Phosphate uptake and PiT-1 protein expression in rat skeletal muscle

Kirk A. Abraham, Jeffrey J. Brault, and Ronald L. Terjung

Department of Physiology, College of Medicine, Department of Biomedical Sciences, College of Veterinary Medicine, and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211

Submitted 17 November 2003 ; accepted in final form 17 February 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (Pi) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of Pi into the cell must equal the rate of efflux, which is expected to be a function of intracellular Pi concentration. We measured 32P-labeled Pi uptake rates in different muscle fiber types to determine whether they are proportional to cellular Pi content. Pi uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 µmol·g–1·h–1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g–1·h–1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g–1·h–1). Reasonably similar rates were obtained in vivo. Pi uptake rates at plasma Pi concentrations of 0.3–1.7 mM confirm that the Pi uptake process is nearly saturated at normal plasma Pi levels. Pi uptake rate correlated with cellular Pi content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of Pi uptake across fiber types implies that other factors are involved in regulating Pi uptake in skeletal muscle. Furthermore, fractional turnover of the cellular Pi pool (0.67, 0.57, and 0.33 h–1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular Pi, likely due to differences in resistance to Pi efflux from the fiber.

inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux


IN EVERY CELL, phosphate is necessary for structural and metabolic needs. In cellular energetics, phosphate forms the high-energy bonds of ATP and phosphocreatine (PCr), and inorganic phosphate (Pi) is a substrate for reactions in glycolysis, tricarboxylic acid cycle, and mitochondrial F0F1 ATPase. Because cellular Pi is a determinant of the free energy of ATP hydrolysis, its concentration is tightly controlled. However, alterations in total phosphate content in skeletal muscle, e.g., expansion of the PCr and/or reduction of the ATP pools, suggest that either Pi uptake or Pi loss from the cell is also modulated.

In skeletal muscle, phosphate is distributed among the Pi, ATP, and PCr pools primarily via ATPases, mitochondrial oxidative phosphorylation, and the creatine kinase reaction (21). Maintenance of the total phosphate contained in these pools depends on the balance of uptake and efflux of phosphate across the sarcolemma. Although most cellular phosphate is contained in organic phosphates, these molecules cannot easily cross the plasma membrane; rather, phosphate is transported as Pi. Because Pi is transported against a concentration and electrical gradient, it must be imported via active transport. This is presumably accomplished by the type III sodium-phosphate (Na-Pi) cotransporter (15) and is driven by the electrochemical sodium gradient (13, 17, 23).

Two isoforms of the type III Na-Pi transporter, termed PiT-1 and PiT-2, have been described. These transporters have a broad tissue distribution, with mRNA expression reported in lung, liver, heart, skeletal muscle, kidney, and brain (15). In skeletal muscle, Kavanaugh et al. (15) showed abundant PiT-2 mRNA expression and little PiT-1 expression. Studies using cultured myoblasts (17), kidney cells (10), cardiac myocytes (13), and Xenopus laevis oocytes transfected with PiT-1 and PiT-2 (15) reported an apparent Km for Pi of 0.09–0.5 mM. This is well below the normal plasma Pi concentration in humans (1 mM) and rats (1.5–2.0 mM), suggesting that these transporters are nearly saturated at physiological plasma Pi concentrations. Thus the rate of Pi uptake should be directly proportional to Na-Pi transporter content. In addition, at steady state, Pi uptake must match Pi efflux. Although the mechanism of Pi efflux is unknown, it is presumed to be a passive process and thus determined primarily by intracellular Pi concentration (16, 25). If these assertions are correct, Pi uptake rate and Na-Pi transporter quantity should be proportional to intracellular Pi content. We tested this hypothesis with isolated hindlimb perfusion in the rat and analyzed different muscle fiber types, which are known to vary in intracellular Pi concentration (18, 20).


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Animal care. Male Sprague-Dawley rats (Taconic, Germantown, NY) weighing 300–400 g were housed two per cage at constant temperature (20–22°C) and a 12:12-h light-dark cycle. They were allowed unrestricted access to food and water. The experiments in this study were approved by the University of Missouri-Columbia Animal Care and Use Committee.

