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CELLULAR |
Dipartimenti di
1 Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia
2 Clinica Medica, Nefrologia e Scienze della Prevenzione
3 Farmaceutico, Universitá degli Studi di Parma, 43100 Parma, Italy
4 Dipartimento di Patologia Sperimentale, Università degli Studi di Bologna, 40126 Bologna, Italy
5 Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino ‘Carlo Bo’, 61029 Urbino, Italy
6 Department of Biochemistry, School of Life Sciences, JMS Building, University of Sussex, Brighton BN1 9QG, UK
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Abstract |
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(Received 9 June 2006;
accepted after revision 24 July 2006;
first published online 27 July 2006)
Corresponding author K. P. Wheeler: Department of Biochemistry, School of Life Sciences, JMS Building, University of Sussex, Brighton BN1 9QG, UK. Email: k.p.wheeler{at}sussex.ac.uk
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Introduction |
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-aminobutyric acid and taurine transporters (Guimbal & Kilimann, 1993; Zorzano et al. 2000; Speer et al. 2004). Creatine kinase catalyses the phosphorylation of cellular creatine and the creatine pool (creatine plus creatine phosphate) depends on the rate of creatine uptake from blood and on the rate of non-enzymic conversion to creatinine (Wyss & Kaddurah-Daouk, 2000). As only about 2% of the creatine pool per day is converted into creatinine, the most important process that controls the creatine pool is the uptake from the extracellular fluid (Murphy et al. 2003a). Normally a high creatine concentration gradient is maintained across muscle cell membranes, the cellular concentration of creatine plus creatine phosphate being 500–1000 times higher than that in the plasma (Beis & Newsholme, 1975; Wyss & Kaddurah-Daouk, 2000). Oral creatine supplementation is used widely by athletes to improve performance and muscle mass (Casey et al. 1996; Volek & Rawson, 2004) and has been extended to the medical field to treat a number of muscular, neurological and cardiovascular diseases such as gyrate atrophy (Sipila et al. 1981; Heinanen et al. 1999), McArdle disease (Vorgerd et al. 2000), Duchenne dystrophy (Felber et al. 2000; Tarnopolsky et al. 2004), myastenia gravis (Stout et al. 2001), amyotrophic lateral sclerosis (Mazzini et al. 2001) and Parkinson's disease (Matthews et al. 1999). The putative benefits of creatine supplementation generally have been attributed to an ergogenic effect: an increase in the concentration of creatine phosphate acting as an ATP buffer. Recently, however, other possibilities have been suggested. For example, creatine shows antioxidant properties (Lawler et al. 2002) and had protective effects against oxidative stress in cultured mammalian cells (Lenz et al. 2005; Sestili et al. 2006). Creatine has also been shown to have anti-inflammatory activity in endothelial cells (Nomura et al. 2003) and creatine supplementation improved neuronal differentiation and dopaminergic cell survival under adverse conditions (Andres et al. 2005). Now we report that supplementation of growth medium with creatine can enable cultured muscle cells to survive exposure to hypertonicity.
Several types of mammalian cells can survive when exposed to a hypertonic environment (up to about 500 mosmol (kg H2O)–1) because of a specific adaptation process that eventually results in their accumulating compatible osmolytes (Burg, 1995; Burg et al. 1997). This adaptation involves changes of gene expression that result in an increased synthesis either of a compatible osmolyte itself (e.g. sorbitol) or of transporters for the osmolytes, such as SNAT-2 for neutral amino acids (Alfieri et al. 2001, 2005), BGT1 for betaine (Petronini et al. 2000), SMIT for myo-inositol and TAUT for taurine (Burg et al. 1997). The usual explanation of this phenomenon is the need to replace the early cellular accumulation of inorganic ions with small organic molecules (compatible osmolytes) that do not perturb macromolecular structures as the increased concentrations of inorganic ions do. These responses were extensively characterized in kidney-derived cells (Beck et al. 1998; Beck & Neuhofer, 2005) but have also been detected in chondrocytes (De Angelis et al. 1999), macrophages (Denkert et al. 1998), endothelial cells (Petronini et al. 2000; Alfieri et al. 2002, 2004) and mesothelial cells (Matsuoka et al. 1999). Relatively few studies of muscle cells have been reported, but hypertonicity was shown to induce amino acid transport system A in vascular smooth muscle cells (Chen & Kempson, 1995), whilst hypotonicity activated taurine efflux from C2C12 myoblasts, in keeping with taurine's role as an organic osmolyte (Manolopoulos et al. 1997). Hypertonicity also caused a stimulation of NKCC (Na+–K+–2Cl– cotransporter) activity in rat L6 skeletal muscle cells (Zhao et al. 2004). Here we show not only that hypertonicity induces creatine transport in C2C12 cells but also that creatine can act like the well-established compatible osmolytes betaine, taurine and myo-inositol in protecting the cells against hypertonic stress.
