ETB receptor activation
leads to activation and phosphorylation of NHE3
Y.
Peng1,
O. W.
Moe1,2,
T.-S.
Chu1,
P. A.
Preisig1,
M.
Yanagisawa3,4, and
R. J.
Alpern1
Departments of 1 Internal
Medicine and 3 Molecular Genetics
and 4 Howard Hughes Medical
Institute, University of Texas Southwestern Medical Center, Dallas,
75235; and 2 Veterans Affairs
Medial Center, Dallas, Texas 75216
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ABSTRACT |
In OKP cells
expressing ETB endothelin
receptors, activation of
Na+/H+
antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation of
Na+/H+
exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation in
cells expressing ETB receptors but
not in cells expressing ETA
receptors. Receptor specificity was not due to demonstrable differences
in receptor-specific activation of tyrosine phosphorylation pathways or
inhibition of adenylyl cyclase. Phosphorylation was associated with a
decrease in mobility on SDS-PAGE, which was reversed by treating
immunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation was
first seen at 5 min and was maximal at 15-30 min. Phosphorylation
was maximal with 10
9 M
ET-1. Phosphorylation occurred on threonine and serine residues at
multiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKP
cells on multiple threonine and serine residues.
ETB receptor specificity, time
course, and concentration dependence are all similar between
ET-1-induced increases in NHE3 activity and phosphorylation, suggesting
that phosphorylation plays a key role in activation.
sodium/hydrogen antiporter; adenylyl cyclase; tyrosine kinases; endothelin; OKP cells
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INTRODUCTION |
THE ENDOTHELINS ARE A family of three 21-amino acid
peptides (ET-1, ET-2, and ET-3) that serve as paracrine/autocrine
factors by interacting with two receptors
(ETA and
ETB). ET-1 has been shown to
increase the activity of the proximal tubule apical membrane Na+/H+
antiporter (13, 15), which mediates the majority of proximal tubular
NaCl and NaHCO3 absorption. Five
plasma membrane
Na+/H+
exchanger (NHE) isoforms have been cloned (NHE1-NHE5). The
proximal tubule apical membrane
Na+/H+
antiporter is encoded predominantly by NHE3, based on localization of
NHE3 protein expression (3, 8), localization of NHE3 mRNA expression
(26, 31), inhibitor kinetics (25, 32, 37), regulation by
glucocorticoids and acidosis (1, 6, 37, 40), and inhibition of 61% of
proximal tubule HCO
3 absorption in
NHE3 knockout mice (29).
OKP cells express NHE3, and in these cells NHE3 is stimulated by
glucocorticoids and acidosis, similar to its regulation in vivo (4, 5).
In OKP cells expressing the ETB
but not the ETA receptor, ET-1
increases
Na+/H+
antiporter activity (12). This agrees with binding studies that suggest
that the ETB receptor is the
predominant endothelin receptor of the renal proximal tubule (30). The
present studies examine whether ET-1-induced
Na+/H+
antiporter activation is associated with NHE3 activation and phosphorylation. The results demonstrate that ET-1 activates NHE3. Binding of ET-1 to ETB receptors,
but not to ETA receptors, causes a
two- to threefold increase in NHE3 phosphorylation with a time and dose
dependence that parallels that of the change in activity. Phosphorylation occurs at multiple sites involving serine and threonine
residues. The nature of the receptor specificity is not due to
receptor-specific activation of tyrosine phosphorylation pathways or
inhibition of adenylyl cyclase.
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METHODS |
Materials.
