Cyclosporin A Inhibits Creatine Uptake by Altering Surface Expression of the Creatine Transporter
2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês
10.1074/jbc.m005636200
ISSN1083-351X
AutoresThanh T. Tran, Wenxuan Dai, Hemanta K. Sarkar,
Tópico(s)Nerve injury and regeneration
ResumoThe 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. 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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 loops (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). Expression of the cloned CRT-1 cDNAs in heterologous expression systems induced a Na+ and Cl−-dependent creatine uptake that was both biochemically and pharmacologically similar to those obtained using cultured cells (for examples, see Refs.18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar, 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). In the present studies, we investigate the effects of CsA on creatine uptake in Xenopus oocytes and cultured muscle cells expressing recombinant and endogenous creatine transporter, respectively. We show that CsA selectively inhibited the activities of both the recombinant and endogenous creatine transporters. Our results further suggest that the inhibition in the creatine uptake is due to an alteration of the relative surface abundance of the creatine transporter. Cyclosporin A (CsA) and FK 506 were generous gifts from Dr. J. Clifford Waldrep and Dr. Albert Chang (Baylor College of Medicine), respectively. [4-14C]Creatine (50 mCi/mmol) and l-[3H(G)]glutamic acid (30 Ci/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO), and 1,2-[3H]taurine was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). All the other reagents were either ACS grade pure or ultrapure, and were purchased from various commercial sources. C2C12 mouse myoblasts (American Type Culture Collection, Rockville, MD) were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated bovine fetal serum and 1% penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37 °C. For uptake studies, cells were seeded in 6-well culture dishes at a density of ∼105cells/well and were incubated for 2–3 days at 37 °C. Differentiation of myoblasts into myotubes was induced by replacing the growth medium of nearly confluent myoblasts with the differentiation medium (Dulbecco's modified Eagle's medium containing 5% heat-inactivated horse serum and 1% penicillin/streptomycin) followed by an additional period of incubation at 37 °C. Drugs were added directly to the differentiation medium 24 h after the cells were exposed to the differentiation medium, following which cells (nascent myotubes) were incubated at 37 °C for at least 24 h before they were used for uptake studies. The construction of the recombinant plasmid pCrT.3 containing human CRT-1 (hCRT-1) cDNA was described previously (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar). Synthetic RNA (cRNA) was prepared from the pCrT3 plasmid using SP6 RNA polymerase (Epicenter Technologies, WI), and was used to microinject (5 ng/oocyte) defolliculated and healthy Xenopus oocytes (24Ruiz M. Egal H. Sarthy V. Qian X. Sarkar H.K. Invest. Ophthalmol. Vis. Sci. 1994; 35: 4039-4048PubMed Google Scholar). The microinjected oocytes were subsequently used for the functional studies as described (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar, 25Vinnakota S. Qian X. Egal H. Sarthy V. Sarkar H.K. J. Neurochem. 1997; 69: 2238-2250Crossref PubMed Scopus (66) Google Scholar). Unless otherwise noted, uptake assays were carried out at least ∼72 h post-injection. Unless stated otherwise, the creatine uptake studies in oocytes were performed essentially as described earlier (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar) using 30 μm final concentration of [14C]creatine (stock solution: 10 mm; specific activity, 10 mCi/mmol) as a substrate. The creatine uptake in cultured C2C12 cells were measured as follows. Cells were first washed with the choline chloride buffer (ChCl-1 buffer: 135 mm choline chloride, 1 mm calcium chloride, 2 mm potassium chloride, 5 mmmagnesium chloride, 5 mm HEPES-Tris (pH 7.5)). The uptake was initiated by replacing the ChCl-1 buffer with the NaCl-1 buffer (same composition as the ChCl-1 buffer except the choline chloride was replaced with an equimolar amount of NaCl) containing 10 μm [14C]creatine. After incubation for a given period of time, the uptake process was terminated by aspirating off the buffer from each well, followed by three quick washes with ice-cold ChCl-1 buffer. Cells were subsequently solubilized in 1 ml of 1% SDS and the amount of radioactivity in the extract was measured using a Beckman LS 3800 scintillation counter. Protein concentration in a portion of the extract was measured using the Pierce