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Methods |
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[14C]Creatine (55 Ci mol–1) was obtained from American Radiolabeled Chemicals Inc. (Saint Louis, MO, USA), and L-[4,5-3H]leucine from Amersham plc (Little Chalfont, UK). Media, fetal calf serum and antibiotics for culturing the cells were purchased from Gibco (Grand Island, NY, USA). Disposable plastics were obtained from Costar (Broadway, Cambridge, MA, USA), and other reagents from Sigma Chemical Co. (St Louis, MO, USA).
Cell Culture
C2C12 mouse myoblasts from the Istituto Zooprofilattico Sperimentale (Brescia, Italy) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with glutamine (2 mmol l–1), fetal calf serum (10%), penicillin (100 U ml–1) and streptomycin (100 µg ml–1). endothelial cells were obtained and cultured as previously described (Petronini et al. 2000). All cultures were kept in an incubator at 37°C in a water-saturated atmosphere of 5% CO2 in air.
Experimental growth media
The media consisted of DMEM supplemented with bovine serum albumin (0.1%, w/v), betaine (0.1 mmol l–1) and myo-inositol (0.1 mmol l–1) (Alfieri et al. 2002). When required, media were made hypertonic by addition of sucrose. The osmolalities of the media were checked with a vapour-pressure osmometer (Wescor) and normal medium was about 0.3 osmol (kg H2O)–1.
Cell viability
Cell density was assayed in terms of the protein content per dish or cell number, determined by cell counting (Alfieri et al. 2002). To test cell viability under hypertonic conditions, cells were first seeded at a density of about 104 cells cm–2 and cultured for 2 days in normal isotonic medium before it was replaced with the appropriate hypertonic media.
Cell protein
Cell protein, precipitated by trichloroacetic acid, was dissolved in 0.2 N NaOH and its concentration measured by a dye-fixation method (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as standard (Bradford, 1976).
Rate of protein synthesis
The rate of protein synthesis was measured as the rate of incorporation of labelled leucine (2.5 mCi mmol–1, 2 µCi ml–1) during a 30 min incubation of the cell monolayers. This procedure has been described in detail elsewhere (Petronini et al. 2000).
Transport measurements
Approximate initial rates of uptake of creatine by C2C12 cells were measured after the latter had been incubated in control (0.3 osmol (kg H2O)–1) or test (0.480 osmol (kg H2O)–1) medium for the desired time. The cell monolayers were quickly washed with Earle's balanced salt solution containing 0.1% glucose and then incubated in this solution for 15 min at 37°C to diminish the cellular pool of osmolytes. The cells were washed again and immediately incubated at 37°C for the desired time in the presence of 14C-labelled creatine (usually 2 µCi µmol–1). The incubations were stopped by removal of the medium and the cells were quickly washed three times with fresh cold medium. Trichloroacetic acid (5%, w/v) was added to denature the cells and the radioactivity in samples of the acid extracts was measured by scintillation counting.
The accumulation of creatine by the cells was monitored with the use of [14C]creatine added to the incubation media. The incubations were stopped after the desired time by removal of the medium and the cell monolayers quickly washed three times with Earle's balanced salt solution (containing 0.1% glucose) before they were denatured and assayed as just described.
Cellular ATP content
This was determined by a luminescence assay system (ATPLiteTM-M, Packard) as previously described (Fumarola et al. 2005).
