ETB receptor activation causes exocytic insertion of NHE3 in OKP cells

Yan Peng1, Morimasa Amemiya2, Xiaojing Yang1, Lingzhi Fan1, Orson W. Moe1,3, Helen Yin4, Patricia A. Preisig1, Masashi Yanagisawa5, and Robert J. Alpern1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelin-1 (ET-1) activates sodium/hydrogen exchanger 3 (NHE3) in opossum kidney clone P (OKP) cells expressing ETB receptors. ET-1 (10-8 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Sralpha 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, beta -glycerophosphate, 50 NaF, 1 Na orthovanadate, 1 phenylmethylsulfonyl fluoride, and 0.5 dithiothreitol, as well as 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 2 µg/ml pepstatin, 5 µg/ml aprotinin, and 5 µg/ml leupeptin], at 4°C × 45 min, and centrifuged at 10,000 g for 15 min. Supernatants were diluted with RIPA buffer to 3 mg protein/ml (Bradford method, Bio-Rad). In some experiments, an aliquot of the supernatant, representing total cellular protein, was mixed with an equal volume of 2× SDS loading buffer (5 mM Tris · HCl, pH 6.8, 1% SDS, 10% glycerol, 1% 2-mercaptoethanol), boiled × 5 min, size fractionated by SDS-PAGE on 7.5% gels, and electrophoretically transferred to nitrocellulose. After blocking with 5% nonfat milk and 0.05% Tween 20 in PBS × 1 h, blots were probed with a polyclonal anti-opossum NHE3 antibody [antiserum 5683, generated against a maltose binding protein/NHE3 (amino acid 484-839) fusion protein] at a dilution of 1:200 (1). Blots were washed in 0.05% Tween 20 in PBS × 15 min × 1, and × 5 min × 2, incubated with a 1:5,000 dilution of horseradish peroxidase-labeled donkey anti-rabbit IgG in 5% nonfat milk and 0.05% Tween 20 in PBS × 1 h, washed as above, and then visualized by enhanced chemiluminescence. This procedure labeled a 90-kDa band that was not seen when the antibody was preincubated with fusion protein or when preimmune serum replaced the anti-NHE3 antiserum (1). The remainder of the supernatant was incubated with streptavidin-coupled agarose in a 1:5 dilution to isolate biotinylated proteins, and the precipitate was subjected to SDS-PAGE and blotting with anti-NHE3 antibodies as above.

Studies similar to those described above were performed with biocytin hydrazide, a labeling reagent that reacts with sugar residues (26, 29). Cells were washed with PBS-Ca-Mg at 4°C and incubated for 30 min with 10 mM NaIO4 in PBS-Ca-Mg at 4°C in the dark. The cells were again washed with PBS-Ca-Mg and labeled with 2 mM biocytin hydrazide in 100 mM sodium acetate (pH 5.5) for 60 min at 4°C in the dark. Cells were then washed with PBS-Ca-Mg and processed as above.

Endocytic internalization. The endocytic internalization of biotinylated NHE3 (biotin-SS-NHE3) was measured by its acquisition of resistance to the membrane-impermeant reducing agent beta -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 10-8 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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Binding of ET-1 to the ETB receptor increases apical membrane NHE3 protein abundance. We previously found that 10-8 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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Fig. 1.   Endothelin-1 (ET-1) increases apical membrane sodium/hydrogen exchanger 3 (NHE3) protein abundance: sulfo-NHS-SS-biotin labeling. Cells were treated with 10-8 M ET-1 or vehicle for 35 min, and cell surface biotinylation was performed with sulfo-NHS-SS-biotin. A: typical blots. Biotinylated proteins were isolated by precipitation with streptavidin-bound agarose, and apical NHE3 protein abundance was measured by immunoblot (biotinylated NHE3). Total cellular NHE3 protein abundance was measured on cell lysate by immunoblot (cellular NHE3). B: summary of results. Con, control. n = 5. *P < 0.05.

