INTRODUCTION

Renal proximal tubular P_i reabsorption is acutely regulated by parathyroid hormone (PTH).¹ This effect involves inhibition of the brush border membrane sodium-dependent P_i transport and is characterized by a decrease in the maximal transport rate (V_max) (1, 2). Two different renal Na⁺/P_i cotransporters have been cloned, classified either as type I Na⁺/P_i cotransporter or as type II Na⁺/P_i cotransporter (3-14). Both are localized at the brush border membrane in proximal tubules. Recent data documented that physiologically and pathophysiologically altered brush border membrane Na⁺/P_i cotransport involves altered brush border expression of the type II Na⁺/P_i cotransporter (15-17).

In the present study we investigated the PTH-mediated regulation of the type II Na⁺/P_i cotransporter in opossum cells (OK cells); these cells have recently been shown to contain such a cotransporter (NaP_i-4; Ref. 8). The validity of the opossum kidney cell model to study proximal tubular Na⁺/P_i cotransport and its regulation has been established (18-23). With respect to PTH-dependent control of Na⁺/P_i cotransport activity, we have reported that the recovery from the PTH-mediated inhibition of Na⁺/P_i cotransport in OK cells is dependent on de novo protein synthesis. This latter observation led to the hypothesis that PTH might lead to the retrieval and degradation of the transporter (27).

The aims of the present study were 2-fold: (i) to study cellular/molecular mechanisms involved in PTH-dependent control of type II Na⁺/P_i cotransporters, (ii) to create by transfection an in vitro model that also permits the study of the PTH control of the rat type II Na⁺/P_i cotransporter. Obviously, the latter approach would then offer a tool to characterize the molecular determinants involved in such regulations. A prerequisite for this approach was the availability of antisera, permitting a distinction between intrinsic (NaP_i-4) and transfected (NaP_i-2) cotransporters.

The results obtained show that both intrinsic (NaP_i-4) and transfected type II Na⁺/P_i cotransporters (NaP_i-2, rat) are functionally (transport) and morphologically (immunofluorescence) located at the apical cell surface, are functionally inhibited in response to PTH addition, are retrieved in a PTH-dependent manner from the apical cell surface (immunofluorescence), and are subsequently degraded (Western blots). These data document that PTH control of the type II Na⁺/P_i cotransporters involves a step of membrane retrieval and degradation. Furthermore, the OK cell system should represent the ideal in vitro model to dissect the cellular/molecular mechanisms participating in regulation of this proximal tubular transport function, which is crucially involved in overall P_i homeostasis.

EXPERIMENTAL PROCEDURES

The generation of the vectors used (pLKneo and NaP_i-2/pLKneo) has been described previously (24, 25). Both vectors code for a geneticin (G418) resistance under a SV40 promoter. In addition, the vector NaP_i-2/pLKneo contains a cDNA coding for the rat renal type II Na⁺/P_i cotransporter (NaP_i-2) under a dexamethasone-inducible promoter.

All cell culture supplies were obtained from Life Technologies, Inc. (Basel, Switzerland). Opossum kidney cells (clone 3B/2) were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1) supplemented with 10% fetal calf serum, 22 mM NaHCO₃, 20 mM Hepes, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO₂, 95% air at 37 °C.

Monolayers on permeable filter supports were grown on Millicell-CM filter inserts (Millipore; 12-mm diameter, 0.45-µm pore size) coated with a very thin film of rat tail collagen (R type, 0.5 mg/ml in 50% ethanol; Serva, Basel, Switzerland). Cells were seeded at approximately 1-2 × 10⁵ cells/filter and were re-fed with fresh medium every 12 h until reaching confluency 24 h after seeding. Transport studies were commenced 36 h after seeding. The tightness of confluent cell monolayers has been measured by resistance measurements as described previously (22).

For transfection, cells grown to a confluency of approximately 60% in 35 mm dishes (Nunc) were incubated for 16 h with 20 µl of a 1:1 mixture of water and Lipofectin (Life Technologies) containing 10 µg of either NaP_i-2/pLKneo or the same amount of empty vector pLKneo. Afterward, cells were trypsinized, split at a ratio of 1:30, and grown in 150-mm dishes (Nunc) in medium containing, in addition, 400 µg/ml active geneticin. After 1-2 weeks, colonies of geneticin-resistant cells were isolated by ring cloning, expanded, and analyzed for the expression of the NaP_i-2 protein by immunoblotting (see below). For experimental purposes, transfected cells were used within 10 passages. Dexamethasone induction was performed by an incubation with 1 µM dexamethasone (Sigma) for 20 h (10,000-fold stock, made in ethanol).