Cellular contents of creatine and creatine phosphate
These were measured by HPLC analysis of cell extracts, obtained as follows The incubation medium was rapidly removed and ice-cold perchloric acid (3.8%) immediately added to the cell monolayer. After 30 min on ice, the acid extract was removed and neutralized with saturated Na2CO3 solution. The HPLC system consisted of two Gilson 305 solvent delivery pumps (Gilson Inc., Middleton, WI, USA), a 20 µl capacity sample injector (Rheodyne LLC, Rohnert Park, CA, USA) and a Gilson UV 115 detector (Gilson Inc.). Chromatographic separation was achieved at room temperature on a Alltima C-18 column (5 µm, 250 x 4.6 mm, Alltech Inc., Columbia, MD, USA). The mobile phase consisted of 70 mmol l–1 phosphate buffer (pH 6.0) containing 7 mmol l–1 tetrabutylammonium hydrogen sulphate. The flow rate was 0.75 ml min–1 and the eluant was monitored at 218 nm. Standard curves for creatine, creatine phosphate and creatinine, constructed by plotting peak area against solute concentration, were linear over the range 5–500 µmol l–1.
RNA isolation, cDNA synthesis and quantitative RT-PCR
Total RNA was extracted from about 2 x 106 cells by the method of Chomczynski & Sacchi (1987) with the use of TRIzol (Gibco BRL, Gaithersburg, MD, USA) and digested with DNAseI (DNA-free kit, Ambion Inc., Austin, TX, USA) to remove any contaminating genomic DNA. RNA quality was checked by electrophoresis on 1% TBE agarose gel, using a denaturing loading buffer (RNA Ladder, New England Biolabs Inc., Beverly, MA, USA). The concentration of RNA was measured with the RiboGreen probe on a fluorescence spectrophotometer Cary Eclipse (Varian Inc., Palo Alto, CA, USA). cDNA was synthesized with the use of 500 ng of RNA, 250 ng of random hexamer primers, 200 U of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 1 U of SUPERase IN (Ambion Inc., Austin, TX, USA), following Invitrogen's recommended experimental conditions.
Gene expression was assessed by quantitative real time PCR with the use of an iCycler iQ Multicolor Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). Reaction mixtures contained 2 µl of template cDNA with 300 nM of forward and reverse primers and 12.5 µl of 2xiQ SybrGreen Supermix (Bio-Rad, Hercules, CA, USA) in a total volume of 25 µl. Triplicate assays were run for each sample and each included a standard curve and a negative control. Specific oligonucleotide primers were designed with the Probe Finder 2.04 programme (Roche Applied Science, F. Hoffmann-La Roche Ltd, Basel, Switzerland) to span the exon–exon junctions of the Mus musculus genes encoding the creatine transporter (GenBank sequence AB077327.1; sense 5'-CTCTCCATGGTGACTGATGGT-3'; antisense 5'-TGCCACTAGCTGAGTAGTAGTCAAA-3') and ß-actin (GenBank sequence NM_007393.1; sense 5'-TGACAGGATGCAGAAGGAGA-3'; antisense 5'-CGCTCAGGAGGAGCAATG-3') to produce 67 and 75 bp products, respectively. The amplification protocol consisted of 3 min at 95°C followed by 40 cycles at 95°C for 30 s, 61°C for 30 s and 72°C for 30 s. Then a final melting step with a gradual increase in temperature from 50°C to 94°C was used to ensure there were no non-specific products. The relative quantitative expression of the creatine transporter, after normalization with ß-actin as housekeeping gene (Murphy et al. 2003b), was calculated as suggested by Pfaffl (2001).
Translation of endogenous mRNA by rabbit reticulocyte lysate
Rabbit reticulocyte lysate was prepared as described by Allen & Schweet (1962). Translation of endogenous mRNA in vitro by the unfractionated rabbit reticulocyte lysate was performed in reaction mixtures (125 µl) containing 30 mmol l–1 Hepes-KOH, pH 7.5, 80 mmol l–1 KCl, 1.8 mmol l–1 magnesium acetate, 50 µmol l–1 of each amino acid except leucine, 2 mmol l–1 ATP, 0.25 mmol l–1 GTP, 0.5 mmol l–1 dithiothreitol, 0.4 mmol l–1 spermidine, 0.24 µmol l–1 (2 µCi) [3H]leucine and 50 µl of lysate. Where indicated, the osmolarity of the reaction mixture was increased by addition of KCl, creatine or betaine. The complete mixture was incubated for 5 min at 28°C and then a 62.5 µl sample was taken and added to 1 ml of 0.1 M KOH, the solution was decolourized with two drops of 35% (w/v) H2O2 and 1 ml of 20% (w/v) trichloroacetic acid added. The precipitate was collected on a Whatman GF/C filter and its radioactivity measured by scintillation counting. The osmolality of the residual reaction mixture was measured with a vapour pressure osmometer (Wescor), the standard mixture being 0.373 osmol (kg H2O)–1. Under these standard conditions the [3H]leucine incorporated was 67368 ± 4883 dpm (mean ± S.D., n = 12).
