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Am J Physiol Heart Circ Physiol
2020 Feb 01;3182:H448-H460. doi: 10.1152/ajpheart.00433.2019.
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Several phosphate transport processes are present in vascular smooth muscle cells.
Hortells L
,
Guillén N
,
Sosa C
,
Sorribas V
.
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We have studied inorganic phosphate (Pi) handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting of 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under nonprecipitating conditions. This work not only shows that the Pi handling by cells is highly complex but also that the transport systems are shared with other ions such as bicarbonate or sulfate.NEW & NOTEWORTHY In addition to the inorganic phosphate (Pi) transporters PiT1 and PiT2, rat vascular smooth muscle cells show a sodium-dependent Pi transport system that is inhibited by DIDS and SITS. A sodium-independent Pi uptake system of high affinity is also expressed, which is inhibited by sulfate, bicarbonate, and arsenate. The exit of excess Pi is through an exchange with extracellular chloride. Whereas the metabolic effects of the inhibitors, if any, cannot be discarded, kinetic analysis during initial velocity suggests competitive inhibition.
Figure 1. Characteristics of Pi transport in confluent VSMC. A. Total uptakes of Pi at increasing concentrations of substrate, in the presence or absence of sodium. The equation fitted to data includes an unsaturable component. Non-visible error bars are smaller than the symbols. B. Theoretical sodium-dependent and sodium-independent (choline chloride) transport components of total Pi uptake, according to the results of panel A. C. Effect of pH in the presence (white bars) and absence (black bars) of sodium. In A and C, every symbol and bar are the means of six data.
Figure 2. Effect of confluence on VSMC. A. Effect on total sodium-dependent and sodium-independent Pi uptakes as a function of tissue culture confluence. Symbols are the mean of 3 experimental values. B. Expression of the indicated RNAs in VSMC at 50% (black bars) and 100% (white bars) confluence. Bars show the means of three data. C. Protein expression of the corresponding RNAs analyzed in B and expressed in relation to ß actin. Representative Western blots of target proteins are shown at the right. When more than one band is present, the specific signal is shown with an arrowhead. All western blots were normalized to ß-Actin, which was unchanged between samples on all blots showing no errors in loading. Full length images of all target genes and ß-Actin for all blots were provided during peer review. Only representative blots of target genes are shown for brevity. In B and C, asterisks mean significant differences with a t-test (p < 0.05). Bars are the means of 3 to 6 values.
Figure 3. Role of chloride in Pi uptake and P efflux. A. Effect of chloride on sodium637 dependent and sodium-independent Pi uptakes under initial velocity conditions (only influx). *, p < 0.05 with a t-test. B. Phosphorus content in cell after 3 hours of incubation 31 with 32 Pi in the presence or absence of chloride for the experiment shown in C and D. The remaining P content after 2 hours of P exit is also shown, in the presence or absence of chloride. The molecular entity of this phosphorus is unknown, and therefore only the mass of P is considered. C. P released from cells after 3 hours of loading with 0.05 mM 32 Pi. 32 Phosphorus was determined in uptake media at the indicated times, with the total P
expressed per milligram of VSMC protein. The slopes and the r2 are shown for this representative experiment. Both regression lines were significantly different. The 95% confidence intervals of both linear regressions are also shown with two dashed confidence bands. D. P exit per unit of time at every time point. Non-regression lines of exponential decay curves are shown to help understand the P exit behavior. The calculated half-life parameters and the decay rate constants are also shown. Inset: net P content in uptake medium at every time point. In all four panels, the number of data per bar or symbol is six.
Figure 4. Inhibition profile of Pi transport. A. Effect of the indicated inhibitors on sodium-dependent and sodium-independent Pi transport. Asterisks indicate significant difference with respect to the Control condition, with an ANOVA and a Tukey post-test (p <0.05). Pi (as inhibitor), sulfate, bicarbonate, arsenate, and PFA were used at 5 mM, oxalate at 10 mM, and DIDS/SITS at 0.1 mM. Bars are the mean of 12 experimental data. B. Microphotographs of VSMC after incubation in uptake solution, with or without calcium for 10 minutes. Bar, 50 µm. C. No effect of DIDS and SITS on Pi transport resulting from the expression of rat PiT1 and PiT2 in Xenopus laevis oocytes. Bars are the mean of 10 experimental data.
Figure 5. Dose-response relationships. The assays were performed at a constant Pi concentration (0.05 mM Pi), while increasing the concentrations of inhibitors. Sulfate, 32 bicarbonate, and arsenate were assayed for dose-responses of sodium-independent Pi transport, and DIDS/SITS were assayed for uptake in the presence of sodium chloride. Figures show representative experiments of the inhibition assays, each of which was performed three times. Every symbol represents the mean of triplicates.
Figure 6. Determination of Ki values. Michaelis-Menten saturation kinetics were performed for sulfate in the absence of sodium (A) and for SITS in the presence of sodium (B). For each saturation experiment a different concentration of inhibitors was used, as indicated in the legends. Left, Lineweaver-Burk linear regressions. Right, non-linear regression fits of a Michaelis-Menten equation to data, plus an unsaturable component. Apparent affinities are indicated in the symbol legends. Every symbol represents the mean of triplicates.
Figure 7. Expression of anion exchangers. A. Quantitative, real-time PCR of the indicated anion exchanger RNAs from VSMC cultivated at 50% or 100% confluence. The significance of different expressions was determined with a t-test. B. Resulting RNA abundance inhibition after treatments with either scrambled or specific siRNAs after 48 hours. C. The resulting Pi transport after anion exchanger RNA interference 48 hours post transfection, in the presence or absence of sodium. Bars of panels A and B are the means of triplicates. Bars of panel C show the means of 6 experimental data.
Figure 8. Effect of VSMC incubation at a high Pi concentration. A. No effect on Pi uptake after 7 days of incubation with either 1 or 2 mM Pi. Bars show means of triplicates. B. No effect on P exit from the cell after 2 hours of incubation with 32 P. The graph represents the accumulation of 32 P as grams of total phosphorus in uptake medium per milligram of VSMC protein. Symbols show the means of six experimental data.
Figure 9. Drawing depicting the Pi transport systems in VSMC. Inward Pi transporters are shown at the left, including the known Slc20 members PiT1 and PiT2. Another sodium dependent Pi transporter that is sensitive to stilbene-derivatives is also shown. Pi uptake systems also include a sodium-independent pathway (shown in the middle of the drawing), which handles both monobasic and dibasic phosphates, and it is weakly inhibited by sulfate. The transport system is most likely coupled to the exit of anions. Finally, the exit of Pi from the cell is partially coupled to the entrance of chloride, and it is resistant to DIDS and SITS.
