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J. Biol. Chem., Vol. 275, Issue 41, 31601-31608, October 13, 2000

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Acute Regulation of Na⁺/H⁺ Exchanger NHE3 by Parathyroid Hormone via NHE3 Phosphorylation and Dynamin-dependent Endocytosis^*

Roberto Collazo, Lingzhi Fan, Ming Chang Hu, Hui Zhao, Michael R. Wiederkehr, and Orson W. Moe^§

From the Medical Service, Department of Veterans Affairs Medical Center and Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Received for publication, January 27, 2000, and in revised form, June 23, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na⁺ transport via its action on the apical membrane Na⁺/H⁺ exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple serines was evident after 5 min of PTH, but decrease in surface NHE3 antigen was not detectable until after 30 min of PTH. The decrease in surface NHE3 antigen was due to increased NHE3 endocytosis. When endocytic trafficking was arrested with a dominant negative dynamin mutant (K44A), the early inhibition (5 min) of NHE3 activity by PTH was not affected, whereas the late inhibition (30 min) and decreased surface NHE3 antigen induced by PTH were abrogated. We conclude that PTH acutely inhibits NHE3 activity in a biphasic fashion by NHE3 phosphorylation followed by dynamin-dependent endocytosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The calcitropic effect of PTH¹ is effected by altering calcium flux between the plasma and the skeleton, intestine, and kidney in a concerted fashion. In the mammalian kidney, PTH is the principal anti-calciuric hormone exerting its action on the proximal tubule, thick ascending limb, and the distal convoluted tubule (1-4). In the renal proximal tubule, PTH is a potent inhibitor of NaHCO₃ absorption (5-11) that results in increased delivery to the distal nephron where NaHCO₃ acts as an important stimulus for active transcellular Ca²⁺ absorption (12-14). In the mammalian proximal tubule, two-thirds of the transcellular NaHCO₃ absorption is mediated by apical membrane Na⁺/H⁺ exchange (15) which is coded by the NHE3 isoform (16-18), a member of the multigene family of mammalian Na⁺/H⁺ exchangers (19-21). Inhibition of proximal tubule HCO₃ absorption by PTH (5-11) has been shown to be mediated by decreased Na⁺/H⁺ exchange activity using suspended tubules (22), isolated in vitro perfused tubules (23), in vivo perfused tubule (24), apical membrane vesicles (25, 26), cultured renal cells (27-34), and non-epithelial cells transfected with the PTH receptor and NHE3 gene (35-36).

Acute inhibition of NHE3 can potentially be mediated by multiple mechanisms including NHE3 phosphorylation, binding to regulatory factors, and alteration of surface NHE3 protein abundance (37, 38). In cultured cells, acute regulation of NHE3 has been shown to be accompanied by NHE3 phosphorylation (39-45), and the functional role of phosphorylation has been demonstrated for PKA and PKC (40, 42, 44). The signal transduction cascade mediating the effect of PTH on NHE3 is complex, likely involving multiple pathways, but at least part of the effect of PTH on NHE3 is PKA-dependent (29-32, 35, 36). In the intact animal, acute infusion of PTH increases NHE3 phosphorylation as assayed by mobility retardation (26) which lends additional support for the importance of NHE3 phosphorylation in its regulation. This paper further characterizes the time course and pattern of NHE3 phosphorylation in OK cells in response to PTH.

