Departments of Medicine and Physiology, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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The Na+/H+ exchangers NHE2 and NHE3 are
involved in epithelial Na+ and HCO
sodium absorption; Caco-2 cells; lysosomes; trafficking
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INTRODUCTION |
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THE NA+/h+ exchangers (NHEs) are a family of membrane transport proteins that catalyze the electroneutral exchange of intracellular H+ for extracellular Na+. The "housekeeping" NHE1 is ubiquitously expressed and is present on the basolateral membrane in virtually all mammalian epithelial cells, where it is involved in maintaining intracellular pH (pHi) and in cell volume regulation. NHE2 and NHE3 have a more restricted tissue distribution, being predominantly located in the apical membrane of epithelia, and are highly expressed in the intestine and in the kidney. They are thought to be involved in Na+, bicarbonate, and water reabsorption (13, 37). However, the relative roles of these two isoforms in these processes are not completely understood. Mouse knockout studies demonstrated the importance of NHE3 in fluid and electrolyte balance in the intestine and kidney, but null NHE2 mice had no perturbations of intestinal or renal acid-base or Na+ homeostasis, and NHE2 was not upregulated in the NHE3 knockout mouse (31). In addition, there did not appear to be a difference in renal proximal tubule function in NHE3 vs. NHE2 plus NHE3 knockout mice (7). The only abnormal physiology that the NHE2 knockout mice did display was loss of viability of gastric parietal cells (30). On the other hand, both NHE2 and NHE3 appear to contribute to basal rabbit and avian ileal brush border Na+/H+ exchange (12, 16, 39), and NHE2 is the major Na+/H+ exchanger in the avian colon, where it is increased by a low-salt diet (12). NHE2 is also highly expressed in human skeletal muscle, where its function is unknown, and in human colon, suggesting that it has a significant role in human colonic Na+ absorption (24).
The subcellular distribution and trafficking of the NHEs give some insights into the physiological roles of these transport proteins. NHE1 is predominantly on the cell surface (4, 9) and is regulated by changes in the sensitivity to intracellular H+ (21). In contrast, only 15% of NHE3 is located on the plasma membrane in PS120 cells (2, 4). The rest is located in a juxtanuclear recycling compartment from where it undergoes basal phosphatidylinositol (PI) 3 kinase-dependent cycling to the plasma membrane (11, 17, 19). The presence of an intracellular pool of NHE3 has also been demonstrated in epithelial cells. In the opossum kidney proximal tubule cell line (OK), the majority of NHE3 is located intracellularly in a subapical compartment (Akhter and Donowitz, unpublished observations), whereas in the colonic adenocarcinoma Caco-2 cells, 80% of NHE3 is located on the plasma membrane, with the rest being located in a subapical recycling compartment (18). Alteration of NHE3 transport rate usually occurs through a change in maximal velocity (Vmax), and this has been shown to occur via changes in the amount of the protein on the plasma membrane and/or changes in the turnover number of the protein by phosphorylation-dependent and -independent mechanisms. Stimulation of NHE3 transport rate by serum and fibroblast growth factor (FGF) causes an increase in the amount of NHE3 on the plasma membrane via an increase in exocytosis in PS120 fibroblasts (2, 17), whereas phorbol 12-myristate 13-acetate inhibits NHE3 by causing an increase in endocytosis and a change in turnover number in Caco-2 cells (18). In OK cells, activation of NHE3 by acidosis is due to an increase in the exocytosis of NHE3 and, thus, an increase of NHE3 on the cell surface (40), whereas parathyroid hormone (PTH) inhibits NHE3 in OK cells by both changing turnover number (by altering phosphorylation) and later decreasing surface NHE3 (8). In rats, PTH decreases cortical Na+/H+ exchange partially through redistribution of NHE3 transporter away from the apical membrane to a nonapical pool (14).
NHE2 exists in two forms in the PS120 cell line, an 85-kDa glycosylated form that is predominantly located on the plasma membrane and an unglycosylated 75-kDa form that is predominantly located intracellularly (4, 36). Little is known about the intracellular trafficking and location of NHE2 in either the basal or regulated state. Functionally, NHE2 responds to agonists through a change in Vmax (21); whether this is due to changes in turnover number and/or the percentage of transporter on the plasma membrane has yet to be determined.
