10
Biochimica et Bioplo.sica Acta, 1137(1992) 10-18 © 1992ElsevierSciencePublishersB.V.All rightsreserved0167-4889/92/S05.00
BBAMCR 13234
Skeletal muscle Pi transpo~ and cellular [Pi] studied in L6 myoblasts and rabbit muscle-membrane vesicles G r a h a m J. K e m p , K i m E. P o l g r e e n a n d G e o r g e K. R a d d a Depannu~ of Biochemislly, Unirersitvof Oxfor~ 04ord and MRC Clinicaland Biocl'~,micalMagnetic Resonance Unit, John Radcliffe Hospital. Oxford (UK)
(Received 6 April 1992) Keywords: Phosphatetmmpon:Cellular phosp~hate;Skeletal mu~-.ccell; ~P-labelling In the rat skeletal myoblast line L6 and in a rabbit skeletal muscle sarealemma/t-tubule vesicle preparation, [32P]PI uptake was largely dependent on the transm.embrane Na gradient. Na-dependent [32PIPi uplake had a hyperbolic relationship to [Pi] and [Na], being half-maximalat 0.2-0.3 mM [Pi] and at 25-40 mM [Na]. In vesicles the Ha-dependence suggests that approx, two Na are transported with each Pi, but the inhibition of [32P]Pi uptake at high pH suggests that the Pl monoanion is the transported form. Together these imply electrogenic transport and this is confirmed by the results of manipulating the vesicle me,-nbrane potential. Thus. electrogenic Na-Pi co*transpo~ exploits both the sodium gradient and the cell membrane potential to maintain muscle cellular [Pi] against an unfavourable electrochemical gradient. The low [Pl] for half-maximal flux may partly explain the small effect of altered extracellular [Pi] on cellular [Pi]- In L6 myoblasts most 32_pwas fit'st detectable in an organic phosphate pool rather than ct:llular Pl, v,'~c the ~ f i c a-,:t-'.,vi~of fell Pi rapidly reached 40% of that of extra~llular Pi and was stable for at least 3 h. These results are discussed in terms of the organisation of cellular p~osphate metabolism. latroduellon Despite the central role of intracellular Pi in bioenergetics and elsewhere [1] and the serious muscular (and other) consequences of clinical Pi disorders [2] tittle is known about how [Pt] is regul~.ted in skeletal muscle, where resting [Pl] is maintained at 2-4 mmol/I, despite a highly unfavourable electrochemical gradient and appears to be held nearly constant when extracellular [Pi] changes [3,4]. In this paper we report studies of cellular [Pi] and Pi Uansport in the cultured rat myoblast line L6 and a more detailed characterisation of Pi transport in sarcolemmal and t-tubule vesicles prepared from rabbit muscle. Some of this work has been presented in preliminary form [5,6]. Materials and 1YT~:rods Cultured cell methods
1.6 cells (passage number 5-20) were grown at 37~C under 5% CO 2 on 9 cm Petti dishes in 10 ml Eagle's
Cotr~pondence to: GJ. Kemp, MRC Clinical and Biochemical Magneti, R.~,anan.=eUnit, John Radcliffe Hospital, Oxford, OX3 9DU, UK. Abbreviations: MEM. Eagle's minimumessential medium; DMEM, Dulbecco'sMEM.
minimum essential medium ( M E M ) with 10% foetal calf serum (Northumbrian Biologicals) or Dulbeco's MEM (DMEM) with 10% horse serum; 2 mM glutamine was added to both. Medium was changed every 3-4 days and cells were passaged (1:10) every 5-7 d a ~ after removal from the plate with 2.5 ml trypsin/EDTA (Gibco BRL Life Technologies, Paisley, UK). For experiments, cells were grown to subconfluence for 1 or 2 days on 12-well culture plates (1 Petri dish, approx. 107 cells, seeding 24-36 wells). Under these conditions L6 cells grow as myoblasts. No attempt was made to fuse into myotubes, as this would require longer culture times, making it difficult to be sure that all extracellular medium was washed away during the cell sampling steps described below. Cells were then incubated at 3"PC for 1-2 h in fresh medium under 5% CO e or in Tris- or Hepes-buffered Ringer solutions at YPC, under air, with 74 kBq (2 /tCi) per ml of 3-O-methyl/D-[l-3H]glucose to allow measurement of cell volume. Ringer solutions contained 122 mM Na (replaced by choline for Na-free experiments), 130 mM Ci, 5.6 mM K, 2 mM Mg, 2 mM Ca, 5 mM glucose and 30 mM Tris or Hepes. At sample times plates were washed rapidly four times with Pi-free Hepes-Ringer solution (plus 10/~M phloretin) and 500/zl of ice-cold 1/3 M perchloric acid added to each well. After scraping with a syringeplunger, 260/tl acid extract were added to 20/ti 1 M silver perchiorate in 2 / 3 M perchloric acid and 2 0 / t l
11 1.8 M NaCI, vortexed for 5 s and centrifuged at 3000 × g for 5 min at 40C. 250/~1 supematant were neutralised with 4.3 M KOH/0.6 M imidazole (plus 2% (w/v) wide-range indicator). 3H activity was measured in 200 ~tl acid extract and 25 pl incubation medium: cell volume per ml neutral extract (/~1) = (3H per ml)/(3H per/~1 of medium), from which cellular [Pi] = (nmol Pi per ml extract)/(ce!! volume per ml extract). No attempt was made to correct for trapped or bound extracellular medium, which is negligible under these condilions. Pi was assayed in the neutrai'-~ed cell extract by a mezhod depending on addition of acidified molybdate, rapid organic ey-traction of resulting phnsphomolybdate, aqueous back-extraction of Pi and formation of phosphomolybdate/Malachite green complex whose absorbance is measured at 660 nm [7]. For labelling experiments, 37 kBq (1/~Ci) of [32p]pi were added per ml of medium at appropriate times. 32p distribution is A T / A t = (32p/3H in cell extract)/ (32p/3 H in medium) where A T is total 32p (cpm/I cell water) and A I is extracellular 32p activity (cpm/l). Inorganic [3eP]Pi (A 2 cpm/1 cell water) and organic 32p (A 3 cpm/.! cell water) were separated by organic extraction of phosphomolybdate (correcting for incomplete recovery of organic phase [8]), and cellular activities calculated using the cell volume (as for cellular [P~] above). To study effects on P~ fluxes, we expressed [3zP]P i uptake at t = 5 rain relative to control: this a good estimate (within 15%) of the true fractional changes in Pi flux. 32p and 3H in medium and neutralised ceil extract were measured by liquid scintillation counting. Membrane vesicle methods Sarcolemmal and t-tubule vesicles were prepared from leg white muscle of female New Zealand White rabbit. The t-tubule fraction was isolated by a modification of a published method [9]. Briefly, minced muscle homogenate in 300 m M sucrose/.5 mM imidazole / 1 0 0 / t M EDTA buffer (all buffers used were at pH 7.4) was centrifuged at 3000 x g for 20 rain, the supernatant centrifuged :~ice at 10000 × g for 20 rain and the resulting supernatant filtered, resnspended in 0.5 M KCi and centrifuged three times at 1501100 × g for 2 h. The resulting supernatant mlcrosomal fraction was resuspended to 40 mg protein per 10 rnl and centrifuged on a 17-35--48% sucrose gradient for 12 h at 7 0 0 0 0 x g ; the 17/35% interface was harvested and similarly centrifuged on a 35-50% sucrose gradient after loading with calcium phosphate to precipitate sarcoplasmic reticulum; the interface was harvested, resuspended and centrifuged three times. Vesicles were stored in sucrose/imidazole buffer in liquid nitrogen. The sarcolemma fraction was isolated by a published method [10]. Briefly, minced muscle, ho-
mogenised in 0.75 M KCi/imidazole, was centrifuged twice at 3000 x g for 20 rain, each time resaspending the pellet, which was then homogenised in sucrose/" imidazole, centrifuged twice and homogenised again. The resulting supematanL recentrifuged and filtered, was centrifuged at 5 0 0 0 0 x g for 2 h and tL ,~ pellet resnspeoded in 0.6 M KCI and layered onto a 17/23% sucrose gradient. Tlais was centrifuged at 700(10 x g for 12 h and vesicles harvested from the interface. Relative to the original microsomal fraction, the appropriate marker enzymes Na, K-ATPase, MgATPase and 5'-nucleutidase were enriched by factors 26, 11 and 24, respectively, in the sarcolemmal fraction and 17, 15 and 21, respectively, in the tubule fraction. In both fractions the sarcoplasmic reticular markers Ca-ATPase and glucose 6-phosphatase were diluted by factors of at least 0.02 and 0.008 and the mitochondrial marker succinate debydrogenase was undetectable. Ti~ere are no specific markers for t-tubules but t h e t-tubule fraction had (as expected) less Na, K-ATPase, more Mg-ATPase and a higher density than sarcolemma [9]. Assessed by ouabain inhibition of ~a,KATPase, both vesicle fractions were 90-95% sealed; from the latency of Na,KoATPase (the effect of ouabain on Na,K-ATPase in the presence and absence of sodium dodecyl sulphate), the sarcolemmal vesicles were 80% right-side-out, the t-tubule vesicles being 43%. Both vesicles were about 2~, nm in diameter by electron microscopy. The overall ~ield of vesicles was 96, 0.6 and 0.l mg protein/g wet wt mu..~le in crude microsomal, t-tubule and sarcolemmal fractions, respectively. Pi transport was measured by addition of 20 /zg vesicle protein to 1.2 ml buffer containing 370 kBq (10 ttCi)/ml [32P]Pi; the reaction was stopped by three rapid washes with ice-cold incubation buffer through a 0.22/~m filter. Fluxes were calculated from labelling at 1 min, at which time labelling was acceptably finear with time. C/s (i.e., extravesicular) buffers contained 135 mM NaC! or choline chloride (chloride sometimes replaced by thiocyanate or gluconate), 30 m M Tris or 2[N-morpholino] ethane sulfonate and 1 mM EDTA; trans (i.e., intravesicular) buffers contained 30 m M "Iris or 2[N-morpholino]ethane suifonate and appropriate salts, with sucrose up to 250 mM. 32p activity in c/s buffer and on the fdters was measured by liquid scintillation counting. Protein concentration in vesicle suspensions were measured by the Lowry method. Data analysis Results are expressed as mean 4- S.E. Statistical significance of differences was assessed by Student's t-test. Straight lines were fitted by linear regression and rate constants for exponential changes obtained from semilogarithmic plots. Kinetic parameters for Pi influx were
12 estimated using the Hanes plot, i.e., [substratc]/rate against [substrate]. The basis of the analysis of ceilular labelling and the control of celh'~!ar [Pi] is briefly summarised in the Appendix.