It is presently unclear how phosphorylation of NHE3 alters its transport activity. One mechanism that has been shown to alter acutely NHE3 transport activity is the change in cell surface protein abundance. This has been demonstrated in cultured cells (46-48) as well as in intact kidney (26, 49-53). Two studies have shown that acute infusion of PTH leads to a decrease in proximal tubule apical membrane NHE3 using immunohistochemistry (53) and membrane vesicles (26). It is unclear whether this acute decrement in apical membrane NHE3 is due to decreased exocytic insertion, endocytotic internalization, or both. Studies in rat kidney suggest that acute changes in apical membrane NHE3 abundance is microtubule-dependent (26) and is partially mediated by the cytochrome P450 system (50). There is evidence in cell culture that NHE3 is recycled via phosphatidylinositol 3-kinase-dependent mechanisms (46, 47) and is internalized through clathrin-mediated pathway in fibroblasts (54). Full expression of NHE3 activity is dependent on the integrity of the cytoskeleton, and NHE3 co-sediments with the actin cytoskeleton (55). This study will define some of the mechanisms by which PTH acutely decreases surface NHE3 in a renal epithelial cell.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection of Cells-- OK cells were passaged in high glucose Dulbecco's modified Eagle's medium, supplemented with Na⁺ pyruvate (1 mM), fetal bovine serum (10%, v/v), penicillin (100 units/ml), and streptomycin (100 mg/ml) and were rendered quiescent post-confluence by serum removal for 48 h prior to experimentation. To examine the role of dynamin on regulation of NHE3 activity by PTH, transient transfections of OK cells were performed with the following plasmids: carboxyl-terminal c-Myc-tagged opossum NHE3, hemagglutinin-tagged wild type (dyn^WT), and dominant negative GTP-binding defective dynamin (dyn^K44A) using LipofectAMINE (dynamin plasmids were kindly provided by Dr. Sandra Schmid, La Jolla, CA) (56). Transfection efficiency was monitored by co-transfection with -galactosidase and staining cells with 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal) as well as staining with anti-hemagglutinin antibody. For experiments examining the effect of dynamin on the regulation of NHE3 by PTH, only cells with transfection efficiency greater than 80% were used.

NHE3 Activity Assay-- NHE3 activity was measured fluorimetrically with the pH-sensitive dye BCECF as Na⁺-dependent pH_i recovery (dpH_i/dt) after an acid load using the K⁺/H⁺ ionophore nigericin as described previously (57). BCECF calibration curves to convert fluorescence to pH_i values were obtained by nigericin permeation at varying levels of extracellular pH. PTH addition had no effect on the buffer capacity, trough pH during Na⁺ addition, or nigericin calibration curves (data not shown). For paired measurements, a base-line dpH_i/dt was obtained after NH₄⁺/NH₃ prepulse/withdrawal (20 mM NH₄Cl) followed by Na⁺ addition (58). PTH or vehicle was then added for the specified time, and a second dpH_i/dt was obtained with NH₄⁺/NH₃ prepulse/withdrawal and Na⁺ addition.

In Vivo NHE3 Phosphorylation-- After incubation in phosphate-free Dulbecco's modified Eagle's medium (60 min), OK cells were pulsed with phosphate-free medium containing [³²P]orthophosphate (300 mCi/ml) for 120 min. PTH (10⁷ M) or vehicle was added, and the cells were washed, lysed with RIPA buffer (in mM, 300 NaCl, 80 NaF, 50 Tris-HCl, pH 7.4, 5 EDTA, 10 EGTA, 25 Na⁺ pyrophosphate, 1 activated Na⁺ orthovanadate, 50 -glycerophosphate, 0.5 dithiothreitol; Triton X-100 1% (v/v), deoxycholate 0.5% (w/v), SDS 0.1% (w/v); in mg/ml, 100 phenylmethylsulfonyl fluoride, 4 leupeptin, 4 aprotinin, 10 pepstatin), and lysate was centrifuged (109,000 × g at r_max, 50,000 rpm, 25 min, 2 °C, Beckman TLX/TLA 100.3 rotor, Fullerton, CA). The supernatant was retrieved and mixed with an equal amount of water, and NHE3 was immunoprecipitated (antiserum:lysate, 5:1000 dilution, v/v) with a polyclonal antisera (number 5683, directed against a chimeric protein consisting of maltose-binding protein and opossum NHE3 amino acids 484-839). Immunoblot was performed with antiserum 5683 (1:1000) and goat anti-rabbit antibody (1:5000) using enhanced chemiluminescence (ECL). The ³²P content of NHE3 was visualized by autoradiography on the same filters after ECL decay. Both signals were quantified by densitometry, and changes in phosphorylation were normalized to the antigenic signal. For tryptic phosphopeptide and phosphoamino acid analysis, the in vivo ³²P-phosphorylated, immunoprecipitated NHE3 was excised and extracted (100 mM acetic acid containing 0.5% polyvinylpyrrolidone at 37 °C for 30 min) from the nitrocellulose filter and digested with 15 mg L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin in 0.05 M NH₄HCO₃ at 37 °C. The released phosphopeptides were washed with water, lyophilized repeatedly, and resuspended in electrophoresis buffer (per liter, 25 ml of 88% (w/v) formic acid, 78 ml of glacial acetic acid, 897 ml of deionized water, pH 1.95) and fractionated with sequential electrophoresis (1.0 kV for 40 min, Hunter Electrophoresis Unit HTLE 7000, CBS Scientific Co., Del Mar, CA) and ascending chromatography (375:250:75:300 (v/v) n-butyl alcohol, pyridine, glacial acetic acid, deionized water) on cellulose TLC plates as described previously (42). Phosphopeptide spots were identified by autoradiography. For phosphoamino acid analysis, purified phosphoproteins were hydrolyzed by boiling in 6 M HCl, and ³²P-labeled amino acids were electrophoretically resolved on a Hunter Unit (first dimension, 2.2% formic acid, 7.8% acetic acid, pH 1.9; second dimension, 5% acetic acid, 0.5% pyridine, pH 3.5) along with the cold Ser(P), Thr(P), and Tyr(P) standards. Phosphoamino acids were identified by autoradiography and aligned with ninhydrin-stained standards.