Hindlimb perfusion. The perfusion medium was Krebs-Henseleit buffer containing 4% bovine serum albumin, bovine red blood cells at a hematocrit of 40, 5 mM glucose, 100 µU/ml insulin, and amino acids at concentrations typical of rat plasma (3). Before use, the perfusate was warmed to 37°C, and the pH was adjusted to 7.40. The standard Pi concentration in Krebs-Henseleit buffer is 1.1 mM; however, this was altered in some experiments to create a plasma Pi concentration range of 0.3–1.7 mM to determine the Pi dependence of uptake. In a second set of experiments, we used cell-free perfusate with and without 1 mM ouabain, an inhibitor of the Na+-K+-ATPase. This was done to determine the importance of the sodium gradient on Pi uptake. The effectiveness of ouabain was verified by an absence of muscle contraction on stimulation.

Rats were anesthetized with pentobarbital sodium, and surgery was performed to isolate the abdominal aorta and vena cava. The hind feet and tail were tied with umbilical tape to concentrate flow to the hindlimb musculature. Once perfusion was established and the catheters were secured, rats were killed with an overdose of pentobarbital sodium delivered into the carotid artery. Perfusion was maintained with a peristaltic pump, and the perfusion medium was equilibrated with 95% O2-5% CO2 before entering the rat.

The flow rate was increased gradually over the first 20 min. The initial 150–200 ml of venous effluent was discarded, after which the perfusion medium was recirculated. When perfusate flow and pressure reached steady values (~38 ml/min and ~60 mmHg perfusion pressure), the perfusion medium was replaced with one containing 0.12 µCi 32P-labeled Pi/ml. This medium was then recirculated for the duration of the experiment. Perfusate pH was monitored periodically, and, although it decreased over time, it was always >7.30. Although the Pi transport activity of type III Na-Pi transporters is altered by decreasing pH (17), a small change in pH from 7.4 to 7.3 would have negligible impact on the Pi uptake rate, as the fraction of H2PO4 would increase from 0.19 to only 0.23. Perfusate samples were collected every 5, 10, or 15 min during experiments of 15-, 30-, or 60-min duration, respectively, and were centrifuged to isolate plasma for analysis of radioactivity and Pi concentration.

Before quick freezing of the leg muscles, radioactivity was cleared from the extracellular space by switching the perfusate to one containing no 32P for 8.5 min. This was done because any [32P]Pi remaining in the extracellular space would contribute additional tissue counts and cause an overestimation of Pi uptake. Removal of radioactivity was verified by collecting samples of venous effluent every 30 s. This perfusate was not recirculated. Muscles were then removed and quickly frozen with tongs cooled in liquid nitrogen. We sampled the soleus muscle (primarily slow-twitch red fibers), the red portion of the gastrocnemius (primarily fast-twitch red fibers), the white portion of the gastrocnemius (primarily fast-twitch white fibers), and the remainder of the gastrocnemius (mixture of fibers) (1).

In vivo Pi uptake. Rats were anesthetized with ketamine, and a catheter was placed in the right carotid artery and exteriorized at the back of the neck. When the rats regained consciousness, they were given an oral dose of [32P]Pi (50 µCi) in water. Every 30 min, blood samples were collected and centrifuged and plasma was isolated for analysis of Pi concentration and radioactivity. After 2.5 h, rats were anesthetized with pentobarbital sodium and muscles were sampled and quickly frozen as described in Hindlimb perfusion.

Tissue analyses. Metabolites from muscle and plasma samples were extracted in cold 3.5% perchloric acid and neutralized with tri-n-octylamine and 1,1,2-trichlorotrifluoroethane (7). These samples were stored at –80°C until being analyzed.

To calculate Pi uptake, we determined the radioactivity in aliquots of muscle and plasma extracts by scintillation counting. The radioactivity in muscle (dpm/g) was then divided by the average specific activity of plasma Pi (dpm/µmol) to yield Pi uptake in micromoles of Pi per gram of muscle.

Tissue contents of adenine nucleotides and PCr were determined with reverse-phase and ion-exchange HPLC, respectively (27, 29). Additionally, fractions of HPLC effluent containing ATP and PCr were collected and counted to detect 32P incorporation into those organic phosphate pools. Plasma Pi concentrations were determined by an enzymatic assay as described previously (8).