Materials Unless stated otherwise, all tissue culture materials were purchased £rom Gil~co or from Flow Laboratories (Rickmanswertb, Herts, UK), all reagents were of analytical grade mid purchased from B D H (Poole, Dorset, UK) o r Sigma (Poole, Dorset, UK). Radioisotopes were purchased from A m e r s h a m International (Amersham, Bucks, UK). L6 wete obtained from the PHLS Centre for Applied Microbiology, Porton Down. Results
O.6
L6 cells Fig. 1 shows the time course of cellular labelling. The a n m u n t of organic labelling was large (Fig. la): even at J = 2 min only 27 + 6 % of cellular 32p was ill the Pi pool. The initial rate of inorganic labelling, expressed as A , / a I (i.e, absolute activity of cellular Pi relative to specific activity of extracellular Pi) can be t a k e n as the apparent Pi flux into the cellular Pi pool (we call this 1): this was 4.9 + 0.8 retool/! cell water p e r h. The initial rate of organic labelling, expressed as A 3 / a I (i.e., the overall absolute activity of cellular
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Time(rain) Fig. l. Time course of [32p]pi uptake in L6 cells. (a) Total labelling (A T) (closed circles) and labelling of cell Pi (A2) (open circles); (b) specific activity of cellular Pi (a2)* all expressed relative to specific activity of extrace[Iular Pi (at)- [32p]Piwas added at t = 0 to cells in MEM/10% foetal calf serum (n = 3).
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Fig. 2- Effects of foetal calf serum and sodium on [32PIPi uptake in I-6 cells. (a) The effects of 24 h omission of foetal calf serum (solid bars) and 24 h omission f o l ~ e d by I h replacement of 10% foetal calf serum (shaded bars) on total [32pIPi uptake (total labelling T), [3-'PIPi uplake into cellular Pi (inorganic labelling I) and on cellular [Pi]. Cells weru grown (for 24 h) and incubated in MEM. Data expressed relative to incubation with 10% foetal calf serum throughout (n = 6). (b) Comparison of cellular [Pi] (black bars) and [32plPi uptake (shaded bars) after I h incubation at zero and 3 mM extrucellular [Pi] (n = 4) in the presence of sodium, and at ! mM [Pi] in the absence of sodium (n =6). CelLswere grown in MEM/10% foetal calf serum and incubated for I h in Tris-Ringer (simile: Na-dependence is seen in ceils grown in DME,L~/10% horse serum). Results expressed relative to incubation at 1 mM [Pi], i-~ mM INa].
organic phosphates relative to specific activity of extracellular Pi) can be taken as the apparent Pi flux into organic phosphates (we call this J): this was 29 _+ 2 m m o l / l cell water p e r h. The specific activity of cellular Pi relative to that of extracellular Pi, a2/al, approached a plateau 0.39 + 0.01 (stable for at least 3 h) with a half-time of 10 + 1 min (Fig. lb). Fig. 2 shows some effects of altered incubation conditions on [3zP]Pi uptake and cellular [P~]. Total cellular labelling, expressed as A T / a ~ (i.e., total absolute cellular activity relative to specific activity of extracellular Pi) can be taken es the total Pi flux into the cell (we call this T = I + J ) . Incubation for 24 h without foetal calf serum reduced T by approx. 50% and this was restored by 1 h incubation with serum. Neither manipulation altered cellular [Pi] (Fig. 2a). A t 1 mM Pi, 1 h incubation without sodium reduced T by approx. 80%, while cellular [Pi] was not significantly affected (Fig. 2b). Incubation for 1 h in the absence of extracel|ular P, reduced cellular [Pi] to 85% and incubation at 3 m M [Pi] increased both T and cellular [Pi] by 10-20%
(Fig. 2b). Absolute cellular [Pi] was 3 . 2 _ 0.7 m m o l / I cell water in cells incubated for 1 h in M E M / 1 0 % foetal calf serum under 5% C O 2 (n = 6) and 4.6 + 0.6 m m o l / ! cell water in Tris-Ringer solution under air (n = 6). Fig. 3 shows more details of the dependence of [32PIPi uptake on [PJ and [Na]. T has a hyperbolic relationship to extracellular [Na] (Fig. 3a): from the Hanes plot (Fig. 3b) T was half-maximal at 40 mM [Na]. The Na-independent component of T was linear with extracellular [P~] (slope 4.1 __ 0.1 h-~). The Na-dependent component of T showed hyperbolic dependence oi: [PJ (Fig. 3e) and from the Hanes plot (Fig. 3d) K m = 0.26 mM and maximum flux = 40 m m o l / l cell w a t e r per h; K m was indistinguishable for ! and T (Fig. 3d).
"t-Tubule" m e m b r m w vesicles Fig. 4 shows some properties of Pi transport in t-tubule membrane vesicles. Fig. 4a shows that in the presence of 135 m M c/s (external) [Na], [32P]Pi uptake
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showed classical overshoot kinetics. Fig. 4b shows that [32P]Pi uptake was negligible in vesicles disrupted by rapid freeze-thawing and was zeduced to 20% of control when c/s [Na] was zero and to 4 0 - 5 0 % of control when c/s and trans (internal) [Na] were set cqual or when the Na-gradient was partially dissipated with 1 /~M monensin. Fig. 4b also shows that [32p]Pi uptake was stimulated by 50% when trans [Fi] was increased to 10 mM and that this overcame the inhibitory effect of 100 mM trans [Na] (but see Fig. 6c). Fig. 5 shows more details of the dependence of Pi transport on [Na] and [Pi]. Fig. 5a shows that [32pIPi uptake had an approximately hyperbolic relationship to [Na], although the sigmoid shape of the plot at low [Na] suggests that more than one Na ion is transported for each Pi ion. In accordance with this, the Hanes plot (Fig. 5b) is markedly nonlinear at low [Na], but becomes linear when [Na] 2 is used in the plot rather than [Na], suggesting a stoichiometry of approx. 2 Na: 1 Pi.'.32P]Pi uptake was half-maximal at 25 mM [Na]. Na-dependent [32p]p i uptake had a hyperbolic relationship to
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Fig. 3. Dependence of [32p]p~uptake on extraceilular [Na] and [Pi] in 1.6 cells. (a) Total [ 32p]pi uptake (T) (expressed relative to its value at 120 mM [Ha]) as a function of lNa]- Cells were grown in DMEM/10% horse serum and incubated for I h in Tris-Ringer (n = 7). (b) A Hanes plot of Na-dependent 132p]Pi uptake and [Na]. Cells weru gro~. in DMEM/10% horse serum and incubated for I h in Tris-Ringer to = 7L (c) Na-dependent (closed circles) and Na-independent (open circtes) components of total [32plp i uptake T (expressed in absolute terms) as a function of extracellular [Pi]. Cells were grown in MEM/10% foetal calf serum and incubated for I h in Hepes-Ringer with 120 riM [Nal. (d) Haoes plots of Na-dependent [32P]Pi uptake (expressed relative to ,.lptake at I mM [PJ) and [Pi]. Closed circles show a Hanes plot for the dependence of Na-depcadent component of total [3:PIPt uptake (T) on extracellular [PJ; crosses show similar data for the Na-dependen[ component of [32plp, uptake into cellular Pi (I). Cells were grown ir MEM/10% foetal calf serum and incubated f~,r 1 h in Hepes-Ringer with 120 mM [Na].
[Pi] (Fig. 5c) and from the Hanes plot K m =0.16 mM and maximum flux= l l p . m o l / m g protein per min (Fig. 5d). Fig. 6 shows the dependence of Na-dependent [32PIPi uptake on pH. Fig. 6a shows that at 0.09 mM cL: [P;] (a nonsaturating concentration) and t r a ~ pH 7.4, in the absence of trans [Pi], Na-dependent [32pIPi uptake decreased with increasing pH (slope = - 0 . 9 + 0.2 # m o l / m g per min per pH unit; P < 0.01). However Fig. 6b shows that when c/s [P~] was manipulated to maintain 0.09 mM c/s [Pi monoanion], c/s pH had little effect. Fig. 6a also shows that when c/s pH was 5.5, [32pIPi uptake was slightly stimulated when trans pH was 5.5 rather than 7.4 ( P < 0.05) and that at c/s pH 9.5 [32PIPi uptake was slightly inh~ited when trans pH was 9_5 aLso ( P < 0.05). Fig. 0c shows that, at constant c/s pH, the effect of trans pH depended on trans [Pi]: at zero trans [Pi] [32PIPi uptake was lower at pH 6.5-7A than at either extreme pH ( P < 0.05); in contrast, at 10 mM trans [Pi] [32PIPi uptake fell steeply with increasing pH (slope, - 1.3 _+0.2 p m o l / m g per min per pH unit; P < 0.001). Thus trans-stimulation was only seen (cf. Fig. 4b) at trans oH 7.4 and below, while above this pH trans Pi inh~ited Pi uptake. ~0]
(a)
Fig. 6d shows that at c/s [Pi] in excess of K m (in contrast to the results in Fig. 6a) Na-dependent [-~2P]Pi uptake increased with increasiag pH (at 0.9 mM ~'~ [Pi], slope, 2.8 +0.1 # m o l / g per rain per pH unit; P < 0.02). In anion-substitution experiments Na-dependent [32PIPi uptake was increased to 220 _+ 12% of control when chloride was replaced by thiocyanate ( P < 0.05; n = 3) and reduced by to 77 + 6% of control when chloride was replaced by glucona~.e ( P < 0.05; n = 3). Ha-dependent [3-'P]P i uptake was reduced by 30% by 10 mM arsenate, but unaffected by 10 mM vanadate (n = 3). Lastly, the effects of pH and [Pi] on Na-independent [3-'P]P i uptake can be summarised briefly. At constant pH it was linear with c/s [P,] (slope, 5.2 + 0.7 ( p m o l / g per m i n ) / ( m m o l / I ) ) ; at 0.09 mM c/s [Pi], c/s pH and trans o H had little effect; at constant 0.02 mM c/s [monoanion], by contrast, it increased with increasing pH in proportion to the increase in total c/s [Pi]; it was substantially inhibited by I0 mM trans [Pi]Sarcolemmal cesides
[3zP]Pi uptake was much less active in the sarcolemmal preparation, and so no characterisation was attempted. At 0.09 mM c/s [Pi], peak uptake (at t = 1 min) in the presence of Na was 1.5 p m o l / g protein (n = 3 ) , only 20% of the equivalent figure for the t-tubule preparation. Discussion Approaches to P~ metabolism
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Steady-state cellular [Pi] depends mainly on active Pi uptake [11] (see Appendix) which is easily studied using 32p-labelliag. Studies on whole cells illuminate (but are complicated by) exchange between cellular Pi and organic phosphates [12] and functional compartmentation [8,13]. The mechanisms of Pi transport are best studied using membrane vesicles, bearing in mind that changes in cellular [Pi] also depend on the permeability to passive back-flux of Pi [11] (see Appendix) and, transiently, on changing organic phosphate pool size and net flu~es across the cell membrane [4]. Effects o f [Pi] a n d [ N a ] on Pi transport
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Fig. 4. [32PIPi uptake in rabbit muscleplasma membranepreparation rich in t-tubule vesicles (all n = 3). (a) The time-course of ['~2pIPi uptake followingaddition of ,zesielesto mediumcontaining[3~"PlPi at t = 0. Open squares, with sodium; open circles, without sodium; closed circles, sodium-dependent uptake. (b) [3'PIPi uptake (left to fight): in vesiclesdisrupted t~" freeze-thawing,at 135 mM c/s (external) [Na], at zero c/s lNa]: with 135 mM c/s and trans [Na]: with 10 mM trans [Pil at 135 mM c/s [Na]: and with l0 mM trans IPi] and 100 mM trans INal at 135 mM c/s lima]. Data expressed relatb.'e to 135 raM cis INa]with zero trans [Na] and lP,l: all 0.09 mM cis lPl].
