Luminal and contraluminal action of 1-34 and 3-34 PTH peptides on renal type IIa Na-Pi cotransporter

Martin Traebert1,*, Harald Völkl2,*, Jürg Biber1, Heini Murer1, and Brigitte Kaissling1

1 Institutes of Anatomy and Physiology, University of Zurich, CH-8057 Zurich, Switzerland; and 2 Institute of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Parathyroid hormone (PTH) inhibits proximal tubular reabsorption of Pi by retrieval of type IIa Na-Pi cotransporters (NaPi-IIa) from the brush-border membrane (BBM). We analyzed by immunohistochemistry whether PTH analogs, signaling through either protein kinase A (PKA) and C (PKC; 1-34 PTH) or only PKC (3-34 PTH), elicit in rat kidney in vivo or in the perfused murine proximal tubule in vitro a retrieval of NaPi-IIa and whether pharmacological agonists or inhibitors of these kinases are able to either mimic or interfere with these PTH effects. Treatment with either 1-34 or 3-34 PTH downregulated NaPi-IIa in rat kidney. In isolated murine proximal tubules 1-34 PTH was effective when added to either the apical or basolateral perfusate, whereas 3-34 PTH acted only via the luminal perfusate. These effects were mimicked by an activation of PKA with 8-bromoadenosine 3',5'-cyclic monophosphate or PKC with 1,2-dioctanoylglycerol. The luminal action of both PTH peptides was blocked by inhibition of the PKC pathway (calphostin C), whereas the basolateral effect of 1-34 PTH was completely abolished by inhibiting both pathways (H-89 and calphostin C). These results suggest that 1) NaPi-IIa can be internalized by cAMP-dependent and -independent signaling mechanisms; 2) functional PTH receptors are located in both membrane domains; and 3) apical PTH receptors may preferentially initiate the effect through a PKC-dependent mechanism.

isolated proximal tubule; immunohistochemistry; parathyroid hormone receptor; type IIa sodium-phosphate transporter; protein kinase A; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RENAL PHOSPHATE REABSORPTION occurs mostly in the renal proximal tubule and involves a brush-border membrane (BBM) type IIa sodium-phosphate cotransporter (NaPi-IIa) (31). Gene-disruption experiments documented that NaPi-IIa accounts for ~70% of BBM Na-Pi cotransport activity. Thus the type IIa Na-Pi cotransporter determines to a major extent renal Pi reabsorption (3) and its regulation. As an example, the phosphaturic effect of parathyroid hormone (PTH) can be explained by the inhibition of BBM Na-Pi cotransport (30, 31), caused by a reduction in the amount of NaPi-IIa protein in the BBM and its subsequent lysosomal degradation (18, 19).

Studies in intact tubules in vivo or in vitro indicated that stimulation of adenylate cyclase and protein kinase A is involved in the PTH-dependent inhibition of BBM Na-Pi cotransport (1, 2, 6, 7, 9). Experiments in purified cortical membranes demonstrated the presence of a PTH-sensitive adenylate cyclase at the basolateral cell surface (for review, see Ref. 28). Although specific PTH-binding sites were found in studies in isolated BBM, no evidence for a coupling to adenylate cyclase of apical PTH receptors has been reported (17). Furthermore, no functional role of apically located PTH receptors is known.

Opossum kidney (OK) cells have been widely used to study cellular mechanisms involved in the control of "proximal tubular" Na-Pi cotransport (29). In these cells, PTH also provokes inhibition of Na-Pi cotransport activity and internalization of NaPi-IIa cotransporters (26, 27, 33-36). In agreement with data obtained in studies with the cloned PTH receptor (16), PTH was found to activate in OK cells both the protein kinase A and protein kinase C regulatory pathway (10, 14, 26, 27, 33, 36). Consequently, the PTH effect on Na-Pi cotransport in this cell line could be mimicked by pharmacological activation of either protein kinases A and/or C (10, 27, 33, 36). We also provided evidence that activation of both pathways by 1-34 and/or 3-34 PTH leads to an inhibition of Na-Pi cotransport, which is caused by an internalization of type IIa cotransporters; also, these effects were mimicked by pharmacological activators of protein kinases A and/or C (20, 33, 36). Furthermore, studies in OK cells grown on permeant filter supports demonstrated that PTH receptors are expressed in the basolateral and in the apical membrane; evidences for different PTH-signaling mechanisms of the differentially located PTH receptors were obtained (37, 38).

