Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN; Department of Biochemistry, University of Oxford, Oxford OX1 3BN, United Kingdom; and Eidgenoessisch-technische Hochschule (ETH)-Zürich, Institute of Cell Biology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland
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ABSTRACT |
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The intracellular creatine
concentration is an important bioenergetic parameter in cardiac muscle.
Although creatine uptake is known to be via a NaCl-dependent creatine
transporter (CrT), its localization and regulation are poorly
understood. We investigated CrT kinetics in isolated perfused hearts
and, by using cardiomyocytes, measured CrT content at the plasma
membrane or in total lysates. Rats were fed control diet or diet
supplemented with creatine or the creatine analog
-guanidinopropionic acid (
-GPA). Creatine transport in control
hearts followed saturation kinetics with a Km of
70 ± 13 mM and a Vmax of 3.7 ± 0.07 nmol · min
1 · g
wet wt
1. Creatine supplementation significantly decreased
the Vmax of the CrT (2.7 ± 0.17 nmol · min
1 · g
wet wt
1). This was matched by an ~35% decrease in the
plasma membrane CrT; the total CrT pool was unchanged. Rats
fed
-GPA exhibited a >80% decrease in tissue creatine and increase
in
-GPAtotal. The Vmax of the CrT
was increased (6.0 ± 0.25 nmol · min
1 · g
wet wt
1) and the Km decreased
(39.8 ± 3.0 mM). The plasma membrane CrT increased about
fivefold, whereas the total CrT pool remained unchanged. We conclude
that, in heart, creatine transport is determined by the content of a
plasma membrane isoform of the CrT but not by the total cellular CrT pool.
creatine transport(er); creatine feeding; creatine depletion; isolated perfused heart; cell-surface biotinylation
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INTRODUCTION |
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THE CONCEPT of the creatine kinase/phosphocreatine (CK/PCr) shuttle describes the functional association of CK isoenzymes with discrete intracellular compartments of ATP production and hydrolysis and the suitability of PCr and creatine to serve as cytosolic energy transducers (2, 3, 27). In this view, the functions of PCr and creatine are to form essential links between sites of ATP synthesis and utilization.
Both PCr and creatine can be spontaneously converted into creatinine,
which leaves the cardiomyocyte and is excreted by the kidney at a rate
of ~2% per day (1). This loss of creatine must be
replenished, either from food or by endogenous synthesis. Creatine is
not synthesized by cardiomyocytes, however (10), but
rather is taken up by the action of a highly specific
Na+Cl-dependent creatine cotransporter (CrT)
(for review see Ref. 25). It has been suggested that CrT
content and/or activity may be a major determinant of the myocardial
creatine and, hence, PCr concentrations (19). For example,
we have reported that decreased CrT content correlates well with
decreased myocardial creatine (and PCr) content in human, rat
(19), and dog (5) failing heart, suggesting
that a decreased energy reserve in the failing heart might be a direct
consequence of loss of CrT protein. Furthermore, the failure of the
myocardium and other tissues to substantially increase intracellular
creatine content in response to elevated plasma creatine levels caused
by chronic creatine feeding has been proposed to be due to either an
inactivation of the CrT or a decrease in CrT content (12,
13).
Many unanswered questions with regard to the biology of the CrT remain, however: it has yet to be shown how CrT activity and creatine content are related, and the precise mechanism by which the subcellular distribution and activity of the CrT are regulated remains unclear. We have recently shown that two distinct pools of the CrT protein exist in the cardiomyocyte (28), one associated with the mitochondrial compartment and a second with the plasma membrane. Studies (25) quantifying what we now know as a predominantly mitochondrial protein must be reassessed to take into account the existence of this discreet membrane-bound CrT isoform. The plasma membrane CrT is likely to be the only CrT pool relevant for the regulation of creatine content in the cardiomyocyte, yet there are currently no studies on this specific CrT pool in the heart. The purpose of this work, therefore, was to study the relationship between intracellular creatine content, subcellular CrT pools, and CrT uptake kinetics under conditions of creatine supplementation or depletion.
