Expression of AE2 anion exchanger in mouse intestine
Seth L.
Alper1,2,3,
Heidi
Rossmann4,
Sabine
Wilhelm1,
Alan K.
Stuart-Tilley1,
Boris E.
Shmukler1,2, and
Ursula
Seidler4
1 Molecular Medicine and Renal
Units, Beth Israel Deaconess Medical Center; Departments of
2 Medicine and
3 Cell Biology, Harvard Medical
School, Boston, Massachusetts 02215; and
4 Department of Medicine,
Eberhard-Karls Universitat, 72076 Tuebingen, Germany
 |
ABSTRACT |
We have characterized expression of anion
exchanger 2 (AE2) mRNA and protein in the mouse intestine. AE2 mRNA
abundance was higher in colon than in more proximal segments. AE2a mRNA
was more abundant than AE2b mRNA throughout the intestine, and AE2c mRNA was expressed at very low levels. This AE2 mRNA pattern contrasted with that in mouse stomach, in which AE2c > AE2b > AE2a. AE2
polypeptide abundance as detected by immunoblot qualitatively
paralleled that of mRNA, whereas AE2 immunostaining exhibited a more
continuous decrease in intensity from colon to duodenum. AE2
polypeptide was more abundant in colonic surface cells than in crypts,
whereas ileal crypts and villi exhibited similar AE2 abundance. AE2 was also observed in mural and vascular smooth muscle. Localization of AE2
epitopes was restricted to the basolateral membranes of epithelial
cells throughout the intestine with three exceptions. Under mild
fixation conditions, anti-AE2 amino acids (aa)
109-122 detected nonpolarized immunostaining of ileal enterocytes
and of Paneth cell granule membranes. An epitope detected by anti-AE2 aa 1224-1237 was also localized to subapical regions of Brunner's gland ducts of duodenum and upper jejunum. These localization studies
will aid in the interpretation of anion exchanger function measured in epithelial sheets, isolated cells, and membrane vesicles.
enterocytes; Paneth cells; Brunner's glands; chloride-bicarbonate
exchange; immunocytochemistry
 |
INTRODUCTION |
THE MAMMALIAN INTESTINE displays axial heterogeneity of
anion transport function along three anatomic axes: the proximal-distal organ axis from duodenum to colon, the villus-crypt epithelial axis
from lumen to serosa, and the apical-basolateral cellular axis.
Cl
/HCO
3
exchange,
Cl
/OH
exchange, and/or other anion exchange functions have been reported throughout the gut along all three axes (26, 27, 30, 32, 33,
41-43). As in other cells,
Cl
/HCO
3
exchange is thought to contribute to the housekeeping function of
intracellular pH (pHi) regulation. In addition, a major
portion of intestinal Na+
reabsorption across the apical microvillar membranes is mediated via
coupled function of
Na+/H+
exchangers with
Cl
/HCO
3
and
Cl
/OH
exchangers. Whereas the former have been defined as NHE3 and NHE2 (34,
46), the molecular identities of the polypeptides that mediate the
anion exchange functions remain uncertain. Apical Cl
/HCO
3
exchange has also been proposed to contribute to cystic fibrosis
transmembrane conductance regulator-dependent cAMP-stimulated
HCO
3 secretion in duodenum (16, 36)
and elsewhere in the gut.
The localization of the anion exchanger 2 (AE2)
Cl
/HCO
3
exchanger, in particular, has been controversial. The first description
of ileal AE2 polypeptide presented immunoblot evidence for an apical
localization in rabbit (12). However, AE2 polypeptide has been
localized to basolateral plasma membranes in gastric parietal cells
(39), choroid plexus epithelium (4), and kidney tubular cells of rat
(3) and mouse (40) and in many other epithelial tissues as well.
Moreover, the properties of
Cl
/HCO
3
exchange in rabbit ileal basolateral membrane vesicles, but not in
apical membrane vesicles (26), resemble those of recombinant AE2
expressed in Xenopus oocytes (18, 19, 47) and in transfected mammalian cells (20, 23).
The AE2 gene has been found to encode at least four transcripts (AE2a,
AE2b, AE2c1, and AE2c2) generated from at least three promoters. These
transcripts encode AE2 polypeptides with three distinct
NH2-terminal amino acid (aa)
sequences, AE2a, AE2b, and AE2c (3, 40, 45). Although these sequence
differences have been proposed to regulate distinct steady-state
subcellular localizations (45), evidence is lacking for the moment.
Similarly lacking is evidence that the alternative amino termini lead
to variation in regulation of anion exchange activity in situ or in
heterologous functional expression systems.
Because genetic experiments in mouse are likely to be helpful in the
resolution of some of the above questions, we have characterized expression of AE2 mRNA and protein in the mouse intestine. The studies
presented here indicate that mouse AE2
1) is expressed in enterocytes and
enteric smooth muscle, 2) is in
greater abundance in colon than in more proximal segments of intestine,
3) appears predominately in the AE2a
isoform, and 4) that, as detected by antibodies to two epitopes, enterocyte AE2 is detected almost exclusively in the basolateral plasma membranes, with three exceptions. First, in ileal enterocytes fixed under mild conditions, the
immunostaining pattern of one AE2 epitope was not polarized. Second, a
different AE2 epitope was localized to the subapical region of duct
cells at the mouths of duodenal and jejunal glands. Third, AE2-related immunoreactivity was also present in Paneth cells.
 |
METHODS |
Materials.
All reagents with no further specification in the text were purchased
from Sigma (St. Louis, MO), Sigma-Aldrich, Fluka (Deisenhofen, Germany)
or Merck (Darmstadt, Germany) at molecular biology grade or the highest
grade available.
RT-PCR.
Mouse stomach, duodenum, ileum, jejunum, colon, and kidney were
resected, and the mucosa of the gastrointestinal organs was scraped
off. Total cellular RNA was prepared from mucosal and submucosal tissue
and kidney cortex with the use of guanidinium isothiocyanate and
phenol-chloroform (Appligene, Heidelberg, Germany) extraction as
described (11). RNA integrity was confirmed in all preparations by
ethidium bromide staining and visualization of rRNA separated on
glyoxal agarose gels.