Assay of NHE3 Surface Antigen and NHE3 Endocytosis-- To measure surface NHE3, OK cells were treated with either agonist or vehicle and then surface-labeled with biotin using a modification of the method of Gottardi et al. (59). After rinsing (PBS-Ca²⁺/Mg²⁺ in mM, 150 NaCl, 10 Na₂HPO₄, pH 7.40, 0.1 CaCl₂, 1 MgCl₂), cells were treated with 2 mg/ml of NHS-SS-biotin (Pierce) in buffer (in mM, 150 NaCl, 10 triethanolamine, pH 7.4, 2 CaCl₂), quenched (PBS-Ca²⁺/Mg²⁺, with 100 mM glycine), and lysed in biotin-RIPA (150 NaCl, 50 Tris-HCl, pH 7.4, 5 EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS). Lysates were clarified by ultracentrifugation (109,000 × g at r_max, 50,000 rpm, 25 min, 2 °C, Beckman TLX/TLA 100.3 rotor, Fullerton, CA), and protein content was quantified by the method Bradford. Equal amounts of cell lysate were equilibrated with streptavidin-agarose (Pierce) at 4 °C. Beads were washed sequentially with solutions A (in mM, 50 Tris-HCl, pH 7.4, 100 NaCl, 5 EDTA), B (in mM, 50 Tris-HCl, pH 7.4, 500 NaCl), and C (50 mM Tris-HCl, pH 7.4), and biotinylated proteins were released by incubation in 100 mM dithiothreitol, reconstituted in Laemmli's buffer, and subjected to SDS-polyacrylamide gel electrophoresis and labeled with either anti-NHE3 antiserum 5683 or for experiments with exogenous c-Myc-tagged NHE3 a monoclonal anti-c-Mxc was used (Invitrogen). Endocytosis was measured by a protocol adapted and modified from the stage-specific Mes/Na⁺-resistant and avidin protection endocytosis assays originally described by Carter and co-workers (60). OK cells were surface-labeled with NHS-SS-biotin and quenched exactly as described above. Cells were then warmed to 37 °C in the presence of 10⁷ M PTH or vehicle to allow endocytosis to occur over 30-40 min. Surface biotin was then saturated with avidin (50 mg/ml PBS) and washed with biocytin (50 mg/ml PBS), or alternatively, surface biotin were cleaved with the small cell-impermeant reducing agent TCEP (100 mM in 50 mM Tris, pH 7.4). The freshly endocytosed biotinylated proteins were protected from either avidin saturation or TCEP cleavage. Cells were then solubilized in RIPA, and biotinylated proteins were retrieved and assayed for NHE3 as described above. Avidin-protected fraction measures early and late endocytosis because avidin cannot enter the constricted necks of clathrin-coated pits. TCEP-protected fraction measures late endocytosis because complete excommunication from the exterior is required to prevent TCEP access.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PTH Inhibits NHE3 Activity-- PTH inhibited NHE3 activity measured as Na⁺-dependent pH_i recovery in OK cells as shown by the representative tracing (Fig. 1A). Dose and time dependence of inhibition of NHE3 activity by PTH are summarized in Fig. 1, B and C. PTH inhibits NHE3 activity as early as 5 min and over the concentration from 10⁸ to 10⁵ M. These findings are very similar to previously reported results (29, 31, 32). We next examined the effect of PTH on NHE3 phosphorylation.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effect of PTH on NHE3 activity in OK cells. A, representative tracing of NHE3 activity assayed fluorimetrically (BCECF) as Na+-dependent intracellular pH recovery (dpHi/dt) after acid load. PTH (107 M) was added for 20 min. B, summary of data for time response of NHE3 activity to 107 M PTH. Symbols and bars depict mean ± S.E. Asterisk indicates significant difference from control (p < 0.05, ANOVA). Control (n = number of experiments) n = 14; 5 min, n = 6; 15 min, n = 9; 30 min n = 12. C, summary of data for dose response of NHE3 to 30 min of PTH. Symbols and bars depict mean ± S.E. Asterisk indicates significant difference from control (p < 0.05, ANOVA). Control (n = number of experiments), n = 14; 109 M, n = 6; 108 M, n = 5; 107 M, n = 12; 106 M, n = 6.

