1 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235; 2 Department of Internal Medicine, Jichi Medical School, Tochigi, Japan 329-0498; 3 Dallas Veterans Affairs Medical Center, Dallas 75216; 4 Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235; and 5 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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Endothelin-1 (ET-1) activates
sodium/hydrogen exchanger 3 (NHE3) in opossum kidney clone P (OKP)
cells expressing ETB receptors. ET-1 (108 M)
caused a two- to threefold increase in apical membrane NHE3 (assessed
by surface biotinylation), in the absence of a change in total cellular
NHE3. A maximal effect was achieved within 15 min. The increase in
apical NHE3 was not blocked by cytochalasin D but was blocked by
latrunculin B, which also prevented the ET-1-induced increase in NHE3
activity. Endocytic internalization of NHE3, measured as protection of
biotinylated NHE3 from the membrane-impermeant, sulfhydryl-reducing
agent MesNa was minimal within 35 min and was not regulated by ET-1.
Exocytic insertion of NHE3, measured as the appearance of biotinylated
NHE3 after the blockade of reactive sites with sulfo-NHS-acetate, was
increased in response to ET-1. These studies demonstrate that ET-1
induces net trafficking of NHE3 to the apical membrane that is mediated
by enhanced exocytic insertion and is required for increased NHE3 activity.
sodium/hydrogen antiporter; sodium/hydrogen exchanger 3; opossum kidney clone 3; endothelin; membrane trafficking; proximal tubule
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INTRODUCTION |
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THE ENDOTHELINS ARE A FAMILY of three 21-amino acid peptides [endothelin-1 (ET-1), ET-2, and ET-3], which serve as autocrine/paracrine factors by interacting with two receptors (ETA and ETB). ET-1 increases the activity of the proximal tubule apical membrane Na/H antiporter (14, 18), which is encoded by sodium/hydrogen exchanger 3 (NHE3) (3, 6). Opossum kidney clone P (OKP) cells express an ethylisopropyl amiloride-resistant Na/H antiporter encoded by NHE3 (4). In OKP cells expressing ETB receptors, ET-1 increases NHE3 activity (12). The purpose of the present studies was to determine the role of trafficking of NHE3 to the apical membrane in its regulation by ET-1. Results demonstrate that ET-1 causes a net increase in apical membrane NHE3 that occurs in the absence of a change in total cellular NHE3. The increase in apical membrane NHE3 induced by ET-1 is due to exocytic insertion of NHE3 and is required for ET-1 to increase NHE3 activity.
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METHODS |
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Materials and supplies. All chemicals were obtained from Sigma (St. Louis, MO), unless otherwise noted, as follows: penicillin and streptomycin were from Whittaker M.A. Bioproducts (Walkersville, MD); culture media were from GIBCO-BRL (Grand Island, NY); 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-acetoxymethyl ester (AM) was from Molecular Probes (Eugene, OR); and EZ-Link sulfo-NHS-SS-biotin, EZ-Link biocytin hydrazide, immunopure immobilized streptavidin, and sulfo-NHS-acetate were from Pierce (Rockford, IL); and latrunculin B from Alexis (San Diego, CA).
Cell culture.
Studies were performed in a clonal cell line generated by stable
transfection of OKP cells with pMEhETB, containing the cDNA for the ETB receptor, driven by an Sr promoter
(OKPETB6 cells; 12). Cells were passaged in high-glucose
(450 mg/dl) DMEM supplemented with 10% fetal bovine serum, penicillin
(100 U/ml), streptomycin (100 µg/ml), and 200 µg/ml G418. For
experimentation, G418 was removed at the time of splitting, 5-7
days before study. When confluent, cells were rendered quiescent for
48 h before study by the removal of serum and placed in
low-glucose (100 mg/dl) DMEM. In studies examining the actin
cytoskeleton, studies were performed at 70% confluence, which improved
image quality. Cells were then treated with 10
8 M ET-1 or
vehicle (0.0002% acetic acid).
Biotinylation and immunoblotting. Apical membrane NHE3 was measured by surface biotinylation, as described (17, 25, 31). Sixty-millimeter plates of confluent OKPETB6 cells were placed on ice and rinsed with ice-cold PBS with 0.1 mM CaCl2 and 1.0 mM MgCl2 (PBS-Ca-Mg) × 3. The apical surface was then exposed to 1.5 mg/ml sulfo-NHS-SS-biotin in 10 mM triethanolamine (pH 7.4), 2 mM CaCl2, and 150 mM NaCl for 1 h with horizontal motion at 4°C. After labeling, cells were rinsed with quenching solution (PBS-Ca-Mg containing 100 mM glycine) for 20 min at 4°C.