First-strand cDNA was synthesized by Super Script II RT (GIBCO BRL,
Eggenstein, Germany) from 4 µg of total RNA with 25 ng of
oligo(dT)12-18 primer and 200 units Super Script II per reaction. Samples (5 µl) of the RT reaction
were used in a 100-µl PCR reaction. Primer sequences and reaction
conditions for amplification of murine AE2a (1022-bp amplimer)
and AE2b mRNAs (974-bp amplimer) were as described (40). The
forward primer for amplification of AE2c was
5'-GTGTCTCTGAGGGGCAAAGCA-3', and the reverse
primer for AE2c amplification was
5'-GAGAGGCTCAGTGACATGAGG-3', yielding amplimers of
1161 bp (AE2c2) and 873 bp (AE2c1). An 18S rRNA fragment was amplified
as an internal control with the use of primers supplied by Ambion
(Austin, TX). Negative controls performed for each primer pair included
RT-PCR amplifications separately omitting RT reagents, substrate RNA,
or reverse transcriptase. Amplified cDNA fragments were transferred to
a nylon membrane and hybridized to a
32P-labeled internal
oligonucleotide from AE2 exon 6, common to all known mouse AE2
transcripts (40).
For semiquantitative analysis, 12.5 µl each of AE2 and rRNA
amplification reactions were combined and coelectrophoresed on 1.8%
agarose gels. The ethidium bromide-stained RT-PCR products were
digitally imaged with the ImageMaster VDS system (Pharmacia, Freiburg,
Germany), and gel band optical densities (OD) were measured with
ImageMaster software. OD values of amplification products were
corrected for relative length, and PCR product amplification efficiency
was determined from the slope of log(OD) plotted against amplification
cycle number (10, 31, 35). AE2 mRNA levels were expressed as values
normalized to levels of 18S rRNA. AE2 cDNAs and 18S rRNA were not
coamplified in one-tube reactions to avoid the common problem of
amplification interference. AE2 internal deletion construct standards
were not used for same-tube competitive PCR to avoid heteroduplex
formation between standard and target (31, 35). All experiments were
performed at least three times.
Relative quantitation of AE2 polypeptide in intestinal tissues.
Clarified NP-40 lysates of mouse duodenum, jejunum, ileum, and colon
(<3 mg protein) were subjected to immunoprecipitation with 2-µl
ascites containing monoclonal antibody to the COOH-terminal 12 residues
of mouse AE2 (48), enough to precipitate the maximal precipitable
amount of AE2 from NP-40 lysates of mouse stomach (3-5 mg protein,
not shown).
Immunoprecipitates obtained from known original amounts of whole tissue
detergent lysate were subjected to SDS-PAGE in adjacent gel lanes,
transferred to nitrocellulose, and probed with polyclonal antibody to
the AE2 COOH-terminus as described (4, 39), with the use of
peroxidase-coupled goat anti-rabbit Ig as secondary antibody (Jackson
ImmunoResearch, West Grove, PA) and enhanced chemiluminescence (ECL)
detection (Amersham, Boston, MA) on Kodak SB-5 film at a series of
exposure times determined empirically to maximize signal at
subsaturation values. Digitally scanned images (Agfa Duoscan,
Wilmington, MA) of immunoblots were saved in TIFF format (Photoshop
4.0, Adobe, Mountainview, CA). Pixel intensities of AE2-specific bands
were measured with the use of NIH Image 1.60, and film background
intensity was subtracted.
Plots of ln(pixel intensity) vs. ln(µg loaded protein) were found
empirically to generate a series of roughly parallel lines. These data
were analyzed as a series of parallel line assays by multiple linear
regression (13). Estimates and standard error values were obtained for
the intercepts of the four lines (one for each tissue) and their common
slope using JMP-IN statistical software (SAS Institute, Cary, NC). In
such an assay, the fold difference (F) between two samples is given by
where
b01 is the first
x-intercept,
b02 is the second
x-intercept, and
b1 is the common slope.
The standard error of F is calculated from the variances and
correlations of the parameter estimates.
Tissue preparation.
Adult male CD1 mice were maintained on a standard diet with free access
to water. Animals anesthetized with diethyl ether or methoxyflurane
(MetoFane, Pittman-Moore) underwent cardiac perfusion for 2 min with
140 mM NaCl-20 mM sodium phosphate, pH 7.4 (PBS). Some mice were then
perfusion-fixed with 2% paraformaldehyde-75 mM lysine-10 mM sodium
periodate (PLP) as previously described (3, 4, 38-40). Other mice
were perfusion-fixed with 2 or 3% paraformaldehyde alone. Perfused
tissues were excised, cut into smaller lumen-exposed segments, further
incubated in the same fixation media (overnight for PLP-fixed tissue
and between 2 and 20 h for paraformaldehyde-fixed tissue), then stored
until further use at 4°C in PBS containing 0.02% sodium azide.
Immunocytochemistry.
Fixed tissue blocks were infiltrated with 30% sucrose in PBS-azide,
frozen in liquid nitrogen, and sectioned at 5- to 7-µm thickness on a
Reichert Frigocut cryostat. Tissue sections were placed on
Superfrost/Plus microscope slides (Fisher) and stored in PBS-azide at
4°C until use.
Affinity-purified rabbit polyclonal antibodies directed against mouse
AE2 aa 1224-1237, aa 102-122, and mouse AE1 aa 917-929 were previously described (3, 40). Secondary antibodies, Cy3-coupled
donkey anti-rabbit Ig, fluorescein-coupled goat anti-rabbit Ig, and
fluorescein-coupled goat anti-mouse Ig were from Jackson ImmunoResearch. Fixed sections to be immunostained with anti-AE2 aa
1224-1237 were pretreated with 1% SDS for 5 min, then washed three times in PBS (8). All sections were preincubated at room temperature in PBS for 10 min, blocked in 1% BSA in PBS for 15 min,
and then incubated at room temperature for 1-2 h with primary antibody. Sections were washed 3 × 5 min in PBS. The sections were then incubated for 1 h with fluorophore-conjugated secondary antibodies (at concentrations of 10-15 µg/ml), followed by three additional 5-min washes in PBS. Sections were mounted in 50% glycerol in 0.2 M Tris · HCl, pH 8.0, containing 2.5%
n-propyl gallate as an antiquenching
agent. Sections were examined and photographed with an Olympus BH-2
photomicroscope equipped for epifluorescence and were photographed with
Kodak TMAX 400 film push-processed to 1600 ASA.
