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Am J Physiol Endocrinol Metab 291: E1250-E1257, 2006. First published July 18, 2006; doi:10.1152/ajpendo.00060.2006
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Electrolysis stimulates creatine transport and transporter cell surface expression in incubated mouse skeletal muscle: potential role of ROS

Wim Derave,1 Nadine Straumann,2 Robert A. Olek,1 and Peter Hespel1

1Research Centre for Exercise and Health, Department of Biomedical Kinesiology, Faculty of Kinesiology and Rehabilitation Sciences, Katholieke Universiteit Leuven, Leuven, Belgium; and 2Institute of Cell Biology, ETH Zürich, Zurich, Switzerland

Submitted 7 February 2006 ; accepted in final form 30 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Electrical field stimulation of isolated, incubated rodent skeletal muscles is a frequently used model to study the effects of contractions on muscle metabolism. In this study, this model was used to investigate the effects of electrically stimulated contractions on creatine transport. Soleus and extensor digitorum longus muscles of male NMRI mice (35–50 g) were incubated in an oxygenated Krebs buffer between platinum electrodes. Muscles were exposed to [14C]creatine for 30 min after either 12 min of repeated tetanic isometric contractions (contractions) or electrical stimulation of only the buffer before incubation of the muscle (electrolysis). Electrolysis was also investigated in the presence of the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD) and catalase. Both contractions and (to a lesser degree) electrolysis stimulated creatine transport severalfold over basal. The amount of electrolysis, but not contractile activity, induced (determined) creatine transport stimulation. Incubation with SOD and catalase at 100 and 200 U/ml decreased electrolysis-induced creatine transport by ~50 and ~100%, respectively. The electrolysis effects on creatine uptake were completely inhibited by beta-guanidino propionic acid, a competitive inhibitor of (creatine for) the creatine transporter (CRT), and were accompanied by increased cell surface expression of CRT. Muscle glucose transport was not affected by electrolysis. The present results indicate that electrical field stimulation of incubated mouse muscles, independently of contractions per se, stimulates creatine transport by a mechanism that depends on electrolysis-induced formation of ROS in the incubation buffer. The increased creatine uptake is paralleled by an increased cell surface expression of the creatine transporter.

reactive oxygen species; exercise; oxidative stress; glucose transport

DURING PHYSICAL ACTIVITY, the active skeletal muscles need additional supply of fuels, such as glucose, fatty acids, and amino acids, to sustain contractile activity. The effects of muscular activity and exercise on muscle metabolism are often studied using the isolated rodent muscle incubation model. While placing isolated rodent muscles (e.g., soleus or epitrochlearis) in an incubation bath between two electrodes, electrical field stimulation can induce nerve-independent contractions. Such in vitro contractions have long been shown to potently stimulate the transport of glucose (14), fatty acids (9), and amino acids (22) in isolated skeletal muscles.

A process that is frequently overlooked in electrical field stimulation experiments with incubated muscles is electrolysis. Exposure of a physiological buffer to a 95% O2-5% CO2 gas mixture results in very high PO2 (~700 mmHg). Such a condition facilitates the formation of reactive oxygen species (ROS) due to electrolysis, predominantly the hydroxyl radical (·OH) and the superoxide anion radical (O2·–) at the side of the anode and cathode, respectively (18). Reactive oxygen metabolites are known to play an important role in the regulation of cell function in general (8) and skeletal muscle in particular (24). In 1984, Lamb and Webb (17) showed that electrolysis of an oxygenated buffer leads to relaxation of isolated arteries and that this effect is inhibited by scavenging enzymes of ROS. Interestingly, further studies have shown that ROS, in particular the superoxide-derived ·OH and R·, play a role in myocardial reperfusion injury (33), and electrolysis became an established method for studying the physiological role of ROS in cardiovascular function. ROS have been shown to stimulate muscle glucose transport in L6 myotubes (16). In incubated rat muscles, direct addition of H2O2 or enzymatic production of ROS using glucose oxidase stimulates glucose transport at micromolar H2O2 concentrations (3, 12). Studies in other cell types (human platelets and cultured rabbit epithelial cells) have shown that sodium-dependent transporter systems are also sensitive to stimulation by ROS (2, 4).