Although these results suggest net trafficking of NHE3 to the apical membrane, another possible explanation is a conformational change in NHE3, exposing reactive lysine and arginine residues to the sulfo-NHS-SS-biotin. ET-1 causes phosphorylation of NHE3 (28), and it is possible that this would induce a conformational change resulting in increased accessibility of sulfo-NHS-SS-biotin to these reactive amino acids. To address this, we repeated the previous experiments by using biocytin hydrazide, which reacts with sugar residues on apical glycoproteins (26, 29). As shown in Fig. 2, by using biocytin hydrazide, ET-1 caused a 98 ± 15% increase in apical membrane NHE3 protein abundance.


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Fig. 2.   ET-1 increases apical membrane NHE3 protein abundance: biocytin hydrazide labeling. A: typical blot. B: summary of results. Cells were treated with 10-8 M ET-1 or vehicle for 35 min. Cell surface biotinylation was performed with biocytin hydrazide, biotinylated proteins were isolated by precipitation with streptavidin-bound agarose, and apical NHE3 protein abundance was measured by immunoblot. C, control. n = 5. *P < 0.005.

These results suggest that there is an intracellular pool of NHE3 that can be inserted into the apical membrane, resulting in a two- to threefold increase in apical membrane NHE3. This implies that at least one-half of cellular NHE3 is located in these intracellular stores. To examine this more directly, cells were treated with sulfo-NHS-SS-biotin. We then performed two sequential streptavidin precipitations followed by immunoprecipitation of NHE3 from the supernatant and measurement of NHE3 abundance in the precipitates by immunoblot. As shown in Fig. 3, the majority of NHE3 is present in the fraction that was not precipitated by streptavidin. As a control we performed similar studies with alkaline phosphatase, a known apical membrane protein and found that the vast majority of alkaline phosphatase was present in the initial streptavidin precipitate. These studies confirm that OKP cells possess a large pool of intracellular NHE3 that can be inserted into the apical membrane.


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Fig. 3.   Opossum kidney clone P (OKP) cells possess a significant intracellular pool (IP) of NHE3. After cell surface biotinylation with sulfo-NHS-SS-biotin, biotinylated proteins were isolated by 2 sequential precipitations (ppt) with streptavidin-bound agarose (SA). NHE3 (left) and alkaline phosphatase (Alk Phos; right) were then immunoprecipitated and labeled by immunoblot in the precipitates.

All remaining studies of NHE3 trafficking were performed by using sulfo-NHS-SS-biotin. The increase in apical membrane NHE3 protein abundance induced by 10-8 M ET-1 was first seen at 5 min and was maximal at 15-35 min (Fig. 4). This time course is similar to that of ET-1 on NHE3 activity where we previously found a small effect at 5 min that became maximal at 12 min (12).


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Fig. 4.   ET-1 increases apical membrane NHE3 protein abundance: time course. A: typical blot. B: summary of results. Cells were treated with 10-8 M ET-1 or vehicle for the indicated times. Cell surface biotinylation was performed with sulfo-NHS-SS-biotin, biotinylated proteins were isolated by precipitation with streptavidin-bound agarose, and apical NHE3 protein abundance was measured by immunoblot. Con, control. n = 4. *P < 0.05.

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 10-8 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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Fig. 5.   ET-1 increases apical membrane NHE3 protein abundance: cytochalasin D. A: typical blot. B: summary of results. Cells were treated with 10-8 M ET-1 or vehicle for 35 min in the presence of 1.25 µM cytochalasin D. Cell surface biotinylation was performed with sulfo-NHS-SS-biotin, biotinylated proteins were isolated by precipitation with streptavidin-bound agarose, and apical NHE3 protein abundance was measured by immunoblot. Con, control. n = 6. *P < 0.05.

To address this further we examined the effect of latrunculin B, another cytoskeletal inhibitor (33). Studies were performed in the absence or presence of 10-5 M latrunculin B. In the absence of latrunculin B, ET-1 caused a 103 ± 23% increase in biotinylated NHE3, while in the presence of latrunculin B, ET-1 had no effect on apical membrane NHE3 protein abundance (+5 ± 13%; Fig. 6).


