Expression of multiple
Na+/H+
exchanger isoforms in rat parotid acinar and ductal
cells
Keerang
Park1,
John A.
Olschowka2,
Linda A.
Richardson1,
Crescence
Bookstein3,
Eugene B.
Chang3, and
James E.
Melvin1,2
1 Center for Oral Biology,
Rochester Institute for Biomedical Sciences, and
2 Department of Neurobiology
and Anatomy, University of Rochester Medical Center, Rochester, New
York 14642; and 3 Department of
Medicine, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
Several members of the
Na+/H+
exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique
functional properties have been cloned from rat epithelial tissues. The
present study examined the molecular and pharmacological properties of
Na+/H+
exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM
Na+),
Na+/H+
exchanger activity was inhibited by low concentrations of the amiloride
derivative ethylisopropyl amiloride (EIPA;
IC50 = 0.014 ± 0.005 µM),
suggesting the expression of amiloride-sensitive isoforms NHE1
and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1
transcripts are most abundant in this cell type. In contrast, the
intermediate sensitivity of ductal cells to EIPA indicated that
inhibitor-sensitive and -resistant
Na+/H+
exchanger isoforms are coexpressed. Ductal cells were about one order
of magnitude more resistant to EIPA
(IC50 = 0.754 ± 0.104 µM) than cell lines expressing NHE1 or NHE2
(IC50 = 0.076 ± 0.013 or 0.055 ± 0.015 µM, respectively). Conversely, ductal cells were nearly
one order of magnitude more sensitive to EIPA than a cell line
expressing the NHE3 isoform (IC50 = 6.25 ± 1.89 µM). Semiquantitative RT-PCR demonstrated that both
NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was
immunolocalized to the basolateral membranes of acinar and ductal
cells, whereas NHE3 was exclusively seen in the apical membrane of
ductal cells. Immunoblotting, immunolocalization, and semiquantitative
RT-PCR experiments failed to detect NHE2 expression in either cell
type. Taken together, our results demonstrate that NHE1 is the dominant
functional
Na+/H+
exchanger in the plasma membrane of rat parotid acinar cells, whereas
NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.
salivary gland; fluid secretion; pH regulation; sodium chloride
absorption
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INTRODUCTION |
FOUR DIFFERENT ISOFORMS of the
Na+/H+
exchanger gene family (NHE1, NHE2, NHE3, and NHE4) have been described
in epithelial tissues (31, 41). A nonepithelial
Na+/H+
exchanger, NHE5 (15), and a mitochondrial
Na+/H+
exchanger, NHE6 (28), have also been identified. Virtually all
mammalian cells utilize
Na+/H+
exchangers to maintain the intracellular pH (for reviews, see Refs. 27,
30, 40, and 43). This function is especially important in salivary
acinar cells, in which upregulation of
Na+/H+
exchange buffers the acidification that results from
HCO
3 secretion in response to
Ca2+-mobilizing agonists (16, 23).
Upregulation of parotid gland cells has two distinct phases. Initially,
cell shrinkage rapidly activates
Na+/H+
exchange independent of the intracellular
Ca2+ concentration as well as
protein kinase C (PKC) and calmodulin activity (22). Secondarily, a
slow, Ca2+-dependent upregulation
(half-time >5 min) occurs that is separate from PKC and calmodulin
activation (20, 21) as well as phosphorylation of NHE1 (35). This
delayed activation of
Na+/H+
exchange is apparently the result of both an increase in the maximum
rate of uptake and a shift in the intracellular pH sensitivity of the
exchanger (20).
The kinetic properties of
Na+/H+
exchanger activity suggest that at least two different NHE isoforms are
functionally expressed in rat parotid glands. In the present study,
molecular and pharmacological techniques were used to address the
possibility that multiple NHE isoforms are involved in salivary gland
function. Our results demonstrate that NHE1 is the dominant NHE isoform
in acinar cells. In contrast, NHE3 is a major contributor to pH
regulation in ductal cells, although this cell type also expresses high
levels of NHE1. NHE1 is located in the basolateral membranes of rat
parotid acinar and ductal cells, whereas NHE3 was only found in the
apical membranes of ductal cells. Some aspects of this work have been
previously presented in abstract form (32).
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METHODS |
Isolation of rat parotid cells.
Parotid glands were removed from male 150- to 250-g Wistar rats, and
single acinar cells were dispersed by trypsin-collagenase digestion as
described previously (3). Salivary gland cells were plated onto
poly-L-lysine-coated glass
coverslips, and individual acinar cells or small acini were isolated by
using glass micropipettes. Ductal cells were isolated as small
fragments. The lack of contamination was confirmed by PCR using acinar
and ductal cell-specific primers (17).
Tissue culture and stable transfection of rat NHE3.
NHE-deficient PS120 cells were selected as previously described (34).
The cells were maintained in DMEM (GIBCO BRL, Grand Island, NY)
supplemented with 10% fetal bovine serum and penicillin (50 U/ml)-streptomycin (50 µg/ml).
To express NHE3, cDNA (RKNHE2-1; see Ref. 31) was amplified
by using a rat NHE3-specific sense primer corresponding to nucleotides (nt)
16 to
37,
5'-acgaagcttCGGTATGCGTGTCGGCTCCTG-3',
and an antisense primer corresponding to nt 4566-4586,
5'-actctagaGAAGTGGGGTTTTGTGAATGA-3'. The
lowercase italic letters indicate the addition of
Hind III and
Xba I restriction sites to 5'
and 3' ends of the PCR products, respectively, to facilitate
subcloning of the amplified NHE3 cDNA into a mammalian expression
vector. The amplified 4640-bp cDNA product was digested with
Hind III and
Xba I, separated by electrophoresis on
a 1% Tris-borate-EDTA (TBE)-agarose gel, and eluted using the QIAquick
gel extraction kit (QIAGEN, Hilden, Germany). The isolated NHE3 cDNA
was inserted into linearized pCMV (cytomegalovirus) vector plasmid
(Tropix, Bedford, MA). This pCMV-NHE3 mammalian expression construct
was stably transfected into the NHE-deficient PS120 cell line by
calcium-phosphate precipitation (10). NHE3 stable transfectants (PS3)
were selected by resistance to the antibiotic Geneticin (G418; GIBCO
BRL) and by an H+-killing method
(11, 40). The NHE3 cell line (PS3), as well as cell lines expressing
recombinant rat NHE1 (PS1C), NHE2 (PS2-5), and NHE4 (PS4-4), was used
to determine the sensitivity of the different rat NHE isoforms to
various
Na+/H+
exchange inhibitors.