So far, it is not established whether the above tissue culture (OK cells) observations of cAMP-dependent vs. -independent and of apical vs. basolateral actions of PTH are also valid for the intact proximal tubule. This prompted us to investigate the effect of the 1-34 and 3-34 PTH peptides on the distribution of NaPi-IIa in rat kidneys. In addition, we studied in isolated perfused proximal tubules the sidedness (apical vs. basolateral) of the action of the two PTH peptides. Furthermore, in studies in isolated perfused proximal tubules the effects of pharmacological activators and inhibitors of the protein kinase A and C pathways on apical expression of NaPi-IIa were examined.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Experimental animals. In vivo studies were performed by using nine male (3 rats/group) Wistar rats (BRL, Basel, Switzerland) weighing 180-200 g. They were kept on a standard laboratory diet and had free access to food and water. Group 1 remained untreated and served as control animals, group 2 was treated with 1-34 PTH, and group 3 was treated with 3-34 PTH (see Application of PTH and fixation).

The perfusion experiments were performed in proximal straight tubules isolated from a total of 40 Swiss mice (BRL) weighing 25-30 g. Mice were kept on a low-phosphate diet (0.1% Pi content) for 1 wk to upregulate NaPi-IIa in the BBM of all proximal tubular segments. All perfusions and subsequent immunohistochemical analysis were repeated at least four times by using proximal tubules isolated from different animals.

Solution and chemicals. All standard chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO) or Fluka (Buchs, Switzerland). PTH (1-34 and 3-34) were obtained from Bachem (Bubendorf, Switzerland). 3',5'-Cyclic monophosphate (8-BrcAMP) and 1,2-dioctanoylglycerol (DOG) were purchased from Sigma Chemical. N-(2{[-3-(4-bromophenyl)-2-propenyl]-amino}-ethyl-5-isoquinosulfonamide and calphostin C were obtained from Calbiochem (Lucerne, Switzerland). Protein kinase A and C activators and inhibitors were diluted in DMSO, except for 8-BrcAMP (H2O), stored at -20°C, and were added to the bath solution immediately before use. At all dilutions the final concentration of DMSO to which the isolated tubules were exposed did not exceed 0.05%.

Application of PTH and fixation. Rats were anesthetized with thiopental (Pentothal, 100 mg/kg body wt) injected intraperitoneally, their abdominal cavity was opened, and the aorta and vena cava were exposed. Animals in group 2 received 100 µg 1-34 PTH, and animals in group 3, 100 µg of 3-34 PTH (Bachem). PTH peptides were injected into the vena cava as a single bolus (250 µl; 0.9% saline). Animals from the control group were injected with 250 µl of 0.9% saline alone. Twenty minutes after injection, all animals were fixed by vascular perfusion via the abdominal aorta at a pressure of 1.38 hp, as described previously (12). The fixative consisted of 3% paraformaldehyde and 0.05% picric acid in 0.06 M cacodylate buffer (pH 7.4; containing 3 mM MgCl2 and adjusted to 300 mosmol with sucrose) and 4% hydroxyethyl starch (HAES, Fresenius, Switzerland). After 5 min the fixative was washed out by perfusion for 5 min with 0.1 M cacodylate buffer.

Perfusion of isolated proximal tubules. Proximal tubule segments of 1-2 mm in length were dissected and perfused according to Burg et al. (5). Modifications of the technique concerning track system and pipette arrangement have been described in previous publications (15, 41). Tubules were perfused at a rate of >10 nl/min with (in mmol/l) 120 NaCl, 5 KCl, 20 NaHCO3, 1.3 CaCl2, 1 MgCl2, 0.1 Na2HPO4, and 5 mannitol. The bath was perfused continuously at a rate of 20 ml/min with the same solution containing no mannitol but (in mmol/l) 1 glucose, L-glutamine, and 1 Na-lactate. Both solutions were kept at 38°C and constantly gassed with a mixture of 95% O2-5% CO.