Cells grown to confluency in 10-cm Petri dishes were incubated either with or without dexamethasone (1 µM) for 20 h and washed twice with TBS (0.9% NaCl, 10 mM Tris-HCl, pH 7.4). 15 ml of TBS containing 4 mM EDTA and 1 mM phenylmethylsulfonyl fluoride was added, and the cells were scraped off the dish. The scraped cells were homogenized 5 times with a 20-ml syringe connected to a 20-gauge needle. This homogenate was centrifuged at 2000 rpm for 10 min at 4 °C (Sorvall centrifuge, SS-34 rotor). The postnuclear supernatant was centrifuged at 16,000 rpm for 40 min at 4 °C (Sorvall centrifuge, SS-34 rotor). The pellet corresponding to a crude membrane preparation was resuspended in 100 µl of 50 mM mannitol, 10 mM Hepes-Tris (pH 7.2).

In experiments in which the PTH-mediated degradation of the Na⁺/P_i cotransporter was investigated, the total cell homogenate was centrifuged at 31,000 rpm (100,000 g) for 60 min at 4 °C (Sorvall ultracentrifuge OTD 50B/T865 rotor) to ensure that all membranes were contained within the pellet. The pellet was resuspended in 200 µl of 50 mM mannitol, 10 mM Hepes-Tris (pH 7.2). The protein concentration was determined by the Bio-Rad protein assay. 50 µg of total protein were used for SDS-polyacrylamide gel electrophoresis (9%) and subsequent transfer to nitrocellulose (Schleicher & Schuell, Inc.; 0.45 µm). Nonspecific binding was blocked by incubating the nitrocellulose at room temperature for 2 h in TBS (0.9% NaCl, 10 mM Tris-HCl, pH 7.4) containing 5% nonfat dry milk and 1% Triton X-100 (Blotto-TX-100, pH 7.4). The transfected NaP_i-2 protein was detected using a polyclonal antiserum raised against the N terminus of the NaP_i-2 protein (14) (antibody dilution, 1/4000). The intrinsic NaP_i-4 protein was detected using a polyclonal antiserum raised against the C-terminal 12 amino acids of the published NaP_i-4 sequence (8) (antibody dilution, 1/2000). Incubation with the primary antibody took place overnight at 4 °C. The nitrocellulose was washed four times with TBS, 10% Blotto-TX-100 (pH 7.4) and incubated for 1 h with Blotto-TX-100 (pH 7.4) at room temperature. Then the nitrocellulose was incubated with a 1:10,000 dilution of an anti-rabbit IgG labeled with horseradish peroxidase (Amersham Life Science, Inc.) in Blotto-TX-100 (pH 7.4) for 2 h at room temperature. The nitrocellulose was washed four times with TBS, and the signals were detected by enhanced chemiluminescense (Amersham) according to manufacturer protocol using Kodak X-Omat AR films. For peptide protection assays, the corresponding antigenic peptide was included at a concentration of 100 µg/ml. Broad range SDS-polyacrylamide gel electrophoresis molecular mass marker proteins (Bio-Rad) were run in parallel.

NaP_i-2-transfected and untransfected 3B/2 OK cells were grown to confluency on coverslips as well as on permeable filter supports, as described previously (22). After washing three times with PBS containing 0.5 mM MgCl₂ and 1 mM CaCl₂, cells were fixed for 10 min at room temperature with PBS supplemented with 3% paraformaldehyde, washed three times with PBS, incubated 10 min with 20 mM L-glycine in PBS, and washed again (3 times) with PBS. Permeabilization was performed by an incubation for 30 min with PBS containing 0.1% saponin (PBS/saponin). After one wash with PBS/saponin, cells were incubated with anti-NaP_i-2 (14) or anti-NaP_i-4 antiserum at a dilution of 1:100 in PBS/saponin for 1 h at room temperature and washed three times with PBS/saponin. Thereafter, the cells were incubated with a fluorescein isothiocyanate-conjugated IgG (Dakopatts, Denmark) (dilution, 1:50) and phalloidine rhodamine (Calbiochem) (dilution, 1:50) in PBS/saponin. After incubation for 30 min in the dark, cells were washed three times with PBS/saponin and once with PBS. Coverslips were mounted using Dako-Glycergel (Dakopatts) plus 2.5% 1,4-diazabicyclo-[2.2.2]octane (Sigma) as a fading retardant. Immunofluorescence was revealed by confocal microscopy (Zeiss laser scan microscope 310; Zeiss, Oberkochen, Germany).

