Phosphophloretin sensitivity of rabbit renal NaPi-IIa and NaPi-Ia

【摘要】  The effect of phosphorylated phloretins on Na + -dependent phosphate uptake into rabbit renal brush-border membrane vesicles (BBMV) was examined. Na + -dependent phosphate uptake into isolated rabbit cortex BBMV was sensitive to 2'-phosphophloretin (2'-PP) and 2'-phospho-4',4,6'-trimethoxy phloretin (PTMP) in a dose-dependent and pH-dependent manner. PTMP inhibition of Na + -dependent phosphate uptake was maximum at alkali pH, and 2'-PP inhibition of Na + -dependent phosphate uptake was maximum at acidic pH. Increasing Na + concentrations did not increase PTMP inhibition of renal cortex BBMV Na + -dependent phosphate uptake at pH 6. The effect of phosphophloretins on Na + -dependent phosphate uptake was examined in BBMV isolated from purified proximal tubules and distal tubules. 2'-PP and PTMP inhibition of Na + -dependent phosphate uptake into BBMV isolated from purified proximal tubules was similar to the inhibition seen with BBMV from renal cortex. 2'-PP, but not PTMP, inhibited Na + -dependent phosphate uptake into BBMV isolated from purified distal tubules. The pH dependence of inhibition, the absence of PTMP inhibition of Na + -dependent phosphate uptake into distal tubule BBMV, and the inhibition of Na + -dependent phosphate uptake into distal tubule BBMV suggest that NaPi-Ia is 2'-PP sensitive and NaPi-IIa is PTMP sensitive.

intestinal brush-border membrane vesicles; renal brush-border membrane vesicles; sodium-phosphate cotransport; phosphate reabsorption; 2'-phosphophloretin; 2'-phospho-4',4,6'-trimethoxy phloretin

【关键词】  Phosphophloretin sensitivity NaPiIIa

THE N A P I FAMILY of proteins is directly involved in phosphate homeostasis. NaPi-IIb catalyzes the uptake of dietary phosphorus in the proximal small intestine. NaPi-IIa and NaPi-Ia reabsorb phosphate in the proximal tubule (NaPi-Ia and NaPi-IIa) and distal tubule (NaPi-Ia). Phosphate uptake by a variety of cell types including brain (NaPi-Ib), lung (NaPi-IIb, HG-80), liver (NaPi-Ia), erythrocytes (NaPi-Ib), and parathyroid gland (NaPi-III, PiT-1) appear to have Na + -dependent components ( 28, 49 ). PiT-1 may have an indirect role in the regulation of serum Ca 2+ through the effect of phosphate on parathyroid gland growth ( 13 ) and parathyroid hormone (PTH) mRNA stability ( 29 ).

Na + -phosphate cotransporter proteins are a diverse group of proteins, which appear to share few functional or structural characteristics other than coupling Na + uptake and phosphate uptake into a variety of cell types. NaPi-IIa and NaPi-IIb differ in structure and transport properties. Structurally, the two proteins differ in molecular mass, I° structure (NaPi-IIa and NaPi-IIb share 55% homology), and the presence of a structurally and functionally essential disulfide bridge ( 21 ). Functionally, the substrate sites of the two proteins appear to differ. NaPi-IIa is thought to transport either H 2 PO 4 or HPO 4, with a preference for HPO 4, has a substrate stoichiometry of 2 or 3, and increases its Na + affinity with increasing pH ( 3 ). NaPi-IIb is thought to transport only H 2 PO 4, may be inhibited by HPO 4 at an internal regulatory site ( 10, 32 ), has a substrate stoichiometry of 2, and increases its Na + affinity with decreasing pH ( 10, 32, 33, 41 ). NaPi-IIa is more sensitive to phosphites (foscarnet) and less sensitive to arsenate ( 43 ) than NaPi-IIb ( 3, 24 ).

NaPi-III (PiT-1 and PiT-2) subfamily members share little homology with either the NaPi-II or NaPi-I proteins ( 28 ). Although NaPi-III family members appear to resemble NaPi-IIb and NaPi-Ia in transport characteristics (substrate affinity, substrate selectivity, and pH dependence of transport), there is little common inhibitor sensitivity or structural similarity ( 28 ).

Based on functional characteristics, NaPi-IIb appears to have more in common with NaPi-Ia than NaPi-IIa. NaPi-Ia and NaPi-IIb have increased Na + affinity with decreasing pH and appear to preferentially or exclusively transport H 2 PO 4 ( 10, 25, 28, 32, 41, 44 ). Comparisons of the structures of the two proteins have not been reported.

Differential inhibitor sensitivity is one method commonly used to distinguish between members of a transport protein family. Inhibitor sensitivity differences have been used to distinguish between members of the Na + /H + exchanger (NHE) family ( 8, 39 ), Cl - channel family ( 18 ), and anion exchange protein family ( 23 ). Previous studies suggested that the members of the NaPi family differ in their inhibitor sensitivity to phosphonoformic acid (PFA) and vanadate ( 24, 49 ). These inhibitors are not specific for the Na + -phosphate cotransporters.

The plant chalcone phloretin is an inhibitor of a variety of transport proteins. The aglucone phloretin is an inhibitor of AE-1 (Band 3, 12, 14), urea transport in the kidney ( 6 ) and erythrocytes ( 46 ), lactate and pyruvate transport in cardiomyocytes ( 48 ), and GLUT-4 ( 2, 12, 14 ). Phloretin has also been reported to be an inhibitor of NaPi-IIa and a weak inhibitor of PiT-1, NaPi-Ib ( 45 ), and NaPi-IIb ( 34 ). The 2'-glycosylated derivative phloridzin inhibits Na + and glucose cotransport in renal and intestinal brush-border membranes (BBM) ( 12 ).

In vitro a phosphorylated chalcone 2'-phosphophloretin (2'-PP) has been reported to specifically inhibit NaPi-IIb in human ( 35 ), rat ( 34 ), and rabbit ( 34 ) intestinal BBM vesicles (BBMV). In vivo 2'-PP decreased serum phosphate in aged adult rats ( 34 ).

Phosphophloretins are of potential interest in the treatment of renal failure and as a tool for studying the NaPi family of proteins. Differential phosphophloretin sensitivity would provide a biochemical assay for the determination of NaPi family distribution in tissues. Phosphophloretins are also of potential use for the study of the phosphate sites of the NaPi family of proteins. We hypothesize that differential phosphate selectivity of the NaPi family of proteins is related to differences in phosphate site solvent exposure or depth in the membrane field. As a starting point, we compared the effect of a watersoluble derivative, 2'-phosphophloretin (2'-PP), to the effect of a poorly water-soluble derivative, 2'-phospho-4,4',6'-trimethoxy-phosphophloretin (PTMP), on Na + -dependent phosphate uptake into intestinal and renal BBMV. Methylation of 2'-PP was selected to alter the water solubility of 2'-PP without adding large bulky groups, which could alter inhibitor potency through steric hindrance. The results indicate that NaPi-IIb and NaPi-Ia were 2'-PP sensitive and that NaPi-IIa was PTMP sensitive. These results are consistent with 2'-PP competing with H 2 PO 4 binding to NaPi-IIb and NaPi-Ia and PTMP competing with HPO 4 for binding to NaPi-IIa.

