Institutes of 2 Physiology and 1 Anatomy, University of Zurich, CH-8057 Zurich, Switzerland
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ABSTRACT |
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Type II Na-Pi cotransporters (type IIa and type IIb) represent apically located Na-Pi cotransporters in epithelia of proximal tubules (type IIa) and small intestine (type IIb). Here we provide evidence that the type IIb (but not the type IIa) Na-Pi cotransporter is also expressed in the lung. With the use of immunohistochemistry, location of the type IIb protein was found exclusively in the apical membrane of type II cells of the alveolar epithelium. Such a location of the type IIb cotransporter suggests an involvement in the reuptake of phosphate necessary for the synthesis of surfactant. A possible regulation of the abundance of the type IIb cotransporter in the lung was studied after adaptation of mice to a low-Pi diet. After a chronic adaptation to a low-Pi diet, no changes in the type IIb protein and the type IIb transcript were observed. These results exclude dietary intake of phosphate as a regulatory factor of the type IIb Na-Pi cotransporter in alveolar type II cells.
sodium-inorganic phosphate cotransporter; immunohistochemistry
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INTRODUCTION |
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THE COMPOSITION of alveolar fluid is maintained by transepithelial transports of a variety of solutes through the alveolar epithelium. In alveolar type II (ATII) cells, several epithelial transport processes such as sodium-dependent transport of glucose (1, 4) as well as transport of amino acids (3, 4) and protons (15) and of sodium through the epithelial sodium channel (13, 20) have been described. It is conceivable that some of these and other transport processes provide substrates necessary for surfactant synthesis taking place in ATII cells (17).
Thus far, sodium-dependent transport of phosphate (Na-Pi cotransport) has been described in primary cultures of lung cells (4) and in an ATII cell line of the rat lung (14). However, the molecular identity of Na-Pi cotransport in these cells has not been established. To date, three mammalian Na-Pi cotransporter protein families have been identified (type I, type II, and type III Na-Pi cotransporters; for a review, see Ref. 22). Type I and type II Na-Pi cotransporters are expressed mainly in the epithelia of the kidney, small intestine, and liver (22). On the other side, the expression pattern of type III cotransporters is ubiquitous, including in the lung (10, 22). Two members of the type II Na-Pi cotransporter family can be distinguished: type IIa and type IIb. In mammals, the former isoform is expressed in renal proximal tubules (6, 12, 22), and expression of the type IIb isoform in the small intestine has recently been reported (8). In addition, based on RT-PCR and Northern blot analysis, evidence was obtained that the type IIb Na-Pi cotransporter is also expressed in lung tissue (8, 9). To date, no evidence for an expression of the type I cotransporter in the lung has been obtained (22 and references therein).
In the present work, we show, using immunohistochemistry, that the type IIb Na-Pi cotransporter is expressed in murine lung ATII cells and further that this Na-Pi cotransporter is located at the apical membrane of these cells. In addition, we investigated the question of whether the abundance of the type IIb cotransporter in the lung is influenced by the dietary content of phosphate (adaptation) similar to, for example, the renal type IIa Na-Pi cotransporter.
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MATERIALS AND METHODS |
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Animals. Male mice (National Medical Research Institute; 8 wk old) were obtained from RCC (Füllinsdorf, Switzerland). All animals received tap water ad libidum and a commercial diet. For the adaptation studies, the animals were fed either a 1.1 (high-Pi) or 0.09% (low-Pi) Pi diet (Kliba) for 5 days. Lung tissue used for the preparation of membranes or isolation of RNA was immediately frozen in liquid nitrogen.
Antisera. Polyclonal antibodies were custom-made (Eurogentec) by injection into rabbits of keyhole limpet hemocyanin-coupled synthetic peptides. Antigenic peptides corresponded to either the NH2 or COOH terminus of the type IIb Na-Pi cotransporter amino acid sequence (8). Results obtained with both antisera were identical.
Membrane preparations. A crude
membrane fraction from the lungs was obtained as follows. Frozen lungs
were thawed on ice, and all the following procedures were carried out
at 4°C. The lung was homogenized with a Polytron emulsifier
(setting 5 for 1 min) in 15 ml of buffer
A (300 mM mannitol, 5 mM EGTA, and 12 mM
Tris · HCl, pH 7.1), and the suspension was
centrifuged at 1,000 g for 10 min. The
supernatant was diluted with 10 ml of ice-cold water and centrifuged at
27,000 g for 30 min. The resulting pellet was resuspended in 20 ml of buffer
A and centrifuged at 27,000 g for 30 min. The final pellet was
resuspended in 2-3 ml of buffer A
by passage through a 26-gauge needle, and aliquots were immediately
frozen in liquid nitrogen and stored at 70°C until used.
