Na+/H+ exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells

S. Akhter1, O. Kovbasnjuk1, X. Li1, M. Cavet1, J. Noel2, M. Arpin3, A. L. Hubbard4, and M. Donowitz1,5

Departments of 1 Medicine, 4 Cell Biology, and 5 Physiology, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195; 2 Department of Physiology, University of Montreal, Montreal, Canada H3C 3J7; and 3 Curie Institute, Paris, France 75231


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell biological approaches were used to examine the location and function of the brush border (BB) Na+/H+ exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as ~400- and ~900-kDa standards, whereas surface NHE3V was enriched in the ~900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from ~400 to ~900 kDa.

sodium/hydrogen exchanger 3; opossum kidney cells; sodium absorption; phosphatidylinositol 3-kinase; recycling endosomes; pH


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THERE ARE CURRENTLY SEVEN cloned mammalian Na+/H+ exchangers (NHEs), which differ from each other in tissue distribution, pharmacological inhibitory profiles, organellar subcellular localization, and function (41, 53). Based on a combination of function and cellular localization, one classification of these exchangers has divided them into housekeeping, epithelial, and intracellular isoforms. For instance, by this classification, NHE1 (ubiquitously expressed) and NHE4 (expressed in kidney, stomach, intestine, uterus, kidney, brain, and skeletal muscle) are considered housekeeper isoforms (43, 46, 52). NHE2 and NHE3 are apical membrane exchangers in small intestine colon (NHE2, NHE3), gallbladder (NHE3), proximal tubule (NHE3), thick ascending limb of Henle (NHE2, NHE3), and distal collecting system (NHE2), among other organs (9, 38). NHE6 and NHE7 are present on the membranes of intracellular organelles; NHE7 is present in the trans-Golgi network (TGN), and NHE6 is in the recycling system although initially suggested as being in mitochondria (5, 37, 38).

A weakness of this classification is that NHE3 is not only present on the plasma membrane but is also present in recycling endosomes. In fact, in PS120 and AP-1 fibroblasts, only ~15% of NHE3 is on the plasma membrane, whereas ~85% is intracellular (1, 11, 19, 20). Furthermore, potent agonists such as serum increase plasma membrane NHE3 activity by a maximum of twofold, leading us to question whether intracellular NHE3 may be serving a second function in addition to being a reservoir for plasma membrane recruitable NHE3. The concept of intracellular NHE3 serving an independent function from transcellular Na+ absorption is strengthened by the observations that, under basal conditions, NHE3 continuously cycles from the cell surface to an endosomal compartment through a phosphatidylinositol (PI) 3-kinase-dependent exocytosis mechanism (10, 14). Furthermore, signals within the COOH-terminus of NHE3 are involved in mediating the rapid basal endocytosis of the exchanger (1). In fact, recent work has demonstrated that endosomal NHE3 in opossum kidney (OK) cells may function to both acidify endosomes and to facilitate vesicular fusion (14, 15).

When considering the role of NHE3 in epithelial cells, it becomes important to identify the subcellular distribution in which NHE3 occurs. For instance, in renal proximal tubule, NHE3 is present in the brush border (BB) and in subapical vesicles, which are likely to represent endosomes, although how much NHE3 exists in each pool and the specific nature of this pool was not reported (4).

In this study, we used proximal tubule-derived polarized OK cells to examine the subcellular distribution of NHE3 and the function of its multiple pools. Our results show that, at steady state, the majority of NHE3 in OK cells resides in an intracellular recycling endosomal compartment. This pool of NHE3 moves to the surface in a PI 3-kinase-dependent manner. NHE3 functions on both the plasma membrane as well as in intracellular endosomes. In addition to its role in transcellular NaCl and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption, plasma membrane NHE3 is responsible for an apical membrane acid microenvironment, whereas intracellular NHE3 is at least partially responsible for acidification of the recycling compartment. Moreover, BB NHE3 is present in a distinct subdomain, appearing to be concentrated within a cluster of microvilli in the center of the apical surface. A significant fraction of OK surface NHE3 is present in very large complexes (~900 kDa) and/or in lipid rafts (LRs) and is restricted to the detergent-insoluble fraction of the BB. This concept, that NHE3 is present in distinct subdomains of the BB and in specific intracellular organelles, indicates careful spatial control of NHE3 localization, which has potential functional consequences.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Cell culture. OK-P cells expressing NHE3 endogenously (generously provided by Dr. O. Moe, University of Texas, Southwestern; Refs. 10, 35, and 55) and OK-E3V cells (stably expressing rat NHE3 epitope tagged on the COOH-terminus with the VSVG epitope as described in Ref. 36; generously provided by Dr. J. Noel, University of Montreal) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 25 mM NaHCO3, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2-95% O2 incubator at 37°C. OK-E3V cells were routinely selected for Na+/H+ exchange activity (every other passage) by exposing cells to an acid load consisting of 50 mM NH4Cl-94 mM NaCl solution for 1 h, followed by an isotonic 2 mM Na+ solution, as described previously (31). Surviving cells were then placed in normal culture medium and allowed to reach confluence. Except where stated, cells were studied after being serum starved for 3 days after reaching confluence, which increases Na+/H+ activity (10).

In some experiments, a fusion protein of NHE3 and enhanced green fluorescent protein (eGFP) at its COOH-terminus was transiently transfected into 50% confluent monolayers of OK-E3V cells using Lipofectamine (Invitrogen, GIBCO BRL), following the manufacturer's recommendations. This NHE3 chimera has been described previously and shown to behave like wild-type NHE3 in terms of percentage expressed on the surface of PS120 cells, basal transport, and fibroblast growth factor-stimulated and wortmannin-inhibited transport (19, 20). Twenty-four hours after transfection, serum was removed, and the cells were studied seventy-two h later.

Measurement of Na+/H+ exchange by fluorometry/BCECF. Cells were seeded on glass coverslips and grown until they reached 100% confluency. Transport was measured as described (31) by using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM; Molecular Probes). In the wortmannin experiments, the cells were incubated with 100 nM wortmannin for 60 min at 37°C. Control and test experiments were done on the same day by using parallel coverslips.

Fluorescence microscopy for pH measurements of surface and juxtanuclear compartments. A confluent cell monolayer on a glass coverslip was used at room temperature in a chamber mounted on the stage of an inverted microscope (25). Wheat germ agglutinin (WGA) conjugated with FITC was employed as the pH indicator and was initially exposed to OK cells for 30 min at 22°C. Excitation-ratio imaging at 440 and 490 nm was achieved with a 75-W xenon lamp, epifluorescence illumination, a ×100/1.3 N.A. objective lens, and excitation filters (Omega Optical, Brattleboro, VT) on a filter wheel. The microscope output port was connected to an intensified charge-coupled device camera (Hamamatsu). Images were digitized at 8 bits and stored on computer for offline analysis. For each monolayer, two pairs of images from the cell surface and in the juxtanuclear area were collected just superior to the nucleus under control conditions and during perfusion with the NHE3 inhibitor S3226 (100 nM; kindly provided by Dr. H. Lang, Aventis). The fluorescent images were analyzed for pH by choosing five to eight representative cells from each pair of images. The intensity ratio for each cell on the cell surface and the intracellular juxtanuclear region (selected visually) was calculated by using image processing MetaMorph software and compared with a pH calibration curve.

A pH calibration curve was generated by measuring the ratio of fluorescence emission intensities with the 440- and 490-nm excitation of WGA-FITC dissolved in buffer solutions with known pH values. A 20-µl drop of solution was placed on a coverslip for each of five different pH calibration standards from pH 5.5 to 8.0, a pair of images was taken, and fluorescence intensity ratio was determined.