MATERIALS AND METHODS

Materials

Reagents and solvents for the synthesis of 2'-PP and PTMP were purchased from Aldrich Chemical (Milwaukee, WI). Electrophoresis supplies were purchased from Bio-Rad (Hercules, CA). [ 32 P]phosphate, [ 3 H]glucose, and [ 35 S]sulfate were purchased from New England Nuclear Life Sciences (Boston, MA). [ 3 H]alanine was purchased from Pharmacia Life Sciences (Piscataway, NJ). HEPES, Tris, MES, MOPS, TAPS, and Percoll were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Fisher Chemical (Houston, TX) and were reagent grade or better. Peptide-specific affinity-purified antibodies were purchased from Alpha Diagnostics (San Antonio, TX). Texas red and fluorescein hydrazine derivatives were purchased from Molecular Probes (Eugene, OR).

Methods

Preparation of BBMV. Renal and intestinal BBMV were prepared by Ca 2+ precipitation and differential centrifugation ( 42 ). Intestinal BBMV purification was determined using the enzyme markers sucrase ( 9 ), alkaline phosphatase ( 15 ), and leucine amino peptidase ( 19 ). Renal BBMV purification was assayed using the brush-border enzyme markers leucine amino peptidase, -glutamyl transpeptidase ( 30 ), and alkaline phosphatase. Protein was determined by the method of Lowry using bovine serum albumin as a standard ( 36 ).

Preparation of Distal Tubules and Proximal Tubules

Distal and proximal tubules from rabbit kidneys were isolated by Percoll gradient centrifugation ( 7 ). Briefly, rabbit kidneys were removed, perfused through the renal artery with 50 ml of PBS, and stored on ice. Kidneys were minced and passed through a 10-ml disposable syringe to normalize piece size. Minced kidney slices were digested with collagenase ( Clostridium histolyticum, 1 mg/ml) in Krebs-Henseleit (KH; 138 mM NaCl, 3.8 mM KCl, 1.4 mM potassium phosphate, 1.4 mM MgSO 4, 1.2 mM CaCl 2, 25 mM NaHCO 3, 60 mM mannitol, 1 mM pyruvic acid, 1 mM glutamate, 10 mM lactic acid, and 10 mM glutamine) media with 0.5 mg/ml BSA for 30 min at 37°C with gentle agitation ( 7 ). After digestion, ice-cold KH media was added (4-fold dilution) and the tissue was filtered through a Buchler funnel, followed by filtration through a nylon mesh with a pore diameter of 0.6 µm and then through a 0.2-µm filter. The effluent was centrifuged at 120 g for 10 min, and the pellets were resuspended in fresh KH media. This step was repeated three times.

After the final wash step, the pellets were resuspended in 150 ml of KH media and Percoll/rabbit (final Percoll concentration 40%) and gently mixed at 8°C for 15 min. Percoll gradient centrifugation was performed in a Beckman JA-17 fixed angle rotor spun at 28 k g for 30 min. The upper band (distal segments) and a second band in the bottom one-third of the tube were removed and washed three times by centrifugation (10,000 g for 15 min) to remove Percoll. Tubules were resuspended in 50 mM KCl and 1 mM Tris·HCl, pH 7, and stored at -80°C until needed. Typically, three tubule preparations were combined for isolation of BBMV.

Proximal tubule BBMV and distal tubule BBMV were prepared by Ca 2+ precipitation and differential centrifugation. Fractions were assayed for protein ( 34, 36 ), leucine amino peptidase activity, alkaline phosphatase activity ( 15 ), succinate dehydrogenase activity ( 51 ), -glutamyl transpeptidase ( 30 ), phloridzin-sensitive Na + -dependent [ 3 H]glucose uptake ( 35 ), and Na + -K + -ATPase activity ( 40 ). Proximal tubule BBMV and distal tubule BBMV enrichments are summarized in Tables 1 and 2.

Table 1. Distal and proximal tubule enrichments

Table 2. Proximal tubule and distal tubule brush-border membrane vesicle purification

Phloretin Derivative Synthesis

2'-PP was synthesized from phloridzin as previously described ( 34 ); 171-172°C, 1 HNMR (d 6 -DMSO) 13.0 (s, 1H), 10.7 (br.s, 1H), 9.2 (br.s, 1H), 7.03 (d, J = 8.6 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 6.63 (dd, J = 1.2, 2.1 Hz, 1H), 2.77 (d, J = 7.6 Hz, 2H). 31 P-NMR in D 2 O yielded a single peak at -4 ppm comprising 98% of the phosphorus signal. 31 P-NMR in DMSO yielded a single peak at -4.3 ppm.

PTMP was synthesized from 2'-PP and dimethyl sulfate ( 27 ). Briefly, potassium carbonate (160 mg) was added to 2'-PP (50 mg in 25 ml acetone). Dimethyl sulfate (140 mg) was added and the mixture was stirred for 30 min at 23°C. Solvent was removed under reduced pressure. PTMP was purified by HPLC on a Waters reverse-phase C 4 column eluted with 25% CH 3 CN and dried under reduced pressure.

PTMP was analyzed by 1 HNMR in deuterated methanol ( 38 ) 7.13 (d, J = 8 Hz, 1H), 7.08 (t, J = 6 Hz, 12 Hz, 1H), 6.82 (d, J = 8 Hz, 1H), 6.79 (d, J = 8 Hz, 2H), 6.76 (d, J = 2 Hz, 1H), 6.3 (s, 1H), 6.27 (s, 1H), 6.2 (s, 1H), 3.8 (q, J = 9 Hz, 23 Hz, 3H), 3.7 (t, J = 7, 12 Hz, 3H), 3.68 (s, 2H), 3.56 (s, 2H).

Na + -dependent uptakes. [ 32 P]phosphate, [ 3 H]glucose, [ 3 H]alanine, and [ 35 S]sulfate uptakes into renal and intestinal BBMV were determined using 100 µg of BBMV protein and a rapid filtration set up at 23°C ( 34, 35, 42 ). Uptake was determined as filter-retained counts from media containing 100 mM NaCl or 100 mM KCl, 100 mM mannitol, 20 mM HEPES/Tris, pH 7.5, and 100 µM substrate {[ 32 P]phosphate, [ 3 H]glucose, [ 3 H]alanine, or [ 35 S]sulfate} and 5-s incubations with uptake media. In some experiments, uptakes were determined from 150 mM NaCl or 150 mM KCl, 20 mM HEPES/Tris, pH 7.5, and 100 µM substrate {[ 32 P]phosphate, [ 3 H]glucose, [ 3 H]alanine, or [ 35 S]sulfate}. Na + -dependent uptake was defined as retained counts in the presence of Na + minus retained counts in the presence of K +. Results are expressed as picomoles per milligram per second and are means ± SE of three to five experiments. Results were corrected for filter-retained counts in the absence of protein. In some experiments, the effect of phosphophloretins on Na + -dependent uptakes was examined. In those experiments, 2'-PP was added in 10 mM borate buffer, pH 7.5, immediately before the start of the experiment. In experiments examining the effect of PTMP on uptake, PTMP was added in 1 µl of DMSO.

In some transport experiments, the effect of medium pH on Na + -dependent transport and the effect of phosphophloretins were examined. In these experiments, uptakes were determined from media containing 150 mM NaCl or 150 mM KCl, 100 µM [ 32 P]phosphate, and 20 mM buffer (HEPES/Tris, pH 7.5; MES/Tris, pH 6.5 and 6.0; Pipes/Tris, pH 7; and TAPS/HCl, pH 8 and 8.5).

In some transport experiments, the effect of Na + concentration on phosphophloretin inhibition of Na + -dependent [ 32 P]phosphate uptake was examined. In these experiments, the Na + concentration was varied between 50 and 250 mM, and the solution osmolality was maintained at 500 mosmol/kgH 2 O by addition of mannitol.