All photomicrographs within a figure presenting results with the same
antibody are from a single antibody incubation session with uniform
reagents and parameters for development and printing, allowing
qualitative comparison of staining intensities within the group, except
in Fig. 8. The figures shown are representative of similar results
obtained in immunostaining experiments of perfusion-fixed intestinal
tissue obtained from at least two male CD1 mice.
 |
RESULTS |
AE2 mRNA expression in mouse intestine.
The AE2 gene is transcribed from at least three promoters, generating
AE2a, AE2b, and AE2c transcripts (45). RT-PCR analysis of mouse colon
RNA (Fig. 1) revealed the presence of AE2a
(lane 1) and AE2b transcripts
(lane 3). Both AE2c1 (RT-PCR band of
873 bp) and AE2c2 transcripts (band of 1161 bp) were present in mouse colon at very low levels (lane 5),
in contrast to mouse kidney, which expressed only AE2c2 (40), and mouse
stomach (lane 6), in which AE2c1
greatly exceeded AE2c2 (40). Figure 2 shows
examples of semiquantitative RT-PCR analysis of AE2a, AE2b, and AE2c1
transcripts from mouse gastric mucosa RNA. Similar analysis comparing
RNA from gastric mucosa with RNA from mouse intestinal tissues is presented in Fig. 3.

View larger version (81K):
[in this window]
[in a new window]
|
Fig. 1.
RT-PCR analysis of anion exchanger 2 (AE2) mRNA variants expressed in
mouse colonic mucosa. A: ethidium
bromide (EtBr)-stained AE2 amplification products (35 cycles) from
mouse colonic mucosa RNA (lanes 1,
3, and
5) and from mouse gastric mucosa RNA
(lanes 2,
4, and
6). Lane
5 is loaded with 8 reaction volume equivalents compared
with lanes 1-4 and
6. Amplimers of 18S rRNA
(lane 7, colon, and
lane 8, stomach) underwent 24-cycle
amplification. B: cDNAs in
A gel were transferred to nylon and
hybridized with a 32P-labeled
internal AE2 oligonucleotide probe encoding a sequence present in all
AE2 variants.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Semiquantitative RT-PCR analysis of AE2 mRNA variants in mouse gastric
mucosa. Change in fluorescence intensity of EtBr-stained amplimers of
AE2a (A), AE2b
(B), and AE2c1
(C) as a function of amplification
cycle number, compared in each case with that of 18S
rRNA. ODI, integrated optical density units.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison of relative expression levels of AE2a
(A), AE2b
(B), and AE2c1
(C) mRNA in mouse gastric and
intestinal tissues, normalized to expression of 18S rRNA. Each bar
represents the mean ± SE of at least 3 experiments.
|
|
In each intestinal tissue examined, AE2a and AE2b mRNA levels
normalized to 18S rRNA differed one from the other by no more than two-
to threefold. AE2 mRNA was only slightly less abundant in colonic
submucosa than in scraped mucosa. The rank order of normalized total
AE2 mRNA abundance in intestine was colon > ileum
duodenum
jejunum. Gastric mucosa was richer in AE2 than were intestinal tissues,
with the exception of AE2a, present in colon at two- to threefold
higher abundance than in stomach (Fig.
3A).
In mouse gastric mucosa, AE2c1 mRNA was present in abundance equal to
or greater than the combined abundance of AE2a and AE2b mRNAs. In
contrast, other intestinal tissues expressed very low or undetectable
levels of AE2c1 mRNA.
AE2 polypeptide expression in mouse intestine.
Anti-AE2 aa 1224-1237 displayed specificity as an AE2 immunoblot
reagent (Fig.
4A) for
both whole colon lysate (lanes 1 and 2) and for monoclonal anti-AE2
immunoprecipitate from the same lysate (lanes
3 and 4). Although
the antibody used in these conditions did not precipitate all
solubilized AE2 (lane 3 vs.
lane 7), ECL signal from every
tissue was nonetheless proportional to the input protein in the
immunoprecipitation (Fig. 4A,
bottom). NP-40 extraction of mouse
intestinal tissues, with or without the further incubation associated
with the immunoprecipitation procedure, led to SDS-resistant oligomerization of AE2. This finding suggests that the oligomeric state
of intestinal AE2 resembles that of AE2 in pig gastric mucosa (5). With
AE2 abundance in ileum valued at 1.0, AE2 relative abundance in colon
(2.03 ± 0.11) was significantly higher (ANOVA with Tukey's
all-pairs comparison) than abundances in jejunum (0.95 ± 0.11), in
duodenum (0.91 ± 0.11; n = 3 for
each), and in cecum (1.13; n = 1)
(Fig. 4B). AE2a and AE2b polypeptide
relative abundance in stomach was >7.0 (not shown). Anti-AE2 aa
109-122 was not usable as an immunoblotting reagent (3,
40).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
Immunoblot analysis of AE2 polypeptide abundance in mouse intestine.
A, top: specificity of AE2 detection
by immunoblot with polyclonal anti-AE2 amino acid (aa) 1224-1237
in presence of irrelevant peptide (lanes
1, 3, and
5-7) or of peptide antigen
(lanes 2 and
4). Lanes
1 and 2 were loaded
with NP-40 lysate of whole colon (100 µg protein),
lanes 3 and
4 with immunoprecipitate pellet
obtained from 250 µg colon lysate protein with use of monoclonal
anti-AE2 aa 1224-1237, and lane 7 with residual supernatant (60 µg). Lane
5 shows the precipitation pellet (from 250 µg
protein) with use of nonimmune IgG and lane
6 with the residual supernatant (60 µg).
A, bottom: polyclonal immunoblot of
monoclonal AE2 immunoprecipitates from indicated amounts of NP-40
lysate (µg protein) from colon, ileum, jejunum, and duodenum; ** and
* indicate AE2 dimer and monomer, respectively.