PTH Increases NHE3 Phosphorylation-- We previously showed that acute inhibition of cortical apical membrane NHE activity in rat kidney by PTH is associated with increased NHE3 phosphorylation using an indirect mobility shift assay. We now use OK cells to characterize further the PTH-induced NHE3 phosphorylation. First, the time course of increased NHE3 phosphorylation paralleled that of NHE3 inhibition with detectable changes in NHE3 phosphorylation as early as 5 min after PTH treatment (Fig. 2, A and B). There is a trend toward lesser phosphorylation by 30 min, but total NHE3 phosphorylation is still greater than control. Second, the increase in NHE3 phosphorylation was blocked by the PKA inhibitor H89 (10⁶ M) suggesting involvement of PKA (Fig. 2, A and B). Third, the majority of phosphate content of NHE3 at base line as well as after PTH was on serine residues (Fig. 2C) with phosphothreonine constituting less than 2% and phosphotyrosine less than 3% of the counts. Fourth, PTH induced increase in NHE3 on multiple serine residues as evident by the increase in intensity of 7 tryptic phosphopeptides (phosphopeptides IV-X in Fig. 3, A and B). Finally, the pattern of phosphorylation of NHE3 induced by 10⁷ M PTH was identical to that of 10⁵ M 8-bromo-cAMP (Fig. 3, B and C). Expression of a carboxyl-terminal cytoplasmic domain truncated c-Myc-tagged (amino acids 1-483) mutant NHE3 produced a protein that was expressed at the cell surface (biotin-accessible anti-c-Myc-reactive) but not phosphorylated at base line or upon PTH addition (data not shown). These studies suggest that PTH acutely leads to increased phosphorylation of multiple cytoplasmic serines on NHE3 via the PKA pathway. In all of the above studies, total cellular NHE3 phosphorylation was studied.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of PTH on NHE3 phosphorylation in OK cells. A, representative experiment of NHE3 phosphorylation ± PKA inhibition. OK cells were pulsed with 32P, and native NHE3 was immunoprecipitated and resolved on SDS-polyacrylamide gel electrophoresis, and NHE3 phosphorylation and antigen were quantified by autoradiography and immunoblotting, respectively. Cells were treated with PTH 107 M (20 min) with or without H89 (106 M). B, summary of all experiments showing NHE3 phosphorylation signal normalized to NHE3 antigen signal. Bars depict mean ± S.E. Asterisk indicates significant difference from control (p < 0.05, ANOVA). Control (n = number of experiments), n = 9; PTH 5 min, n = 9; PTH 30 min, n = 9; H89, n = 3; H89 + 5 min PTH, n = 5; H89 + 30 min PTH, n = 4. C, phosphoamino acid analysis. In vivo phosphorylation of NHE3 was performed as described above with either PTH or vehicle. The 32P-labeled immunoprecipitated NHE3 was hydrolyzed to single amino acids and resolved by two-dimensional chromatography. Location of standard phosphoamino acids (PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine) is indicated. Two experiments showed similar results.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Effect of PTH on tryptic phosphopeptide map of NHE3. OK cells were pulsed with 32P and treated with the appropriate agonist, and native NHE3 was immunoprecipitated, digested by trypsin, and resolved in two dimensions. 32P-Labeled phosphopeptides were phosphorimaged and identified by roman numerals (I-X). Conditions were as follows: control (Con, A), PTH 107 M × 10 min (B), and 8-Br-cAMP 104 M × 10 min (C). Three sets of experiments showed similar results.

