1 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235; 2 Department of Internal Medicine, Jichi Medical School, Tochigi, Japan 329-0498; and 3 Dallas Veterans Affairs Medical Center, Dallas, Texas 75216
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ABSTRACT |
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Incubation of opossum kidney clone P (OKP) cells in acid media (pH 6.8) causes activation of Na+/H+ exchanger 3 (NHE3) at 6, 12, and 24 h. OKP cell NHE3 protein abundance was increased by 45% at 24 h of acid incubation but was unaffected at 3-12 h. By contrast, apical membrane NHE3, measured by surface biotinylation, increased approximately twofold at 6, 12, and 24 h, mirroring the increase in activity. Acid incubation caused a 76% increase in exocytic insertion of NHE3 into the apical membrane but had no effect on endocytic internalization at 6 h. Latrunculin B, an inhibitor of microfilament organization, inhibited the acid-induced increases in apical membrane NHE3, exocytic insertion of NHE3, and NHE3 activity at 6 h. These studies demonstrate two mechanisms for acid-induced increases in NHE3 activity. Beginning at 6 h, there is an increase in apical membrane NHE3 that is due to stimulated exocytic insertion and is required for increased NHE3 activity. At 24 h, there is an additional increase in total cellular NHE3.
metabolic acidosis; sodium/hydrogen antiporter; proximal tubule
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INTRODUCTION |
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CHRONIC METABOLIC ACIDOSIS causes parallel increases in the activities of the proximal tubule apical membrane Na+/H+ antiporter and the basolateral membrane Na+/HCO3/CO3 symporter (1, 20). The mechanisms responsible for this adaptation have been studied in cell culture, where it has been demonstrated that acidification of the extracellular fluid is sufficient to reproduce the adaptation in the Na+/H+ antiporter. In cultured opossum kidney clone P (OKP) cells, incubation in acid media for 24 h leads to an increase in the activity of Na+/H+ exchanger 3 (NHE3) (6), the Na+/H+ antiporter isoform expressed on the renal proximal tubule apical membrane (5, 7). This increase in NHE3 activity is associated with an increase in NHE3 mRNA abundance at 24 h (6). Considered as a function of pH, Na+/H+ antiporter activity and mRNA abundance increase in parallel at 24 h. However, whereas incubation in acid media increases NHE3 activity within 6 h, the increase in NHE3 mRNA abundance is not seen until 12 h (6). In addition, the effect of acid media on NHE3 activity is not completely blocked by inhibition of protein synthesis (4, 6). This suggests at least two mechanisms of antiporter activation by acid, one related to increases in NHE3 mRNA abundance, and a second related to posttranslational regulation.
The purpose of the present studies was to determine the roles of NHE3 protein abundance and trafficking of NHE3 to the apical membrane, in the regulation of NHE3 by acid. Results demonstrate that acid incubation for 6 h causes a net increase in apical membrane NHE3 that occurs in the absence of a change in total cellular NHE3 protein abundance. At 24 h, there are increases in total cellular and apical NHE3. The increase in apical NHE3 at 6 h is due to stimulated exocytic insertion of NHE3.
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METHODS |
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Materials and supplies. All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted as follows: penicillin and streptomycin from Whittaker M. A. Bioproducts (Walkersville, MD), culture media from GIBCO BRL (Grand Island, NY), 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM from Molecular Probes (Eugene, OR), EZ-Link sulfo-NHS-SS-biotin, Immunopure immobilized streptavidin, and sulfo-NHS-acetate from Pierce (Rockford, IL), anti-actin antibody from Boehringer Mannheim (Indianapolis, IN), and latrunculin B from Alexis (San Diego, CA).
Cell culture.
OKP cells (11) were passaged in high-glucose (450 mg/dl)
DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml),
and streptomycin (100 µg/ml). For experimentation, confluent cells
were rendered quiescent by removal of serum, and media were changed to
a 1:1 mixture of low-glucose (100 mg/dl) DMEM and Ham's F-12 with
109 M hydrocortisone for 24 h. Cells were then
studied in the same media at pH 7.4 or 6.8. Media were acidified by HCl
addition and equilibrated in the incubator before addition to cells.
Immunoblot.