Apart from a report of vitamin D-stimulated Ha-dependent [32p]pl uptake in chick muscle sarc~le.rmnal vesicles [14] these results provide the fast characterisation of membrane P~ transport in skeletal muscle. The larger part o1 [~:P]P~ uptake depends on extracellular [Na] in cells (Fig. 21)) and vesicles (Fig. 4b) and in membrane vesicles it shows classical overshoot kinetics (Fig. 4a) and requires an intact membrane and transmembrane Na gradient (Fig. 4b). All of these suggest that co-transport of Na and P~ is responsible for the cellular accumulation of P~ against its electrochemical
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Fig. 5. D~.pendenceof [32PIPi uptake on [Pi] and [Nal in rabbit muscle plasma membra~.epreparation rich in t-tubule vesicles(all n = 3). (a) [32P]Pi uptake as a function of c/s [Hal. (b) A simple Hanes plot (closed circles)of [ 32pIpi uptake and c/s [Na],with a regressionline whichomits the anomalous point at 7.5 mM [Na] (which is shown with its standard error): a modified Hanes plot (open circles) (i.e., [substrate]2/velocity against [substrate]2,arbitrarily scaled to fit on the samc axes)on the assumptionthat the Hill coefficient of Na is 2. with a regressionline passing through all the points.(c) Ha-dependent [32p]p~uptahe as a functionof e/s lPl], (d) Hanes plot of Na-dependent 132plPl uptake and c/s [Pl]gradient in skeletal muscle (see Introduction), as in several other cell types [15]. The "/('m of Na-dependent P~ transport for c/s [Pi] is similar (0.2-0.3 raM) in the rat cell line L6 cells and in rabbit skeletal muscle vesicles (compare Fig. 3c and d with Fig. 5c and d). A low K m is consistent with the need to preserve cellular [Pi] against changes in extracellular [Pi] [11] (see Appendix) and similar values of K m are seen in heart muscle [16], osteoblasts [13], renal tubular cells and others [15]. The absolute rates of Pi uptake ~n L6 are comparable to values reported for, e.g., osteoblasts [13] and renal tubular ceils [17]. Effects o f p H on Na-dependent P~ transport At 0.09 mM c/s [Pi], [32p]Pi uptake decreases with c/s pH. but is unaffected by c/s pH when [monoanion] is held constant (Fig. 6a and b). These results, the opposite of those in rat renal tubular brush border vesicles [18], suggest that the Pi monoanion is the form binding to the c/s face of the transporter. By contrast, the increase in [32p]p~ uptake with increasing pH, seen at saturating c/s [P~] (Fi~ 6d), presumably reflects an intrinsic property of the transporter. The evidence of Fig. 6a that, at given c/s pH [32P]P i
uptake is slightly stimulated by alkaline trans pH and inhibited by acid trans pH, suggests that a small component of Na-dependent Pi uptake may be driven by the proton gradient, as reported in renal tubular brush border vesicles [18]. At 10 mM trans [Pi], increasing trans pH inhibits [32P]Pi uptake (Fig. 6(:), suggesting that increasing trans [monoanion] favou~ trans-to-cis translocation of the transporter. This, which is the opposite result and conclusion to that obtained in renal tubular brush border vesicles [18], is to be expected from the evidence above that it is the monoanion which binds to the c/s side, since half the vesicles are inside-out (see Results). However, at zero trans [Pi] a different respouse is seen, probably reflecting the intrinsic properties of the carrier: [32P]Pi uptake reaches a minimum at pH 6_5-7.4, which presumably least favours cis-to-trans translocation of binding sites (Fig. 0c). The result of these effects is that, at high trans pH, the expected trans-stimulation of [z2p]pi uptake (Fig. 4b) is converted to a trans-inhibition (Fig. (c). In general trans-stimulation suggests that carrier-P i complex translocates to the cis face faster than free carrier, and also, since trans Pi can overcome the inhibition by trans Na (Fig. 4b), faster than the carrier-Na complex
16 [18]. It would appear, however, that, at high trans pH, the transloeation of the unloaded carrier is faster than that of carrier-Pi in this system.
Stoichi~,netry o f Na-dependou Pi transport Tl:ere is some evidence in rabbi t. muscle vesicles (Hg~ 5a and b), although not yet in L6 ceils (Figs. 3 and b), that approx. 2 Na inns are transported per P~ ion. If the transported form is the Pi monoaninn (see above), this impfios electrogenic transport (positive charge movement inwards), as in cardiac muscle [16] and in some reports of renal tubular brush border membranes [19]. This is supported by the result of substitution of chloride in the incubation medium: [32p]p~ uptake is stimulated by replacement with the more diffusinie anion thiocyanate, which induces an inside-negative potential, and inhibited by the less-dLffusinle gluconate anion, which induces an/,as/de-positive potential. This makes physiological sense, as clectrogenic (positive inward) transport would ~_,se the membrane potential rather than oppose it [1~. N a - ~ e p e n d t m P~ transport In I.,6 cells (Fig. 2b) and in rabbit muscle vesicles Na-independent [32pIPi uptake is near-first-order with respect to c/s [Pi], which is the usual finding in mam-
(a)
malian cells e.g., Refs. [13,17,18]. In vesicles, its independence of cis and trans pH (at constant cis [Pi]) is shw21ar [18], while its inhibition by trans Pits opposite, to the findings in renal tubular brush border vesicles [19].
Organisation o f c¢llu!ar phosphate metabolism The failure in 1_6 cells of the cellular Pi pool to reach isotopic steady-state b.v 3 h (Fig. lb) could be explained, as in euthrocytes [8] and osteoblast-like cells [13], as a consequence of exchange with the bulk of the organic phosphates [12] (see Appendix). As in an ostcoblast line [13], at least 3 / 4 of the Pi entering 1.6 cells by the Ha-dependent transporter is detectable in a large organic phosphate pool, which labels faster than could be explained by exchange with detectable cellular Pi (Fig. 1). One possible explanation is that a high-specific-activity subpool of Pi has access to an enzyme catalysing its incorporation into an organic pool. If this is so, at 1 mM [P~] the effective flux from extracelhilar Pi to cell Pi appears to be 5 mmol/I cell water per h compared to 30 mmol/i cell water per h into the o~ganic pool; subtra~ing the estimated contrinution of direct exchange with bulk cellular Pi (estimated from a2/a I using the simple model described in the Appendix) from measured organic la-
i
e,
i
._
1°11 (b)
~pH
cis~
~"~sr~H
~ pH
Fig. 6. EffectsofpH on Na-dependent [~2P]Pi uptake in rabbit muscleplasmamembranepreparation rich in t-tubule vesicles(all n=3). (a) Effectsof c/.vpH at 0.09 mM c/s [Pi],when 'runs pH is 7.5 (o~encircles)and when trans pH is equal to c/s pH (closedcircles).(b) Effectsof c/s pH at constant0.02 mM c/s [Pi monuanion],when zrans pH is 7.5 (opencircles)and when trans pH equals¢/s pH (closedcircles).(¢) Effectsof trans pH at ¢/s ptl 7.4 and 0.09 mM c/s IPi].when traas[P,Iis zero (closedsquares)~n~ when trans[Pil is 10 mM (opensquares).(d) Effectsof c/s pH at tra~ pH 7.5, whenc/s [Pi]is 0.09 raM(circles),0.9 mM(diamonds)and 1.5 mM(triangles).
17 belling allows the size of such a rapid-labelling organic pool, if this is the explanation, to be estimated as approx. 15 mmol/I. There is .some precedent for such a mechanism: the erythrocyte has a Na-linked Pi transporter which feeds most of its Pi directly into a membrane-associated nucleotide pool [20]. The Na-dependenee of this organic labelling appears to suggest that such a flux in L6 (and osteoblasts [13]) &_curs via the Na-Pi co-transporter (also supported by the similar K m for organic and in,~rganic labelling - Fig. 3d). However, if the sarcolemmal Na,K-ATPase consumes a large part of ATP turnover then its inhibition by Na-withdrawal could reduce organic incorporation of [32P]P i, as is observed. Nevertheless, even in the absence of sodium the rate of organic labelling is higher than could be explained by exchange solely with what the present methods detect as cellular Pe The finding that it is the 't-tubule' vesicle fraction from mature skeletal muscle which has significant Nalinked P~ uptake is unexpected (though it should be noted that this preparation does not include the triads at the apex of the t-tubules). However, we cannot exclude an artifact, e.g., damage to the transporter by the more elaborate preparation of the sareolemmal vesicles (although other transporte~ can survive this [10]). This finding, if real, suggests that the external microenvironment of the transporter may be quite different from that at the sarcolemmal surface. In particular, the diffusion limitations might make Pi leaking through out of the cell more access~le to cellular re-uptake.