All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless
otherwise noted as follows. Penicillin and streptomycin were from
Whittaker MA Bioproducts (Walkersville, MD); culture media and G418
were from GIBCO BRL (Grand Island, NY); Triton X-100 and protein
G-agarose were from Calbiochem (La Jolla, CA); horseradish peroxidase
(HRP)-labeled anti-mouse IgG, enhanced chemiluminescence (ECL) kit, and
cAMP RIA kit were from Amersham (Arlington Heights, IL);
[35S]methionine-cystine
was from DuPont NEN (Boston, MA); anti-phosphotyrosine monoclonal
antibody PY20 was from Santa Cruz Biotechnology (Santa Cruz, CA);
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM and ethylisopropyl amiloride (EIPA) were from Molecular
Probes (Eugene, OR); and ET-1 was from Peptides International
(Louisville, KY).
Cell culture.
Studies were performed in clonal cell lines generated by stable
transfection of OKP cells with either
pMEhETA or
pMEhETB plasmids, which contain
the cDNAs for the ETA and
ETB receptors, respectively, driven by an Sr
promoter
(OKPETA and
OKPETB cells, respectively) (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 cells were studied. 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.
To measure levels of cAMP production, cells were treated with 2 mM IBMX
during treatment with ET-1 or vehicle. After protein precipitation with
addition of 100 µl TCA/well, the supernatant was extracted with
water-saturated ether and used to measure cAMP by RIA, as described
(12).
Na+/H+
antiporter activity.
Cell pH was measured in a temperature-controlled spectrofluorometer
(SLM 8000C) as the ratio of BCECF fluorescence with excitation at 500 nm to that at 450 nm (530-nm emission wavelength), as previously described (17). To measure
Na+/H+
antiporter activity, cells were acidified by addition of nigericin in
Na+-free media.
Na+/H+
antiporter activity was then measured as the initial rate of cell
alkalinization in response to Na+
addition, as previously described (10).
Western blotting.
Cultured cells were washed in PBS three times, scraped in RIPA buffer
[in mM: 150 NaCl, 50 Tris · HCl (pH 7.4), 2.5 EDTA, 5 EGTA, 50
-glycerophosphate, 50 NaF, 1 sodium orthovanadate, 1 phenylmethylsulfonyl fluoride (PMSF), 0.5 dithiothreitol, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS, 2 µg/ml pepstatin, 5 µg/ml leupeptin, and 5 µg/ml aprotinin], incubated at 4°C
for 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),
then mixed with an equal volume of 2× SDS loading buffer [5
mM Tris · HCl (pH 6.8), 1% SDS, 10% glycerol, and
1%
-mercaptoethanol], boiled 5 min, size fractionated by
SDS-PAGE on 7.5% gels, and electrophoretically transferred to
nitrocellulose. After being blocked with 5% nonfat milk and 0.05%
Tween 20 in PBS for 1 h, blots were probed with a polyclonal
anti-opossum NHE3 antibody [antiserum 5683, generated against a
maltose binding protein-NHE3 (amino acids 484-839) fusion
protein] at a dilution of 1:200 (2). Blots were washed in 0.05%
Tween 20 in PBS once for 15 min and twice for 5 min, incubated with a
1:5,000 dilution of HRP-labeled donkey anti-rabbit IgG in 5% nonfat
milk and 0.05% Tween 20 in PBS for 1 h, washed as above, and then
visualized by ECL. This procedure labeled a 90-kDa band that was not
seen when antibody was preincubated with fusion protein or when
preimmune serum replaced the anti-NHE3 antiserum (2). To measure
protein tyrosine phosphorylation, cell extracts were treated as above
but blotted with 1 µg/ml mouse monoclonal anti-phosphotyrosine
antibody PY20 and HRP-labeled sheep anti-mouse IgG.
Immunoprecipitation.
Cells were extracted in RIPA buffer as above; 700 µl of cell extract
were mixed with 15 µl of polyclonal anti-NHE3 antiserum, rocked
overnight at 4°C, mixed with 25 µl protein G-agarose, rocked for
2 h, pelleted at 10,000 g for 30 s,
and washed three times with RIPA buffer. The pellet was suspended in 60 µl of 1× SDS loading buffer, boiled for 5 min, and subjected to
SDS-PAGE.