The PTH peptides 1-34 and 3-34 were applied by perfusion with the luminal solution at a concentration of 10-6 M or added to the bath solution, resulting in a concentration of 10-6 M. The pharmacological inhibitors were added to the bath solution at least 5 min before the PTH peptides, 8-BrcAMP, or DOG. Tubules of the control group were perfused or incubated with the respective luminal or bath solution without PTH for the same time period. After 20 min of perfusion of the tubules, they were transferred for 5 min into the same fixative as described above. Afterward, the tubules were transferred into droplets of 10% gelatin (Fluka) in PBS and stored in the fixative overnight.

Immunohistochemistry. Slices of fixed rat kidneys were frozen in liquid propane and mounted onto thin cork slices. The gelatin droplets containing the tubules were washed for 2 h in PBS and cut into cubes before freezing. Cryosections of 3-µm thickness were mounted on chromalum/gelatine-coated glass slides, thawed, and stored in PBS until use. For immunofluorescence, sections were pretreated with 3% milk powder in PBS for 10 min and incubated overnight at 4°C with a rabbit anti-rat polyclonal antiserum against the NaPi-IIa protein (11) diluted 1:500 in 3% milk powder in PBS containing 0.3% Triton X-100 or with a rabbit anti-rat polyclonal antiserum against Na-sulfate cotransporter (NaSi-1) protein (23) diluted 1:500 in the same solution. Sections were then rinsed three times with PBS and covered for 45 min at 4°C with the secondary antibody (swine anti-rabbit IgG conjugated to fluorescein isothiocyanate; Dakopatts, Glostrup, Denmark) diluted 1:50 in PBS/milk powder. Double staining of beta -actin filaments and NaPi-IIa was achieved by adding rhodamine-phalloidin (Molecular Probes, Eugene, OR) at a dilution of 1:50. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma Chemical) diluted 1:500 in the solution containing the secondary antibodies. Finally, the sections were rinsed three times with distilled water, plated on coverslips by using DAKO-Glycergel (Dakopatts) containing 2.5% 1,4-diazabicyclo (2.2.2.) octane (DABCO; Sigma Chemical) as a fading retardant and studied by an epifluorescense microscope (Polyvar, Reichert-Jung). For control of the specificity of the NaPi-IIa antiserum, sections of isolated murine proximal tubules were incubated with preimmune serum. Unspecific binding to the tissue of the secondary antibodies was tested by omitting the primary antibody (not shown). All control incubations were clearly negative.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of 1-34 PTH and 3-34 PTH on the distribution of the NaPi-IIa cotransporter in rat kidneys. Overviews of sections through the kidney cortex of control animals showed that the NaPi-IIa protein was expressed in the BBM of proximal tubules throughout the cortex (Fig. 1A). As reported earlier, the intensity of the NaPi-IIa immunostaining was strongest in proximal convoluted tubules of juxtamedullary nephrons (11, 18, 39). A more detailed view of proximal tubules of the midcortical region (Fig. 1D) revealed that the strongest immunostaining for NaPi-IIa is visible in the BBM and weaker at some intracellular sites.


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Fig. 1.   Immunohistochemical detection of type IIa Na-Pi cotransporter (NaPi-IIa) protein in kidneys and midcortical proximal tubules of control rats (A and D), of rats treated for 20 min with 1-34 parathyroid hormone (PTH; B and E) and rats treated with 3-34 PTH (C and F). 1-34 PTH seems to provoke stronger downregulation of brush-border membrane (BBM) NaPi-IIa than does 3-34 PTH. Bars: A-C, ~200 µm; D-F, ~40 µm.

After injection of 1-34 PTH, the NaPi-IIa protein-specific immunofluorescence was scarcely detectable in midcortical and superficial region, whereas in proximal tubules of juxtamedullary nephrons some apical expression of NaPi-IIa was still observed (Fig. 1B). Cross sections of midcortical proximal tubules (Fig. 1E) illustrate that NaPi-IIa staining in the proximal tubular BBM was drastically downregulated and that a fluorescent rim below the BBM became apparent. In parallel, the intensity of the intracellular staining below this subapical region was increased. These data are in agreement with those previously published (18, 25).