Western blotting. SDS-PAGE was performed on 10% polyacrylamide gels by the method of Laemmli ( 20 ). Protein was transferred to nitrocellulose paper by semidry electrophoretic blotting. Transferred gels were probed with primary antibodies at a 1:3,000 dilution and then with horseradish peroxidase-coupled anti-IgG secondary antibody according to the manufacturer's instructions. In some experiments, 25 µg of renal BBMV, BBMV from purified proximal tubules, and BBMV from purified distal tubules were used. In some experiments, applied BBMV protein was varied to titrate Npt antibodies.

Renal BBMV were probed with NaPi-IIa peptide-specific antibodies raised against the amino acid sequences SGILLWYPVPC (Npt 25) or PATPSPRLALPAHHNAC (Npt 21). NaPi-Ia peptide-specific antibodies were raised against the amino acid sequence CKWAPPLERGRLTSMS (Npt 11). Peptide sequences were confirmed by MSMS and amino acid sequencing at the Protein Chemistry Laboratory (UTMB, Galveston, TX). Specificity of the antibodies for the specified sequences was examined by preblot reaction of the antibody with the peptide used to generate the peptide sequence-specific antibodies.

Western blot and antibody sensitivity were determined by analysis of antibody binding to renal cortex BBMV protein (2.5 to 50 µg), distal tubule-enriched BBM protein (5 to 25 µg), and proximal tubule-enriched BBM protein (2.5 to 25 µg) separated by SDS-PAGE ( 20 ). Protein was transferred to nitrocellulose filters, and gels were stained with Ponceau S stain to determine protein transfer efficiency. NaPi-IIa and NaPi-Ia labeling were determined as described above. Standard curves for each antibody were generated as a function of protein concentration from 2.5 to 25 µg.

Immunohistochemistry

Antibodies specific for NaPi-Ia (Npt-11) and antibodies specific for NaPi-IIa (Npt-21) were labeled with fluorescent hydrazine derivatives at their carbohydrate moieties ( 31 ). The appropriate antibody (200 µg) was reacted with 100 mM sodium acetate, pH 5.5, and 10 mM NaIO 4 for 30 min at 4°C. Carbohydrate oxidation was stopped by addition of 50 mM sodium sulfite and incubation for 1 h at 4°C. The antibody was dialyzed for 48 h against 100 mm sodium acetate, pH 5.5, with three buffer changes.

Antibodies were labeled with 5 mM Texas red hydrazide (Npt-11) or 5-{(2-methyl)thio} acetyl aminofluorescein (Npt-21) in 100 mM sodium acetate, pH 5.5, for 2 h at 23°C. Antibodies were purified by gel filtration chromatography through Sephadex G-25 columns (5-ml syringe) eluted with PBS. Fractions containing antibodies were dialyzed for 1 wk against PBS. After dialysis, antibodies were assayed for protein ( 36 ) and lyophilized. Labeled antibodies were resuspended in PBS at a final concentration of 1 µg/µl.

Immunohistochemistry of purified distal tubules and proximal tubule preparations. Tubule-enriched fractions were immobilized on polylysine (50 µg/ml)-coated glass coverslips ( 50 ) and air-dried. Immobilized tubules were washed three times with PBS, two times with water, and finally with 70% ice-cold ethanol. Coverslips were reacted with fluorescently labeled antibodies at a 1:100 dilution (Ab:PBS) for 2 h at 23°C. Antibody was removed by suction, and the coverslips were washed three times with PBS and two times with water.

Tubule labeling was examined on a Nikon 800 fluorescence microscope, Optical Imaging Laboratory, Department of Physiology and Biophysics, UTMB. Coverslips were labeled with Npt-11, Npt-21, or double-labeled with Npt-21 followed by Npt-11. Due to fluorescence channel overlap, double-labeling with the Texas red-conjugated antibody (Npt-11) and the fluorescein-conjugated antibody (Npt-21) was performed by first labeling with the fluorescein-conjugated antibody, followed by the Texas red-conjugated antibody.

Analysis of antibody binding to coverslip-immobilized tubules was performed by epifluorescence on the Nikon 800 fluorescence microscope. Fluorescein- and Texas red-labeled structures were counted in 10 to 15 fields/coverslip and 8 to 10 coverslips/tubule preparation. Three tubule preparations were examined. Structures labeled with both Texas red and fluorescein were counted as proximal tubules, structures labeled with only Texas red were counted as distal tubules, and structures not labeled with either antibody were counted as nontubule distal segments.

Renal sections. Rabbit kidneys were removed, perfused with PBS, followed by 50 ml of 10% buffered formalin. Kidneys were placed in fresh formalin overnight at 8°C. Thick sections were prepared by the Immunohistochemistry Laboratory, Department of Human Biochemistry, and Cellular Genetics, UTMB. Sections were stained with hematoxylin and eosin or fluorescent antibodies at a 1:75 dilution (Npt-11) or 1:100 dilution (Npt-21). Sections were examined on a Nikon 800 fluorescence microscope at the Optical Imaging Laboratory (UTMB, Galveston, TX).

RESULTS

Effect of Phosphophloretins on Na + -Dependent Phosphate Uptake

The effect of phloretin and phloretin derivatives on rabbit renal BBMV Na + -dependent phosphate uptake is shown in Fig. 1. Phloretin (, broken line) inhibited Na + -dependent phosphate uptake 20% at 10 µM. PTMP (, solid line) inhibited 70% at 10 -7 M PTMP. 2'-PP (, solid line) inhibited Na + -dependent phosphate uptake into renal BBMV <30% at pH 7.5.

Fig. 1. Effect of phloretin and phosphophloretins on renal cortex brush-border membrane vesicles (BBMV) Na + -dependent phosphate uptake. [ 32 P]phosphate uptake into renal cortical BBMV was determined as described in MATERIALS AND METHODS at pH 7.5. [ 32 P]phosphate uptake was determined using a membrane filtration assay through 0.45-µm filters and 5-s incubation times at 23°C as described in MATERIALS AND METHODS. Phosphate uptake into renal BBMV in the presence of Na + was determined as filter-retained counts from 100 mM NaCl, 100 mM mannitol, 10 mM HEPES/Tris, pH 7.5, and 100 µM [ 32 P]phosphate. Phosphate uptake in the absence of Na + was determined as filter-retained counts from 100 mM KCl, 100 mM mannitol, 10 mM HEPES/Tris, pH 7.5, and 100 µM [ 32 P]phosphate uptake media. Na + -dependent phosphate uptake was defined as uptake in the presence of a 100-mM external NaCl gradient minus uptake in the presence of a 100-mM external KCl gradient. Uptake experiments were performed using 100 µg of BBMV protein. Immediately before the start of the experiment, the indicated amount of 2'-phosphophloretin (2'-PP;, solid line), phloretin (, broken line), or 2'-phospho-4',4,6'-trimethoxy phloretin (PTMP;, solid line) was added followed by protein. Results shown are from a single experiment performed in triplicate and representative of 4 separate experiments.

A similar series of experiments was performed with intestinal BBMV to compare the effect of phloretin derivatives on NaPi-IIb activity. The effect of 2'-PP and PTMP on Na + -dependent phosphate uptake into intestinal BBMV is shown in Fig. 2. 2'-PP inhibited Na + -dependent phosphate uptake into intestinal BBMV (, solid line) 94 ± 3% with an IC 50 of 40 ± 4 nM ( n = 5). PTMP (, solid line) was a weak inhibitor of Na + -dependent phosphate uptake into intestinal BBMV. PTMP inhibition of Na + -dependent phosphate uptake into intestinal BBMV was similar to phloretin (, broken line) accounting for <30% (29.6 ± 6%, n = 5) at inhibitor concentrations of 10 -5 M.

