From the Department of Pharmacology and Brain Korea
21 Project for Medical Sciences, Yonsei University College of Medicine,
Seoul 120-752, Korea, the Departments of § Physiology and
¶ Internal Medicine, University of Texas Southwestern Medical
Center, Dallas, Texas 75235, and the
Department of Cell and
Molecular Physiology, University of North Carolina,
Chapel Hill, North Carolina 27599
Received for publication, December 28, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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The pancreatic duct expresses cystic
fibrosis transmembrane conductance regulator (CFTR) and
HCO Fluid secretion and the control of the ionic
composition of biological fluids are essential for the function of many
secretory epithelia, including the respiratory, digestive, and
reproductive systems. HCO CF is caused by mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR). CFTR functions as a Cl An intriguing feature of HCO In the present work, we used biochemical and functional approaches to
study the interaction of CFTR and NHE3 with EBP50 and the functional
significance of this interaction in heterologous expression systems and
the native pancreatic duct. In pancreatic duct and PS120 cells
expressing NHE3 and EBP50, expression of CFTR augmented the
cAMP-dependent inhibition of NHE3 activity. Most notably,
CFTR in the pancreatic duct affected not only the activity but also the
expression of NHE3 protein in the luminal membrane. These findings
reveal a new mechanism by which CFTR regulates ion transport at the
luminal membrane and highlight the multiple functions of CFTR in
regulating the overall HCO Materials, Antibodies, and Solutions--
BCECF-AM was purchased
from Molecular Probes, Inc. (Eugene, OR). Polyclonal antiserum directed
against human EBP50 was generated in rabbits as described previously
(15). Rabbit antisera 1566 and 1568 specific for NHE3 were generated as
described previously (16). Monoclonal antibodies against an R domain
and a C terminus of CFTR were purchased from R & D Systems
(Minneapolis, MN). The solution A, used for microdissection and
perfusion, contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH 7.4 with NaOH),
and 10 glucose. Na+-free solutions were prepared by
replacing Na+ with
N-methyl-D-glucamine+ from solution A.
Cell Culture and Transfection--
An NHE-deficient cell line
PS120, originally developed by Pouyssegur et al. (17), and
the pCMV-NHE3 mammalian expression vector (18) were provided by Drs. K. Park and J. E. Melvin (University of Rochester). Mammalian
expressing pCMV-CFTR (pCMVNot6.2) vector was a gift from Dr. J. Rommens
at the Hospital for Sick Children (Toronto, Canada). Mammalian
expressing Ad-CFTR virus was purchased from the Institute of Human Gene
Therapy (Philadelphia, PA), and pcDNA3.1-EBP50 was generated by
subcloning the full-length EBP50 cDNA from pET-EBP50 (15) to
pcDNA3.1 (Invitrogen, Groningen, The Netherlands).
PS120 cells were maintained in DMEM-HG (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum and penicillin (50 units/ml)/streptomycin (50 µg/ml). The pCMV-NHE3 construct was stably
transfected into a PS120 cell line using LipofectAMINE Plus Reagent
(Life Technologies). NHE3 stable transfectants (PS120/NHE3) were
selected by resistance to the antibiotic Geneticin (G418; Life
Technologies, Inc.) and by an H+-killing method (19).
Site-directed Mutagenesis--
Oligonucleotide-directed
mutagenesis using the GeneEditor mutagenesis kit (Promega, Madison, WI)
was performed in the CFTR expression vector pCMVNot6.2 to delete the
C-terminal 4 ( RT-PCR--
mRNA transcripts of mouse EBP50, E3KARP, and
PDZK1 (20) were analyzed in pancreatic and PS120 cells using RT-PCR.
Among several primer sets designed based on the mouse sequences, at least one set for each protein detected the correct band in samples from hamster as well as mouse tissues. The primer sequences were as
follows: 1) mEBP50, sense (5'-CTA AGC CAG GCC AGT TCA TCC GAG CAG T-3')
and antisense (5'-TGG GGT CAG AGG AGG AGG AGG AGG TAG A-3'), size of
PCR product 447 base pairs; 2) mE3KARP, sense (5'-GAG GCC CGG CTG CTG
GTA GTC G-3') and antisense (5'-CAT CTG TGG TGC CCG CTT GTT GA-3'),
size of PCR product 312 base pairs; 3) mPDZK1, sense (5'-GAC AAG GCT
GGG CTG GAG AAT GAG GAC-3') and antisense (5'-CGA AGA GTG CGA GGC TGT
GCT GAG AGT-3'), size of PCR product 297 base pairs. RNA was extracted
from the tissue using Trizol solution (Life Technologies, Inc.) and
reverse transcribed using random hexamer primers and an RNase
H Immunoprecipitation and Immunoblotting--
Precleared
pancreatic or PS120 lysates (~2 mg of protein) were mixed with the
appropriate antibodies and incubated overnight at 4 °C in lysis
buffer. Immune complexes were collected by binding to protein G-
(monoclonal antibody against R domain of CFTR) or protein A- (all other
antibodies) Sepharose and washing four times with lysis buffer prior to
electrophoresis. The immunoprecipitates or lysates (20 µg of protein)
were suspended in SDS sample buffer and separated by SDS-polyacrylamide
gel electrophoresis. The separated proteins were transferred to
polyvinylidene difluoride membranes, and the membranes were blocked by
a 1-h incubation at room temperature in 5% nonfat dry milk in a
solution containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20. The proteins were detected by
1-h incubations with the appropriate primary and secondary antibodies.