Protein Assay kit (Pierce, Rockford, IL). To determine the effect of CsA and FK506 on creatine uptake, C2C12 cells or microinjected oocytes were incubated in the presence of a given concentration of the drug for various time periods. The drug was added directly to the cell culture medium, or to the oocyte bathing medium (Barth's solution: 88 mm NaCl, 1 mm KCl, 2.4 mmNaHCO3, 0.33 mmCa(NO3)2, 0.82 mmMgSO4, 0.41 mm CaCl2, 7.5 mm Tris-HCl (pH 7.6) supplemented with 1 mmsodium pyruvate and 0.01% penicillin/streptomycin). Control cells or oocytes were incubated in parallel with an equivalent amount of the vehicle solvent (ethanol). Pretreated cells and oocytes were subsequently used for uptake assays. K m andV max values for creatine uptake were determined as described previously (18Dai W. Vinnakota S. Qian X. Kunze D. Sarkar H.K. Arch. Biochem. Biophys. 1999; 361: 75-84Crossref PubMed Scopus (70) Google Scholar) by measuring creatine uptake at various external concentrations of [14C]creatine. A polyclonal antibody against a synthetic peptide (LEYRAQDADVRG), corresponding to an internal portion of the creatine transporter C-terminal tail, was raised in rabbits using the commercial services offered by Research Genetics, Inc. (Huntsville, AL). Immunoserum from one of the rabbits showed high antibody titer against the synthetic peptide in an enzyme-linked immunosorbent assay. This immunoserum containing anti-C creatine transporter (anti-CrT-C) antibodies was used for immunoblot assay (26Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4354-4355Crossref Scopus (44924) Google Scholar) without further purification. Cell surface expression of the creatine transporter in control and CsA-treated C2C12 cells was determined using the membrane impermeable biotinylation reagent NHS-SS-biotin (Pierce) as described (27Daniels G.M. Amara S.G. Methods Enzymol. 1998; 296: 307-318Crossref PubMed Scopus (52) Google Scholar, 28Stephan M.M. Chen M.A. Penado K.M.Y. Rudnick G. Biochemistry. 1997; 36: 1322-1328Crossref PubMed Scopus (66) Google Scholar). Briefly, control and CsA-treated myotubes (∼3 × 105 cells; passages 15–18) were grown as described above, following which cells were washed with phosphate-buffered saline containing 0.1 mm CaCl2 and 1.0 mmMgCl2 (phosphate-buffered saline/CM). Biotinylation was carried out using 1.0 mg/ml NHS-SS-biotin in biotinylation buffer (100 mm NaCl, 2 mm CaCl2, 10 mm triethanolamine, pH 8.0) for 20–25 min at 4 °C with gentle shaking. After labeling, cells were washed twice with ice-cold 100 mm glycine in phosphate-buffered saline/CM to quench the residual NHS-SS-biotin. Subsequently, cells were solubilized in lysis buffer (1% Triton X-100, 150 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl, pH 7.5) containing CompleteTM protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) by gently shaking on ice for approximately 1 h. The extracts were clarified by centrifugation at 14,000 × g for 10 min at 4 °C and a portion of the extract (300 μl of extract containing 390 μg of protein) was incubated overnight with 50 μl of streptavidin-agarose beads (Sigma) at 4 °C with gentle agitation. After incubation, beads were washed three times with lysis buffer, twice with high salt wash buffer (0.1% Triton X-100, 500 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl, pH 7.5), and finally once with no-salt wash buffer (50 mm Tris-HCl, pH 7.5). The captured biotinylated proteins were eluted from the beads with 40 μl of 2 × SDS sample buffer and a portion of it (20 μl) was analyzed by immunoblot assay. Total cell extracts and captured biotinylated proteins were separated on a 12% SDS-polyacrylamide gel and subsequently electrotransferred onto a Hybond-P polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was blocked with 3% bovine serum albumin and 2% non-fat dry milk in TBST buffer (137 mm NaCl, 2.6 mm KCl, 25 mm Tris-HCl, pH 7.4, and 0.1% Tween 20) for 1 h at room temperature and incubated with 1:2000 dilution of the anti-CrT-C antibody for 1–3 h at room temperature. After washing with TBST buffer, the blot was incubated with a 1:15,000 dilution of the horseradish peroxidase-conjugated anti-rabbit antibody (Pierce) for 1–2 h at room temperature. The immunoreactive bands were visualized using the ECL-Plus chemiluminescence detection kit (Amersham Pharmacia Biotech) and subsequently quantified by densitometric scanning of autoradiograms using the AlphaImager 2000 (Alpha Innotech Co., San Leandro, CA). To determine the effect of cyclosporin on creatine uptake CsA (1–30 μM final concentration) or ethanol (vehicle solvent, control) was added to the oocyte bathing medium 24 h after the oocytes were injected with the hCRT-1 cRNA. The oocytes were incubated in the absence and presence of CsA