Cells were rinsed with ice-cold PBS three times and dounce
homogenized in membrane buffer [in mM: 150 NaCl, 50 Tris · HCl (pH 7.5), and 5 EDTA] containing proteinase inhibitors [in µg/ml: 100 phenylmethylsulfonyl fluoride (PMSF), 4 aprotinin, and 4 leupeptin]. Nuclei were removed by centrifugation at 13,000 g at 4°C. Membranes were pelleted by centrifugation at
109,000 g for 20 min (Beckman TLX: TLA 100.3 rotor; 50,000 rpm). The resulting pellet was resuspended in membrane buffer and total
protein content determined by the method of Bradford. Twenty micrograms
of protein were diluted 1:5 in 5× SDS loading buffer (1 mM
Tris · HCl, pH 6.8, 1% SDS, 10% glycerol, and 1%
-mercaptoethanol), size fractionated by SDS-PAGE (7.5% gel), and
electrophoretically transferred to nitrocellulose. Blots were then
probed sequentially with rabbit polyclonal anti-opossum NHE3 antiserum
[antiserum 5683, generated against a maltose binding protein/NHE3
(amino acids 484-839) fusion protein] at a dilution of
1:500 and peroxidase-labeled donkey anti-rabbit IgG at a 1:5,000 dilution, as described previously (2). Labeling was
visualized by enhanced chemiluminescence, and NHE3 protein abundance
quantitated by densitometry (Molecular Dynamics Image Quant Software,
version 3.3). This procedure labeled a 90-kDa band that was not seen
when the antibody was preincubated with fusion protein or when
preimmune serum replaced anti-NHE3 antiserum (2).
Biotinylation. To measure apical membrane NHE3, we used surface biotinylation (14, 18, 21). One hundred-millimeter plates of confluent OKP cells were rinsed three times with PBS with 0.1 mM CaCl2 and 1.0 mM MgCl2 (PBS-Ca-Mg) at 4°C. 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 with 100 mM glycine) for 20 min at 4°C. Cells were then lysed in RIPA buffer [150 mM NaCl, 50 mM Tris · HCl (pH 7.4), 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin] × 30 min at 4°C, and centrifuged at 103,000 g × 10 min. The supernatant was diluted to 3 mg of protein/ml, biotinylated proteins precipitated with 120 µl of streptavidin-coupled agarose in a total volume of 600 µl, and the precipitate was subjected to SDS-PAGE and blotting with anti-NHE3 antibodies, as described in Immunoblot.
Internalization assay. The internalization of biotinylated NHE3 (biotin-SS-NHE3) was measured by its acquisition of resistance to the membrane-impermeant reducing agent, mesna (2-mercaptoethanesulfonic acid sodium salt) (10). Cells were labeled with sulfo-NHS-SS-biotin and quenched, as described in Biotinylation. Cells were then rinsed with PBS at 37°C and incubated at 37°C in serum-free media at pH 7.4 or 6.8 for 6 h. Plates were then rinsed with Tris-buffered saline (TBS) [100 mM Tris · HCl (pH 7.5), 150 mM NaCl] at 4°C twice and incubated at 4°C in 2 ml of 10 mM mesna (in 100 mM NaCl, 1 mM EDTA, 50 mM Tris, 0.2% BSA, pH 8.6), prepared just before addition. At 30 min, an additional 0.5 ml of 50 mM mesna was added to each plate, and at 60 min, an additional 0.65 ml of 50 mM mesna was added. At 90 min, mesna was oxidized by addition of 1 ml of 500 mM iodoacetic acid for 10 min, and cells were lysed with RIPA buffer, biotinylated proteins were precipitated with streptavidin-coupled agarose, and the precipitate was subjected to SDS-PAGE and blotting with anti-NHE3 antiserum, as described in Immunoblot. Results are expressed as percentage of apical NHE3 (percentage of biotinylated NHE3 in cells incubated for 6 h under control conditions and not treated with mesna).
Exocytic insertion assay.
Cells were rinsed with PBS-Ca-Mg three times at
room temperature, and the apical surface was 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 apical
membrane proteins were blocked, cells were washed with quenching
solution for 20 min at room temperature. Cells were then rinsed with
PBS at 37°C and incubated in serum-free media at pH 7.4 or 6.8 at
37°C for 6 h. Cells were then labeled with 1.5 mg/ml
sulfo-NHS-SS-biotin and lysed with RIPA buffer, biotinylated proteins
were precipitated with streptavidin-coupled agarose, and the
precipitate was subjected to SDS-PAGE and blotting with anti-NHE3
antiserum, as described in Biotinylation.