Immunohistochemistry and Quantitation of Staining in Confocal
Images--
The pancreata from several wild type (WT) and homozygous
When desired, NHE3 expression in the LM of the pancreatic duct
was compared between samples from wild type and Animals and Preparation of Pancreatic Ducts--
A cystic
fibrosis mouse model in which the Intracellular pH Measurements--
For measurement of
pHi in PS120/NHE3 cells, the glass coverslips with cells
attached to them were washed once with solution A and assembled to form
the bottom of a perfusion chamber. The cells were loaded with BCECF by
a 10-min incubation at room temperature in solution A containing 2.5 µM BCECF-AM. After dye loading, the cells were
perfused with appropriate solutions, and pHi was
measured by photon counting using a fluorescence measuring system
(Delta Ram; PTI Inc., South Brunswick, NJ). In the case of CFTR
transfection with pCMVNot6.2 or its C-terminal deleted mutants, a green
fluorescent protein-expressing plasmid (Life Technologies) was
co-transfected with the CFTR constructs, and pHi measurements
were performed with cells expressing high levels of green fluorescent
protein as previously reported (5). For pHi measurement in the
microdissected ducts, the cannulated ducts were transferred to a
perfusion chamber placed on the stage of an inverted microscope. The
bath was perfused at a flow rate of 6 ml/min, and the lumen was
perfused at a flow rate of 150 µl/min using solution A that was
maintained at 37 °C. BCECF was loaded by including 2.5 µM BCECF-AM in the luminal perfusate for 5 min, and dye
loading was monitored. After completion of dye loading, the lumen was
washed by perfusing solution A, and pHi measurements were
performed according to the specified protocols. The fluorescence ratios
of 490/440 nm were calibrated intracellularly by perfusing the cells or
the ducts with solutions containing 145 mM KCl, 10 mM HEPES, and 5 µM nigericin with pH adjusted
to 6.2-7.8.
Na+/H+ exchange activity was measured using a
standard protocol (23). The cells were acidified by an
NH CFTR·EBP50·NHE3 Complexes in PS120 Cells and the Mouse
Pancreas--
To set the stage for analyzing the interaction between
CFTR and NHE3 in model systems and native cells, we first determined which of the adapter proteins known to interact with CFTR are expressed
in these cells (10, 12). These include EBP50 and E3KARP, both of which
contain two PDZ domains and associate with ezrin via the C terminus. We
also examined the expression of the related mRNA encoding PDZK1
(also called CAP70), a protein that contains four PDZ domains (20).
Although PDZK1 is known to associate with CFTR (24), it is not known
whether it also associates with NHE3 or ezrin via an ERM binding
domain. The RT-PCR analysis shown in Fig.
1A reveals expression of
mRNA for EBP50, E3KARP (13), and PDZK1 (20) in hamster lung and the
mouse pancreas. On the other hand, only mRNA for EBP50 was detected
in PS120 cells.
Expression of EBP50 in PS120 cells was unexpected, since a previous
study reported that this cell line does not express EBP50 (13). To
substantiate the RT-PCR findings, expression of EBP50 in the hamster
fibroblast-derived PS120 clone was first verified by sequencing the
amplified RT-PCR product. As shown in Fig. 1B, EBP50 in
PS120 cells showed the highest similarity to Chinese hamster EBP50 with
99% homology based on nucleotide sequences (GenBankTM
accession numbers AF307992 and AF307993). The 1% sequence difference
is probably due to the use of mutagenic agents during the selection of
the NHE-deficient cells (17) or due to substrain differences.
Expression of EBP50 protein in our PS120 cells was examined by Western
blotting using a polyclonal antiserum generated against full-length
human EBP50. Fig. 1C shows expression of EBP50 protein in
both PS120 cells and the mouse pancreas.