Muscle water content was determined by drying a 150- to 200-mg portion of each mixed gastrocnemius section at 60°C. Metabolite concentrations and Pi uptake rates were calculated to a common water content of 76%, which is typical of rat skeletal muscle (22).

Western blotting. Protein isolation and processing were carried out as described previously (10), with slight modifications. Frozen muscles were homogenized in a buffer of (in mM) 5 Tris·HCl, pH 7.4, 1 EDTA, and 0.2 phenylmethylsulfonyl fluoride, a protease inhibitor, and centrifuged for 10 min at 1,000 g. The supernatant was retained.

Total protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Protein (40 µg) from each fiber type and the heart were separated by SDS-PAGE (8% gel) and transferred to a nitrocellulose membrane. For detection of PiT-1, blots were blocked for 1 h with milk and 0.2% Tween 20 and then incubated overnight at 4°C with a 1:2,000 dilution of rabbit anti-PiT-1 antiserum. Raised against a portion of human PiT-1 Na-Pi transporter ranging from amino acids 408 to 421, these polyclonal antibodies have been described previously (4). For detection of actin, the membranes were incubated with a pan-actin antibody (Neomarkers, Fremont, CA) diluted 1:2,000. After three washes, the membranes were incubated in 50 mM Tris·HCl buffer, pH 7.5, with 0.2% Tween 20, milk, and goat anti-rabbit secondary antibody at room temperature for 1 h. After additional washing, the membranes were covered with a chemiluminescent substrate (ECL Plus; Amersham Pharmacia Biotech) and exposed to film for 15 s to 2 min.

Analysis of band densities was performed with NIH Image version 1.62. Similar protein loading in each sample was verified with actin, and PiT-1 band densities are expressed relative to that of the heart for each gel.

Statistics. All data are expressed as means ± SE. Statistical differences in Pi uptake rates and relative band densities from the Western blot were determined with one-way analysis of variance and the Tukey post hoc test, as appropriate. Statistical significance is defined as P < 0.05.


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Pi uptake rates. Clearance of extracellular 32P was effectively achieved during the washout period, as radioactivity in the venous effluent decreased to ~6% of the initial value after 8.5 min. With values of extracellular space previously determined in the perfused hindlimb (12) and assuming equilibration of the vascular and interstitial spaces, extracellular radioactivity constitutes 1% of the total radioactivity in soleus and red gastrocnemius and 3% in white gastrocnemius.

Although the washout duration was 8.5 min, we calculated Pi uptake rates ending at 2.5 min into the washout period (6) (e.g., the washout began at 57.5 min of a 60-min experiment). Venous effluent 32P radioactivity was ~25% of the initial value at 2.5 min of washout and declined thereafter. Thus any uptake of label during this period would be small and expected to contribute little to overall [32P]Pi uptake. Similarly, loss of label during this last 6 min of washout would have a negligible effect on the overall uptake rates. If we assume that the rate of Pi efflux matches that of Pi uptake and that these remain constant for the entire perfusion duration, label lost during washout represents 1.1%, 0.3%, and 0.1% of total radioactivity in soleus, red gastrocnemius, and white gastrocnemius, respectively.

Pi uptake was linear up to 60 min, and Pi uptake rates differed among muscle fiber types (n = 11), being highest in soleus (2.42 ± 0.17 µmol·g–1·h–1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g–1·h–1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g–1·h–1) (Fig. 1). Comparable rates were obtained in vivo (1.22 ± 0.14, 0.80 ± 0.08, and 0.32 ± 0.04 µmol·g–1·h–1 in soleus, red gastrocnemius, and white gastrocnemius, respectively). To analyze the importance of the sodium gradient, we performed additional perfusions with cell-free perfusate, either with or without 1 mM ouabain. Figure 1 shows that the presence or absence of red blood cells in the perfusion medium had no effect on Pi uptake in soleus and red gastrocnemius; however, Pi uptake was reduced with cell-free medium in the white gastrocnemius for reasons that are not clear. More importantly, in the presence of ouabain, Pi uptake was 55%, 37%, and 48% lower than in cell-free controls in the soleus, red gastrocnemius, and white gastrocnemius, respectively (Fig. 1).