Creatine and phosphocreatine form an important phosphagen system in mammalian cells with high and fluctuating energy demands. Creatine is directly linked to the resynthesis of ATP through the creatine kinase reaction and the phosphocreatine shuttle (30). Elevation of the muscular and neuronal creatine content by oral creatine supplementation has recently been shown to improve muscular performance and to protect against certain muscular and neurodegenerative disorders (13, 31). Creatine transport into muscle cells is facilitated by a specific Na+- and Cl–-dependent creatine transporter (CRT). CRT regulation, however, is poorly understood (19). Elegant studies using one-legged cycling in humans (11, 27) showed that, during a 1-wk oral creatine supplementation period, muscle creatine accumulation was more pronounced in the exercised leg than in the contralateral leg. However, whether this effect is due to enhanced muscle perfusion and/or to direct stimulation of muscle creatine uptake by contractions is still unclear. Evidence of locally regulated metabolite uptake in active muscles has previously been shown for glucose (1) and was confirmed in contemporary in vitro experiments in isolated rodent muscles (14). In fact, later studies showed that muscle contractions stimulate the translocation and activation of the main glucose transporter (GLUT4) to the plasma membrane by intracellular signals (21; reviewed in Ref. 25).

The present study hypothesized that isolated muscle contractions stimulate creatine transport in vitro. For this purpose, we exposed isolated, incubated mouse soleus and extensor digitorum longus (EDL) muscles to [14C]creatine during electrical field stimulation. Additionally, we investigated the possible confounding effect of electrolysis in the study of electrically stimulated contractions in muscle creatine transport and the potential contribution of ROS herein. Finally, we also explored the possible contribution of the sarcolemmal CRT content in this process.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Muscle preparation. Male NMRI mice (n = 118) weighing 35–50 g were purchased from Janvier Breeding Center (Le Genest St.-Isle, France). The experimental protocol was approved by the Ethics Committee for Animal Research at K. U. Leuven. The mice were anesthetized with an intraperitoneal injection (50 mg/kg body wt) of pentobarbital sodium (Nembutal). Hindlimbs were exposed, and soleus and extensor digitorum longus (EDL) muscles from both legs were excised. After dissection of the muscles, the mice were killed by cervical dislocation. The muscles were incubated in an organ bath containing 8 ml (unless otherwise stated) of a Krebs-Henseleit solution (concentrations in mmol/l: 118 NaCl, 25 NaHCO3, 5 KCl, 1 MgSO4, 1 KH2PO4, 2.5 CaCl2, 8 glucose, and 32 mannitol). The incubation medium was continuously gassed with a mixture of 95% O2-5% CO2 (resulting in a buffer PO2 of 600–700 mmHg) and maintained at 30°C, and the muscles were allowed to recover without tension for 20 min following the isolation prior to intervention.

Intervention protocols. Incubated muscles were exposed to either electrically induced muscle contractions or electrolyzed incubation medium in the absence of contractions. In the contraction experiments, wires were attached to the tendons, and muscles were mounted vertically in an incubation bath between two platinum surface electrodes (Hugo Sachs Electronik, March, Germany) with one tendon attached to a force transducer. Electrical field stimulation was obtained by direct currents (100 mA) delivered in tetanic trains of 350 ms duration with a pulse frequency of 50 Hz for soleus and 100 Hz for EDL and a pulse duration of 1 ms (unless otherwise stated). Four minutes after the initial determination of the optimal muscle length (L0), muscles were tetanically stimulated for 12 min in six consecutive blocks of 2 min each. In the first block, the rest interval between tetani was 3.8 s, and this interval decreased with each block (3.1, 2.6, 2.1, 1.6, and 1.3 s for the subsequent five blocks). During electrolysis experiments, the electrical stimulation protocol was performed in the incubation medium prior to mounting of the muscle, whereafter the muscle was incubated within 1 min after completion of the electrolysis. Whenever possible, the intervention (contractions, electrolysis) was performed on the muscles of one leg and the muscles of the other leg served as simultaneous nonstimulated controls, and effects were then analyzed by paired statistical comparison.