Previous works reported that EBP50 can associate with CFTR or NHE3 (10,
11). We first confirmed these findings in our PS120 cells that were
transfected with either CFTR or NHE3 (not shown). Subsequently, we
asked whether CFTR and NHE3 exist in the same complex when expressed in
PS120 cells and in the in vivo situation. For these
experiments, NHE3 was stably expressed in PS120 cells to generate the
PS120/NHE3 clone, and a portion of PS120/NHE3 cells was infected with
Ad-CFTR. The blot in Fig. 2A shows that expression of CFTR had no measurable effect on expression of
NHE3 protein in PS120/NHE3 cells. Lysates were prepared from control
and CFTR-expressing PS120/NHE3 cells, and an antibody recognizing the R
domain of CFTR was used to immunoprecipitate CFTR from PS120/NHE3 cells
and PS120/NHE3 cells co-expressing CFTR. NHE3 was found in the CFTR
immunoprecipitates from the cells infected with Ad-CFTR (Fig.
2C), demonstrating that exogenously expressed CFTR and NHE3
may associate in a stable complex in PS120 cells.
To determine whether CFTR and NHE3 also associate in native cells, we
performed similar experiments using mouse pancreata from WT and Functional Interaction between CFTR and NHE3--
To study the
effect of CFTR on NHE3 activity, we first determined the optimal
conditions to study this interaction. In previous work, the
cAMP-dependent and EBP50-mediated inhibition of NHE3 activity was studied in serum-deprived,
Go/G1-arrested PS120 cells that were
transfected with EBP50 (13). Fig. 3 shows
the results of similar experiments in our PS120/NHE3 cells. Serum
deprivation for 18 h increased NHE3 activity by ~41%. This is
attributable to an increase in NHE3 expression under these
conditions.2 Exogenous
overexpression of EBP50 in PS120/NHE3 cells reduced NHE3 activity in
unstimulated cells by ~35%. Preliminary studies showed that the
optimal experimental condition to consistently observe regulation of
NHE3 by CFTR is to maintain the PS120/NHE3 cells in 10% serum and to
rely on the EBP50 already expressed in the cells.
Having established optimal conditions for measuring NHE3
activity in PS120 fibroblasts, we next examined the effect of increased cAMP on NHE3 activity of control PS120/NHE3 cells and cells transfected with CFTR (Fig. 4). Treatment of
PS120/NHE3 cells with forskolin dose-dependently inhibited
NHE3 activity. At 0.1 and 10 µM forskolin, NHE3 activity
of PS120/NHE3 cells was 91.6 ± 6.1 and 70.8 ± 5.1% of
control, respectively. Importantly, expression of CFTR had no effect on
basal NHE3 activity (rates of pHi recovery: 0.86 ± 0.09
Fig. 4 shows that CFTR markedly augments the inhibition of
NHE3 by cAMP. CFTR binds to EBP50 through its C-terminal DTRL sequence, a signature PDZ domain-binding motif (10). Therefore, we examined whether the enhanced cAMP-dependent inhibition of NHE3
required association of CFTR with PDZ-containing scaffolding proteins. Fig. 5 provides biochemical and
functional evidence that the COOH terminus of CFTR is required for
association of CFTR with NHE3 in the same multiprotein complex. Thus,
when we expressed CFTR mutants lacking the final 4 amino acids involved
in binding PDZ proteins ( Biochemical and Functional Association of CFTR with NHE3 in the
Mouse Pancreatic Duct--
CFTR and NHE3 association in the pancreas
of WT but not
We next determined whether CFTR inhibits the
Na+-dependent H+ efflux (or
OH
When the activity of the luminal Na+-dependent
pHi recovery was measured in pancreatic ducts from
The reduction in NHE3 activity in ducts from The main function of the pancreatic duct is the secretion of fluid
rich in HCO In the present work, we studied the regulatory interaction between CFTR
and NHE3 in heterologous systems and in the native mouse pancreatic
duct. Functional studies, backed by molecular, biochemical, and
immunocytological evidence suggest that CFTR controls NHE3 activity by
two mechanisms: 1) CFTR increases expression of NHE3 in the luminal
membrane of the pancreatic duct, and 2) CFTR acutely augments the
cAMP-dependent inhibition of NHE3.
Probably the most striking finding of the present work is the reduced
levels of NHE3 protein and activity in the pancreatic duct of The age-independent reduction in NHE3 expression in ducts of In addition to increasing NHE3 expression in the luminal membrane of
the pancreatic duct, CFTR acutely augments the inhibition of NHE3
activity by a cAMP-dependent mechanism. The present work provides evidence to show that such a regulation is mediated by binding
of CFTR and NHE3 to EBP50 or other related PDZ scaffolding proteins.