PTH Decreases Surface NHE3 Antigen by Increasing NHE3 Endocytosis-- Although increases in NHE3 phosphorylation were evident after 5 min of PTH treatment, changes in cell surface NHE3 antigen defined as the biotin-accessible fraction of total cellular NHE3 were not detectable until 30 min after PTH treatment (Fig. 4, A and B). Total cellular NHE3 remained unchanged (Fig. 4A). To examine whether the decrease in surface antigen was due to increased endocytosis, we used the avidin protection and TCEP resistance assays to examine late and total endocytosis, respectively. Control experiments performed at 4 °C where endocytosis was arrested revealed a background signal of approximately 10-20% that from cells at 37 °C. This likely represents biotinylated surface proteins that were not completely cleaved by TCEP or saturated with avidin. This background signal did not appear to be consistently altered by PTH treatment. PTH increased both the avidin-protected and TCEP-protected fractions (Fig. 5) indicating that the PTH stimulates both early and late NHE3 endocytosis.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of PTH on surface NHE3 antigen. OK cells were treated with 107 M PTH for the indicated period. Surface proteins were biotinylated and retrieved from solubilized cell lysate by streptavidin-affinity precipitation, and NHE3 antigen was quantified by immunoblot. Total cellular NHE3 was quantified by immunoprecipitating NHE3 from whole cell lysates. A, typical blot. B, summary of all experiments. Symbols and bars depict mean ± S.E. Asterisk indicates significant difference from control (p < 0.05, ANOVA). Control (n = number of experiments), n = 10; 5 min PTH, n = 8; 15 min PTH, n = 4; 30 min PTH, n = 12.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of PTH on NHE3 endocytosis. OK cells were surface-biotinylated, warmed to 37 °C for 40 min in the presence or absence of 107 M PTH for endocytosis, and the remaining surface-accessible biotin was either saturated with avidin or cleaved with TCEP (see text for details). The freshly internalized biotin-bearing proteins were retrieved by streptavidin-agarose precipitation, and NHE3 antigen was quantified by immunoblot. Some cells were kept at 4 °C throughout the procedure to arrest endocytosis, and the signal represents the degree of failure of either complete cleavage by TCEP or saturation by avidin. Four independent experiments showed identical results.

Dependence on Dynamin-- Endocytosis can proceed via multiple pathways (61, 62). Two of these pathways, the caveolin-mediated and clathrin-coated vesicle pathways, are dependent on the GTPase dynamin (63). A GTP binding-deficient dominant negative mutant dynamin (dyn^K44N) was transiently expressed to block endogenous dynamin function. In all the studies examining NHE3, only cells with ~80% transfection efficiency were used. Although the inhibition of NHE3 activity by PTH after 5 min was not blocked by dyn^K44A, PTH inhibition after 30 min was mostly abolished (Fig. 6A). A small but statistically insignificant inhibition was noted. Transfection with wild type dyn^WT had no effect on the PTH-induced inhibition at either 5 or 30 min (Fig. 6A). Note that in the presence of dyn^K44A, base-line NHE3 activity was more variable from plate to plate (Fig. 6A). To rule out data scattering as a cause of the lack of effect of 30 min of PTH on NHE3 activity in the presence of dyn^K44A, we examined PTH inhibition on NHE3 activity in a paired fashion using each plate as its own control. Fig. 6B shows that dyn^K44A abrogated whereas dyn^WT did not affect the inhibition of NHE3 activity by PTH.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of the dominant negative dynamin (dynK44A) on the PTH-induced inhibition of NHE3 activity. OK cells were transfected with either wild type (dynWT) or dynK44A. A, unpaired measurements. The effect of either 5 or 30 min of 107 M PTH on NHE3 activity was measured fluorimetrically (BCECF) as Na+-dependent pHi recovery after an acid load (nigericin). Bars depict mean ± S.E. Asterisk indicates significant difference from control (p < 0.05, ANOVA). Control + dynWT, n = 4; PTH + dynWT, n = 6; control + dynK44A, n = 12; PTH + dynK44A, n = 12 (n = number of experiments). B, paired measurements. NHE3 activity was measured fluorimetrically (BCECF) as Na+-dependent pHi recovery after an acid load (NH3/NH4+ pulse/withdrawal); PTH (107 M × 30 min) was then added, and NHE3 activity was measured again in the same cells on the same coverslip. Individual paired data points are connected. Symbols and bars depict mean ± S.E. Asterisk indicates significant difference from control (p < 0.05, paired t test). n = number of pairs. dynWT, n = 4; dynK44A, n = 10.