To assess the effect of dephosphorylation on NHE3 mobility,
immunoprecipitated NHE3 was washed in 1 ml of reaction buffer [50
mM Tris (pH 8.5), 2 mM PMSF, 0.1%
-mercaptoethanol, and 8 mM
MgCl2], and suspended in 30 µl of reaction buffer with 30 units of alkaline phosphatase (type
VII-T from bovine intestine) at 37°C for 1 h. (Native brush-border
alkaline phosphatase does not interfere with this reaction, as it is
inactivated during extraction in RIPA buffer.) The
reaction was quenched by diluting the sample 1:2 in 2× SDS
buffer, and the sample was subjected to SDS-PAGE and Western blotting
with NHE3 antisera.
To measure protein tyrosine phosphorylation, cells were washed in
methionine- and cystine-free medium for 1 h and metabolically labeled
by incubation with 275 µCi
[35S]methionine-cystine
in the same medium for 2 h before study. After treatment with ET-1 or
vehicle (0.1% acetic acid), extracts from metabolically labeled cells
were prepared as described above, and proteins were immunoprecipitated
with 6 µg/ml mouse monoclonal anti-phosphotyrosine antibody PY20.
Tyrosine phosphorylation was then determined by autoradiography.
Phosphorylation.
Cells were washed in TBS [137 mM NaCl, 2.7 mM KCl, and 25 mM Tris
(pH 7.4)] three times, incubated in phosphate-free DMEM for 60 min, washed in TBS twice, and incubated with
[32P]orthophosphate
(200 mCi/ml) in phosphate-free DMEM for 2.5 h. Cells were then treated
with 10
8 M ET-1 or vehicle
for 35 min and washed with TBS three times, and NHE3 was
immunoprecipitated as above. The immunoprecipitate was subjected to
SDS-PAGE, 32P incorporation was
measured by autoradiography, and results were normalized for NHE3
abundance as measured by Western blot. All phosphorylation experiments
were performed in triplicate.
Phosphoamino acid analysis.
After autoradiography of polyacrylamide gels, the NHE3 band was
extracted in 50 mM
NH4HCO3
with 0.1% (wt/vol) SDS and 50 µl/ml
-mercaptoethanol
as described (9), TCA precipitated, washed with 90% ethanol and then
acetone, and air dried. The phosphoprotein was then hydrolyzed in 200 µl of 6 N HCl for 2 h, lyophilized, suspended in 10 µl of loading
buffer [15 parts pH 1.9 electrophoresis buffer (50 ml 88% formic
acid and 156 ml glacial acetic acid in 2 liters deionized water) and 1 part 1 mg/ml phosphoserine, phosphothreonine, and phosphotyrosine
standards] and centrifuged at 10,000 rpm for 1 min, and 8 µl of
supernatant were spotted onto the TLC plate. The sample was then
electrophoresed in the first dimension in pH 1.9 buffer (1.5 kV; Hunter
thin-layer electrophoresis system) and in the second dimension in pH
3.5 buffer (100 ml glacial acetic acid and 10 ml pyridine in 2 liters
of water; 1.3 kV), and labeled phosphoamino acids were identified by
phosphorimaging and alignment with ninhydrin standards.
Phosphopeptide analysis.
Phosphopeptide analysis was performed by two-dimensional gel
electrophoresis-chromatography following trypsin digestion, as described (9). After 32P
incorporation, immunoprecipitation, SDS-PAGE, and transfer of the
sample to nitrocellulose, the filter was washed in 0.5%
polyvinylpyrrolidone-360 in glacial acetic acid at 37°C for 30 min,
then in 2 ml of water four times, and then in 2 ml of 50 mM
NH4HCO3
twice. The sample was then incubated four times for 2 h each time in
200 µl of 50 mM
NH4HCO3
with 15 µl of trypsin (1 mg/ml 0.1 M HCl) at 37°C; 300 µl of
water were added, the membrane was removed, and the phosphopeptide was
subjected to cycles of 500-µl water washes and lyophilization until
no salt residues were visible. The sample was then washed in 200 µl
of pH 1.9 electrophoresis buffer, lyophilized, resuspended in 20 µl
of the same buffer, and spotted onto a TLC plate. The sample was
electrophoresed in the first dimension in pH 1.9 buffer for 30 min and then subjected to chromatography in the second dimension for 12 h in 375 ml n-butanol, 250 ml
pyridine, 75 ml glacial acetic acid, and 300 ml water, and peptides
were localized by phosphorimaging.