Semiquantitative RT-PCR amplification of rat parotid acini and
ducts.
Salivary glands are complex tissues primarily composed of acinar cells,
although other cell types contribute as much as 20% to the composition
of the gland. Therefore, to eliminate nonacinar cells from the RNA
preparation, single parotid acinar cells were enzymatically dispersed
and isolated using a glass micropipette for subsequent single-cell
RT-PCR. Because ductal cells are difficult to positively identify at
the single-cell level in parotid glands, several dozen cells were
typically isolated as short intact stretches of duct.
Total RNA was prepared from 5-10 single acinar cells or 2-4
ductal segments using TRIzol reagent (GIBCO BRL). RNA was
reverse-transcribed to cDNA using oligo(dT) and random hexamer primers
according to the manufacturer's instructions (1st-STRAND cDNA
synthesis kit; Clontech, Palo Alto, CA).
The cDNA was amplified using PCR primer sets that recognize specific
NHE isoforms. PCR amplification was performed in a DNA thermal cycler
(PTC-200 Peltier thermal cycler; MJ Research, Watertown, MA) using
AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). In initial
studies, PCR amplification products were separated on 2% TBE-agarose
gel, and the identity of the products was verified by restriction
endonucleases and DNA sequencing (36).
To verify that the cDNA preparations were free of genomic DNA, a
-actin sense primer corresponding to nt 2451-2480,
CCCTAGACTTCGAGCAAGAGATGGCCACTG, and an antisense primer corresponding
to nt 2749-2720, CGGATGTCAACGTCACACTTCATGATGGAA, were used to
amplify two neighboring exons containing an 88-bp intron (NCBI data
bank accession no. J00691). Rat genomic DNA was prepared from liver as
previously described (13). Genomic DNA generated a 299-bp product,
whereas cDNA produced a 211-bp product. Contamination of cDNA
preparations by genomic DNA was not observed, even when amplified as
many as 60 PCR cycles (data not shown).
On the basis of published sequences (31, 41), PCR primers were
designed to amplify products with similar sizes and melting temperatures from unique 3'-untranslated regions. NHE
isoform-specific primer pairs were synthesized as follows:
1) NHE1:
5'-(3049)ATTCAACAGTGGAGTGACTTGGGTGATGA(3077)-3' and
5'-(3238) GACTGGCAGGGAAGATTCAAAGGGTCTAAA(3209)-3';
2) NHE2: 5'-(2994)TGACGGTATTAGGGCACAGGTTGGAATGTA(3023)-3' and
5'-(3189)AAATTGGGACAGAGGCGGGGGTAAG(3165)-3'; 3) NHE3:
5'-(2923)CAACGCACCCTAGGAGTTTTAATGCATAGC(2952)-3' and 5'-(3121)TCTCCTCTCAGAATAAGGGTGGCAAACACT(3092)-3'; and
4) NHE4: 5'-(2273)TCTGAGGGTAGGGATGATTAATTGGTCACA(2302)-3' and
5'-(2398)GCATTGGCCTGTTTCAACATTTCTGA(2373)-3'. The cDNA PCR
products for NHE1, NHE2, NHE3 and NHE4 using these primers were 190-, 196-, 199- and 126-bp, respectively. The isolated PCR cDNA products
were inserted into the pCR2.1 TA cloning vector (Invitrogen, Carlsbad, CA).
The isoform-specific pCR2.1 constructs (pCR2.1-NHE1, pCR2.1-NHE2,
pCR2.1-NHE3, and pCR2.1-NHE4) were used to generate standard curves for
quantitative PCR (for an example, see Fig. 6). The DNA concentration of
each isoform-specific plasmid was normalized by first estimating the
amount of DNA present in a given plasmid DNA preparation by
spectrophotometry (Ultrospec III; Pharmacia LKB Biochrom, Piscataway,
NJ). The DNA concentrations of the different NHE isoform-specific
pCR2.1 constructs were then adjusted to equal amounts by comparing the
quantity of PCR product generated with serial dilutions of estimated
amounts of DNA (6, 60, 600, and 6,000 molecules) using pCR2.1-specific
primers. The pCR2.1-specific sense primer corresponded to nt
2579-2603, 5'-GAACCGGAGCTGAATGAAGCCATAC-3', and the
antisense primer corresponded to nt 2703-2732,
5'-CAACTTTATCCGCCTCCATCCAGTCTATTA-3'. The size of the
pCR2.1 amplification product was 154 bp. The normalized DNA
concentration for each isoform-specific plasmid was then amplified by
its respective set of NHE isoform-specific primers to create standard
curves for semiquantitative PCR.
PCR products were quantified by one of two techniques. In the first
method, the PCR products were resolved by 3% TBE-agarose gel and
stained with ethidium bromide. The intensity of PCR products was
measured using a densitometer (IS1000 Digital Imaging System; Alpha
Innotech, San Leandro, CA) and was plotted to generate standard curves
and estimate the amount of NHE-specific cDNA in the acinar and ductal
cell preparations. PCR reaction conditions were optimized using
different numbers of amplification cycles as previously described (25,
45).
Enhanced sensitivity for resolving amplification products was obtained
in the second detection method by labeling the PCR primers with
infrared IRD41 fluorophore and determining the amount of PCR product on
a LI-COR 4000 automated sequencer (LI-COR, Lincoln, NE). The
IRD41-labeled primers were the same as those described above.
The IRD41-labeled pCR2.1 primers were used to normalize the DNA
concentrations for each NHE isoform-specific plasmid DNA. PCR products
were resolved by 4% polyacrylamide gel-8 M urea, and the fluorescence
images of PCR products were generated on the LI-COR automated
sequencer. The intensity of PCR products was quantified using the
IS1000 digital imaging system.
Preparation of polyclonal antibody to the COOH-terminal region of
rat NHE1.