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MATERIALS AND METHODS |
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Experimental animals.
Male Wistar rats were obtained at ~250 g from Harlan UK (Bicester,
UK). These were housed (3 animals to a cage) in a standard experimental
animal laboratory (12:12-h light-dark schedule, room temperature
22°C), with free access to drinking water and a creatine-free powdered diet (RM1 diet; Special Diet Services, Witham, Essex, UK).
Rats were randomly assigned for 6 wk to one of three feeding regimens:
1) Control diet; 2) diet + 3% creatine
(Apin Chemicals, Abingdon, UK); and 3) diet + the
creatine analog -guanidinopropionic acid (
-GPA 1.5%). Rats were
subsequently anesthetized with an intraperitoneal injection of sodium
pentobarbitone (75 mg/kg body wt). Blood samples were removed from the
femoral vein, and hearts were rapidly excised and arrested in ice-cold
buffer (see Radiolabel studies). Hearts were cannulated via
the ascending aorta for retrograde perfusion for radiolabeled
[14C]creatine uptake studies, myocyte isolation, or
metabolite assays. In addition to these feeding regimens, follow-up
experiments were performed to investigate both the time course and dose
dependence of the creatine feeding effect. In these studies, animals
were fed 3% creatine for 2, 4, and 12 wk and 0.6 or 7% creatine for 6 wk.
Radiolabel studies.
Hearts were perfused with Krebs-Henseleit buffer containing (in mmol/l)
110 NaCl, 4.7 KCl, 1.2 MgSO4 7 H2O, 1.75 CaCl2 · 2 H2O, 11 glucose, 2 KH2PO4, 25 NaHCO3, 0.5 Na2EDTA, 0.5 Na-lactate, and 4.5 Na-pyruvate. The buffer,
aerated with 95% O2-5% CO2 to give a pH of
7.4 at 37°C, was perfused through the hearts in a recirculating mode
(recirculating volume of ~400 ml) at a constant pressure of 100 mmHg.
Recirculating buffer was supplemented with creatine (final [creatine] = 25, 100, and 500 µM); this was shown previously to have no effect
on cardiac function, and hearts were stable for the duration of the
protocol (1 h). Cardiac contractile function was monitored throughout
the protocol as previously described (12).
Isolation of cardiac myocytes and cell surface biotinylation. Cardiac myocytes were isolated according to the method of Powell et al. (22). Briefly, after excision, hearts were perfused for 5 min by retrograde coronary perfusion with an isolation Tyrode buffer, containing (in mM) 130 NaCl, 5.4 KCl, 0.4 NaH2PO4 · 2 H2O, 3.5 MgCl2, 10 glucose, 5.0 HEPES, and 20 taurine, pH 7.4, at 37°C. This was followed by 10 min with isolation buffer supplemented with collagenase (1 mg/ml), protease (0.1 mg/ml), and CaCl2 (100 mM). The atria were removed, and the ventricles were placed in a stirred incubation solution (isolation buffer containing 1 mg/ml collagenase and 100 mM CaCl2) for 10 min at 37°C. The supernatant was filtered through a gauze mesh and made up to 10 ml with wash solution (10 mg/ml albumin and 500 µM CaCl2). Cell samples were centrifuged for 1 min at 20-30 g and then filtered through the gauze mesh, made up to 10 ml with wash solution, and centrifuged again for 1 min at 20-30 g. Cells were stored in storage solution containing DMEM (20 g/ml), NaH2CO3 (1 mM), and UltraSer G (2 mg/ml) at pH 7.35 until required.
