in which cells were in without the not suggesting that surface is for the of the by the the was not in the we that of the creatine transporter is not expressed on the surface of the C2C12 cells. suggest that the creatine uptake in the C2C12 cells and the surface expression of the protein Our results also suggest that CsA treatment decreased the abundance of the surface expressed creatine which paralleled the decrease in max of creatine uptake in CsA-treated cells. concentration CsA with its cellular and the function of the protein J. Jr., J. I. 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E.A. 1992; PubMed Scopus Google Scholar), did not creatine uptake. of CsA to the transport the uptake also did not the creatine suggesting that the effect of CsA on creatine uptake was not to a of the creatine transporter. The of a peptidyl-prolyl may as the of a for the or the function of a Commensurate with idea, and S. A. PubMed Scopus Google Scholar) a of of the peptidyl-prolyl in of found in the sequence of the creatine transporter to be in the transmembrane domains of its (19Guimbal C. Kilimann M.W. J. Biol. Chem. 1993; 268: 8418-8421Abstract Full Text PDF PubMed Google Scholar, 20Nash S.R. Giros B. Kingsmore S.F. Rochelle J.M. Suter S.T. Gregor P. Seldin M.F. Caron M.G. Receptors and Channels. 1994; 2: 165-174PubMed Google Scholar). is that CsA also the of the endogenous S. 1992; PubMed Scopus Google Scholar) and the and H. K. a protein that contains to found in the creatine transporter S.R. Giros B. Kingsmore S.F. Rochelle J.M. Suter S.T. Gregor P. Seldin M.F. Caron M.G. Receptors and Channels. 1994; 2: 165-174PubMed Google Scholar). Although not these results to suggest that the mechanism by which CsA creatine and transport might be is that CsA the of an associated cyclophilin, which in turn the a peptidyl-prolyl of the an transporter Our results that the V max of creatine uptake in CsA-treated cells decreased without theK m for by which of the creatine transporter may lead to an apparent decrease in the V max of creatine uptake. possibility is that the transporter is not all to the plasma resulting in an apparent decrease in surface abundance of the transporter. A possibility is that both the and the of the creatine transporter to the plasma resulting in an apparent decrease in the V altering the number of the surface expressed A possibility is that the by resulting in a reduction in the expression level of the transporter. on our we the possibility the of the protein and both in and CsA-treated muscle cells. We also the possibility our results that the number of the surface expressed creatine decreased significantly in the CsA-treated cells than that in the cells. The of our results is that CsA to affect the state of the creatine transporter the plasma by with its targeting Our however, the possibility that the decrease in the V max is to CsA-induced of surface expressed to In organ transplant CsA is used a of and the treatment from to D. T. L. Lancet. 1989; PubMed Scopus Google Scholar, F. Krupp P. Lancet. 1991; 337: 915Abstract PubMed Scopus (33) Google Scholar). disorders associated with CsA and on chronic treatment (5Arellano F. Krupp P. Lancet. 1991; 337: 915Abstract PubMed Scopus (33) Google Scholar). the of CsA in the of organ transplant between and of and higher also have reported D.J. Clin. Chem. 1990; PubMed Scopus Google Scholar, J.M. Transplant. Scholar). in treatment with CsA for inhibited the creatine uptake in cultured muscle cells by the to the cells were with CsA for not a concentration of treatment with CsA inhibited the creatine uptake by The were creatine concentration to the normal μm) of plasma creatine concentration (14Beis I. Newsholme E.A. Biochem. J. 1975; 152: 23-32Crossref PubMed Scopus (363) Google Scholar). Taken together, our results suggest the or its impairs creatine of its of our that CsA creatine transport may have clinical with to its toxic side The CRT-1 creatine transporter found in a variety of is most abundantly expressed in the skeletal muscle S.R. Giros B. Kingsmore S.F. Rochelle J.M. Suter S.T. Gregor P. Seldin M.F. Caron M.G. Receptors and Channels. 1994; 2: 165-174PubMed Google Scholar). This distribution of the creatine transporter mRNA is with the that of the total body creatine is found in the it is as a of energy (11Walker J.B. Adv. Enzymol. 1979; 50: 177-242PubMed Google Scholar, 13Wyss M. Wallimann T. Mol. Cell. Biochem. 1994; 133/134: 51-66Crossref Scopus (98) Google Scholar). in muscles the phosphocreatine to maintain muscle energy In the muscle, phosphocreatine is in a equilibrium with its the muscle the creatine transporter is a that equilibrium between the phosphocreatine and Thus, an of the creatine transporter function to exposure to CsA may chronically muscle creatine may adversely affect the muscle energy (13Wyss M. Wallimann T. Mol. Cell. Biochem. 1994; 133/134: 51-66Crossref Scopus (98) Google Scholar) and may lead to the development of CsA-associated clinical myopathies in organ patients. We for help in cell and and for