Creatine and glucose transport determination. After the different interventions, creatine transport was measured by exposing muscles to radiolabeled creatine for 30 min with a medium concentration of 0.5 mM creatine [except in the experiments with beta-guanidino propionic acid (beta-GPA), where creatine concentration was 0.05 mM and beta-GPA 2 mM], 105 nCi/ml [4-14C]creatine (American Radiolabeled Chemicals, St. Louis, MO), and 105 nCi/ml [3H]inulin, for determination of extracellular space. Glucose transport was measured by 10-min exposure to 1 mM 2-deoxy-D-glucose, 1 mM D-mannitol, 675 nCi/ml 2-deoxy-D-[2,6-3H]glucose, and 365 nCi/ml D-[1-14C]mannitol (Amersham Biosciences). After the exposure to radiolabeled metabolites, muscles were blotted dry, weighed, and dissolved in 0.5 ml of tissue solubilizer (Solvable; PerkinElmer, Boston, MA). Uptake of creatine and 2-deoxy-D-glucose was calculated as the difference between the total tissue radioactivity (dpm) and the amount of radioactivity present in the tissue extracellular (inulin or mannitol) space and expressed per milligram wet muscle weight. In preliminary experiments, the linearity of creatine transport over incubation time was assessed in two muscles at 30, 60, and 90 min of exposure to radiolabeled creatine and appeared to be linear (41, 64, and 106 nmol/g, respectively).

Biotinylation experiments and CTR quantification. Soleus and EDL muscles from both legs were excised and incubated in previously electrolyzed or control medium, as described above. Muscles were incubated for 30 min in sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin; Pierce) with a final concentration of 0.5 mg/ml in Krebs-Henseleit buffer. Nonreacted biotinylation reagent was removed by washing the muscles twice for 1 min in glycine buffer (100 mM glycine, 0.1 mM CaCl2, 1 mM MgCl2 in PBS). Muscles (one soleus and one EDL per sample) were then homogenized in 500 µl of ice-cold homogenization buffer containing 20 mM HEPES, 250 mM sucrose, 1 mM EDTA, and a protease inhibitor cocktail (Mini-Complete) in a Polytron homogenizer at full speed for 30 s. Homogenates were spun at 2,000 g for 10 min (4°C), and Triton X-100 was added to the supernatant in a final concentration of 1% (vol/vol). An aliquot of supernatant containing 400 µg of total protein (quantitated with the BCA Protein Assay Kit; Pierce) 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 homogenization 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 in 50 mM dithiothreitol for 2 h, and CRT expression was quantified as previously described (28). In short, biotynilated proteins were separated using the NuPAGE polyacrylamide 4–12% Bis-Tris gels system (Invitrogen) and transferred to Protran nitrocellulose membranes by semidry Western blotting. Membranes were subsequently probed for 1 h with a 1:5,000 dilution of rabbit anti-NH2-terminal CRT antibody (Alpha Diagnostics) in TBS containing 0.1% Tween-20 and 5% skim milk powder. The secondary goat anti-rabbit horseradish peroxidase (HRP) antibody (Calbiochem) was diluted 1:10,000 in TBS containing 0.1% Tween-20 and 5% skim milk powder. The immunoreactive bands were visualized with a chemiluminescence detection kit (Axon Lab, Switzerland).