To gain insight into the relative functional roles of NHE2 and NHE3, we designed this study to investigate the cellular trafficking and processing of NHE2 compared with NHE3 and NHE1. Initially, we studied the plasma membrane half-life of the NHEs and determined that NHE2 has a short half-life compared with NHE1 and NHE3. Because changing synthesis and degradation rates for a protein with a short half-life is much more effective at regulating protein levels than for a protein with a long half-life, we then investigated the involvement of synthesis and proteolytic pathways in the regulation of the amount of cellular NHE2. Inhibiting the synthetic pathway with Brefeldin A decreases the amount of plasma membrane NHE2. There are two main proteolytic systems within cells for the intracellular degradation of proteins, the lysosomal system and the proteosomal system (3, 25, 34). We demonstrated that inhibition of the lysosomes causes an increase in the amount of endocytosed NHE2, suggesting that NHE2 is degraded by lysosomes. In contrast to NHE3, NHE2 does not appear to undergo basal recycling. This suggests that for NHE2, protein degradation and/or protein synthesis probably plays an important role in basal and regulated states. The cellular pathways of trafficking of NHE2 and NHE3 expressed in the same cell type appear to be fundamentally different, and this may underlie the specialized role of NHE2 vs. NHE3.
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MATERIALS AND METHODS |
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Cell culture. A Chinese hamster lung fibroblast cell line (PS120) previously selected to lack all endogenous Na+/H+ exchangers was stably transfected, using Lipofectin (GIBCO BRL), with human NHE1 or rabbit NHE2 or NHE3 cDNAs tagged on the COOH termini with a previously described 11-amino acid epitope of vesicular stomatitis virus G protein (VSVG) plus an 8-amino acid spacer (NHE1V, NHE2V, or NHE3V) (41). Cells were grown in medium (DMEM) supplemented with 25 mM NaHCO3, 10 mM HEPES, pH 7.4, 50 IU/ml penicillin, 50 µg/ml streptomycin, 10% fetal bovine serum (FBS), and 800 µg/ml G418 in a 5% CO2-95% O2 incubator at 37°C. The colonic adenocarcinoma-derived Caco-2 clonal cell line TC-7 (6) was stably transfected with rabbit NHE2V. Clonal NHE2V cells were selected by using acid loading (see below) and 30 µM HOE-694 to inhibit NHE1. Caco-2 cells were grown in DMEM supplemented with 0.1 mM nonessential amino acids, 1 mM pyruvate, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% FBS in a 10% CO2-humidified incubator at 37°C.
Both cell types were acid loaded once a week to maintain a high level of Na+/H+ exchange activity, as described (21). Briefly, cells were placed in NH4Cl solution (in mM: 50 NH4Cl, 70 choline-Cl, 5 KCl, 1 MgCl2, 2 CaCl2, 5 mM glucose, and 20 HEPES/Tris, pH 7.4) for 1 h, followed by an isotonic 2 mM Na+ solution for a further 1 h. Surviving cells were then placed in normal culture medium. Cells were used between passages 4 and 25 posttransfection.Cell surface biotinylation to determine half-life of plasma
membrane NHEs.