NHE1-specific primers were used to amplify the COOH-terminal region
corresponding to amino acids Pro-602 to Glu-740. The rat NHE1-specific
sense primer corresponded to nt 1804-1831,
5'-acacggatCCATCTGCCGTCTCAACTGTCTCTATGC-3', and the antisense primer corresponded to nt 2199-2220,
5'-acacaagcttaCTCCTCATTCACCAGGTCCACA-3'. The lowercase italic letters indicate the bases added to the primers to
generate a BamH I flanking sequence at
the 5' end and a Hind III
flanking sequence and a stop codon at the 3' end. The 436-bp product was generated using pcDNA2-NHE1 as a template (31). The PCR
product was digested with BamH I and
Hind III and then ligated into the
pRSET-B bacterial expression vector (Invitrogen). The resultant 21-kDa
NHE1 fusion protein (BNHE1) was purified by
Ni2+-affinity chromatography
(Invitrogen) and dialyzed against water, and the amino acid sequence
was verified by protein sequencing (model 473A; Applied Biosystems,
Norwalk, CT). The NHE1 fusion protein was then used to generate a
rabbit polyclonal antibody as previously described (24). The
NHE1-specific antibody was purified using a BNHE1 antigen-linked
affinity column.
Specificity of the antibody was verified by Western analysis of the
truncated NHE1 expressed protein (BNHE1) used to raise the antibody and
membrane preparations from PS120 cells expressing the different NHE
isoforms. Figure
1A,
lane 1, shows the size of the BNHE1
protein stained with Coomassie blue. Incubation with preimmune serum
failed to detect the protein (lane
2), whereas the affinity-purified antibody detected
the 21-kDa fusion protein (lane 3).
Furthermore, Fig. 1B shows that the
anti-BNHE1 antibody did not cross-react with NHE2, NHE3, and NHE4
expressed in PS120 cells (lanes 2,
3, and
4, respectively) or with proteins
isolated from the NHE-deficient cell line PS120 (lane
5). However, anti-BNHE1 detected a 95- to 105-kDa
protein in the PS1C cell line (Fig. 1B, lane
1), which displays stable expression of NHE1.

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Fig. 1.
Characterization of BNHE-1 antibody by Western blotting. Purified
antigen of truncated COOH-terminal region of NHE1 (BNHE1) and membrane
proteins from PS120 cells expressing different
Na+/H+
exchanger (NHE) isoforms were analyzed to verify specificity of
affinity-purified anti-BNHE1 antibody.
A: immunoblotting with anti-BNHE1
antibody was performed as described in
METHODS. Lane
M, protein molecular mass standards stained with
Coomassie blue; lane 1, Coomassie blue
staining of BNHE1; lane 2, immunologic
detection of BNHE1 performed with preimmune serum;
lane 3, immunologic detection of BNHE1
with anti-BNHE1 antibody. B:
immunoblot of membrane proteins (70 µg protein/lane) from the NHE1
expressing PS1C cell line (lane 1),
the NHE2 expressing cell line PS2-5 (lane
2), the NHE3 expressing cell line PS3
(lane 3), the NHE4 expressing cell
line PS4-4 (lane 4), and the
NHE-deficient PS120 cell line (lane
5).
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Membrane preparation.
Crude membranes were prepared from rat parotid glands (19) and from
tissue culture cells (1) as previously described. Aliquots were quickly
frozen in liquid N2 and stored at
85°C until use. Protein concentration was determined using
the bicinchoninic acid protein assay (Pierce, Rockford, IL).
Western blot analysis.
The purified antigen (BNHE1), membranes from rat parotid glands, and
membranes from NHE-expressing cell lines (70 µg/lane) were resolved
by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride
membranes (Bio-Rad, Hercules, CA) as previously described (33).
Membranes were then incubated with primary antibody (anti-BNHE1 and
anti-NHE2) or preimmune serum overnight at 4°C, followed by detection with a horseradish peroxidase-linked goat anti-rabbit IgG
antibody (Jackson ImmunoResearch Laboratory, West Grove, PA) and
enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Immunocytochemistry.
Glands were immediately frozen in 2-methylbutane on dry ice, and
sections were cut at 10 µm on a cryostat (HistoSTAT microtome, Scientific Instruments). The sections were treated as previously described (17) with polyclonal anti-NHE1 antibody (anti-BNHE1), preimmune serum, antisera for NHE2 (2M5; see Ref. 7), or NHE3 (no.
1314; see Ref. 1). The NHE2 and NHE3 anitsera were preabsorbed for 1 h
at room temperature with parotid gland homogenate prepared from NHE2
and NHE3 knockout mice, respectively. Sections were then treated with
FITC-labeled secondary antibody (goat anti-rabbit; Jackson
ImmunoResearch Laboratory) at room temperature for 1 h. Samples were
viewed and analyzed on a Zeiss Axioplan microscope using a ×40
neofluor objective (Carl Ziess, Oberkochen, Germany).
Intracellular pH determinations.
Intracellular pH was monitored using the pH-sensitive fluorescent dye
seminaphthorodafluor-1 (SNARF-1; Molecular Probes, Eugene, OR) as
previously described (4). NHE-expressing cell lines were grown on glass
coverslips for 24-72 h before experiments. Immediately before the
start of an experiment, rat parotid acinar and ductal cells were
attached to a glass coverslip mounted in a superfusion chamber on the
microscope stage of an Ultima confocal laser cytometer (Meridian
Instruments, Okemos, MI).
SNARF-1-loaded cells were acidified by using a
NH4Cl prepulse technique
essentially as previously described (29). Briefly, cells were exposed
to 60 mM NH4Cl in a physiological
salt solution (NaCl was replaced by
NH4Cl) for 10 min, which was then
switched to an Na+-free salt
solution (Na+ was replaced by
N-methyl-D-glucamine).
Approximately 5 min later, the extracellular
Na+ was restored to a
physiological concentration to initiate the Na+/H+
exchanger-mediated intracellular pH recovery (44). The physiological salt solution contained (in mM) 145 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.33 NaH2PO4,
10 glucose, 20 HEPES, 1.2 CaCl2,
and 0.8 MgSO4 (pH 7.4). The
intracellular pH was estimated by the high potassium-nigericin technique as previously described (44). The estimated intracellular pH
after acid loading was ~6.0. After the readdition of extracellular Na+, the intracellular pH rose to
~7.5. The raw data are presented.
 |
RESULTS |
Differences in EIPA sensitivity of
Na+/H+
exchanger activity in parotid acinar and ductal cells.