Brush-border membrane vesicles (BBMVs) from renal proximal tubules were
prepared as previously described (2).
Western blot analyses. Membrane proteins (70 µg/lane) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Before electrophoresis, the membranes were denaturated in the presence of 2% SDS by heating for 2 min at 95°C in the absence of a reducing agent. Incubation with primary antibodies (dilution 1:4,000) was performed as previously described (6, 8). As a secondary antibody, a goat anti-rabbit IgG conjugated to horseradish peroxidase (Amersham) was used, and immunodetection was carried out by enhanced chemiluminescence (Pierce). The type IIa Na-Pi cotransporter of isolated renal BBMVs was assessed as previously described (6).
RNA isolation and Northern blot
analyses. Total RNA and
poly(A)+ RNA were isolated with
TRIzol Reagent (GIBCO BRL) and polyATtract (Promega) according to the
manufacturers' protocols. After electrophoresis on a 1.2%
agarose-formaldehyde gel, mRNAs were transferred onto nylon membranes
(Biodyn). The blots were hybridized with a type IIb full-length probe
(8) that was labeled by random priming in the presence of
[-32P]dCTP. A probe
for the ribosomal protein L28 served as a loading control.
Hybridization was performed in 6× saline-sodium citrate (SSC),
5× Denhardt's solution, 0.5% SDS, and 100 mg/ml of herring sperm DNA at 65°C. The blots were washed sequentially with 2× SSC-0.1% SDS (10 min at room temperature), 1× SSC-0.1% SDS (10 min at 40°C), and 0.5× SSC-0.1% SDS (20 min at 55°C).
Immunohistochemistry. After anesthesia
(100 mg/kg body wt of thiopental intraperitoneally), the mice were
perfused with a hypodermic syringe via the right cardiac ventricle. The
fixative consisted of 3% paraformaldehyde and 0.05% picric acid in
0.1 M cacodylate buffer (pH 7.4; adjusted to 300 mosM with sucrose), 3 mM MgCl2, and 10% hydroxyethyl
starch. For immunofluorescence, perfused lung tissue was stored in
cacodylate buffer for 2 h. Lung tissue designated for immunogold
electron microscopy was postfixed for 2 h in the same fixative
described above to which 0.1% glutaraldehyde was added. Lung tissue
was cut into slices that were mounted onto thin cork plates and
immediately frozen in liquid propane cooled with liquid nitrogen.
Cryosections of 4 µm thickness were mounted on chrome
alum-gelatin-coated glass slides, thawed, and stored in cold (4°C)
phosphate-buffered saline (PBS) until used. Before addition of the
first antibody, sections were pretreated with 10% normal goat serum in
PBS for 10 min. Anti-type IIb antiserum was added at a dilution of
1:500 in the presence of 3% nonfat milk powder in PBS containing 0.3%
Triton X-100 and incubated overnight at 4°C. The sections were then
rinsed three times with PBS and covered for 45 min at 4°C with a
secondary antibody (swine anti-rabbit IgG conjugated to fluorescein
isothiocyanate; Dakopatts, Glostrup, Denmark) that was diluted 1:50 in
PBS-3% milk powder. Double staining for -actin was achieved by
adding rhodamine-phalloidin (Molecular Probes, Eugene, OR) at a
dilution of 1:50. Finally, the sections were rinsed three times with
distilled water, covered with a coverslip with DAKO-Glycergel
(Dakopatts) containing 2.5%
1,4-diaza-bicyclo[2.2.2]octane (Sigma), and studied with
an epifluorescence microscope (Polyvar, Reichert-Jung).
For controls, sections were incubated with either preimmune serum or anti-Na-Pi type IIb antiserum that was pretreated for 30 min with the corresponding antigenic peptide (100 µg/ml). Nonspecific binding to the tissue of the secondary antibodies was tested by omitting the primary antibody (data not shown).
Immunogold electron microscopy. Ultrathin cryosections (80 nm) were prepared according to Griffiths et al. (7) with a Leica Ultracut UCT. The sections were transferred to polyvinyl chloride-polyvinyl acetate-covered nickel grids and stored on 1% gelatin at 4°C overnight. To quench free aldehyde groups, the grids were incubated for 5 min in 50 mM NH4Cl and afterward incubated for 10 min in PBS containing 3% bovine serum albumin, 0.02% Triton X-100, and 0.01% Tween 20 (solution A). Thereafter, the grids were transferred for 1 h at 37°C with the primary antibodies (dilution 1:500 in solution A), washed in PBS, and incubated for 1 h at room temperature with goat anti-rabbit IgG coupled to 8-nm gold particles at a dilution (in solution A) corresponding to an optical density at 525 nm of 0.06. Conjugation to gold particles was performed according to the method of Slot and Geuze (18). Finally, the grids were rinsed with PBS; postfixed for 10 min in 1% glutaraldehyde in PBS; and rinsed again with PBS, then with cacodylate buffer, and finally with distilled water. The sections were contrasted in 2% (wt /vol) methylcellulose containing 0.2% uranyl acetate for 10 min and examined on a Phillips CM 100 electron microscope.