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Fig. 6.   ET-1 increases apical membrane NHE3 protein abundance: latrunculin B. A: typical blot. B: summary of results. Cells were treated with 10-8 M ET-1 or vehicle for 35 min in the absence or presence of 10-5 M latrunculin B. Cell surface biotinylation was performed with sulfo-NHS-SS-biotin, biotinylated proteins were isolated by precipitation with streptavidin-bound agarose, and apical NHE3 protein abundance was measured by immunoblot. C, control. -Latrunculin B, n = 6; +latrunculin B, n = 8. *P < 0.05.

The different results with latrunculin B and cytochalasin D are likely due to different mechanisms of actin inhibition (see DISCUSSION). To address this we examined the effect of endothelin on the actin cytoskeleton in the absence and presence of cytochalasin D or latrunculin B. The actin cytoskeleton was visualized by staining with FITC-labeled phalloidin (Fig. 7). In the absence of cytoskeletal inhibitor, 10-8 M ET-1 caused the formation of stress fibers (Fig. 7, A and B). In the presence of latrunculin B, no stress fibers were formed and phalloidin labeling was minimal (Fig. 7, C and D). Cytochalasin D also inhibited stress fiber formation, but punctate actin labeling was seen in the perinuclear area and at the cell periphery (Fig. 7, E and F).


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Fig. 7.   Effects of ET-1 on the actin cytoskeleton in the absence and presence of cytochalasin D or latrunculin B. Cells were treated with vehicle (A, C, E) or 10-8 M ET-1 (B, D, F) for 35 min in the absence of cytoskeletal inhibitors (A, B), in the presence of 10-5 M latrunculin B (C, D), or in the presence of 1.25 µM cytochalasin D (E, F). Filamentous actin was then labeled with FITC-conjugated phalloidin.

Given that latrunculin B inhibits NHE3 trafficking, it offers the opportunity to determine the role of trafficking in ET-1-induced increases in NHE3 activity. As shown in Fig. 8, in the absence of latrunculin B ET-1 treatment increased NHE3 activity by 83%, whereas in the presence of latrunculin B ET-1 was without effect.1 These results suggest that the increase in NHE3 activity requires trafficking of NHE3 to the apical membrane.


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Fig. 8.   Latrunculin B inhibits ET-1-induced NHE3 activation. Cells were treated with 10-8 M ET-1 or vehicle for 35 min, in the absence or presence of 10-5 M latrunculin B. NHE3 activity was then assessed as the rate of Na-dependent cell pH recovery from an acid load. dpHi/dt, intracellular pH (pHi) increase. n = 8. *P < 0.001.

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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Fig. 9.   ET-1 does not affect NHE3 internalization. A: typical blot. B: summary of results. Cell surface biotinylation was performed with sulfo-NHS-SS-biotin, after which cells were maintained at 4°C, or were treated with vehicle or ET-1 for 35 min at 37°C. Cells were then treated with beta -mercaptoethane sulfonate, sodium salt (MesNa), biotinylated proteins were isolated by precipitation with streptavidin-bound agarose, and protected NHE3 protein abundance was measured by immunoblot. Labeling was compared with cells not treated with MesNa and is expressed as %NHE3 protected from MesNa. C, control. Horizontal line: protection of labeling in cells maintained at 4°C to inhibit endocytosis. 4°C, n = 3; 37°C, n = 6. P NS.

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 10-8 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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Fig. 10.   ET-1 increases exocytic insertion of NHE3 into the apical membrane. A: typical blot. B: summary of results. Cells were labeled with sulfo-NHS-acetate and then treated with 10-8 M ET-1 or vehicle for 35 min. Cell surface biotinylation was then performed with sulfo-NHS-SS-biotin, biotinylated proteins were isolated by precipitation with streptavidin-bound agarose, and apical NHE3 protein abundance was measured by immunoblot. Con, control. n = 5. *P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

Technical assistance was provided by Kavita Mahti and Ebtesam Abdel-Salam.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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