Cell surface biotinylation. OK cells were grown to 100% confluence in 10-cm petri dishes. For wortmannin studies, 100 nM wortmannin were added for 60 min at 37°C. All subsequent manipulations were performed at 4°C. Cells were washed twice in phosphate-buffered saline (PBS; 150 mM NaCl and 20 mM Na2HPO4, pH 7.4) and once in borate buffer (154 mM NaCl, 10 mM boric acid, 7.2 mM KCl, and 1.8 mM CaCl2, pH 9.0). The surface plasma membrane proteins were then biotinylated by gently shaking the cells for 40 min with 3 ml of borate buffer containing NHS-SS-biotin (0.75 mg/ml; Pierce), with a fresh 3 ml of the same biotinylation solution added at 20 min. The cells were then washed extensively with the quenching buffer (20 mM Tris and 120 mM NaCl, pH 7.4) to scavenge the unreacted biotin and subsequently washed twice with PBS.

Cells were scraped and solubilized with 1 ml of N+ buffer (60 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM KCl, 5 mM EDTA trisodium, 3 mM EGTA, 1 mM Na3VO4, and 1% Triton X-100), sonicated for 20 s, agitated on a rotating rocker at 4°C for 30 min, and then centrifuged at 5,000 g for 10 min to remove insoluble cellular debris.

To determine the percentage of cell surface NHE3V, biotinylated surface proteins were precipitated by two 2-h incubations with streptavidin-agarose beads at 4°C. The depletion of biotinylated proteins was ascertained by the absence of proteins in a further avidin precipitation. Biotinylated proteins bound to the avidin-agarose beads were eluted by heating in Laemmli sample buffer and were subsequently analyzed in an immunoblot (1). Biotinylated NHE3V was quantitated by using monoclonal antibodies to the VSVG epitope (P5D4 hybridoma tissue culture supernatant). The amount of surface NHE3 was expressed as a percentage of total cellular NHE3, which was determined by using Western analysis of whole cell lysates (1).

In some experiments, NHE3V was immunoprecipitated from the biotinylated cell extract by using anti-VSVG monoclonal antibodies and visualized through Western analysis. Surface NHE3V was recognized by using horseradish peroxidase-conjugated streptavidin (HRP-streptavidin; Molecular Probes), whereas total NHE3V was recognized by using anti-VSVG monoclonal antibodies followed by exposure to enhanced chemiluminescent solution.

Cell fractionation into detergent-soluble and detergent-insoluble fractions. Surface NHE3 was separated into detergent-soluble (DS) and detergent-insoluble (DI) fractions. Cells were grown to confluency in 10-cm Petri dishes and biotinylated at 4°C, as described in Cell surface biotinylation. Cells were then solubilized in 50 mM MES buffer, pH 6.4, containing 60 mM NaCl, 3 mM EGTA, 5 mM MgCl2, 1% Triton X-100, and 1 mM vanadate plus 1:1,000 protein-inhibitor cocktail (Sigma), sonicated, and spun briefly to remove unbroken cells, nuclei, and cell debris. The resulting supernatant was subsequently centrifuged at 100,000 g for 30 min to recover the DI fraction, while the supernatant was designated as the DS fraction. The DI pellet was solubilized in RIPA buffer [150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris · HCl, pH 8.0]. Surface NHE3V was avidin precipitated as described in Cell surface biotinylation. Equal volumes of surface DS and DI fractions and total DS/DI fractions were separated by SDS-PAGE.

Sucrose gradient centrifugation. Cells were grown to confluency in 10-cm petri dishes. Cells were then solubilized in 1 ml of N+ buffer (see Cell surface biotinylation), sonicated, and spun briefly to remove unbroken cells, nuclei, and cell debris. Solubilized cell extracts (4°C or 1°C in 1% Triton X-100) were applied to the top of discontinuous 5-25% sucrose gradients (increasing at increments of 2.5% sucrose containing 0.1% Triton X-100). After centrifugation for 16-18 h at 4°C at 150,000 g in an Sw41 rotor, the gradients were fractionated (0.6 ml) from the bottom with a pump and NHE3 immunoprecipitated with anti-VSVG monoclonal antibody from each fraction. Complex size was estimated by comparison to standard proteins (beta -amylase = 200 kDa, apoferritin = 443 kDa, and thyroglobulin = 669 kDa) run on parallel identical sucrose gradients.

Immunofluorescence. Cells were seeded on glass coverslips, and grown until they reached 100% confluency, and then studied 72 h after serum was removed. Cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature. The residual paraformaldehyde was neutralized with 20 mM glycine in PBS for 10 min. Cells were then permeabilized for 30 min in 0.1% saponin/PBS before being blocked for 30 min in 1% BSA/PBS supplemented with 10% FBS. Cells were incubated in primary antibody (at the indicated concentrations) in PBS for 60 min at room temperature. After three 10-min washes in 0.1% saponin/PBS, secondary antibodies (Alexa conjugates, Molecular Probes) were added at 1:100 in PBS, incubated for 30 min, and again washed three times for 10 min. Cells were then mounted on slides with Prolong (Molecular Probes) antifade reagent and viewed on a Zeiss LSM 410 confocal fluorescent microscope. For lectin experiments, cells were labeled at 4°C with 100 µg/ml fluorescently labeled lectins for 30 min before fixation [WGA, phytohemagluttin E (PHA-E), cholera toxin B (CTB) subunit, or peanut agglutinin; Sigma].

Antibodies. Anti-VSVG monoclonal antibodies were generated from P5D4 hybridoma supernatant (26) and used at a dilution of 1:5 in PBS for immunoprecipitation. Polyclonal anti-VSVG antibodies 1648 were as described (14) and used at a dilution of 1:1,000. Polyclonal antibodies to endogenous OK NHE3 (Ab 5683) were a generous gift from Dr. Moe and were used at a dilution of 1:2,000 (10). Rab11 (monoclonal) and EEA1 (polyclonal) antibodies were purchased from Transduction Laboratories (Lexington, KY) and used at dilutions of 1:1,000. Monoclonal antibodies to Na+-K+-ATPase were a gift from Dr. Fambrough (Department of Biology, Johns Hopkins University School of Medicine) (13) and were used at a dilution of 1:1,000. Monoclonal anti-Biotin conjugated with HRP was from Jackson ImmunoResearch Lab (200-032-096).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NHE3V in OK-E3V cells is expressed at a similar level as endogenous NHE3 in OK-P cells. OK-P cells express endogenous NHE3 as their only NHE isoform. In preliminary immunofluorescence experiments, antibodies to endogenous NHE3 (Ab 5683) did not give a clean signal. Accordingly, we studied OK cells stably expressing epitope-tagged NHE3 (OK-E3V), which was identified with anti-VSVG monoclonal antibodies (P5D4). OK-E3V cells were derived as previously described (36). Briefly, a population of OK cells (OK-Tina) was subjected to repeated cycles of acid suicide to reduce expression of endogenous NHE3. Transport experiments demonstrated that expression of endogenous NHE3 was reduced to ~10% of the wild-type cell line. The low-expressing cells were subsequently stably transfected with COOH-terminal VSVG-tagged rat NHE3 (NHE3V). Western analysis of OK-P and OK-E3V cell lysates using antibodies to endogenous NHE3 demonstrated that endogenous OK NHE3 migrated as a 90-kDa band, whereas NHE3V migrated as a slightly smaller 85-kDa band. Only the smaller band was recognized by antibodies to the VSVG epitope (Fig. 1A). Comparisons of the NHE3 expression levels in the two cell lines showed that OK-P and OK-E3V cells expressed approximately equal levels of NHE3 (see Fig. 1A).