for an additional period of 72 h prior to using them for uptake assays. As shown in Fig.1 A, the creatine uptake in oocytes treated with 1 or 5 μm CsA for 72 h remained unchanged from that of the control uptake level. At higher concentrations, however, CsA inhibited the creatine uptake (Fig.1 A). Thus, in three independent experiments, treatment with 30 μm CsA for 72 h inhibited the creatine uptake in oocytes by ∼38.6 ± 4.5% (mean ± S.E.; results not shown). Fig. 1 B shows that the uptake level in oocytes pretreated with 30 μm CsA for 24 h remained unchanged from that of the control (ethanol, 24 h) uptake. However, the uptake was inhibited by ∼36% in oocytes treated with 30 μm CsA for 48 and 72 h, but not in vehicle solvent-treated (control) oocytes. Addition of 30 μm CsA directly to the uptake buffer of the untreated oocytes also had no effect on the induced creatine uptake (results not shown). To test the specificity of CsA, we examined the effects of cyclosporin C (CsC), a cyclosporin A analog, and FK506, a structurally unrelated immunosuppressive drug (29Ochiai T. Nakajima K. Nagat M. Transplant Proc. 1987; 19: 1284-1286PubMed Google Scholar), on creatine uptake. As shown in Fig.2, neither CsC nor FK506 inhibited the creatine uptake. We used only 3 μm concentration of FK506 because at this concentration FK506 is known to inhibit the PPIase activity of FKBP, and at higher concentration FK506 is toxic for the oocytes (30Helekar S.A. Char D. Neff S. Patrick J.A. Neuron. 1994; 12: 179-189Abstract Full Text PDF PubMed Scopus (99) Google Scholar). In our previous experiments, CsA was added to the oocyte bathing medium 24 h post-injection, and the inhibitory effect of CsA was not apparent for at least another 24 h. To gain further insight into the mechanism of CsA-mediated inhibition of creatine uptake, we examined the relative expression level of the creatine transporter in untreated control oocytes by measuring the appearance of the creatine uptake as a function of time. The creatine uptake levels at 24, 48, 72, and 96 h after microinjection were 47.7 ± 10.2 (mean ± S.E.; n = 7 oocytes), 148.1 ± 14.4, 195.8 ± 21.8, and 302.7 ± 38.3 pmol/60 min/oocyte, respectively (results not shown). Since the creatine uptake continued to increase during the time course of the experiment, it is reasonable to assume that the transporters were functionally assembled and targeted to the oocyte cell surface beyond 48 h. Thus, these results raise the possibility that, at least in oocytes, CsA might affect the targeting of the creatine transporter to the cell surface. To rule out the possibility that the effect of CsA on creatine uptake was cell specific, we examined the effect of CsA on the endogenous creatine uptake in cultured C2C12 muscle cells. Creatine uptake in C2C12 cells almost doubled after the myoblasts were differentiated into myotubes by serum deprivation (results not shown). The increased creatine uptake in myotubes is probably due to an increase in the creatine transporter expression as a result of muscle cell differentiation. We did not observe any significant morphological difference between CsA-treated and control C2C12 myoblast (results not shown), which is in agreement with the previous report that CsA minimally affected C2C12 myoblast 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). Visual inspection of C2C12 cells treated with CsA for 24 h appeared slightly larger in size than the control myotubes, an effect that was more apparent at higher CsA concentration (results not shown). Moreover, less number of multinucleated differentiated myotubes were found when the differentiated cells were treated with 30 μm CsA in differentiation medium (results not shown), which is also consistent with the earlier observation by Abbott et al. (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). At 10 μm concentration of CsA, however, visually the number and the appearance of C2C12 myotubes were virtually similar to those in the control plate (results not shown). Therefore, we used differentiating C2C12 cells and 10 μm CsA for most of our studies. The Na+-dependent creatine uptake in C2C12 nascent myotubes decreased when cells were treated with 10 μm CsA 48 h (Fig.3, compare 1 versus 2). In parallel experiments, CsA treatment also inhibited the Na+-dependent taurine uptake significantly (Fig. 3, compare 3 versus 4), while the Na+-dependent glutamate uptake remained mostly unaffected (Fig. 3, compare 5 versus 6) in C2C12 myoblasts. Since the transport of creatine, glutamate, and taurine via their respective Na+-dependent transporters are driven by the sodium electrochemical gradient of the cell, our observation rules out the possibility that the effect of CsA on creatine uptake was due to a general decrease