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
UI.
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.
+_
0
0
20
40
60
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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