After treatment with 3-34 PTH the distribution pattern of NaPi-IIa protein was similar to that observed after treatment with 1-34 PTH (Fig. 1C). NaPi-IIa was clearly downregulated in superficial proximal tubules, whereas downregulation of NaPi-IIa in proximal tubules of the midcortical region seemed to be less pronounced compared with the NaPi-IIa immunofluorescence after treatment with 1-34 PTH. Comparable to the effect of 1-34 PTH, NaPi-IIa staining was increased in the subapical region (Fig. 1F). However, this effect appeared less pronounced compared with the extent of internalization of NaPi-IIa by 1-34 PTH.

Effect of 1-34 PTH and 3-34 PTH on the expression of NaPi-IIa cotransporter in isolated perfused proximal tubules of mice. Proximal tubules were isolated from mice kept for 1 wk on a low-phosphate diet. Previous studies have shown that this condition leads to a marked upregulation of the type IIa Na-Pi cotransporter in the BBM (4, 21). Thus the abundance of NaPi-IIa in BBM is similar in all nephron generations and along all proximal tubular segments. We chose this condition to obtain a better starting point for studying downregulatory events induced by PTH. All perfusion experiments were performed with proximal tubules from the midcortical region. The results for the different treatments were homogenous in each experimental group.

As illustrated in Fig. 2, after 20 min of perfusion of the isolated proximal tubules with a control solution, the Na-Pi cotransporter was expressed almost exclusively in the brush borders. Intracellular staining was hardly detectable. By using a preimmune serum, no immunoreaction was observed, indicating that our antiserum specifically detected the NaPi-IIa protein. The same tubules were also stained for beta -actin to check for the maintenance of structural organization and of the integrity of the brush-border region. The results shown in Fig. 2 indicate that after 20 min of perfusion cell polarity of the proximal tubule cells was well preserved.


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Fig. 2.   Sections of isolated untreated murine proximal tubules. Triple labeling by anti- NaPi-IIa antiserum, rhodamine-phalloidine for beta -actin, and 4,6-diamidino-2-phenylindole for nuclei. Incubation with preimmune serum is shown (bottom). Bar: ~15 µm.

The basolateral vs. luminal effects of 1-34 PTH on NaPi-IIa were tested (Fig. 3, rows 1 and 2). Luminal perfusion with 10-6 M 1-34 PTH for 20 min provoked a complete disappearance of NaPi-IIa staining in the BBM. Similarly, basolateral incubation of the proximal tubules with 10-6 M 1-34 PTH for 20 min led to a comparable disappearance of the NaPi-IIa staining. The same experimental design was repeated with 3-34 PTH (Fig. 3, rows 3 and 4). Luminal perfusion with 3-34 PTH (10-6 M) for 20 min resulted in a downregulation of NaPi-IIa. However, basolateral incubation with 3-34 PTH for the same time period did not affect the expression of NaPi-IIa.


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Fig. 3.   NaPi-IIa immunostaining and beta -actin labeling in isolated murine proximal tubules treated with 1-34 PTH or 3-34 PTH from luminal or basolateral side. Bar: ~15 µm.

Under all conditions described above, the brush border was well preserved, as indicated by the beta -actin staining. Preservation of the brush borders was further documented by an analysis of the location of NaSi-1. This cotransporter has been shown to be localized exclusively in proximal tubular brush borders, and evidence was obtained that PTH did not affect its distribution (18, 23). The apical abundance of the NaSi-1 protein was not altered by the treatments of the isolated proximal tubules with either 1-34 PTH or 3-34 PTH (Fig. 4).


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Fig. 4.   Immunodetection of type 1 Na-sulfate cotransporter (NaSi-1) in isolated murine proximal tubules under control conditions (top) and treated with 1-34 PTH from luminal and basolateral side (bottom). The same result was obtained by bipolar treatment with 3-34 PTH (bottom). Bar: ~15 µm.