in the driving force.Figure 1CsA inhibits creatine uptake in a dose- and time-dependent manner. A, dose dependence: a day after the oocytes were injected with hCRT-1 cRNA (∼5 ng/oocyte), CsA or ethanol was added directly to the medium bathing the oocytes, and the oocytes were incubated in the absence or presence of CsA for an additional ∼72 h at 18 °C. Subsequently, the oocytes were used for a 30-min uptake assay at room temperature as described under "Experimental Procedures." Results are expressed as mean creatine uptake ± S.E. (n = 6–8 oocytes). Uninjected oocytes were used as negative control. *,p < .0001. B, time dependence: microinjected oocytes were treated with CsA (30 μm) or vehicle solvent (ethanol; control) for 24, 48, and 72 h. In all cases, CsA was added a 24 h after the oocytes were injected. The control and CsA-treated oocytes were subsequently used for a 30-min creatine uptake assay. Results are expressed as mean uptake ± S.E. (n = 6 oocytes). *, p < .005.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of CsA treatment on creatine, glutamate, and taurine uptake in C2C12 nascent myotubes. Twenty-four hours after the C2C12 myoblasts were subjected to differentiation by serum deprivation, 10 μm CsA was added directly to the differentiation medium and the cells were incubated for an additional 48 h at 37 °C. Ethanol-treated control (1, 3, and 5) and CsA-treated (2, 4, and6) cells were subsequently used for a 60-min uptake assay in NaCl-1 buffer containing 10 μm[14C]creatine (1 and 2), 5 μm [3H]taurine (3 and4), or 5 μm [3H]glutamate (5 and 6) as a substrate. Results (mean of duplicate measurement ± deviation) are expressed as % of control uptake.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CsA inhibited the endogenous creatine uptake in C2C12 cells both in a concentration- and time-dependent manner (Fig.4). As shown in Fig. 4 A, treatment with various concentrations of CsA for 24 h resulted in decreased creatine uptake, and the decrease in uptake was higher as the CsA concentration was increased. The inhibitory effect of CsA also increased with time of treatment. At 1 μm CsA, the inhibition in uptake was ∼10% (n = 3;p < 0.04) at 24 h (Fig. 4A), which increased to ∼28% (n = 3; p < 0.05) at 96 h (results not shown). At 10 μm CsA, the uptake was inhibited by ∼40 and ∼65% at 24 and 48 h, respectively (Fig.4 B). Compared with the results obtained with oocytes expressing the hCRT-1 cRNA, a lower dose and a shorter incubation time were required for CsA to inhibit the endogenous creatine uptake in the C2C12 muscle cells. This discrepancy may be due to a difference in the amount and/or rate of diffusion of CsA into these two different cell types. FK506, another potent immunosuppressive drug that acts via its own receptor FKBP, did not mimic the effect of CsA even at a concentration of 3 μm, which is significantly higher than its immunosuppressive dose. These results, in general, are similar to the results obtained using the oocytes expressing the hCRT-1 cDNA. We further examined whether CsA inhibited the creatine uptake by altering the K m and/or theV max of creatine transport. To this end, we measured the rate of uptake as a function of various extracellular concentrations of [14C]creatine. In both CsA-treated or ethanol-treated (control) oocytes expressing the hCRT-1 cDNA, the rate of uptake increased hyperbolically as the external creatine concentration was increased (Fig. 5). Similar results were also obtained using the control and the CsA-treated C2C12 cells (results not shown). These results were further analyzed using the Michaelis-Menten equation and the results are summarized in TableI. As shown, CsA treatment did not affect the K m for creatine in both oocytes and C2C12 cells. Moreover, the observedK m (20–24 μm) value is very similar to the K m value we reported earlier for the recombinant 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). However, CsA treatment resulted in reducing the V max for creatine uptake in CsA-treated C2C12 cells by ∼72% and in CsA-treated oocytes by ∼36%. Currently, we do not know why CsA reducedV max to different extents in cells and oocytes. One explanation is that the observed discrepancy might be simply due a difference in the cell type (mammalian versus amphibian). Nevertheless, these results suggest that theV max effect is probably due to a reduction of the amount of transporters in the cell surface (i.e. steady state levels at the plasma membrane).Table IEffect of CsA on Km and Vmax of creatine uptakeC2C12 cellsOocyteControlCsA-treatedControlCsA-treatedK m(μm)24.820.722.322.6% Decrease inV max7236Creatine concentration-dependent creatine uptake was measured in CsA-treated (10 μm, 2
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