Cells were washed with ice-cold PBS × 3, lysed in radioimmunoprecipitation assay (RIPA) buffer [in mM: 150 NaCl, 50 Tris · HCl (pH 7.4), 2.5 EDTA, 5 EGTA 50,Endocytic internalization.
The endocytic internalization of biotinylated NHE3 (biotin-SS-NHE3) was
measured by its acquisition of resistance to the membrane-impermeant reducing agent -mercaptoethane sulfonate, sodium salt (MesNa; 9).
Cells were labeled with 1.5 mg/ml sulfo-NHS-SS-biotin and quenched as
above. Cells were then treated with 10
8 M ET-1 or
vehicle × 35 min at 37°C, and rinsed with Tris-buffered saline
[100 mM Tris · HCl (pH 7.5), 150 mM NaCl] × 2 at 4°C. The cells were then incubated at 4°C in 2 ml of 10 mM MesNa in 100 mM
NaCl, 1 mM EDTA, 50 mM Tris, and 0.2% BSA, and pH 8.6 (prepared just
before each addition). An additional 0.5 ml of 50 mM MesNa was added to
each plate at 30 min, and an additional 0.65 ml was added at 60 min.
MesNa was then oxidized by the addition of 1 ml of 500 mM iodoacetic
acid × 10 min, and cells were lysed with RIPA buffer. The
biotinylated fraction, which represents nascent endocytosed surface
protein, was precipitated with streptavidin-coupled agarose, and the
precipitate was subjected to SDS-PAGE and blotting with anti-NHE3
antibodies, as above. To assess the efficiency of MesNa cleavage,
experiments were performed where, after biotinylation, cells were
maintained at 4°C to prevent endocytosis and then treated with MesNa.
Exocytic insertion.
To measure exocytic insertion of NHE3, NHS reactive sites on the cell
surface were blocked by pretreatment with sulfo-NHS-acetate (30). Cells were rinsed with PBS-Ca-Mg × 3 at room
temperature. The apical surface was then exposed to 1.5 mg/ml
sulfo-NHS-acetate in 0.1 M sodium phosphate (pH 7.5), and 0.15 M NaCl
for 2 h with horizontal motion at room temperature. After
quenching for 20 min at room temperature, cells were rinsed with PBS at
37°C and treated with 108 M ET-1 or vehicle for 35 min.
Cells were then labeled with 1.5 mg/ml sulfo-NHS-SS-biotin and lysed
with RIPA buffer. The biotinylated fraction, which represents newly
inserted surface proteins, was precipitated with streptavidin-coupled
agarose, and the precipitate was subjected to SDS-PAGE and blotting
with anti-NHE3 antibodies, as above.
Measurement of intracellular pH and Na/H antiporter activity. Continuous measurement of cytoplasmic pH (pHi) was accomplished by using the intracellularly trapped pH-sensitive dye BCECF, as previously described (8). Cells were loaded with 10 µM BCECF-AM for 35 min at 37°C, and pHi was estimated from the ratio of fluorescence with excitation at wavelengths of 500 and 450 nm, with 530-nm emission in a computer-controlled spectrofluorimeter. Na/H antiporter activity was assayed as the initial rate of Na+-dependent pHi increase after an acid load (13 µM nigericin in Na+-free solution) in the absence of CO2/HCO3, as previously described (8). The initial rate of pHi increase (dpHi/dt) on Na+ addition (140 mM) was calculated by drawing a line tangent to the initial deflection. ET-1 has no effect on buffer capacity (12); results are therefore reported as dpHi/dt.
Stress fiber staining. Cells were washed with 2× PBS, fixed with 3.7% formaldehyde × 20 min on ice, and washed with 2× PBS. Cells were then permeabilized in 0.1% Triton X-100 × 7 min at 4°C, and rinsed with 2× PBS. Cells were then blocked with 1% BSA × 20 min, incubated with 5 µg/ml FITC-labeled phalloidin × 1 h at room temperature, and rinsed with PBS × 15 min × 4. Fluorescence was visualized on an Axiovert 135 (Zeiss, Germany).