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Fig. 1.   Expression of NHE3V in opossum kidney (OK) cells. A: OK cells were grown on glass coverslips as described (10). Cells were solubilized in 1% Triton X-100, and 40 µg of cell extract were separated on a 10% SDS-PAGE gel and immunoblotted with either anti-NHE3 Ab 5683 or anti-VSVG P5D4 hybridoma supernatant and visualized through enhanced chemiluminescence. Similar amounts of NHE3 and NHE3V were recognized by Ab 5638. B: Na+/H+ exchange was measured in OK cells grown on glass coverslips. Cells were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein for 30 min before being mounted in a spectrofluorometer cuvette. Cells were acidified by brief exposure to 40 mM NH4Cl and then rinsing with TMA+ solution. Intracellular pH was allowed to recover through NHE3 activity by the addition of Na+ solution. Intracellular pH was calibrated by using the K+ ionophore nigericin (31).

Na+/H+ exchange was measured in OK-P and OK-E3V cells by using the pH-sensitive dye BCECF. Representative traces of NHE3 activity in OK-P and OK-E3V cells are shown in Fig. 1B. To confirm the isoform of expressed NHE, we verified that Na+/H+ exchange in OK-P and OK-E3V cells was completely abrogated, in a fully reversible manner, in the presence of 100 nM of the specific NHE3 inhibitor S3226 (Ref. 48; data not shown). Transport rates of NHE3 in OK-P and OK-E3V were very similar (Fig. 1B), consistent with similar expression levels of NHE3 in the two cell lines. Furthermore, regulation of NHE3 by such agents as cAMP and angiotensin were reproduced in OK-E3V cells (M. Cavet and M. Donowitz, unpublished results). On the basis of expression levels, transport rates, and regulation, we concluded that OK-E3V cells do not represent an overexpression model for the study of NHE3 in polarized epithelial cells.

A minor fraction of both endogenous NHE3 and NHE3V targeted to the OK apical membrane-BB NHE3V is predominantly present in a central apical surface location. Surface expression of endogenous NHE3 and transfected NHE3V in OK cells was monitored by using cell surface biotinylation. Western analysis of total and surface biotinylated proteins using both endogenous NHE3 antibodies and anti-VSVG antibodies showed that only a minor fraction of both NHE3 and NHE3V (~10-15%) was successfully biotinylated (Fig. 2A). Because we could not eliminate the possibility that surface biotinylation in OK cells was incomplete, we next examined the cellular location of NHE3 with confocal microscopy. To mark the BB, fluorescently labeled lectins and CTB subunit were applied to the apical surface of live cells at 4°C. OK cells grown on glass coverslips were polarized with BB facing the free solution. These cells mimicked the binding specificities of native proximal tubule cells in that their apical membranes stained positively with PHA-E, CTB subunit, and WGA but not peanut agglutinin (data not shown) (Fig. 2, B, D, and F; Ref. 8). The positive lectins and CTB exhibited different surface-binding patterns. PHA-E and CTB labeled the microvilli diffusely and also labeled the junctional areas of cells (Fig. 2, B and D). WGA had less junctional labeling and less staining of the most lateral microvilli compared with PHA-E and CTB but also otherwise labeled microvilli diffusely. NHE3 was visualized in the same cells (Fig. 2, C, E, and G). NHE3 was present in the BB where it had predominantly a central apical location, although some diffuse apical NHE3 was present. The fact that NHE3 was not diffusely present in the microvilli is shown in Fig. 2, C, F, and G, in which peripheral microvilli as marked by lectins and CTB lack NHE3.


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Fig. 2.   Lectin and NHE3V staining of OK cells. A: OK-E3V and OK-P cells grown on plastic dishes were biotinylated from the surface at 4°C, lysates were then prepared, and avidin was precipitated at 4°C. Avidin precipitates and lysates were separated on SDS-PAGE, and Western analysis was performed with anti-NHE3 hybridoma supernatant P5D4 and anti-NHE3 antibody Ab 5683. Approximately 10-15% of NHE3 was present on the surface of both cell lines. This is a single representative experiment repeated 5 times. B-G: OK-E3V cell monolayers grown on plastic dishes were incubated with 100 µg/ml fluorescent lectin or cholera toxin B (CTB) for 30 min at 4°C (B, D, and F). Cells were rinsed with cold PBS and then fixed, permeabilized, and incubated with antibodies to NHE3V (C, E, and G). Images are all 0.5-µm confocal sections at the level of the most superior aspects of the apical surface of OK cells, except F and G, which were 0.5-um confocal sections taken ~1 um below the cell apex. Magnification was increased in F and G to allow demonstration of individual microvilli. A single confocal section is shown of OK cells labeled with phytohemagluttin E (PHA-E; B) and NHE3V (C); CTB (D) and NHE3V (E); wheat germ agglutinin (WGA; F) and NHE3V (G).

Fibroblasts expressing NHE3 target the majority of the exchanger to a discrete intracellular compartment that colocalizes with markers of recycling endosomes including cellubrevin and the transferrin receptor (11). The localization of NHE3 in OK cells also consists of surface plus intracellular, juxtanuclear staining as seen in fibroblasts. Because our biochemical studies indicated the presence of a large intracellular pool of NHE3 as well as a surface pool, further studies were undertaken to characterize these populations, including determination of the location of intracellular NHE3 as well as the centrally located, apical NHE3.

Two confocal microscopic approaches were used. The first was done in living cells and examined eGFP chimeras of NHE3 transiently transfected into OK cells. Localization of NHE3 was compared with location of surface WGA, marked with TRITC (Fig. 3A). The second method colocalized NHE3 and a marker of the general recycling compartment (Rab11) and of the plasma membrane (WGA at 4°C ; Fig. 3, B and C). Figure 3A shows the subcellular distribution of NHE3 in living OK cells. To visualize the exchanger, NHE3 was tagged at its COOH-terminus with eGFP (NHE3-eGFP), as previously described (19, 20), and transiently transfected into OK cells as described in MATERIALS AND METHODS. The BB of OK cells was labeled with fluorescent WGA, and the cells were examined by using confocal microscopy, all kept at 4°C. Serial X-Y sections (1 µm) are shown and demonstrate that the majority of NHE3-eGFP fluorescence is present in an apical juxtanuclear compartment that is labeled in an area devoid of lectin. A small fraction of the exchanger colocalizes with WGA with the overlapping area concentrated in a central apical domain. The expression of NHE3-eGFP in OK cells confirms that only a small fraction of the exchanger is found on the cell surface, with the majority of the protein in an intracellular juxtanuclear location.