Cell surface expression of the CrT was determined by using the membrane-impermeable biotinylation reagent Sulpho-NHS-LC-biotin (Pierce, Rockford, IL) (6, 26). Isolated myocytes were washed three times with ice-cold PBS and then incubated with PBS containing 1.0 mg/ml Sulfo-NHS-LC-biotin for 45 min with constant shaking (4°C). After labeling, the cells were washed three times in ice-cold PBS containing glycine (15 mM) to quench the residual Sulpho-NHS-LC-biotin. Subsequently, cells were solubilized by resuspension in solubilization buffer [containing (in mM) 150 NaCl, 20 HEPES, 2 phenylmethylsulfonyl fluoride, 1 EDTA, 0.1% Triton X-100, and anti-protease cocktail (Sigma, 1:100 dilution)] and gentle shaking for 1 h. Extracts were clarified by centrifugation at 13,000 g for 5 min (4°C), and a portion of the lysate was taken for incubation with streptavidin beads (Sigma Chemical). The remaining lysate was kept for protein determination and immunoblotting of the total cell extract. Streptavidin beads (150 µl) were washed three times in wash buffer [containing (in mM) 150 NaCl and 10 Tris, pH 7.0] by rapid centrifugation. Lysate (2,000 µg protein) was added to the beads, incubated on ice, and rotated/shaken overnight. Samples were then washed six times by rapid centrifugation with wash buffer plus anti-proteases. To elute the biotinylated protein from the beads, 80 µl of urea buffer (containing 9 M urea, 2% SDS, and 100 mM Tris, pH 6.8) plus anti-proteases were added and incubated (with constant shaking) for 45 min at 95°C. Finally, tubes were centrifuged at 13,000 g, and the supernatant was collected.Blood preparation.
Blood samples were centrifuged at 12,000 rpm for 10 min. The pellet was
washed with isosmotic buffer (MOPS-buffered saline) three times by
rapid centrifugation. Approximately 250 µl were resuspended in
solubilization buffer (see above) and incubated with shaking for 30 min
at 4°C. The supernatant was used for the determination of plasma
concentration of creatine or -GPA by HPLC, as has been previously
described (18). The pellet was used for immunoblotting to
observe the erythrocyte CrT (data not shown).
Western blotting. Total cell extracts (myocyte and erythrocyte at 20 and 100 µg protein/lane, respectively) and captured biotinylated proteins (100 µg protein/lane) were separated on a 10% SDS-polyacrylamide gel and subsequently transferred onto a nitrocellulose membrane (Gelman Laboratories). The membrane was blocked with 5% fat-free milk powder in PBS-T buffer (Tween, 0.1%) for 1 h at room temperature followed by 1 h in PBS supplemented with avidin (Sigma) at a concentration of 20 µg/ml. After washing for 1 h, membranes were incubated with a 1:3,000 dilution of the anti-CrT antibody overnight at 4°C. After washing with PBS-T buffer, the blot was incubated with a 1:5,000 dilution of the HRP-conjugated anti-rabbit antibody (Amersham Pharmacia Biotech). The immunoreactive bands were visualized using the ECL-Plus chemiluminescence detection kit (Amersham Pharmacia Biotech) and quantified by scanning densitometry. The intensity values for both CrT bands were added together for calculation of absolute CrT content.
Biochemical analysis.
Hearts were perfused for 10 min before being rapidly freeze-clamped
using Wollenberger tongs. For HPLC analysis, powdered tissue was
homogenized in 0.4 N perchloric acid at 0°C, and aliquots of
homogenates were removed for protein determination. The homogenate was
neutralized and centrifuged for 5 min. The supernatant was used for
measuring -GPA (where appropriate), PCr, free Cr, and ATP, as
previously described (18). Noncollagen protein was
measured by the method of Lowry et al. (14). Tissue
concentrations were expressed as nanomoles per milligram protein. To
investigate any possible coregulation of creatine kinase (CK) with the
CrT, hearts were analyzed for total CK activity and isoenzyme specific
activity (17, 18).
Statistical analysis. The data are expressed as means ± SD. To determine statistical significance between feeding groups, a one-way analysis of variance was used followed by a Bonferroni post hoc test.
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RESULTS |
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Heart weights, body weights, and cardiac function.
Table 1 shows heart weight (HW) and body
weight (BW) data from the three main feeding groups. Creatine
feeding had no effect on BW. HWs and HW-to-BW ratios (HW/BW) were
similarly unaffected. -GPA-fed rats displayed a small decrease in BW
(P < 0.01) and a small increase in both HW and HW/BW
(P < 0.01), suggesting the development of mild
myocardial hypertrophy. Table 1 also shows that there was no difference
in cardiac function between control and fed animals.