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RESULTS |
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Binding of ET-1 to the
ETB receptor increases apical membrane NHE3
protein abundance.
We previously found that 108 M ET-1 increased NHE3
activity in OKPETB6 cells. To determine whether this
increase in activity is due to an increase in apical membrane NHE3 we
used surface biotinylation. Apical membrane proteins were biotinylated
by reaction with sulfo-NHS-SS-biotin, and isolated by precipitation
with streptavidin-bound agarose. Apical membrane NHE3 was then
identified by immunoblot. As shown in Fig.
1, addition of 10
8 M ET-1
to OKPETB6 cells for 35 min caused a 254 ± 103%
increase in apical membrane NHE3 protein abundance. This ET-1-induced
increase in apical NHE3 protein abundance occurred in the absence of an increase in total cellular NHE3 abundance.
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Role of the cytoskeleton in ET-1-induced increases
in apical membrane NHE3 protein abundance and antiporter activity.
In a previous study, we found that cytochalasin D, an inhibitor of
actin polymerization, did not inhibit the ET-1-induced increase in NHE3
activity (11). To examine whether the actin cytoskeleton
is required for trafficking of NHE3, we examined the effect of
cytochalasin D on this process. Cytochalasin D (1.25 µM) was added
with 108 M ET-1 or vehicle for 35 min. As shown in Fig.
5, ET-1 caused a 179 ± 63%
increase in apical NHE3 in the presence of cytochalasin D. These
results suggest that the actin cytoskeleton is not required for NHE3
trafficking.
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ET-1-induced increase in apical NHE3 is not due to decreased endocytic internalization. The net increase in apical membrane NHE3 abundance could be due to a decrease in endocytic internalization or an increase in exocytic insertion of NHE3. To measure endocytic internalization, apical membrane proteins were labeled with sulfo-NHS-SS-biotin before treatment with ET-1 or vehicle. After treatment for 35 min, cells were exposed to the cell-impermeant, sulfhydrl-reducing reagent MesNa, which removes biotin from proteins exposed to the apical fluid. Biotinylated NHE3 was then identified as above. With this protocol, biotinylated NHE3 represents NHE3 initially present on the apical membrane, and then endocytosed to protect it from MesNa.
To determine the effectiveness of MesNa cleavage, biotinylated NHE3 abundance was compared in cells not treated with MesNa (total apical NHE3) and in cells maintained at 4°C (to inhibit endocytosis) and treated with MesNa immediately after biotinylation (Fig. 9A, lanes 6 and 1, respectively). By using this protocol, 4% of biotinylated NHE3 was not cleaved by MesNa (horizontal line, Fig. 9B), showing that MesNa-induced cleavage of biotin from apical NHE3 is 96% effective. Compared with cells not treated with MesNa (Fig. 9A, lane 6), 9% of biotinylated NHE3 was protected from MesNa after 35 min of incubation with vehicle at 37°C (Fig. 9A, lanes 2 and 4), and 9% was protected after treatment with ET-1 (Fig. 9A, lanes 3 and 5). When corrected for a 96% effectiveness of MesNa cleavage, it is concluded that 5% of NHE3 is internalized in 35 min at 37°C and that ET-1 has no effect on this process (Fig. 9B).
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ET-1-induced increase in apical NHE3
is due to increased exocytic insertion.
To measure exocytic insertion, we blocked all apical protein reactive
sites by pretreatment with sulfo-NHS-acetate. Cells were then treated
with 108 M ET-1 or vehicle × 35 min, and membrane
biotinylation was performed as above. Biotinylated NHE3 represents NHE3
that was located intracellularly at the beginning of the experiment
(protected from sulfo-NHS-acetate) and on the apical membrane at the
end of the experiment. After ET-1 treatment for 35 min, there was a
70 ± 30% increase in labeled NHE3 (Fig.
10). Thus ET-1 causes an increase in
the rate of exocytic insertion of NHE3 into the apical membrane.