B: ln/ln plot of pixel intensity
(optical density, OD) of AE2 dimer vs. input protein (µg) for
immunoprecipitation reaction, as portrayed in A,
bottom. Immunoprecipitations were performed with
detergent extract from membrane fractions of colon ( ), ileum ( ),
jejunum ( ), and duodenum ( ). C:
abundance of AE2 polypeptide in intestinal segments, normalized to AE2
abundance in ileum. Except for cecum
(n = 1), data are depicted with mean
values, 75%, and 95% confidence limits
(n = 3).
|
|
Immunolocalization of AE2 polypeptide in intestine.
Figure 5 confirms the specificity of
anti-AE2 aa 1224-1237 as an immunocytochemical reagent in semithin
cryosections of mouse colon. This antibody, after preincubation with
the irrelevant peptide AE2 aa 109-122, detected not only AE2,
evident in the mucosal enterocytes, but also crossreacted with AE1 in
the trapped red blood cells (Fig. 5a), both of which
signals are abolished in the presence of excess peptide antigen (Fig.
5b). However, whereas antibody preincubated with the
crossreactive peptide AE1 917-929 completely lost reactivity with
red blood cells, substantial immunoreactivity was retained with colonic
mucosa (Fig. 5, c and d). Because this AE1
COOH-terminal peptide used in the preincubation also partially depletes
immunoreactivity of this antibody tested against recombinant AE2, and
because multiple red blood cell-reactive anti-AE1 antibodies did not
immunostain colon (not shown), this reduced intensity signal is
consistent with the presence of AE2 in colonic mucosa of mouse, as is
also likely the case in rat (30).

View larger version (83K):
[in this window]
[in a new window]
|
Fig. 5.
Specificity of polyclonal anti-AE2 aa 1224-1237 as
immunocytochemical reagent for colon. Anti-AE2 aa 1224-1237
previously depleted with agarose beads carrying either AE2 peptide aa
109-122 (a and b) or with AE1
COOH-terminal peptide aa 917-929 (c and d)
were incubated with semithin sections of overnight 2%
paraformaldehyde-75 mM lysine-10 mM sodium periodate (PLP)-fixed mouse
colon in the presence of irrelevant peptide (a and
c) or peptide antigen (b and d).
rbc, Red blood cells. Bar = 50 µm.
|
|
The basolateral plasma membrane localization of AE2 suggested in Fig. 5
was evident in all segments of mouse colon (Fig.
6) with anti-AE2 aa 1224-1237
(right panels) and with anti-AE2 aa 109-122 (left
panels). No apical staining was evident. In all colonic segments, surface enterocyte staining was much more intense than crypt staining. Anti-AE2 aa 109-122 also detected AE2 in muscularis mucosae and in the thicker smooth muscle layers, whereas conditions were not found with which anti-AE2 aa 1224-1237
recognized smooth muscle AE2. Anti-AE2 aa 109-122 immunostaining
also coincided with that of anti-AE2 aa 1224-1237 in gastric
parietal cells and in choroid plexus epithelial cells, which express
the highest levels of AE2 (not shown).

View larger version (85K):
[in this window]
[in a new window]
|
Fig. 6.
Colocalization of 2 AE2 epitopes in mouse colon enterocytes. Anti-AE2
aa 109-122 (a,
c, e,
and g) or anti-AE2 aa 1224-1237
(b,
d, f,
and h) were incubated with
cryosections of tissue fixed 2 h in 3% paraformaldehyde, in the
presence of peptide antigen (c and
d) or irrelevant peptide
[a; b; c, inset (magnified from
a);
d, inset (magnified from
b);
e-h]. Distal colon
(a-d),
proximal colon (e and
f), cecum (g
and h) are shown. ap, Apical
membrane; bl, basolateral membrane. Bar = 50 µm.
|
|
Figure 7 illustrates that the AE2
immunostaining intensity decreased gradually from highest levels in
colon (a), through medium levels in
ileum (b), to lowest levels in
duodenum (c). Figure 7b shows that AE2 was present in both
surface and crypt cells of ileum, in contrast to the low staining
intensity in colonic crypt cells
(a). AE2 in duodenum was also
present both in columnar cells of the villi and the cuboidal cells of
Brunner's gland-like structures. In all intestinal segments, AE2
immunostaining of enterocytes was restricted to a basolateral
distribution, with three exceptions to be described below. Whereas
anti-AE2 aa 1224-1237 did not detect AE2 in colonic muscularis,
immunostaining was evident in ileal smooth muscle (Fig.
7b, bottom
left).

View larger version (98K):
[in this window]
[in a new window]
|
Fig. 7.
Comparison of AE2 expression in colon, ileum, and upper jejunum.
Cryosections of colon (a), ileum
(b), and duodenum
(c) were immunostained with anti-AE2
aa 1224-1237. s, Luminal surface; cr, crypt(s); v, villus; gl,
glands. Bar = 50 µm.
|
|
Exceptions to basolateral immunolocalization of intestinal AE2
polypeptide.
The first two exceptions to basolateral localization of AE2, presented
in Fig. 8, are restricted to the epitope
detected by anti-AE2 aa 109-122. Figure 8, a, c,
e, and g,
show prominent staining of ileal Paneth cells in PLP-fixed tissue,
whether Epon-embedded (Fig. 8, a and
b) or frozen (Fig. 8,
c and
d). The AE2-related epitope was
present in the plasma membrane but more abundantly in the large
secretory granules. In ileum fixed 2 h with 2% paraformaldehyde, the
Paneth cell staining was rather faint (Fig.
8e), but, after 24-h fixation, it
was prominent in the granule content (Fig.
8g). Paneth cell immunostaining in
all these preparations was competed completely by peptide antigen (Fig.
8, b, d, f, and
h; Fig.
9, e and
f).

View larger version (82K):
[in this window]
[in a new window]
|
Fig. 8.
Fixation condition dependence of AE2 epitope aa 109-122 in ileum.
Etched Epon sections (1 µm, a and
b) or 5-µm cryosections
(c-h) from jejunum were fixed overnight
(a-d) or fixed for 2 h (e and
f) or 24 h (g and
h) in 2% paraformaldehyde, in
presence of irrelevant peptide (a, c,
e, and g) or peptide
antigen (b, d, f, and
h);
e and
f are underexposed compared with other
panels. sm, Smooth muscle. Bar = 50 µm.
|
|

View larger version (90K):
[in this window]
[in a new window]
|
Fig. 9.
