Dual Mechanisms of Regulation of Na/H Exchanger NHE-3 by
Parathyroid Hormone in Rat Kidney*
Lingzhi
Fan
,
Michael R.
Wiederkehr
,
Roberto
Collazo
§,
Huamin
Wang¶,
Ladonna A.
Crowder
, and
Orson W.
Moe
¶
From the ¶ Medical Service, Department of Veterans Affairs
Medical Center and the
Department of Internal Medicine,
University of Texas Southwestern Medical Center,
Dallas, Texas 75225-8856
 |
ABSTRACT |
Parathyroid hormone (PTH) is a potent inhibitor
of mammalian renal proximal tubule sodium absorption via suppression of
the apical membrane Na/H exchanger (NHE-3). We examined the mechanisms by which PTH inhibits NHE-3 activity by giving an acute intravenous PTH
bolus to parathyroidectomized rats. Parathyroidectomy per se increased apical membrane NHE-3 activity and antigen. Acute infusion of PTH caused a time-dependent decrease in NHE-3
activity as early as 30 min. Decrease in NHE-3 activity at 30 and 60 min was accompanied by increased NHE-3 phosphorylation. In contrast to
the rapid changes in NHE-3 activity and phosphorylation, decrease in
apical membrane NHE-3 antigen was not detectable until 4-12 h after
the PTH bolus. The decrease in apical membrane NHE-3 occurred in the
absence of changes in total renal cortical NHE-3 antigen. Pretreatment
of the animals with the microtubule-disrupting agent colchicine blocked
the PTH-induced decrease in apical NHE-3 antigen. We propose that PTH
acutely cause a decrease in NHE-3 intrinsic transport activity possibly
via a phosphorylation-dependent mechanism followed by a
decrease in apical membrane NHE-3 antigen via changes in protein trafficking.
 |
INTRODUCTION |
PTH1 plays a paramount
role in mammalian calcium homeostasis. The calcitropic actions of PTH
include direct stimulation of bone turnover (1) and renal Ca absorption
(2) and indirect enhancement of intestinal Ca absorption via its action
on 1,25-vitamin D3 (3). In the kidney, PTH exerts direct
action on the proximal tubule, thick ascending limb, distal convoluted
tubule, and connecting tubule (4, 5). In the proximal tubule, PTH is a
potent inhibitor of NaHCO3 absorption (6-12). Because
proximal tubule calcium and sodium absorption are tightly coupled (6,
13), the potent inhibitory action of PTH on proximal NaHCO3
transport appears to be counterproductive for an anticalciuric hormone.
Moreover, the acute inhibition of proximal tubule NaHCO3
absorption seems to serve little purpose because the HCO3
exiting the proximal tubule is largely reclaimed in the distal nephron
as evident by the fact that acute PTH only cause modest reductions in
plasma HCO3 concentration (14-16). However, in the distal
convoluted tubule, luminal HCO3 is an important stimulus
for transcellular calcium absorption (17-19). Thus the shift of
NaHCO3 absorption from proximal to distal nephron results
in minimal net change in acid-base balance but serves as a key enhancer
of the anticalciuric effect of PTH.
In the mammalian proximal tubule, two-thirds of the transcellular
NaHCO3 absorption is mediated by apical membrane Na/H
exchange (20). Immunohistochemical (21-23), pharmacokinetic (24), and genetic (25) data all indicate that the NHE-3 isoform is predominantly responsible for proximal tubule apical membrane Na/H exchange. Direct
inhibition of proximal tubule HCO3 absorption by PTH
(6-12) is effected at least in part by inhibition of apical membrane Na/H exchange activity, which has been demonstrated in the suspended tubules (26), isolated perfused tubule (27), in vivo
perfused tubule (28), apical membrane vesicles (29), cultured renal cells (30-35), and nonepithelial cells transfected with the PTH receptor and NHE-3 gene (36, 37). The mechanisms by which PTH inhibits
NHE-3 activity has not been examined to date. Phosphorylation of NHE-3
has been shown to play a role in its acute regulation by protein kinase
A (PKA) (38-40), and part of the effect of PTH on NHE-3 is
PKA-dependent (32-36). One study showed that the
PTH-induced decrease in apical membrane Na/H exchange activity was
accompanied by commensurate increase in Na/H exchange activity in a
different membrane fraction on a density gradient (41). This suggests redistribution of NHE-3 protein as a mechanism of its acute regulation, although there were no antigenic data in this study. NHE-3 protein has
been shown to exist both in the apical membrane and in subapical vesicles (23). In a model of pressure natriuresis, the acute inhibition
of apical membrane Na/H exchange was associated with redistribution of
NHE-3 antigen from the apical to subapical region. (42, 43).