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DISCUSSION |
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At low concentrations, ET-1 stimulates volume and presumably Na+ absorption in the proximal tubule (16). This effect is likely related to the observation that ET-1 activates the proximal tubule apical membrane Na/H antiporter (14, 18), encoded by NHE3 (3, 6). In OKP cells expressing the ETB receptor, ET-1 leads to activation of NHE3 (12) and trafficking of NHE3 to the apical membrane. The increases in NHE3 activity and apical membrane NHE3 abundance have similar timecourses. In addition, latrunculin B inhibits both the ET-1-induced trafficking of NHE3 to the apical membrane and the increase in NHE3 activity. These results strongly suggest that trafficking of NHE3 to the apical membrane plays a key role in the increase in NHE3 activity induced by ET-1.
In the biotinylation studies we used two reagents to label apical proteins, sulfo-NHS-SS-biotin, which labels lysines and arginines, and biocytin hydrazide, which labels sugar residues. Both of these reagents showed similar results, a two- to threefold increase in apical NHE3 in response to ET-1. However, there is a question as to whether NHE3 is a glycoprotein. Previous results have suggested that rat and canine NHE3 are not detectably glycosylated whereas rabbit NHE3 is a glycoprotein (7, 13). To determine whether OKP NHE3 is a glycoprotein, we examined the effect of PNGase F on the mobility of immunoprecipitated OKP NHE3 (5). We found no mobility shift, suggesting that OKP NHE3 is not a glycoprotein (data not shown). However, the mechanism responsible for the labeling of NHE3 by biocytin hydrazide remains unclear. It is possible that there are a small number of sugar residues that are sufficient for labeling but not sufficient to cause a mobility shift. The alternative explanation is that biocytin hydrazide is labeling another residue on NHE3. Whatever the explanation, given that biocytin hydrazide is unable to enter cells, it remains useful for confirming the results with sulfo-NHS-SS-biotin.
The ET-1-induced increase in apical membrane NHE3 could be due to decreased endocytic internalization or increased exocytic insertion. To measure NHE3 endocytic internalization, we prelabeled apical proteins and measured the ability of ET-1 treatment to cause NHE3 to move to a location inaccessible to MesNa. These studies showed minimal internalization of NHE3 in 35 min and no effect of ET-1 on this process. In other studies we have found that 40% of NHE3 is internalized over 6 h (36). In the present studies, 5% of NHE3 was internalized over 35 min.
To measure exocytic insertion of NHE3, we preblocked apical proteins and then measured the ability to label new apical NHE3 with biotin. The results showed that ET-1 induced a 70% increase in exocytic insertion of NHE3 into the apical membrane. By using an identical protocol, when biotinylation was performed immediately after acetate blockade, 84% of biotinylation was blocked, demonstrating that acetate blockade is effective (36). When results of the present studies are normalized by apical NHE3 abundance and corrected for the effectiveness of acetate blockade (36), it is calculated that 4% of apical NHE3 is inserted over 35 min under control conditions, and 20% is inserted after ET-1 addition, a fivefold increase. However, given that the basal exocytosis rate is only 4% of apical NHE3, even the fivefold increase demonstrated here is not sufficient to explain the two- to threefold increase in apical NHE3 protein abundance that we observed. One possible explanation for this quantitative discrepancy is that preblockade with sulfo-NHS-acetate alters cell function and affects the quantitative aspects of trafficking. We previously showed that biotinylation of NHE3 with sulfo-NHS-SS-biotin inactivates NHE3 (36). Changes in the function of this and other apical proteins may alter cell function and rates of trafficking. Nevertheless, these results do support the qualitative demonstration that ET-1 increases the rate of exocytic insertion of NHE3.
In support of trafficking as a regulator of NHE3, we demonstrated a large pool of NHE3 that is not accessible to biotinylating reagents, and is presumably located on intracellular vesicles. In agreement, exogenously expressed NHE3 has been shown to localize to recycling endosomes (10). In addition, trafficking to and from the apical membrane has been proposed to mediate regulation of NHE3 in other settings. In the in vivo proximal tubule, acute increases in blood pressure cause NHE3 to shift from the apical membrane to higher density membranes enriched in intracellular membrane markers (38). Similarly, by using immunohistochemistry, NHE3 was shown to shift from the apical membrane to an intracellular location in acute and chronic hypertension (37). PTH also causes a shift of NHE3 from the apical membrane to an intracellular compartment (15, 39). Similarly, protein kinase C activation causes internalization of NHE3 in Caco-2 cells (19).