AE2 epitope in Paneth cells. Cryosections of two jejunal crypts are
shown, one viewed in phase (a) and
the second stained with Dolichos biflorus agglutinin
(b). Sequential sections of each crypt are immunostained
with anti-AE2 aa 109-122 in presence of irrelevant peptide
(c and
d) or of peptide antigen
(e and
f). Bar = 50 µm.
|
|
Additional examination of Paneth cell immunostaining (Fig. 9) confirmed
that the AE2-related epitope was present most prominently in granule
content (Fig. 9, c and
d) but is also likely in granule membrane as defined by Dolicho's biflorus agglutinin lectin
staining (b). Similarly,
immunospecific Paneth cell staining with the same antibody was evident
in jejunum (Fig. 10,
e and
f).

View larger version (92K):
[in this window]
[in a new window]
|
Fig. 10.
AE2 epitopes in duodenum and upper jejunum. PLP-fixed cryosections of
duodenum (sequential sections a and
b) and upper jejunum crypt
(c,
e, and
f) or villus
(d) were immunostained with anti-AE2
aa 1224-1237
(a-d)
or with anti-AE2 aa 109-122 (sequential sections
e and
f) in presence of irrelevant peptide
(a,
c, d,
and e) or peptide antigen
(b and
f). Triple arrow in
c demarcates basal surfaces of crypt
or gland neck epithelial cells. Bar = 50 µm.
|
|
Whereas in PLP-fixed ileum, anti-AE2 aa 109-122 detected little or
no immunoreactivity in surface enterocytes, tissue fixed for 2 h in 2%
paraformaldehyde revealed AE2 immunostaining not only in basolateral
membranes of villar enterocytes but equally prominently in the apical
microvillar membrane (Fig. 8e).
Staining at each site was fully competed by excess peptide antigen
(Fig. 8f). Interestingly,
immunostaining with this antibody in both basolateral and apical
membranes was destroyed by 24-h fixation in paraformaldehyde (Fig.
8g).
Brunner's gland-like structures of the duodenum showed two types of
AE2 immunostaining. AE2 was basolateral in the branching convolutions
of the glands (Fig. 10a). As
reported by some others (6), we found these Brunner's gland-like
structures not to immunostain positive for epidermal growth factor.
However, the gland necks that empty into the duodenal crypts revealed
punctate staining in the apical pole of the cell (Fig.
10a, top
center). Both the basolateral membrane and the apical
pole staining patterns were competed by excess peptide antigen (Fig.
10b). Similar staining at or near
the apical pole was evident in deep gland necks of the upper jejunum
(Fig. 10c), in which more
superficial villar enterocytes were stained only in their basolateral
membranes (Fig. 10d).
 |
DISCUSSION |
We have examined expression of AE2 in mouse intestine to provide a
context in which to interpret studies of intestinal physiology in
wild-type and mutant mouse strains. AE2 gene products were analyzed by
RT-PCR, immunoblot, immunoprecipitation, and immunocytochemistry. Total
AE2 mRNA was most abundant in colon and was of intermediate abundance
in ileum and duodenum. In these regions, AE2a mRNA was approximately
two- to threefold more abundant than AE2b with respect to rRNA in
colon, ileum, and duodenum. Total AE2 mRNA was least abundant in
jejunum, in which AE2a and AE2b were expressed at equal levels (Fig.
3).
AE2 polypeptide detected by semiquantitative immunoblot (AE2a + AE2b)
migrated as an SDS-resistant dimer (5) and was approximately twofold
more abundant in colon than in cecum, ileum, jejunum, or duodenum (Fig.
4). Because the AE2c mRNAs encode a polypeptide ~20 kDa shorter than
those encoded by AE2a and AE2b mRNAs, the finding that the predominant
gastric mucosal AE2 mRNA transcript is AE2c1 (Fig. 3) suggests that the
reproducible observation of 165- and 145-kDa AE2 polypeptides in
stomach of multiple species (39, 48, 49) reflects the translation of
multiple AE2 transcripts more than it does proteolytic processing or degradation.
Immunolocalization studies with antibodies to two AE2 epitopes detected
AE2 exclusively in basolateral membranes of colonic surface
enterocytes, with low levels in colonic crypt enterocytes. In contrast,
AE2 immunostaining intensity was comparable in villus and crypt
enterocytes of ileum, jejunum, and duodenum. AE2 abundance in surface
enterocytes declined from colon to duodenum. This basolateral localization conformed to the patterns previously detected for AE2 in
other epithelial tissues (3, 4, 8, 14, 15, 29, 38-40). Basolateral
AE2 likely contributes to pHi regulation and to cell volume
regulation during transepithelial reabsorption of NaCl, and potentially
contributes in ileal crypts to
Cl
secretion by basolateral
Cl
loading.
AE2 or a protein carrying a closely related epitope was also detected
in four locations previously undescribed or unsubstantiated. The first
was in the mural smooth muscle of the gut, in both muscularis mucosae
and in thicker outer layers. AE3, previously observed in vascular
smooth muscle (7), is also present in intestinal smooth muscle (not
shown). Although
Cl
/HCO
3
exchange is likely to contribute to regulation of smooth myocyte
pHi and excitability, regulation
of pHi in intestinal smooth muscle
has been little studied.
The second novel location was in surface and internal membranes of
Paneth cells in duodenum, jejunum, and ileum, detected by antibody to
only one AE2 epitope, aa 109-122. Paneth cells secrete antibiotic
secretagogue cryptdins, as well as other protein components of the
innate immune system (28). Lectin costaining suggested that some of the
AE2 epitope resided in granule membranes but could not rule out its
presence in granule matrix. AE2 or a related protein in Paneth cells
may facilitate protein exocytosis at the granular membrane or cell
surface. However, despite the operational immunospecificity of Paneth
cell AE2-related immunostaining, interpretation of the Paneth cell AE2
epitope requires caution. This is especially true in view of inability
to stain Paneth cells with anti-AE2 aa 1224-1237 and the
reputation of Paneth cells to stain nonspecifically with a wide range
of in situ and immune reagents.