Statistical analysis
Unless noted otherwise, the results are expressed as mean values ± S.D. for the indicated number of measurements. The significance of differences between the mean values recorded for different experimental conditions was calculated by Student's t test and P-values are indicated where appropriate in the figures and their legends. Curves were fitted to experimental values with the use of ‘Kaleidagraph’ (Synergy Software, Reading, PA, USA).
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Results |
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Figure 1A shows the rate of uptake of creatine by C2C12 cells that had been incubated for 16 h in media with osmolalities ranging from 0.3 to 0.5 osmol (kg H2O)–1. The rate clearly increased in parallel with the imposed hypertonicity, reaching a peak at about 0.45 osmol (kg H2O)–1. Figure 1B shows that although an increase in the rate of creatine influx was detectable only 4 h after exposing the cells to hypertonic medium, the rate continued to increase for several hours and the maximum value was reached much later, after about 24 h.
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Approximate initial rates of uptake of creatine by C2C12 cells were measured from a range of creatine concentrations, and in the presence and absence of Na+, after the cells had been exposed for 24 h to isotonic (0.3 osmol (kg H2O)–1) or hypertonic (0.48 osmol (kg H2O)–1) conditions. After subtraction of the linear Na+-independent components of influx, the remaining saturable, Na+-dependent, components could be expressed as Michaelis-Menten equations (Fig. 2). The lines shown, from the ‘Kaleidagraph’ curve-fitting program, are for Vmax values of 4.5 ± 0.4 and 7.1 ± 0.6 nmol (15 min)–1 (mg protein)–1 with Km values of 75 ± 22 and 77 ± 19 µmol l–1 for cells exposed to isotonic and hypertonic conditions, respectively. Thus the increase in the rate of uptake was caused by an increase in Vmax, with no significant change in the Km value, which was in the micromolar range, as reported in the literature for Na+-dependent creatine transport (Guimbal & Kilimann, 1993; Tran et al. 2000; Peral et al. 2002).
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Dependence on RNA and protein synthesis
Since this last conclusion is consistent with a mechanism involving an increase in the number of active transporters in the cell membrane, the dependence of the hypertonicity-induced changes in creatine transport on RNA and protein synthesis was examined. As shown in Fig. 3, induction of creatine transport activity was prevented by the addition of either actinomycin D (0.8 µmol l–1) or cycloheximide (35 µmol l–1) to the hypertonic culture medium, indicating that it requires both RNA and protein synthesis. In keeping with this finding, quantitative real-time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity (Fig. 4).
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To see if creatine could behave as a compatible osmolyte, cell survival was monitored after the C2C12 cells had been incubated in the presence or absence of creatine (0.1 mmol l–1), first in isotonic (0.3 osmol (kg H2O)–1) medium for 16 h, and then for 24 h in media ranging in osmolality from 0.30 to 0.56 osmol (kg H2O)–1. As shown in Fig. 5A, cell survival in the absence of creatine decreased with exposure to hypertonicity, with a drastic loss at osmolalities above about 0.55 osmol (kg H2O)–1. The addition of the creatine, however, largely prevented the loss of cells, changing a survival of only 28 ± 3% to one of 68 ± 9% with cells exposed to 0.56 osmol (kg H2O)–1. In a similar experiment, cell survival was checked after 24 h exposure to 0.55 osmol (kg H2O)–1 in the presence of added creatine at concentrations ranging from 0.1 to 20 mmol l–1, again after a preliminary 16 h incubation in isotonic medium with the same creatine concentrations (Fig. 5B). It is clear that cell survival increased as the concentration of added creatine increased. Significant protection of the cells occurred only after they had been incubated for at least 4 h in creatine-supplemented isotonic medium before exposure to hypertonicity, suggesting that accumulation of creatine by the cells is important (results not shown). Finally, Fig. 6 shows that cell survival in hypertonic medium supplemented with creatine was comparable to that observed in the presence of the well known compatible osmolytes betaine, taurine and myo-inositol (Petronini et al. 2000; Alfieri et al. 2002).