Formation of the CFTR·NHE3 complex was dependent on an intact PDZ
binding motif of CFTR (Fig. 5), and EBP50, as well as CFTR and NHE3,
was co-localized at the luminal pole of pancreatic ducts (Fig. 6).
EBP50 has two PDZ domains and an ERM-binding domain. CFTR binds to
PDZ1, (10), while NHE3 binds the C terminus of EBP50 including PDZ2
(11). In PS120 fibroblasts, EBP50 was required for cAMP-induced
inhibition of NHE3 (13). The present work extends these findings to
show that stimulation of CFTR with cAMP further inhibits NHE3 activity,
beyond the inhibition of NHE3 activity observed in cells expressing
NHE3 and EBP50 alone (Figs. 3 and 4). The EBP50-associated protein
ezrin is known to possess an amphipathic helix that can associate
in vitro with the regulatory subunit of protein kinase A
(29). Therefore, it will be interesting to determine whether protein
kinase A is indeed contained within CFTR·NHE3 protein complexes by
association with ezrin or other cellular A kinase anchoring proteins.
In addition, it will be important to identify other activities that
require the presence of EBP50, E3KARP, or PDZK1, since these proteins
appear to be expressed in excess over the ion channels and transporters
whose activities they are known to regulate.
Measurement of Na+-dependent
H+/OH The physiological significance of regulating the overall
Na+-dependent
HCOF508 mice. Accordingly, luminal
Na+-dependent and HOE694- sensitive recovery
from an acid load was reduced by 60% in ducts of
F508 mice. CFTR
and NHE3 were co-immunoprecipitated from PS120 cells expressing both
proteins and the pancreatic duct of wild type mice but not from PS120
cells lacking CFTR or the pancreas of
F508 mice. The interaction
between CFTR and NHE3 required the COOH-terminal PDZ binding motif of
CFTR, and mutant CFTR proteins lacking the C terminus were not
co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120
cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ
binding motif, augmented cAMP-dependent inhibition of NHE3
activity by 31%. These findings reveal that CFTR controls overall
HCO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
HCO
channel, and many mutations causing CF reduce or abolish
Cl
channel activity (2). However, several CFTR mutations
retain normal or even elevated Cl
channel activity
although they are associated with mild or severe forms of CF (3). This
suggests that CFTR may have other functions, besides conducting
Cl
, that are essential for normal fluid and electrolyte
transport. Indeed, CFTR regulates the activity of the epithelial
Na+ channel ENaC (4), and CFTR supports
Cl
/HCO
channel activity correlates
with the pancreatic status of the CF phenotype (7). Another
HCO
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DTRL) or 26 (S1455X) amino acids. Briefly, mutants
were selected based upon the incorporation of a second-site mutation in
-lactamase, which alters its substrate specificity, allowing
resistance of transformed bacteria to cefotaxime and cerftriaxone in
addition to ampicillin. Incorporation of the mutation was verified by
DNA sequencing. The mutagenesis primers were as follows:
DTRL,
5'-GGA GAC AGA AGA AGA GGT GTA AGA TAC AAG GCT
TTA GAG AG-3'; S1455X, 5'-GCT CTT TCC CCA CCG GAA CTG
AAG CAA GTG CAA GTC TAA GCC-3'.
reverse transcriptase (Life Technologies). The cDNA
was amplified using specific primers and a Taq polymerase
(Promega), and the products were separated on a 1.5% agarose gel
containing 0.1 µg/ml ethidium bromide. The identities of all
amplified products were verified by nucleotide sequencing.
F508 (
F) mice were embedded in OCT (Miles, Elkhart, IN), frozen in liquid N2, and cut into 4-µm sections. Immunostaining
of frozen sections was performed as previously reported (8). Briefly, the sections were fixed and permeabilized by incubation with cold methanol for 10 min at
20 °C. After removal of the methanol, slices were washed twice with phosphate-buffered saline, and the tissue
area was encircled using a hydrophobic marker (Pap Pen; Zymed Laboratories Inc., South San Francisco, CA).
Nonspecific binding sites were blocked by incubation for 1 h at
room temperature with 0.1 ml of phosphate-buffered saline containing
5% goat serum, 1% bovine serum albumin, and 0.1% gelatin (blocking
medium). After blocking, the sections were stained by incubation with
the appropriate primary antiserum followed by fluorophore-tagged
secondary antibodies. For double labeling experiments, these primary
and secondary incubations were repeated with antibodies against the
second protein of interest. Then the sections were incubated in a
bisbenzimide (10 µg/ml in phosphate-buffered saline) solution to
stain DNA and sealed with a coverslip using a Mowiol-based (Calbiochem)
mounting medium. Images were collected with a Leica TCS-NT confocal microscope.