The different NHE isoforms display unique sensitivities to various
Na+/H+
exchange inhibitors including the 5-amino acid derivative of amiloride
(EIPA). NHE1 and NHE2 are sensitive, whereas NHE3 and NHE4 are
resistant, to inhibition by EIPA in heterologous expression systems (9,
29, 42). Inhibitor sensitivities were thus used to characterize
Na+/H+
exchanger activity in native parotid cells.
The EIPA sensitivity was determined in rat parotid acinar and ductal
cells by monitoring the intracellular pH recovery after an acid load
generated by an NH4Cl prepulse.
Figure 2 shows typical examples of the
intracellular pH recovery from an acid load for parotid acinar and
ductal cells. EIPA (200 nM) blocks the initial rate of the
intracellular pH recovery by >90% in acinar cells (Fig.
2A). In contrast, EIPA
inhibits the pH recovery by <15% in ductal cells (Fig.
2B). Washout of EIPA restored
Na+/H+
exchanger activity to control rates (data not shown). Figure 3 summarizes the effects of the EIPA
concentration on the intracellular pH recovery in rat parotid acinar
cells and ductal cells. The EIPA sensitivity of ductal cells
(IC50 = 0.754 ± 0.104 µM)
was ~50-fold less sensitive than that of acinar cells
(IC50 = 0.014 ± 0.005 µM).

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Fig. 2.
Ethylisopropyl amiloride (EIPA) sensitivity of NHE activity in rat
parotid acinar and ductal cells. Seminaphthorodafluor-1
(SNARF-1)-loaded acinar (A) and
ductal (B) cells were acidified
using the
NH+4/NH3
prepulse technique (see METHODS).
Recovery from an acid load was initiated by readdition of a
physiological concentration of Na+
at time indicated by arrow. Rate of recovery was determined in presence
and absence of 0.2 µM EIPA. Data are from representative examples
with 8 or more cells per condition. pHi, intracellular
pH.
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Fig. 3.
Dependence of NHE inhibition on EIPA concentration ([EIPA])
in rat parotid acinar and ductal cells. Concentration dependence of
EIPA inhibition was tested using concentrations ranging from 2 nM to 30 µM EIPA for acinar cells and from 0.02 µM to 30 µM EIPA for
ductal cells. Dotted line is a sigmoidal fit to acinar cell ( ) data
(IC50 = 0.014 ± 0.005 µM), and solid line is a sigmoidal fit to ductal cell ( )
data (IC50 = 0.754 ± 0.104 µM). Number of cells tested for each EIPA concentration was 26 or
more per condition. In all cases, SE was <5% of mean.
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EIPA sensitivity of recombinant rat NHE1, NHE2, and NHE3 expressed
in PS120 cells.
The conditions for monitoring the EIPA sensitivity of parotid
Na+/H+
exchanger activity in the present studies are different from those used
earlier to characterize recombinant rat NHE1, NHE2, and NHE3 (29, 42).
Previously, the EIPA sensitivity of
22Na uptake was determined in low
external Na+ (<1 mM). In
contrast, we monitored the EIPA sensitivity of the NHE-dependent pH
recovery in a physiological Na+
concentration (145 mM). Na+ is
known to compete with amiloride (2); thus the potency of EIPA for the
different rat NHE isoforms was unknown under our experimental
conditions. Figure 4 shows that, when
conditions identical to those used with acinar and ductal cells were
utilized, recombinant rat NHE1 was very sensitive to 1 µM EIPA (Fig.
4A), whereas NHE3 was very resistant
(Fig. 4B).

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Fig. 4.
EIPA sensitivity of NHE activity in PS120 cells expressing either rat
NHE1 (PS1C) or rat NHE3 (PS3). SNARF-1-loaded PS1C
(A) and PS3
(B) cells were acidified using
NH+4/NH3
prepulse technique (see METHODS).
Recovery from an acid load was initiated by readdition of a
physiological concentration of Na+
at time indicated by arrow. Rate of recovery from an acid load was
determined in presence and absence of 1 µM EIPA. Data are from
representative examples with 10 or more cells per condition.
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Figure 5 summarizes the effects of the EIPA
concentration on the intracellular pH recovery in PS120 cells
expressing rat recombinant NHE1, NHE2, and NHE3. The EIPA sensitivity
of cells expressing NHE3 (IC50 = 6.25 ± 1.89 µM) was about two orders of magnitude less sensitive
compared with that of cells expressing NHE1 or NHE2. In contrast, the
EIPA sensitivity was not significantly different between rat NHE1 and
NHE2 (IC50 = 0.076 ± 0.013 and 0.055 ± 0.015 µM, respectively).

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Fig. 5.
Dependence of NHE inhibition on EIPA concentration in PS120 cells
expressing either rat NHE1, NHE2, or NHE3. Concentration dependence of
EIPA inhibition was tested using concentrations ranging from 2 nM to 30 µM EIPA for PS1C ( ) and PS2-5 ( ) cells and from 1 to 800 µM
EIPA for PS3 cells ( ). Dotted line is a sigmoidal fit to NHE3 data
(IC50 = 6.25 ± 1.89 µM),
dashed line is fit to NHE1 data
(IC50 = 0.076 ± 0.013 µM),
and solid line is fit to NHE2 data
(IC50 = 0.055 ± 0.015 µM).
Number of cells tested for each EIPA concentration was 35 or more per
condition. In all cases, SE was <5% of mean.
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NHE4 activity is very resistant to EIPA (9). Therefore, the relative
resistance of ductal cells to EIPA suggests that NHE4 expression may
potentially contribute to intracellular pH regulation in these cells
(see Fig. 2). However, under our experimental conditions, Na+/H+
exchanger activity was not detected in the PS4-4 cell line expressing the NHE4 isoform (data not shown), even when hypertonic conditions were
used to activate the exchanger as previously reported (6). Chambrey et
al. (8, 9) have also reported difficulty in detecting NHE4 activity in
an expression system as well as in native cells. Thus the failure of
the NHE4 cell line to express detectable
Na+/H+
exchange activity most likely indicates that NHE4 may be quiescent in
acinar and ductal cells under the conditions of our experiments.
Semiquantitative RT-PCR in isolated acinar and ductal cells.
The
Na+/H+
exchange inhibitor EIPA was unable to distinguish between rat
recombinant NHE1 and NHE2 when expressed in NHE-deficient PS120 cells.