In this study, we present evidence in intact animals that PTH inhibits
renal cortical apical membrane Na/H exchange by dual mechanisms:
immediate inhibition of the intrinsic transport activity of NHE-3
associated with an increase in NHE-3 phosphorylation followed by
redistribution of NHE-3 transporter away from the apical membrane to a
nonapical pool.
 |
EXPERIMENTAL PROCEDURES |
Animal Model and Membrane Preparations--
Parathyroidectomized
or sham-operated Sprague-Dawley rats (150-200 g, Charles River,
Wilmington, MA) were given free access to food supplement with 4%
(w/v) calcium gluconate drinking water until 2 days prior to
experiments when they were switched to tap water. Animals were
anesthetized by thiopental (100 mg/kg body weight intraperitoneal;
Abbot Laboratories, Abbot Park, IL), the femoral vein was exposed, and
either PTH (100 µg/kg body weight; 0.5 mg/ml in 10 mM
acetic acid, 0.4 mM dithiothreitol, 1% bovine serum
albumin; Peninsula, San Carlos, CA) or the vehicle was injected intravenously. For the 30-60-min time points, animals were kept under
anesthesia on a heating pad until bilateral nephrectomy performed
through an anterior abdominal incision. For longer time points, the
inguinal incision was sutured, and animals were allowed to recover. At
the appropriate time points, anesthesia was again induced and kidneys
were harvested. Renal cortex was dissected and homogenized on ice in
membrane buffer (150 mM NaCl, 80 mM NaF, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM EGTA, 25 mM sodium pyrophosphate, 1 mM sodium vanadate, 100 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 2 µg/ml
pepstatin A) (Brinkman polytron, Westbury, NY), and apical membrane
vesicles were prepared from the cortical homogenate by exposing
cortical membranes to three consecutive precipitations by 15 mM MgCl2, and the final apical
membrane-enriched vesicles were pelleted from the supernatant (Beckman
J2-21 M, JA-20 rotor, 20,000 rpm, 40 min 4 °C; Beckman,
Fullerton, CA). For experiments with colchicine, parathyroidectomy
(PTX) animals were given colchicine (10 µg/kg body weight) 3 h
prior to infusion of PTH or vehicle.
Na/H Exchange Activity and Antigen on Membrane Vesicles--
For
measurement of Na/H exchange activity in rat renal apical membranes,
200 µg of vesicles were acid loaded (300 mM mannitol, 20 mM MES, pH 5.5, at 4 °C for 2 h) and exposed to
10× volume uptake solution (300 mM mannitol, 20 mM Tris, pH 7.5, 0.1 mM 22NaCl) to
initiate transport at 20 °C for 10 s. Uptake was stopped by
dilution with stop solution (150 mM NaCl, 20 mM
Tris, pH 7.5) at 4 °C, and 22Na uptake was quantified by
rapid filtration on 0.65 µM Millipore filters (Millipore,
Bedford, MA) and scintillation counting. For determination of NHE-3
antigen, either 20 µg of rat renal cortical apical membranes or 50 µg of cortical membranes were solubilized in SDS buffer, fractionated
by SDS-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose filters. Anti-rat NHE-3 antisera were used: 1568 (against epitope DSFLQADGPEEQLQ at 1:1000) (20) to quantified NHE-3
antigen. Controls with the anti-Type II sodium phosphate co-transporter
NaPi-2 antisera (1:1000 dilution, gift from Drs. Biber and Murer,
Zürich, Switzerland) were performed as described previously (44).
After incubation with horseradish peroxidase-coupled mouse anti-rabbit
secondary antibody, signals were detected by enhanced chemiluminescence
(Amersham Pharmacia Biotech) and quantified by densitometry.