Fig. 2. Effect of phosphophloretins on intestinal BBMV Na + -dependent phosphate uptake. [ 32 P]phosphate uptake was determined using membrane filtration through 0.45-µm filters and 5-s incubation times at 23°C as described in MATERIALS AND METHODS. Phosphate uptake into intestinal BBMV in the presence of Na + was determined as filter-retained counts from 100 mM NaCl, 100 mM mannitol, 10 mM HEPES/Tris, pH 7.5, and 100 µM [ 32 P]phosphate. Phosphate uptake in the absence of Na + was determined as filter-retained counts from 100 mM KCl, 100 mM mannitol, 10 mM HEPES/Tris, pH 7.5, and 100 µM[ 32 P]phosphate uptake media. Na + -dependent phosphate uptake was defined as uptake in the presence of a 100-mM external NaCl gradient minus uptake in the presence of a 100-mM external KCl gradient. Uptake experiments were performed using 100 µg of intestinal BBMV protein. Immediately before the start of the experiment, the indicated amount of 2'-PP (, solid line), phloretin (, broken line), or PTMP (, solid line) was added followed by protein. Results shown are from a single experiment performed in triplicate and representative of 5 separate experiments.

Phloretin is a nonspecific inhibitor of a variety of transport proteins including band 3 ( 14 ), the urea transporter ( 6 ), and pyruvate and lactate transporter of cardiomyocytes ( 48 ). The specificity of PTMP inhibition of NaPi transporters was examined by comparing renal cortex BBMV Na + -dependent phosphate uptake to PTMP inhibition of renal cortex BBMV Na + -dependent glucose, Na + -dependent alanine, and Na + -dependent sulfate uptake. The results are shown in Fig. 3.

Fig. 3. Effect of PTMP on Na + -dependent uptake into renal cortex BBMV. Na + -dependent [ 32 P]phosphate uptake (, broken line), [ 3 H]alanine uptake (, solid line), [ 3 H]glucose uptake (, broken line), and [ 35 S]sulfate uptake (, solid line) into renal cortex BBMV were determined as described in Fig. 1 legend and in MATERIALS AND METHODS. Na + -dependent phosphate uptake was performed at pH 8.5. Results are means ± SE of triplicate determinations and representative of 3 experiments (sulfate) or 4 experiments (glucose, alanine, and phosphate).

Figure 3 compares the effect of PTMP on Na + -dependent phosphate uptake (, dashed line), Na + -dependent glucose uptake (, dashed line), Na + -dependent alanine uptake (, solid line), and Na + -dependent sulfate (, solid line) uptake into renal cortex BBMV. PTMP did not alter glucose, sulfate, or alanine uptakes into renal BBMV at PTMP concentrations up to 10 µM. These results are similar to the results seen with 2'-PP inhibition of intestinal Na + -organic substrate cotransport ( 34 ) and consistent with a selective inhibition of Na + -dependent phosphate uptake into renal cortex BBMV by PTMP.

Effect of pH on PTMP Inhibition of Renal BBMV Na + -Dependent Phosphate Uptake

The presence of two Na + -dependent phosphate cotransporter pathways into renal cortex BBMV limits analysis of phosphophloretin derivative inhibition of NaPi-mediated phosphate uptake into renal cortex BBMV. Alkaline pH may be used to minimize NaPi-Ia activity, or acid pH at low-Na + concentrations may be used to minimize NaPi-IIa-mediated Na + -dependent phosphate uptake ( 28 ). To examine the effect of phosphophloretins on NaPi-IIa-mediated and NaPi-Ia-mediated Na + -dependent phosphate uptake into renal cortex BBMV, the effect of pH on PTMP and 2'-PP inhibition of Na + -dependent phosphate uptake was examined between pH 5.5 and 8.5. The results of these studies are summarized in Fig. 4.

Fig. 4. Effect of pH on PTMP inhibition of renal cortex Na + -dependent phosphate uptake. Na + -dependent [ 32 P]phosphate uptake into renal cortex BBMV was determined as described in Fig. 1 legend and in MATERIALS AND METHODS. The pH of the uptake media was varied between pH 5.5 and 8.5. PTMP (, broken line) concentration was varied between 5 nM and 1 µM. 2'-PP (, solid line) concentration was varied between 1 and 500 nM. Results are means ± SE of triplicate determinations and representative of 4 experiments.

PTMP inhibition of renal cortex BBMV Na + -dependent phosphate uptake (, broken line) was maximal at alkaline pH. At 100 mM NaCl, the apparent pKa was 7.5 ± 0.3 pH units ( n = 3). The IC 50 for PTMP inhibition of Na + -dependent phosphate uptake was slightly affected by pH (IC 50 = 23 ± 7 nM between pH 5.5 and 8.5) in 150 mM NaCl (results not shown). Alkaline pH sensitivity of PTMP inhibition of Na + -dependent phosphate uptake was similar to the effect of pH on NaPi-IIa activity ( 3, 44 ). In contrast, 2'-PP inhibition of renal cortex BBMV Na + -dependent phosphate uptake (, solid line) was decreased at alkaline pH. At medium pH values above pH 7, 2'-PP inhibition of Na + -dependent phosphate uptake was <10% (5 ± 3%). The apparent pK a for 2'-PP inhibition of Na + -dependent phosphate uptake into renal cortex BBMV was 6.2 ± 0.4 ( n = 3). The effect of pH on phosphophloretin inhibition of renal cortex BBMV Na + -dependent phosphate uptake is consistent with 2'-PP inhibition of NaPi-Ia activity and PTMP inhibition of NaPi-IIa activity.

The selectivity of PTMP for NaPi-IIa-mediated Na + -dependent phosphate uptake was tested by comparing the effect of pH on phosphophloretin inhibition of Na + -dependent phosphate uptake into renal cortex BBMV as a function of Na + concentration. The results of these studies are shown in Fig. 5.

Fig. 5. Effect of Na + concentration on PTMP inhibition of renal cortex BBMV Na + -dependent phosphate uptake. Na + -dependent [ 32 P]phosphate uptakes into renal cortex BBMV were determined as described in MATERIALS AND METHODS at pH 6 (, solid line) and pH 8 (, broken line). Na + concentration was varied from 0 to 250 mM. PTMP concentration was 100 nM. Results are means ± SE of triplicate determinations and representative of 3 experiments.

If PTMP inhibition of renal cortex BBMV Na + -dependent phosphate uptake was Na + concentration sensitive at pH 6, then the low-PTMP inhibition of Na + -dependent phosphate uptake might be the result of low-NaPi-IIa Na + affinity at acid pH and not differential inhibition of NaPi-IIa and NaPi-Ia. However, if Na + concentration did not increase PTMP inhibition at acid pH, then PTMP has little effect on NaPi-Ia activity. Figure 5 shows PTMP inhibition at pH 6 (, solid line) was unaffected by increasing Na + concentration. At pH 8, PTMP inhibition of Na + -dependent phosphate uptake was Na + concentration sensitive (, broken line). This result suggests that PTMP inhibition of phosphate uptake at acid pH was not limited by reduced Na + affinity of NaPi-IIa. A similar series of experiments substituting 2'-PP for PTMP was more difficult to interpret due to very low 2'-PP-sensitive uptakes at alkaline pH. At acid pH and 50 mM NaCl, 2'-PP inhibition was 45 ± 10% ( n = 3) of the Na + -dependent phosphate uptake and unaffected by increasing the Na + concentration to 250 mM. These results are consistent with PTMP being a potent inhibitor of NaPi-IIa, with little effect on NaPi-Ia, and 2'-PP being a potent inhibitor of NaPi-Ia, with little effect on NaPi-IIa activity.