H2O2 quantification. H2O2 concentration was determined fluorometrically by the method of Guilbault et al. (10), which is based on the oxidation of nonfluorescent substrate p-hydroxyphenylacetic acid (PHPA) to the stable fluorescent product, named 2,2'-dihydroxybiphenyl-5,5'-diacetic acid (PHPA)2, by H2O2 in the presence of HRP (EC 1.11.1.7 [EC] ). (PHPA)2 fluorescence was measured at an excitation maximum of 320 nm and emission maximum of 400 nm. The reaction mixture consisted of Krebs-Henseleit buffer, 166 µg/ml PHPA, and 10 U of HRP/ml. H2O2 generation was estimated at 6 and 12 min during and in 10-min intervals following electrical stimulation. Known concentrations of H2O2 were used to construct a standard curve.

Statistics. Effects of interventions were statistically analyzed with paired (contralateral muscle within one mouse) or unpaired (between mice) Student's t-tests or factorial analysis of variance (ANOVA) depending on the intervention examined. Post hoc analyses of significant ANOVA effects were performed with Tukey tests. In some circumstances, when no statistical difference was observed between effects in soleus and EDL, data from both muscle types were pooled for statistical evaluation. Statistical significance was assumed when P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Effects of electrical stimulation. A standard stimulation protocol of 12 min of repeated tetanic stimulations was applied to evaluate the effects of muscle contractions on creatine transport. As shown in Fig. 1A, electrically stimulated muscle contractions stimulated creatine transport two- and fourfold in soleus and EDL, respectively. However, the magnitude of creatine transport stimulation depended on the incubation volume: higher volume resulted in a smaller degree of stimulation (Fig. 1B). This finding suggested that the stimulating factor of creatine transport is dilutable and located extracellularly.


Figure 1
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  		Fig. 1. Effect of 12-min repeated tetanic contractions [350 ms train duration, 1 ms pulse duration, 50 Hz for soleus and 100 Hz for extensor digitorum longus (EDL) muscles, decreasing rest interval between tetani: 3.8, 3.1, 2.6, 2.1, 1.6, and 1.3 s on muscle creatine transport rate (nmol·g–1·h–1)] in an incubation volume of 10 ml (A) and of 5 or 15 ml (B). Data from soleus and EDL were pooled in B. Data are means ± SE of 9 (A) and 5–8 (B) observations. *Different (P > 0.05) vs. electrical stimulation and rest; §different (P > 0.05) vs. 5 ml.

 
Therefore, we decided to perform two series of control experiments to investigate whether the electrical stimulation by the electrodes, and the inherent electrolysis of the oxygenated physiological buffer, would generate the creatine transport-stimulating factor. A schematic representation of the different intervention protocols is visualized in Fig. 2A, and the results are shown in Fig. 2B. First, a fixed volume of well-oxygenated buffer was exposed to electrical stimulation (12 min of repeated tetanic stimulation) in the absence of a muscle. Thereafter, a muscle was placed in the previously electrolyzed buffer, and creatine transport was measured in the absence of electrical stimulation. As shown in Fig. 2B, this intervention (named electrolysis) significantly increased muscle creatine transport twofold (P < 0.05), although the muscle had not contracted once. Second, muscles were subjected to contractions as in Fig. 1A, but after completion of the contraction series the incubation medium was discarded (intended to remove the stimulating factor from the bath), and creatine transport in the previously contracted muscle was determined in a fresh incubation medium. By doing this (contractions alone), no stimulation of creatine transport was observed (Fig. 2B). When pulse durations of 0.2 ms instead of 1.0 ms were applied, the stimulating effects of electrolysis and electrolysis plus contractions on creatine transport were no longer observed. This finding suggests that creatine transport stimulation is proportional to the amount of electrolysis. In the next experiment, the tetanic train duration was increased from 350 to 700 ms, leading to a further stimulation of creatine transport in EDL from about two- to about sixfold over basal (P < 0.05, data not shown).