As an alternative approach, we analyzed the transcript levels for NHE1,
NHE2, NHE3, and NHE4 by semiquantitative RT-PCR methods to determine
the expression levels of the different NHE isoforms in acinar and
ductal cells. Figure 6 shows an example of
control amplifications of the NHE1-containing plasmid run in duplicate
at different concentrations to generate a standard curve. Standard
curves were then used to empirically estimate the concentrations of the
different NHE isoforms derived from acinar and ductal
cells.

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Fig. 6.
Standard curve for semiquantitative PCR of pCR2.1-NHE1. The amount of
NHE-specific cDNA from acinar and ductal cell preparations was
empirically calculated using the corresponding standard curve.
A: standard curve generated by
plotting no. of molecules added vs. arbitrary fluorescence units
(×102) for amplification
of pCR2.1-NHE1 construct using NHE1-specific primers. This procedure
was repeated for each NHE isoform (see
METHODS).
B: amplification products from
different concentrations of pCR2.1-NHE1 construct were run in
duplicate. Fluorescence intensity was measured by densitometry. Size of
pCR2.1-NHE1 amplification product was 190 bp. Lane
M is a 100-bp ladder molecular weight size standard;
estimated amounts of DNA were 6, 60, 600, or 6,000 molecules.
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Figure 7 shows the results from a
representative experiment (n
4 for
each isoform-specific set of primers). NHE1 was expressed most
abundantly in acinar (Fig. 7A,
lane 1) and ductal cells (Fig. 7B, lane
1). NHE4 transcripts were also expressed in both cell types (Fig. 7, A and
B, lane
4). In contrast, NHE3 transcripts were detected at
levels less than that observed for either NHE1 or NHE4 in ductal cells
(Fig. 7B, lane
3), but NHE3 mRNA was not present in acinar cells
(Fig. 7A, lane
3). NHE2 transcripts were below the detection limit
of this assay in both acinar and ductal cells (Fig. 7,
A and
B, lane
2).

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Fig. 7.
Semiquantitative RT-PCR amplification of NHE1, NHE2, NHE3, and NHE4
from rat parotid acinar cells and ductal cells. RT-PCR products were
analyzed as described in METHODS. cDNA
was reverse-transcribed from acinar
(A) and ductal
(B) preparations using random
hexamer and oligo(dT) primers. Each preparation was amplified for 40 cycles using either NHE1 (lane 1, 190 bp), NHE2 (lane 2, 196 bp), NHE3
(lane 3, 199 bp), or NHE4
(lane 4, 126 bp) isoform-specific
primers. Lane M represents a 100-bp
ladder DNA size marker.
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In additional experiments similar to those shown in Fig. 7, the number
of PCR cycles was increased beyond the linear range of the assay (up to
60 cycles). Amplification products were observed for all four
epithelial NHE isoforms in both acinar and ductal cells (data not
shown). DNA sequencing confirmed their identity as NHE1, NHE2, NHE3,
and NHE4. The cDNA prepared from individual acinar cells and duct
fragments were free of genomic DNA (see METHODS). Although not quantitative,
these data demonstrate that NHE2 mRNAs are present in both cell types,
but at very low levels, likely orders of magnitude less than those of
NHE1 and NHE4.
Western analysis of NHE proteins.
Pharmacological characterization and semiquantitative RT-PCR analysis
suggest that NHE1 is the most abundant
Na+/H+
exchanger isoform in rat parotid gland. To verify that the NHE1 protein
is expressed in the membranes of this tissue, a rabbit polyclonal
antibody was prepared against a nonconserved 139-amino acid region of
the COOH-terminal domain of rat NHE1, and the specificity was verified
(see METHODS; Fig. 1). Western blot
analysis of rat parotid membranes detected a 90- to 100-kDa protein
with the BNHE1 polyclonal antibody (Fig.
8A,
lane 1), whereas preimmune serum failed to label protein (lane 2).
The size of the NHE1 protein shown is similar to that previously
described in rabbit kidney (5). Based on the primary sequence, the NHE1
protein is predicted to be ~91 kDa.

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Fig. 8.
Immunoblot analysis of parotid and submandibular membrane proteins
using anti-BNHE1 and anti-NHE2 antibodies. Immunoblotting of membrane
proteins (70 µg/lane) from rat salivary glands was performed as
described in METHODS.
A: a rat parotid gland immunoblot was
probed with either anti-BNHE1 antibody (lane
1) or preimmune serum (lane
2). B: an immunoblot
containing rat submandibular (lane
1), rat parotid (lane
2), wild-type mouse parotid (lane
3), and NHE2-deficient parotid (lane
4) membrane proteins were probed with anti-NHE2
antibody 2M5.
|
|
Na+/H+
exchanger activity in acinar cells was very sensitive to inhibition by
EIPA, suggesting that these cells functionally express NHE1
and/or NHE2. However, RT-PCR failed to detect significant levels of NHE2 mRNA. In agreement with the semiquantitative RT-PCR experiments, the NHE2-specific antibody 2M5 did not detect a 90-kDa protein in a rat parotid gland membrane preparation (Fig.
8B, lane
2). In contrast, the NHE2-specific antibody detected
an ~90-kDa protein in rat submandibular glands (Fig.
8B, lane
1 at arrow). The size of the NHE2 protein in rat
submandibular gland is similar to that previously described in rat
intestine and kidney (7) and is consistent with the predicted molecular
mass (90 kDa). However, nonspecific bands are also labeled with the
NHE2 antibody. To verify that the 90-kDa band labeled in the rat
submandibular protein preparation is NHE2, Western blots of proteins
isolated from parotid glands of mice lacking NHE2 expression were
probed (38). Comparable to rat submandibular glands, parotid proteins isolated from wild-type mice contained a 90-kDa protein labeled by the
NHE2 antibody (Fig. 8B,
lane 3). However, the band
representing NHE2 was absent in NHE2 knockout mice (Fig.
8B, lane
4), whereas the staining intensity of proteins
labeled nonspecifically remained constant.
Immunolocalization of NHE proteins.
NHE1 has been localized to the basolateral membrane of epithelial cells
(5), and this includes rat submandibular and parotid glands (12, 35).
The NHE1-specific polyclonal antibody, anti-BNHE1, was used to confirm
the site of this protein in the plasma membrane of rat parotid glands.
Figure 9A
shows that NHE1 is localized to the basolateral membrane of both acinar
cells (less intense meshwork of staining) and ductal cells (intense
labeling). NHE1 expression is considerably more abundant in ductal
cells (arrows). Figure 9B is a
Nomarski image of the same field as in Fig.