Next, we correlated the functional data with antigenic data. We did these studies two ways. First, we examined the effect of dyn^WT or dyn^K44A on native opossum NHE3 using conditions exactly as described above for the activity assay using only transfections with ~80% efficiency. Fig. 7, A and B, showed that dyn^K44Acompletely blocked the PTH-induced decrease in surface NHE3, whereas dyn^WT had no effect. Second, we co-transfected the dyn^WT or dyn^K44A construct along with carboxyl-terminal c-Myc-tagged opossum NHE3 and performed the surface biotinylation assay with anti-c-Myc antisera. This assay selects out only the cells that were transfected. The data were virtually identical to that presented in Fig. 7, A and B, using native NHE3 as a read-out (data not shown).

View larger version (41K): [in this window] [in a new window]   Fig. 7.   Effect of the dominant negative dynamin (dynK44A) on the PTH-induced decrease of surface NHE3 antigen. OK cells were transfected with either wild type (dynWT) or dynK44A. Cells were treated with either PTH (107 M, 40 min) or vehicle for 40 min. Surface proteins were biotinylated and retrieved from solubilized cell lysate streptavidin affinity precipitation, and NHE3 antigen was quantified by immunoblot. A, typical experiment. B, summary of 7 experiments. Bars depict mean ± S.E. Asterisk indicates significant difference from control (p < 0.05, unpaired t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

When parathyroidectomized rats received an acute infusion of PTH, a biphasic response was seen when early (1-2 h) inhibition of NHE3 activity was accompanied by increased NHE3 phosphorylation and later (>2 h) inhibition was associated with decreased apical membrane NHE3 antigen (26). More detailed definition of this dual response is difficult in intact animals. In OK cells, a similar biphasic response was observed with slightly different kinetics. The reasons for the disparity is unclear but likely relates to differences between intact animals where multiple factors can mediate and modulate the acute PTH response and in cell culture where a pure PTH effect is seen. The timing of agonist delivery and exposure may also differ as the PTH was given intravenously in the whole animal, whereas cells were exposed instantly to the desired concentration. Nonetheless, the biphasic response of regulation is clearly present in OK cells and thus provides a model for further study of the mechanism of the response.

Early inhibition of NHE3 activity by PTH is associated with increased NHE3 phosphorylation on multiple serines on the cytoplasmic domain of NHE3. Phosphorylation of multiple cytoplasmic serines on NHE3 has been described with PKA activation in fibroblasts transfected with NHE3 (39, 40, 42, 45) or native NHE3 in OK cells (42), acute addition of endothelin in OK cells (43), and PKC activation in transfected cells in one study (44) but not in another study (41). The functional importance of specific phosphoserines has previously been documented (38, 40, 42, 44). In both OK cells and in the intact animal treated with PTH, changes in NHE3 phosphorylation and activity follow the same time course. This finding further supports the importance of phosphorylation in NHE3 functional regulation. The kinases involved in the inhibition for NHE3 by PTH has been controversial with current data suggesting a dual PKA/PKC pathway (29-36). In this study, NHE3 phosphorylation was completely blocked by the PKA inhibitor H89 and NHE3 phosphorylation pattern from cells treated with PTH or 8-Br-cAMP were identical when compared side-by-side. This strongly suggests that the PTH-induced phosphorylation of NHE3 is mediated by PKA. In the whole animal only PKA-activating PTH peptides were competent in inducing NHE3 trafficking (53). However, this does not rule out a contributory role for PKC since PKC activation can alter NHE3 activity by mechanisms independent of changes in NHE3 phosphorylation (41, 44). There was a trend toward lesser total cellular NHE3 whole protein phosphorylation at 30 min but the phospho content was still higher than control. We did not specifically examine surface protein phosphorylation in this study. It is possible that there may be some reversal of surface NHE3 phosphorylation at 30 min. The small but statistically insignificant inhibition by PTH at 30 min when dynamin was inhibited is also compatible with reversal of modification of surface NHE3.