In previous studies we found that the increase in NHE3 activity induced by ET-1 was not inhibited by cytochalasin D (11). This result suggested that the actin cytoskeleton and NHE3 trafficking were not required for regulation of NHE3 by ET-1. However, in the present studies we found that cytochalasin D does not inhibit NHE3 trafficking whereas latrunculin B does. These results are very similar to those reported previously for receptor-mediated endocytosis of transferrin where cytochalasin D had no effect, whereas the latrunculins inhibited the process (23). Cytochalasin D binds to the growing barbed ends of F-actin, whereas the latrunculins function by sequestering actin monomers (33). Latrunculins are significantly more potent inhibitors of the actin cytoskeleton than cytochalasin D. It has been postulated that the lack of inhibition of receptor-mediated endocytosis by cytochalasin D may be due to the fact that cortical actin filaments are more resistant to its effects (23). We found that whereas latrunculin B caused a complete disappearance of actin filaments, cytochalasin D caused actin filaments to clump, mostly around the nucleus. Similar observations have been reported in NIL8 fibroblasts (33). In permeabilized pancreatic acinar cells, low levels of actin depolymerization enhance exocytosis, whereas extensive depolymerization inhibits exocytosis (27). This result suggests that exocytic secretion requires actin depolymerization and an additional effect of the actin cytoskeleton. The specific role played in exocytic insertion of NHE3 may be similar.
In previous studies we demonstrated ET-1-induced NHE3 phosphorylation on multiple serine and threonine residues (28). This occurred with a time course similar to that observed for NHE3 activation and trafficking (12). Vasopressin-induced insertion of aquaporin-2-containing vesicles into the apical membrane of the renal collecting duct is accompanied by phosphorylation of aquaporin-2 (21, 24). In addition, mutation of the phosphorylated residue Ser-256 inhibits trafficking of aquaporin-2 (20). Similarly, phosphorylation of NHE3 may serve as a potential signal for ET-1-induced exocytic insertion. ET-1-induced phosphorylation of NHE3 is associated with decreased mobility of NHE3 on SDS-PAGE, resulting in an apparent size shift (28). ET-1 also caused an apparent size shift in apical membrane NHE3 (Figs. 1, 2, and 4), implying that both hypo- and hyperphosphorylated forms of NHE3 are resident in the apical membrane. Last, we found that treatment with latrunculin B did not block the size shift in apical NHE3 (data not shown), suggesting that NHE3 phosphorylation does not require the actin cytoskeleton.
Metabolic acidosis induces an increase in proximal tubule apical membrane NHE3 activity, which is associated with an increase in apical membrane NHE3 protein abundance (2, 35). Thus far, studies have failed to demonstrate an increase in renal cortical NHE3 protein abundance or NHE3 mRNA abundance, suggesting that the major mechanism responsible for increased apical membrane NHE3 abundance may be trafficking to the apical membrane. In OKP cells, incubation in acid media induces increased exocytic insertion of NHE3 into the apical membrane (36), similar to the effects of ET-1. CO2-induced intracellular acidification induces exocytosis in perfused proximal tubules (32). By regulating trafficking of NHE3, the endothelins could mediate the effects of acidosis on NHE3. Renal interstitial levels of ET-1 are increased in acidosis, and blockade of ETB receptors impairs regulation of distal tubule function in acidosis (34). We have found recently that while acid feeding increases renal cortical apical membrane Na/H antiporter activity in wild-type mice, it has no effect in ETB receptor deficient mice (22). Thus acidosis-induced increases in ET-1 expression likely signal trafficking and activation of NHE3.
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ACKNOWLEDGEMENTS |
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Technical assistance was provided by Kavita Mahti and Ebtesam Abdel-Salam.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-39298 and DK-48482, by the Advanced Research Program of the state of Texas, and by the Department of Veterans Affairs. M. Yanagisawa is an investigator of the Howard Hughes Medical Institute.
Address for reprint requests and other correspondence: R. J. Alpern, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9003 (E-mail: Robert.Alpern{at}utsouthwestern.edu).
1 In these studies, the magnitude of the change in apical NHE3 protein abundance tends to be greater than the change in NHE3 activity. The assay for Na/H antiporter activity measures the initial rate of cell pH increase on addition of Na. In that this measurement is made over a few seconds, the "initial rate" is slowed by increases in cell pH and cell Na concentration. This effect becomes more significant at greater antiporter rates, and thus will tend to decrease the apparent magnitude of a change in activity.
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 8 December 1999; accepted in final form 20 September 2000.
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