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Fig. 3.   ---Continued Colocalization of NHE3 with surface and intracellular organelle markers in OK cells. A: WGA and NHE3-eGFP in OK cells. OK cells were transiently transfected with NHE3-eGFP by using Lipofectamine. Monolayers were incubated with 100 µg/ml/TRITC-WGA for 30 min at 4°C. Slides were mounted on a cooled microscope stage (4°C), and serial 1-µm X-Y sections were obtained from the level of the nucleus (i) to the apical surface of the cells (v) at levels indicated in inset. Red marks WGA binding and green denotes NHE3-eGFP. There was colocalization of NHE3 and WGA at the central part of the apical surface, as indicated by yellow, but not more laterally in the brush border (BB). The contrast to visualize the intensity of WGA binding was elevated above saturation equally in all sections to ensure that any intracellular WGA was detected. No intracellular WGA was detected. Inset: an interpretation of NHE3 location in green based on its location in i-v. B: NHE3V, Rab11, and EEA1 in OK-E3V cells. Images are single 0.5-µm X-Y confocal sections of OK-E3V cells taken above the level of the nucleus as shown in inset in A. i-iii: OK cells were stained with antibodies to NHE3V (i; red) and Rab11 (ii; green). Composite images are shown in iii. The majority of, but not all, intracellular NHE3V colocalized with Rab11. iv-vi: OK cells were stained with antibodies to NHE3V (iv; red) and EEA1 (v; green). Coposite images are shown in vi. Most NHE3V was in vesicles that appeared different from those containing EEA1, although there appeared to be some overlap, indicated by yellow. C: NHE3 colocalizes with surface WGA and intracellular Rab11 in OK-E3V cells. Images are X-Z reconstructions of serial 0.5-µm confocal sections of OK cells taken from the surface to above the level of the nucleus. OK cells were stained with secondary antibodies tagged with Alexa-568 to NHE3V (i; red), Alexa-488 to Rab11 (ii; green), and Alexa-350 to WGA (iii; blue). Composite images are shown of NHE3V/WGA (iv) and NHE3V/Rab11 (v). Two cells are shown with diminished staining of NHE3V in BB toward the areas of cell-cell contact. Moreover, 2 distinct pools of NHE3 are demonstrated (purple for NHE3 and WGA and yellow for NHE3 and Rab11). D: intracellular, supranuclear colocalization of NHE3 and Rab11 in OK-E3V cells. X-Z reconstruction of serial 0.5-µm confocal X-Y sections as in C, except the nuclei are stained with Hoechst 33245 (blue) and WGA staining is absent. Note colocalization of NHE3V and Rab11 (yellow) in a juxtanuclear location and external NHE3V in a central BB location (red). E: localization of NHE3V and Na+-K+-ATPase in OK-E3V cells. X-Z reconstruction of serial 0.5-µm confocal X-Y sections. NHE3V is stained with Alexa-568-tagged antirabbit secondary antibody to polyclonal anti-VSVG antibody 1648 (red), and Na+-K+-ATPase is stained with Alexa-488 (green)-tagged anti-mouse secondary antibody to mouse monoclonal antibody. Note lateral but not basal staining of Na+-K+-ATPase and no colocalization of NHE3V and Na+-K+-ATPase.

The overall distribution of intracellular NHE3V in a juxtanuclear subapical location suggested that the exchanger was localized to a population of intracellular endosomes. We used Rab11 as a marker for recycling endosomes (6) and found that the distribution of intracellular NHE3V in a 0.5-µm X-Z optical section closely resembled the staining for Rab11 (Fig. 3B). The distribution of early endosomes was examined by using antibodies directed against EEA1 (early endosomal antigen-1), which specifically associates with early endosomes (54). EEA1 marked punctuate structures dispersed throughout the apical cytoplasm of OK cells (only sections above the nucleus were collected), whereas NHE3V maintained a more constrained juxtanuclear distribution (Fig. 3B). Although some intracellular organelles appeared to stain for both NHE3V and EEA1, the general staining patterns of the two proteins were dissimilar, suggesting that NHE3V was preferentially localized to recycling endosomes (Rab 11 positive) vs. early endosomes under the steady-state conditions of these studies.

In separate studies, OK-E3V monolayers, with the BB marked with WGA (blue), were stained for Rab11 (green) and NHE3V (red). X-Z sections are shown in Fig. 3C. NHE3V on the apical surface was present maximally in a central location with less NHE3V near the cell junctions. Two pools of NHE3V were demonstrated: NHE3V and WGA colocalized in a central apical distribution, whereas NHE3 and Rab11 localized in a more internal but still centralized distribution. This qualitative separation allowed us to differentiate between the two pools of NHE3V within one cell. Figure 3D shows colocalization of intracellular NHE3V (red), Rab11 (green), and nuclei (blue), again demonstrating colocalization of an intracellular pool of NHE3V and Rab11 in a juxtanuclear location, plus an external pool of NHE3V with central BB localization. The total height of the OK monolayer is shown by marking the lateral cell border with an antibody to Na+-K+-ATPase (Fig. 3E). The central apical surface location of NHE3V is demonstrated, as is the apical distribution of NHE3V that exceeds the BB thickness and is made up of BB plus supranuclear (Rab11 containing) NHE3V.

Characteristics of surface NHE3 in OK-E3V cells: surface NHE3 is in a large DI complex. The central clustered distribution of BB NHE3V suggested that it might be tethered to prevent diffuse BB distribution. Studies of renal proximal tubule have shown that endogenous NHE3 has limited solubility with various detergents, further suggesting the link between NHE3 and cytoskeleton (3). To investigate these potential interactions, the solubility characteristics of both total and surface NHE3V in OK cells were examined. Surface NHE3V was biotinylated before separating DS and DI fractions. Figure 4 demonstrates that, whereas the majority of total NHE3V is in the DS pool, surface NHE3 is only ~10% in the DS (~90% in the DI) fraction.


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Fig. 4.   Detergent solubility of total and surface NHE3V. OK-E3V cells were surface biotinylated at 4°C, and detergent-soluble (DS) and -insoluble (DI) fractions were separated as described in MATERIALS AND METHODS. NHE3V was immunoprecipitated from equal volumes of DS and DI fractions and then separated on a 10% SDS-PAGE gel. Total NHE3V was visualized by using anti-VSVG antibodies before the same blot was stripped and surface NHE3V visualized by using horseradish peroxidase-conjugated (HRP)-streptavidin. Results from 2 separate OK-E3V preparations studied simultaneously are shown. Total NHE3 is almost entirely in the DS, whereas surface NHE3 is ~90% in the DI fraction.

Sucrose gradient centrifugation studies examining the complex size of NHE3 in solubilized renal microsomes have shown that the protein migrates in a broad distribution with a significantly higher sedimentation coefficient than that for NHE1 (2, 3). We investigated the complex size for both endogenous and transfected NHE3 in OK cells. Cells were solubilized in 1% Triton X-100, the extract was applied to the top of a 5-25% sucrose gradient, and, after centrifugation for 16-18 h, the resulting fractions were collected and analyzed by immunoblotting. Figure 5Aa shows fractions of OK-P cells immunoblotted with antibodies to endogenous NHE3. As in the proximal tubule, endogenous NHE3 migrates as a broad peak near the 400-kDa standard, indicating that it is not only part of a large protein complex but is heterogeneous in size. Sucrose gradient centrifugation experiments of biotinylated OK-E3V cells with NHE3V immunoprecipitated from each fraction are shown in Fig. 5A, b and c. Total NHE3V (Fig. 5Ab) visualized by using anti-VSVG antibodies, like endogenous NHE3, migrates as a similar-sized broad band, indicating that the transfected protein behaves like the endogenous protein in terms of its location in large protein complexes. Figure 5Ac shows the same gradient probed with HRP-streptavidin, allowing the specific visualization of surface NHE3V. Although the majority of total NHE3V migrates at a ~400-kDa peak, a small fraction of the exchanger migrates as ~900-kDa complexes. Examinations of surface complex sizes demonstrated, however, that surface NHE3V is significantly enriched in the ~900-kDa complex, although some ~400-kDa complexes were also present on the plasma membrane (Fig. 5Ac). Because of insufficient antibodies, we were unable to immunoprecipitate endogenous NHE3 from OK-P cells. The inability to visualize the small fraction of ~900-kDa NHE3-containing complex in OK-P cells may have resulted from our inability to specifically immunoprecipitate, and hence concentrate, NHE3.