(biotin-NHE3Blockade/Incubation). To measure the efficiency
of blockade, we performed experiments wherein membrane biotinylation
was performed immediately after blockade with sulfo-NHS-acetate (biotin-NHE3Blockade/No incubation). NHE3 inserted is
expressed as the percentage of basal apical NHE3, corrected for lack of completeness of blockade, using the following formula
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Measurement of intracellular pH and Na+/H+ antiporter activity. Continuous measurement of cytoplasmic pH (pHi) was accomplished using the intracellularly trapped pH-sensitive dye BCECF, as previously described (9). Cells grown on glass coverslips were loaded with 10 µM AM of BCECF 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 (140 mM Na+) pHi increase after an acid load (13 µM nigericin in Na+-free solution) in the absence of CO2/HCO3, as previously described (9). Initial rates were determined over the pH range of 6.4-6.5, measured over 15-30 s. Changes in media pH have no effect on buffer capacity (6). Results are, therefore, reported as dpHi/dt.
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RESULTS |
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Acid incubation increases NHE3 protein abundance.
NHE3 protein abundance was measured by Western blot at 3, 6, 12, and
24 h. Compared with pH 7.4, incubation at pH 6.8 increased NHE3
protein abundance by 45 ± 12% at 24 h (Fig.
1). There was no effect at 3, 6, or 12 h. This time course is slightly delayed from that for the
effect of acid incubation on NHE3 mRNA abundance, which increases at
12 h (6).
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Acid incubation increases apical membrane NHE3 protein abundance.
We previously found that NHE3 activity was increased by acid incubation
at 6 h, before NHE3 mRNA or protein abundance were increased. We
next used surface biotinylation to address whether this early increase
in activity is due to a selective increase in apical membrane NHE3
protein. As shown in Fig. 2, apical
membrane NHE3 protein abundance increased progressively over time in
response to acid incubation. At 3 h, acid-incubated cells
demonstrated a 52 ± 37% increase in apical NHE3 [not
statistically significant (NS)], at 6 h a 96 ± 44% increase, at 12 h a 126 ± 48% increase, and at 24 h a 126 ± 35% increase. At 6 and 12 h, the increase in
apical NHE3 protein abundance occurred in the absence of an increase in
total cellular NHE3 abundance (Fig. 1). The time course for the effect
of acid incubation on apical NHE3 protein abundance mirrors the time
course for the increase in NHE3 activity (6).
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Latrunculin B blocks acid-induced increases in apical membrane NHE3
protein abundance and
Na+/H+
antiporter activity at 6 h.
To determine the role of trafficking in the acid-induced increase in
NHE3 activity, we attempted to inhibit trafficking by cytoskeletal
disruption with latrunculin B. In the absence of latrunculin B, acid
incubation for 6 h increased biotinylated NHE3 by 94 ± 28%
(Fig. 4). Latrunculin B
(105 M) caused a 23% decrease in apical NHE3 abundance,
and in the presence of latrunculin B, acid incubation decreased
biotinylated NHE3 by 40 ± 7% (Fig. 4). Thus latrunculin B
inhibits the acid-induced increase in apical NHE3. The mechanism
responsible for the acid-induced decrease in apical NHE3 in the
presence of latrunculin B is unclear.
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The acid-induced increase in apical NHE3 is not due to decreased
endocytic internalization.
The increase in apical membrane NHE3 abundance could be due to
decreased endocytic internalization or increased exocytic insertion of
NHE3 into the apical membrane. To measure endocytic internalization, apical membrane proteins were labeled with sulfo-NHS-SS-biotin before
incubation at pH 7.4 or 6.8 for 6 h. Cells were then exposed to
the cell-impermeant sulfhydryl reducing reagent, mesna, which cleaves
off biotin from any proteins remaining on the apical membrane. After
this, biotinylated NHE3 was identified by precipitation and Western
blotting, as described in Biotinylation. With this protocol, biotinylated NHE3 represents internalized NHE3 (present on
the apical membrane initially and endocytosed during the 6 h
incubation to protect it from mesna). In cells maintained at 4°C to
inhibit endocytosis, mesna cleaved off 96% of biotin from apical NHE3
(unpublished observations), indicating that mesna is effective in
reducing the disulfide bond when NHE3 remains exposed to the apical
fluid. Compared with cells not treated with mesna, 42 ± 9% of
biotin labeling was protected from mesna in control cells incubated at
pH 7.4 for 6 h. This indicates that 42% of apical NHE3 undergoes
endocytosis over 6 h. In other experiments, we found that 5% of
apical NHE3 is internalized over 35 min (unpublished observations).
Extrapolating this result to 6 h suggests that endocytic
internalization is near linear over 6 h. After acid incubation for
6 h, 37 ± 7% of biotin labeling was protected from mesna
(NS), indicating no effect of acid incubation on endocytic retrieval
(Fig. 7).