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Fig. 1. Inorganic phosphate (Pi) uptake rates during isolated hindlimb perfusion with red blood cell (RBC)-containing perfusate (n = 11), cell-free perfusate (n = 5), and cell-free perfusate with 1 mM ouabain (n = 4). *Significantly lower than corresponding value with RBC-containing perfusate; #significantly lower than corresponding value with cell-free perfusate.

 
The dependence of Pi uptake on plasma Pi concentration is shown in Fig. 2. Assuming Michaelis-Menten kinetics, the calculated Km values are 0.45, 0.42, and 0.51 mM for the soleus, red gastrocnemius, and white gastrocnemius, respectively. These values are consistent with results from studies using cultured cells, which have reported the apparent Km of Pi transport in type III Na-Pi transporters to be 0.09–0.5 mM Pi (13, 15, 17).



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Fig. 2. Pi uptake rates as a function of perfusate Pi concentration in the isolated hindlimb preparation; n = 2 at 0.32 mM, 3 at 0.45 mM, 2 at 0.71 mM, 11 at 1.22 mM, and 3 at 1.76 mM.

 
PiT-1 protein expression. PiT-1 protein abundance was greatest in the soleus and red gastrocnemius and least in the white gastrocnemius. Relative to heart, PiT-1 expression in soleus was 0.42 ± 0.05, expression in red gastrocnemius was 0.46 ± 0.05, and expression in white gastrocnemius was 0.06 ± 0.01. Although there is a general relationship between Pi uptake rate and PiT-1 protein expression across the three muscle fiber types, it is apparent that PiT-1 expression in red gastrocnemius is high relative to its Pi uptake rate (Fig. 3).



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Fig. 3. Pi uptake rates as a function of PiT-1 expression in each fiber section. The value of Pi uptake in heart is taken from Medina and Illingworth (19). Western blot data are normalized to heart PiT-1 protein expression. The line is drawn from the origin to the heart data point; white gastrocnemius and soleus data fall on this line, suggesting a correlation between PiT-1 expression and Pi uptake in these muscles. In contrast, red gastrocnemius data are not on this line. Inset: representative Western blot for PiT-1.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Management of phosphate and the phosphate-containing PCr and ATP pools is important for maintaining optimal cellular function. Intracellular phosphate content is dependent on the balance of Pi uptake and efflux, the latter of which is thought to be a function of intracellular Pi concentration. Because the Pi uptake process is nearly saturated at normal plasma Pi concentration, uptake rates should be primarily determined by the quantity of Na-Pi transporters.

This is the first report of Pi uptake rates and PiT-1 protein expression in the three different skeletal muscle fiber types. Pi uptake rates were significantly different across fiber types, with the highest rate measured in soleus (2.42 ± 0.17 µmol·g–1·h–1), a lower rate in red gastrocnemius (1.31 ± 0.11 µmol·g–1·h–1), and the lowest rate in white gastrocnemius (0.49 ± 0.06 µmol·g–1·h–1). PiT-1 protein expression in soleus and red gastrocnemius was seven- to eightfold higher than in the white gastrocnemius. The maximal Pi uptake rate we measured in soleus (2.42 ± 0.17 µmol·g–1·h–1) is somewhat higher than the rate of 1.9 mmol·l cell water–1·h–1 (estimated to be 1.0 µmol·g–1·h–1) reported by Polgreen et al. (23) in the incubated rat soleus. This is likely due to enhanced delivery of Pi via the intact vasculature in our system. Previous studies measured Pi transport rates in cardiac sarcolemmal vesicles (13), cultured rat myoblasts (17, 23), and membrane vesicles from rabbit skeletal muscle (17) and reported the apparent Km of Pi transport as 0.09–0.5 mM.

Although we did not measure Pi transport across the sarcolemma, but rather Pi uptake in the tissue in situ, our results show a surprisingly good match with these previous reports, as our estimated Km values ranged from 0.42 to 0.51 mM (Fig. 2). These results lend support for the notion that the Pi transport process in skeletal muscle is nearly saturated at normal Pi concentrations.