 |
RESULTS |
Binding of ET-1 to the ETB receptor
activates NHE3.
We previously showed that
10
8 M ET-1 causes a
25-40% increase in
Na+/H+
antiporter activity in clonal OKP cells stably transfected with a cDNA
encoding the ETB receptor
(OKPETB cells) (12). Although resting OKP cells express an EIPA-resistant
Na+/H+
antiporter encoded by NHE3, it is possible that endothelin activates a
different NHE isoform that is not detectable at baseline. To address
this, we examined the EIPA sensitivity of
Na+/H+
antiporter activity in the absence and presence of
10
8 M ET-1 in
OKPETB6 cells
(OKPETB cells, clone 6).
Studies were performed in the presence of 15 mM
Na+ and in the absence or presence
of 10
7 M EIPA. With 15 mM
Na+, this concentration of EIPA
should inhibit the amiloride-sensitive antiporter
isoforms, NHE1 and NHE2, but should not inhibit the amiloride-resistant NHE3. As shown in Fig.
1,
10
7 M EIPA did not inhibit
Na+/H+
antiporter activity in the absence or presence of ET-1. In addition, 10
7 M EIPA did not inhibit
the ET-1-induced increase in
Na+/H+
antiporter activity. In the absence of EIPA ET-1 increased
Na+/H+
antiporter activity by 39%, whereas in the presence of EIPA ET-1 increased
Na+/H+
antiporter activity by 43%. Thus ET-1 increases the activity of NHE3
in OKP cells.

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Fig. 1.
Endothelin-1 (ET-1) activates
Na+/H+
exchanger isoform 3 (NHE3) in
OKPETB6 cells.
OKPETB6 cells were treated with
10 8 M ET-1 or vehicle
(control) for 35 min.
Na+/H+
antiporter activity was then measured as initial rate of
Na+-dependent (15 mM
Na+) intracellular pH recovery
(dpHi/dt)
following an acid load. Studies were performed in absence or presence
of 10 7 M ethylisopropyl
amiloride (EIPA). * P < 0.05 vs. control; ** P < 0.005 vs.
control.
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Binding of ET-1 to the ETB receptor
causes NHE3 phosphorylation.
To examine whether phosphorylation of NHE3 plays a role in the
ET-1-induced increase in NHE3 activity, clonal OKP cell lines expressing the ETB receptor were
preincubated in
32PO4
and treated with vehicle or ET-1, and NHE3 was immunoprecipitated. Phosphorylation was measured by autoradiography and normalized for NHE3
abundance as measured by Western blot (Fig.
2). Addition of
10
8 M ET-1 to
OKPETB6 cells for 35 min caused a
2.3 ± 0.6-fold increase in NHE3 phosphorylation
(P < 0.03), which was associated
with a decrease in mobility on SDS-PAGE (Fig.
2A). Similar results were obtained
in OKPETB5 cells (Fig.
2B), in which ET-1 caused a 2.1 ± 0.4-fold increase in NHE3 phosphorylation
(P < 0.03).

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Fig. 2.
ET-1 leads to phosphorylation of NHE3 in
OKPETB cells. Cells were treated
with 10 8 M ET-1 or vehicle
(control) for 35 min. After immunoprecipitation of NHE3 and SDS-PAGE,
proteins were transferred to nitrocellulose, and
32P incorporation into NHE3 was
assessed by autoradiography (top),
and NHE3 was abundance assessed by immunoblot
(bottom).