Effect of Phosphophloretins on Na + -Dependent Phosphate Uptakes into Distal Tubule-Enriched BBMV and Proximal Tubule-Enriched BBMV

Phosphophloretin derivative inhibition of NaPi-Ia- and NaPi-IIa-mediated Na + -dependent phosphate uptakes into BBMV purified from proximal tubule-enriched and from distal tubule-enriched fractions was examined to further define inhibitor sensitivity. Proximal tubule-enriched fractions and distal tubule-enriched fractions were purified by collagenase digestion and a combination of differential sieving and Percoll gradient centrifugation. Table 1 summarizes biochemical analysis of collagenase digested and Percoll gradient centrifugation purification of proximal tubules and distal tubules. Table 2 summarizes biochemical analysis of BBMV isolated from purified tubule fractions. Table 1 indicates that distal tubule fractions were three- to fourfold deenriched in the brush-border enzyme markers, alkaline phosphatase, leucine aminopeptidase, and -glutamyl transpeptidase compared with the renal cortex. Compared with the renal cortex total homogenate, proximal tubule fractions were enriched two- to threefold. Table 2 indicates that distal tubule-enriched BBMV were slightly enriched in BBM marker enzymes compared with the renal cortex homogenate, whereas proximal tubule-enriched BBMV were 12-fold enriched in BBM marker enzyme activities. These results are consistent with previous studies indicating that distal segments contain alkaline phosphatase and -glutamyl transpeptidase ( 37 ) at lower expression levels than proximal tubule apical membranes.

Distal tubule and proximal tubule Na + /phosphate cotransporter expression was performed by Western blot analysis of antipeptide antibodies to examine tubule expression of NaPi-IIa and NaPi-Ia and BBMV purification. Figure 6 is a Western blot analysis of BBMV purified from distal tubules ( lanes 2 - 4 ) and BBMV purified from proximal tubules ( lanes 5 - 8 ). Peptide-blocked Npt-11 antibody ( lane 1 ) and peptide-blocked Npt-25 antibody ( lane 9 ) are also shown. Varying amounts of distal tubule BBMV protein were added to the gel ( lane 2 contained 5 µg, lane 3 contained 10 µg, and lane 4 contained 25 µg of protein) to examine blot sensitivity as a measure of proximal tubule contamination of the distal tubule-enriched BBMV. Variable amounts of proximal tubule BBMV protein were added to the gel ( lane 8 contained 2.5 µg, lane 7 contained 5 µg, lane 6 contained 10 µg, and lane 5 contained 25 µg of protein) to examine the sensitivity of Npt-25 as a reporter NaPi-IIa expression. BBMV were probed with Npt-11 (NaPi-Ia-specific polyclonal antibody) followed by Npt-25 (NaPi-IIa-specific polyclonal antibody). Npt-25 ( top arrow) recognized an 87-kDa polypeptide in proximal tubule-enriched BBMV ( lanes 5 - 9 ) but not in distal tubule-enriched BBMV ( lanes 1 - 4 ). Npt-11 ( bottom arrow) recognized a 65-kDa polypeptide in both proximal tubule-enriched BBMV and distal tubule-enriched BBMV. Similar results were seen when blots were probed with Npt-25 followed by Npt-11 (results not shown). These results indicate that NaPi-Ia is expressed in the distal tubule-enriched fractions and suggest that Npt-25 is a sensitive reporter of NaPi-IIa and proximal tubule contamination of distal tubule fractions to a lower limit of 5% contamination of the distal tubule-enriched BBMV.

Fig. 6. Western blot of BBMV isolated from purified distal tubules and proximal tubules. Ca 2+ -precipitated BBM protein was resolved on 10% polyacrylamide gels by the method of Laemmli ( 20 ). Gels were transferred to nitrocellulose as described in MATERIALS AND METHODS. NaPi-IIa ( top arrow) was visualized with polyclonal antibody Npt-25, the nitrocellulose paper was stripped, and NaPi-Ia ( bottom arrow) was visualized with Npt-11. Distal tubule-enriched BBM protein was varied from 5 µg ( lane 2 ) to 25 µg ( lane 4 ). Proximal tubule-enriched BBM protein was varied between 2.5 µg ( lane 8 ) to 25 µg ( lane 5 ). Peptide-blocked Npt-11 ( lane 1 ) and peptide-blocked Npt-25 ( lane 9 ) are also shown. Results are from a single experiment and representative of 3 experiments.

Immunohistochemistry was performed on coverslips containing polylysine-immobilized, distal tubule-enriched fractions and proximal tubule-enriched fractions. Antibodies were directly labeled at their carbohydrate moieties to simplify washes and to improve reporter group access. Npt-11 labeled 70 ± 8% ( n = 4) of the structures on polylysine-coated coverslips containing distal tubule-enriched fractions. Npt-11 and Npt-21 (NaPi-IIa-specific antibody) labeled 6 ± 3% of the structures, and neither antibody recognized 19 ± 7% of the structures. Labeling experiments were also performed on renal sections with fluorescently labeled Npt-11. The results from one experiment are shown in Fig. 7.

Fig. 7. Immunohistochemistry of rabbit renal cortex stained with Texas red-conjugated Npt-11. Rabbit renal cortex slices were prepared for light microscopy and fluorescent antibody staining as described in MATERIALS AND METHODS. Texas red-conjugated Npt-11 labeling of renal cortex was examined on a Nikon 800 fluorescence microscope. DT, distal tubules; PT, proximal tubules. Texas red-conjugated Npt-11 labeling is shown as light areas around a black background. In some tubules, cells have collapsed into the tubule lumen.

Figure 7 shows Texas red-conjugated Npt-11 labeling of both proximal tubules (PT) and distal tubules (DT) at their luminal membranes. There was no labeling of the glomerulus or connecting tubules. Figures 6 and 7 and Tables 1 and 2 indicate that BBMV purified from distal tubule-enriched fractions isolated from rabbit renal cortex are at least 90% distal nephron and contain 5% proximal tubule contamination. Na + -dependent phosphate uptake experiments using BBMV purified from distal tubule-enriched, Ca 2+ -precipitated BBMV and into Ca 2+ -precipitated, proximal tubule-enriched were performed to test the suitability of BBMV for phosphophloretin inhibition experiments. Na + -dependent phosphate uptake into distal tubule-enriched BBMV and proximal tubule enriched BBMV as a function of time was examined. Phosphate uptake into distal tubule-enriched BBMV and into proximal tubule-enriched BBMV as a function of time is shown in Fig. 8.

Fig. 8. Time course of [ 32 P]phosphate uptake into distal tubule-enriched BBMV and proximal tubule-enriched BBMV. [ 32 P]phosphate uptake into distal tubule-enriched BBMV and proximal tubule-enriched BBMV was measured as described in MATERIALS AND METHODS. Distal tubule-enriched phosphate uptake in the presence of NaCl (, solid line) or in the presence of KCl (, broken line) is shown as a function of time from 3 s to 30 min. Proximal tubule-enriched phosphate uptake in the presence of NaCl (, solid line) or in the presence of KCl (, broken line) is shown as a function of time from 3 s to 60 min. Results are means ± SE of triplicate determinations and 3 experiments.

In the presence of NaCl, distal tubule-enriched BBMV phosphate uptake (, solid line) was maximal at 60 s reaching an internal concentration 2 x equilibrium values. In the presence of NaCl, phosphate uptake into proximal tubule-enriched (, solid line) BBMV was maximal at 120 s, reaching an internal concentration 3 x equilibrium phosphate concentration. These results indicate that BBMV prepared from distal tubule-enriched fractions and proximal tubule-enriched fractions show a modest overshoot consistent with Na + -coupled phosphate uptake. On the basis of these experiments, 5-s uptakes were chosen for phosphophloretin inhibition studies using initial rates.