Transfected PS120 cells were grown to 70-80% confluence in 10-cm
petri dishes. Cells were then placed at 4°C. Cells were washed twice
in phosphate-buffered saline (PBS) and once in borate buffer (in mM:
154 NaCl, 10 boric acid, 7.2 KCl, and 1.8 CaCl2, pH 9.0). The plasma membrane proteins were then biotinylated by gently shaking
the cells for 20 min with 3 ml of borate buffer containing 1.5 mg of
sulfo-NHS-LC-biotin (biotinylation solution). An additional 3 ml of the
same biotinylation solution was then added, and the cells were rocked
for an additional 20 min. The cells were washed extensively with
quenching buffer (in mM: 120 NaCl and 20 Tris, pH 7.4) to remove excess
NHS-LC-biotin, and then the cells were washed twice with PBS. Cells
were then returned to complete PS120 medium at 37°C for various times
before being frozen at 80°C until further processing. Cells were
then lysed in 1 ml of N+ buffer (in mM: 60 HEPES, pH 7.4, 150 NaCl, 3 KCl, 5 EDTA trisodium, 3 EGTA, and 1% Triton X-100) and sonicated to
clarity on ice with a Branson 450 probe sonicator. The supernatant was
subjected to two consecutive avidin precipitations. The avidin-agarose
beads were washed five times in N+ buffer, and bound proteins were
solubilized in sample buffer yielding the surface fraction. Samples
were separated on a 9% gel by SDS-PAGE, and Western analysis was then
performed using anti-VSVG antibody. Bands were visualized with enhanced chemiluminescence (ECL), exposed to preflashed X-ray film within the
linear range of ECL signal, and quantified using a densitometer and
Imagequant software. Half-life of plasma membrane NHEs was determined
by fitting the data to an exponential decay curve using Origin software.
Internalization assay.
Cells were pretreated with vehicle or lysosomal, proteosomal
inhibitors. Cells were biotinylated as described above except for the
use of NHS-SS-biotin rather than sulfo-NHS-LC biotin. Cells were then
incubated in complete medium at 37°C for 1 h with and without
lysosomal/proteosomal inhibitors. Cells were then returned to 4 °C,
and all remaining surface biotin was removed by the fresh addition of
10 mM 2-mercaptoethanesulfonic acid (MESNA) for three 30-min washes in
stripping buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 8.6, and 2 mg/ml BSA). All unreacted MESNA was quenched with 100 mM iodoacetamide
in PBS for 10 min. One sample of cells was stripped without a 37°C
incubation to check stripping efficiency, which was 90%. Cells were
then lysed and treated as described above.
Pulse-chase labeling of cultured cells.
Cells were rinsed once and incubated with methionine- and cysteine-free
DMEM for 1 h to deplete intracellular pools. The cells were then
incubated with methionine- and cysteine-free DMEM containing 0.2 mCi/ml
[35S]methionine/cysteine cell-labeling mix (Amersham) for
4 h. To chase, we aspirated this labeling solution and rinsed the
cells four times with PBS. Cells were then incubated with DMEM
containing 10% serum, 2 mM methionine, and 2 mM cysteine for between 2 and 26 h. The cells were then rinsed four times in ice-cold PBS
and stored at 70°C until further processing. Cells were lysed in 1 ml of N+ buffer, homogenized through a 26-gauge needle, and agitated on
a rotating rocker at 4°C for 30 min, followed by centrifugation at
12,000 g for 30 min, to remove insoluble cellular debris.
The supernatant was precleared by using 20 µl of protein A-Sepharose beads cross-linked to anti-mouse IgG (PAM; Jackson). NHE1V was then
immunoprecipitated from the precleared supernatant by incubation with
anti-VSVG P5D4 monoclonal antibody, followed by two rounds of
incubation with PAM. Immunoprecipitated pellets were washed five times
with N+ buffer. The precipitated proteins were eluted from the beads
with SDS-sample buffer (110 mM Tris · HCl, 0.9% SDS, 0.8%
EDTA, 5% glycerol, 1% 2-mercaptoethanol, and bromphenol blue) by
incubation at 95°C for 3 min, followed by SDS-PAGE on 9% gels.
Proteins were transferred to nitrocellulose and exposed to Amersham
Hyperfilm for 2 days. The nitrocellulose was then subjected to Western
analysis with anti-VSVG antibody to ensure that an equal amount of NHE
protein was precipitated at each time point.
Na+/H+ exchange Vmax by fluorimetry. Cells were seeded on glass coverslips and grown until they reached 50-70% confluency. The cells were loaded with the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM; 5 µM) in "Na+ medium" (in mM: 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 glucose, and 20 HEPES, pH 7.4) for 20 min at 22°C and then washed with "TMA+ medium" (in mM: 130 tetramethylammonium chloride, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 glucose, and 20 HEPES, pH 7.4) to remove the extracellular dye, and the coverslip was mounted at an angle of 60° in a 100-µl fluorometer cuvette designed for perfusion and thermostatted at 37°C. The cells were pulsed with 40 mM NH4Cl in TMA+ medium for 3 min, followed by TMA+ medium, which resulted in the acidification of the cells. Na+ medium was then added, which induced alkalinization of the cells.