Control o f cellular [Pi] Accurate measurement of cellular [Pi] requires minimal hydrolysis of organic phosphates during extraction and assay. In the present experiments on L6 cells this should be achieved by the speed of perchloric acid extraction and organic extraction of phosphomolybdate in the Pi assay [7]. Chemically measured cellular [Pi] includes at least cytosolic, nuclear and mitochondrial Pi. Nevertheless, the total cellular [Pi] in this study is close to NMR measurements [3] of cytosolic [Pi] in vivo. Many cells can apparently regulate [4] intracellular [Pi] in the face of changes in extracellular [P~]. la I.,6 cells incubated for ! h a t zero or 3 mM [Pi], the fractional change in cellular [P~] relative to cells incubated at I mM [Pi] is smaller than that in extracellular [Pi] (Fig. 2b). This gives an apparent rega',dtion index (see Appendix) of approx. 0.9, compared to 0.5-1.0 in a variety of other cells and situations [4,21] and specifically with 0.7 in skeletal muscle in vivo [3]. Little is known about the details of this regulation [4,11]. In the present work the small effect of increased extracellular [Pi] (Fig. 2b) is consistent with the low K m of Na-de-
pendent P~ uptake (Fig. 3c), as under these conditions both Pi uptake and cellular [Pi] increase by a similar small fraction. The failure of Pi-free incubation to deplete cellular [Pi] (Fig. 2b) could be wholly or partly the result of slow equilibration of the Pi pool (as muscle membrane permeability to net effiux of Pi is very low in vivo [4]), or of non-exchangeable (and perhaps non-cytosolic) Pi pools. Much longer incubations will be required to resolve this. The dependence of Pi uptake and cellular [Pi] on [Na] has rarely been compared, but in a study of Ehrlich cells [22] sodium deprivation reduced Pi influx by 65% but cellular [Pi] by only 15%. This, and the similar discrepancy in L6 (Fig. 2b), may also result either from failure to reach steady-state, from temporary maintenance of cell [Pi] by the slow collapse of an organic pool or from changes in Pi back-flux permeability [4,11].
Net flux attd exchange flux We have argued elsewhere [231 that we should expect an approximate equality between permeability of a cell membrane to small net fluxes of Pi and the 'back-flux' rate constant IA/C 2 (i.e., the ratio of active influx to cellular [Pi])- Such equality seems to hold, approximately, in the small number of cases where suitable data exist [4,23]. In I.,6 cells the rate of Na-linked Pi uptake is large so that IA/C 2 is about 1 h - I (considering only labelling of cellular Pi) or 5-10 h -I (considering total cellular labelling). However, the net flux permeability of skeletal muscle sarcolemma is very low (0.1 h -I or less) [4]. This is presumably a special adaptation to prevent significant Pi loss during contraction, in which intracellular [Pi] can increase up to 10-fold. However, we do not yet know whether exchange fluxes of Pi are as ia:'ge as in L6 cells across the sarcolemma of normal muscle cells in vivo. In conclusion, we have demonstrated Na-dependent P~ uptake in a rat myoblast line and in rabbit skeletal muscle-membrane vesicles. This has properties consistent witi~ i~ role in maintaining cellular [Pi] in the face of a large negative membrane potential, namely a low K m and an electrogenic mechanism. Strangely, most of this transport activity is found in the t-tubule membrane fraction, although an artifact cannot yet be excluded. We have identified an unexpectedly rapid exchange between incoming Pi and cellular organic phosphate pools, and noted an apparent discrepancy between the relatively large exchange rates of Pi across the L6 cell membrane and the low permeability of skeletal muscle to net P~ effiux. Acknowledgements GJK was supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland and KEP by the British I-~eart Foundation.
18 AmDend~x (See Materials a n d M e t h o d s f o r e x p l a n a t i o n o f n o t a tion)
Cellular labelling In t h e simplest m o d e l o f cellular Pi m e t a b o l i s m [12] extracellular Pi e x c h a n g e s with cellular Pi ( c o n c e n t r a tion C 2) a t t r a n s m e m b r a n e flux r a t e 1 = kC,, a n d with o r g a n i c p h o s p h a t e s a t r a t e G = 3"kC2. If o r g a n i c pool size is l a r g e t h e n a f t e r a d d i t i o n o f [32P]Pi the relative specific activity o f cell Pi (a2/al) increases e x p o n c n tionally with initial r a t e k a n d r a t e c o n s t a n t k(1 + 3') t o w a r d s a t e m p o r a r y ' p l a t e a u " 1 / ( 1 + 3')- O r g a n i c labelling (A3/a ~) is l i n e a r w h e n a2/a , has r e a c h e d p l a t e a u with slope 5 = 1/[(1/1)+ ( l / G ) ] (which a p p r o x i m a t e s I ff 3" is large). In t h e e x p e r i m e n t s r e p o r t e d h e r e S exceeds L O n e e x p l a n a t i o n is t h a t a n o r g a n i c pool ( c o n c e n t r a t i o n C3~) e x c h a n g e s with a pool a t a p p r o x i m a t e l y extracellular specific activity ( p e r h a p s a n u n d e t e c t a b l e subpool o f cellular P~) a t a r a t e .I: the initial r a t e a n d e x p o n e n tional r a t e c o n s t a n t o f its labelling (A3Ja t) w o u l d b e J and J/C3~, respectively. S u c h a pool 3 a w o u l d m a k e t h e largest c o n m ' b u t i o n t o overall /13, so the r a t e o f increase o f A3/a t (approx. AT/a l) estimates ./ while t h a t o f A2/a t estimates L
r a t i o o f C 2 a t C I = 0 ( m e a s u r e d o r e x t r a p o l a t e d ) to its value a t C , = 1 m M [4,21]. References 1 Bevington, A., Kemp, G l and Russell, R.G.G. (1992) Clin. Chem. Enzya~l. Commur,. 4, 235-257. 2 Beviogton. A.. Kemp, GJ. and Russell, R.G.G. (1990) Cohen, R.D, Lewis, B, Alberli, K.G.M.M. and Denman, A.M. (Eds.), in The Metabolic and Molecular Basis of Acquired Disease, pp. 1097-1123, Bm311ereTiodall. London.
3 Bevington. A.. Mundy, K-L Yates, AJ.P., K,-ulis.J.A. Russell, R.G.G, Taylor, D J , Rajagopalan, B. and Radda, G.K. (1987) Gin. Sci. 71, 729-735. 4 Kemp, GJ. and Bevington, A. (1992_) Clio. Chem. Enz~mol. Commtm. 4, 209-233. 5 Kemp, GJ., Polgreen, K.E. and. Radda, G.K. (1990) Biochem. Tra~s. 18, 625-626. 6 Polgreen, K.F_~ Kemp, GJ. and Radda. G.IL (1992) BiocherrL Soc. Trans. 200.168S. 7 Betington, A-. Angler, C.M., Kemp. GJ. and Russell, •.G.G. (1989) Anal. B m c h ~ 181,130-134. 8 Kemp, G J , Bev~.gtou, A~ IGu~ja, D , Challa, A.C. and Russell,
ILG.G. (1989) B~ochem.J. 264, 729-734. 9 Rosemblatt, M, Hidalgo, Co, Vergara, COand lkemoto, N, (1981) .L Biol. Chert, 256, 81404148. 19 Seller. S. and Fleischer, S. (1982) J. BIOLChem. 257,13862-13871. ! | Kemp, GJ. and Bevinglon, A. (1989) Biochem. Soc. Trans. 17, 1092_ 12 Kemp, G.J, Beviogton. A. and Russell, ILG.G. (1988) J. Theor. BioL 134. 351-364. 13 Kemp, G J , K]muja, ILl, Bevington, A. and Russell, R.G.G. (1989) l~acl~m. Soc. Trans. 17, 521-522.
Control of cellular [Pi] W i t h o u t active t r a n s p o r t intracellular [Pi] w o u l d , given t h e m e m b r a n e potential o f a r o u n d - 7 0 m V in muscle, b e negligible. If active i ~ l u x (IA) obeys h y p e r bolic ( M i c h a e l i s - M e n t e n ) kinetics a n d if passive efflux (E) is n e a r f i r s t - o r d e r so t h a t E = 21C2, say, t h e n at steady-state cellular [Pi] (C2) h a s a hyperbolic d e p e n d e n c e o n extracellular [Pi] (Cl), a p p r o a c h i n g a maxim u m C2m = ( m a x i m u m IA)/A w h e n C t is l a r g e [12]. C 2 is t h e n half-maximal a t s o m e C t , w h i c h is close to o r exceeds K m f o r the active P~ t r a n s p o r t e r [12]. C 2 = 0 a t C , = 0 u r J c s s cellular [Pt] is t e m p o r a r i l y b u f f e r e d at t h e expense o f a n o r g a n i c p h o s p h a t e pool o r t h e r e is a n o n e x c h a n g e a b l e c o m p o n e n t o f P~ [8]. ! n practice, regulation c a u b e m e a s u r e d by a n index defined as t h e
14 De Boland, A.R, Crallego. S. and Boland. 17,.(1983} Biochim. Bioph~ Acla 733, 2fi4-273. 15 Wehrle. J.P an0 Pedessen, P.L. (1989) J. Membr. Biol. IlL 199-213. 16 Jack, M.G. and Askari, H.. (19F,B) J. BioL Chem. 264, 3904-3908. 17 Biber, J , Brown, CoDA. and Muter, IL (1983) Biochim. Biophys. Acta 735, 325-330. 18 Strevey. J , Giroux, S. and Belbeau, It. (1990) Biochem. J. 271, 687-692. ~9 Belb-eau, R. and Strev~, J. (1988) Am. J. Physiol. 254, F329-F336. 20 Shoemaker. D.G, Bender, CA. and Gunn, R.B. (1989) J. Gen. Physiol. 92, 449--474. 21 Bevington, A , Kemp, GJ. and Rns~ell, R.G.G. (1989) Bmchem. Soc. Trans. 17,124-125. 22 Bowen, $.W. and Levinson, Co (1982) J. Cell. Physiul. 110, 149154. 23 Kemp, OJ. and Bevington, A. (1991} Bioc.~em. Soc. Trans. 19. 176S.