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Fig. 5.   Size of total and surface NHE3V. OK cells were biotinylated, and cells were lysed in 1% Triton X-100 at either 4 or 15°C. Cell extracts were layered on top of a discontinuous 5-25% sucrose gradient and centrifuged at 150,000 g for 16-18 h at 4°C. Fractions were collected in 0.6-ml aliquots from the bottom of the gradient. A: most surface NHE3V exists in larger complexes than that in intracellular pools. a: equivalent volumes of each fraction from OK-P cells were separated on a 10% SDS-PAGE gel, and endogenous NHE3 were visualized by using Ab 5683. b and c: NHE3V was immunoprecipitated from each fraction, and total NHE3V was visualized by using antibodies to anti-VSVG (b). The blot was then stripped and surface NHE3V was visualized with HRP-streptavidin for surface NHE3V (c). Triton X-100 lysis was done at 4°C for 30 min. B: sizes of NHE3V complexes were not significantly changed whether OK-E3V cells were lysed in 1% of Triton at 15°C (a and c) or 4°C (b and d). Equivalent volumes of each fraction from OK-E3V cells were separated on a 10% SDS-PAGE gel, and NHE3V was visualized either by using anti-VSVG for total NHE3V (a and b) or antibiotin for surface NHE3V (c and d). NHE3V detected by antibiotin was confirmed by immunoprecipitation with anti-VSVG Ab. Estimations of complex sizes were based on addition of the standards shown on an identical sucrose gradient studied simultaneously.

We have previously reported that in rabbit ileal BB, a significant percentage of NHE3 is in LR (32). Thus it was important to consider whether the large NHE3 apical complexes in OK cells included LR. In these studies we assumed that LR would be soluble in Triton X-100 at 15°C, whereas non-LR protein complexes would remain stable at this temperature. This was because the definition of LR includes insolubility in Triton X-100 at 4°C. Shown in Fig. 5B, NHE3 on the surface of OK cells distributed very similarly whether solubilized at 4 or 15°C. In fact, there was a small shift to slightly larger complexes at 15°C, suggesting that most of the large NHE3-containing surface complexes present under basal conditions did not involve LR. A warning, however, is that this temperature comparison has not been used frequently to define the role of LR in membrane complex formation.

Wortmannin treatment reduces both transport rates and surface levels of NHE3. NHE3 stably transfected into both PS120 and AP-1 fibroblasts is found both in an intracellular recycling endosome compartment as well as on the plasma membrane. Under basal conditions, the exchanger rapidly trafficks between the recycling endosomes and the cell surface in a PI 3-kinase-dependent manner. In fibroblasts, wortmannin, a potent inhibitor of PI 3-kinase, reduces the transport rates and surface levels of NHE3 by inhibiting the exocytosis of the exchanger back to the plasma membrane (19, 27). Because NHE3V in OK cells also appeared to be targeted to both cell surface and intracellular sites, it was determined whether NHE3, in the context of a polarized epithelial cell, also underwent rapid basal recycling. Wortmannin treatment (100 nM, 60 min, 37°C) significantly reduced Na+/H+ exchange rates in both OK-P and OK-E3V cells (Fig. 6). Wortmannin reduced NHE3 transport by 57% in OK-P cells and 51% in OK-E3V cells. Cell surface biotinylation indicated that the reduction in transport rates was accompanied by a corresponding reduction in surface NHE3 levels. Surface levels of endogenous NHE3 in OK-P cells were reduced by ~50% with 1 h of wortmannin treatment. Surface levels of NHE3V in OK-E3V cells showed a similar reduction (Fig. 6).


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Fig. 6.   Wortmannin treatment of OK cells decreased NHE3 activity and surface expression. OK cells were incubated with 100 nM wortmannin for 60 min at 37°C. NHE3 activity was measured as described. Vmax Na+/H+ exchange rates are shown, calculated as described (31). Surface levels of NHE3 were determined by biotinylating cells at 4°C using avidin-agarose. Avidin-agarose pellets were solubilized in sample buffer, and equivalent volumes from control and wortmannin-treated cells were separated on a 10% SDS-PAGE gel. NHE3V was visualized by using antibodies against VSVG.

NHE3V functions in both surface and intracellular locations. Studies were undertaken to determine whether NHE3 functioned in both its major cellular locations, on the cell surface and in an intracellular Rab11-positive endosomal pool. pH in these compartments was measured by exposing the OK cells to FITC-WGA at 22°C for 60 min, allowing the cells to take up the WGA. Of note, after 60 min at 22°C, WGA, NHE3V, and Rab11 had similar locations based on confocal colocalization studies (compare Figs. 3C and 7A). To ascertain the role of functional NHE3V, the exchanger was inhibited by exposure to the specific blocker S3226 (100 nM; 48), and the pH on the surface as well as inside the cell was compared before and after NHE3V inhibition (Fig. 7B). The surface pH after S3226 exposure as measured by FITC-WGA was alkalinized, suggesting that NHE3 activity contributed to the low pH of the BB surface. The intracellular compartment marked with WGA also alkalinized with S3226 exposure. Because FITC-WGA was inside this intracellular compartment, this result indicated that the function of NHE3 in that compartment was to acidify it.


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Fig. 7.   A: distribution of NHE3, WGA, and Rab11 after 30 min at 22°C. Cells and antibody staining are as in Fig. 3C, except cells were kept at 22°C for 30 min after initial exposure to WGA at 4°C. Shown are X-Z reconstruction of serial X-Y (0.5 µm) sections from surface to level of nucleus. The same colors are used to mark NHE3V (red), Rab11 (green), and WGA (blue) as in Fig. 3C. Note large NHE3V, WGA overlap (purple), and large overlap of all three (white), which differs from the WGA localization at 4°C shown in Fig. 3C. These are the conditions used for FITC-WGA measurement of pH in B. B: pH measurements on the surface and in the juxtanuclear region of OK cells using FITC-WGA. Cells were allowed to take up WGA for 30 min, and pH was measured by using the FITC bound to the WGA. Studies were performed in the absence of ~5 min after exposure to S3226 (100 nM). In all cases, inhibiting NHE3 led to alkalinization, both of the surface and in the juxtanuclear location. 1, surface pH; 2, intracellular pH. Filled bars, pH before S3226; open bars, pH after 5-min perfusion with S3226. * P < 0.001; paired t-test; n = 18 cells. ** P < 0.001; paired t-test; n = 18 cells. The difference between surface and intracellular pH with and without S3226 is not statistically significant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although BB membrane Na+/H+ exchange in epithelial cells has been partially characterized, studies have not specifically used cell biological approaches to examine the subcellular localization of the apical Na+/H+ exchanger isoform, NHE3. Studies of NHE3 transfected into fibroblasts have shown that the protein is expressed in a surprising pattern for an exchanger postulated to work only on the plasma membrane. Specifically, only a minor fraction of NHE3 in two unrelated fibroblast lines is targeted to the plasma membrane, whereas the vast majority is retained in a recycling endosome compartment (11, 19). There are other reasons to suspect that NHE3 is present in multiple subcellular locations. We have previously shown that the COOH terminus of NHE3 contains at least two independent signals that determine the endocytic rate and, consequently, the plasma membrane levels of the exchanger (1). Furthermore, a number of studies have demonstrated a role for membrane trafficking in the regulation of NHE3 function in both polarized epithelial cells and native tissue (18, 21, 42, 55-58). Finally, electron microscopy of NHE3 in kidney proximal tubule has shown some vesicular staining in addition to BB membrane and intermicrovillar cleft populations (3, 4). Despite the evidence suggesting the existence of multiple pools of NHE3 in kidney and intestine, little has been reported on the subcellular distribution of NHE3 in physiological settings. Unlike the situation of other transport proteins such as GLUT4, gastric H+-K+ ATPase, or aquaporin-2, which are present largely in intracellular pools under basal conditions and respond to physiological stimuli by massive mobilization to the plasma membrane (12, 17, 40), the more subtle peak stimulation of NHE3 (amount of plasma membrane NHE3 increases ~2 times) prompted us to question what other function the intracellular exchanger might have (1, 19, 32).