A:
OKPETB6 cells.
B:
OKPETB5 cells. Ag, antigen.
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Binding of ET-1 to the ETA receptor does
not lead to NHE3 phosphorylation.
In contrast to cells expressing the
ETB receptor, ET-1 did not
regulate
Na+/H+
antiporter activity in clonal OKP cells transfected with a cDNA encoding the ETA receptor
(OKPETA6 and
OKPETA9 cells) (12). To
examine whether this correlated with NHE3 phosphorylation, these clones
were studied. ET-1 (10
8 M)
had no effect on phosphorylation as assessed by
32P content or mobility shift in
OKPETA9 cells (Fig.
3A) or
OKPETA6 cells (Fig.
3B). In
OKPETA9 cells ET-1 induced a 2 ± 1% increase, and in OKPETA6
cells ET-1 induced an 18 ± 28% decrease, in NHE3 phosphorylation
(not significant). Thus binding of ET-1 to the ETB receptor leads to activation
and phosphorylation of NHE3, whereas binding of ET-1 to the
ETA receptor does not result in either of these effects.

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Fig. 3.
ET-1 does not cause phosphorylation of NHE3 in
OKPETA cells. Cells were treated
with 10 8 M ET-1 or vehicle
(control) for 35 min. After immunoprecipitation of NHE3 and SDS-PAGE,
proteins were transferred to nitrocellulose, and
32P incorporation into NHE3 was
assessed by autoradiography (top),
and NHE3 abundance was assessed by immunoblot
(bottom).
A:
OKPETA9 cells.
B:
OKPETA6 cells.
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Signaling pathways responsible for receptor specificity.
The mechanism responsible for the receptor specificity of NHE3
activation and phosphorylation is not clear. We previously showed that
addition of ET-1 to OKP cells expressing
ETB receptors caused an increase
in intracellular Ca2+
concentration, increased protein tyrosine phosphorylation, and decreased cAMP generation (11, 12). One possible explanation for
receptor specificity is that only
ETB receptors activate these signaling pathways. We previously showed that addition of ET-1 to
ETA-expressing cells caused
similar increases in cell Ca2+
concentration (12).
To examine whether failure of the
ETA receptor to induce tyrosine
phosphorylation was responsible for receptor specificity, we performed
Western blots with anti-phosphotyrosine antibodies in
OKPETA9 cells. As shown in Fig.
4,
10
8 M ET-1 induced tyrosine
phosphorylation of proteins of 210, 130, 125, 110, and 68 kDa. This
pattern is similar to that seen with addition of ET-1 to
OKPETB6 cells (11) and is typical
of a focal adhesion kinase pattern.

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Fig. 4.
ET-1 elicits tyrosine phosphorylation in
OKPETA9 cells: Western blotting
with anti-phosphotyrosine antibody. Cells were treated with
10 8 M ET-1 for indicated
times. Tyrosine phosphorylation was then assessed by Western blotting
with anti-phosphotyrosine antibody PY20. Sizes at
right are in kDa.
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To further examine this, we metabolically labeled cells with
[35S]methionine and
measured tyrosine phosphorylation by immunoprecipitation with
anti-phosphotyrosine antibodies. As shown in Fig.
5, similar patterns of phosphorylation were
seen in OKPETB6 (Fig.
5A) and OKPETA9 (Fig.
5B) cells.

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Fig. 5.
ET-1 elicits similar patterns of protein tyrosine phosphorylation in
OKPETB6 and
OKPETA9 cells: immunoprecipitation
with anti-phosphotyrosine antibodies. Cells were metabolically labeled
with [35S]methionine
and then treated with 10 8 M
ET-1 for indicated times. C, control. Tyrosine phosphorylation was then
assessed by immunoprecipitation with anti-phosphotyrosine antibodies.