Na+/H+ exchange rates (H+ efflux) were calculated, as described previously (21), as the product of Na+-dependent change in pHi and the buffering capacity at each pHi and were analyzed with the use of a nonlinear regression data analysis program (Origin) that allowed fitting of data to a general allosteric model described by the Hill equation (v = Vmax · [S]napp/K'+[S]napp, where [S] is substrate concentration, v is velocity, K is affinity constant, and napp is apparent Hill coefficient.) with estimates for Vmax and K'[H+]i and their respective errors (SE).Immunofluorescence. Cells were seeded on glass coverslips until they reached 80% confluency. Cells were fixed in 3% paraformaldehyde/PBS for 30 min at 4°C, and the paraformaldehyde was neutralized in 20 mM glycine for 10 min. Cells were permeabilized for 30 min in 0.1% saponin/PBS and then blocked for 30 min in 1% BSA/PBS supplemented with 10% FBS. The cells were incubated in anti-VSVG P5D4 monoclonal antibody for 1 h, followed by three washes in 0.1% saponin/PBS. Cells were then incubated with secondary antibodies (Alexa conjugates; Molecular Probes) and Hoechst 33342 for 1 h. After three 10-min washes in 0.1% saponin/PBS, cells were mounted in Prolong antifade reagent before being viewed on a Zeiss LSM 410 confocal microscope. Images were analyzed using Metamorph software (Universal Imaging).
Materials. Human NHE1 was a kind gift from Drs. J. Noel and J. P. Pouyssegur (Dept. of Physiology, University of Montreal, Canada, and Dept. Biochemistry, University of Nice, France). [35S]methionine/cysteine cell-labeling mix was from Amersham, and NHS-SS-biotin, NHS-LC-biotin, and avidin-agarose were from Pierce. All other chemicals were from Sigma unless stated otherwise.
Statistical analysis. Results are expressed as means ± SE. Statistical significance was evaluated using Student's t-test. Significance was accepted at P < 0.05.
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RESULTS |
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Half-life of plasma membrane NHEs.
Half-lives of Na+/H+ exchanger isoforms
NHE1-3 appeared in PS120 fibroblasts that were initially on
the plasma membrane and were compared by determining the amount of
biotinylated NHE proteins remaining within the cells 0-24 h after
initial biotinylation of surface proteins. These results (Fig.
1) demonstrate that plasma membrane NHEs
have different degradation rates. The 110-kDa form of NHE1 had a
half-life of 23.1 ± 2.3 h (n = 4) (Fig.
1A). NHE3 also had a long half-life of 13.8 ± 1.6 h (n = 4) (Fig. 1C). In contrast, the
half-life of the 85-kDa form of NHE2 was much shorter at 3.2 ± 0.3 h (n = 5). The 75-kDa form of NHE2 had a
similar short half-life of 3.1 ± 0.2 h (n = 5) (Fig. 1B).
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NHE2 and NHE3 have divergent subcellular distribution and
trafficking mechanisms in PS120 cells.
The significantly faster degradation rate of plasma membrane NHE2
compared with NHE3 was of interest and suggests a fundamental difference in the cellular processing of these isoforms. NHE2 and NHE3
are both epithelial isoforms whose relative roles in Na+
absorption are not well understood. The current study has demonstrated that one fundamental difference between NHE2 and NHE3 is the short half-life of NHE2. One possible explanation for differences in plasma
membrane half-life is differences in the rates of degradation. Differences in degradation rates may suggest differences in the cellular processing and trafficking of these two isoforms. Whereas NHE3
is known to undergo a basal PI-3 kinase-dependent recycling between the
cell surface and an intracellular compartment (11, 17,
19), subcellular trafficking of NHE2 has not been described. We
studied the effect of the PI-3 kinase inhibitor wortmannin (100 nM,
1 h) on basal NHE2 transport in PS120 cells. The
Vmax of H+ efflux for NHE2 was not
affected by wortmannin [control: 3,790 ± 90 µM/s, wortmannin:
3,533 ± 90 µM/s, P = not significant (NS), Fig.