For these studies, the OK polarized proximal tubule cell line was chosen because these cells express endogenous NHE3 that is correctly targeted to the apical membrane and is sensitive to regulation by such physiological agents as dopamine, endothelin, and parathyroid hormone (PTH; Refs. 10, 18, 29, and 36). Furthermore, the existence of stably transfected NHE3V-expressing cells allowed us to study the expression patterns of the exchanger in a setting that demonstrated similar transport rates to the endogenous exchanger (Fig. 1). Both cell surface biotinylation studies and confocal microscopy experiments showed that the minority of total cell NHE3V is on the BB (10-15% of total), whereas the majority of NHE3V is intracellular, with most in the Rab11-associated recycling compartment (Figs. 2A and 3, A and B). Moreover, as also seen in the fibroblast cell lines, NHE3V cycles under basal conditions from the cell surface to an endosomal population through a mechanism that is at least partially regulated by PI 3-kinase dependent exocytosis. Inhibition of PI 3-kinase by wortmannin decreases both the percentage of NHE3V on the plasma membrane and the transport rates of the protein (Fig. 6). The PI 3-kinase-dependent cycling of NHE3 under basal conditions has been found in all cell types studied, polarized and symmetrical, and appears to be a general property of NHE3.

Although some NHE3V colocalized with EEA1, a marker for early endosomes, the distribution of NHE3V more closely mirrored that of Rab11, a marker for recycling endosomes (Fig. 3B). Although NHE3 is endocytosed under basal conditions, the greater colocalization with Rab11 would suggest that NHE3 is either preferentially retained in the Rab11-positive compartment or internalized through a population of EEA1-negative endosomes. Studies with the apical Na+-phosphate cotransporters NaPi-2 and NaPi-4 in OK cells have shown that both proteins are rapidly internalized with PTH stimulation and then degraded in subapical lysosomes (23, 24). It remains to be determined what signals mediate why endocytosed NHE3 is targeted to be recycled, whereas NaPi-2/4 are degraded.

We characterized the BB expression of NHE3 in OK cell monolayers. Several fluorescent-tagged lectins and CTB were used to label the microvillar surface. PHA-E and CTB both showed a diffuse surface distribution with clear labeling of the junctional areas. In contrast, although WGA also labeled microvilli, it appeared to be excluded from peripheral microvilli and the junctional areas. Staining of NHE3V in the same 0.5-µM X-Y confocal sections showed that the exchanger was not distributed diffusely on the OK apical surface. Rather, NHE3 seems to be in a cluster of centrally localized microvilli on the apical surface of most OK cells (Fig. 2, B--G). In a small fraction of cells, NHE3 demonstrated a more uniform distribution in clumped microvilli, an observation also made by Moe and colleagues for NHE3 and similar to distribution of NaPi-2 and NaPi-4 in OK cells (Refs. 18, 22; S. Akhter and M. Donowitz, unpublished results). Although NHE3 was concentrated toward the center of the apical cell surface, there was detectable staining throughout the BB, including toward the junctional complexes. Of note, the difference in the microvillar distribution of NHE3 compared with the lectins and CTB showed that it is NHE3 that is in a centralized location rather than the centralized location of the microvilli. The surface expression of NHE3V may reflect subdomains of protein expression on the BB of OK cells. Thus epithelial cell proteins may be targeted not only to the apical or basolateral domains but may be more specifically delivered to or retained at physical subdomains within these larger areas. Several other transport proteins have been shown to have restricted distributions on the surface of epithelial cells. 1) Functional studies by Oberleithner demonstrated that Na+ channels in Madin-Darby canine kidney (MDCK) cells had such a central localization (36, 39); 2) K+ channel, Kv1.4, clustered at the leading edge of migrating MDCK cells (47); 3) NHE1 is localized to pseudopodial protrusions in invasive MDCK cells (28); and 4) Sorribas et al. (50) showed that NaPi-2 and NaPi-4 in OK cells were expressed only in the clumped microvilli and were largely excluded from the intermicrovillar clefts. The existence of plasma membrane subdomains has been clearly demonstrated in neurons. Although the mechanism underlying these plasma membrane subdomains in epithelial cells in unknown in neurons, isoform-specific targeting of K+ channels in the dendrite is mediated through such diverse mechanisms as interactions with binding proteins, associations with the cytoskeleton, or targeting to LRs or caveolae (33, 34).

On a biochemical level as well, BB NHE3 was shown to be made up of multiple pools and not to consist of a single population of exchanger. This was demonstrated by 1) the fact that NHE3 exists in OK cells in DS and DI populations; although total NHE3V is largely soluble in detergent, surface NHE3V is almost entirely insoluble (Fig. 4); and 2) sucrose density gradient centrifugation of total cell protein. The total NHE3V is present in complexes of multiple sizes, with two predominant peaks of ~900 and ~400 kDa. Of interest, total NHE3V consists almost entirely of the 400-kDa pool, whereas surface NHE3V was fairly evenly distributed between the 900- and 400-kDa populations (Fig. 5A). Whether this represents NHE3 in multiprotein complexes and/or NHE3 presence in LRs, as we have documented occurs (32), is not known. The preliminary studies reported in Fig. 5B comparing complex size in apical surface NHE3 exposed to Triton X-100 at 4 and 15°C (difference interpreted as LR contribution) did not reveal a role for LRs in large surface NHE3 complexes under basal conditions. However, it will be important to extend these studies at least by using other ways to define LR and with stimulated NHE3.

In addition, enrichment of NHE3V in large DI complexes on the apical surface of OK cells suggests that the different pools of NHE3 may fulfill distinct functional roles. The endocytosis of albumin in the kidney proximal tubule, for instance, is mediated through binding such large scavenger receptors as megalin and cubilin (7). Independent studies have shown that 1) inhibition of NHE3 virtually eliminates receptor-mediated albumin uptake by OK cells (14, 15), and 2) proximal tubule NHE3 tightly associates with megalin, denoting a megalin-associated, inactive form of the exchanger which is targeted to the intermicrovillar microdomain of the apical plasma membrane (2, 3). Whether the 900-kDa surface NHE3 in OK cells also include a scavenger receptor isoform and whether it is this association that mediates the NHE3-dependent albumin uptake is not known.

A postulated function for the presence of BB NHE3 in large restricted complexes and/or LRs is suggested by the functional association between NHE3 and H+/peptide transport. Thwaites et al. (51) have demonstrated that inhibition of NHE3 in intestinal Caco-2 cells leads to a reduction in the rates of H+/peptide uptake, suggesting a coordinated activity of H+-solute symport with apical Na+/H+ exchange. Such an association, if correlated with physical proximity of the exchangers, would allow the creation of acidic microdomains on the surface of epithelial cells and, consequently, would optimize the efficient absorption of nutrients and Na+ while maintaining intracellular pH homeostasis and the ionic gradients involved in driving transport. Our measurements of surface pH using FITC-WGA did, in fact, verify that NHE3 substantially reduced the pH on the microvillar surface (Fig. 7B). Measurements of surface pH in both CaCo-2 monolayers and native tissue have shown the presence of an acidic environment, and in Caco-2 cells, this environment is significantly alkalinized in the presence of blockers of NHE3 (16, 24). The level of resolution of our measurements did not allow us to determine whether the BB central NHE3-rich area of OK cell surface was more acidic than the periphery. It also remains to be determined whether H+-solute transporters or other transport proteins driven by a H+ gradient are associated with NHE3 in the large surface complexes.