OK cell monolayers grown on collagen-coated porous filter supports were prefixed with 0.25% glutaraldehyde in 0.16 M cacodylate buffer (pH 7.2) for 30 min at room temperature, postfixed with 2% glutaraldehyde in 0.16 M cacodylate buffer also for 30 min at room temperature, and washed three times with 0.16 M cacodylate buffer (pH 7.2) for 5-10 min. After that, the monolayers were osmicated with 1% OsO₄ in 0.16 M cacodylate buffer (pH 7.2) for 1 h at 37 °C, washed three times with 0.16 M cacodylate buffer, dehydrated in an acetone series, and dried by the critical point method. The specimens were then examined in a scanning electron microscope 505 (Philips, Eindhoven).

Na⁺-dependent and -independent transport of phosphate was determined in cells grown to confluency on either plastic dishes (35 mm; Nunc) or on permeant filter supports (8 mm), as described previously (22). Briefly, uptake solutions consisted of 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl₂, 1.2 mM MgSO₄, 10 mM Hepes-Tris (pH 7.4), and 0.1 mM KH₂³²PO₄ (1 µCi/ml). For Na⁺-independant uptake, NaCl was replaced equimolarly by N-methyl-D-glucamine·HCl. Routine uptake on plastic dishes was performed at room temperature for 6 min and then stopped by washing the cells four times with ice-cold stop solution (137 mM NaCl, 10 mM Tris-HCl, pH 7.2). Cells were solubilized with 1% Triton X-100, and radioactivity was determined by liquid scintillation. Transport rates are expressed as nmol of P_i taken up/mg of total cellular protein, which was determined by the Bio-Rad protein assay.

For transport assays on permeant filter supports, growth medium was aspirated, and both sides of the monolayer were gently rinsed twice in substrate-free uptake solution. Filter insert monolayers were then placed in a 24-well culture plate (Nunclon) for uptake measurements. Substrate-free uptake solution (500 µl) was added to the appropriate filter insert compartment, and N-methyl-D-glucamine·HCl-uptake solution was added to the opposite compartment. Transport was initiated by mixing 50 µl of the same uptake solution containing an 11-fold concentration of the desired radioactive substrate to the uptake solution already present in the filter insert compartment. Uptake was stopped as described above. Nonspecific binding (blanks) was assessed measuring zero time uptake in N-methyl-D-glucamine·HCl-uptake solution by starting uptake and immediately aspirating the uptake solution. Nonspecific binding was <10% that of radioactivity associated with any experimental point. Total radioactivity incorporated into the monolayer was measured by liquid scintillation counting of the whole filter insert.

Incubation of OK cells with PTH has been described previously (22). Treatment of OK cells with cycloheximide to prevent protein synthesis has also been described previously (27).

Statistical results are expressed as mean ± S.E. for three dishes. Significance was accepted at p < 0.05. Experiments were repeated at least twice, and one representative experiment was choosen for presentation. The results presented concerning the transfected NaP_i-2 were obtained with one single clone of NaP_i-2-transfected 3B/2 OK cells. Qualitatively, the same results were obtained with two other clones of stably NaP_i-2-transfected 3B/2 OK cells.

RESULTS

To study transport function, transfected OK cells were grown to confluency on plastic Petri dishes and exposed to 1 µM dexamethasone for 20 h to induce the expression of the transfected NaP_i-2. As illustrated in Fig. 1, induction of NaP_i-2 expression in transfected OK cells by dexamethasone led to an approximately 2-fold stimulation of the Na⁺-dependent P_i transport, whereas dexamethasone had no significant effect on the Na⁺/P_i cotransport activity in empty vector-transfected and in untransfected OK cells. Furthermore, dexamethasone had no effect in any of the tested cell lines on the Na⁺-independent P_i transport (Fig. 1 and data not shown). Corresponding experiments have been carried out with NaP_i-2-transfected OK cells grown to confluency on collagen-coated porous filter supports. By measuring the P_i transport at the apical and basolateral membrane separately in induced and noninduced NaP_i-2-transfected OK cells, it was found that the additional P_i uptake, as observed in cells grown on Petri dishes (Fig. 1), is entirely restricted to the apical membrane (data not shown).