Figure 2
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  		Fig. 2. A: schematic representation of the 4 different interventions prior to creatine transport measurements (results in B). The medium or the muscle is shaded when exposed to electrical stimulation. Different conditions during electrical stimulation included electrolysis (electrical stimulation of buffer prior to incubation of muscle), contractions + electrolysis (electrical stimulation of incubated muscle, as in Fig. 1), and contractions (electrical stimulation of incubated muscle, but subsequent removal of electrolyzed buffer). B: creatine transport stimulation (basal rate set at 1) following a 12-min protocol of repeated tetanic stimulation (350-ms trains, 100 Hz) with pulse durations of 1 or 0.2 ms in incubated EDL muscles. Data are means ± SE of 4–7 observations. *Different (P > 0.05) vs. rest.

 
Involvement of ROS. To investigate whether the formation of ROS is involved in the electrolysis-induced stimulation of muscle creatine transport, we tested the effect of the ROS scavenging enzymes SOD and catalase. As shown in Fig. 3A, the addition of 100 U/ml each of SOD (catalyzes the formation of H2O2 from O2·–) and catalase (catalyzes the formation of H2O and O2 from H2O2) during electrolysis reduced stimulation of muscle creatine transport by ~50%. Doubling the concentration of the scavenging enzymes (200 U/ml) completely negated the stimulation, whereas the creatine transport rate in muscles incubated in a control solution (not exposed to electrolysis) was not affected by the scavenging enzymes. The effect of catalase alone (200 U/ml) was smaller than with both SOD and catalase present, indicating that both the superoxide and the hydroxyl radical are likely involved in creatine transport stimulation.


Figure 3
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  		Fig. 3. A: creatine transport rate (nmol·g–1·h–1) in soleus and EDL at rest and following prior 12-min electrical stimulation of the medium (stimulation protocol as in Fig. 1, except train duration is set at 700 ms instead of 350 ms). In the different experiments, superoxide dismutase (SOD) and catalase (CAT) concentrations are, respectively, 0 and 0 U/ml (control), 100 and 100 U/ml (SOD + CAT 100), 200 and 200 U/ml (SOD + CAT 200), and 0 and 200 U/ml (CAT 200). Data from soleus and EDL are pooled and represent means ± SE of 8–14 observations. B: H2O2 concentration (µM) in the medium. During the first 12 min, the medium was electrically stimulated. Stimulation protocol and addition of SOD and CAT as in A. Data represent means ± SE of 6 or 12 (in Control group) observations. C: correlation of electrolysis-induced creatine transport (A) in H2O2 concentration (B) data in different conditions of reactive oxygen species (ROS)-scavenging enzyme additions; r = 0.96 (P < 0.05).

 
We evaluated the formation of ROS by measuring the H2O2 concentration in the electrolyzed medium (Fig. 3B). The H2O2 concentration gradually increased to ~160 µM during the 12 min of electrical stimuation (control) and remained relatively constant over the subsequent 30 min. The addition of scavenging enzymes partially (in the case of 200 U/ml catalase addition or 100 U/ml catalase + 100 U/ml SOD addition) or nearly completely (in the case of 200 U/ml catalase + 200 U/ml SOD addition) inhibited the H2O2 generation. Figure 3C indicates that the peak H2O2 concentration (at 12 min) shown in Fig. 3B correlated positively (r = 0.96, P < 0.05) with the corresponding creatine transport rates (Fig. 3A).

We tested whether the direct addition of H2O2 in a concentration of 160 µM would stimulate creatine transport. The data in Fig. 4 indicate that this was not the case. Furthermore, we investigated the stability of the unknown factor responsible for stimulation of creatine transport in the electrolyzed medium. Two experiments were performed in series in the same incubation bath. As outlined in Fig. 5A, muscle A was exposed to radiolabeled creatine for 30 min either at rest or with electrical stimulation during the first 12 min. Muscle A was then removed and counted for radioactivity, and a fresh muscle B was added to the same medium. Muscle B was exposed to radiolabeled creatine for 30 min either at rest or with electrical stimulation during the first 12 min. Then muscle B was counted. The results show that in the rest condition there was no effect of serial incubations in the same medium, whereas in the contractions plus electrolysis condition, the second muscle (B) had significantly higher creatine transport than the first muscle (A) (Fig. 5B). This indicates that there is a residual effect of the first electrical stimulation period on the second experiment. Thus the factor causing stimulation of creatine transport appears to be stable in the medium for at least half an hour.