OK Cells--
OK cells expressing native NHE-3 (47) were used as
a control experiment to validate the mobility shift assay for NHE-3
phosphorylation. OK cells were maintained in Dulbecco's modified Eagle
medium (Life Technologies, Inc.) supplemented with 4.5 mg/ml glucose,
100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal
bovine serum (removed 48 h prior to study). Phosphorylation
experiments were done as described previously (40). In brief, confluent
monolayers were incubated in phosphate-free Dulbecco's modified Eagle
medium and pulsed with [32P]orthophosphate (500 µCi/ml;
120 min). After addition of, 200 µM 8Br-cAMP, cells were
lysed with RIPA buffer (300 mM NaCl, 80 mM NaF,
50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM EGTA, 25 mM sodium pyrophosphate, 1 mM activated sodium orthovanadate, 50 mM
-glycerophosphate, 0.5 mM dithiothreitol, Triton X-100
1% (v/v), deoxycholate 0.5% (w/v), SDS 0.1% (w/v), 100 µg/ml
phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, 4 µg/ml
aprotinin, 10 µg/ml pepstatin), centrifuged (109,000 × g at rmax, 50,000 rpm, 25 min, 2 °C, Beckman
TLX ultracentrifuge, TLA 100.3 rotor, Fullerton, CA), and NHE-3 was
immunoprecipitated (antiserum 5683 against a fusion protein of maltose
binding protein and OK NHE-3 amino acid 484-839) (1:250 dilution, v/v)
from the supernatant. Immunoblots were performed with antiserum 5683)
(1:1000) and goat anti-rabbit antibody (1:5000) using ECL (Amersham
Pharmacia Biotech). The 32P content of NHE-3 was visualized
by autoradiography on the same filters after decay of ECL.
NHE-3 Mobility Shift Assay and in Vitro Treatment with Alkaline
Phosphatase--
Mobility shift was detected by immunoblot after
SDS-PAGE (6% gel with 80-kDa marker ran to the edge of the gel). For
treatment with alkaline phosphatase, we modified a protocol described
by Jou et al. (48). 20 µg of apical membrane was washed
with reaction buffer (50 mM Tris-HCl, pH 8.5, 2 mM phenylmethylsulfonyl fluoride, 8 mM
MgCl2, 0.1%
-mercaptoethanol (v/v)) three times and
resuspended in 45 µl of reaction buffer containing 10 units of
calf-intestine alkaline phosphatase. After incubation at 37 °C for
90 min, the reaction was terminated by adding polyacrylamide gel
loading buffer.
 |
RESULTS |
Effect of PTX on NHE-3--
Parathyroidectomy was complete as
plasma PTH levels were undetectable in PTX animals (data not shown).
Compared with sham operation, parathyroidectomy per se
increased apical NHE-3 activity (35 ± 8%, n = 4, p < 0.05) and antigen (75 ± 8%,
n = 4, p < 0.05) and total renal
cortical NHE-3 antigen (55 ± 8%, n = 4, p < 0.05) (Fig. 1). This
suggests that PTH has a tonic suppressive effect on NHE-3 protein
expression.

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Fig. 1.
Effect of parathyroidectomy on renal
NHE-3. Sham-operated rats (Sham; n = 4)
were compared with parathyroidectomized (PTX;
n = 4) rats. A, NHE activity was measured in
apical membrane vesicles as pH-driven 22Na flux. The
asterisk indicates p < 0.05 (t
test). B, NHE-3 antigen was measured in apical brush border
membrane (BBM) and total cortical membranes (Cx)
by immunoblot. Mobility on SDS-PAGE is shown in kDa.
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Effect of PTH on Renal Apical Membrane NHE-3 Activity--
Fig.
2 shows a typical experiment where acute
intravenous infusion of PTH decreased apical membrane NHE-3 activity in
PTX rats in a time-dependent fashion, whereas vehicle
injection had no effect. Fig. 3
summarizes all the experiments. Significant inhibition was detected as
early as 30 min after the bolus of PTH and persisted up to 20 h
(percentage of decrease compared with vehicle time control: 30 min,
14 ± 4%; 1 h, 21 ± 6%, 2 h; 28 ± 7%;
4 h, 38 ± 6; 8 h, 35 ± 7%; 12 h, 33 ± 10%; 20 h, 14 ± 9%; 20 h, 15 ± 9%; 24 h,
7 ± 7%; all p < 0.05 except 24 h).

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Fig. 2.