Pharmacological activation and inhibition of the protein kinase A and C pathway in isolated proximal tubules. First, we tested whether activation of the protein kinase A pathway by 8-BrcAMP or the protein kinase C pathway by DOG alters the expression of NaPi-IIa in the BBM (Fig. 5). After 20 min of incubating the proximal tubules in bath solution containing 10-4 M 8-BrcAMP, the NaPi-IIa immunofluorescence is drastically downregulated in the BBM compared with the untreated tubulus. The same effect on NaPi-IIa expression is visible after treatment of the proximal tubules for 20 min with 10-5 M DOG. The structural integrity of the BBM under these conditions (8-BrcAMP, DOG) is well preserved, as assessed by beta -actin staining (Fig. 5, top row). Exposure of the tubules to the protein kinase inhibitors H-89 and calphostin C had no apparent effect on the expression of the type IIa Na-Pi cotransporter. However, the protein kinase A inhibitor H-89 (10-6 M) prevented the 8-BrcAMP-induced downregulation of NaPi-IIa. Similarly, the DOG-induced downregulation of NaPi-IIa was prevented by the protein kinase C inhibitor calphostin C (10-8 M).


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Fig. 5.   NaPi-IIa immunostaining and beta -actin labeling (top row) in isolated murine proximal tubules treated with pharmacological activators of protein kinase A [PKA; 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP)] or protein kinase C [PKC; 1,2-dioctanoylglycerol (DOG)] and their inhibitors [N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl-5-isoquinosulfonamide (H-89), calphostin C]. Bar: ~15 µm.

To characterize the involvement of protein kinases A and/or C within the basolateral and/or luminal effects of the PTH fragments, we performed perfusion experiments with the respective PTH peptides in the absence or presence of the protein kinase inhibitors (Fig. 6). Luminal perfusion with 3-34 PTH for 20 min (Fig. 6, left column) in the presence of the protein kinase A inhibitor H-89 (10-6 M) did not abolish the downregulation of NaPi-IIa. In contrast, in the presence of the protein kinase C inhibitor calphostin C (10-8 M), or in the presence of H-89 and calphostin C, the NaPi-IIa immunostaining was strongly detectable after luminal perfusion with 3-34 PTH. In the presence of calphostin C (10-8 M) or of calphostin C and H-89, the immunofluorescence of NaPi-IIa in the BBM was also not affected by an application of 1-34 PTH to the luminal side (Fig. 6, middle column). Addition of 1-34 PTH to the basolateral side (Fig. 6, right column) in the presence of H-89 (10-6 M) led only to a weak downregulation of NaPi-IIa. In contrast to the experiments with luminal 3-34 and 1-34 PTH, NaPi-IIa was downregulated by basolateral incubation with 1-34 PTH in the presence of calphostin C (10-8 M). Only the incubation with H-89 and calphostin C could completely prevent the downregulatory effect of basolateral incubation with 1-34 PTH on NaPi-IIa expression in the BBM.


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Fig. 6.   Immunodetection of NaPi-IIa in untreated isolated murine proximal tubules and treated with 3-34 and 1-34 PTH in presence of H-89 and/or calphostin C. Bar: ~15 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PTH leads to an inhibition of phosphate transport across the proximal tubular BBM by reducing the maximal velocity of Na-Pi cotransport activity (30, 31, 32). This effect of PTH is explained by a retrieval from the BBM and lysosomal degradation of Na-Pi -IIa cotransporters. (18, 19, 35). Studies on intact kidneys and isolated tubules suggested a preferential role of the adenylate cyclase-kinase A regulatory pathway in PTH action on proximal tubular Pi reabsorption (1, 2, 6, 9).

The intracellular signaling mechanisms in PTH action on Na-Pi cotransport have been widely studied by using OK cells (10, 26, 27, 33, 36). In agreement with studies in cloned PTH receptors (16), these studies provided evidence for a stimulation of adenylate cyclase-protein kinase A and phospholipase C-protein kinase C pathways by the 1-34 PTH analog, whereas the 3-34 PTH analog activates a cAMP-independent, mainly a phospholipase C-protein kinase C, pathway (8, 10). Both 1-34 and 3-34 PTH have been shown to inhibit Na-Pi cotransport in OK cells (10, 20, 33), and, importantly, both PTH peptides also led to an internalization of NaPi-IIa cotransporters (33; see also Ref. 20). However, before the present study it had not been established whether the data obtained in OK cells are also relevant for intact tubules, i.e., whether the signaling pathway via protein kinase C is also important for the in vivo regulation of Na-Pi cotransport by PTH.