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The acid-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. After this, cells were
incubated at pH 7.4 or 6.8 for 6 h and membrane biotinylation was
performed as described in Biotinylation. Any labeled NHE3
should represent 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. As a control, we performed
experiments wherein membrane biotinylation was performed immediately
after blockade with sulfo-NHS-acetate. In this setting, labeling of
NHE3 was inhibited by 84 ± 5% (Fig. 8), demonstrating an 84% efficiency of
blockade. NHE3 inserted into the apical membrane after control or acid
incubation is expressed as the percentage of basal apical NHE3,
corrected for the lack of completeness of acetate blockade, as
described in METHODS (Fig. 9). Exocytic insertion of NHE3 was
41 ± 8% of apical NHE3 over 6 h under control conditions,
and increased to 72 ± 13% under acid conditions.
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DISCUSSION |
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Chronic metabolic acidosis elicits a series of homeostatic adaptations that serve to ameliorate the change in pH. One component of this adaptation involves an increase in the activity of the proximal tubule apical membrane Na+/H+ antiporter, encoded by NHE3 (1, 5, 7, 20, 24). The increase in apical NHE3 activity is accompanied by an increase in renal cortical apical membrane NHE3 protein abundance (3, 24).
Incubation of OKP cells in acid media elicits an increase in NHE3
activity similar to that seen in vivo (6). The increase in
NHE3 activity is first seen between 3 and 6 h and persists over
24 h. It is of significant interest that the increase in NHE3
activity and the increase in apical membrane NHE3 abundance have
similar time courses, both increasing between 3 and 6 h, and then
plateauing (Fig. 11). This suggests
that the increase in apical membrane NHE3 is responsible for the
increase in activity. It should be noted that the increase in apical
protein abundance is greater (100%) than the increase in activity
(30-50%). This may be due to the fact that a fraction of inserted
NHE3 is inactive or, more likely, the assay for activity is not truly
linear.1
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On the basis of the present and previous results, the mechanism of the increase in apical membrane NHE3 abundance includes at least 2 components that are temporally distinct. The first component, seen at 6 h, involves a twofold increase in apical membrane NHE3 in the absence of changes in whole cell NHE3 protein abundance and in the absence of a change in NHE3 mRNA abundance (6) (Fig. 11). At 3 h, a 50% increase in apical NHE3 was seen, but this did not achieve statistical significance, and it is difficult to be certain of its significance. There is no increase in NHE3 activity at 3 h (6).
To further prove that NHE3 trafficking to the apical membrane mediates the increase in NHE3 activity at 6 h, we used latrunculin B. The latrunculins are marine compounds isolated from the Red Sea sponge Latrunculia magnifica, which disrupt microfilament organization by sequestering actin monomers and inhibit microfilament-mediated processes (17, 23). Incubation of OKP cells in latrunculin B for 6 h caused a 23% decrease in apical NHE3 abundance and a 15% decrease in NHE3 activity (Figs. 4 and 5). This could represent a toxic effect of latrunculin B or could represent a physiological effect of cytoskeletal disruption. The cells appeared healthy after 6 h of exposure to latrunculin B.
Addition of 105 M latrunculin B inhibited trafficking of
NHE3 to the apical membrane and inhibited the acid-induced increase in
NHE3 activity at 6 h. This result suggests that trafficking of
NHE3 to the apical membrane is responsible for the increase in NHE3
activity at 6 h. Given that cytoskeletal disruption may inhibit
many cellular functions, it remains possible that other mechanisms
contribute to this early increase in NHE3 activity. We have previously
attempted to measure the effect of acid incubation on NHE3
phosphorylation, but results were not interpretable due to acid-induced
inhibition of phosphate uptake into OKP cells. The observation that
colchicine, a microtubule inhibitor, also inhibits the early increase
in NHE3 activity provides further support for a role of membrane trafficking.
The acid-induced net increase in apical membrane NHE3 at 6 h could be due to decreased endocytic internalization or increased exocytic insertion. To measure endocytic internalization of NHE3, we prelabeled NHE3 with sulfo-NHS-SS-biotin and then measured the ability of acid incubation to cause NHE3 to shift to a location where the disulfide bond would be protected from mesna. These studies showed that 40% of apical NHE3 is internalized over 6 h and that acid incubation had no effect on the process. To measure exocytic insertion of NHE3, we preblocked apical NHE3, and then measured the ability to label new NHE3 with biotin. These studies showed that ~40% of apical NHE3 is inserted over 6 h and that this rate increases twofold on acid incubation. Latrunculin B also blocked the acid-induced increase in exocytic insertion of NHE3.