A:
OKPETB6 cells.
B:
OKPETA9 cells. Sizes at
right are in kDa.
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We previously showed that ET-1 inhibited cAMP production in
OKPETB6 cells (12). In the present
studies, we examined whether ET-1 had a similar effect in cells
expressing ETA receptors. As shown
in Fig. 6,
10
8 M ET-1 caused similar
inhibition of cAMP production in
OKPETB6 and
OKPETA9 cells.

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Fig. 6.
ET-1 inhibits cAMP production similarly in
OKPETB6 and
OKPETA9 cells. Cells were treated
with 10 8 M ET-1 or vehicle
(control) for 35 min in presence of 2 mM IBMX, and cAMP levels
(pmol · mg
protein 1 · h 1)
were measured in cell extracts.
* P < 0.05.
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Time course and concentration dependence.
All remaining studies were performed in
OKPETB6 cells. To examine whether
the apparent decrease in mobility induced by ET-1 was due to
phosphorylation, immunoprecipitates were treated with alkaline
phosphatase. As shown in Fig. 7, treatment
with alkaline phosphatase significantly decreased the change in
mobility on SDS-PAGE, confirming that it was due to phosphate
incorporation. The upper band in Fig. 7 is a nonspecific band that is
occasionally seen on anti-NHE3 Western blots with our antibody.

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Fig. 7.
ET-1-induced phosphorylation causes decreased mobility in
OKPETB6 cells. Cells were treated
with 10 8 M ET-1 or vehicle
(control; C) for 35 min. Immunoprecipitated NHE3 was incubated with
alkaline phosphatase (AP) or buffer alone and then subjected to
SDS-PAGE. Proteins were transferred to nitrocellulose, and NHE3
mobility was assessed by immunoblot. E,
10 8 M ET-1. Sizes at
right are in kDa.
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Using the change in mobility as an index of phosphorylation, we
examined the time course and dose dependence of phosphorylation. ET-1
(10
8 M) induced a mobility
shift that was first seen at 5 min and was maximal at 15-30 min
(Fig. 8). This is similar to the time course of ET-1 on NHE3 activity in which we previously found a small
effect at 5 min that became maximal at 12 min (12).

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Fig. 8.
ET-1-induced phosphorylation of NHE3 in
OKPETB6 cells: time course. Cells
were treated with 10 8 M
ET-1 or vehicle (control; C) for various times as indicated. After
SDS-PAGE, proteins were transferred to nitrocellulose, and NHE3
mobility (kDa) was assessed by immunoblot.
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As shown in Fig. 9, the ET-1-induced
mobility shift at 35 min was not seen with
10
10 M ET-1 and was maximal
between 10
9 and
10
7 M ET-1. Again, this is
similar to the concentration dependence observed by measuring the
effect of ET-1 on NHE3 activity in these cells, in which no effect was
seen with 10
10 M ET-1 and
maximal stimulation was seen with
10
9 M and
10
8 M ET-1 (12).

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Fig. 9.
ET-1-induced phosphorylation of NHE3 in
OKPETB6 cells: concentration
dependence. Cells were treated with various concentrations of ET-1 or
vehicle (control; C) for 35 min, as indicated. After SDS-PAGE, proteins
were transferred to nitrocellulose, and NHE3 mobility (kDa) was
assessed by immunoblot.
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1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA), which inhibits cell
Ca2+ increases, and herbimycin A,
a tyrosine kinase inhibitor, inhibit ET-1-induced NHE3 activation (11).
When added together, BAPTA and herbimycin A inhibited ET-1-induced
32P incorporation into NHE3
(control, 2.7-fold increase; inhibitors, 20% decrease).
Phosphoamino acid analysis and phosphopeptide mapping.