Phosphophloretin inhibition of Na + -dependent phosphate uptake into distal tubule-enriched BBMV was examined to test the hypothesis that NaPi-Ia is 2'-PP sensitive and not PTMP sensitive. Na + -dependent phosphate uptake into distal tubule-enriched BBMV and proximal tubule-enriched BBMV was examined. The results of these experiments are shown in Fig. 9.

Fig. 9. Effect of 2'-PP and PTMP on Na + -dependent phosphate uptake into distal tubule-enriched BBMV. Na + -dependent [ 32 P]phosphate uptake into distal tubule-enriched BBMV was examined at pH 6.0 (open symbols) or pH 8.5 (filled symbols) as a function of 2'-PP concentration (solid lines) or PTMP concentration (broken lines). Results are means ± SE of triplicate determinations and representative of 3 experiments.

Na + -dependent phosphate uptake into distal tubule-enriched BBMV was sensitive to 2'-PP in a concentration-dependent manner. At pH 6 (, solid lines), the IC 50 for 2'-PP inhibition of phosphate uptake was 35 ± 8 nM ( n = 3). At pH 8.5 (, solid line), the IC 50 was 48 ± 6 nM ( n = 3). Na + -dependent phosphate uptake into distal tubule-enriched BBMV was not sensitive to PTMP at pH 6 (, dashed line) or at pH 8.5 (, dashed line).

Similar experiments in proximal tubule-enriched BBMV were similar to phosphoploretin inhibition experiments with BBMV isolated from renal cortex. 2'-PP inhibition of Na + -dependent phosphate uptake into BBMV isolated from proximal tubule-enriched fractions decreased with increasing pH. Maximum inhibition was 34% (%I max = 34 ± 8%, n = 3) at pH 6 to 3% (3.4 ± 2%, n = 3) of the Na + -dependent phosphate uptake into proximal tubule-enriched BBMV at pH 8.5. PTMP was a potent inhibitor of Na + -dependent phosphate uptake into proximal tubule-enriched BBMV at all pH values studied. The IC 50 for PTMP inhibition of Na + -dependent phosphate uptake into proximal tubule-enriched BBMV was 22 ± 6 nM ( n = 4).

DISCUSSION

Within a membrane protein family differences in inhibitor sensitivity are a common characteristic, which has been exploited to distinguish between family members in different tissues. Similar to the use of amiloride and NHE family members, and stilbene disulfonates and the anion exchange protein family, differences in phosphophloretin derivative sensitivity may prove helpful in defining NaPi family member expression. In addition, differences in phosphophloretin sensitivity may be related to differences in the cotransporter phosphate sites. Comparing the NaPi family of proteins ( 11, 28, 44, 45 ), the intestinal BBM Na + -phosphate cotransporter (NaPi-IIb) and the renal cortex PTH-insensitive Na + /phosphate cotransporter (NaPi-Ia) share functional characteristics including a preference for H 2 PO 4. The proximal tubule PTH-sensitive BBM Na + /phosphate cotransporter (NaPi-IIa) differs from NaPi-IIb and NaPi-Ia with respect to phosphate valence state preference; vanadate, PFA, and phloretin sensitivity; a preference for HPO 4; and the effect of H + concentration on Na + affinity.

Previous studies suggested a correlation between vanadate and PFA sensitivity and cotransporter preference for HPO 4 ( 24, 43, 45 ). Based on these studies with phosphate site analogs, we decided to examine NaPi family sensitivity to phosphophloretin derivatives. In addition, solvent polarity has a marked effect on phosphate dissociation state ( 26 ). We hypothesized that differences in phosphate valence state preference within the NaPi family of proteins may be related to substrate site polarity. To test this hypothesis and to develop selective phosphophloretin derivatives, the effect of water-soluble and poorly water-soluble phosphophloretins on three members of the NaPi family of proteins with different phosphate valence state preferences was examined.

Na + -dependent phosphate uptakes into BBMV prepared from rabbit kidney cortex were sensitive to 2'-PP and PTMP ( Fig. 1 ). The phloretin. 2'-PP inhibition of Na + -dependent phosphate uptake into renal cortex BBMV was variable but always significantly greater than phloretin sensitivity. In contrast, intestinal BBMV Na + -dependent phosphate uptake ( Fig. 2 ) was sensitive to 2'-PP and insensitive to PTMP. Intestinal BBMV Na + -dependent phosphate uptake was inhibited more than 90% at 500 nM 2'-PP with an IC 50 of 40 ± 4 nM ( Fig. 2 ). 2'-PP inhibition of intestinal BBMV Na + -dependent phosphate uptake was greater at acidic pH than at alkaline pH. The pH dependence of 2'-PP inhibition of intestinal Na + -dependent phosphate uptake was consistent with the pH dependence of Na + binding to the cotransporter ( 10, 28, 33, 41 ). These results are similar to previous studies ( 28, 34, 35 ). Based on these studies, phloretin derivative sensitivity PTMP phloretin. Further studies attempted to define NaPi-IIa and NaPi-Ia phloretin derivative sensitivity.

Assigning inhibition in rabbit renal cortex BBMV is complicated by the presence of two Na + -phosphate cotransporters in BBM, NaPi-IIa and NaPi-Ia. To examine Na + -phosphate cotransporter inhibitor sensitivity, conditions were altered to minimize each cotransporter's contribution to Na + -dependent phosphate uptake in membrane vesicles. NaPi-IIa activity was selectively examined at alkaline pH. NaPi-Ia activity was selectively examined at acidic pH and in BBMV purified from distal tubule-enriched fractions.

The pH dependence of PTMP inhibition of renal cortex BBMV Na + -dependent phosphate uptake suggested PTMP inhibition of NaPi-IIa activity ( Fig. 4 ). The IC 50 of PTMP inhibition of renal cortex BBMV Na + -dependent phosphate uptake was independent of pH (results not shown). The magnitude of PTMP inhibition of renal cortex BBMV Na + -dependent phosphate uptake ( Fig. 4,, broken line) was greater at alkaline pH than at acidic pH, consistent with Na + activation of NaPi-IIa-mediated phosphate uptake and Na + activation of a Na + -dependent inhibitor of phosphate uptake.

The decreased inhibition of Na + -dependent phosphate uptake at acid pH was not due to reduced NaPi-IIa Na + affinity at acid pH reducing the effect of a Na + -dependent inhibitor like PTMP. Increasing the Na + concentration to about three times the K M (Na + ) did not increase PTMP inhibition of Na + -dependent phosphate uptake into renal cortex BBMV at acid pH ( Fig. 5 ). This result is consistent with PTMP inhibition of renal cortex Na + -dependent phosphate uptake being limited to NaPi-IIa and not due to reduced NaPi-IIa Na + affinity at acidic pH ( Fig. 5 ).

Renal cortex BBMV was less sensitive to 2'-PP than to PTMP. 2'-PP inhibition of renal BBMV Na + -dependent phosphate uptake was 45% at acid pH and decreased with increasing pH to 5% at alkaline pH ( Fig. 4,, solid line). The effect of pH on 2'-PP inhibition as a function of pH is inconsistent with inhibition of NaPi-IIa, consistent with the pH dependence of NaPi-Ia activity, and consistent with 2'-PP inhibition of NaPi-Ia.