Figure 4
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  		Fig. 4. Creatine transport rate (nmol·g–1·h–1) in soleus and EDL without (open bars) or with (filled bars) addition of 160 µM H2O2 to the medium. Each bar represents means ± SE of 6 observations.

 

Figure 5
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  		Fig. 5. Effect of consecutive experiments in the same incubation medium. A: the protocol. Shaded means electrically stimulated as in Fig. 1. B: results of creatine transport (n = 4; data combined from soleus and EDL).

 
Involvement of CRT. Experiments were performed to investigate whether electrolysis stimulated creatine transport across the sarcolemma through the CRT. The creatine analog GPA is a known substrate competitor of creatine for the creatine transporter. As shown in Fig. 6A, electrolysis did not stimulate muscle creatine transport in the presence of GPA, indicating that the effect is mediated through the CRT. To further explore this, we investigated the sarcolemmal protein content of CRT by exposing the muscles to a cell-impermeable biotinylation compound. As shown in Fig. 6B, electrolysis stimulated the amount of biotinylated CRT approximately twofold, indicative of increased sarcolemmal expression of CRT.


Figure 6
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  		Fig. 6. A: creatine transport rate (nmol·g–1·h–1) in soleus and EDL at rest and following prior electrical stimulation of the medium (stimulation protocol as in Fig. 4A) in the presence of 0.05 mM creatine and 0 (Control) or 2 mM beta-guanidino propionic acid (GPA). Data from soleus and EDL are pooled and represent means ± SE of 8–10 observations. *Different (P > 0.05) vs. rest; §different (P > 0.05) vs. Control. B: cell surface expression of the 58-kDa band of creatine transporter (CRT) in soleus and EDL muscles incubated in control (Rest, R) and electrolyzed (E) medium. Data represent means ± SE of 3 observations. Individual blots of the biotinylated fractions are shown.

 
Glucose transport. To evaluate whether the effect of electrolysis is specific for creatine transport or also applies to other membrane transport systems, we investigated the effects of muscle contractions and electrolysis on glucose transport. The data presented in Fig. 7 show that electrolysis to a degree that stimulated creatine transport sixfold did not stimulate glucose transport, whereas electrically induced muscles contractions resulted in a two- to threefold stimulation of glucose transport.


Figure 7
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  		Fig. 7. Glucose transport rate (µmol·g–1·h–1) at rest, following prior electrical stimulation of the medium, and following muscle contractions (stimulation protocol as in Fig. 4A) in soleus and EDL muscles. Data represent means ± SE of 6–7 observations. *Different (P > 0.05) vs. Rest.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Electrical stimulation of incubated frog or rodent muscles is a common method to investigate muscle metabolism during contractions. An a priori assumption in this approach is that the observed effects, indeed, are caused by the contractile activity resulting from the electrical stimulation. Driven by recent indirect evidence for a possible effect of muscular activity on muscle creatine accumulation in humans (5, 11, 27), we applied this technique to investigate the effects of contractions on creatine transport in isolated mouse skeletal muscles. Interestingly, we found that a protocol of electrically stimulated tetanic muscle contractions, routinely used to study muscle fatigue (6), potently stimulated creatine transport in both soleus (slow-twitch type) and EDL (fast-twitch type) muscles (severalfold over basal). However, a number of observations indicated that the activation of muscle creatine transport was not elicited by the contractions per se but was due to the electrolysis intrinsic to electrical field stimulation: 1) the magnitude of creatine transport stimulation was dependent on the incubation volume, indicating that the stimulating factor is extramuscular and can be diluted by the buffer; 2) creatine transport stimulation is not observed when the buffer is discarded and renewed following muscle contractions and prior to the addition of radiolabeled creatine; 3) creatine transport is enhanced in a nonstimulated muscle when placed in a bath that was previously exposed to electrical stimulation in the absence of a muscle; and 4) the magnitude of creatine transport stimulation is directly proportional to the amount of electrical stimulation but not to the amount of muscle tension development. These observations prove that the effect of electrical stimulation on muscle creatine transport is contraction independent. Instead, electrolysis resulting from the electrical currents between stimulation electrodes is the stimulatory factor involved.

