1 Institute of Physiology and 2 Institute of Anatomy, University of Zurich, CH-8057 Zürich, Switzerland
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ABSTRACT |
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Parathyroid hormone (PTH) leads to the
inhibition of Na-Pi cotransport
activity and to the downregulation of the number of type II
Na-Pi cotransporters in proximal
tubules, as well as in opossum kidney (OK) cells. PTH is known also to
lead to an activation of adenylate cyclase and phospholipase C in
proximal tubular preparations, as well as in OK cells. In the present
study, we investigated the involvement of these two regulatory pathways
in OK cells in the PTH-dependent downregulation of the number of type
II Na-Pi cotransporters. We have
addressed this issue by using pharmacological activators of protein
kinase A (PKA) and protein kinase C (PKC), i.e., 8-bromo-cAMP
(8-BrcAMP) and
-12-O-tetradecanoylphorbol 13-acetate (
-TPA), respectively, as well as by the use of synthetic peptide fragments of PTH that activate adenylate cyclase and/or phospholipase C, i.e., PTH-(1-34) and PTH-(3-34),
respectively. Our results show that PTH signal
transduction via cAMP-dependent, as well as cAMP-independent, pathways
leads to a membrane retrieval and degradation of type II
Na-Pi cotransporters and, thereby, to the inhibition of Na-Pi
cotransport activity. Thereby, the cAMP-independent regulatory pathway
leads only to partial effects (~50%).
opossum kidney cells; protein kinase A; protein kinase C; phorbol ester; parathyroid hormone-(3-34)
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INTRODUCTION |
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PARATHYROID HORMONE (PTH) is an important regulator of renal proximal tubular Pi reabsorption. By interacting with the PTH/PTH-related peptide receptor, PTH leads to the acute inhibition of the sodium-dependent Pi transport across the proximal tubular brush-border membrane (11, 12). Inhibition of the proximal tubular Na-Pi cotransport by PTH is characterized by a decrease in the maximal transport rate (Vmax), as well as by a decreased expression of the type II Na-Pi cotransporter in the brush borders of renal proximal tubules (6). Recent experiments on opossum kidney (OK) cells, a cell line exhibiting many of the characteristics of renal proximal tubule cells (2, 14-16, 19-24), provided insight into mechanisms involved in PTH-dependent regulation of Na-Pi cotransport activity. It was found that PTH leads to the endocytosis and lysosomal degradation of the type II Na-Pi cotransporter and, thereby, to the inhibition of the apical Na-Pi cotransport activity (8, 15, 16). Recent experiments provided evidence that PTH also leads to the endocytosis and lysosomal degradation of the type II Na-Pi cotransporter in vivo in rat renal proximal tubules (7).
Intracellular signaling pathways leading to the inhibition of the
proximal tubular Na-Pi cotransport
and to the downregulation of the number of type II
Na-Pi cotransporters by PTH are
incompletely understood. Experiments on renal proximal tubular
preparations and OK cells provided firm evidence for PTH-mediated
stimulation of adenylate cyclase (1, 13) and phospholipase C/protein kinase C (PKC) (1, 5, 13). Moreover, it was found that, in OK cells,
the inhibitory effect of PTH on apical
Na-Pi cotransport activity can be
mimicked by 8-bromo-cAMP (8-BrcAMP) and phorbol esters, pharmacological
activators of protein kinase A (PKA) and PKC, respectively (3, 4, 8, 9,
17). It has also been shown that the PTH fragment PTH-(3-34) is
able to inhibit OK cell Na-Pi
cotransport activity through cAMP-independent pathways; however,
maximal inhibition induced by PTH-(3-34) is significantly less
(50%) than that obtained by exposure to PTH-(1-34) (3, 4, 8).
From these data, it has been inferred that inhibition of the apical
Na-Pi cotransport by PTH is likely
to involve activation of adenylate cyclase/PKA and/or phosholipase
C/PKC. In the present study, we examined the involvement of these two
regulatory pathways in the PTH-dependent downregulation of the number
of type II Na-Pi cotransporters in
OK cells.
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MATERIALS AND METHODS |
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Cells. All cell culture supplies were obtained from Life Technologies (Basel, Switzerland). OK cells (clone 3B/2) were maintained in DMEM/Ham's F-12 medium (1:1), supplemented with 10% FCS, 22 mM NaHCO3, 20 mM HEPES, and 2 mM L-glutamine in a humidified atmosphere of 95% air-5% CO2 at 37°C.