The results from our renal cortex BBMV Na + -phosphate cotransporter inhibitor studies were consistent with NaPi-IIa being PTMP sensitive and NaPi-Ia being 2'-PP sensitive. To define NaPi-Ia inhibition of Na + -dependent phosphate uptake in the kidney, distal tubules and proximal tubules were prepared from renal cortex by collagenase digestion, molecular sieving, and Percoll gradient centrifugation.

The distribution of NaPi-IIa, NaPi-Ia, and brush-border marker enzymes along the nephron is controversial. NaPi-IIa and NaPi-Ia are present in the apical membrane of the proximal tubule ( 28 ). However, there is little agreement concerning the presence of phosphate reabsorption, -glutamyl transpeptidase, and leucine aminopeptidase at more distal sites.

Four lines of evidence suggest that NaPi-Ia is present in distal tubule fractions purified from rabbit renal cortex. Tables 1 and 2 indicate that following Percoll gradient centrifugation and Ca 2+ precipitation, distal tubule-enriched BBMV are four-fold deenriched in the BBM markers, leucine aminopeptidase, and -glutamyl transpeptidase relative to the renal cortex. Based on marker enzymes and transport activities, the distal BBMV is at least 10-fold deenriched in proximal tubule activities.

Western blot analysis shown in Fig. 6 indicates that NaPi-IIa was not detected in BBMV purified from the distal tubule-enriched fractions ( Fig. 6, lanes 2 - 4 ). Western blot analysis of serial dilutions of renal cortex BBM protein indicated that lower limit of Npt-25 sensitivity was <2.5 µg or approximately a 10-fold dilution of NaPi-IIa (results not shown). Similar studies with Npt-11 indicated that the lower limit for Npt-11 recognition of NaPi-Ia in renal cortex or proximal tubule-enriched BBMV was 10 µg. These results indicate that a 10% proximal tubule contamination of distal tubule BBMV would be detected with our antibodies. Immunocyto-chemistry of polylysine-coated coverslips with immobilized distal tubules and proximal tubule renal sections indicated that 70% of the structures were labeled with the NaPi-Ia-specific antibody (Npt-11) and 6% of the structures were labeled with the NaPi-IIa-specific antibody and the NaPi-Ia-specific antibody.

Immunohistochemistry of renal slices were consistent with NaPi-Ia localization in both proximal tubules and distal segments ( Fig. 7 ). The apical membranes of distal tubules contained NaPi-Ia and lacked NaPi-IIa ( 4, 5, 22, 28, 44 ). These results are in agreement with previous reports ( 1, 4, 5, 16, 17, 22, 28 ) of phosphate handling in distal nephron segments. These results indicate that at least 70% of distal tubule-enriched BBMV were from distal tubule-derived structures.

Finally, phosphate uptake into BBMV isolated from distal tubule-enriched fractions displayed an overshoot of equilibrium phosphate uptake in NaCl media ( Fig. 8 ). The presence of an overshoot in Na + is characteristic of Na + -dependent organic solute cotransporters, indicating active accumulation of phosphate above intervesicular equilibrium due to continued uptake of Na + ( 47 ). The presence of Na + -dependent phosphate uptake in BBMV from distal tubule-enriched fractions ( Fig. 8 ) and apparent absence of NaPi-IIa in distal tubule-derived BBMV ( Fig. 6 ) strongly suggests that NaPi-Ia is present in the distal tubule-enriched BBMV and that these membrane vesicles may be useful in studies of phosphophloretin sensitivity of NaPi-Ia.

PTMP inhibition of Na + -dependent phosphate uptake into distal tubule-enriched BBMV was not statistically different from phloretin inhibition ( Fig. 9 ). PTMP inhibited <10% of the Na + -dependent phosphate uptake into these membrane vesicles. 2'-PP inhibited Na + -dependent phosphate uptake into BBMV purified from distal tubules in a concentration-dependent manner with an IC 50 of 38 nM at pH 6 and 48 nM at pH 8.5 ( Fig. 9 ). These results are consistent with 2'-PP inhibition of NaPi-Ia in renal BBMV. The results indicate that NaPi-IIa is PTMP sensitive and NaPi-Ia and NaPi-IIb are 2'-PP sensitive. Based on our studies, we suggest that PTMP and 2'-PP may be useful tools for the analysis of NaPi protein expression.

With regard to the molecular mechanism responsible for differential phosphophloretin sensitivity in renal and intestinal BBMV, we hypothesize that differences in the phosphate sites of the cotransporters are the most likely cause of phosphophloretin sensitivity differences within the NaPi family. Phosphate valence state preference appeared to correlate with cotransporter phosphophloretin derivative sensitivity. Na + /phosphate cotransporters preferring H 2 PO 4 (NaPi-Ia and NaPi-IIb) were sensitive to 2'-PP. A preference for HPO 4 was associated with a greater sensitivity to PTMP.

Differences in phosphophloretin derivative sensitivity were consistent with differences in the degree of phosphate site-solvent accessibility within the NaPi family. 2'-PP is water soluble and an acid in water. PTMP is poorly water soluble and slightly basic in water. PTMP sensitivity of NaPi-IIa may indicate a more hydrophobic phosphate site. In this regard, changes in solvent polarity, such as mixed organic solvent: water solutions, have been shown to alter phosphate dissociation ( 26 ). A less polar phosphate site may contribute to cotransporter preference for HPO 4, and a more polar phosphate site may contribute to a preference for H 2 PO 4 transport. The selective inhibition by phosphophloretin derivatives is consistent with this interpretation.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the expert assistance of E. Ezell in performing the NMR experiments. The authors acknowledge S. Smith and T. Hoag for performing amino acid sequencing of the peptides used to raise the polyclonal antibodies. Immunohistochemistry was performed at the Immunohistochemistry Core Laboratory (UTMB, Galveston, TX).

【参考文献】
  Amiel C, Kuntziger H, and Richet G. Micropuncture study of handling of phosphate by proximal and distal nephron in normal and parathyroidectomized rat. Evidence for distal reabsorption. Pflügers Arch 317: 93-109, 1970.

Basketter DA and Widdas WF. Asymmetry of the hexose transfer system in human erythrocytes. Comparison of the effects of cytochalasin B, phloretin and maltose as competitive inhibitors. J Physiol 278: 389-401, 1978.

Bernier W, Kinne R, and Murer H. Phosphate transport into brushborder membrane vesicles isolated from rat small intestine. Biochem J 160: 467-474, 1976.

Biber J, Custer M, Werner A, Kaissling B, and Murer H. Localization of NaPi-1, a cotransporter in rabbit kidney proximal tubules. II. Localization by immunohistochemistry. Pflügers Arch 424: 210-215, 1993.

Chong SS, Kozak CA, Liu L, Kristjansson K, Dunn ST, Bourdeau JE, and Hughes MR. Cloning, genetic mapping, and expression analysis of a mouse renal sodium-dependent phosphate cotransporter. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1038-F1045, 1995.

Chou CL and Knepper MA. Inhibition of urea transport in inner medullary collecting duct by phloretin and urea analogues. Am J Physiol Renal Fluid Electrolyte Physiol 257: F359-F365, 1989.

Claveau D, Pellerin I, LeClerc M, and Brunette MG. Characterization of the Na + /H + exchanger in the luminal membrane of the distal nephron. J Membr Biol 165: 265-274, 1998.

Counillon L and Pouyssegur J. The expanding family of eukaryotic Na + /H + exchangers. J Biol Chem 275: 1-4, 2000.

Dalquist A. Method for assay of intestinal disaccharidases. Anal Biochem 7: 18-25, 1964.

Danisi G, Murer H, and Staub RW. Effect of pH on phosphate transport into intestinal brush-border membrane vesicles. Am J Physiol Gastrointest Liver Physiol 246: G180-G186, 1984.