Given the high PO2 of the incubation buffer, generation of oxygen free radicals or ROS is the most likely mechanism by which electrolysis could initiate the stimulatory effect on creatine transport. Indeed, as shown in Fig. 3B, the currently applied electrical stimulation protocol causes substantial accumulation of H2O2 in the medium. A ROS-mediated artifact of electrical stimulation has previously been described in the study of smooth muscle contractions in isolated artery segments and of mechanical function of the isolated heart (15, 17, 18). ROS formation starts with the reduction of molecular oxygen to form the relatively unstable superoxide (O2·–), which is further catalyzed by dismutation to the more stable hydrogen peroxide (H2O2). H2O2 can be spontaneously reduced by O2·– to form the hydroxyl radical (·OH).

Evidence for the involvement of ROS in the stimulation of muscle creatine transport comes from our experiments with the ROS-scavenging enzymes SOD and catalase. Our data show that both the generation of H2O2 and the stimulation of creatine transport during electrical stimulation can be blocked in a dose-dependent manner, leading to a nearly complete inhibition with the addition of 200 U/ml SOD and 200 U/ml catalase. Catalase alone (200 U/ml), however, induced only half-maximal inhibition. Incubation with SOD and catalase in the absence of electrolysis did not affect muscle creatine transport, indicating that the observed effects are due to prevention of ROS generation rather than a direct effect of these proteins on the muscle. Furthermore, electrolysis-induced creatine transport correlated strongly with the amount of accumulated H2O2 in the medium (Fig. 3C). On the basis of this strong correlation and the finding that the creatine transport stimulating factor is stable in the medium for at least 30 min (see Fig. 5), one would expect H2O2 (a relatively stable ROS) to be the responsible factor. However, the direct addition of H2O2 did not stimulate creatine transport (Fig. 4). Lecour et al. (18) have directly measured concentrations of the less stable ROS during electrolysis of a Krebs-Henseleit buffer using electron spin resonance techniques. They concluded that both hydroxyl (·OH) and superoxide (O2·–) were formed in a dose-dependent manner during electrolysis (18). In the present experiments, inhibition of electrolysis-induced creatine transport was more pronounced in the presence of combined SOD and catalase than with catalase alone. Therefore, it is hypothesized that, although H2O2 strongly correlates with creatine transport stimulation, the additional and simultaneous generation of other ROS is required to stimulate muscle creatine transport.

The physiological relevance of these findings remains to be elucidated, yet lies beyond the scope of this study. However, the results in Fig. 2B show that creatine transport is higher when electrical stimulation is performed in the presence than in the absence of muscle tissue in the incubation medium. This could indicate that muscle contractions produced additional ROS (24), which further enhanced creatine transport. However, the present data point to a methodological artifact of electrical stimulation and therefore cannot predict whether in vivo there is a role of ROS in the induction of metabolic processes in muscle by contractile activity.