Effect of acute PTH on apical membrane NHE
activity: typical experiment. PTH or vehicle was given
intravenously into parathyroidectomized (PTX) animals, renal
cortical apical membranes were harvested at the indicated time points,
and NHE activity was measured by pH-driven 22Na flux.
Data from one typical experiment are shown with bars and
error bars depicting the means ± S.E. from three
animals/time point. The asterisks indicate p < 0.05 (t test; PTH versus vehicle).
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Fig. 3.
Effect of acute PTH on apical membrane NHE
activity: summary of all experiments. PTH or vehicle was given
intravenously into parathyroidectomized (PTX) animals, renal
cortical apical membranes were harvested at the indicated time points,
and NHE activity was measured by pH-driven 22Na flux.
NHE-3 activity of PTH-treated animals are expressed as percentages of
the vehicle-injected animals for each time point. Number of animals
(vehicle/PTH): 30 min, 8/10; 1 h, 8/10; 2 h, 8/8; 4 h,
8/12; 8 h, 10/10; 29 h, 6/8; 24 h, 8/12).
Bars and error bars depict the means ± S.E.
The asterisks indicate p < 0.05 (t test; PTH versus vehicle).
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Effect of PTH on Renal Apical Membrane NHE-3
Phosphorylation--
Acute regulation of NHE-3 by activation of
kinases is associated with changes in NHE-3 phosphorylation in cultured
cells (38-40). To examine whether PTH acutely modifies NHE-3
phosphorylation in the intact animal, we utilized a mobility shift
assay. Fig. 4A illustrates
data validating this assay. We have shown that protein kinase A
activation increases NHE-3 phosphorylation in OK cells (40). Fig.
4A shows that the cAMP-induced increase in NHE-3
phosphorylation was associated with deceased NHE-3 mobility on SDS-PAGE
(Fig. 4A). Treatment of the same samples with alkaline phosphatase removed the 32P label, increased the mobility
of NHE-3, and eliminated the cAMP-induced NHE-3 mobility shift (Fig.
4A). Fig. 4B shows a similar experiment performed
in rat renal cortical apical membranes. An intravenous bolus of
8Br-cAMP (250 µl of 0.43 mg/ml 8Br-cAMP in saline injected into the
suprarenal abdominal aorta with transient infrarenal aortic clamping)
induced a mobility shift of NHE-3 that was completely abolished by
in vitro treatment with alkaline phosphatase. Fig. 4C shows a typical experiment of the effect of PTH on NHE-3
phosphorylation in the intact animal. Acute PTH infusion increased
NHE-3 phosphorylation as early as 30 min.

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Fig. 4.
Mobility shift assay for phosphorylation and
the effect of acute PTH on NHE-3 phosphorylation in intact
animals. A, OK cells were pulsed with 32P
and treated with 500 µM 8Br-cAMP or vehicle, and
32P-NHE-3 was immunoprecipitated and resolved by SDS-PAGE.
Phosphorylation (32P-NHE3) and antigenic (NHE3
Ag) signals were determined in the same filters by autoradiography
and immunoblot, respectively. The right lanes show the same
samples after dephosphorylation with alkaline phosphatase (Alk
Phos) in vitro. Mobility in kDa are indicated on the
left margin. The arrow marks the mobility of
dephosphorylated NHE-3. Con, control. B, rats
were given either 8Br-cAMP or vehicle via the aorta, and renal cortical
apical membranes were isolated and analyzed by SDS-PAGE and immunoblot.
The right two lanes shows the same samples after
dephosphorylation with alkaline phosphatase in vitro. The
arrow marks the mobility of the control and phosphatased
sample. C, apical membranes were prepared from rats after an
acute infusion of PTH and subjected to immunoblotting by anti-NHE-3.
Mobility in kDa is shown on the right margin. The
arrow marks the mobility of the control sample. Results from
two experiments are shown. Total number of experiments = 4.
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Effect of PTH on NHE-3 Protein Distribution--
Next, we examined
whether the decrease in NHE-3 activity is associated with changes in
NHE-3 protein abundance in apical and total cortical membranes. Fig.
5A shows a typical experiment. Vehicle injection did not alter NHE-3 protein abundance in the apical
or total cortical membranes from 30 min throughout to 16 h,
indicating that the anesthesia and intravenous catheterization did not
lead to redistribution of NHE-3. From 30 min to 1 h after PTH
infusion, although NHE-3 activity was clearly decreased (Fig. 3), there
was no detectable change in apical membrane NHE-3 antigen (Fig.