In lightly fixed tissue, anti-AE2 aa 109-122 detected
immunostaining of both basolateral and apical membranes of ileal
enterocytes in both villi and crypts. In both membranes the epitope
appeared equally susceptible to paraformaldehyde concentration,
exposure time, and postfixation storage. However, anti-AE2 aa
1224-1237 detected no apical AE2 immunostaining in mouse ileum,
consistent with immunoblot analysis of rabbit ileum (32) and duodenum
(33), in which AE2 detected by antibody to aa 1224-1237 was
present only in basolateral membrane vesicles and not in apical vesicles.
Two previous studies have presented evidence of AE2 in apical membranes
of polarized epithelial cells. In the first, guinea pig polyclonal
antibody raised against a glutathione
S-transferase fusion protein
containing AE2 aa 396-499 detected a 160-kDa band in immunoblots
of rabbit ileal brush-border membrane vesicles but not basolateral
membrane vesicles (12). Neither specificity tests with recombinant AE3
nor use in immunocytochemical localization was reported. The AE2 region
used as antigen consists of two portions: the COOH-terminal aa
433-499 share only 16% identity with rabbit AE3 (AF031650) but
the internal aa 396-432 share 69% identity and 86% similarity
with rabbit AE3. Transcripts and/or polypeptides encoding alternative
AE3 transcripts are expressed throughout gut mucosae of rat (22), human
(42), and mouse (unpublished observations).
In the second instance, a mouse monoclonal IgM raised against the
species-specific human AE2 peptide sequence aa 882-895 (25), located in the largest exofacial loop between the fifth and sixth putative transmembrane spans and containing an
N-glycosylation site at its
NH2-terminal end (48),
immunostained apical canalicular membranes of hepatocytes and small
bile ducts in human liver (24) and in both apical and basolateral
membranes of human parotid gland interlobular ducts (44). Gastric
parietal cells, choroid plexus (24), and salivary gland striated ducts
(44) all exhibited basolateral immunostaining patterns with this antibody.
Unlike the apical AE2 epitope detectable only by anti-AE2 aa
109-122, the subapical/apical AE2 epitope detected in the necks of
Brunner's gland-like structures in the duodunum and upper jejunum was
detected only by anti-AE2 1224-1237. Brunner's glands have been
thought to secrete HCO
3, although
their abundance is not correlated with secretory rate (1). A
subapical/apical pole AE2 or AE2-related polypeptide could mediate
HCO
3 secretion, perhaps enhancing
glandular secretion of lysozyme, defensins, and mucins. In contrast,
the same antibody detected only basolateral AE2 immunostaining in the
deep, branching portion of these glands, where it presumably
contributes to Cl
secretion.
This is the first subapical/apical pole distribution described for an
epitope detected by anti-AE2 1224-1237. This epitope (as well as
that detected only by anti-AE2 aa 109-122 in ileal apical
membrane) may represent a novel AE2 or AE1 isoform, a product of a
novel AE-related gene, or cell type-specific sorting or retention of
either AE2a or AE2b. Study with additional antibodies to colinear AE2
epitopes will be required to resolve these possibilities.
Although some form of epitope-shielded AE2 polypeptide may plausibly
contribute to apical
Cl
/HCO
3
exchange in enterocytes, rabbit ileal brush-border
Cl
/Cl
exchange displays the lack of osmotic stimulation and shallow pH
dependence (26) characteristic of recombinant AE3 expressed in
Xenopus oocytes (unpublished
observations). This pattern differs from the steep pH dependence and
hyperosmotic activation of
Cl
/Cl
exchange in gastric parietal cell basolateral vesicles (26) rich in AE2
polypeptide (5, 48, 49) and in Xenopus
oocytes expressing AE2 (18, 19, 47).
A major portion of apical NaCl reabsorption in ileum and proximal colon
is mediated by or requires the downregulated in adenoma (DRA) gene
product (17, 37). Homozygosity for mutations in this protein is
associated with congenital Cl
-losing diarrhea. Although
shown thus far only to mediate sulfate flux across the plasma membrane
(9, 37), DRA likely mediates or potentiates
Cl
/HCO
3
or
Cl
/OH
exchange. Homology of DRA with other putative sulfate transporters also
suggests the possibility that one or more of those may also mediate or
potentiate apical
Cl
/HCO
3
exchange in some portions of gut. However, the DRA-related transporter,
Sat-1, prefers sulfate to Cl
for exchange with
bicarbonate or oxalate and is basolaterally, not apically, situated in
renal proximal tubular epithelial cells (21).
Further understanding of the contribution of
Cl
/anion exchange to
intestinal HCO
3 secretion and NaCl
reabsorption will require identification and localization of additional
AE anion exchangers, clarification of the function of putative anion exchangers from different gene families, and application of that knowledge to appropriate mouse knockout models. Development of pharmacological inhibitors of anion exchange more selective than those
currently available (2) would accelerate the pace of progress.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
DK-43495 (S. L. Alper), HL-09853 (B. E. Shmukler), and DK-34854 (The
Harvard Digestive Diseases Center), by Deutsche Forschungesgemeinschaft Grants Se 460/2-5 and 9-1 (U. Seidler), by the
Fortune-Programm of Eberhard-Karls University Nr. 219 (H. Rossmann),
and by the Duisberg Foundation (S. Wilhelm).
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. L. Alper,
Molecular Medicine Unit RW763, Beth Israel Deaconess Med. Ctr., 330 Brookline Ave., Boston, MA 02215 (E-mail: salper{at}caregroup.harvard.edu).
Received 12 January 1999; accepted in final form 23 April 1999.
 |
REFERENCES |
1.
Ainsworth, M. A.,
M. A. Koss,
D. L. Hogan,
and
J. I. Isenberg.
Higher proximal duodenal mucosal bicarbonate secretion is independent of Brunner's glands in rats and rabbits.
Gastroenterology
109:
1160-1166,
1995[Medline].
2.
Alper, S. L.,
M. N. Chernova,
J. Williams,
M. Zasloff,
F.-Y. Law,
and
P. A. Knauf.
Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic, squalamine.
Biochem. Cell Biol.
76:
799-806,
1998[Medline].
3.
Alper, S. L.,
A. K. Stuart-Tilley,
D. Biemesderfer,
B. Shmukler,
and
D. Brown.
Immunolocalization of AE2 anion exchanger in rat kidney.
Am. J. Physiol.