For both plasma membrane and intracellular NHEs, the directionality of Na+ and H+ transport is determined by the gradients of the ions across the phospholipid membrane. In most physiological settings, plasma membrane NHEs extrude intracellular H+ coupled to the inward movement of Na+ down its concentration gradient. The transport function of vesicular NHE3 would be expected to be dependent on the cytoplasmic and intravesicular Na+ and H+ concentrations. Although the intraendosomal pH concentration is expected to be acidic because of V-ATPase function, previous reports of endosomal NHE3 function in fibroblasts and OK cells have demonstrated that NHE3 works to further acidify the endosome (11, 14, 15). The required levels of intravesicular Na+ to drive H+ uptake could be derived either from the transport of Na+ via functional Na+-K+-ATPase in the endosomal membrane or through the delivery of Na+ via endocytic vesicles carrying Na+-rich extracellular fluid. In polarized epithelial cells, Na+-K+-ATPase is a basolateral membrane protein and would not be expected to be present in the apical membrane or to be available in the apical recycling endosomes. In this study, we measured intravesicular pH using internalized FITC-WGA and found that inhibition of NHE3 by S3226 raised endosomal pH, further supporting the role of NHE3 in acidifying endosomes (Fig. 7). WGA was chosen because it behaves similarly to NHE3, being endocytosed and distributing in the recycling endosome before trafficking back to the surface but does not distribute significantly in lysosomes (45, 49). Gekle et al. (15) have shown that inhibition of endosomal NHE3 in OK cells functions not only to alkalinize endosomes but also to reduce the fusion of vesicles early in the endocytic process. It is unclear whether NHE3 works to acidify both early and recycling endosomes. Because our measurement of vesicular pH by WGA targeted all vesicular compartments labeled during 30 min of uptake at room temperature, it is conceivable that the change in pH we detected was restricted to the early endosome population. Although it seems unlikely that endosomes later in the membrane trafficking system would retain a sufficient gradient of Na+ to fuel the uptake of H+, we cannot rule out this possibility without measurements of the Na+ concentration within these vesicles.

In conclusion, the presence of NHE3 in multiple apical plasma membrane subdomains and in multiprotein complexes of multiple sizes and/or in LRs suggests that a single transport protein may be present in several separate subcellular and functional pools. How this separation occurs and the specific functional role for these distinct NHE3 populations will be important to understand.


    ACKNOWLEDGEMENTS

We acknowledge the expert editorial assistance of H. McCann.


    FOOTNOTES

These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases RO1 DK-26523, PO1 DK-44484, T32 DK-07632, the Wellcome Foundation, the Hopkins Center for Epithelial Disorders, and the Hopkins CMM Graduate Program.

Portions of this paper are presented in S. Akhter's PhD dissertation.

Address for reprint requests and other correspondence: M. Donowitz, Johns Hopkins Univ. School of Medicine, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2195 (E-mail: mdonowit{at}jhmi.edu).

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.

May 22, 2002;10.1152/ajpcell.00613.2001

Received 26 December 2001; accepted in final form 13 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akhter, S, Cavet ME, Tse M, and Donowitz M. C-terminal domains of Na+/H+ exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger. Biochemistry 39: 1990-2000, 2000[ISI][Medline].

2.   Biemesderfer, D, Degray B, and Aronson PS. Active (9.6 s) and inactive (21 s) oligomers of NHE3 in microdomains of the renal brush border. J Biol Chem 276: 10161-10167, 2001[Abstract/Free Full Text].

3.   Biemesderfer, D, Nagy T, DeGray B, and Aronson PS. Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule. J Biol Chem 274: 17518-17524, 1999[Abstract/Free Full Text].

4.   Biemesderfer, D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997[Abstract/Free Full Text].

5.   Brett, CL, Wei Y, Donowitz M, and Rao R. Human Na+/H+ exchanger isoform 6 is found in the recycling endosomes of cells, not mitochondria. Am J Physiol Cell Physiol 282: C1031-C1041, 2002[Abstract/Free Full Text].

6.   Brown, PS, Wang E, Aroeti B, Chapin SJ, Mostov KE, and Dunn KW. Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling. Traffic 1: 124-140, 2000[ISI][Medline].

7.   Brunskill, NJ. Albumin handling by proximal tubular cells: mechanisms and mediators. Nephrol Dial Transplant 6: 39-40, 2000.

8.   Castagnaro, M. Lectin histochemistry of rabbit nephron. Biol Struct Morphog 3: 20-26, 1990[Medline].

9.   Chow, CW. Regulation and intracellular localization of the epithelial isoforms of the Na+/H+ exchangers NHE2 and NHE3. Clin Invest Med 22: 195-206, 1999[ISI][Medline].

10.   Collazo, R, Fan L, Hu MC, Zhao H, Wiederkehr MR, and Moe OW. Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis. J Biol Chem 275: 31601-31608, 2000[Abstract/Free Full Text].

11.   D'Souza, S, Garcia-Cabado A, Yu F, Teter K, Lukacs G, Skorecki K, Moore HP, Orlowski J, and Grinstein S. The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes. J Biol Chem 273: 2035-2043, 1998[Abstract/Free Full Text].

12.   Deen, PM, and Knoers NV. Vasopressin type-2 receptor and aquaporin-2 water channel mutants in nephrogenic diabetes insipidus. Am J Med Sci 316: 300-309, 1998[ISI][Medline].

13.   Fambrough, DM, and Bayne EK. Multiple forms of Na+/K+/ATPase in the chicken. Selection detection of the major nerve, skeletal muscle, and kidney form by a monoclonal antibody. J Biol Chem 258: 3926-3935, 1983[Abstract/Free Full Text].

14.   Gekle, M, Drumm K, Mildenberger S, Freudinger R, Gassner B, and Silbernagl S. Inhibition of Na+/H+ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum. J Physiol 520: 709-721, 1999[Abstract/Free Full Text].

15.   Gekle, M, Freudinger R, and Mildenberger S. Inhibition of Na+/H+ exchanger-3 interferes with apical receptor-mediated endocytosis via vesicle fusion. J Physiol 531: 619-629, 2001[Abstract/Free Full Text].

16.   Gonda, T, Maouyo D, Rees SE, and Montrose MH. Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid. Am J Physiol Gastrointest Liver Physiol 276: G259-G270, 1999[Abstract/Free Full Text].

17.   Holman, GD, and Sandoval, IV Moving the insulin-regulated glucose transporter GLUT4 into and out of storage. Trends Cell Biol 11: 173-179, 2001[ISI][Medline].

18.   Hu, MC, Fan L, Crowder LA, Karim-Jimenez Z, Murer H, and Moe OW. Dopamine acutely stimulates Na+/H+ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation. J Biol Chem 276: 26906-26915, 2001[Abstract/Free Full Text].

19.   Janecki, AJ, Janecki M, Akhter S, and Donowitz M. Basic fibroblast growth factor stimulates surface expression and activity of Na+/H+ exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase. J Biol Chem 275: 8133-8142, 2000[Abstract/Free Full Text].

20.   Janecki, AJ, Janecki M, Akhter S, and Donowitz M. Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts. J Histochem Cytochem 48: 1479-1492, 2000[Abstract/Free Full Text].

21.   Janecki, AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM, Khurana S, and Donowitz M. Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 273: 8790-8798, 1998[Abstract/Free Full Text].

22.   Karim-Jimenez, Z, Hernando N, Biber J, and Murer H. Requirement of a leucine residue for (apical) membrane expression of type IIb NaPi co-transporters. Proc Natl Acad Sci USA 97: 2916-2921, 2000[Abstract/Free Full Text].

23.   Kempson, SA, Helmle C, and Murer H. Endocytosis and phosphate transport in OK epithelial cells. Renal Physiol Biochem 12: 359-364, 1989[ISI][Medline].

24.   Kovbasnjuk, O, and Donowitz M. Apical glycocalyx pH measurements in Caco-2 epithelial cells reveal acidic microdomains. FASEB J 14: A113, 2000.