5A). PTH acutely shifts the type IIa sodium phosphate
cotransporter NaPi-2 from an apical to a subapical location (44,
45).2 As a positive control
for our negative finding in NHE-3 trafficking after 30 min and 1 h
of PTH, we probed the same apical membrane samples after 30 min and
1 h of PTH with an antiserum against NaPi-2. Fig. 5B
shows that although there was no change in NHE-3 antigen, NaPi-2
underwent dramatic endocytosis after 30 min of PTH as previously shown
(44, 45).2 Although the early decrease in apical NHE-3
activity was not accompanied by decreased NHE-3 antigen, after 2 h
of PTH treatment, NHE-3 antigen was significantly decreased compared
with vehicle-treated controls (Fig. 5A). Fig.
6 summarizes all the experiments. No significant decrease of NHE-3 was detected in apical membrane in 30-60
min of PTH, and a trend toward decrease was noted at 2 h (12 ± 7%, p = 0.07). From 4 to 12 h, PTH
significantly decreased apical membrane NHE-3 antigen with recovery by
24 h (percentage of decrease, PTH compared with vehicle time
control: 30 min, 6 ± 8%; 1 h, 1 ± 4%; 2 h,
12 ± 7%; 4 h, 33 ± 7; 8 h, 38 ± 10%;
12 h, 36 ± 8%; 24 h, 16 ± 12%; all
p < 0.05 except 30 min and 1, 2, and 24 h). No
change in total cortical NHE-3 antigen was noted in response to acute
PTH, although a statistically insignificant decrease occurred after
12 h (15 ± 10% decrease, p = 0.07). In concert, the data suggest that the decrease in apical membrane NHE-3
protein is due to redistribution to a nonapical compartment rather than
protein degradation.

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Fig. 5.
Effect of PTH on NHE-3 antigen: typical
immunoblots. PTH or vehicle was given intravenously to PTX
animals, renal total cortical (Cx) (50 µg) apical brush
border (BBM) membranes (20 µg) were harvested at the
indicated time points, resolved by SDS-PAGE, and studied by immunoblot
for NHE-3 protein abundance for all the time points (A) and NHE-3 and
NaPi-2 protein abundance for 30 min and 1 h (B).
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Fig. 6.
Effect of PTH on NHE-3 antigen: summary of
all experiments. NHE-3 Ag in PTH-treated animals was expressed as
a percentage of NHE-3 Ag in vehicle-treated animals for each time
points. Open circles represent renal cortical membranes
(Cortex). Closed circles represent brush border
membranes (BBM). Bars and error bars
depict the means ± S.E. Number of animals (vehicle/PTH): 30 min,
6/8; 1 h, 8/8; 2 h, 8/8; 4 h, 8/8; 8 h, 10/10;
12 h, 10/10; 24 h, 10/10. Bars and error
bars represent the means ± S.E. The asterisks
indicate p < 0.05, PTH versus
vehicle.
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Effect of Colchicine on the PTH-induced Shift in NHE-3
Antigen--
To examine whether this redistribution of NHE-3 involves
microtubule-dependent protein trafficking, we pretreated
the rats with the microtubule disrupting agent colchicine prior to
infusion of PTH. Fig. 7 shows one
experiment. Colchicine per se appeared to increase the
baseline apical membrane NHE-3 abundance. In the background of
colchicine treatment. PTH failed to induce redistribution of NHE-3
(percentage of decrease compared with vehicle time control: 2 h,
3 ± 7%; 4 h, 8 ± 7; 8 h, 5 ± 14%; 24 h, 5 ± 10%; all not significant), suggesting that the decrease
in apical membrane NHE-3 is microtubule-dependent (Fig.
7B).

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Fig. 7.
Effect of PTH on NHE-3 antigen: pretreatment
with colchicine. Animals were given colchicine prior to PTH
infusion. A, immunoblot from a typical experiment.