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RESULTS |
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Based on RT-PCR and Northern blotting, expression of the type IIb
Na-Pi cotransporter in the lung
tissue of mice and Xenopus laevis,
respectively, has been previously suggested (8, 9). With Northern blot
analysis performed with poly(A)+
RNA isolated from mouse lungs, we now have confirmed the presence of
type IIb mRNA (Fig.
1A). A
type IIb-specific probe hybridized with lung
poly(A)+ RNA at a position of ~4
kb, corresponding to the size of the type IIb transcript present in the
small intestine (8). By using a probe specific for the renal type IIa
Na-Pi cotransporter, a weak
reaction at 4 kb was detected (data not shown; see Ref. 12). This
reaction, however, has to be regarded as a cross-hybridization with
type IIb mRNA because it has been shown earlier with RT-PCR that in the
lung the type IIa cotransporter gene is not expressed (8) and,
moreover, that the type IIa protein could not be detected on Western
blots (see below).
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Further evidence for the expression of the type IIb Na-Pi cotransporter in the lung was obtained by immunoblotting of a "crude" membrane fraction. With an anti-NH2 terminus antiserum, a specific immunoreaction with a band corresponding to a molecular mass of ~108 kDa was observed (Fig. 1B). Specificity of this immunoreaction was confirmed by inclusion of the antigenic peptide. The same result was obtained with an antiserum directed against the COOH terminus (8) of the type IIb cotransporter (data not shown). No immunoreaction was observed with an anti-type IIa antibody (Fig. 1B), further indicating that the renal type IIa Na-Pi cotransporter is not expressed in the lung.
The location of the type IIb cotransporter protein in mouse lung
tissue was assessed by immunofluorescence (Fig.
2) and immunogold electron microscopy (Fig.
3). Throughout the lung, tissue-specific type IIb Na-Pi
cotransporter-mediated immunostaining was detected only in the
respiratory tissue and was limited to large, roughly cuboidal cells
where the alveolar walls unite and form angles. No specific
immunostaining was detected in other cells of the alveolar
epithelium or in the epithelium of the bronchi (Fig. 2E). Costaining for -actin
revealed that the type IIb protein is located at sites of high
abundance of
-actin, representing the apical pole of type II
alveolar cells (Fig. 2, C and
D).
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With immunogold electron microscopy, the type IIb protein was detected along the short microvilli present at the apical pole of large cuboidal cells (Fig. 3). The vacuolated subapical area and the tight junctions in the margins clearly identify these cells as ATII cells (21). Besides, in microvilli, type IIb protein-associated gold particles were also rarely observed in vacuoles present in the subapical cytoplasm but were absent in the so-called multilamellar bodies that are critically involved in surfactant storage and secretion (16).
Under conditions of reduced dietary intake of phosphate
(low-Pi diet),
Na-Pi cotransport in the small
intestine and proximal tubules is upregulated (5, 11). In both tissues,
increased transepithelial Pi
transport is due to an increase in the amount of type II
Na-Pi cotransporters in the apical
membrane (5, 11; Hattenhauer, Traebert, Murer, and Biber, unpublished
data). To answer the question of whether the dietary
content of Pi also affects the
amount of type IIb cotransporter in the lung, mice were fed a
low-Pi diet and, as a control, a
high-Pi diet (chronic adaptation)
for 5 days. As indicated in Fig. 4, no
evidence for an upregulation of the type IIb protein with a
low-Pi diet was obtained. As a
control for the physiological response of the diets given, the amount
of renal type IIa Na-Pi
cotransporter was determined by Western blots in isolated renal
proximal tubular BBMVs (Fig. 4A). As indicated, a
low-Pi diet resulted in a large
increase in the amount of type IIa protein compared with that in BBMVs isolated from control (high-Pi
diet) animals and was similar in size to that reported in another study
(11). Furthermore, as documented by Northern blotting, a
low-Pi diet also did not result in
a change in the amount of the type IIb transcript (Fig.
4B).
