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. 2006 Oct 15;576(Pt 2):391-401.
doi: 10.1113/jphysiol.2006.115006. Epub 2006 Jul 27.

Creatine as a Compatible Osmolyte in Muscle Cells Exposed to Hypertonic Stress

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Creatine as a Compatible Osmolyte in Muscle Cells Exposed to Hypertonic Stress

Roberta R Alfieri et al. J Physiol. .
Free PMC article

Abstract

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H(2)O)(-1). This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na(+) dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in V(max) of the Na(+)-dependent component, with no significant change in the K(m) value of about 75 mumol l(-1). Quantitative real-time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo-inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l(-1) to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.

Figures

Figure 1
Figure 1. Effect of hypertonicity on creatine uptake by C2C12 cells
Cells were seeded and cultured for 48 h in isotonic (0.3 osmol (kg H2O)−1) medium. A, samples were incubated in isotonic medium or hypertonic medium (0.36–0.50 osmol (kg H2O)−1) for 16 h before the initial rate of creatine uptake by the cells was measured from a solution containing 0.1 mmol l−1 creatine. B, samples were incubated in isotonic medium or hypertonic medium (0.48 osmol (kg H2O)−1) and creatine influx was measured, as described above, at the indicated time points. Mean values (±s.d.) of three measurements are given. ^, isotonic; •, hypertonic.
Figure 2
Figure 2. Effect of hypertonicity on the kinetics of creatine uptake
C2C12 cells were incubated for 24 h in either isotonic (0.3 osmol (kg H2O)−1) medium (controls) or hypertonic (0.48 osmol (kg H2O)−1) medium (test cells) before their initial rate of creatine uptake was measured in the presence and absence of Na+, with creatine concentrations ranging from 0.01 to 0.5 mmol l−1. Mean values (±s.e.m.) for the Na+-dependent influx are given for 4 independent duplicate measurements. The curves, drawn with the use of ‘Kaleidagraph’ (Synergy Software, Reading, PA, USA), fit ‘Michaelis-Menton’ equations with kinetic parameters Km= 75 ± 22 μmol l−1, Vmax= 4.5 ± 0.4 nmol (15 min)−1 (mg protein)−1 for control cells (^) and Km= 77 ± 19 μmol l−1, Vmax= 7.1 ± 0.6 nmol (15 min)−1 (mg protein)−1 for test cells (•).
Figure 3
Figure 3. Effects of actinomycin D and cycloheximide on creatine uptake
Cells were seeded and cultured for 48 h in isotonic (0.30 osmol (kg H2O)−1) medium and then incubated for 16 h in isotonic medium or hypertonic medium (0.48 osmol (kg H2O)−1) with or without the addition of 0.8 μmol l−1 actinomycin D (Act D) or 35 μmol l−1 cycloheximide (CHX). Then creatine influx was measured from solutions containing 0.1 mmol l−1 creatine. Mean values (±s.d.) of three measurements are given. Open bars, isotonic condition; filled bars, hypertonic conditions.
Figure 4
Figure 4. Expression of CT1 mRNA in C2C12 cells exposed to hypertonicity
Cells were seeded and cultured for 48 h in isotonic (0.3 osmol (kg H2O)−1) medium and then samples were incubated in isotonic or in hypertonic medium (0.48 osmol (kg H2O)−1) for 16 h and 24 h. The amounts of CT1 mRNA and β-actin mRNA in cell extracts were then measured with the use of ‘Real Time RT-PCR’. Values of the expression of CT mRNA were normalized to the corresponding values for β-actin mRNA and the results for the cells exposed to hypertonicity are given relative to those from the control (isotonic) cells. Mean values (±s.d.) from three experiments are given. Open bar, isotonic; filled bar, hypertonic.
Figure 5
Figure 5. Effect of creatine on cell survival under hypertonic conditions