Experimental groups are as described: PTX,
parathyroidectomy; Colchicine, animals pretreated with
colchicine prior to PTH bolus; PTH or Veh, either
PTH or vehicle was given for the stated time points. B,
summary of all experiments. NHE-3 antigen in renal cortical apical
brsuh border membranes (BBM) in PTH-treated animals was
expressed as a percentage of NHE-3 antigen in vehicle-treated animals
for each time point. Bars and error bars depict
the means ± S.E. Number of animals (vehicle/PTH): 2 h, 4/4;
4 h, 6/6; 8 h, 8/8; 24 h, 6/6.
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DISCUSSION |
The acute inhibition of renal proximal apical membrane Na/H
exchange activity by PTH has been shown in renal tubules, membrane vesicles, and cultured cells (26-37), but the mechanisms for this decrease has not been defined. We found that PTX per se
increased apical membrane NHE-3 activity as described previously (49), and PTX increased NHE-3 antigen in total renal cortical as well as
apical membrane vesicles. This suggests that PTH has a chronic suppressive effect on NHE-3 protein expression. The increase in renal
net acid excretion seen in chronic hyperparathyroidism (14-16) is
likely due to heightened distal rather proximal tubule acidification. With a single PTH bolus, we did not observed a statistically
significant decrease in total cortical NHE-3 antigen in the PTX
animals, although a slight trend was observed at 20 h. It is
possible that more sustained or repeated doses of PTH are required to
inhibit total cortical NHE-3 protein expression.
Both protein kinase C and PKA pathways have been implicated in
mediating the effects on PTH on the proximal tubule (27-29, 32-37).
Acute regulation of NHE-3 activity by PKA activation has been shown to
be associated with NHE-3 phosphorylation (38-40), and mutation of
specific phosphorylated serines aborted functional regulation by PKA
activation (39, 40). The requirement of the Na/H exchanger regulatory
factors NHERF and E3KARP as putative functional A kinase anchoring
proteins to effect NHE-3 inhibition and phosphorylation further
consolidate the importance of NHE-3 phosphorylation in its functional
regulation (50-53). All of these studies were done either in
fibroblasts transfected with NHE-3 (38-40, 51, 52) or in cultured
epithelial cells expressing native NHE-3 (40, 51, 53). This study is
the first demonstration that NHE-3 is indeed phosphorylated as a native
protein in an intact animal and that NHE-3 phosphorylation is regulated
acutely by administration of a hormone. The association of increased
NHE-3 phosphorylation with decreased NHE-3 activity in the intact
kidney after 30 min of PTH further substantiate the importance of NHE-3 phosphorylation in its functional regulation.
The time profile of inhibition of NHE-3 activity did not parallel that
of NHE-3 antigen in the apical membrane. The decrease in NHE-3 activity
within 2 h of PTH administration was not associated with decreased
apical membrane NHE-3 antigen. There are other examples where NHE-3
activity is regulated likely without changes in surface NHE-3 antigen.
Na/H exchange activity is inhibited by direct addition of cAMP
analogues or hormones coupled to adenylyl cyclase to apical membrane
vesicles, which are unlikely to retain competence for protein
trafficking (54-56). Weinman and co-workers found decrease in Na/H
exchange activity in proteoliposomes reconstituted from solubilized
apical membrane proteins phosphorylated in solution by PKA in
vitro (57-59). In OK cells, 15 min of PTH application altered the
pHi sensitivity (KH) of native NHE-3 in
addition to a Vmax effect (31), and a recent
study showed similar findings with cAMP addition to fibroblasts
expressing NHE-3 (53). A change in the KH of NHE-3 is
compatible with the hypothesis that mechanisms other than changes in
plasma membrane NHE-3 protein may be operational in modifying NHE-3
activity. Our data from intact animals support the notion that changes
in the intrinsic activity of NHE-3 may be one mechanism responsible for
physiologic regulation of NHE-3 function. Substrate kinetics were not
examined in this study, but with the imposed gradients on the vesicles
(internal pH, 5.5; external, pH 7.5; external [sodium], 0.1 mM), one is unlikely to be observing a pure KH
effect. In fibroblasts transfected with NHE-3 and in OK cells,
activation of PKA can acutely decrease NHE-3 activity in the intact
cell without changing NHE-3 antigen abundance on the plasma
membrane.3,4
One preliminary report indicated early inhibition of native NHE-3 by
PTH in OK cells can also occur without changes in plasma membrane NHE-3
antigen (60).