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The accumulation of [14C]creatine in C2C12 cells amounted to 488 ± 30 nmol (mg protein)–1 after 16 h in isotonic (0.31 osmol (kg H2O)–1) medium containing 20 mmol l–1 creatine, and increased to 836 ± 128 nmol (mg protein)–1 after a subsequent 24 h in hypertonic (0.53 osmol (kg H2O)–1) medium. To distinguish between creatine and phosphocreatine, this experiment was repeated with unlabelled creatine and the cell contents were analysed with the use of HPLC. This revealed that although the cell content of both creatine and creatine phosphate increased after the 16 h isotonic incubation, only the amount of creatine increased further after the subsequent hypertonic treatment (Fig. 9). These increases in content of creatine and creatine phosphate, however, were not accompanied by any increase in the amount of ATP in the C2C12 cells, under either isotonic or hypertonic conditions. On the contrary, hypertonic incubation, with or without added creatine, caused a marked decrease (about 40%) in cellular ATP (Fig. 10).
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The stimulation of creatine uptake by hypertonicity is not restricted to C2C12 cells: parallel induction of creatine transport activity (not shown) and creatine accumulation (Fig. 11) occur in the porcine endothelial cells that we have used extensively for previous studies of responses to hypertonicity (Petronini et al. 2000; Alfieri et al. 2001, 2002, 2004).
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Discussion |
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The finding that the ATP concentration in C2C12 cells was not affected by changes in the concentrations of creatine and phosphocreatine (Fig. 10) parallel the observations of Nomura et al. (2003) with endothelial cells, as well those of Ceddia & Sweeney (2004) with L6 rat skeletal muscle cells. The decrease in ATP concentration under hypertonic conditions (Fig. 10) could result from an increased ATP consumption due to the stimulation of the Na+,K+-ATPase during the first hours of hypertonic treatment (Ferrer-Martinez et al. 1996).
Although it seems clear that muscles cells, like endothelial cells, are not normally subjected to such drastic hypertonic conditions as those used here, physiologically significant changes in plasma osmolality can occur in certain circumstances. For example, the increase in blood glucose concentration caused by diabetes can increase plasma osmolality to 0.33–0.38 osmol (kg H2O)–1 (Powers, 2005), within the range that gave a detectable effect with C2C12 muscle cells (Fig. 1A). Similarly, a 4% increase in plasma osmolality was reported to accompany an increase in plasma taurine content in human subjects doing vigorous exercise with no fluid intake (Cuisinier et al. 2002). Such a relatively small change in osmolality, although physiologically important, would not produce significant effects in the kind of experiments described here, so it is not clear if the accumulation of compatible osmolytes would become important for cell survival under such conditions. Similarly, it is not clear if the accumulation of creatine in muscle tissue that occurs when creatine is taken as a food supplement could be beneficial simply as a compatible osmolyte under conditions such as those described by Cuisinier et al. (2002). On the other hand, the apparent safety of the consumption of relatively high concentrations of compounds such as creatine and betaine, in the form of dietary supplements might be explained by their being compatible osmolytes. This property may be relevant to the recent finding, in a study of a mouse model for stroke, that dietary creatine supplementation improved cerebral blood flow and had a clear neuro-protective effect, with no detectable change in the energy status of brain tissue (Prass et al. 2006).
Another possibility is that these responses to hypertonicity by osmolyte transporters in non-renal tissues are relics from earlier requirements. The accumulation of compatible osmolytes might well have been a very early property of cells that enabled them to survive under temporary hypertonic conditions. This requirement could subsequently have become redundant in cells in controlled tissue environments in multicelluar organisms, and these osmolytes gradually used for other more specific purposes, such as neurotransmitters (glycine and GABA), components of second messengers (inositol) and metabolic intermediates (betaine, creatine). If this is the case, it seems possible that other compounds, such as the catecholamines and L-carnitine, might fall into the same category.
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References |
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, isotonic; •, hypertonic.