25.   Kovbasnjuk, ON, and Spring KR. The apical membrane glycocalyx of MDCK cells. J Membr Biol 176: 19-29, 2000[ISI][Medline].

26.   Kreis, TE. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J 5: 931-941, 1986[Abstract].

27.   Kurashima, K, Szabo EZ, Lukacs G, Orlowski J, and Grinstein S. Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway. J Biol Chem 273: 20828-20836, 1998[Abstract/Free Full Text].

28.   Lagana, A, Vadnais J, PU, Le Nguyen TN, Laprade R, Nabi IR, and Noel J. Regulation of the formation of tumor cell pseudopodia by the Na+/H+ exchanger NHE1. J Cell Sci 113: 3649-3662, 2000[Abstract/Free Full Text].

29.   Laghmani, K, Preisig PA., Moe OW, Yanagisawa M, and Alpern RJ. Endothelin-1/endothelin-B receptor-mediated increases in NHE3 activity in chronic metabolic acidosis. J Clin Invest 107: 1563-1569, 2001[Abstract/Free Full Text].

31.   Levine, SA, Montrose MH, Tse CM, and Donowitz M. Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line. J Biol Chem 268: 25527-25535, 1993[Abstract/Free Full Text].

32.   Li, X, Galli T, Leu S, Wade JB, Weinman EJ, Leung G, Cheong A, Louvard D, and Donowitz M. Na+/H+ exchanger 3 is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3. J Physiol 537: 537-552, 2001[Abstract/Free Full Text].

33.   Martens, JR, Navarro-Polanco R, EA, Coppock Nishiyama A, Parshley L, Grobaski TD, and Tamkun MM. Differential targeting of Shaker-like potassium channels to lipid rafts. J Biol Chem 275: 7443-7446, 2000[Abstract/Free Full Text].

34.   Martens, JR, Sakamoto N, Sullivan SA, Grobaski TD, and Tamkun MM. Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv15 to caveolae. J Biol Chem 276: 8409-8414, 2001[Abstract/Free Full Text].

35.   Moe, OW. Acute regulation of proximal tubule apical membrane Na/H exchanger NHE-3: role of phosphorylation, protein trafficking, and regulatory factors. J Am Soc Nephrol 10: 2412-2425, 1999[Free Full Text].

36.   Noel, J, Roux D, and Pouyssegur J. Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines. J Cell Sci 109: 929-939, 1996[Abstract/Free Full Text].

37.   Numata, M, and Orlowski J. Molecular cloning and characterization of a novel Na+/K+/H+ exchanger localized to the trans-Golgi network. J Biol Chem 276: 17387-17394, 2001[Abstract/Free Full Text].

38.   Numata, M, Petrecca K, Lake N, and Orlowski J. Identification of a mitochondrial Na+/H+ exchanger. J Biol Chem 273: 6951-6959, 1998[Abstract/Free Full Text].

39.   Oberleithner, H, Wunsch S, and Schneider S. Patchy accumulation of apical Na+ transporters allows cross talk between extracellular space and cell nucleus. Proc Natl Acad Sci USA 89: 241-245, 1992[Abstract].

40.   Okamoto, CT, and Forte JG. Vesicular trafficking machinery, the actin cytoskeleton, and H+-K+-ATPase recycling in the gastric parietal cell. J Physiol 532: 287-296, 2001[Abstract/Free Full Text].

41.   Orlowski, J, and Grinstein S. Na+/H+ exchangers of mammalian cells. J Biol Chem 272: 22373-22376, 1997[Free Full Text].

42.   Peng, Y, Amemiya M, Yang X, Fan L, Moe OW, Yin H, Preisig PA, Yanagisawa M, and Alpern RJ. ETB receptor activation causes exocytic insertion of NHE3 in OKP cells. Am J Physiol Renal Physiol 280: F34-F42, 2001[Abstract/Free Full Text].

43.   Pizzonia, JH, Biemesderfer D, Abu-Alfa AK, Wu MS, Exner M, Isenring P, Igarashi P, and Aronson PS. Immunochemical characterization of Na+/H+ exchanger isoform NHE4. Am J Physiol Renal Physiol 275: F510-F517, 1998[Abstract/Free Full Text].

45.   Raub, TJ, Koroly MJ, and Roberts RM. Rapid endocytosis and recycling of wheat germ agglutinin binding sites on CHO cells: evidence for two compartments in a nondegradative pathway. J Cell Physiol 144: 52-61, 1990[ISI][Medline].

46.   Rossmann, H, Sonnentag T, Heinzmann A, Seidler B, Bachmann O, Vieillard-Baron D, Gregor M, and Seidler U. Differential expression and regulation of Na+/H+ exchanger isoforms in rabbit parietal and mucous cells. Am J Physiol Gastrointest Liver Physiol 281: G447-G458, 2001[Abstract/Free Full Text].

47.   Schwab, A, Gabriel K, Finsterwalder F, Folprecht G, Greger R, Kramer A, and Oberleithner H. Polarized ion transport during migration of transformed Madin-Darby canine kidney cells. Pflügers Arch 430: 802-807, 1995[ISI][Medline].

48.   Schwark, JR, Jansen HW, Lang HJ, Krick W, Burckhardt G, and Hropot M. S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types. Pflügers Arch 436: 797-800, 1998[ISI][Medline].

49.   Shogomori, H, and Futerman AH. Cholesterol depletion by methyl-beta -cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons. J Neurochem 78: 991-999, 2001[ISI][Medline].

50.   Sorribas, V, Markovich D, Hayes G, Stange G, Forgo J, Biber J, and Murer H. Cloning of a Na/Pi co-transporter from opossum kidney cells. J Biol Chem 269: 6615-6621, 1994[Abstract/Free Full Text].

51.   Thwaites, DT, Ford D, Glanville M, and Simmons NL. H+ solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells. J Clin Invest 104: 629-635, 1999[Abstract/Free Full Text].

52.   Wakabayashi, S, Sardet C, Fafournoux P, Counillon L, Meloche S, Pages G, and Pouyssegur J. Structure function of the growth factor-activatable Na+/H+ exchanger (NHE1). Rev Physiol Biochem Pharmacol 119: 157-186, 1992[ISI][Medline].

53.   Wakabayashi, S, Shigekawa M, and Pouyssegur J. Molecular physiology of vertebrate Na+/H+ exchangers. Physiol Rev 77: 51-74, 1997[Abstract/Free Full Text].

54.   Wilson, JM, de hoop M, Zorzi N, Toh BH, Dotti CG, and Parton RG. EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts. Mol Biol Cell 11: 2657-2671, 2000[Abstract/Free Full Text].

55.   Yang, X, Amemiya M, Peng Y, Moe OW, Preisig PA, and Alpern RJ. Acid incubation causes exocytic insertion of NHE3 in OKP cells. Am J Physiol Cell Physiol 279: C410-C419, 2000[Abstract/Free Full Text].

56.   Yip, KP, Tse CM, McDonough A, and Marsh DJ. Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension. Am J Physiol Renal Physiol 275: F565-F575, 1998[Abstract/Free Full Text].

57.   Zhang, Y, Magyar CE, Norian JM, Holstein-Rathlou NH, Mircheff AK, and McDonough AA. Reversible effects of acute hypertension on proximal tubule sodium transporters. Am J Physiol Cell Physiol 274: C1090-C1100, 1998[Abstract/Free Full Text].

58.   Zhang, Y, Mircheff AK, Hensley CB, Magyar CE, Warnock DG, Chambrey R, Yip KP, Marsh DJ, Holstein-Rathlou NH, and McDonough AA. Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis. Am J Physiol Renal Fluid Electrolyte Physiol 270: F1004-F1014, 1996[Abstract/Free Full Text].


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