Biochimica et Bioplo.sica Acta, 1137(1992) 10-18 © 1992ElsevierSciencePublishersB.V.All rightsreserved0167-4889/92/S05.00
BBAMCR 13234
Skeletal muscle Pi transpo~ and cellular [Pi] studied in L6 myoblasts and rabbit muscle-membrane vesicles G r a h a m J. K e m p , K i m E. P o l g r e e n a n d G e o r g e K. R a d d a Depannu~ of Biochemislly, Unirersitvof Oxfor~ 04ord and MRC Clinicaland Biocl'~,micalMagnetic Resonance Unit, John Radcliffe Hospital. Oxford (UK)
(Received 6 April 1992) Keywords: Phosphatetmmpon:Cellular phosp~hate;Skeletal mu~-.ccell; ~P-labelling In the rat skeletal myoblast line L6 and in a rabbit skeletal muscle sarealemma/t-tubule vesicle preparation, [32P]PI uptake was largely dependent on the transm.embrane Na gradient. Na-dependent [32PIPi uplake had a hyperbolic relationship to [Pi] and [Na], being half-maximalat 0.2-0.3 mM [Pi] and at 25-40 mM [Na]. In vesicles the Ha-dependence suggests that approx, two Na are transported with each Pi, but the inhibition of [32P]Pi uptake at high pH suggests that the Pl monoanion is the transported form. Together these imply electrogenic transport and this is confirmed by the results of manipulating the vesicle me,-nbrane potential. Thus. electrogenic Na-Pi co*transpo~ exploits both the sodium gradient and the cell membrane potential to maintain muscle cellular [Pi] against an unfavourable electrochemical gradient. The low [Pl] for half-maximal flux may partly explain the small effect of altered extracellular [Pi] on cellular [Pi]- In L6 myoblasts most 32_pwas fit'st detectable in an organic phosphate pool rather than ct:llular Pl, v,'~c the ~ f i c a-,:t-'.,vi~of fell Pi rapidly reached 40% of that of extra~llular Pi and was stable for at least 3 h. These results are discussed in terms of the organisation of cellular p~osphate metabolism. latroduellon Despite the central role of intracellular Pi in bioenergetics and elsewhere [1] and the serious muscular (and other) consequences of clinical Pi disorders [2] tittle is known about how [Pt] is regul~.ted in skeletal muscle, where resting [Pl] is maintained at 2-4 mmol/I, despite a highly unfavourable electrochemical gradient and appears to be held nearly constant when extracellular [Pi] changes [3,4]. In this paper we report studies of cellular [Pi] and Pi Uansport in the cultured rat myoblast line L6 and a more detailed characterisation of Pi transport in sarcolemmal and t-tubule vesicles prepared from rabbit muscle. Some of this work has been presented in preliminary form [5,6]. Materials and 1YT~:rods Cultured cell methods
1.6 cells (passage number 5-20) were grown at 37~C under 5% CO 2 on 9 cm Petti dishes in 10 ml Eagle's
Cotr~pondence to: GJ. Kemp, MRC Clinical and Biochemical Magneti, R.~,anan.=eUnit, John Radcliffe Hospital, Oxford, OX3 9DU, UK. Abbreviations: MEM. Eagle's minimumessential medium; DMEM, Dulbecco'sMEM.
minimum essential medium ( M E M ) with 10% foetal calf serum (Northumbrian Biologicals) or Dulbeco's MEM (DMEM) with 10% horse serum; 2 mM glutamine was added to both. Medium was changed every 3-4 days and cells were passaged (1:10) every 5-7 d a ~ after removal from the plate with 2.5 ml trypsin/EDTA (Gibco BRL Life Technologies, Paisley, UK). For experiments, cells were grown to subconfluence for 1 or 2 days on 12-well culture plates (1 Petri dish, approx. 107 cells, seeding 24-36 wells). Under these conditions L6 cells grow as myoblasts. No attempt was made to fuse into myotubes, as this would require longer culture times, making it difficult to be sure that all extracellular medium was washed away during the cell sampling steps described below. Cells were then incubated at 3"PC for 1-2 h in fresh medium under 5% CO e or in Tris- or Hepes-buffered Ringer solutions at YPC, under air, with 74 kBq (2 /tCi) per ml of 3-O-methyl/D-[l-3H]glucose to allow measurement of cell volume. Ringer solutions contained 122 mM Na (replaced by choline for Na-free experiments), 130 mM Ci, 5.6 mM K, 2 mM Mg, 2 mM Ca, 5 mM glucose and 30 mM Tris or Hepes. At sample times plates were washed rapidly four times with Pi-free Hepes-Ringer solution (plus 10/~M phloretin) and 500/zl of ice-cold 1/3 M perchloric acid added to each well. After scraping with a syringeplunger, 260/tl acid extract were added to 20/ti 1 M silver perchiorate in 2 / 3 M perchloric acid and 2 0 / t l
11 1.8 M NaCI, vortexed for 5 s and centrifuged at 3000 × g for 5 min at 40C. 250/~1 supematant were neutralised with 4.3 M KOH/0.6 M imidazole (plus 2% (w/v) wide-range indicator). 3H activity was measured in 200 ~tl acid extract and 25 pl incubation medium: cell volume per ml neutral extract (/~1) = (3H per ml)/(3H per/~1 of medium), from which cellular [Pi] = (nmol Pi per ml extract)/(ce!! volume per ml extract). No attempt was made to correct for trapped or bound extracellular medium, which is negligible under these condilions. Pi was assayed in the neutrai'-~ed cell extract by a mezhod depending on addition of acidified molybdate, rapid organic ey-traction of resulting phnsphomolybdate, aqueous back-extraction of Pi and formation of phosphomolybdate/Malachite green complex whose absorbance is measured at 660 nm [7]. For labelling experiments, 37 kBq (1/~Ci) of [32p]pi were added per ml of medium at appropriate times. 32p distribution is A T / A t = (32p/3H in cell extract)/ (32p/3 H in medium) where A T is total 32p (cpm/I cell water) and A I is extracellular 32p activity (cpm/l). Inorganic [3eP]Pi (A 2 cpm/1 cell water) and organic 32p (A 3 cpm/.! cell water) were separated by organic extraction of phosphomolybdate (correcting for incomplete recovery of organic phase [8]), and cellular activities calculated using the cell volume (as for cellular [P~] above). To study effects on P~ fluxes, we expressed [3zP]P i uptake at t = 5 rain relative to control: this a good estimate (within 15%) of the true fractional changes in Pi flux. 32p and 3H in medium and neutralised ceil extract were measured by liquid scintillation counting. Membrane vesicle methods Sarcolemmal and t-tubule vesicles were prepared from leg white muscle of female New Zealand White rabbit. The t-tubule fraction was isolated by a modification of a published method [9]. Briefly, minced muscle homogenate in 300 m M sucrose/.5 mM imidazole / 1 0 0 / t M EDTA buffer (all buffers used were at pH 7.4) was centrifuged at 3000 x g for 20 rain, the supernatant centrifuged :~ice at 10000 × g for 20 rain and the resulting supernatant filtered, resnspended in 0.5 M KCi and centrifuged three times at 1501100 × g for 2 h. The resulting supernatant mlcrosomal fraction was resuspended to 40 mg protein per 10 rnl and centrifuged on a 17-35--48% sucrose gradient for 12 h at 7 0 0 0 0 x g ; the 17/35% interface was harvested and similarly centrifuged on a 35-50% sucrose gradient after loading with calcium phosphate to precipitate sarcoplasmic reticulum; the interface was harvested, resuspended and centrifuged three times. Vesicles were stored in sucrose/imidazole buffer in liquid nitrogen. The sarcolemma fraction was isolated by a published method [10]. Briefly, minced muscle, ho-
mogenised in 0.75 M KCi/imidazole, was centrifuged twice at 3000 x g for 20 rain, each time resaspending the pellet, which was then homogenised in sucrose/" imidazole, centrifuged twice and homogenised again. The resulting supematanL recentrifuged and filtered, was centrifuged at 5 0 0 0 0 x g for 2 h and tL ,~ pellet resnspeoded in 0.6 M KCI and layered onto a 17/23% sucrose gradient. Tlais was centrifuged at 700(10 x g for 12 h and vesicles harvested from the interface. Relative to the original microsomal fraction, the appropriate marker enzymes Na, K-ATPase, MgATPase and 5'-nucleutidase were enriched by factors 26, 11 and 24, respectively, in the sarcolemmal fraction and 17, 15 and 21, respectively, in the tubule fraction. In both fractions the sarcoplasmic reticular markers Ca-ATPase and glucose 6-phosphatase were diluted by factors of at least 0.02 and 0.008 and the mitochondrial marker succinate debydrogenase was undetectable. Ti~ere are no specific markers for t-tubules but t h e t-tubule fraction had (as expected) less Na, K-ATPase, more Mg-ATPase and a higher density than sarcolemma [9]. Assessed by ouabain inhibition of ~a,KATPase, both vesicle fractions were 90-95% sealed; from the latency of Na,KoATPase (the effect of ouabain on Na,K-ATPase in the presence and absence of sodium dodecyl sulphate), the sarcolemmal vesicles were 80% right-side-out, the t-tubule vesicles being 43%. Both vesicles were about 2~, nm in diameter by electron microscopy. The overall ~ield of vesicles was 96, 0.6 and 0.l mg protein/g wet wt mu..~le in crude microsomal, t-tubule and sarcolemmal fractions, respectively. Pi transport was measured by addition of 20 /zg vesicle protein to 1.2 ml buffer containing 370 kBq (10 ttCi)/ml [32P]Pi; the reaction was stopped by three rapid washes with ice-cold incubation buffer through a 0.22/~m filter. Fluxes were calculated from labelling at 1 min, at which time labelling was acceptably finear with time. C/s (i.e., extravesicular) buffers contained 135 mM NaC! or choline chloride (chloride sometimes replaced by thiocyanate or gluconate), 30 m M Tris or 2[N-morpholino] ethane sulfonate and 1 mM EDTA; trans (i.e., intravesicular) buffers contained 30 m M "Iris or 2[N-morpholino]ethane suifonate and appropriate salts, with sucrose up to 250 mM. 32p activity in c/s buffer and on the fdters was measured by liquid scintillation counting. Protein concentration in vesicle suspensions were measured by the Lowry method. Data analysis Results are expressed as mean 4- S.E. Statistical significance of differences was assessed by Student's t-test. Straight lines were fitted by linear regression and rate constants for exponential changes obtained from semilogarithmic plots. Kinetic parameters for Pi influx were
12 estimated using the Hanes plot, i.e., [substratc]/rate against [substrate]. The basis of the analysis of ceilular labelling and the control of celh'~!ar [Pi] is briefly summarised in the Appendix.