The importance of the sodium gradient to Pi uptake was demonstrated by the 45–63% reduction in Pi uptake rates in the presence of ouabain, an inhibitor of Na+-K+-ATPase (Fig. 1). This finding is similar to the observations of Escoubet et al. (9), who reported a 35% reduction in Pi uptake rate in cultured cardiac myocytes after treatment with ouabain. Jack et al. (13) showed that gradual dissipation of the sodium gradient resulted in progressive decreases in Pi transport in cardiac sarcolemmal vesicles. However, total dissipation of the sodium gradient only reduced the Pi transport rate by ~70%, indicating that sodium flux across the membrane was still present. Additionally, Pi uptake can be increased by stimulation of Na+-K+-ATPase. Atkinson and Butterworth (2) reported that treatment of hepatocytes with 10–8 M insulin, which activates Na+-K+-ATPase, increased Pi uptake in these cells by 50–100%. Thus sodium flux across the cell membrane seems to be an important factor in regard to Pi uptake rates.

Critique of methods. A potential confounding factor in our calculation of uptake rates is that we do not account for Pi efflux that occurred during the experiment, which would cause an underestimation of Pi uptake. However, we believe this underestimation to be very small because the specific activity of cellular Pi remained substantially lower than plasma Pi and a large majority, ~70%, of the incoming Pi was incorporated into ATP and PCr, molecules that do not readily pass through the plasma membrane. We estimate that loss of label from the muscles, and thus underestimation of uptake, ranged from 0.5% to 4.5%, depending on fiber type.

Implications for cellular phosphate handling. In the steady state, the rate of uptake of Pi into the cell must equal the rate of efflux from the cell. The rate of Pi efflux is expected to be a function of intracellular Pi concentration (16). Because the driving force for Pi efflux is quite strong, it is considered to be a passive process, with the largest determinant of efflux rate being intracellular Pi concentration, especially in cells with large membrane potentials such as skeletal muscle (16). Thus, in skeletal muscle fiber types, which have similar membrane potentials of –90 to –95 mV (24), efflux rates should differ based on cellular Pi content. At steady state, Pi uptake must match efflux and, therefore, should also correlate with cellular Pi content.

To test our hypothesis regarding the relationship between intracellular Pi concentration and Pi uptake, we must know the intracellular Pi concentration during measurement of uptake. Unfortunately, Pi contents measured in freeze-clamped muscles are substantially higher than values measured via 31P nuclear magnetic resonance (NMR) spectroscopy (18, 20), because of artifactual hydrolysis of ATP during freezing (5). Therefore, we have estimated resting Pi contents in soleus and red and white gastrocnemius to be 3.6, 2.3, and 1.5 µmol/g, respectively, from the Pi contents measured in resting muscles by NMR (18) and the fiber type distribution in rat skeletal muscles (1). With these values, there is a linear relationship between Pi content and Pi uptake rate (Fig. 4), thus confirming one aspect of our hypothesis.



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Fig. 4. Pi uptake rates in each muscle fiber type plotted as a function of Pi content. Pi contents were derived from Refs. 1 and 18.

 
Although Pi uptake rates correlated with cellular Pi content, turnover of the Pi pool varied across fiber types (Table 1). In soleus and red gastrocnemius, fractional turnover rates were 0.67 and 0.57 h–1, respectively, whereas in the white gastrocnemius, Pi turnover was 0.33 h–1. This variation in turnover rates implies that the resistance to Pi efflux is different among fiber types. If the relationship between Pi uptake and muscle Pi content represents steady-state conditions, then Pi efflux is predicted to be zero when Pi content of the muscle is ~1.0 µmol/g (cf. Fig. 4). This implies either that the electrochemical gradient for Pi efflux has dissipated or that ~1.0 µmol/g of Pi is not "free" in solution and thus not available for efflux. Both of these possibilities are highly unlikely, because a dominant driving force leading to Pi efflux is thought to be the high negative membrane potential (16). Furthermore, the possibility of a sequestered pool of Pi is not reasonable, because the muscle Pi content used in the relationship shown in Fig. 4 is the available Pi pool determined by NMR (18). On the other hand, the assumption that our muscle was at steady state is probably reasonable, because the muscle maintains normally high ATP and PCr contents (Table 1) and is capable of excellent contractile performance (30). Management of the total phosphate pool within muscle is also different among fiber sections. The soleus, which has the lowest total phosphate content, has the highest uptake rate and therefore the highest total phosphate fractional turnover (Table 1). The opposite is true for the white gastrocnemius, which has the highest total phosphate content and the lowest Pi uptake rate. Thus this variation in fractional turnover of the total phosphate pool is determined in large part by differences in the adenine nucleotide and PCr contents of the muscle. Although it is presently unknown what determines the size of the adenine nucleotide and total creatine (PCr + creatine) pools within muscle, they represent a "sink" of Pi that can rapidly exchange with the inorganic Pi pool.