F mice. In this
case, all of the images were taken at the same laser intensity, and the
same recording conditions and staining intensities of specified regions
were analyzed using an imaging software (MCID version 3.0; Brook
University, St. Catharines, Ontario, Canada). Briefly, the pixel counts
of a 1-µm square region in the nucleus (FNuc; background)
and a midportion of the LM (FLM) of the same cell were
measured. The ratio of (FLM
FNuc)/FNuc from multiple cells taken from at
least three separate sections and from separate animals were averaged
and compared between each group.
F508 mutation was introduced in
the mouse CFTR gene targeting in embryonic stem cells (21) was
obtained from Dr. K. R. Thomas (University of Utah). The mice were
maintained on a standard diet, and genotyping was carried out during
days 7-14 postpartum, as described (22). The procedure for preparation
and perfusion of the main pancreatic duct was identical to that
described previously (8). Briefly, the mice were anesthetized, the
abdomen was opened, and the duct lumen was cannulated using a modified
31-gauge needle. After ligating the proximal end of the common duct,
the pancreas was removed into a dish containing ice-cold solution A
supplemented with 0.02% soybean trypsin inhibitor and 0.1% bovine
serum albumin. The main duct was cleared of acini and connective
tissue, and the proximal end of the main duct was cut in order to
facilitate retrograde luminal perfusion. After transferring to the
perfusion chamber and during pHi measurement, the ducts were
continuously perfused with separate bath and luminal solutions.
pHi in response to 5-20
mM NH4Cl pulses. In each experiment, the buffer
capacity (
i) showed a negative linear relationship with
pHi between 6.4 and 7.3. The
i of PS120 cells
(30.2 ± 2.1 mM/pH unit at pHi 7.0) was lower
than that of the pancreatic duct cells (48.6 ± 6.2). However,
forskolin treatment or any gene modulation did not significantly change
i. Therefore, all the results of NHE activity are expressed as
pH/min, and this value was directly analyzed without compensating for
i. The results are presented as mean ± S.E., and, when appropriate, statistical analysis was determined using Student's t test or analysis of variance. p < 0.05 was considered statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of EBP50 in PS120 cells and the
mouse pancreas. A, mRNA was prepared from PS120
cells, Chinese hamster lung (H. Lung), and the mouse
pancreas (M. Panc.), and primers listed under
"Experimental Procedures" were used to amplify a sequence from
EBP50, E3KARP, and PDZK1 from all mRNA preparations. B,
phylogenetic analysis of EBP50 expressed in the five species indicated
in the tree and in PS120 cells. The 408-base pair nucleotide sequences
(corresponding to 672-1079 of mouse EBP50) were aligned, and a
neighbor-joining tree was drawn. The bootstrap probabilities, as
determined for 1000 resamplings, are given in percentages
beside the internal branches. *, the sequences of PS120
(GenBankTM accession number AF307992) and hamster
(AF307993) were sequenced in this study, and others were selected from
registered sequences in GenBankTM (human, NM004252; rabbit,
U19815; rat, AF154336; mouse, U74079). C, Western blot
analysis of EBP50 in lysates prepared from PS120 cells and the mouse
pancreas.
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Fig. 2.
Immunoprecipitation of CFTR·NHE3 complexes
from PS120 cells and the mouse pancreas. Lysates were prepared
from PS120/NHE3, PS120/NHE3 infected with Ad-CFTR (A), and
the pancreas of WT and F508 mice (B). The lysates were
analyzed by blotting with NHE3 antisera to ensure the same amount of
NHE3 protein in the extracts during immunoprecipitation (A
and B). For immunoprecipitation, precleared extracts from
PS120/NHE3 or the mouse pancreas were incubated with monoclonal
antibodies recognizing the R domain of CFTR. The immunoprecipitates
were separated by SDS-polyacrylamide gel electrophoresis, transferred
to membrane, and blotted with the anti-NHE3 antibody 1568 (PS120/NHE3 cells (C) and pancreatic extracts
(D)). IP, immunoprecipitation.
F
mice. For these experiments, the amount of lysate used was adjusted to
contain the same amount of NHE3 (Fig. 2B). NHE3 was found in
CFTR immunoprecipitates from the pancreas of WT mouse (Fig.
2D). In contrast, only a very small amount of NHE3 was found
in CFTR immunoprecipitates from the pancreas of
F mouse. The small
amount of NHE3 found to associate with
F508 CFTR can be accounted
for by the small amount of CFTR that is found in the luminal membrane
of these mice (22). The findings in Fig. 2 provide the first evidence
that NHE3 and CFTR exist in the same multiprotein complex, both in
model systems and in native cells.