273 (Renal Physiol. 42):
F601-F614,
1997[Abstract/Free Full Text].
4.
Alper, S. L.,
A. Stuart-Tilley,
C. F. Simmons,
D. Brown,
and
D. Drenckhahn.
The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+,K+-ATPase rather than with basolateral anion exchanger AE2.
J. Clin. Invest.
93:
1430-1438,
1994[Medline].
5.
Alper, S. L., and A. S. Zolotarev.
Chemical crosslinking demonstrates homooligomeric interaction of
AE2 anion exchanger polypeptide in pig gastric membranes.
Biochemistry. In press.
6.
Beerstecher, J. J.,
C. Huiskens-van der Meij,
and
S. O. Warnaar.
An immunohistochemical study performed with monoclonal and polyclonal antibodies to mouse epidermal growth factor.
J. Histochem. Cytochem.
36:
1153-1160,
1988[Abstract].
7.
Brosius, F. C.,
R. L. Pisoni,
P. H. Kim,
X. Cao,
G. Deshmukh,
A. K. Stuart-Tilley,
C. Haller,
and
S. L. Alper.
Anion transporter mRNA and protein expression in rat vascular smooth muscle cells, aorta, and renal microvessels.
Am. J. Physiol.
273 (Renal Physiol. 42):
F1039-F1047,
1997[Abstract/Free Full Text].
8.
Brown, D.,
J. Lydon,
M. McLaughlin,
A. Stuart-Tilley,
R. Tyszkowski,
and
S. L. Alper.
Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate.
Histochem. Cell Biol.
105:
261-267,
1996[Medline].
9.
Byeon, M. K.,
A. Frankel,
T. S. Papas,
K. W. Henderson,
and
C. W. Schweinfest.
Human DRA functions as a sulfate transporter in Sf9 insect cells.
Protein Expr. Purif.
12:
67-77,
1998[Medline].
10.
Chelly, J.,
J. C. Kaplan,
P. Maire,
S. Gautron,
and
A. Kahn.
Transcription of the dystrophin gene in human muscle and non-muscle tissue.
Nature
333:
858-860,
1988[Medline].
11.
Chomczynski, P,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
12.
Chow, A.,
J. W. Dobbins,
P. S. Aronson,
and
P. Igarashi.
cDNA cloning and localization of a band 3-related protein from ileum.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G345-G352,
1992[Abstract/Free Full Text].
13.
Colquhoun, D.
Lectures on Biostatistics. Oxford, UK: Clarendon, 1971, chapt. 13, p. 279-343.
14.
Grayck, E. N.,
C. A. Piantodosi,
J. van Adelsberg,
S. L. Alper,
and
Y.-C. T. Huang.
Protection of perfused lung from oxidant injury by inhibitors of anion exchange.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L296-L304,
1997[Abstract/Free Full Text].
15.
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].
16.
Hogan, D. L.,
D. L. Crombie,
J. I. Isenberg,
P. Svendsen,
O. B. Schaffalitzky de Muckadell,
and
M. A. Ainsworth.
CFTR mediates cAMP- and Ca2+-activated duodenal epithelial HCO
3 secretion.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G872-G878,
1997[Abstract/Free Full Text].
17.
Hoglund, P.,
S. Haila,
J. T. Socha,
L. Tomaszewski,
U. Saarialho-Kere,
M.-L. Karjalainen-Lindsberg,
K. Airola,
C. Holmberg,
A. de la Chapelle,
and
J. Kere.
Mutations of the down-regulated in adenoma (DRA) gene cause congenital diarrhea.
Nat. Genet.
14:
316-319,
1996[Medline].
18.
Humphreys, B. D.,
L. Jiang,
M. Chernova,
and
S. L. Alper.
Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1295-C1307,
1994.
19.
Humphreys, B. D.,
L. Jiang,
M. Chernova,
and
S. L. Alper.
Activation of murine AE2 anion exchanger in Xenopus oocytes by increased pHi secondary to hypertonic activation of Na+/H+ exchange.
Am. J. Physiol.
268 (Cell Physiol. 37):
C201-C209,
1995[Abstract/Free Full Text].
20.
Jiang, L.,
A. K. Stuart-Tilley,
J. Parkash,
and
S. L. Alper.
AE2-mediated Cl
/HCO
3 exchange in CHOP cells of defined, transient transfection status is regulated by pHi and serum.
Am. J. Physiol.
266 (Cell Physiol. 35):
C845-C856,
1994.
21.
Karniski, L. P.,
M. Lotscher,
M. Fucentese,
H. Hilfiker,
J. Biber,
and
H. Murer.
Immunolocalization of sat-1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney.
Am. J. Physiol.
275 (Renal Physiol. 44):
F79-F87,
1998[Abstract/Free Full Text].
22.
Kudrycki, K. E.,
P. R. Newman,
and
G. E. Shull.
cDNA cloning and tissue distribution of mRNAs for two proteins that are related to the band 3 Cl
/HCO
3 exchanger.
J. Biol. Chem.
265:
462-471,
1990[Abstract/Free Full Text].
23.
Lee, B. S.,
R. B. Gunn,
and
R. R. Kopito.
Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line.
J. Biol. Chem.
266:
11448-11454,
1991[Abstract/Free Full Text].
24.
Martinez-Anso, E.,
J. E. Castillo,
J. Diez,
J. F. Medina,
and
J. Prieto.
Immunohistochemical detection of chloride/bicarbonate exchangers in human liver.
Hepatology
19:
1400-1406,
1994[Medline].
25.
Medina, J. F.,
A. Acin,
and
J. F. Prieto.
Molecular cloning and characterization of the human AE2 anion exchanger (SLC4A2) gene.
Genomics
39:
74-85,
1997[Medline].
26.
Nader, M.,
G. Lamprecht,
M. Classen,
and
B. Seidler.
Different regulation by pHi and osmolarity of the rabbit ileum brush border and parietal cell anion exchanger.
J. Physiol. (Lond.)
481:
605-615,
1994[Abstract].
27.
Orsenigo, M. N.,
M. Tosco,
and
A. Faelli.
Cl/HCO3 exchange in the basolateral membrane domain of rat jejunal enterocytes.
J. Membr. Biol.
124:
13-19,
1991[Medline].
28.
Ouellette, A. J.