4A], in contrast to the 33%
inhibition obtained with NHE3 (control: 4,095 ± 92 µM/s,
wortmannin: 2,754 ± 112 µM/s, P < 0.0001, Fig.
4B). This finding demonstrates that NHE2 is not
undergoing PI-3 kinase-dependent basal recycling. Furthermore,
immunolocalization demonstrates that the subcellular distribution of
NHE2 and NHE3 is different. The subcellular distribution of NHE2 and
NHE3 was compared in PS120 fibroblasts. While NHE3 is predominantly
localized in a juxtanuclear compartment and on the plasma membrane as
previously described (Fig. 5A)
(11), the pattern of staining for NHE2 was different.
Intracellular NHE2 (Fig. 5B) had a more diffuse cytoplasmic and perinuclear localization.
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The amount of recoverable endocytosed plasma membrane NHE2 is
increased by inhibiting the lysosomes.
Proteins with a short half-life are much more effectively regulated by
changing synthesis and degradation rates than are proteins with a long
half-life. Therefore, we investigated the role of the lysosomes in the
short-term degradation of NHE2 in PS120 cells. The amount of
endocytosed plasma membrane NHE2 protein detectable by Western blotting
was compared in the presence and absence of lysosomal inhibitor.
Degradation by the lysosomal pathway was inhibited using the inhibitor
leupeptin (100 µg/ml) (22) and the lysosomotropic agents
ammonium chloride (20 mM) and chloroquine (50 µM) (29,
32). Cells expressing NHE2 were treated for 3 h with each
blocker, after which cell surface proteins were labeled with biotin and
allowed to internalize for 1 h in the continued presence of
lysosomal inhibitor. Residual cell surface biotinylated protein was
stripped with the reducing agent, which is efficient in removing >90%
of surface biotin in PS120 fibroblasts (2) (data not
shown), after which the amount of endocytosed NHE protein was
determined. Treatment of cells with lysosomal inhibitors (leupeptin, chloroquine, and NH4Cl) led to an increase in NHE2 (both 85 and 75 kDa) as detected by Western blotting (Fig.
6, A-C). Densitometric analysis revealed that treatment of NHE2 with lysosomal inhibitors led
to an ~100% increase in NHE2 internalized protein. In contrast, internalized NHE3 was not significantly altered after treatment with
leupeptin (Fig. 6D).
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Brefeldin A, an inhibitor of the synthetic pathway, inhibits NHE2
transport.
We used Brefeldin A (BFA), which blocks Golgi-to-plasma membrane
trafficking by causing tubulation and redistribution of the Golgi into
the ER (23, 26), to determine the importance of the
synthetic pathway in determining plasma membrane levels of NHE2. PS120
cells were exposed to 5 µg/ml BFA for 3 h before study. The
Vmax of H+ efflux for NHE2 was
reduced by 38% from 2,575 ± 78 to 1,598 ± 38 µM
(P < 0.0001) in the BFA-treated cells, whereas the
transport of NHE1, which has the longest half-life of the NHEs studied, was unchanged (control: 5,015 ± 61 µM, BFA treatment:
4,755 ± 76 µM, P = NS, Fig.
7A). To verify that reduction
of NHE2 transport was due to a decrease in surface NHE2, we used cell
surface biotinylation to measure NHE2 surface protein with and without
BFA (Fig. 7B). Densitometric analysis demonstrated that BFA
decreased the amount of the 85-kDa NHE2 protein on the cell surface by
~50%.