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Plasma levels of creatine and -GPA.
The mean plasma creatine and
-GPA concentrations from the main
feeding groups are shown in Table 1. Creatine supplementation led to an
about sevenfold significant increase in plasma creatine concentration.
Feeding
-GPA for 6 wk resulted in a plasma
-GPA concentration of
1.5 ± 0.9 mM. There was a small but significant increase in
plasma creatine in
-GPA-fed rats (P < 0.01).
CrT kinetics in perfused heart.
Figure 1 shows creatine uptake rates
in the three main feeding groups. Uptake of creatine in control hearts
followed first-order saturation kinetics. Rates of
[14C]creatine uptake were dependent on the extracellular
creatine concentration and reached saturation by 500 µM. The apparent
Km and Vmax for
[14C]creatine uptake in isolated perfused control hearts
were calculated as 69.6 ± 13.1 µM and 3.7 ± 0.07 nmol · min1 · g
wet wt
1, respectively. Feeding 3% creatine for 6 wk led
to an ~30% significant decrease in the Vmax
(P < 0.001), despite no change in the calculated Km. Feeding 1.5%
-GPA for 6 wk induced an
~70% significant increase in the Vmax
(P < 0.001), as well as a significant decrease in the
calculated Km (P < 0.05).
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Quantification of total and membrane-bound CrT.
Two major protein bands with an apparent mobility of 72 and 52 kDa were
consistently recognized in the whole lysate preparations from all three
experimental groups (Fig. 2). There was
no significant difference in the total content of CrT between rats fed
control, creatine (Fig. 2, A and B), or -GPA
diets (Fig. 2, C and D).
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Intracellular high-energy phosphates and CK.
Table 2 shows high-energy phosphate and
creatine concentrations, as well as CK activity, for the three main
experimental groups. PCr, Cr, and ATP levels did not change
significantly in the creatine-fed group. Total creatine content
(PCr + creatinefree), however, showed a significant
11% increase in creatine-fed rats.
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Time and concentration dependence of creatine.
To investigate whether the effect of feeding 3% creatine for 6 wk was
maximal, a further series of animals was studied at different time
points and with different doses of creatine. Plasma creatine was
measured together with the maximal rate of creatine transport. Data
from these experiments are presented in Fig.
4. Feeding 0.6% creatine for 6 wk, or
3% for 2 wk, had small effects on both plasma creatine and creatine
transport compared with control animals. After 4 wk, the effect of 3%
creatine was not significantly different from that after 6 wk. Feeding
3% creatine for 12 wk led to a decreasing trend in the plasma creatine
concentration compared with 6 wk, although the
Vmax was unchanged. Feeding creatine at a higher
dose (7%) for 6 wk failed to have any additional effect on plasma
creatine or the Vmax of the CrT compared with
3%. Heart weights, body weights, and cardiac function were unaffected
in these additional feeding protocols (data not shown). Thus, by feeding 3% creatine for 6 wk, we observed a maximal effect on creatine
absorption, retention, and transport.
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DISCUSSION |
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The results presented in this study show that creatine
supplementation leads to a decrease in the
Vmax of creatine transport in the heart and to a
concomitant decrease in the content of the plasma membrane CrT pool.
Creatine depletion by -GPA feeding, on the other hand, leads to an
increase in the Vmax of creatine transport and a
concomitant increase in the plasma membrane CrT pool. To the best of
our knowledge, these are the first data to show directly a chronic
coregulation of CrT activity and plasma CrT content in the intact
perfused tissue.
Creatine and -GPA metabolism in rats.
The creatine doses were chosen on the basis of the recommended daily
maximum in humans of 20 g/day (0.6%) and our previous work (3 and 7%)
(12). HWs and HW/BW ratios in all creatine-fed groups were
not different from those of controls. Animals appeared to tolerate the
feeding regimens well, with no signs of distress even at the highest
creatine dose, which was equivalent to ~200 g/day in humans.