Paneth cells and innate immunity in the crypt microenvironment.
Gastroenterology
113:
1779-1784,
1997[Medline].
29.
Perrone, R. D.,
S. A. Grubman,
S. L. Murray,
D. S. Lee,
E. Moy,
S. L. Alper,
and
D. M. Jefferson.
Decreased function of anion exchanger in continuous epithelial cell lines from ADPKD liver cysts.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1748-C1756,
1997[Abstract/Free Full Text].
30.
Rajendran, V.,
P. Sangan,
M. Mann,
M. Kashgarian,
S. Alper,
J. Black,
and
H. Binder.
Tissue-specific expression of basolateral anion exchange (AE2) isoform in rat colon (Abstract).
Gastroenterology
114:
A408,
1998.
31.
Rossmann, H.,
O. Bachmann,
D. Vieillard-Baron,
M. Gregor,
and
U. Seidler.
Na+-HCO
3 cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal cells and mucous cells.
Gastroenterology
116:
1389-1398,
1999[Medline].
32.
Rossmann, H.,
M. Nader,
M. Classen,
S. Alper,
M. Gregor,
and
U. Seidler.
Evidence for the existence of two different anion exchangers in rabbit duodenum (Abstract).
Gastroenterology
110:
A356,
1996.
33.
Rossmann, H.,
M. Nader,
U. Seidler,
M. Classen,
and
S. Alper.
Basolateral membrane localization of the AE2 isoform of the anion exchanger family in both stomach and ileum (Abstract).
Gastroenterology
108:
A319,
1995.
34.
Schultheis, P. J.,
L. L. Clarke,
P. Meneton,
M. L. Miller,
M. Soleimani,
L. R. Gawenis,
T. M. Riddle,
J. J. Duffy,
T. Doetschman,
T. Wang,
G. Giebisch,
P. S. Aronson,
J. N. 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].
35.
Seibert, P. D.
Quantitative RT-PCR: Methods, and Applications Book 3. Palo Alto, CA: Clontech Laboratories, 1993.
36.
Seidler, U.,
I. Blumenstein,
A. Kretz,
D. Viellard-Baron,
H. Rossman,
W. H. Colledge,
M. Evans,
R. Ratcliff,
and
M. Gregor.
A functional CFTR protein is required for mouse intestinal cAMP-, cGMP-, and Ca2+-dependent HCO
3 secretion.
J. Physiol. (Lond.)
505:
411-423,
1997[Abstract].
37.
Silberg, D. G.,
W. Wang,
R. H. Moseley,
and
P. G. Traber.
The down regulated in adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein.
J. Biol. Chem.
270:
11897-11902,
1995[Abstract/Free Full Text].
38.
Stankovic, K. M.,
D. Brown,
S. L. Alper,
and
J. C. Adams.
Localization of pH regulating proteins H+ ATPase and Cl
/HCO
3 exchanger in the guinea pig inner ear.
Hear. Res.
114:
21-34,
1997[Medline] [Corrigenda. Hear. Res. 124: p. 191-192.]
39.
Stuart-Tilley, A.,
C. Sardet,
J. Pouyssegur,
M. A. Schwartz,
D. Brown,
and
S. L. Alper.
Immunolocalization of anion exchanger AE2 and cation exchanger NHE1 in distinct, adjacent cells of gastric mucosa.
Am. J. Physiol.
266 (Cell Physiol. 35):
C559-C568,
1994[Abstract/Free Full Text].
40.
Stuart-Tilley, A. K.,
B. E. Shmukler,
D. Brown,
and
S. L. Alper.
Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney.
J. Am. Soc. Nephrol.
9:
946-959,
1998[Abstract].
41.
Sundaram, U.,
R. G. Knickelbein,
and
J. W. Dobbins.
pH regulation in ileum: Na+/H+ and Cl
/HCO
3 exchange in isolated crypt and villus cells.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G440-G449,
1991[Abstract/Free Full Text].
42.
Tyagi, S.,
M. M. Ali,
P. Malik,
T. J. Layden,
K. Ramaswamy,
and
P. K. Dudeja.
Expression of anion exchanger isoform AE-3 along the length of the human gastrointestinal tract (Abstract).
Gastroenterology
110:
A369,
1996.
43.
Vaandrager, A. B.,
and
H. R. de Jonge.
A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO
3 and SITS in rat intestinal epithelium.
Biochim. Biophys. Acta
939:
305-314,
1988[Medline].
44.
Vazquez, J. J.,
M. Vazquez,
M. A. Idoate,
L. Montuenga,
E. Martinez-Anso,
J. E. Castillo,
N. Garcia,
J. F. Medina,
and
J. F. Prieto.
Anion exchanger immunoreactivity in human salivary glands in health and Sjogren's Syndrome.
Am. J. Pathol.
146:
1422-1432,
1995[Abstract].
45.
Wang, Z.,
P. J. Schultheis,
and
G. E. Shull.
Three N-terminal variants of the AE2 Cl
/HCO
3 exchanger are encoded by mRNAs transcribed from alternative promoters.
J. Biol. Chem.
271:
7835-7843,
1996[Abstract/Free Full Text].
46.
Wormmeester, L.,
F. Sanchez de Medina,
F. Kokke,
C.-M. Tse,
S. Khurana,
J. Bowser,
M. E. Cohen,
and
M. Donowitz.
Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1261-C1272,
1998[Abstract/Free Full Text].
47.
Zhang, Y.,
M. Chernova,
A. Stuart-Tilley,
L. Jiang,
and
S. L. Alper.
The cytoplasmic and transmembrane domains of AE2 both contribute to regulation of anion exchange by pH.
J. Biol. Chem.
271:
5741-5749,
1996[Abstract/Free Full Text].
48.
Zolotarev, A. S.,
M. N. Chernova,
D. Yannoukakos,
and
S. L. Alper.
Proteolytic cleavage sites of native AE2 anion exchanger in gastric mucosal membranes.
Biochemistry
35:
10367-10376,
1996[Medline].
49.
Zolotarev, A. S.,
R. R. Townsend,
A. Stuart-Tilley,
and
S. L. Alper.
HCO
3-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G311-G321,
1996[Abstract/Free Full Text].
Am J Physiol Gastroint Liver Physiol 277(2):G321-G332
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society