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Exposure of NHE2 to hyperosmolarity for 3 h does not alter the half-life of plasma membrane NHE2. The short half-life of NHE2 suggested that physiological regulation might involve changes in the half-life. Because NHE2 is present in the apical membrane of renal distal tubule and of duodenal absorptive cells and thus is exposed to hyperosmolarity, including for prolonged periods, we determined whether prolonged exposure to hyperosmolarity altered NHE2 function and plasma membrane half-life. In these studies, PS120/NHE2 cells were exposed to Na+ medium or Na+ medium plus 150 mM mannitol for 3 h, and NHE2 activity was determined. After 3 h of exposure to hyperosmolarity, NHE2 activity was still inhibited compared with control (43% inhibition, n = 3, data not shown). After 3 h of exposure to hyperosmolarity, half-life of plasma membrane NHE2 was determined by use of biotinylation and was studied 0, 2.5, 5, 7, 10, and 19 h later (i.e., plus 3 h), being maintained in the same hyperosmolar conditions that inhibited NHE2. After 3 h of exposure to hyperosmolarity, the half-life of plasma membrane NHE2 was not altered (difference in half-life of plasma membrane p85 exposed to hyperosmolarity vs. time control 1.1 ± 1.2 h, n = 3, not significant).
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DISCUSSION |
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In this study we have characterized the half-life of plasma membrane NHE1-3 and have demonstrated that the half-life of plasma membrane NHE2 is relatively short (3 h) in both fibroblasts (PS120) and epithelial (Caco-2) cells compared with that of NHE3 and NHE1 (14 h and 24 h, respectively). Both 85- and 75-kDa forms of NHE2 had similar plasma membrane half-lives, suggesting that both forms that reach the plasma membrane are undergoing similar cellular trafficking pathways. The short half-life of plasma membrane NHE2 is supported by the fact that inhibiting both the synthetic pathway and degradation rapidly alters the transport activity of NHE2. Transport and surface levels of NHE2 are reduced by 3-h treatment with inhibition of the synthetic pathway using Brefeldin A. This suggests the importance of new synthesis in maintaining plasma membrane of NHE2 over a short time period. Furthermore, it appears that the degradation of NHE2 is mediated, at least in part, by the lysosomes. Treatment with the lysosomal inhibitor leupeptin (22, 32) and the lysosomotropic agents chloroquine and NH4Cl (29) increased the amount of detectable endocytosed NHE2 protein, presumably by inhibiting its breakdown in the lysosomal compartment, over a 3-h time period. The rapid degradation of NHE2 appears to be predominantly by the lysosomes, because inhibition of the proteosomal pathway did not affect the amount of endocytosed NHE2 and NHE2 does not appear to be ubiquitinated.
This study demonstrates fundamental differences in the cellular processing of NHE2 and NHE3. NHE3 is known to undergo a dynamic process of basal recycling between the plasma membrane and an endosomal recycling compartment that is PI 3-kinase dependent (11, 19). However, in this study we have demonstrated that basal NHE2 transport is not affected by the PI 3-kinase inhibitor wortmannin. Furthermore, immunofluorescence studies showed that NHE3 has a discrete juxtanuclear localization that has previously been shown to be the recycling endosome on the basis of colocalization with the transferrin receptor and the SNARE protein cellubrevin (11, 17). In contrast, NHE2 had a more diffuse cytoplasmic and perinuclear localization. Although the wortmannin data do not rule out non-PI 3-kinase-dependent cycling for NHE2, as occurs with part of FGF stimulation of NHE3 in PS120 fibroblasts (17), this, combined with the contrasting subcellular localization with NHE3, suggests a lack of basal recycling of NHE2.
Both NHE2 and NHE3 are regulated in part by a change in their Vmax (21). One of the mechanisms of NHE3 regulation is changes in the amount of total NHE3 protein on the plasma membrane vs. the intracellular compartment, and this often involves changes in the rate of endocytosis and exocytosis. Similarly for NHE2, there is a large amount of the 75-kDa form (80%) of NHE2 within the cell, whereas 36% of the 85-kDa form is present in the intracellular compartment (4). Given the short plasma membrane half-life of NHE2, this intracellular population may represent protein associated with the biosynthetic pathway. However, it remains to be determined whether trafficking of a subpopulation of intracellular NHE2 plays a role in the regulated state in either a PI 3-kinase-dependent or -independent manner.