Materials Unless stated otherwise, all tissue culture materials were purchased £rom Gil~co or from Flow Laboratories (Rickmanswertb, Herts, UK), all reagents were of analytical grade mid purchased from B D H (Poole, Dorset, UK) o r Sigma (Poole, Dorset, UK). Radioisotopes were purchased from A m e r s h a m International (Amersham, Bucks, UK). L6 wete obtained from the PHLS Centre for Applied Microbiology, Porton Down. Results
O.6
L6 cells Fig. 1 shows the time course of cellular labelling. The a n m u n t of organic labelling was large (Fig. la): even at J = 2 min only 27 + 6 % of cellular 32p was ill the Pi pool. The initial rate of inorganic labelling, expressed as A , / a I (i.e, absolute activity of cellular Pi relative to specific activity of extracellular Pi) can be t a k e n as the apparent Pi flux into the cellular Pi pool (we call this 1): this was 4.9 + 0.8 retool/! cell water p e r h. The initial rate of organic labelling, expressed as A 3 / a I (i.e., the overall absolute activity of cellular
° :1,., o ~ o° o
3o
0 60
0
0
90
120
0 150
180
,;0
,~o
Time(min)
:t i
(b)
0
30
.'0
9"o
,~o
Time(rain) Fig. l. Time course of [32p]pi uptake in L6 cells. (a) Total labelling (A T) (closed circles) and labelling of cell Pi (A2) (open circles); (b) specific activity of cellular Pi (a2)* all expressed relative to specific activity of extrace[Iular Pi (at)- [32p]Piwas added at t = 0 to cells in MEM/10% foetal calf serum (n = 3).
0.4
rc
io[
o t,,reM~l~J 3 mM[~]
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Fig. 2- Effects of foetal calf serum and sodium on [32PIPi uptake in I-6 cells. (a) The effects of 24 h omission of foetal calf serum (solid bars) and 24 h omission f o l ~ e d by I h replacement of 10% foetal calf serum (shaded bars) on total [32pIPi uptake (total labelling T), [3-'PIPi uplake into cellular Pi (inorganic labelling I) and on cellular [Pi]. Cells weru grown (for 24 h) and incubated in MEM. Data expressed relative to incubation with 10% foetal calf serum throughout (n = 6). (b) Comparison of cellular [Pi] (black bars) and [32plPi uptake (shaded bars) after I h incubation at zero and 3 mM extrucellular [Pi] (n = 4) in the presence of sodium, and at ! mM [Pi] in the absence of sodium (n =6). CelLswere grown in MEM/10% foetal calf serum and incubated for I h in Tris-Ringer (simile: Na-dependence is seen in ceils grown in DME,L~/10% horse serum). Results expressed relative to incubation at 1 mM [Pi], i-~ mM INa].
organic phosphates relative to specific activity of extracellular Pi) can be taken as the apparent Pi flux into organic phosphates (we call this J): this was 29 _+ 2 m m o l / l cell water p e r h. The specific activity of cellular Pi relative to that of extracellular Pi, a2/al, approached a plateau 0.39 + 0.01 (stable for at least 3 h) with a half-time of 10 + 1 min (Fig. lb). Fig. 2 shows some effects of altered incubation conditions on [3zP]Pi uptake and cellular [P~]. Total cellular labelling, expressed as A T / a ~ (i.e., total absolute cellular activity relative to specific activity of extracellular Pi) can be taken es the total Pi flux into the cell (we call this T = I + J ) . Incubation for 24 h without foetal calf serum reduced T by approx. 50% and this was restored by 1 h incubation with serum. Neither manipulation altered cellular [Pi] (Fig. 2a). A t 1 mM Pi, 1 h incubation without sodium reduced T by approx. 80%, while cellular [Pi] was not significantly affected (Fig. 2b). Incubation for 1 h in the absence of extracel|ular P, reduced cellular [Pi] to 85% and incubation at 3 m M [Pi] increased both T and cellular [Pi] by 10-20%
(Fig. 2b). Absolute cellular [Pi] was 3 . 2 _ 0.7 m m o l / I cell water in cells incubated for 1 h in M E M / 1 0 % foetal calf serum under 5% C O 2 (n = 6) and 4.6 + 0.6 m m o l / ! cell water in Tris-Ringer solution under air (n = 6). Fig. 3 shows more details of the dependence of [32PIPi uptake on [PJ and [Na]. T has a hyperbolic relationship to extracellular [Na] (Fig. 3a): from the Hanes plot (Fig. 3b) T was half-maximal at 40 mM [Na]. The Na-independent component of T was linear with extracellular [P~] (slope 4.1 __ 0.1 h-~). The Na-dependent component of T showed hyperbolic dependence oi: [PJ (Fig. 3e) and from the Hanes plot (Fig. 3d) K m = 0.26 mM and maximum flux = 40 m m o l / l cell w a t e r per h; K m was indistinguishable for ! and T (Fig. 3d).
"t-Tubule" m e m b r m w vesicles Fig. 4 shows some properties of Pi transport in t-tubule membrane vesicles. Fig. 4a shows that in the presence of 135 m M c/s (external) [Na], [32P]Pi uptake
11 1.0
(ll)
•
o.e
i
0.6 o.4
•
0.2 o.o
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210
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showed classical overshoot kinetics. Fig. 4b shows that [32P]Pi uptake was negligible in vesicles disrupted by rapid freeze-thawing and was zeduced to 20% of control when c/s [Na] was zero and to 4 0 - 5 0 % of control when c/s and trans (internal) [Na] were set cqual or when the Na-gradient was partially dissipated with 1 /~M monensin. Fig. 4b also shows that [32p]Pi uptake was stimulated by 50% when trans [Fi] was increased to 10 mM and that this overcame the inhibitory effect of 100 mM trans [Na] (but see Fig. 6c). Fig. 5 shows more details of the dependence of Pi transport on [Na] and [Pi]. Fig. 5a shows that [32pIPi uptake had an approximately hyperbolic relationship to [Na], although the sigmoid shape of the plot at low [Na] suggests that more than one Na ion is transported for each Pi ion. In accordance with this, the Hanes plot (Fig. 5b) is markedly nonlinear at low [Na], but becomes linear when [Na] 2 is used in the plot rather than [Na], suggesting a stoichiometry of approx. 2 Na: 1 Pi.'.32P]Pi uptake was half-maximal at 25 mM [Na]. Na-dependent [32p]p i uptake had a hyperbolic relationship to
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Fig. 3. Dependence of [32p]p~uptake on extraceilular [Na] and [Pi] in 1.6 cells. (a) Total [ 32p]pi uptake (T) (expressed relative to its value at 120 mM [Ha]) as a function of lNa]- Cells were grown in DMEM/10% horse serum and incubated for I h in Tris-Ringer (n = 7). (b) A Hanes plot of Na-dependent 132p]Pi uptake and [Na]. Cells weru gro~. in DMEM/10% horse serum and incubated for I h in Tris-Ringer to = 7L (c) Na-dependent (closed circles) and Na-independent (open circtes) components of total [32plp i uptake T (expressed in absolute terms) as a function of extracellular [Pi]. Cells were grown in MEM/10% foetal calf serum and incubated for I h in Hepes-Ringer with 120 riM [Nal. (d) Haoes plots of Na-dependent [32P]Pi uptake (expressed relative to ,.lptake at I mM [PJ) and [Pi]. Closed circles show a Hanes plot for the dependence of Na-depcadent component of total [3:PIPt uptake (T) on extracellular [PJ; crosses show similar data for the Na-dependen[ component of [32plp, uptake into cellular Pi (I). Cells were grown ir MEM/10% foetal calf serum and incubated f~,r 1 h in Hepes-Ringer with 120 mM [Na].
[Pi] (Fig. 5c) and from the Hanes plot K m =0.16 mM and maximum flux= l l p . m o l / m g protein per min (Fig. 5d). Fig. 6 shows the dependence of Na-dependent [32PIPi uptake on pH. Fig. 6a shows that at 0.09 mM cL: [P;] (a nonsaturating concentration) and t r a ~ pH 7.4, in the absence of trans [Pi], Na-dependent [32pIPi uptake decreased with increasing pH (slope = - 0 . 9 + 0.2 # m o l / m g per min per pH unit; P < 0.01). However Fig. 6b shows that when c/s [P~] was manipulated to maintain 0.09 mM c/s [Pi monoanion], c/s pH had little effect. Fig. 6a also shows that when c/s pH was 5.5, [32pIPi uptake was slightly stimulated when trans pH was 5.5 rather than 7.4 ( P < 0.05) and that at c/s pH 9.5 [32PIPi uptake was slightly inh~ited when trans pH was 9_5 aLso ( P < 0.05). Fig. 0c shows that, at constant c/s pH, the effect of trans pH depended on trans [Pi]: at zero trans [Pi] [32PIPi uptake was lower at pH 6.5-7A than at either extreme pH ( P < 0.05); in contrast, at 10 mM trans [Pi] [32PIPi uptake fell steeply with increasing pH (slope, - 1.3 _+0.2 p m o l / m g per min per pH unit; P < 0.001). Thus trans-stimulation was only seen (cf. Fig. 4b) at trans oH 7.4 and below, while above this pH trans Pi inh~ited Pi uptake. ~0]
(a)
Fig. 6d shows that at c/s [Pi] in excess of K m (in contrast to the results in Fig. 6a) Na-dependent [-~2P]Pi uptake increased with increasiag pH (at 0.9 mM ~'~ [Pi], slope, 2.8 +0.1 # m o l / g per rain per pH unit; P < 0.02). In anion-substitution experiments Na-dependent [32PIPi uptake was increased to 220 _+ 12% of control when chloride was replaced by thiocyanate ( P < 0.05; n = 3) and reduced by to 77 + 6% of control when chloride was replaced by glucona~.e ( P < 0.05; n = 3). Ha-dependent [3-'P]P i uptake was reduced by 30% by 10 mM arsenate, but unaffected by 10 mM vanadate (n = 3). Lastly, the effects of pH and [Pi] on Na-independent [3-'P]P i uptake can be summarised briefly. At constant pH it was linear with c/s [P,] (slope, 5.2 + 0.7 ( p m o l / g per m i n ) / ( m m o l / I ) ) ; at 0.09 mM c/s [Pi], c/s pH and trans o H had little effect; at constant 0.02 mM c/s [monoanion], by contrast, it increased with increasing pH in proportion to the increase in total c/s [Pi]; it was substantially inhibited by I0 mM trans [Pi]Sarcolemmal cesides
[3zP]Pi uptake was much less active in the sarcolemmal preparation, and so no characterisation was attempted. At 0.09 mM c/s [Pi], peak uptake (at t = 1 min) in the presence of Na was 1.5 p m o l / g protein (n = 3 ) , only 20% of the equivalent figure for the t-tubule preparation. Discussion Approaches to P~ metabolism
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Steady-state cellular [Pi] depends mainly on active Pi uptake [11] (see Appendix) which is easily studied using 32p-labelliag. Studies on whole cells illuminate (but are complicated by) exchange between cellular Pi and organic phosphates [12] and functional compartmentation [8,13]. The mechanisms of Pi transport are best studied using membrane vesicles, bearing in mind that changes in cellular [Pi] also depend on the permeability to passive back-flux of Pi [11] (see Appendix) and, transiently, on changing organic phosphate pool size and net flu~es across the cell membrane [4]. Effects o f [Pi] a n d [ N a ] on Pi transport
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Fig. 4. [32PIPi uptake in rabbit muscleplasma membranepreparation rich in t-tubule vesicles (all n = 3). (a) The time-course of ['~2pIPi uptake followingaddition of ,zesielesto mediumcontaining[3~"PlPi at t = 0. Open squares, with sodium; open circles, without sodium; closed circles, sodium-dependent uptake. (b) [3'PIPi uptake (left to fight): in vesiclesdisrupted t~" freeze-thawing,at 135 mM c/s (external) [Na], at zero c/s lNa]: with 135 mM c/s and trans [Na]: with 10 mM trans [Pil at 135 mM c/s [Na]: and with l0 mM trans IPi] and 100 mM trans INal at 135 mM c/s lima]. Data expressed relatb.'e to 135 raM cis INa]with zero trans [Na] and lP,l: all 0.09 mM cis lPl].