Transfected OK cells were also analyzed for expression of the NaP_i-2 protein. Induction of NaP_i-2-transfected OK cells by dexamethasone (1 µM, 20 h) led to the expression of a protein with an apparent molecular mass of 95-120 kDa (Fig. 2). An excess of the corresponding antigenic peptide (100 µg/ml) prevented the appearance of this band completely. The band seen above the NaP_i-2 protein is unspecific: it is neither induced by dexamethasone nor protected by the antigenic peptide (Fig. 2A). The intrinsic type II transporter protein (NaP_i-4) was detected by the use of an antiserum directed against the C terminus of NaP_i-4. Fig. 2B shows the NaP_i-4-specific signal detected with membranes obtained from untransfected OK cells. Specificity of the anti-NaP_i-4 antiserum was established by peptide protection assay with the corresponding antigenic peptide (100 µg/ml; Fig. 2B). Comparing Fig. 2A with Fig. 2B shows that the intrinsic NaP_i-4 and the transfected NaP_i-2 have approximately the same molecular mass (95-120 kDa). Furthermore it is seen that the anti-NaP_i-2 antiserum does not cross-react with the intrinsic NaP_i-4 (Fig. 2A, transfected cells in the absence of dexamethasone).

Expression of the type II transporter was also analyzed by immunofluorescence. Immunofluorescence pictures obtained by confocal microscopy showed that both the intrinsic NaP_i-4 (Fig. 3A) and the transfected NaP_i-2 (Fig. 3B) are localized at the apical membrane within distinct clusters of a diameter of about 1-2 µm, whereas no type II Na⁺/P_i cotransporter-specific staining was seen at the basolateral membrane. The immunohistochemical staining of the transfected NaP_i-2 as well as of the intrinsic NaP_i-4 could be specifically abolished by the corresponding antigenic peptide (100 µg/ml; data not shown). In Figs. 3A and 3B, the parallel staining for -actin (a component of the microvillar cytoskeleton) is shown. It is apparent that NaP_i-4-specific (Fig. 3A) and NaP_i-2-specific (Fig. 3B) staining on the apical cell surface coincides with the -actin staining. In addition to the above apical staining, -actin is also present at the basolateral cell surfaces (Fig. 3A and 3B). Visualization of the apical surface of OK cells by scanning electron microscopy showed that microvilli are expressed at the apical surface, forming distinct clusters (Fig. 3C); the diameter of the clusters corresponds well with the diameter of the clusters seen by immunofluorescence double staining for -actin and the corresponding type II Na⁺/P_i cotransporter. Therefore the data given in Fig. 3 (A-C) documents that intrinsic and transfected type II Na⁺/P_i cotransporters are predominantly expressed at the apical cell surface (most likely within microvilli).

Fig. 4 summarizes the effect of PTH (10⁸ M; 4 h) on the Na⁺-dependent P_i transport in NaP_i-2-transfected OK cells as well as in untransfected OK cells. PTH inhibited the Na⁺-dependent P_i transport activity in control cells by about 60%. A similar inhibitory effect of PTH was observed in NaP_i-2-transfected cells that were not treated with dexamethasone. In cells expressing the NaP_i-2 transporter (induced by dexamethasone), PTH also inhibited the additionally expressed Na⁺/P_i cotransport activity. Interestingly, the residual transport activity after PTH treatment was similar in all cells tested. This latter observation is in agreement with earlier studies (21, 27) demonstrating a PTH-insensitive Na⁺/P_i cotransport activity in OK cells.

Parathyroid hormone (10⁸ M h) leads to the inhibition of the intrinsic and the transfection-mediated Na⁺/P_i cotransport activity in transfected OK cells.

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The effect of PTH on the type II Na⁺/P_i cotransporter protein content was investigated by immunoblotting. Fig. 5 shows that incubating OK cells for increasing times with PTH (10⁸ M) leads to a time-dependent decrease of NaP_i-4 and NaP_i-2 protein expressed in OK cells. We conclude that the whole protein was degraded due to PTH action and that both transfected (NaP_i-2) and intrinsic transporter (NaP_i-4) behave very similar, i.e. are degraded. The finding that PTH leads to the degradation of the type II Na⁺/P_i cotransporter is in agreement with our previous observation that the recovery of the Na⁺/P_i cotransport after PTH-mediated inhibition is dependent on de novo protein synthesis (27, 28). To evaluate whether the recovery of the transport was due to de novo synthesis of type II Na⁺/P_i cotransporter, cell monolayers were exposed to 10⁸ M PTH for 4 h, PTH was removed, and cell monolayers were incubated with normal medium in the presence or absence of 20 µM cycloheximide for 2 or 4 h. The results presented in Fig. 6 show that the reappearance of the transporter is dependent on de novo protein synthesis. It is also seen that 4 h after PTH removal, the reappearance of the transporter is not completed. Parallel measurements of the Na⁺/P_i cotransport showed that after 4 h of PTH removal, the recovery of the Na⁺/P_i cotransport is also incomplete. This is in agreement with our previous observation that complete recovery of Na⁺/P_i cotransport from PTH inhibition takes about 10-12 h (28).