In these studies, treatment with surface-labeling reagents such as sulfo-NHS-SS-biotin or sulfo-NHS-acetate may alter activity, which may secondarily affect trafficking. To address this, we measured NHE3 activity after biotinylation and a subsequent 6 h of incubation at pH 7.4. Compared with cells preincubated in biotinylation buffer alone, cells treated with sulfo-NHS-SS-biotin demonstrated a 50% decrease in NHE3 activity. This suggests that biotinylation inhibits NHE3 activity. In that changes in activity may have secondary effects on trafficking, it is possible that measured rates of exocytic insertion and endocytic internalization may be quantitatively affected. However, this does not invalidate the qualitative comparison in that all control and acid-incubated cells were treated similarly. In addition, it is reassuring that under conditions of incubation at pH 7.4, rates of endocytic internalization and exocytic insertion are similar. Biotinylation had no effect on cell pH, either baseline or after an acid load.
Trafficking to and from the apical membrane has been proposed to mediate regulation of NHE3 in other settings. In the in vivo proximal tubule, an acute increase in blood pressure causes a rapid shift of NHE3 from the apical membrane fraction to higher density membranes enriched in intracellular membrane markers (26). A similar response was shown using immunohistochemistry to localize NHE3 in acute and chronic hypertension (25). In vivo administration of parathyroid hormone also causes a shift of NHE3 from the apical membrane to an intracellular compartment (12, 27). Activation of protein kinase C in Caco-2 cells inhibits NHE3 by causing endocytic retrieval (15).
Changes in intracellular pH have been shown to regulate movement of late endosomes along microtubules (19). Exocytic insertion has been shown to mediate CO2-induced increases in apical membrane H-ATPase activity. In the turtle urinary bladder, increases in CO2 tension lead to an increase in H+ secretion that is accompanied by an increase in exocytosis (13). Colchicine pretreatment inhibits the increase in exocytosis and the increase in H+ transport. CO2-induced exocytosis is mediated by intracellular acidification with secondary increases in cell Ca2+ concentration (8). CO2-induced exocytosis has also been demonstrated in perfused proximal and collecting tubules (22). Exocytosis thus may provide a common mechanism for regulation of apical membrane transporters by changes in intracellular pH.
The second component of the acid-induced increase in NHE3 activity involves an increase in NHE3 mRNA and whole cell NHE3 protein abundance. The increase in mRNA abundance is first seen at 12 h (6), preceding the increase in protein abundance (Fig. 11). We have previously shown that the increase in NHE3 protein abundance at 24 h is inhibited by cycloheximide, suggesting that it is due to increased NHE3 protein synthesis (4), likely due to the increase in NHE3 mRNA abundance. In addition, we found that the increase in protein synthesis is synergistically increased by glucocorticoids in the absence of a synergistic effect on NHE3 mRNA (4). In that the increase in whole cell NHE3 protein at 24 h is just 45%, whereas the increase in apical membrane NHE3 is 126%, trafficking likely contributes to increased apical NHE3 even at 24 h. In support of this, we previously found that acid incubation causes a 33% increase in apical NHE3 protein abundance in the absence of protein synthesis (cycloheximide) (4).
One question of significant importance is whether one or both of these components of NHE3 regulation are relevant to the in vivo response of the kidney to metabolic acidosis. Chronic metabolic acidosis induces an increase in proximal tubule apical membrane NHE3 activity, which is associated with an increase in apical membrane NHE3 abundance (3, 24). These studies 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 is trafficking to the apical membrane. However, it is possible that small increases in NHE3 mRNA and protein abundance have been missed because of their small magnitudes. Of interest, in the thick ascending limb, NHE3 protein and mRNA abundances are increased in chronic metabolic acidosis (16). Thus, of the two components of the acid response found in the present studies, trafficking may be more important in the proximal tubule, and increased mRNA and protein abundance may be more important in the thick ascending limb.
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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 and by the Veterans Administration.
Address for reprint requests and other correspondence: R. J. Al-pern, Southwestern Medical School, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9003 (E-mail: Robert.Alpern{at}emailswmed.edu).
1 The assay for Na+/H+ antiporter activity involves measuring the initial rate of cell pH increase in response to Na+ addition. If a rate later than the initial rate is measured, it will be slowed by increases in cell pH and cell Na+ concentration. This will be a more significant problem when antiporter activity is greater and thus will tend to decrease the apparent magnitude of an increase 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. §1734 solely to indicate this fact.
Received 12 August 1999; accepted in final form 29 February 2000.
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