Apart from a report of vitamin D-stimulated Ha-dependent [32p]pl uptake in chick muscle sarc~le.rmnal vesicles [14] these results provide the fast characterisation of membrane P~ transport in skeletal muscle. The larger part o1 [~:P]P~ uptake depends on extracellular [Na] in cells (Fig. 21)) and vesicles (Fig. 4b) and in membrane vesicles it shows classical overshoot kinetics (Fig. 4a) and requires an intact membrane and transmembrane Na gradient (Fig. 4b). All of these suggest that co-transport of Na and P~ is responsible for the cellular accumulation of P~ against its electrochemical
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Fig. 5. D~.pendenceof [32PIPi uptake on [Pi] and [Nal in rabbit muscle plasma membra~.epreparation rich in t-tubule vesicles(all n = 3). (a) [32P]Pi uptake as a function of c/s [Hal. (b) A simple Hanes plot (closed circles)of [ 32pIpi uptake and c/s [Na],with a regressionline whichomits the anomalous point at 7.5 mM [Na] (which is shown with its standard error): a modified Hanes plot (open circles) (i.e., [substrate]2/velocity against [substrate]2,arbitrarily scaled to fit on the samc axes)on the assumptionthat the Hill coefficient of Na is 2. with a regressionline passing through all the points.(c) Ha-dependent [32p]p~uptahe as a functionof e/s lPl], (d) Hanes plot of Na-dependent 132plPl uptake and c/s [Pl]gradient in skeletal muscle (see Introduction), as in several other cell types [15]. The "/('m of Na-dependent P~ transport for c/s [Pi] is similar (0.2-0.3 raM) in the rat cell line L6 cells and in rabbit skeletal muscle vesicles (compare Fig. 3c and d with Fig. 5c and d). A low K m is consistent with the need to preserve cellular [Pi] against changes in extracellular [Pi] [11] (see Appendix) and similar values of K m are seen in heart muscle [16], osteoblasts [13], renal tubular cells and others [15]. The absolute rates of Pi uptake ~n L6 are comparable to values reported for, e.g., osteoblasts [13] and renal tubular ceils [17]. Effects o f p H on Na-dependent P~ transport At 0.09 mM c/s [Pi], [32p]Pi uptake decreases with c/s pH. but is unaffected by c/s pH when [monoanion] is held constant (Fig. 6a and b). These results, the opposite of those in rat renal tubular brush border vesicles [18], suggest that the Pi monoanion is the form binding to the c/s face of the transporter. By contrast, the increase in [32p]p~ uptake with increasing pH, seen at saturating c/s [P~] (Fi~ 6d), presumably reflects an intrinsic property of the transporter. The evidence of Fig. 6a that, at given c/s pH [32P]P i
uptake is slightly stimulated by alkaline trans pH and inhibited by acid trans pH, suggests that a small component of Na-dependent Pi uptake may be driven by the proton gradient, as reported in renal tubular brush border vesicles [18]. At 10 mM trans [Pi], increasing trans pH inhibits [32P]Pi uptake (Fig. 6(:), suggesting that increasing trans [monoanion] favou~ trans-to-cis translocation of the transporter. This, which is the opposite result and conclusion to that obtained in renal tubular brush border vesicles [18], is to be expected from the evidence above that it is the monoanion which binds to the c/s side, since half the vesicles are inside-out (see Results). However, at zero trans [Pi] a different respouse is seen, probably reflecting the intrinsic properties of the carrier: [32P]Pi uptake reaches a minimum at pH 6_5-7.4, which presumably least favours cis-to-trans translocation of binding sites (Fig. 0c). The result of these effects is that, at high trans pH, the expected trans-stimulation of [z2p]pi uptake (Fig. 4b) is converted to a trans-inhibition (Fig. (c). In general trans-stimulation suggests that carrier-P i complex translocates to the cis face faster than free carrier, and also, since trans Pi can overcome the inhibition by trans Na (Fig. 4b), faster than the carrier-Na complex
16 [18]. It would appear, however, that, at high trans pH, the transloeation of the unloaded carrier is faster than that of carrier-Pi in this system.
Stoichi~,netry o f Na-dependou Pi transport Tl:ere is some evidence in rabbi t. muscle vesicles (Hg~ 5a and b), although not yet in L6 ceils (Figs. 3 and b), that approx. 2 Na inns are transported per P~ ion. If the transported form is the Pi monoaninn (see above), this impfios electrogenic transport (positive charge movement inwards), as in cardiac muscle [16] and in some reports of renal tubular brush border membranes [19]. This is supported by the result of substitution of chloride in the incubation medium: [32p]p~ uptake is stimulated by replacement with the more diffusinie anion thiocyanate, which induces an inside-negative potential, and inhibited by the less-dLffusinle gluconate anion, which induces an/,as/de-positive potential. This makes physiological sense, as clectrogenic (positive inward) transport would ~_,se the membrane potential rather than oppose it [1~. N a - ~ e p e n d t m P~ transport In I.,6 cells (Fig. 2b) and in rabbit muscle vesicles Na-independent [32pIPi uptake is near-first-order with respect to c/s [Pi], which is the usual finding in mam-
(a)
malian cells e.g., Refs. [13,17,18]. In vesicles, its independence of cis and trans pH (at constant cis [Pi]) is shw21ar [18], while its inhibition by trans Pits opposite, to the findings in renal tubular brush border vesicles [19].
Organisation o f c¢llu!ar phosphate metabolism The failure in 1_6 cells of the cellular Pi pool to reach isotopic steady-state b.v 3 h (Fig. lb) could be explained, as in euthrocytes [8] and osteoblast-like cells [13], as a consequence of exchange with the bulk of the organic phosphates [12] (see Appendix). As in an ostcoblast line [13], at least 3 / 4 of the Pi entering 1.6 cells by the Ha-dependent transporter is detectable in a large organic phosphate pool, which labels faster than could be explained by exchange with detectable cellular Pi (Fig. 1). One possible explanation is that a high-specific-activity subpool of Pi has access to an enzyme catalysing its incorporation into an organic pool. If this is so, at 1 mM [P~] the effective flux from extracelhilar Pi to cell Pi appears to be 5 mmol/I cell water per h compared to 30 mmol/i cell water per h into the o~ganic pool; subtra~ing the estimated contrinution of direct exchange with bulk cellular Pi (estimated from a2/a I using the simple model described in the Appendix) from measured organic la-
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Fig. 6. EffectsofpH on Na-dependent [~2P]Pi uptake in rabbit muscleplasmamembranepreparation rich in t-tubule vesicles(all n=3). (a) Effectsof c/.vpH at 0.09 mM c/s [Pi],when 'runs pH is 7.5 (o~encircles)and when trans pH is equal to c/s pH (closedcircles).(b) Effectsof c/s pH at constant0.02 mM c/s [Pi monuanion],when zrans pH is 7.5 (opencircles)and when trans pH equals¢/s pH (closedcircles).(¢) Effectsof trans pH at ¢/s ptl 7.4 and 0.09 mM c/s IPi].when traas[P,Iis zero (closedsquares)~n~ when trans[Pil is 10 mM (opensquares).(d) Effectsof c/s pH at tra~ pH 7.5, whenc/s [Pi]is 0.09 raM(circles),0.9 mM(diamonds)and 1.5 mM(triangles).
17 belling allows the size of such a rapid-labelling organic pool, if this is the explanation, to be estimated as approx. 15 mmol/I. There is .some precedent for such a mechanism: the erythrocyte has a Na-linked Pi transporter which feeds most of its Pi directly into a membrane-associated nucleotide pool [20]. The Na-dependenee of this organic labelling appears to suggest that such a flux in L6 (and osteoblasts [13]) &_curs via the Na-Pi co-transporter (also supported by the similar K m for organic and in,~rganic labelling - Fig. 3d). However, if the sarcolemmal Na,K-ATPase consumes a large part of ATP turnover then its inhibition by Na-withdrawal could reduce organic incorporation of [32P]P i, as is observed. Nevertheless, even in the absence of sodium the rate of organic labelling is higher than could be explained by exchange solely with what the present methods detect as cellular Pe The finding that it is the 't-tubule' vesicle fraction from mature skeletal muscle which has significant Nalinked P~ uptake is unexpected (though it should be noted that this preparation does not include the triads at the apex of the t-tubules). However, we cannot exclude an artifact, e.g., damage to the transporter by the more elaborate preparation of the sareolemmal vesicles (although other transporte~ can survive this [10]). This finding, if real, suggests that the external microenvironment of the transporter may be quite different from that at the sarcolemmal surface. In particular, the diffusion limitations might make Pi leaking through out of the cell more access~le to cellular re-uptake.