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Fig. 3.
Effect of serum deprivation and
overexpression of EBP50 on NHE3 activity in PS120/NHE3 cells.
PS120/NHE3 cells were serum-deprived or transfected with EBP50. The
experimental protocol of acidifying cells with an
NH
pH/min (n = 6) in control and 0.94 ± 0.10
pH/min (n = 9) in CTFR-expressing PS120/NHE3 cells).
Incubation of CFTR-expressing PS120/NHE3 cells with forskolin also
inhibited NHE3 activity in a dose-dependent manner.
However, the inhibition was nearly maximal at 0.1 µM
forskolin; at 0.1 and 10 µM forskolin, NHE3 activity in
CFTR-expressing cells was 60.3 ± 5.1 and 53.4 ± 6.2% of
control, respectively. Thus, we conclude that activation of CFTR
augments cAMP-mediated inhibition of NHE3.
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Fig. 4.
Expression of CFTR augments cAMP-mediated
inhibition of NHE3 activity in PS120/NHE3 cells. PS120/NHE3 cells
infected with Ad-LacZ (A, control) or Ad-CFTR (B)
were acidified twice, before and after stimulation with forskolin, and
Na+-dependent pH recovery from acidification
was measured. The ratios of the slopes of forskolin-stimulated to
control periods (b/a) were calculated and plotted
in C to evaluate the effect of CFTR on NHE3 activity.
Stimulation of CFTR with 0.1 µM forskolin and above
significantly inhibited NHE3 activity beyond that observed in control
cells. **, p < 0.01; *, p < 0.05 compared with control.
DTRL), CFTR and NHE3 were no longer
co-immunoprecipitated (Fig. 5, A and B).
Furthermore, in cells expressing CFTR-
DTRL, activation of CFTR with
forskolin did not augment the cAMP-mediated inhibition of NHE3 (Fig.
5C). It is important to note that deletion of the DTRL
sequence has no effect on the function of CFTR as a Cl
channel (25). Another finding of note in Fig. 5 is the behavior of a
mutant CFTR protein lacking the final C-terminal 26 amino acids
(S1455X). This mutation, which is associated with impaired fluid
secretion (26), appears to have no effect on CFTR Cl
channel activity (25) but rather results in inefficient targeting of
CFTR to the luminal membrane of polarized cells (27). Similar to the
findings with the CFTR-
DTRL, the CFTR-S1455X did not associate with
NHE3 (Fig. 5B) and had no effect on NHE3 activity (Fig.
5C). Taken together, these data indicate that CFTR and NHE3
exist within a multiprotein complex in PS120 cells. Furthermore, the
data indicate that binding of CFTR to EBP50 (or other EBP50-related
protein) is required for association of CFTR with NHE3 and for
CFTR-mediated inhibition of NHE3.
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Fig. 5.
The C terminus of CFTR is required for
formation of CFTR·NHE3 complexes and regulation of NHE3 activity by
CFTR. PS120/NHE3 cells were transfected with mammalian expressing
vectors for WT-CFTR, CFTR- DTRL, and CFTR-S1455X. Lysates were
prepared from all cells, and lysates containing an equal amount of CFTR
constructs (A) were used to immunoprecipitate
(IP) CFTR with antibodies recognizing the R domain of CFTR
(B). The same cells were used to measure the effect of cAMP
stimulation (0.1 µM forskolin) on NHE3 activity using
protocols in Fig. 4 (C). *, p < 0.05 compared with control.
F mice (Fig. 2) suggested that the two proteins
exist in the same complex and are co-localized in the pancreatic duct.
This prediction was verified by immunolocalization studies in the mouse
pancreas. Fig. 6, A-C, shows
that CFTR and NHE3 are co-localized in the luminal membrane of the
mouse pancreatic duct. In view of the results in Fig. 5, it was of
interest to determine whether EBP50 is also localized at the luminal
pole of the pancreatic duct. Fig. 6, D-F, shows that EBP50
can be found in the lateral and luminal regions of pancreatic duct
cells and that there is substantial co-localization between EBP50 and
CFTR in the luminal pole of these cells.
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Fig. 6.
Immunolocalization of CFTR, NHE3, and EBP50
in pancreatic ducts. Frozen sections were prepared from the mouse
pancreas, fixed, and double stained with anti-CFTR and anti-NHE3
antibodies (A-C) or anti-CFTR and anti-EBP50
antibodies (D-F). Similar results were observed in at least
four separate experiments.
influx) activity in the luminal membrane of the
perfused pancreatic duct. We previously showed that 50% of the luminal
Na+-dependent pHi increase is mediated
by NHE3 and about 50% by a novel,
Na+-dependent, and amiloride-sensitive
mechanism different from any known NHE isoform (8). The
Na+-dependent pHi increase was measured
in perfused ducts from WT and
F mice. Fig.