Proteins with a short half-life are much more effectively regulated by changing rates of protein synthesis and degradation than are proteins with longer half-lives. Because NHE3 is known to undergo rapid internalization and recycling back to the plasma membrane, the long half-life of plasma membrane NHE3 suggests that this isoform avoids targeting to the late endosomes and lysosomes. This is demonstrated in the present study by the lack of effect of the lysosomal inhibitor leupeptin on the amount of endocytosed NHE3. The long half-lives of NHE1 and NHE3 suggest that regulation of basal levels of degradation/synthesis is not an efficient mechanism for rapid modulation of levels of NHE1 and NHE3 on the plasma membrane. In contrast, the short half-life of NHE2 suggests that changing synthesis or degradation rates can much more effectively regulate the amount of this isoform on the plasma membrane.
One possible function of NHE2, given its short plasma membrane
half-life, is to respond to stimuli through a change in its synthesis
and degradation. Other transport proteins that have rapid plasma
membrane turnover include the ENaC and the type II Na+/Pi cotransporter. For both of these
proteins, degradation plays an important role in regulating the
transport rate. In HEK-293 cells, ENaC is ubiquitinated by the binding
of Nedd4 to the COOH termini of the - and
-subunits, rapidly
targeting the constitutively active channel for proteosomal and
lysosomal degradation. In the condition Liddle's syndrome, Nedd4
cannot bind to the channel subunits; thus degradation is slowed and
hypertension results because of increased surface expression of ENaC
and increased absorption of Na+ (1, 33). In
the case of the Na/Pi cotransporter, in rat renal brush
border and in OK cells, this transporter is broken down under basal
conditions by the lysosomal pathway, and upon stimulation with PTH the
rate of degradation is substantially increased, leading to an
inhibition of Na+/Pi cotransport activity
(27, 28). By analogy, the short half-life of NHE2 could be
important in maintaining basal levels of this transporter and also for
altering transporter levels in the regulated state.
The findings in this study may give insight into the relative specialized cellular roles of NHE2 and NHE3. Although both are located on the apical surface of renal and intestinal epithelial cells, in many species their relative roles have not yet been fully defined. Knockout studies in mice suggest that NHE3 is the only isoform carrying out intestinal basal Na+/H+ exchange, whereas the role of NHE2 is not well understood (7, 31). On the other hand, NHE2 appears to be important for basal ileal brush border Na+/H+ exchange in some species (12, 16, 39). In support of a specialized function for NHE2 in Na+ absorption, two studies have demonstrated the presence of NHE2 in renal segments that do not contain NHE3. In rat and mouse kidney, a study showed that NHE2 was present in the inner medullary collecting duct (35). Another study in mouse demonstrated that NHE2 is present in the cortical thick ascending limbs, distal convoluted tubules, and connecting tubules (5). In the rat epididymis, NHE2 also is found in the cauda epididymis, whereas NHE3 is located in the efferent duct (20). NHE2 also is expressed in parietal cells and skeletal muscle where NHE3 is not localized (24, 30), but its role in these tissues is not yet understood. The differences in the cellular processing mechanisms described in this study between NHE2 and NHE3 may underlie the specialized functions as well as tissue and subtissue distributions of these two Na+/H+ exchanger isoforms and may provide insights to further understand the physiological function of NHE2.
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ACKNOWLEDGEMENTS |
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M. E. Cavet was supported in part by a Wellcome International Traveling Fellowship (United Kingdom). F. S. de Medina was supported by a grant from the Spanish Ministry of Education. These studies were also supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-26525, PO1-DK-44484, and RO1-DK-51116 and by The Hopkins Center for Epithelial Disorders.
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FOOTNOTES |
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Present address of M. E. Cavet: Center for Cardiovascular Research, University of Rochester, Box 679, 601 Elmwood Ave., Rochester, NY 14618.
Present address of F. S. de Medina: Department of Pharmacology, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain.
Address for reprint requests and other correspondence: M. Donowitz, Dept. of Medicine, Division of Gastroenterology, Johns Hopkins Univ., 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.
Received 18 December 2000; accepted in final form 20 August 2001.
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