Control o f cellular [Pi] Accurate measurement of cellular [Pi] requires minimal hydrolysis of organic phosphates during extraction and assay. In the present experiments on L6 cells this should be achieved by the speed of perchloric acid extraction and organic extraction of phosphomolybdate in the Pi assay [7]. Chemically measured cellular [Pi] includes at least cytosolic, nuclear and mitochondrial Pi. Nevertheless, the total cellular [Pi] in this study is close to NMR measurements [3] of cytosolic [Pi] in vivo. Many cells can apparently regulate [4] intracellular [Pi] in the face of changes in extracellular [P~]. la I.,6 cells incubated for ! h a t zero or 3 mM [Pi], the fractional change in cellular [P~] relative to cells incubated at I mM [Pi] is smaller than that in extracellular [Pi] (Fig. 2b). This gives an apparent rega',dtion index (see Appendix) of approx. 0.9, compared to 0.5-1.0 in a variety of other cells and situations [4,21] and specifically with 0.7 in skeletal muscle in vivo [3]. Little is known about the details of this regulation [4,11]. In the present work the small effect of increased extracellular [Pi] (Fig. 2b) is consistent with the low K m of Na-de-
pendent P~ uptake (Fig. 3c), as under these conditions both Pi uptake and cellular [Pi] increase by a similar small fraction. The failure of Pi-free incubation to deplete cellular [Pi] (Fig. 2b) could be wholly or partly the result of slow equilibration of the Pi pool (as muscle membrane permeability to net effiux of Pi is very low in vivo [4]), or of non-exchangeable (and perhaps non-cytosolic) Pi pools. Much longer incubations will be required to resolve this. The dependence of Pi uptake and cellular [Pi] on [Na] has rarely been compared, but in a study of Ehrlich cells [22] sodium deprivation reduced Pi influx by 65% but cellular [Pi] by only 15%. This, and the similar discrepancy in L6 (Fig. 2b), may also result either from failure to reach steady-state, from temporary maintenance of cell [Pi] by the slow collapse of an organic pool or from changes in Pi back-flux permeability [4,11].
Net flux attd exchange flux We have argued elsewhere [231 that we should expect an approximate equality between permeability of a cell membrane to small net fluxes of Pi and the 'back-flux' rate constant IA/C 2 (i.e., the ratio of active influx to cellular [Pi])- Such equality seems to hold, approximately, in the small number of cases where suitable data exist [4,23]. In I.,6 cells the rate of Na-linked Pi uptake is large so that IA/C 2 is about 1 h - I (considering only labelling of cellular Pi) or 5-10 h -I (considering total cellular labelling). However, the net flux permeability of skeletal muscle sarcolemma is very low (0.1 h -I or less) [4]. This is presumably a special adaptation to prevent significant Pi loss during contraction, in which intracellular [Pi] can increase up to 10-fold. However, we do not yet know whether exchange fluxes of Pi are as ia:'ge as in L6 cells across the sarcolemma of normal muscle cells in vivo. In conclusion, we have demonstrated Na-dependent P~ uptake in a rat myoblast line and in rabbit skeletal muscle-membrane vesicles. This has properties consistent witi~ i~ role in maintaining cellular [Pi] in the face of a large negative membrane potential, namely a low K m and an electrogenic mechanism. Strangely, most of this transport activity is found in the t-tubule membrane fraction, although an artifact cannot yet be excluded. We have identified an unexpectedly rapid exchange between incoming Pi and cellular organic phosphate pools, and noted an apparent discrepancy between the relatively large exchange rates of Pi across the L6 cell membrane and the low permeability of skeletal muscle to net P~ effiux. Acknowledgements GJK was supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland and KEP by the British I-~eart Foundation.
18 AmDend~x (See Materials a n d M e t h o d s f o r e x p l a n a t i o n o f n o t a tion)
Cellular labelling In t h e simplest m o d e l o f cellular Pi m e t a b o l i s m [12] extracellular Pi e x c h a n g e s with cellular Pi ( c o n c e n t r a tion C 2) a t t r a n s m e m b r a n e flux r a t e 1 = kC,, a n d with o r g a n i c p h o s p h a t e s a t r a t e G = 3"kC2. If o r g a n i c pool size is l a r g e t h e n a f t e r a d d i t i o n o f [32P]Pi the relative specific activity o f cell Pi (a2/al) increases e x p o n c n tionally with initial r a t e k a n d r a t e c o n s t a n t k(1 + 3') t o w a r d s a t e m p o r a r y ' p l a t e a u " 1 / ( 1 + 3')- O r g a n i c labelling (A3/a ~) is l i n e a r w h e n a2/a , has r e a c h e d p l a t e a u with slope 5 = 1/[(1/1)+ ( l / G ) ] (which a p p r o x i m a t e s I ff 3" is large). In t h e e x p e r i m e n t s r e p o r t e d h e r e S exceeds L O n e e x p l a n a t i o n is t h a t a n o r g a n i c pool ( c o n c e n t r a t i o n C3~) e x c h a n g e s with a pool a t a p p r o x i m a t e l y extracellular specific activity ( p e r h a p s a n u n d e t e c t a b l e subpool o f cellular P~) a t a r a t e .I: the initial r a t e a n d e x p o n e n tional r a t e c o n s t a n t o f its labelling (A3Ja t) w o u l d b e J and J/C3~, respectively. S u c h a pool 3 a w o u l d m a k e t h e largest c o n m ' b u t i o n t o overall /13, so the r a t e o f increase o f A3/a t (approx. AT/a l) estimates ./ while t h a t o f A2/a t estimates L
r a t i o o f C 2 a t C I = 0 ( m e a s u r e d o r e x t r a p o l a t e d ) to its value a t C , = 1 m M [4,21]. References 1 Bevington, A., Kemp, G l and Russell, R.G.G. (1992) Clin. Chem. Enzya~l. Commur,. 4, 235-257. 2 Beviogton. A.. Kemp, GJ. and Russell, R.G.G. (1990) Cohen, R.D, Lewis, B, Alberli, K.G.M.M. and Denman, A.M. (Eds.), in The Metabolic and Molecular Basis of Acquired Disease, pp. 1097-1123, Bm311ereTiodall. London.
3 Bevington. A.. Mundy, K-L Yates, AJ.P., K,-ulis.J.A. Russell, R.G.G, Taylor, D J , Rajagopalan, B. and Radda, G.K. (1987) Gin. Sci. 71, 729-735. 4 Kemp, GJ. and Bevington, A. (1992_) Clio. Chem. Enz~mol. Commtm. 4, 209-233. 5 Kemp, GJ., Polgreen, K.E. and. Radda, G.K. (1990) Biochem. Tra~s. 18, 625-626. 6 Polgreen, K.F_~ Kemp, GJ. and Radda. G.IL (1992) BiocherrL Soc. Trans. 200.168S. 7 Betington, A-. Angler, C.M., Kemp. GJ. and Russell, •.G.G. (1989) Anal. B m c h ~ 181,130-134. 8 Kemp, G J , Bev~.gtou, A~ IGu~ja, D , Challa, A.C. and Russell,
ILG.G. (1989) B~ochem.J. 264, 729-734. 9 Rosemblatt, M, Hidalgo, Co, Vergara, COand lkemoto, N, (1981) .L Biol. Chert, 256, 81404148. 19 Seller. S. and Fleischer, S. (1982) J. BIOLChem. 257,13862-13871. ! | Kemp, GJ. and Bevinglon, A. (1989) Biochem. Soc. Trans. 17, 1092_ 12 Kemp, G.J, Beviogton. A. and Russell, ILG.G. (1988) J. Theor. BioL 134. 351-364. 13 Kemp, G J , K]muja, ILl, Bevington, A. and Russell, R.G.G. (1989) l~acl~m. Soc. Trans. 17, 521-522.
Control of cellular [Pi] W i t h o u t active t r a n s p o r t intracellular [Pi] w o u l d , given t h e m e m b r a n e potential o f a r o u n d - 7 0 m V in muscle, b e negligible. If active i ~ l u x (IA) obeys h y p e r bolic ( M i c h a e l i s - M e n t e n ) kinetics a n d if passive efflux (E) is n e a r f i r s t - o r d e r so t h a t E = 21C2, say, t h e n at steady-state cellular [Pi] (C2) h a s a hyperbolic d e p e n d e n c e o n extracellular [Pi] (Cl), a p p r o a c h i n g a maxim u m C2m = ( m a x i m u m IA)/A w h e n C t is l a r g e [12]. C 2 is t h e n half-maximal a t s o m e C t , w h i c h is close to o r exceeds K m f o r the active P~ t r a n s p o r t e r [12]. C 2 = 0 a t C , = 0 u r J c s s cellular [Pt] is t e m p o r a r i l y b u f f e r e d at t h e expense o f a n o r g a n i c p h o s p h a t e pool o r t h e r e is a n o n e x c h a n g e a b l e c o m p o n e n t o f P~ [8]. ! n practice, regulation c a u b e m e a s u r e d by a n index defined as t h e
14 De Boland, A.R, Crallego. S. and Boland. 17,.(1983} Biochim. Bioph~ Acla 733, 2fi4-273. 15 Wehrle. J.P an0 Pedessen, P.L. (1989) J. Membr. Biol. IlL 199-213. 16 Jack, M.G. and Askari, H.. (19F,B) J. BioL Chem. 264, 3904-3908. 17 Biber, J , Brown, CoDA. and Muter, IL (1983) Biochim. Biophys. Acta 735, 325-330. 18 Strevey. J , Giroux, S. and Belbeau, It. (1990) Biochem. J. 271, 687-692. ~9 Belb-eau, R. and Strev~, J. (1988) Am. J. Physiol. 254, F329-F336. 20 Shoemaker. D.G, Bender, CA. and Gunn, R.B. (1989) J. Gen. Physiol. 92, 449--474. 21 Bevington, A , Kemp, GJ. and Rns~ell, R.G.G. (1989) Bmchem. Soc. Trans. 17,124-125. 22 Bowen, $.W. and Levinson, Co (1982) J. Cell. Physiul. 110, 149154. 23 Kemp, OJ. and Bevington, A. (1991} Bioc.~em. Soc. Trans. 19. 176S.