ET-1 induced a 3.2-fold increase in phosphothreonine content and a
2.5-fold increase in phosphoserine content, with only a 1.4-fold
increase in phosphotyrosine content (Fig.
10). Phosphopeptide mapping identified
increased phosphorylation in three to five peptides (Fig.
11).
32P incorporation into
peptides A, I, and
J [as designated by Wiederkehr et al. (36)]
was seen only in ET-1 treated cells.
32P incorporation into
peptides C and D was
seen in control cells but appeared to increase in ET-1-treated cells
(Fig. 11). Thus ET-1 induces phosphorylation of NHE3 at multiple sites
on threonine and serine residues.

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Fig. 10.
ET-1-induced phosphorylation of NHE3 in
OKPETB6 cells: phosphoamino acid
analysis. Cells were treated with
10 8 M ET-1 or vehicle
(control) for 35 min. After immunoprecipitation and acid hydrolysis of
NHE3, phosphoamino acids were separated by 2-dimensional gel
electrophoresis, and amount of 32P
incorporation was determined by phosphorimaging. PS, phosphoserine; PT,
phosphothreonine; PY, phosphotyrosine.
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Fig. 11.
ET-1-induced phosphorylation of NHE3 in
OKPETB6 cells: phosphopeptide map.
Cells were treated with 10 8
M ET-1 or vehicle (control) for 35 min. After immunoprecipitation of
NHE3 and digestion with trypsin, phosphopeptides were separated by
2-dimensional gel electrophoresis-chromatography, and amount of
32P incorporation was determined
by phosphorimaging. Peptides are labeled as per Wiederkehr et al.
(36).
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 |
DISCUSSION |
At low concentrations, ET-1 stimulates volume and presumably
Na+ absorption in the proximal
tubule (14). This effect is likely related to the observation that ET-1
activates the proximal tubule apical membrane
Na+/H+
antiporter, encoded by NHE3 (13, 15). In OKP cells expressing ETB receptors ET-1 increases
Na+/H+
antiporter activity, whereas in cells expressing
ETA receptors there is no effect
on antiporter activity (12).
The present studies demonstrate that ET-1 binding to the
ETB receptor leads to activation
of NHE3 and phosphorylation of NHE3 involving multiple serine and
threonine residues. Phosphorylation is associated with a decrease in
NHE3 mobility on SDS-PAGE. Although the present studies do not prove
that phosphorylation plays a role in the increase in NHE3 activity, the
many similarities between NHE3 phosphorylation and activation suggest a
relationship. Both ET-1-induced increases in activity and
phosphorylation occur in cells expressing
ETB receptors but not in cells
expressing ETA receptors. In
addition, activation and phosphorylation have a similar ET-1
concentration dependence and time course. Last, BAPTA and herbimycin A
inhibit activation and phosphorylation of NHE3.
The specificity for the ETB
receptor agrees with the observation that proximal tubules express
ETB receptors (30). Nevertheless, the mechanism responsible for this specificity is unclear. We previously showed that 10
8
M ET-1 caused an increase in cell
Ca2+, an increase in protein
tyrosine phosphorylation, and inhibition of cAMP production in OKP
cells expressing ETB receptors
(11, 12). Previous studies showed that the increase in cell
Ca2+ also occurred in
ETA-expressing cells (12). The
present studies demonstrate that ET-1 causes similar patterns of
protein tyrosine phosphorylation and adenylyl cyclase inhibition in
ETA- and
ETB-expressing cells.
Thus the mechanism for the specificity of the
ETB receptor in causing
phosphorylation and activation of NHE3 is not immediately clear. A
number of possibilities exist. First, it is possible that the
ETB and
ETA receptors cause spatially
distinct cell Ca2+ increases.