Creatine transporter kinetics. Our data are in close agreement with the one previous study investigating creatine transport kinetics in the rat heart (23). The uptake data for control mice fit well to a Michaelis-Menten plot with a calculated Km of ~70 µM. As this is lower than the plasma creatine concentration, the CrT is working at ~70% of its calculated Vmax.
The maximal rate of creatine transport was significantly decreased after feeding 3% creatine for 6 wk; there was no significant change in Km. This can classically be explained by a decrease in the CrT protein content without kinetic regulation. Although the ~30% decrease in Vmax is small, under these fed conditions the actual calculated rate of creatine transport is ~2.7 nmol · minCrT content. The plasma CrT and total mitochondrial CrT appeared as two bands. This is different from what was previously observed in biotinylated mouse cardiomyocytes, where only one band was observed in the membrane fraction (28). The two plasma membrane bands were at a higher molecular mass than those observed in whole myocyte preparations. We do not believe that this was a result of the biotinylation process, because biotinylation of whole cell lysates followed by immunoblotting did not result in any change in the apparent molecular mass of the CrT proteins (data not shown). In addition, isolated rat erythrocytes that possess only a plasma membrane CrT exhibit bands identical to those found in membrane preparations here (data not shown). It is possible that the membrane-bound form of the CrT is glycosylated, as has been previously suggested (25). However, the question as to why the plasma membrane CrTs from rat and mouse differ warrants further study.
Feeding 3% creatine for 6 wk consistently resulted in an ~30% decrease in the content of the plasma CrT compared with controls. As this decrease was similar to that found in CrT activity, we may conclude that the regulation of creatine transport during creatine supplementation is due to a decrease in the number of active CrTs rather than any kinetic regulation, such as phosphorylation (25). We found no difference in the total CrT pool, which agrees with the understanding that creatine feeding has no effect on mitochondrial content or function. FeedingRegulation of intracellular creatine content.
The total creatine content of a cardiomyocyte is dependent on the
rates of creatine uptake, creatine retention, and creatine loss
via creatinine. Production of creatinine is spontaneous and nonenzymatic and occurs at a rate of ~0.2
µmol · g wet
wt1 · day
1 (2% of
the total pool). The calculated creatine uptake rate measured in this
study in control hearts is equivalent to ~4.0
µmol · g wet
wt
1 · day
1. Because
intracellular creatine is normally constant, a steady state must be
attained by the simultaneous transport of creatine into the cell and
efflux of creatine from the cell. As the rate of creatine loss as
creatinine is relatively low, creatine efflux from the cell must be by
a second mechanism at a rate approaching that of creatine influx. The
mechanism by which creatine can simultaneously be transported into and
out of the cell is unclear. Dodd et al. (7) demonstrated
that cells with a preloaded pool of radiolabeled creatine did not lose
label to the medium unless creatine was provided extracellularly. They
showed that, in the presence of extracellular creatine, creatine efflux
was catalyzed at a rate equal to that of creatine influx, suggesting
that the creatine concentration is maintained at a steady state under
normal conditions by a simultaneous transport into the cell and efflux
from the cell, both processes catalyzed by the CrT.
Conclusions. Using new techniques for measuring both the plasma membrane content of the CrT and its activity under physiological conditions, we show a coregulation of the plasma membrane CrT content and activity under conditions of creatine supplementation and depletion. A predominantly mitochondrial pool of CrT remains constant. This has important implications for studies in heart and other tissues that have reported changes in the total CrT content only (25). To further understand the biology of the CrT, future studies must endeavor to combine measurements of CrT activity and CrT isoform localization in healthy and diseased myocardium.
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ACKNOWLEDGEMENTS |
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-Guanidinopropionic acid was provided as a gift from Dr. Ralf
Jäger, Degussa Health and Nutrition, Freiburg, Germany.
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FOOTNOTES |
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This study was supported by the British Heart Foundation.