Cells were seeded and cultured for 48 h in isotonic (0.3 osmol (kg H2O)−1) medium. A, samples were incubated for 24 h in hypertonic media (0.44–0.56 osmol (kg H2O)−1) in the presence or absence of 0.1 mmol l−1 creatine before cell survival was estimated by cell counting. Mean values (±s.d.) from three measurements are given. Open bars, without added creatine; filled bars, with added creatine. B, samples were incubated for 16 h in isotonic medium in the presence of creatine at the indicated concentrations before being transferred to hypertonic medium (0.53 osmol (kg H2O)−1) containing the same creatine concentrations and incubated for a further 24 h. Cell survival was then estimated by cell counting. Mean values (±s.d.) from three measurements are given. Open bar, isotonic conditions; filled bars, hypertonic conditions.
Figure 6
Figure 6. Similar effects of creatine and established compatible osmolytes
C2C12 muscle cells were seeded and cultured for 48 h in isotonic (0.3 osmol (kg H2O)−1) medium and then incubated in hypertonic medium (0.53 osmol (kg H2O)−1), with or without the addition of 20 mmol l−1 betaine (Be), myo-inositol (In), taurine (Ta) or creatine (Cr). Culture was continued for 24 h and cell growth was estimated by cell counting. Mean values (±s.d.) from three measurements are given. Open bar, isotonic conditions; filled bars, hypertonic conditions.
Figure 7
Figure 7. Effect of creatine on the rate of protein synthesis under hypertonic conditions
The rate of protein synthesis was measured as the incorporation of radio-labelled l-leucine into C2C12 cell proteins during a 30-min pulse after the cells had been incubated for the indicated times in hypertonic (0.48 osmol (kg H2O)−1) medium in the absence or presence of creatine (20 mmol l−1). Mean values (±s.d.) from four measurements are given. Open bars, without added creatine; filled bars, with added creatine.
Figure 8
Figure 8. Effects of increased osmolarity, produced by different solutes, on the translation of globin mRNA by rabbit reticulocyte lysate
Translation was measured as the rate of incorporation of radio-labelled leucine into proteins, as described in Methods, with one of KCl, betaine or creatine added to give the indicated osmolalities. The results are expressed relative to the value obtained with the standard reaction mixture (0.37 osmol (kg H2O) −1).
Figure 9
Figure 9. Cellular accumulation of creatine and phosphocreatine
Cells were seeded and cultured for 48 h in isotonic (0.3 osmol (kg H2O)−1) medium and then samples incubated for 16 h in isotonic medium containing 20 mmol l−1 creatine. Some of these cells were then analysed for creatine and phosphocreatine whilst other samples were transferred to hypertonic medium (0.53 osmol (kg H2O)−1), also containing 20 mmol l−1 creatine, and incubated for a further 24 h. Cell content of creatine and phosphocreatine was determined with the use of HPLC as described in Methods. Mean values (±s.d.) from three measurements are given. Open bars, isotonic; light grey bars, isotonic plus creatine; cross-hatched bars, hypertonic plus creatine.
Figure 10
Figure 10. Intracellular ATP content
C2C12 cells were seeded and cultured in isotonic (0.3 osmol (kg H2O)−1) medium for 24 h and then incubated for 16 h in isotonic medium in the presence or absence of creatine (20 mmol l−1). Samples were taken for the measurement of ATP content and the rest of the cells transferred to hypertonic medium (0.53 osmol (kg H2O)−1), again with or without the addition of creatine (20 mmol l−1) for a further 5 h incubation, after which cell ATP content was again measured. Mean values (±s.d.) from four independent experiments are given. Open bars, without added creatine; filled bars, with added creatine.
Figure 11
Figure 11. Hypertonic stimulation of creatine uptake by endothelial cells
Endothelial cells were incubated in isotonic (0.3 osmol (kg H2O)−1) or hypertonic medium (0.5 osmol (kg H2O)−1) containing 0.05 mmol l−1 radiolabelled creatine and the cellular content of creatine was measured at the indicated times. Mean values (±s.d.) from three measurements are given. ^, isotonic; •, hypertonic.

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