With respect to the distribution of PTH receptors in renal proximal tubules, hormone binding was seen in basolateral as well as in apical membrane preparations. However, stimulation of adenylate cyclase by PTH could be observed only in basolateral membranes (17), and no functional role has yet been ascribed to apically localized PTH receptors. Studies performed with OK cells grown on permeant filter supports provided evidence for a bipolar expression of PTH receptors because addition of PTH to either side resulted in an inhibition of Na-Pi cotransport (37, 38). Furthermore, the studies in OK cells provided some evidence for functional differences in control of Na-Pi cotransport between apically and basolaterally located PTH receptors (38).

The results obtained by the presently described in vivo and in vitro studies in intact tubules indicate that both 1-34 and 3-34 PTH induce a downregulation of NaPi-IIa cotransporter protein in proximal tubules. As described earlier for 1-34 PTH (18, 25, 40), downregulation induced by 3-34 PTH was also found to be associated with an accumulation of NaPi cotransporters in the subapical compartment. Activation of protein kinase A by 8-BrcAMP or an activation of the protein kinase C pathway by DOG is able to mimic the action of the PTH fragments. Both effects could be blocked by the use of H-89 or calphostin C. These results indicate that a separate activation of either the protein kinase A or the protein kinase C pathway by PTH is sufficient for an internalization of the NaPi-IIa cotransporter. Compared with 1-34 PTH, the degree of downregulation of the NaP-IIa cotransporter induced by 3-34 PTH was somewhat less. This is in agreement with results from OK cells demonstrating that at a maximal concentration of 3-34 PTH Na-Pi cotransport inhibition and NaPi-IIa cotransporter downregulation was ~50% of the effect induced by 1-34 PTH (10, 33).

We provide evidence that the NaPi-IIa cotransporter can be regulated via PTH receptors located in both the basolateral and apical membrane. Application of 1-34 PTH to either site resulted in a strong downregulation of the NaPi-IIa protein. However, although 1-34 PTH was active from both sites, 3-34 PTH was only effective when applied from the luminal side. Additionally, the luminal actions of both PTH peptides were abolished by the inhibition of the protein kinase C pathway with calphostin C, but not by inhibition of the protein kinase A pathway with H-89. Thus luminal effects by the PTH-fragments might be preferentially mediated by protein kinase C. The basolateral action of 1-34 PTH could only be completely prevented by inhibiting the protein kinase A and C pathway, suggesting that both signaling pathways might be active under these conditions. Our results could be explained by the presence of different PTH-receptor subtypes and/or coupling of the same PTH-receptor to different signaling cascades, depending on its cellular location. It is of note that a bipolar location in proximal tubules has also been described for other receptors, e.g., for angiotensin II receptors within regulation of bicarbonate reabsorption (13, 22).

Because PTH is in part cleared from the circulation by glomerular filtration, the functional importance of apical PTH receptors may be dependent on the capacity of PTH- degradative mechanisms at the luminal surface. Clearly, at early tubular sites PTH fragments capable of activating PTH receptors may be present in the tubular fluid and therefore may contribute to transport regulation such as Na-Pi cotransport or Na/H exchange.

In summary, this study shows that in intact proximal tubules the BBM type IIa Na-Pi cotransporter can be regulated via internalization by cAMP-dependent and -independent mechanisms. Furthermore we provide evidence that functional PTH receptors are located in the basolateral as well as in the luminal membrane of proximal tubules and that apical PTH receptors may preferentially be coupled to a cAMP-independent signaling pathway.


    ACKNOWLEDGEMENTS

The study was supported by Swiss National Science Foundation Grants 31-47742-96 (B. Kaissling) and 31-46523-95 (H. Murer).


    FOOTNOTES

* M. Traebert and H. Völkl contributed equally to this study.

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: B. Kaissling, Institute of Anatomy, Univ. Zuerich-Irchel, Winterthurerstr. 190, CH-8057 Zurich, Switzerland (E-mail: bkaissl{at}anatom.unizh.ch).

Received 9 August 1999; accepted in final form 30 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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