Address for reprint requests and other correspondence: E. Boehm, Wellcome Trust Centre for Human Genetics, Univ. of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom (E-mail: ernie.boehm{at}bioch.ox.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00259.2002
Received 11 June 2002; accepted in final form 31 July 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Balsom, PD,
Soderlund K,
and
Ekblom B.
Creatine in humans with special reference to creatine supplementation.
Sports Med
18:
268-280,
1994[ISI][Medline].
2.
Bessman, SP,
and
Carpenter CL.
The creatine-creatine phosphate energy shuttle.
Annu Rev Biochem
54:
831-862,
1985[ISI][Medline].
3.
Bessman, SP,
and
Geiger PJ.
Transport of energy in muscle: the phosphorylcreatine shuttle.
Science
211:
448-452,
1982[ISI].
4.
Bloch, K,
Schoenheimer R,
and
Rittenberg D.
Rate of formation and disappearance of body creatine in normal animals.
J Biol Chem
138:
155-166,
1941.
5.
Boehm, EA,
Chan S,
Monfared M,
Zhang J,
Liu J,
and
Neubauer S.
Simultaneous decreases in the creatine transporter, creatine content and creatine kinase in an experimental dog model of heart failure (Abstract).
Circulation
104:
II-119,
2001.
6.
Daniels, GM,
and
Amara SG.
Selective labeling of neurotransmitter transporters at the cell surface.
Methods Enzymol
296:
307-318,
1998[Medline].
7.
Dodd, JR,
Zheng T,
and
Christie DL.
Creatine accumulation and exchange by HEK293 cells stably expressing high levels of a creatine transporter.
Biochim Biophys Acta
1472:
128-136,
1999[ISI][Medline].
8.
Fitch, CD,
Shields RP,
Payne WF,
and
Dacus JM.
Creatine metabolism in skeletal muscle. 3. Specificity of the creatine entry process.
J Biol Chem
243:
2024-2027,
1968
9.
Garcia-Delgado, M,
Peral MJ,
Cano M,
Calonge ML,
and
Ilundain AA.
Creatine transport in brush-border membrane vesicles isolated from rat kidney cortex.
J Am Soc Nephrol
12:
1819-1825,
2001
10.
Guerrero-Ontiveros, ML,
and
Wallimann T.
Creatine supplementation in health and disease. Effects of chronic creatine ingestion in vivo: down-regulation of the expression of creatine transporter isoforms in skeletal muscle.
Mol Cell Biochem
184:
427-437,
1998[ISI][Medline].
11.
Harris, RC,
Soderlund K,
and
Hultman E.
Elevation of creatine in resting and exercised muscle of normal subjects by creatine supplementation.
Clin Sci (Colch)
83:
367-374,
1992[ISI][Medline].
12.
Horn, M,
Frantz S,
Remkes H,
Laser A,
Urban B,
Mettenleiter A,
Schnackerz K,
and
Neubauer S.
Effects of chronic dietary creatine feeding on cardiac energy metabolism and on creatine content in heart, skeletal muscle, brain, liver and kidney.
J Mol Cell Cardiol
30:
277-284,
1998[ISI][Medline].
13.
Loike, JD,
Zalutsky DL,
Kaback E,
Miranda AF,
and
Silverstein SC.
Extracellular creatine regulates creatine transport in rat and human-muscle cells.
Proc Natl Acad Sci USA
85:
807-811,
1988[Abstract].
14.
Lowry, OH,
Rosebrough MJ,
Farr AL,
and
Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem
193:
265-275,
1951
15.
Mekhfi, H,
Hoerter J,
Lauer C,
Wisnewsky C,
Schwartz K,
and
Ventura Clapier R.
Myocardial adaptation to creatine deficiency in rats fed with -guanidinopropionic acid, a creatine analogue.
Am J Physiol Heart Circ Physiol
258:
H1151-H1158,
1990
16.
Moller, A,
and
Hamprecht B.
Creatine transport in cultured cells of rat and mouse brain.
J Neurochem
52:
544-550,
1989[ISI][Medline].
17.
