Modulation of inorganic phosphate uptake into a mouse myoblast cell line by extracellular creatine. KIM E. POLGREEN, GRAHAM J. KEMP, and GEORGE K.
440s Biochemical Society Transactlons ( 1 993) 21 Modulation of inorganic phosphate uptake into a mouse myoblast cell line by extracellular creatine.
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KIM E. POLGREEN, GRAHAM J. KEMP, and GEORGE K. RADDA. Department of Biochemistry, University of Oxford, Oxford OX1 3QU. We have previously characterised inorganic phosphate (Pi) transport in a skeletal muscle cell line 6 6 ) and in a t-tubule membrane vesicle preparation from rabbit skeletal muscle [ 11. We have also demonstrated similar activity in a more differentiated muscle cell line (G8) [2] which contains creatine kinase [Y. Anderson, personal comunication] and now use this to examine relationships between transport of Pi and creatine. As in several other tissues, Pi uptake into skeletal muscle is largely dependent on the transmembrane Na gradient [l]. Creatine exists in muscle cells both free and as phosphocreatine (PCr). Free creatine is taken up into the cell via a Na-dependent mechanism [3]. It has been shown that dietary creatine loading in human subjects increases muscle concentrations of total creatine (TO, i.e. creatine plus PCr) [4]. In muscle cells, the concentrations of creatine, PCr, ATP and ADP are linked by the equilibrium reaction catalysed by creatine kinase [5]. In order to hold free cytosolic [ADP] constant during a rise in [TCr], then [PCr] must rise proportionately. This would entail net consumption of inorganic phosphate (Pi) by the expanding PCr pool. Unless this demand is met by concomitant decrease in the concentrations of other intracellular phosphates, this would require a net influx of Pi. We therefore tested the hypothesis that Pi uptake may be. stimulated by creatine loading to supply Pi for the synthesis of phosphocreatine (pcr). The techniques used are as previously described [l]. G8 cells were cultured in DMEM supplemented with 10% foetal calf serum and 10% horse serum. Cells were grown as myoblasts (i.e. unfused cells) to near-confluence. Cell monolayers were then incubated for varying periods with added creatine. Following this, [32P]Pi uptake was measured either over 5 min to estimate the initial rate of Pi uptake across the cell membrane, or over 2 h, to measure the combined rates of Pi transport and incorporation into organic phosphates such as phosphocreatine (PCr). In all cases, the final 1 h of incubation in Tris-Ringer was performed in the presence of [3H] 2-deoxymethylglucoseused to measure cell water
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To examine effects of extracellular creatine, we used concentrations higher than the normal human plasma [creatine] of around 30 IM, which should saturate the Na-linked creatine transporter, assuming this to have the low Km characteristic of the similar cell line, L6 [6]. Two kinds of protocols were used (1) 24 h incubations in DMEM with added creatine, followed by incubation with [3H] 2-deoxymethylglucose in a Tris-Ringer solution containing no creatine, at the end of which [32P]Pi uptake was measured over 5 min. (2) Incubations of up to 2 h in Tris-Ringer with added creatine, in which [32P]F'i uptake was measured over 5 min or for the full 2 h (n = 6 for all experiments). Unless otherwise stated reported changes are of Na-dependent [32P]Piuptake. In short term incubations (1 h), addition of creatine to the incubation medium caused a marked stimulation of [32P]Pi uptake. With 0.5 mM creatine (Fig. 1). the degree of stimulation depended on the length of incubation with creatine: maximal stimulation was seen at 20 min, but at 60 min [32P]Pi uptake was still double the basal value. However, in cells incubated for 24 h with 0.5 mM creatine there was no significant effect on the initial rate of [32P]Pi uptake when this was measured at the end of 1 h incubation in creatine-free Tris-Ringer, nor was there any change in the incorporation of [3*P]Pi in creatine-free Tris-Ringer over 2 h. This appears to suggest that the stimulation was dependent on the presence of creatine during the uptake measurement, rather than being an effect of creatine loading. However, it is possible that the stimulation simply declines after 24 h. Curiously, preincubation with 20 mM for 24 h caused a decrease in both the initial rate of [32P]Pi uptake (a 72 13% decrease), and in the 2 h incorporation into organic phosphates (a 40 k 21 % decrease) when these were measured in creatine-free Tris-Ringer. No effects of creatine on the rate of Na-independent [32P]Piuptake were observed. +_
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Length of exposure to creatine (min) Fig. 1 Stimulation of initial rate of [32P]Pi uptake by exposure to
0.5 mM creatine for various intervals.
Metabolic incorporation of phosphate has been postulated to explain the stimulation by insulin of Pi uptake into isolated hepatocytes [6] and the isolated perfused heart [7]. Incorporation of Pi into an expanding organic pool would cause cytosolic [Pi] to fall, and therfore decrease the rate of Pi efflux. The result would be net Pi influx without any change in the rate of the (active) influx component of transmembrane Pi exchange. This was the expected mechanism for incorporation of phosphate following creatine loading and subsequent PCr synthesis. Our results do not preclude this possibility. But we have demonstrated here that the influx component of Pi exchange (i.e initial [32P]Pi uptake) is increased, and within a short time from the start of exposure, probably before appreciable loading of creatine into the cell has occured. This result was unexpected and suggests an interaction between the two Nadependent transport mechanisms. Incubation in the absence of Pi for 24 h, led to a 270 k 98 % increase in the initial rate of subsequent [32P]Pi uptake. A similar finding has been observed in several cell types [8], and may represent a protective response against intracellular Pi depletion. Interestingly, [32P]Pi uptake was further enhanced (an increase of 41 1 f 175 %) when 0.5 mM creatine was also present during the Pi-free incubation. This suggests, perhaps, that a 'need' for Pi uptake during creatine-loading is capable of stimulating Pi uptake up to 1 h (the length of the incubation for Pi uptake measurement) after Pi is resupplied if that 'need has not been previously satisfied. As initial [32P]Pi uptake is a measure of the influx component of Pi exchange, both these results suggest that an intracellular deficiency can also stimulate the influx component of the exchange flux as well as decrease efflux. The mechanism by which such a stimulation may be modulated is unknown.
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