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Table 1. Metabolite contents, Pi uptake rates, and phosphate turnover in different muscle fiber sections

 
Although the mechanisms responsible for different phosphate turnover rates and resistance to Pi efflux across fiber types are unknown, these phenotypic characteristics may be beneficial in fast white muscle fibers for maintaining cellular phosphate levels. During contractions, these fibers can produce very high intracellular Pi concentrations that would be expected to produce elevated rates of Pi efflux and net loss of phosphate from the cell. Thus a high resistance to Pi efflux may aid in preserving cellular phosphate during periods of intense contractions.

One aspect of our hypothesis that was not confirmed was the prediction that Pi uptake would relate directly to PiT-1 protein content. Although there is a general relationship between Pi uptake rates and PiT-1 protein content, it is apparent that this relationship is not fully consistent with the different Pi uptake rates across fiber types, as shown in Fig. 3. There may be several reasons for this. First, PiT-1 may not be the only phosphate transporter expressed by skeletal muscle. Indeed, Kavanaugh et al. (15) and Tatsumi et al. (26) reported modest mRNA expression of PiT-2 in rat skeletal muscle but relatively low PiT-1 mRNA expression. PiT-1 protein expression has been demonstrated in the mouse, with heart and skeletal muscle having similar PiT-1 contents (4). Unfortunately, PiT-2 protein expression in skeletal muscle has not been reported. If protein expression is proportional to mRNA abundance, PiT-2 may be the dominant Na-Pi cotransporter isoform in skeletal muscle. Thus the relationship between Pi uptake rate and transporter protein would require a comparison to both PiT-1 and PiT-2 together.

Second, there may be an acute modulation of PiT-1 activity independent of total protein content. For example, Pi uptake rates may be regulated by posttranslational modification of Na-Pi transporters (10). Although phosphorylation of Na-Pi transporters has not been reported, PiT-1 and PiT-2 contain multiple consensus sequence phosphorylation sites for protein kinase C, which modulates Pi uptake rates in many cell types (10, 14, 28). Thus a different phosphorylation status of PiT-1 in red gastrocnemius may explain its relatively low Pi uptake rate compared with its abundant PiT-1 expression. A second means of acutely modifying transporter activity is to vary the proportion of total cellular PiT-1 protein resident in the plasma membrane. Although this possibility has not been reported for type III Na-Pi transporters, inhibition of Pi transport in kidney cells by parathyroid hormone is associated with type II Na-Pi transporter endocytosis (11). This may be an unlikely scenario in skeletal muscle, however, because type II and type III transporters have different Pi transport characteristics, and type II transporters are located in the brush-border membranes of kidney proximal tubules.

Third, it is known that there is a sodium-independent component of Pi uptake that does not rely on a Na-Pi transporter. We think this is also an unlikely explanation because, as mentioned above, experiments in cell culture have shown that only 7–10% of the total Pi uptake is sodium independent (10, 17).

In conclusion, we have shown that Pi uptake rates differ among skeletal muscle fiber types and correlate with cellular Pi content. However, despite results that suggest that phosphate transporters are nearly saturated at normal plasma Pi concentrations, the relationship between Pi uptake rate and PiT-1 protein content was not strong, indicating that other factors may be important in determining Pi uptake rates. Finally, turnover of the cellular Pi and total phosphate pools varies between fiber sections, suggesting differential regulation of phosphate among skeletal muscle fiber types.


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This study was supported by National Institutes of Health Grants AR-21617 and HL-07094.


    ACKNOWLEDGMENTS
 
We appreciate the excellent technical assistance of Hong Song, Jackie Love, and Yuhua Xiao. In addition, we thank Dr. Richard Beliveau from the Membrane Transport Research Group, University of Quebec, Montreal, Quebec, Canada, for providing the PiT-1 antibodies.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. L. Terjung, Biomedical Sciences, College of Veterinary Medicine, E102 Veterinary Medicine Bldg., Univ. of Missouri, Columbia, MO 65211 (E-mail: terjungr{at}missouri.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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