Similar to the data from rat renal cortex, the immediate inhibition of NHE3 in OK cells was not accompanied by changes in surface NHE3 antigen. The inability of dominant negative dynamin to block the early inhibition further supports the notion that changes in NHE3 intrinsic activity may mediate the immediate inhibition. Numerous examples in either native membrane vesicles or reconstituted proteoliposomes where there is no possible NHE3 trafficking have shown regulation of transport activity (38, 64-71). Substrate kinetic analysis of the effect of PTH on native NHE3 in OK cells and of cAMP on NHE3-transfected fibroblasts has revealed changes in K_Na or K_H of NHE3 (28, 72), a finding that is not compatible with changes in surface NHE3 antigen. Several groups have shown changes in NHE3 activity that are not accounted for by changes in surface NHE3 antigen in response to PKA activation (38), PKC activation (48), and modification of the actin cytoskeleton (55). All NHEs appear to have comparable turnover rates at base line when expressed in fibroblasts (73). However, the mechanism by which changes in turnover rate of NHE3 is effected is currently unclear. A number of NHE binding partners have been described such as calcineurin-homologous protein and calmodulin for NHE1 (74-77), calmodulin and a variety of Src homology 3-containing proteins for NHE2 (78, 79), and certain members of the Na⁺/H⁺ exchanger regulatory factor (NHERF) family of proteins, ezrin and megalin for NHE3 (45, 70-72, 80, 83, 84). The current model proposed by Yun, Donowitz, and Weinman and co-workers (72, 80, 82, 83) suggests that although NHERF phosphorylation is not regulated by PKA, NHERF functions to relay PKA to access its substrate NHE3. Currently there is no evidence suggesting that NHERF binding to NHE3 per se alters NHE3 activity. Biemesderfer and co-workers (84) have proposed that association of NHE3 with megalin can be a mechanism of altering NHE3 activity without changes in NHE3 protein abundance in the plasma membrane.

Data supporting regulation of NHE3 trafficking have been described in the rat kidney with acute and chronic hypertension (49-52), acute PTH infusion (26, 53), and chronic metabolic acidosis (85) in fibroblasts transfected with NHE3 (46-48, 54, 55) and in OK cells exposed to acid pH (86) or endothelin (81). We now show that OK cells incubated with PTH for 30 min caused inhibition of NHE3 activity and decrease in surface NHE3 antigen. Decrease in steady state surface NHE3 can be due to decreased rate of exocytic insertion or increased rate of endocytic retrieval. In this study, we provide biochemical evidence that PTH stimulates endocytic retrieval of NHE3 from the plasma membrane. Some caution is warranted regarding the interpretation of biochemical data of trafficking. The biotinylation was performed at 4 °C which should arrest all trafficking events, although we cannot completely rule out a small persistent recycling during the labeling which will render the assay not purely indicative of endocytotic retrieval. Another unlikely but theoretically conceivable scenario is that accessibility of reagents such as NHS-SS-biotin, avidin, and TCEP to NHE3 may be partially masked by neighboring proteins. We have not ruled out the possibility that binding of NHE3 by a protein such as megalin may "mimic" the biochemical data suggesting regulation of NHE3 trafficking. However, the fact that decreased apical membrane NHE3 antigen was detected in rats given PTH (26, 53) strongly supports the trafficking model. In addition, the fact that the GTP binding-defective dominant negative dynamin blocked the 30-min inhibition of NHE3 activity is congruent with endocytotic retrieval of NHE3 as a mechanism of inhibition. The dependence on dynamin suggests that NHE3 is endocytosed via either the caveolin pathway, clathrin-coated vesicle pathway, or both (61, 62). Chow and co-workers (54) have previously shown that a different dominant negative mutant dynamin (dyn^S45N) blocks endocytosis of NHE3 and documented a functional role for e-COP protein in NHE3 trafficking in fibroblasts. Chow and co-workers (54) have co-purified NHE3 with clathrin light and heavy chains and AP-1 and AP-2 adaptins in liver and ileal villi. In concert, the current body of data supports regulation of NHE3 trafficking by the clathrin-coated vesicles pathway. We have not ruled decreased exocytosis and increased endocytosis as joint mechanisms mediating decreased surface NHE3.