In their study on perfused isolated rabbit hearts, Jackson et al. (15) have shown that electrolysis-induced ROS stimulate the uptake of radiolabeled albumin in ventricle tissue and lead to increased vascular permeability to macromolecules. To exclude the possibility that creatine transport was increased by virtue of an unspecific increase in membrane permeability, we have studied the effect of electrolysis on creatine transport while blocking the Na+- and Cl–-dependent CTR by GPA. Our findings show that GPA can completely block the electrolysis-induced increase in creatine transport, which raises the hypothesis that ROS initiate an activation of Na+- and Cl–-dependent CRTs by a specific mechanism.

In our biotinylation experiments (Fig. 6B), we showed for the first time that increased creatine transport in muscles was associated with increased sarcolemmal expression of CRT. Given that the increased expression occurred rapidly (30 min), translocation from an intracellular pool to the sarcolemma, rather than de novo CRT protein synthesis, probably explains this effect. Such a translocation process toward the sarcolemma would be analogous with other transport proteins found in muscle cells, such as the glucose transporter (GLUT4) and fatty acid transporter (FAT) (7, 20). Interestingly, Tran et al. (29) recently showed that cyclosporin A inhibits creatine uptake and the cell surface expression of CRT in C2C12 muscle cells. These and our data thus suggest that regulation of creatine uptake in muscle cells is effected by up-/downregulation of cell surface expression of the CRT.

The solute transmembrane transport system, for which the technique of electrical field stimulation of incubated muscles is most frequently used, is undoubtedly the glucose transport/GLUT4 system. Several reports have indicated that ROS exert a stimulatory effect on muscle glucose transport (3, 12, 16). Therefore, we have investigated whether electrolysis stimulates muscle glucose transport. Our data clearly show that the amount of electrolysis leading to H2O2 concentrations of 160 µM and a substantial increase of creatine transport did not affect glucose transport. Similar concentrations of H2O2, generated by glucose oxidase, have previously been shown to be associated with increased glucose transport in incubated skeletal muscles of Zucker rats (12). The reason for this discrepancy is presently unclear but may relate to species differences or the difference in the way the ROS are generated (electrolysis, direct addition of H2O2, or enzymatic generation with oxidases).

Other Na+-dependent transmembrane transporters are perhaps more likely to be as sensitive to ROS stimulation as the CRT. Numerous transporters present in muscle act in a Na+-dependent way (32). The CRT is a member of a superfamily of neurotransmitter transporters, with GABA, betaine, and taurine transporters as its closest relatives (23). It remains to be investigated whether other family members are also sensitive to ROS and to elucidate by which cellular mechanism this stimulation is effected (26).

In conclusion, we here show that electrical stimulation of incubated mouse skeletal muscles stimulates creatine transport. However, this effect is not caused by muscle contractions per se but is conceivably initiated by electrolysis-induced generation of ROS, inherent to electrical discharge between two electrodes immersed in a physiological well-oxygenated buffer. This phenomenon is a potentially important source of error when one is aiming to study the effects of muscle contractions with this experimental technique. The electrolysis-induced increase in creatine uptake is paralleled by an increased cell surface expression of the creatine transporter.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study is supported by grants from Onderzoeksraad K. U.-Leuven (Grant no. OT99/38) and from the Flemish Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO-Vlaanderen Grant nos. G.0255.01 and 1.5172.02N). W. Derave is a recipient of a postdoctoral fellowship from the Flemish Fonds voor Wetenschappelijk Onderzoek Vlaanderen. N. Straumann is a recipient of a doctoral fellowship of the Swiss Society for Research on Muscle Diseases and Swiss Cardiovascular Research and Training Network.


   ACKNOWLEDGMENTS
 
The skillful assistance of Monique Ramaekers is greatly acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Hespel, Dept. of Biomedical Kinesiology, FaBer - K. U. Leuven, Tervuursevest 101, B-3001 Leuven, Belgium (e-mail: Peter.Hespel{at}faber.kuleuven.be)

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.

Present affiliation of W. Derave: Department of Movement and Sport Sciences, Ghent University, Ghent, Belgium (wim.derave{at}ugent.be).


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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