9A. In contrast, the polyclonal
NHE3-specific antibody no. 1314 (1) recognized the apical membrane of
ductal cells (Fig. 9, C and D, arrows) but failed to label acinar
cells.

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Fig. 9.
Immunolocalization of NHE1 and NHE3 in rat parotid gland. Frozen
sections of rat parotid glands were fixed and permeablized with cold
methanol. Sections were incubated with polyclonal anti-NHE1 antibody or
antisera no. 1314 for NHE3 and then treated with FITC-labeled secondary
antibody. A: sections treated with
anti-NHE1 antibody showed strong staining of basolateral membranes of
ductal cells (arrow) and less intense staining of basolateral membranes
of acinar cells. B: Nomarski image of
same field as in A.
C: sections stained with NHE3-specific
antibody labeled the apical membranes of ductal cells (arrow).
D: Nomarski image of same
field as in C.
|
|
Our results suggest the relative absence of NHE2 mRNA or protein in rat
parotid acinar and ductal cells (see Figs. 7 and 8). Consistent with
these observations, immunolocalization studies in which an
NHE2-specific antibody was used failed to detect NHE2 protein in rat
parotid glands (Fig. 10,
A and
B). However, as previously reported
(12), the apical membranes of rat submandibular ductal cells were
labeled with the NHE2-specific antibody (Fig. 10,
C and
D, arrows).

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Fig. 10.
Immunolocalization of NHE2 in rat parotid and submandibular glands.
Frozen sections of rat parotid and submandibular glands were fixed and
permeablized with cold methanol. Sections were incubated with
polyclonal anti-NHE2 antibody and antisera 2M5 and then treated with
FITC-labeled secondary antibody. A:
parotid gland sections treated with anti-NHE2 antibody showing no
specific staining. B: Nomarski image
of same field as in A.
C: submandibular tissue treated with
anti-NHE2 antibody showing strong staining of apical membranes of
ductal cells (arrows) and little or no staining of acinar cells.
D: Nomarski image of same field as in
C.
|
|
 |
DISCUSSION |
Na+/H+
exchange plays an important role in regulating the intracellular pH of
salivary gland cells. When acinar cells are stimulated to secrete,
HCO
3 efflux produces a drop in the intracellular pH that is rapidly buffered by the activation of Na+/H+
exchangers (16, 23, 44). A recent report suggests that NHE1 is most
likely the
Na+/H+
exchanger isoform involved in this process (35). However, earlier studies indicate that upregulation of
Na+/H+
exchange in this gland is different compared with recombinant NHE1.
NHE1 is regulated by protein kinase A (PKA) and PKC pathways (14, 18,
37) as well as calmodulin (26). In contrast, upregulation of
Na+/H+
exchanger activity in parotid cells is not associated with PKA or PKC
pathways (20, 21) and is not blocked by calmodulin antagonists (22).
The present study therefore investigated further the properties of the
Na+/H+
exchangers regulating intracellular pH in the rat parotid gland by
using a combination of molecular, pharmacological, and immunochemical techniques. It has been previously suggested that the activities of
NHE1 and NHE2 can be distinguished on the basis of their sensitivities to the
Na+/H+
exchange inhibitor EIPA (40, 43). In basolateral membrane vesicles
prepared from rat parotid glands, Manganel and Turner (19) found that
amiloride inhibited
Na+/H+
exchange with an IC50 of ~1.6
µM. This was the first observation suggesting that
Na+/H+
exchanger activity in the rat parotid gland is due to NHE1
and/or NHE2 expression. In the present study, the sensitivity
of rat parotid acinar cells to EIPA (Fig. 3) was comparable to the
sensitivity of expressed recombinant rat NHE1 and NHE2 proteins (Fig.
5). These results are consistent with previous reports for rat NHE1 and
NHE2 (8, 9, 29, 42).
Neither proteins (Figs. 8 and 10) nor transcripts (Fig. 7) for NHE2 and
NHE3 were detected in acinar cells. NHE1 and NHE4 transcripts were
observed in single parotid acinar cells. In contrast to NHE1, NHE4
activity is very resistant to EIPA
(IC50 > 10 µM; see Ref. 8).
Although NHE4 transcripts were relatively abundant in parotid acinar
cells, the total block of
Na+/H+
exchanger activity by low concentrations of EIPA indicates that NHE4 is
not actively involved in intracellular pH regulation in these cells.
The explanation for this observation may be that NHE4 is quiescent
under most physiological conditions (6, 8, 9). Although NHE4 appears to
be inactive, we cannot rule out the possibility that this NHE isoform
has an unknown mode of regulation that uniquely activates this
exchanger in salivary cells.
In ductal cells, transcripts and protein for NHE3 were seen in addition
to those for NHE1 and NHE4. These results are consistent with an
earlier report suggesting that NHE1 is most likely the primary
Na+/H+
exchanger in parotid acinar cells (35), but the results differ in that
NHE3 and NHE4 are also expressed in rat parotid salivary glands. One
potential reason for this apparent inconsistency might be the loss of
ductal cells during the isolation procedure (35). Alternatively, the
efficiency of the PCR primers used in the two studies may be different.
Indeed, we failed to get a PCR product when we attempted to amplify our
cDNA preparations with the NHE3 and NHE4 primers previously described
(35).
Although we could not distinguish between NHE1 and NHE2 with EIPA,
PCR-based and immunochemical methods demonstrate that NHE1 is by far
the dominant exchanger for regulating the intracellular pH in rat
parotid acinar cells. In contrast to acinar cells, ductal cells were
much more resistant to EIPA, indicating functional expression of
multiple NHE isoforms. Ductal cells were less sensitive to EIPA than
the cell lines expressing recombinant rat NHE1, but they were more
sensitive to EIPA than NHE3. In agreement with these observations, NHE1
and NHE3 transcripts (Fig. 7) and proteins (Figs. 8 and 9) are
expressed in ductal cells. Therefore, both NHE1 and NHE3 contribute
significantly to intracellular pH regulation in ductal cells. The
apical location of NHE3 suggests a potential role for this
Na+/H+
exchanger isoform in NaCl reabsorption by ductal cells, as has been
recently demonstrated (39) in other NaCl conserving tissues such as
mouse kidney and intestine.
 |
ACKNOWLEDGEMENTS |
We thank Drs. L. A. Tabak and F. Hagen for constructive discussions
and the use of facilities throughout the course of these studies. We
also thank Laurie Koek, Heather M. Lantz, Daniel Frank, Brian
VanWuyckhuyse, and Xuhong Wang for technical assistance.
 |
FOOTNOTES |
The antisera (no. 1314) for localization of NHE3 was kindly provided by
Dr. O. W. Moe. Parotid tissue from NHE2 and NHE3 knockout mice used for
preabsorbing the NHE2 and NHE3 antibodies, respectively, and plasmids
containing the different NHE isoforms were furnished by Dr. G. E. Shull.