SDS-PAGE and immunoblotting. SDS-PAGE and immunoblotting were performed as previously described (16). For preparation of total cellular homogenates, cells were grown to confluency on 6-cm petri dishes (Corning), washed twice with PBS, and scraped into 2 ml of PBS. The scraped cells were centrifuged for 10 min at 3,000 rpm in an Eppendorf centrifuge at 4°C. The supernatant was sucked off and 250 µl of 50 mM mannitol, 10 mM HEPES-Tris, pH 7.2, was added to each sample. The samples were homogenized 10 times with a 1-ml syringe connected to a 25-gauge needle. Twenty-five micrograms of total protein of cell homogenates were used for SDS-PAGE (9%) and subsequent transfer to nitrocellulose (0.45 µm; Schleicher and Schuell). We blocked nonspecific binding 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). We detected the type II Na-Pi cotransporter (NaPi-4) using a polyclonal antiserum raised against the COOH-terminal 12 amino acids of the published NaPi-4 sequence (antiserum dilution 1:4,000) (15, 16, 24). 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. Thereafter, the nitrocellulose was incubated with a 1:10,000 dilution of an anti-rabbit IgG labeled with horseradish peroxidase (Amersham) 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 chemiluminescence (Amersham), according to the manufacturer's protocol, with the use of Kodak X-OMAT AR films.
Immunofluorescence. Immunofluorescence was performed as previously described (8, 15, 16). We grew 3B/2 OK cells to confluency on coverslips. After cells were washed 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 three 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-NaPi-4 antiserum (8, 15, 16) 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 anti-rabbit IgG (dilution 1:50; Dakopatts, Denmark) and phalloidin rhodamine (dilution 1:50; Calbiochem) in PBS-saponin. After incubation for 30 min in the dark, cells were washed three times with PBS-saponin and once with PBS. We mounted coverslips using Dako-Glycergel (Dakopatts, Denmark) plus 2.5% 1,4-diazabicyclo[2.2.2]octane (Sigma) as a fading retardant. Immunofluorescence was revealed by confocal microscopy (Zeiss laser-scanning microscope 310; Zeiss, Oberkochen, Germany).
Scanning electron microscopy. OK cell monolayers grown on coverslips 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 for 30 min at room temperature, and washed three times with 0.16 M cacodylate buffer (pH 7.2) for 5-10 min. Thereafter, 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 (Philips, Eindhoven).
Phosphate uptake measurements. Na+-dependent transport of phosphate was measured as previously described (15).
Agonist treatment of OK cells.
Incubation of OK cells with PTH, 8-BrcAMP, and
-12-O-tetradecanoylphorbol
13-acetate (
-TPA) was performed as previously described (9, 20).
Briefly, 10
8 M PTH
(dissolved in 10 mM acetic acid), 200 nM TPA (dissolved in DMSO),
10
4 M 8-BrcAMP (dissolved
in water), or the corresponding amount of vehicle (controls) was
directly added to the culture medium, and cells were incubated with
these agonists for the appropriate time.
cAMP determination. Cell monolayers were exposed to hormone for 5 min in the presence of 1 mM 3-isobutyl-1-methylxanthine (IBMX). Cells were washed and disrupted as previously described (9). We determined total intracellular cAMP concentration by using the test kit from NEN Life Science Products (NEK033).
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RESULTS AND DISCUSSION |
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Pharmacological activation of PKA or PKC mimics the inhibition of the
apical Na-Pi cotransport activity
by PTH in OK cells (3, 4, 8, 9, 17, 18). The PTH analog PTH-(3-34) is able to partially mimic the action of PTH-(1-34) (3, 4). However, PTH-(3-34)-induced inhibition of
Na-Pi cotransport occurs in the
complete absence of cellular accumulation of cAMP and is only ~50%
(at a concentration of 106
M) compared with transport inhibition obtained at maximal
concentrations of PTH (10
8
M) (3, 4). In addition to the aforementioned effects on Na-Pi cotransport, it was observed
that PTH-(3-34) leads to a dose-dependent stimulation of PKC
activity in OK cells in the absence of any stimulation of adenylate
cyclase activity (1). PTH-(1-34)-induced inhibition of
Na-Pi cotransport in OK cells was
dependent on "intact" PKA (10, 23) and PKC (18) regulatory pathways. More recently, it was shown that PTH-(1-34)-induced inhibition of OK cell apical Na-Pi
cotransport is related to membrane retrieval of the specific type II
Na-Pi cotransporter protein (15),
followed by its lysosomal degradation (8, 16).
In the present study, we examined whether pharmacological activation of
PKA or PKC mimics the downregulation of the number of type II
Na-Pi cotransporters by PTH.
Confluent OK cell monolayers were treated for 4 h with either
108 M PTH-(1-34) or
10
6 M PTH-(3-34), 4 h
with 10
4 M 8-BrcAMP, 2 h
with 200 nM
-TPA, or with the corresponding vehicle for the
appropriate time. After these treatments, we compared Na-Pi cotransport activity with
the expression of type II Na-Pi cotransporter protein.
In Fig. 1, it can be seen that treating
cells with 8-BrcAMP, as well as with -TPA, mimics not only the
PTH-dependent inhibition of Na-Pi
cotransport activity but also the PTH-dependent reduction in the amount
of the type II Na-Pi cotransporter
protein. Parallel immunofluorescence experiments (Fig.