After 4 h of PTH administration, we clearly observed a decrease in
apical membrane NHE-3 without changes in total cortical NHE-3
compatible with redistribution of NHE-3 protein from the apical
membrane to another pool. Protein trafficking has been implied to
explain regulation of apical membrane Na/H exchange (41, 61). Hensley
and co-workers (41) fractionated homogenates from renal tubules treated
with PTH and showed that PTH decreased Na/H exchange activity in a
fraction enriched with alkaline phosphatase and increased Na/H exchange
activity in a fraction enriched with acid phosphatase and
galactosyltransferase. Although no antigenic data was available, this
finding is compatible with trafficking of NHE-3 from an apical to an
intracellular compartment. There are two major discrepancies between
this study and that of Hensley and co-workers. First, the time course
of redistribution NHE-3 antigen is much slower in this study than that
inferred by activity measurements in Hensley's study. This may be due
to systemic administration of PTH in this study versus
direct incubation of tubules with PTH in Hensley's paper. Second, the
Hensley experiment suggests that the redistributed Na/H exchanger is
rapidly destroyed due to loss of Na/H exchange activity in the acid
phosphatase fraction, whereas the present study shows minimal decrease
in whole cortex NHE-3 after PTH infusion. It is difficult to correlate
Na/H exchange activity in various membrane fractions to NHE-3 antigen.
The measured Na/H exchange activities can be due to NHE-3, other NHE
isoforms, or parallel Na and H conductances. Biemesderfer and
co-workers (21) have shown NHE-3 residing in subapical vesicles in
native rat kidneys. Zhang and co-workers (42, 43) showed that the acute
decrease in proximal tubule apical membrane NHE-3 activity in a rat
model of pressure natriuresis is associated with redistribution of
NHE-3 antigen from the apical membrane to a subapical region. Preliminary reports have shown that the acute activation of NHE-3 activity by acidosis extracellular pH or endothelin are both associated with increase in plasma membrane NHE-3 antigen (46, 62). PTH induces
dramatic change in the distribution of the type II sodium phosphate
cotransporter NaPi-2 (44, 45).2 There are marked
differences between the responses of NHE-3 and NaPi-2 to PTH. Within 30 min of PTH infusion when no change in NHE-3 antigen can be detected,
apical membrane NaPi-2 antigen is dramatically reduced. In addition to
the difference in kinetics, the internalized NaPi-2 is rapidly targeted
for lysosomal degradation, whereas no detectable decrease in total
cortical NHE-3 antigen occurred in the study period. These findings
suggest that PTH modulates distinct trafficking pathways for NaPi-2 and
NHE-3 involving different vesicle populations destined for different fates.
The present study shows that immediate inhibition of renal cortical
apical membrane NHE-3 activity is associated with phosphorylation of
NHE-3 without detectable changes in apical membrane NHE-3 antigen. With
sustained PTH, NHE-3 is distributed away from the apical membrane with
no change in total cortical NHE-3 antigen. At present, we cannot
establish the role of NHE-3 phosphorylation in mediating the change in
apical membrane NHE-3 antigen, nor do we know whether the decrease in
apical membrane NHE-3 antigen is mediated by decreased insertion or
increased removal. With chronic changes in PTH levels such as
parathyroidectomy, total renal NHE-3 antigen is altered.
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ACKNOWLEDGEMENTS |
We are grateful to Dr. Moshe Levi and Dr.
Robert Alpern for valuable discussions throughout these studies.
 |
FOOTNOTES |
*
This work was supported by funds from the Research Service
of the Department of Veterans Affairs and by National Institutes of
Health Grant DK48482 and American Heart Association Texas Affiliate Grant 98G-052.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.
§
Recipient of National Institutes of Health Training Grant T32
DK07257-17.
To whom correspondence should be addressed: Dept of Internal
Medicine, University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75235-8856. Tel.: 214-648-3152; Fax:
214-648-2071; E-mail: omoe{at}mednet.swmed.edu.
2
M. Lötscher, Y. Scarpetta, M. Levi, H. Wang, H. K. Zajicek, J. Biber, H. Murer, and B. Kaissling,
submitted for publication.
3
O. W. Moe, Unpublished observation.
4
S. Grinstein, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
PTH, parathyroid hormone(s);
PKA, protein kinase A;
PTX, parathyroidectomy;
MES, 4-morpholineethanesulfonic acid;
PAGE, polyacrylamide gel
electrophoresis.
 |
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