This work was supported in part by National Institute of Dental
Research Grants DE-09692 and DE-08921.
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: J. E. Melvin, Center for Oral Biology,
Box 611, Rochester Institute for Biomedical Sciences, Univ. of
Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642-8611.
Received 25 June 1998; accepted in final form 27 October 1998.
 |
REFERENCES |
1.
Amemiya, M.,
J. Loffing,
M. Lotscher,
B. Kaissling,
R. J. Alpern,
and
O. W. Moe.
Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb.
Kidney Int.
48:
1206-1215,
1995[Medline].
2.
Aronson, P. S.
Kinetic properties of the plasma membrane Na+-H+ exchanger.
Annu. Rev. Physiol.
47:
545-560,
1985[Medline].
3.
Arreola, J.,
K. Park,
J. E. Melvin,
and
T. Begenisich.
Three distinct chloride channels control anion movements in rat parotid acinar cells.
J. Physiol. (Lond.)
490:
351-362,
1996[Abstract].
4.
Bassnett, S.,
L. Reinisch,
and
D. C. Beebe.
Intracellular pH measurement using single excitation-dual emission fluorescence ratios.
Am. J. Physiol.
258 (Cell Physiol. 27):
C171-C178,
1990[Abstract/Free Full Text].
5.
Biemesderfer, D.,
R. F. Reilly,
M. Exner,
P. Igarashi,
and
P. S. Aronson.
Immunocytochemical characterization of Na+-H+ exchanger isoform NHE-1 in rabbit kidney.
Am. J. Physiol.
263 (Renal Fluid Electrolyte Physiol. 32):
F833-F840,
1992[Abstract/Free Full Text].
6.
Bookstein, C.,
M. W. Musch,
A. DePaoli,
Y. Xie,
M. Villereal,
M. C. Rao,
and
E. B. Chang.
A unique sodium-hydrogen exchange isoform (NHE-4) of the inner medulla of the rat kidney is induced by hyperosmolarity.
J. Biol. Chem.
269:
29704-29709,
1994[Abstract/Free Full Text].
7.
Bookstein, C.,
Y. Xie,
K. Rabenau,
M. W. Musch,
R. L. McSwine,
M. C. Rao,
and
E. B. Chang.
Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1496-C1505,
1997[Medline].
8.
Chambrey, R.,
J. M. Achard,
P. L. St. John,
D. R. Abrahamson,
and
D. G. Warnock.
Evidence for an amiloride-insensitive Na+/H+ exchanger in rat renal cortical tubules.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1064-C1074,
1997[Abstract/Free Full Text].
9.
Chambrey, R.,
J. M. Achard,
and
D. G. Warnock.
Heterologous expression of rat NHE4: a highly amiloride-resistant Na+/H+ exchanger isoform.
Am. J. Physiol.
272 (Cell Physiol. 41):
C90-C98,
1997[Abstract/Free Full Text].
10.
Chen, C.,
and
H. Okayama.
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol. Cell. Biol.
7:
2745-2752,
1987[Medline].
11.
Franchi, A.,
D. Perucca-Lostanlen,
and
J. Pouysségur.
Functional expression of a human Na+/H+ antiporter gene transfected into antiporter-deficient mouse L cells.
Proc. Natl. Acad. Sci. USA
83:
9388-9392,
1986[Abstract].
12.
He, X.,
C.-M. Tse,
M. Donowitz,
S. L. Alper,
S. E. Gabriel,
and
B. J. Baum.
Polarized distribution of key membrane transport proteins in the rat submandibular gland.
Pflügers Arch.
433:
260-268,
1997[Medline].
13.
Janssen, K.
Current Protocols in Molecular Biology. New York: Greene and Wiley, 1993, p. 17.13.5-17.13.6.
14.
Kandasamy, R. A.,
F. H. Yu,
R. Harris,
A. Boucher,
J. W. Hanrahan,
and
J. Orlowski.
Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.
J. Biol. Chem.
270:
29209-29216,
1995[Abstract/Free Full Text].
15.
Klanke, C. A.,
Y. R. Su,
D. F. Callen,
Z. Wang,
P. Meneton,
N. Baird,
R. A. Kandasamy,
J. Orlowski,
B. E. Otterud,
M. Leppert,
G. E. Shull,
and
A. G. Menon.
Molecular cloning and physical and genetic mapping of a novel human Na+/H+ exchanger (NHE5/SLC9A5) to chromosome 16q22.1.
Genomics
25:
615-622,
1995[Medline].
16.
Lau, K. R.,
A. C. Elliot,
and
P. D. Brown.
Acetylcholine-induced intracellular acidosis in rabbit salivary gland acinar cells.
Am. J. Physiol.
256 (Cell Physiol. 25):
C288-C295,
1989[Abstract/Free Full Text].
17.
Lee, M. G.,
P. J. Schultheis,
M. Yan,
G. E. Shull,
C. Bookstein,
E. Chang,
M. Tse,
M. Donowitz,
K. Park,
and
S. Muallem.
Membrane-limited expression and regulation of Na+-H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct.
J. Physiol. (Lond.)
513:
341-357,
1998[Abstract/Free Full Text].
18.
Levine, S. A.,
M. M. Montrose,
C.-M. Tse,
and
M. Donowitz.
Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line.
J. Biol. Chem.
268:
25527-25535,
1993[Abstract/Free Full Text].
19.
Manganel, M.,
and
R. J. Turner.
Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles.
J. Membr. Biol.
102:
247-254,
1988[Medline].
20.
Manganel, M.,
and
R. J. Turner.
Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells.
J. Membr. Biol.
111:
191-198,
1989[Medline].
21.
Manganel, M.,
and
R. J. Turner.
Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells is dependent on calcium but not on protein kinase C.
