Creatina

Am J Physiol Endocrinol Metab 283: E390–E401, 2002. First published February 26, 2002; 10.1152/ajpendo.00428.2001.

New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle
BERND WALZEL,1* OLIVER SPEER,1* ERNIE BOEHM,3 SØREN KRISTIANSEN,2 SHARON CHAN,4 KIERIAN CLARKE,4 JOSEPH P. MAGYAR,1 ERIK A. RICHTER,2 AND THEO WALLIMANN1 1 Institute of CellBiology, Eidgenossische Technische Hochschule-Zurich, Honggerberg, ¨ ¨ CH-8093 Zurich, Switzerland; 2Human Physiology, Copenhagen Muscle Research Center, Institute of Exercise and Sport Sciences, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark; 3Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN; and 4Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United KingdomReceived 24 September 2001; accepted in final form 14 February 2002

Walzel, Bernd, Oliver Speer, Ernie Boehm, Søren Kristiansen, Sharon Chan, Kierian Clarke, Joseph P. Magyar, Erik A. Richter, and Theo Wallimann. New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle. Am J Physiol Endocrinol Metab 283: E390–E401, 2002. First published February 26, 2002;10.1152/ajpendo.00428.2001.—Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giantvesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 9.4 M and maximal velocity value 17.3 3.1 pmol min 1 mg vesicle protein 1), which cotransports Cr into skeletal muscle together with Na and Cl ions. The regulation of Cr transport in giant vesicles by substrates,analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH- and NH2-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase isabsent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of 58 kDa is present in the sarcolemma, whereas the previously identified majorCRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [14C]PCr/Cr measurements in vivo as well as from recent in vivo NMRstudies.
* These authors contributed equally to this study. Address for reprint requests and other correspondence: T. Wallimann, Institute of Cell Biology, ETH-Honggerberg HPM F42, CH¨ 8093 Zurich, Switzerland (E-mail: theo.wallimann@cell.biol.ethz.ch). E390

plasma membrane giant vesicles; sarcolemma; mitochondria; subcellular compartmentation of creatine; bioenergetics
CLINICAL APPLICATIONSOF CREATINE (Cr) supplementation have recently attracted considerable attention, and a series of intriguing new discoveries about potential therapeutic effects of Cr have been made. Cr analogs have been proven to be prospective anticancer agents that act synergistically with currently used chemotherapeutics (3, 25, 35–37, 53), and beneficial effects of Cr supplementation have been reported for...