2) revealed that, under control conditions,
the type II Na-Pi cotransporter is
localized at the apical membrane, within distinct clusters, and that
the immunofluorescence staining for the transporter coincides with the
F-actin staining at the apical surface (15). Treating cells with
PTH-(1-34) (10
8 M, 4 h) led to an almost complete reduction in the expression of the
Na-Pi cotransporter at the apical
membrane of OK cells (Fig. 2). These PTH effects were mimicked in cells
treated with 8-BrcAMP (10
4
M, 4 h) or with
-TPA (200 nM, 4 h; Fig. 2).
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A comparison of the transport activity with the transporter protein
content (Fig. 1) shows that PTH-(1-34) and 8-BrcAMP led to an
almost complete disappearance of the transporter protein, with
remaining significant transport activity. Therefore, the residual
transport activity after treatment with either PTH-(1-34) or
8-BrcAMP is related to a Na-Pi
cotransporter different from the type II-transporter protein, which is
not under the control of this hormone. Consequently, -TPA led only
to a partial transport inhibition and membrane retrieval of the type II
Na-Pi cotransporter under the
conditions used, e.g., 200 nM
-TPA for 2 h (Fig. 1).
The F-actin staining in cells treated with -TPA was markedly
different from the corresponding F-actin stainings seen in cells treated either with PTH-(1-34) or 8-BrcAMP, respectively (Fig. 2).
These observations suggested that treating cells with
-TPA had
profound effects on cytoskeletal structures, including actin filaments,
thereby also affecting the expression of the type II Na-Pi cotransporter. This was
directly tested by confocal microscopy. Each set of pictures in Fig.
3 showing staining for F-actin (control and
-TPA) consists of an apical section, representing a focal plane at
the apical membrane, and a basal section, representing a focal plane at
the basal membrane. Under control conditions, the apical section
reveals distinct clusters related to clustered microvilli (15). The
corresponding basal section reveals the stress fibers. In cells treated
with 200 nM
-TPA, virtually no specific staining for F-actin can be
detected at the apical membrane. On the basolateral side, an amorphous
F-actin staining is observed. Stress fibers are no more detectable
after treatment with
-TPA. As
-TPA had no effect (data not
shown), these effects of
-TPA were likely due to pharmacological
activation of PKC. Therefore, the observed
-TPA effects on
transporter activity and expression (Figs. 1 and 2) could be secondary
to the cytoskeletal alterations, e.g., to a reduced expression of
microvilli. Indeed, a
-TPA-dependent reduction in the number of
microvilli could be documented by the use of scanning electron
microscopy (Fig. 4). Moreover, Fig. 4 reveals that
-TPA-treated OK cells have a spherical shape.
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To test for the role of a cAMP-independent pathway in the PTH control
of the type II Na-Pi
cotransporter, without using phorbol esters (-TPA), we used the PTH
analog PTH-(3-34). We treated OK cells for 4 h with
10
6 M PTH-(3-34). In
accordance with previous reports (3, 4, 8, 9), our results show that
treating OK cells with PTH-(3-34) (10
6 M, 4 h) led to a
half-maximal inhibition of Na-Pi
cotransport activity (Fig.
5A), in
the absence of an increase in the total cellular cAMP level (Fig.
5B). In parallel
experiments, PTH-(1-34) (10
8 M, 4 h) lead to a
maximal inhibition of Na-Pi
cotransport activity (Fig. 5A), in
the presence of a marked increase in the cellular cAMP level (Fig.
5B). Treating OK cells with
PTH-(3-34) (10
6 M, 4 h) led to a downregulation of the number of type II
Na-Pi cotransporters by ~50%
(Fig. 6), whereas treating OK cells with PTH-(1-34) (10
8 M, 4 h) led to the almost complete downregulation of the number of
transporters. Also, immunofluorescence pictures, shown in Fig. 7, demonstrate that treating OK cells with
PTH-(3-34) (10
6 M, 4 h) leads to a downregulation of the number of type II
Na-Pi cotransporters. Results
presented in Figs. 5-7 provide evidence for a cAMP-independent
signal transduction pathway that can account for the inhibition of
Na-Pi cotransport activity, as
well as for the downregulation of the number of type II
Na-Pi cotransporters.
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We conclude that PTH signal transduction via cAMP-dependent, as well as cAMP-independent, pathways leads to a membrane retrieval and degradation of the number of type II Na-Pi cotransporters and, thereby, to the inhibition of Na-Pi cotransport activity. The cAMP-independent pathway itself leads only to a partial inhibition and related membrane retrieval of the Na-Pi cotransporter. This might be explained by the previously suggested interdependence of PKA and PKC regulatory pathways in PTH-induced control of OK cell apical Na-Pi cotransporter (10, 18, 23).
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ACKNOWLEDGEMENTS |
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We thank C. Gasser for assistance in preparing the figures for this paper.
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FOOTNOTES |
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This work was financially supported by Swiss National Science Foundation Grant 31.46523 (to H. Murer). Antibodies against OK cell Na-Pi cotransporter protein were a generous gift from E. Lederer.
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. Zürich-Irchel, Winterthurerstr. 190, CH-8057 Zürich, Switzerland (E-mail: biber{at}physiol.unizh.ch).
Received 9 September 1998; accepted in final form 12 February 1999.
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