J. Biol. Chem.
265:
4284-4289,
1990[Abstract/Free Full Text].
22.
Manganel, M.,
and
R. J. Turner.
Rapid secretagogue-induced activation of Na+/H+ exchange in rat parotid acinar cells. Possible interrelationship between volume regulation and stimulus-secretion coupling.
J. Biol. Chem.
266:
10182-10188,
1991[Abstract/Free Full Text].
23.
Melvin, J. E.,
A. Moran,
and
R. J. Turner.
The role of HCO
3 and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation.
J. Biol. Chem.
263:
19564-19569,
1988[Abstract/Free Full Text].
24.
Moreira, J. E.,
L. A. Tabak,
G. S. Bedi,
D. J. Culp,
and
A. R. Hand.
Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins.
J. Histochem. Cytochem.
37:
515-528,
1989[Abstract].
25.
Murphy, G. M., Jr.,
X.-C. Jia,
A. C. H. Yu,
Y. L. Lee,
J. R. Tinklenberg,
and
L. F. Eng.
Macrophage inflammatory protein 1-alpha mRNA expression in an immortalized microglial cell line and cortical astrocyte cultures.
J. Neurosci. Res.
35:
643-651,
1993[Medline].
26.
Nath, S. K.,
C. Y. Hang,
S. A. Levine,
C. H. Yun,
M. H. Montrose,
M. Donowitz,
and
C.-M. Tse.
Hyperosmolarity inhibits the Na+/H+ exchanger isoforms NHE2 and NHE3: an effect opposite to that on NHE1.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G431-G441,
1996[Abstract/Free Full Text].
27.
Noel, J.,
and
J. Pouysségur.
Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms.
Am. J. Physiol.
268 (Cell Physiol. 37):
C283-C296,
1995[Abstract/Free Full Text].
28.
Numata, M.,
K. Petrecca,
N. Lake,
and
J. Orlowski.
Identification of a mitochondrial Na+/H+ exchanger.
J. Biol. Chem.
273:
6951-6959,
1998[Abstract/Free Full Text].
29.
Orlowski, J.
Heterologous expression and functional properties of amiloride high affinity (NHE-1) and low affinity (NHE-3) isoforms of the rat Na/H exchanger.
J. Biol. Chem.
268:
16369-16377,
1993[Abstract/Free Full Text].
30.
Orlowski, J.,
and
S. Grinstein.
Na+/H+ exchangers of mammalian cells.
J. Biol. Chem.
272:
22373-22376,
1997[Free Full Text].
31.
Orlowski, J.,
R. A. Kandasamy,
and
G. E. Shull.
Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.
J. Biol. Chem.
267:
9331-9339,
1992[Abstract/Free Full Text].
32.
Park, K.,
O. Aguirre,
D. Pathmanathan,
L. Koek,
L. A. Tabak,
and
J. E. Melvin.
NHE1 is the dominant Na+/H+ exchanger in rat parotid acinar cells (Abstract).
FASEB J.
10:
A1432,
1996.
33.
Park, K.,
J. Arreola,
T. Begenisich,
and
J. E. Melvin.
Comparison of voltage-activated Cl
channels in rat parotid acinar cells with ClC-2 in a mammalian expression system.
J. Membr. Biol.
163:
87-95,
1998[Medline].
34.
Pouysségur, J.,
C. Sardet,
A. Franchi,
G. L'Allemain,
and
S. Paris.
A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.
Proc. Natl. Acad. Sci. USA
81:
4833-4837,
1984[Abstract].
35.
Robertson, M. A.,
M. Woodside,
J. K. Foskett,
J. Orlowski,
and
S. Grinstein.
Muscarinic agonists induce phosphorylation-independent activation of the NHE-1 isoform of the Na+/H+ antiporter in salivary acinar cells.
J. Biol. Chem.
272:
287-294,
1997[Abstract/Free Full Text].
36.
Sanger, F.,
S. Nicklen,
and
A. R. Coulson.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:
5463-5467,
1977[Abstract].
37.
Sardet, C.,
L. Counillon,
A. Franchi,
and
J. Pouysségur.
Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD.
Science
247:
723-726,
1990[Medline].
38.
Schultheis, P. J.,
L. L. Clarke,
P. Meneton,
M. Harline,
G. P. Boivin,
G. Stemmermann,
J. Duffy,
T. Doetschman,
M. L. Miller,
and
G. E. Shull.
Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion.
J. Clin. Invest.
101:
1243-1253,
1998[Abstract/Free Full Text].
39.
Schultheis, P. J.,
L. L. Clarke,
P. Meneton,
M. L. Miller,
M. Soleimani,
M. Harline,
T. Riddle,
J. Duffy,
T. Doetschman,
T. Wang,
G. Giebisch,
P. Aronson,
J. Lorenz,
and
G. E. Shull.
Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.
Nat. Genet.
19:
282-285,
1998[Medline].
40.
Wakabayashi, S.,
M. Shigekawa,
and
J. Pouysségur.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol. Rev.
77:
51-74,
1997[Abstract/Free Full Text].
41.
Wang, Z.,
J. Orlowski,
and
G. E. Shull.
Primary structure and functional expression of a novel gastrointestinal isoform of the rat Na/H exchanger.
J. Biol. Chem.
268:
11925-11928,
1993[Abstract/Free Full Text].
42.
Yu, F. H.,
G. E. Shull,
and
J. Orlowski.
Functional properties of the rat Na/H exchanger NHE-2 isoform expressed in Na/H exchanger-deficient Chinese hamster ovary cells.
J. Biol. Chem.
268:
25536-25541,
1993[Abstract/Free Full Text].
43.
Yun, C. H.,
C.-M. Tse,
S. K. Nath,
S. A. Levine,
S. R. Brant,
and
M. Donowitz.
Mammalian Na+/H+ exchanger gene family: structure and function studies.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G1-G11,
1995[Abstract/Free Full Text].
44.
Zhang, G. H.,
E. J. Cragoe, Jr.,
and
J. E. Melvin.
Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation.
J. Membr. Biol.
129:
311-321,
1992[Medline].
45.
Zhou, J.,
and
E. P. Hoffmann.
Pathophysiology of sodium channelopathies. Studies of sodium channel expression by quantitative multiplex fluorescence polymerase chain reaction.
J. Biol. Chem.
269:
18563-18571,
1994[Abstract/Free Full Text].
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