Second, it is possible that a key tyrosine kinase substrate that is
only phosphorylated in response to
ETB receptor activation and is
responsible for NHE3 activation does not appear in Figs. 4 and 5. Last,
it is possible that receptor specificity lies in an additional
signaling pathway that is unique to the ETB receptor. One possibility is
that the ETB receptor, but not the
ETA receptor, binds an NHE3
regulatory protein. This would be analogous to regulation of NHE3 by
the
2-adrenergic receptor. Binding of agonist causes the receptor to bind and sequester NHE3 regulatory factor (NHE-RF), leading to activation of NHE3
(16). Activation of NHE3 by ET-1 could require activation of calmodulin kinase and a tyrosine kinase and binding of NHE-RF or a related protein
to the ETB receptor. This would
explain the requirements for these signaling pathways and for the
ETB receptor.
Regulation of
Na+/H+
antiporter activity by phosphorylation was first demonstrated for NHE1
activation by growth factors (27). Activation of distinct signaling
pathways resulted in similar patterns of NHE1 phosphorylation at five
sites in the cytoplasmic domain (28). In PS120 cells, regulation of
NHE3 by growth factors and phorbol esters was not accompanied by
changes in phosphorylation, whereas serum did lead to phosphorylation
at two sites (39). In contrast, in AP-1 cells, phorbol esters and cAMP
led to phosphorylation of NHE3 (20, 24, 36).
On the basis of results with inhibitors, regulation of NHE3 by
endothelin is mediated by
Ca2+/calmodulin kinase and
tyrosine kinase pathways rather than protein kinase A- or protein
kinase C-related pathways (11, 12). Activation of NHE1 by
Ca2+/calmodulin involves binding
of the complex to amino acids 636-656 of the cytoplasmic domain of
NHE1 (7, 33). It is not clear whether a similar interaction plays a
role in regulation of NHE3 (23, 34). In addition, our studies suggest
that, although tyrosine kinase pathways play a significant role, NHE3
phosphorylation on tyrosine is not significant. Thus tyrosine kinases
most likely activate upstream pathways that converge on one or more
serine/threonine kinases that phosphorylate NHE3.
Ca2+/calmodulin kinase may also
activate upstream pathways or may interact directly with NHE3. In the
present studies, we found that BAPTA and herbimycin A together
inhibited ET-1-induced NHE3 phosphorylation.
Phosphorylation could increase activity by activating NHE3 resident in
the apical membrane or could regulate trafficking of NHE3 into and out
of the apical membrane. Vasopressin-induced insertion of aquaporin-2
into the apical membrane is accompanied by phosphorylation of
aquaporin-2 (21, 22). Mutation of the phosphorylated serine
(Ser-256) leads to inhibition of trafficking (19). In
preliminary studies, we have found that ET-1 leads to trafficking of
NHE3 to the plasma membrane in
OKPETB6 cells (unpublished observation).
By regulating NHE3, the endothelins could play important roles in
acid-base regulation. Acidosis leads to increased expression of c-Fos
and c-Jun, transcription factors that are key regulators of ET-1
expression (18, 38). Renal interstitial levels of ET-1 are increased in
acidosis, and blockade of ETB
receptors impairs regulation of distal tubule function in acidosis
(35). In preliminary studies, we have found that acid feeding increases renal cortical apical membrane
Na+/H+
antiporter activity in wild-type mice but has no effect in
ETB receptor-deficient mice
(unpublished observation). Thus acidosis-induced increases in ET-1
expression could lead to phosphorylation and activation of NHE3.
 |
ACKNOWLEDGEMENTS |
We thank Martha Ferguson for technical assistance.
 |
FOOTNOTES |
These studies were supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-39298 and DK-48482, the
Department of Veterans Affairs, and a grant from the Texas Coordinating
Board. M. Yanagisawa is an investigator of the Howard Hughes Medical Institute.
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.
Address for reprint requests and other correspondence: R. J. Alpern,
Office of the Dean, Southwestern Medical School, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX
75235-9003 (E-mail: robert.alpern{at}emailswmed.edu).
Received 20 October 1998; accepted in final form 8 January 1999.
 |
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