Parathyroid hormone (10⁸ M) leads to the disappearance of the NaP_i-4-specific (A) and NaP_i-2-specific (B) staining on immunoblots.

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The retrieval of the intrinsic (NaP_i-4) and the transfected cotransporter (NaP_i-2) upon addition of PTH could also be documented by immunofluorescence. Consistent with the data presented above (Figs. 4, 5, 6), we found that incubating OK cells with PTH (10⁸ M) leads to a time-dependent decrease of NaP_i-4-dependent immunofluorescence and NaP_i-2-dependent immunofluorescence at the apical membrane of OK cells (data not shown). After 4 h of PTH treatment (10⁸ M), the Na⁺/P_i cotransporter type II-specific immunofluorescence staining in untransfected (Fig. 7A) and NaP_i-2-transfected OK cells (Fig. 7B) was virtually absent. Corresponding immunofluorescence pictures (double immunofluorescence) stained for -actin clearly showed that under these conditions the microvilli were still present at the apical surface of OK cells. With the aim to detect the retrieved Na⁺/P_i cotransporters within intracellular vesicles, we have treated OK cells for increasing times with PTH (0, 0.5, 1, 2, and 4 h; data not shown). As mentioned above, we observed a time-dependent decrease of the NaP_i-4-specific staining and NaP_i-2-specific staining at the apical surface. However, we were not able to detect a significant amount of NaP_i-4 protein or NaP_i-2 protein within intracellular vesicles at any time points (data not shown). As already suggested by the experiments presented in Fig. 5, a delay between a PTH-dependent membrane retrieval and intracellular degradation seems to be absent or minimal.

Parathyroid hormone (10⁸ M, 4 h) leads to the disappearance of the NaP_i-2- and NaP_i-4-specific immunohistochemical staining within the microvilli at the apical membrane.

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DISCUSSION

Previous studies on rats have suggested that inhibition of Na⁺/P_i cotransport in renal proximal tubules by PTH involves a retrieval of the type II Na⁺/P_i cotransporter (NaP_i-2) from the brush border membrane (15). The recent cloning of the type II Na⁺/P_i cotransporter of OK cells (NaP_i-4; Ref. 8) and the obvious limitations of an in vivo system to study molecular mechanisms prompted us to investigate the regulation of this transporter by PTH in OK cells. OK cells are a renal epithelial cell line that has been used successfully to investigate mechanisms involved in the control of proximal tubular Na⁺/P_i cotransport by PTH (21, 27). In the present study, we stably transfected OK cells with a cDNA coding for the rat type II Na⁺/P_i cotransporter (NaP_i-2) and investigated whether similar mechanisms are involved in the PTH-mediated regulation of the intrinsic (NaP_i-4) and the transfected transporters (NaP_i-2).

On immunoblots, appearance of the transfected (NaP_i-2) and the intrinsic (NaP_i-4) cotransporter proteins resembled each other closely. The broad staining pattern (95-120 kDa) of these transporters likely represents different degrees of glycosylation, which for the NaP_i-2 protein, has recently been demonstrated (29). The expression of the rat type II Na⁺/P_i cotransporter was also demonstrated by transport experiments. Dexamethasone-induced NaP_i-2-transfected OK cells exhibited an approximately 2-fold-stimulated transport activity compared with uninduced NaP_i-2-transfected or control cells (untransfected and empty vector-transfected). Immunofluorescence experiments demonstrated an exclusive apical localization for both Na⁺/P_i cotransporters (intrinsic and transfected), that, in the case of the intrinsic NaP_i-4, is in agreement with earlier transport studies performed with OK cells grown on permeant filter supports (22). Furthermore, immunofluorescence demonstrated a concentration of the intrinsic as well as of the transfected Na⁺/P_i cotransporters within clustered microvillar structures. It is suggested that such a distinct concentration of the Na⁺/P_i cotransporters within the microvilli may be due to a yet unknown interaction of these transporters with components of the microvillar cytoskeleton. Clearly no immunostaining related to these transporters was detected in the basolateral membrane, suggesting a specific apical sorting mechanism for the type II Na⁺/P_i cotransporters in OK cells. In contrast, we recently demonstrated that the NaP_i-2, when transfected into Madin-Darby canine kidney cells, is expressed to equal amounts at the apical and basolateral membrane (25). The observed different sorting behavior of the same transporter in two different cell lines indicates that not only molecular determinants are decisive for a polarized sorting but that also the cellular context is of importance. This has also been documented for other proteins and cell systems. For example, it has been shown that aquaporin-2, when transfected into LLC-PK₁ cells, is, upon cAMP stimulation, inserted into the basolateral membrane rather than into the apical membrane as suggested from studies on native renal epithelia (30).