One quantitative aspect of the data is noteworthy. More than two-thirds of the maximal inhibition of NHE3 activity by PTH is achieved by 15 min (Fig. 1B) when no detectable decreased surface NHE3 protein was evident. After 30 min, when the activation of NHE3 endocytosis decreased surface NHE3 (Fig. 4B), a modest additional decrease in NHE3 activity occurred (Fig. 1B). Although the activity and surface antigen data were not obtained in the same cells, a semi-quantitative analysis is presented in Table I where an attempt is made to estimate surface NHE3 activity normalized to surface NHE3 antigen. It is evident that the decrease in NHE3 activity at 15 min is all due to decreased intrinsic NHE3 transport activity. As indicated above, this may be mediated by NHE3 phosphorylation, binding to regulatory proteins, or both. At 30 min when surface NHE3 decreased, NHE3 activity per surface protein appears to have partially recovered. We postulated two hypothetical models that are consistent with the empiric observations (Fig. 8). In model 1, only a fraction of surface NHE3 undergoes phosphorylation and dramatic reduction in transport via yet undefined mechanisms at 15 min. At 30 min, the phosphorylated fraction undergoes endocytosis leaving the remaining surface transporters running at about the same rate as under control conditions. In model 2, phosphorylation increases in all surface NHE3 at 15 min resulting in decrease in NHE3 transport activity. A restricted population of surface NHE3 then undergoes endocytosis. The surface transporters that do not get internalized partially recover their function via reversal of phosphorylation, binding to cofactors, or both. In both models, total cellular NHE3 phosphorylation remains elevated at 30 min due mainly to the intracellular endocytosed pool of NHE3. The ability of dyn^K44A to block completely inhibition of NHE3 at 30 min does suggest a time-dependent reversal of surface NHE3, although a caveat of this interpretation is that dyn^K44A may theoretically interfere with the inhibition of NHE3 surface protein. Both models proposed remain conjectural at present. They are by no means proven, but they are compatible with the existing data.

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Two hypothetical models consistent with the experimental observations. Approximate relative NHE3 activities and surface NHE3 antigens are shown for 0, 15, and 30 min after PTH addition.

In summary, the data from rat renal cortex (43) and from OK cells suggest a model of biphasic acute regulation of NHE3 activity by PTH involving dual mechanisms. Immediate inhibition is effected by changes in intrinsic activity of NHE3 and associated with phosphorylation. The above two models are purely conjectural at the moment, but they are consistent with the experimental findings of multiple serines on the cytoplasmic membrane via PKA. More sustained inhibition is mediated by endocytic retrieval of NHE3 from the plasma membrane via a dynamin-dependent pathway. Presently, the fates of the remaining surface transporters and the ones that have undergone endocytosis are unknown.

	ACKNOWLEDGEMENTS

We acknowledge the technical expertise of Ladonna A. Crowder. We are grateful to Dr. Michel Baum and Dr. Robert Alpern for their helpful discussions and Dr. Sandy Schmid for providing us with reagents.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants DK-48482 and DK-54396, the Department of Veterans Affairs Research Service, the American Heart Association Texas Affiliate Grant 98G-052, and the National Kidney Foundation of Texas.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. Section 1734 solely to indicate this fact.

Recipient of National Institutes of Health Training Grant T32 DK07257-17.

^§ To whom correspondence should be addressed: Dept. Of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8856. Tel.: 214-648-3152; Fax: 214-648-2071; E-mail: orson.moe@vtsouthwestern.edu.

Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M000600200

	ABBREVIATIONS

The abbreviations used are: PTH, parathyroid hormone; OK, opossum kidney; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; Mes, 4-morpholineethanesulfonic acid; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; PBS, phosphate-buffered saline; dyn, dynamin; WT, wild type; ANOVA, analysis of variance; NHERF, Na⁺/H⁺ exchanger regulatory factor; BCECF, 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein.

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