7A shows that exposing the
luminal membrane of an acidified duct from the WT pancreas to 140 mM Na+ resulted in a pHi recovery at a
rate of 1.02 ± 0.09 pH units/min (n = 13). The
subsequent addition of Na+ to the bath had no further
effect on pHi, indicating that luminal Na+ caused
maximal recovery of pHi. As we reported previously (8),
stimulation of the duct with forskolin dose-dependently inhibited the luminal Na+-dependent pHi
recovery in ducts from WT mice.
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Fig. 7.
NHE activity in the luminal membrane of
pancreatic ducts from WT and F mice.
Pancreatic ducts were microdissected from WT (A) and
F
mice (B), cannulated, and used to measure luminal
Na+-dependent recovery from an acid load before
and after forskolin stimulation of the same ducts. In all experiments,
the effect of basolateral Na+ on pHi recovery was
measured after measurement of the luminal activity under resting
conditions. The results of multiple experiments are summarized in
C. **, p < 0.01; *, p < 0.05 compared with control.
F mice,
it was immediately evident that the basal activity was significantly lower than that in ducts from WT mice (Fig. 7B). In fact,
Na+-dependent pHi recovery in
pancreatic ducts from
F mice occurred at a rate of 0.42 ± 0.1 pH units/min (n = 9), which is only 40% of that
measured in ducts from WT mice. Furthermore, luminal Na+
only partially recovered pHi of acidified ducts from
F
animals and exposing the basolateral membrane of the ducts to
Na+ after completion of luminal-dependent
pHi recovery resulted in an increase in pHi back to
resting levels. In additional experiments, we compared the basolateral
Na+-dependent pHi recovery of ducts
from the pancreas of WT and
F mice and found them to be the same
(not shown). Hence, only the luminal
Na+-dependent pHi recovery was reduced
by ~60% in pancreatic ducts from
F mice. Another significant
finding in Fig. 7B and the summary shown in Fig.
7C is that forskolin stimulation did not inhibit the
residual Na+-dependent pHi recovery in
the luminal membrane of the pancreatic duct.
F animals can be due to
down-regulation of NHE3 activity or NHE3 protein expression. To
distinguish between these possibilities, we compared the level of NHE3
protein in pancreatic tissues from WT and
F mice by Western blot. In
nine separate experiments, the Western blotting showed significant
variability in the level of NHE3. Examples of preparation from WT and
three preparations from
F animals are shown in Fig. 8A. The levels of NHE3 in
tissues from
F animals varied between 103 and 28% of WT. This might
reflect a variable amount of duct cells, in particular large ducts in
preparations. Nevertheless, averaging the nine experiments showed
31 ± 11% (p = 0.128) less NHE3 protein in
pancreata from
F relative to WT mice. More reliable results were
obtained when the level of NHE3 expression was analyzed in confocal
images collected from ducts stained with two separate anti-NHE3
antibodies. Fig. 8B shows that expression of NHE3 protein in
the luminal membrane of the pancreatic duct of
F mice was markedly
reduced. Similar results were obtained with two different Abs (1566 and
1568) that recognize different epitopes of NHE3 and in several pairs of
animals of ages ranging between 10 days and 6 months old (not shown).
Analysis of 22 images from WT and 20 images from
F mice showed that
expression of NHE3 was reduced in the luminal membrane of pancreatic
ducts from
F ducts by 53 ± 4% (p < 0.01) (Fig. 8C). This reduction does not appear to be due
to increased endocytosis of NHE3, since no evidence for an increased
intracellular pool of NHE3 could be detected (for example, see Fig.
8B). Hence, it seems that CFTR stabilizes expression of NHE3
in the luminal membrane of the pancreatic duct.
View larger version (26K):
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Fig. 8.
Analyses of NHE3 expression in pancreatic
ducts from WT and F mice. The main
pancreatic ducts were dissected from WT and
F mice and used for
Western blot analysis of NHE3 (A). Band intensities were
determined by densitometry. In the second protocol, frozen sections
were prepared from the mouse pancreas of WT and
F mice, fixed, and
stained with anti-NHE3 antibodies (B). The sections were
used to measure intensity of NHE3 staining in the nuclear (background)
and luminal membrane (experimental) regions, as marked in B.