In previous studies it has been demonstrated that the intrinsic apical Na⁺/P_i cotransport activity in OK cells is inhibited by PTH (21, 27). In the present study we extended this observation by showing that the inhibition of the Na⁺/P_i cotransport by PTH in OK cells is paralleled by the disappearance of the apical, Na⁺/P_i cotransporter-specific immunofluorescence staining within the microvilli. It is also shown that PTH leads to the degradation of the type II Na⁺/P_i cotransporter in OK cells. This finding is in agreement with our earlier observation demonstrating that the recovery of Na⁺/P_i cotransport activity from PTH inhibition in OK cells is dependent on de novo protein synthesis. Correspondingly, we found that the recovery of Na⁺/P_i cotransport activity from PTH inhibition was paralleled on immunoblots by the reappearance of the type II Na⁺/P_i cotransporter protein. In the presence of cycloheximide, there was neither a recovery of transport activity nor a reappearance of the specific transporter protein. As documented by immunoblots and by immunohistochemical studies presented in this paper, PTH leads to an almost complete retrieval and degradation of the type II Na⁺/P_i cotransporter (Figs. 5 and 7). However, even after prolonged exposures to PTH, a "refractory" residual Na⁺/P_i cotransport activity is observed (Fig. 4 and Ref. 21). This implies that this residual activity (~40% of total transport activity) is not related to the type II Na⁺/P_i cotransporter but could be associated with, for example, the type I transporter. Such conclusions are also valid for the Na⁺/P_i cotransport activity in rat brush border membranes; 2 h of PTH treatment leads to a much higher reduction of the NaP_i-2 protein compared with the reduction in Na⁺/P_i cotransport activity (15).

The present study clearly indicates that the regulation of the type II Na⁺/P_i cotransporter in renal proximal tubules and in OK cells is very similar. One apparent difference exists: after PTH treatment of OK cells for various lengths (0.5, 1, 2 , and 4 h) we were not able to detect a significant transient intracellular staining specific for the type II Na⁺/P_i cotransporter. In rat proximal tubules, the degradation of the NaP_i-2 protein seems to be delayed (compared with the OK cell system), permitting a visualization of an increased intracellular NaP_i-2 protein content after short (15 min to 1 h) but not after prolonged treatments with PTH (15). Despite this difference, the similarities in the regulation of this transporter by PTH in OK cells and in rat renal proximal tubules are evident. Furthermore, in this report we demonstrated that the transfected (NaP_i-2) and intrinsic type II Na⁺/P_i cotransporters (NaP_i-4) are regulated in OK cells in the same way by PTH. OK cells are therefore a physiologically relevant in vitro system for the study of the regulation of the type II Na⁺/P_i cotransporter type II. They are a useful tool to dissect the molecular/cellular mechanisms involved in the PTH-mediated internalization and subsequent degradation of the transporter. Although it is tempting to assume a final breakdown of these Na⁺/P_i cotransporters within lysosomes, the mechanisms involved in internalization, trafficking, and subsequent degradation are completely unknown.

In summary, the present study shows (i) that the intrinsic (NaP_i-4) and the transfected rat type II Na⁺/P_i cotransporter (NaP_i-2) are functionally and morphologically localized at the apical membrane, (ii) that the intrinsic as well as the transfection-mediated (NaP_i-2) Na⁺/P_i cotransport activities are inhibited by PTH, (iii) that PTH leads to the disappearance of both cotransporters from the apical membrane, and (iv) that both cotransporters are rapidly degraded in response to PTH. These results suggest that PTH leads to the endocytosis of the type II Na⁺/P_i cotransporters from the apical membrane and to their subsequent degradation, thereby leading to the down-regulation of the Na⁺/P_i cotransport.