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
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ABSTRACT |
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inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux
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 0.090.5 mM. This is well below the normal plasma Pi concentration in humans (1 mM) and rats (1.52.0 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).
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METHODS |
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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 0.31.7 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 150200 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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RESULTS |
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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·g1·h1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g1·h1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g1·h1) (Fig. 1). Comparable rates were obtained in vivo (1.22 ± 0.14, 0.80 ± 0.08, and 0.32 ± 0.04 µmol·g1·h1 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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DISCUSSION |
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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·g1·h1), a lower rate in red gastrocnemius (1.31 ± 0.11 µmol·g1·h1), and the lowest rate in white gastrocnemius (0.49 ± 0.06 µmol·g1·h1). 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·g1·h1) is somewhat higher than the rate of 1.9 mmol·l cell water1·h1 (estimated to be 1.0 µmol·g1·h1) 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 0.090.5 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 4563% 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 108 M insulin, which activates Na+-K+-ATPase, increased Pi uptake in these cells by 50100%. 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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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 710% 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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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