De la Horra C, Hernando N, Lambert G, Forster I, Biber J, and Murer H. Molecular determinants of pH sensitivity of the type II a Na/Pi cotransporter. J Biol Chem 275: 6284-6287, 2000.

Diedrich DF. Photoaffinity labeling analogs of phloridzin and phloretin: synthesis and effects on cell membranes. Methods Enzymol 191: 755-780, 1990.

Dusso AS, Pavlopoulos T, Naumovich L, Lu Y, Finch J, Brown AJ, Morrissey J, and Slatopolsky E. p21 (WAF1) and transforming growth factor mediate dietary phosphate regulation of parathyroid cell growth. Kidney Int 59: 855-865, 2001.

Forman SA, Verkman AS, Dix JA, and Solomon AK. Interaction of phloretin with the anion transport protein of the red blood cell membrane. Biochim Biophys Acta 689: 531-538, 1982.

Forstner GC, Sabesin S, and Isselbacher KJ. Rat intestinal microvillus membranes. Purification and biochemical characterization. Biochem J 106: 381-390, 1968.

Gesek FA and Friedman PA. Sodium entry mechanisms in distal convoluted tubules. Am J Physiol Renal Fluid Electrolyte Physiol 268: F89-F98, 1995.

Hoag HM, Martel J, Gauthier C, and Tennenhouse HS. Effects of Npt2 gene ablation on renal Na + /phosphate cotransport and cotransporter expression. J Clin Invest 104: 679-686, 1999.

Jentsch TJ, Stein V, Weinreich F, and Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503-568, 2001.

Kimura T, Shiosaka S, and Yoshida A. Effect of dietary amino acids on jejunal sucrase and leucineaminopeptidase activities in rats. J Nutr 108: 1087-1097, 1978.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970.

Lambert G, Traebert M, Biber J, and Murer H. Cleavage of disulfide bonds leads to inactivation and degradation of the type II a, but not type II b sodium phosphate cotransporter expressed in Xenopus laevis oocytes. J Membr Biol 176: 143-149, 2000.

Lassiter WE and Colindres RE. Phosphate reabsorption in the distal convoluted tubule. Adv Exp Med Biol 151: 21-32, 1982.

Lee BS, Gunn RB, and Kopito RR. Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line. J Biol Chem 266: 11448-11454, 1991.

Logham-Adham M, Szczepanska-Konkel M, Yusufi AN, Van Scoy M, and Dousa TP. Inhibition of Na + -Pi cotransporter in small gut brush border by phosphonocarboxylic acids. Am J Physiol Gastrointest Liver Physiol 252: G244-G249, 1987.

Magaganin S, Werner A, Markovich D, Sorribas V, Stange G, Biber J, and Murer H. Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc Natl Acad Sci USA 90: 5979-5983, 1993.

Maurel P, Hui Bon Hoa G, and Douzou P. The pH dependence of the tryptic hydrolysis of benzoyl- L -arginine ethyl ester in cooled mixed solvents. J Biol Chem 250: 1376-1382, 1975.

Mirrington RN and Feutrill GI. Orcinol monoethyl ether. Organic Synthesis Coll 6: 858-861, 1988.

Murer H, Hernandez N, Forster I, and Biber J. Proximal tubule phosphate reabsorption: molecular mechanisms. Physiol Rev 80: 1373-1409, 2000.

Naveh-Many T, Rahamimov R, Livni N, and Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate and vitamin D. J Clin Invest 96: 1786-1793, 1995.

Orlowski M and Meister A. -Glutamyl nitroanilide: a new convenient substrate for determination and study of L -, D -, -glutamyltranspeptidase activities. Biochim Biophys Acta 73: 679-681, 1963.

O'Shannessy DJ and Quarles RH. Specific conjugation of the oligosaccharide moieties of immunoglobulins. J Appl Biochem 7: 347-355, 1985.

Peerce BE. Examination of the substrate stoichiometry of the intestinal Na + /phosphate cotransporter. J Membr Biol 110: 189-197, 1989.

Peerce BE. A 40 kDa polypeptide from papain digestion of the rabbit intestinal Na + /phosphate cotransporter retains Na + and phosphate cotransport. Arch Biochem Biophys 401: 1-10, 2002.

Peerce BE and Clarke RD. A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na + -dependent phosphate absorption. Am J Physiol Gastrointest Liver Physiol 283: G848-G855, 2002.

Peerce BE, Fleming RYD, and Clarke RD. Inhibition of human intestinal brush border membrane vesicle Na + -dependent phosphate uptake by phosphophloretin derivatives. Biochem Biophys Res Commun 301: 8-12, 2003.

Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83: 346-356, 1977.

Pfaller W, Gstraunthaler G, Kotanko P, Wolf H, and Curthoys NP. Immunocytochemical localization of -glutamyl-transferase on isolated renal cortical tubular fragments. Histochemistry 80: 289-293, 1984.

Pouchard CJ. Aldrich Library of NMR Spectra (2nd ed.). Milwaukee, WI: Aldrich Chemical, 1983, vol. 1, p. 833A.

Putney LK, Denher SP, and Basher DL. The changing face of the Na + /H + exchanger, NHE 1: structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol 42: 527-552, 2002.

Scharschmidt BF, Keeffe EB, Blankenship NM, and Ockner RK. Validation of a recording spectrophotometric method for measurement of membrane-associated Mg and Na-K-ATPase activity. J Lab Clin Med 93: 790-799, 1979.

Shirazi-Beechey SP, Gorvel JP, and Beechey RB. Phosphate transport in intestinal brush border membranes. J Bioenerg Biomembr 20: 273-288, 1988.

Stevens BR, Ross HJ, and Wright EM. Multiple transport pathways for neutral amino acids in rabbit jejunal brush border membranes. J Membr Biol 66: 213-225, 1982.

Szczepanska-Konkel M, Yusufi ANK, VanScoy M, Webster SK, and Dousa TP. Phosphonocarboxylic acids as specific inhibitors of Na + -dependent transport of phosphate across renal brush border membrane. J Biol Chem 261: 6375-6383, 1986.

Tenenhouse HS, Gauthier C, Martel J, Gesek FA, Countermarsh BA, and Friedman PA. Na + -phosphate cotransport in the mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression. J Bone Miner Res 13: 590-597, 1998.

Timmer RT and Gunn RB. Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells. Am J Physiol Cell Physiol 274: C757-C769, 1998.

Toon MR and Solomon AK. Modulation of water and urea transport in human red cells: effects of pH and phloretin. J Membr Biol 99: 157-164, 1987.

Turner RJ. Quantitative studies of cotransport systems: models and vesicles. J Membr Biol 76: 1-15, 1983.

Wang X, Poole RC, Halestrap AP, and Levi AJ. Characterization of the inhibition by stilbene disulfonates and phloretin of lactate and pyruvate transport into rat and guinea-pig cardiac myocytes suggests the presence of two kinetically distinct carriers in heart cells. Biochem J 290: 249-258, 1993.

Wesson LG. Homeostasis of phosphate revisited. Nephron 77: 249-266, 1997.

Wills NK, Weng T, Mo L, Hellmich HL, Yu A, Wang T, Bucheit S, and Godley BF. Chloride channel expression in cultured human fetal RPE cells: response to oxidative stress. Invest Ophthalmol Vis Sci 41: 4247-4255, 2000.

Yu CA, Yu L, and King TE. Spectral evidence of multiple cytochromes b present in succinate: cytochrome c reductase. Biochim Biophys Acta 267: 300-308, 1972.

作者单位：Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641