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DISCUSSION |
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In agreement with previously reported results obtained by Northern blot analysis (9) and RT-PCR (8), the data presented indicate that the type IIb Na-Pi cotransporter is expressed in the lung tissue of mice. By Northern and Western blots, mRNA and protein species of the same sizes as observed in the small intestine (8) were observed. Furthermore, by immunocytochemistry, the type IIb Na-Pi cotransporter was identified as an apical protein of ATII cells. This cell type of the alveolar epithelium is characterized by formation of tight junctions, a high concentration of actin in the microvilli, and multilamellar bodies involved in surfactant synthesis (16, 19, 21). In all cases, type IIb-associated immunoreactions were found only in such cells, suggesting that the type IIb Na-Pi cotransporter is a specific marker for ATII cells and, moreover, for the apical membrane of these cells. No evidence for a location different (e.g., the bronchial epithelium) from type II cells was obtained.
Thus far, three different mammalian Na-Pi cotransporters have been identified and were grouped into three families: type I, type II, and type III cotransporters (22). Expression of type I cotransporters in the lung seems unlikely because no evidence for type I gene expression was obtained in earlier studies (22 and references therein). Also, in preliminary studies, we could not detect specific immunostaining with an anti-type I antibody (data not shown). With respect to type II Na-Pi cotransporters, two subfamilies were distinguished: type IIa and type IIb. In mammals, expression of the type IIa isoform is largely restricted to the proximal tubules (22). Expression of the type IIb isoform has been demonstrated in the small intestine and (based on RT-PCR) was suggested to occur in other tissues as well (8). In this study, expression of the type IIb cotransporter in the lung is described. Expression of the type IIa cotransporter was not evident as shown by the lack of immunoreaction with a type IIa-specific antibody. This is in agreement with an earlier study (8) that indicated, by RT-PCR, that the type IIa gene is not expressed in the lung. On the other hand, expression of the type III Na-Pi cotransporter in lung tissue seems to be very likely. Expression of this Na-Pi cotransporter/retrovirus receptor family was shown to be ubiquitous, including in the lung (10, 22). Due to the lack of suitable antibodies, the exact cellular location of the type III Na-Pi cotransporter in different tissues and/or cells is not yet known. The broad expression pattern, however, suggests that the location of the type III protein is not cell specific.
The precise role of the type IIb Na-Pi cotransporter located in the apical membrane of ATII cells is not known. The alveolar surface fluid consists of an aqueous and a lipid phase acting as a surfactant. The latter contains high amounts of phosphatidylcholine, phosphoglycerol, and cholesterol (17). Thus because phosphate is a major constituent of surfactant and, therefore, is also needed in high amounts for surfactant synthesis, one may assume that Pi liberated in the alveolar fluid compartment is efficiently taken up (recycled) by ATII cells via the type IIb Na-Pi cotransporter.
Extracellular concentration of Pi is largely controlled by the rate of renal proximal tubular Pi reabsorption and also by the capacity of the small intestine to reabsorb Pi. It has been demonstrated that in both epithelia the rate of transepithelial Pi transport is determined by the abundance of type II Na-Pi cotransporters residing in the apical membranes. Among other factors, limited intake of phosphate via the diet (low-Pi diet) results in an increased amount of type II Na-Pi cotransporters in brush borders of proximal tubules (11) and enterocytes (5; Hattenhauer, Traebert, Murer, and Biber, unpublished data). Although, in the kidney, upregulation occurs, to a large part, independent of other factors such as parathyroid hormone (11), upregulation of the type IIb cotransporter in the small intestine is mediated via 1,25-dihydroxyvitamin D3 (5). To investigate whether restriction of dietary Pi may also lead to an upregulation of the amount of type IIb cotransporters in the lung, the relative abundance of the type IIb protein and transcript was analyzed after a chronic low-Pi diet. Although, by this condition, upregulation of the renal type IIa cotransporter was clearly detected, no evidence for upregulation of the amount of the type IIb Na-Pi cotransporter in the lung was obtained. This suggests that the type IIb Na-Pi cotransporter in ATII cells is not regulated by the dietary content of phosphate directly or indirectly via 1,25-dihydroxyvitamin D3. It remains to be determined whether the amount of type IIb Na-Pi cotransporters in ATII cells is regulated at all or if this Na-Pi cotransporter is constitutively expressed in these cells.
In summary, we have shown that the type IIb Na-Pi cotransporter is specifically expressed in the apical membranes of ATII cells. Furthermore, evidence was obtained that this transporter is not regulated by changes in the dietary content of Pi. Although the exact role of Na-Pi cotransport through the apical membrane of the ATII cells remains to be determined, a role in surfactant synthesis is suggested.
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ACKNOWLEDGEMENTS |
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The first two authors contributed equally to this work.
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FOOTNOTES |
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This work was supported by Swiss National Fund Grants 31-052853.97 (to J. Biber) and 31-47742-96 (to B. Kaissling).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Biber, Institute of Physiology, Univ. of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland (E-mail: Biber{at}Physiol.unizh.ch).
Received 9 March 1999; accepted in final form 19 July 1999.
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