Neubauer, S,
Horn M,
Naumann A,
Tian R,
Hu K,
Laser M,
Friedrich J,
Gaudron P,
Schnackerz K,
Ingwall JS,
and
Ertl G.
Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction.
J Clin Invest
95:
1092-1100,
1995[ISI][Medline].
18.
Neubauer, S,
Hu K,
Horn M,
Remkes H,
Hoffmann KD,
Schmidt C,
Schmidt TJ,
Schnackerz K,
and
Ertl G.
Functional and energetic consequences of chronic myocardial creatine depletion by beta-guanidinopropionate in perfused hearts and in intact rats.
J Mol Cell Cardiol
31:
1845-1855,
1999[ISI][Medline].
19.
Neubauer, S,
Remkes H,
Spindler M,
Horn M,
Wiesmann F,
Prestle J,
Walzel B,
Ertl G,
Hasenfuss G,
and
Wallimann T.
Downregulation of the Na+-creatine cotransporter in failing human myocardium and in experimental heart failure.
Circulation
100:
1847-1850,
1999
20.
Ogorman, E,
Beutner G,
Wallimann T,
and
Brdiczka D.
Differential effects of creatine depletion on the regulation of enzyme activities and on creatine-stimulated mitochondrial respiration in skeletal muscle, heart, and brain.
Biochim Biophys Acta
1276:
161-170,
1996[ISI][Medline].
21.
Ponticos, M,
Lu QL,
Morgan JE,
Hardie DG,
Partridge TA,
and
Carling D.
Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle.
EMBO J
17:
1688-1699,
1998
22.
Powell, T,
Noma A,
and
Severs NJ.
Isolation and culture of adult cardiac myocytes.
In: Cell Biology: A Laboratory Handbook (2nd ed.). New York: Academic, 1998, p. 125-132.
23.
Seppet, EK,
Adoyaan AJ,
Kallikorm AP,
Chernousova GB,
Lyulina NV,
Sharov VG,
Severin VV,
Popovich MI,
and
Saks VA.
Hormone regulation of cardiac energy metabolism. I. Creatine transport across cell membranes of euthyroid and hyperthyroid rat heart.
Biochem Med
34:
267-279,
1985[ISI][Medline].
24.
Shoubridge, EA,
Challiss RA,
Hayes DJ,
and
Radda GK.
Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.
Biochem J
232:
125-131,
1985[ISI][Medline].
25.
Snow, RJ,
and
Murphy RM.
Creatine and the creatine transporter: a review.
Mol Cell Biochem
224:
169-181,
2001[ISI][Medline].
26.
Stephan, MM,
Chen MA,
Penado KM,
and
Rudnick G.
An extracellular loop region of the serotonin transporter may be involved in the translocation mechanism.
Biochemistry
36:
1322-1328,
1997[ISI][Medline].
27.
Wallimann, T,
Wyss M,
Brdiczka D,
Nicolay K,
and
Eppenberger HM.
Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the "phosphocreatine circuit" for cellular energy homeostasis.
Biochem J
281:
21-40,
1992[ISI][Medline].
28.
Walzel, B,
Speer O,
Boehm E,
Kristiansen S,
Chan S,
Clarke K,
Magyar JP,
Richter EA,
and
Wallimann T.
New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle.
Am J Physiol Endocrinol Metab
283:
E390-E401,
2002
29.
Willott, CA,
Young ME,
Leighton B,
Kemp GJ,
Boehm EA,
Radda GK,
and
Clarke K.
Creatine uptake in isolated soleus muscle: kinetics and dependence on sodium, but not on insulin.
Acta Physiol Scand
166:
99-104,
1999[ISI][Medline].
30.
Wyss, M,
and
Kaddurah Daouk R.
Creatine and creatinine metabolism.
Physiol Rev
80:
1107-1213,
2000
31.
Zweier, JL,
Jacobus WE,
Korecky B,
and
Brandejs Barry Y.
Bioenergetic consequences of cardiac phosphocreatine depletion induced by creatine analogue feeding.
J Biol Chem
266:
20296-20304,
1991