Results of all experiments are summarized in C. **,
p < 0.01 compared with control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
influx mechanisms in the luminal membrane (8). Because
both CFTR and NHE3 bind to the scaffolding proteins EBP50 and E3KARP, it was of interest to determine whether CFTR exists in the same complex
as NHE3 and whether CFTR can regulate
HCO
F
mouse. Reduced expression of NHE3 was observed by Western blot and by
reduced staining of the luminal membrane of the duct. Furthermore,
measurement of total Na+-dependent recovery
from acidification revealed nearly 60% reduction in the rate of
pHi recovery in ducts of
F mice. Reduced expression of NHE3
in the pancreatic duct was observed in mice as young as 10 days old. In
our colony, 57% of
F mice died between 3 weeks and 3 months of age,
while the surviving mice lived well for at least 6 months. The
pancreatic duct of
F mice of all ages showed reduced NHE3 activity
relative to the ducts of age-matched WT mice. This suggests that
reduction in NHE3 expression in the
F mice occurs early in
development rather than being an adaptive response to the lack of
function of CFTR.
F mice
suggests that the most plausible mechanism for control of NHE3
expression by CFTR is stabilization of NHE3 protein expression. CFTR
may increase transcription of NHE3 mRNA or the half-life of NHE3
mRNA to increase expression of the protein. Alternatively, by
forming a complex including CFTR, EBP50, and NHE3, CFTR may enhance the
stability of the expressed NHE3 by either preventing its degradation or
by enhancing its delivery to the luminal membrane of the pancreatic
duct. It is interesting to note that expression of CFTR in the
PS120/NHE3 cells had no measurable effect on the levels of NHE3 protein
or on basal NHE3 activity in these cells (Figs. 2 and 4). Hence, of the
acute and chronic regulation of NHE3 activity by CFTR in
vivo, only the acute regulation could be reproduced in
vitro. This may reflect differences in sorting mechanisms and/or
targeted expression of NHE3 between the in vivo (pancreas)
and model systems (PS120/NHE3 cells) and highlight the importance of
confirming observations made in model systems in the in vivo situation.
fluxes showed that the pancreatic duct
of
F mice retained only 40% of the activity measured in ducts from
WT mice. In the WT duct, NHE3 mediates only 50% of NHE activity,
whereas the remaining activity is mediated by a novel
Na+-dependent mechanism (8). Furthermore, the
residual NHE3 expressed in the duct of
F mice is likely to mediate
part of the residual luminal NHE activity. This suggests that CFTR
regulates the expression and/or activity of both NHE3 and the novel
Na+-dependent H+/OH
transport mechanism in the luminal membrane of the pancreatic duct.
Once this mechanism is identified with certainty, it will be of
particular interest to determine whether it is indeed regulated by
CFTR.
fluxes by CFTR. At the same time, CFTR stimulates
HCO
/HCO
View larger version (13K):
[in a new window]
Fig. 9.
A model of
HCO
The present findings may have significant importance in understanding
the overall role of CFTR in epithelial physiology and in CF. Not only
do we report a new role for CFTR in regulating HCO transport
proteins in the luminal membrane of the pancreatic duct. In this
respect, recently Wheat et al. (30) reported that CFTR
up-regulates mRNA expression of the colonic
Cl
/HCO
channels. Rather, the more
we understand the role of CFTR in epithelial transport, the more it
becomes evident that expression of CFTR in the luminal membrane of
fluid-secreting epithelia is critical for epithelial cell function and
normal fluid-electrolyte homeostasis.
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FOOTNOTES |
---|
* This work was supported by Basic Research Program of the Korea Science and Engineering Foundation Grant 2000-2-21400-002-1 (to M. G. L.) and National Institutes of Health Grants DE12309 and DK38938 (to S. M.) and HL63755 (to S. L. M.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF307992 and AF307993.
** To whom correspondence may be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: 214 648 2593; Fax: 214 648 8879; E-mail: Shmuel.Muallem@UTSouthwestern.edu.
To whom correspondence may be addressed: Dept. of Pharmacology,
Yonsei University College of Medicine, 134 Sinchon-Dong, Seoul 120-752, South Korea. Tel.: 82 2 361 5221; Fax: 82 2 313 1894; E-mail:
mlee@yumc.yonsei.ac.kr.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M011763200
2 O. W. Moe, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
CF, cystic fibrosis;
CFTR, cystic fibrosis transmembrane conductance regulator;
NHE, Na+/H+ exchanger;
EBP50, ERM-binding
phosphoprotein 50;
BCECF, 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
AM, acetoxymethyl ester;
PCR, polymerase chain reaction;
RT-PCR, reverse
transcription-PCR;
WT, wild type;
F,
F508;
LM, luminal membrane;
VIP, vasoactive intestinal polypeptide.
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