Development of an endogenous epithelial
Na+/H+
exchanger (NHE3) in three clones of Caco-2 cells
Andrzej J.
Janecki1,
Marshall
H.
Montrose1,
C. Ming
Tse1,
Fermin Sanchez
de
Medina1,
Alain
Zweibaum2, and
Mark
Donowitz1
1 Departments of Medicine and
Physiology, Division of Gastroenterology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205; and
2 Unité de Recherches sur la
Différenciation Cellulaire Intestinale, Institut National de la
Santé et de la Recherche Médicale U178, Paris,
France
 |
ABSTRACT |
Expression of
endogenous
Na+/H+
exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL)
membrane domains was investigated in three clones (ATCC, PF-11, and
TC-7) derived from the human adenocarcinoma cell line Caco-2. In all
three clones, NHE1 was the only isoform detected at the BL domain
during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1
activity increased up to 7 PCD and remained stable thereafter. Both
NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP
Na+/H+
exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 µM
H+/s, whereas AP NHE1 activity
decreased from 192 to 18 µM H+/s
during that time. Similar results were obtained with the ATCC clone,
whereas very little AP NHE3 activity was observed in clone TC-7.
Surface biotinylation and indirect immunofluorescence confirmed these
results and also suggested an increase in the number of cells
expressing NHE3 being the major mechanism of the observed overall
increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 µM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the
protein kinase C (PKC) inhibitor
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement
of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of
inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD,
suggesting the development of regulatory mechanisms in the early
postconfluent period. Taken together, these data suggest a close
similarity of membrane targeting and regulation of endogenous NHE3
between Caco-2 cells and native small intestinal epithelial cells and
support the usefulness of some Caco-2 cell clones as an in vitro model
for studies on physiology of NHE3 in the intestinal epithelium.
phorbol ester; protein kinase C; epidermal growth factor
 |
INTRODUCTION |
CONTROL OF INTRACELLULAR ion composition is a
fundamental requirement of all living organisms. As a result of
specialization, epithelial cells use their ion transporting machinery
not only for "housekeeping" purposes, but also for regulation of
transport of ions and water across the epithelium. One example of such
specialization is the family of
Na+/H+
exchangers (NHEs). Six isoforms of NHE have been cloned to date (33,
47). All of the isoforms are membrane proteins that perform Na+/H+
exchange with stoichiometry 1:1. The two isoforms best characterized to
date are NHE1 and NHE3. NHE1, the ubiquitously present exchanger, is
believed to perform housekeeping functions, which include regulation of
intracellular pH (pHi), cell
volume, and cell proliferation. In most polarized
epithelial cells, NHE1 is located at the basolateral (BL) membrane
domain (9, 31). The other well-characterized isoform is NHE3. It has
been found predominantly in the brush border (BB) of renal, intestinal,
and salivary gland epithelium (4, 14, 36). In the kidney, NHE3 is
involved in Na+,
HCO
3, and probably
NH4 absorption (35). In the
intestine, NHE3 is believed to play a major role in
1) neutral NaCl absorption (23),
2) the increased ileal
Na+ absorption, which is
stimulated via neurohormonal signals after meals (24, 50), and
3) the decreased
Na+ absorption, which contributes
to secretory diarrhea (10). NHE3 is present in jejunal and ileal villus
cells, colonic surface cells, and in the upper crypt cells of small
intestine and colon in humans, rabbit, and rat (9).
Most of our knowledge concerning the regulation of NHE3 activity comes
from studies of the exchanger molecules exogenously expressed in
nonepithelial and some epithelial cell lines. The results of these
studies indicate that most of the regulation of NHE3 activity occurs
via changes in maximal velocity
(Vmax) of the
exchanger, in contrast to the regulation of NHE1, which occurs mostly
by changes in K'(H+).
Growth factors, serum, and okadaic acid stimulate NHE3 activity, whereas protein kinase C (PKC), carbachol, and hyperosmolality inhibit
the exchanger (9). Recent studies of regulation of NHE3 in native ileum
indicate that activation of some signal transduction pathways is highly
polarized in expression (16, 17). Moreover, some evidence suggests that
the regulation of NHE3 in polarized epithelial cells involves
cytoskeletal proteins (e.g., villin), which are unique to some
specialized epithelial cell types (18). These facts may create problems
when extrapolating the results of in vitro studies on NHE3 regulation
to native tissue. Published studies of regulation of endogenous NHE3 in
epithelial cells are limited to kidney-derived OK cells, and most in
vitro studies have been performed using cells transfected with NHE3
cDNA. The latter approach may be complicated by a separate set of
problems, including those related to possible overexpression and
mistargeting of membrane proteins. In this communication we describe a
gradual development of activity, membrane targeting, and regulation of endogenous NHE3 in three clones of the human colonic adenocarcinoma cell line Caco-2. This gradual development was clone specific and
appeared to result from an increasing number of cells expressing NHE3
at the BB and not from parallel, progressive differentiation of
targeting and regulatory mechanisms within individual cells. In mature
monolayers of two of three tested Caco-2 clones, both the polarity of
expression as well as the regulation of NHE3 by PKC and epidermal
growth factor (EGF) closely resembled those described for native
mammalian intestinal epithelial cells.
 |
MATERIALS AND METHODS |
Cell culture.
Three clones of Caco-2 cell line originally derived from human colonic
adenocarcinoma were studied. Clone ATCC was purchased from American
Type Culture Collection (Rockville, MD) and was used between
passages 27 and 47. Two other clonal lines
derived from the original Caco-2 cell population, PF-11 and TC-7, were characterized previously (7). Clonal line PF-11 was originally an
"early" passage (passage 29) and was studied between
35 and 45 subsequent passages. Clonal line TC-7 was originally a
"late" passage (passage 198) and was studied between
42 and 49 subsequent passages. These two clonal lines differ
significantly in monolayer morphology, glucose consumption rate,
glycogen accumulation, and sucrase-isomaltase expression at the BB (7).
All cells were plated at a density 5-8 × 104
cells/cm2 into tissue culture
inserts equipped with 0.45-µm pore size, high-pore density Falcon PET
membranes (Becton Dickinson Labware, Franklin Lakes, NJ) and grown in
DMEM supplemented with 0.1 mM nonessential amino acids, 1 mM pyruvate,
penicillin (50 IU/ml), streptomycin (50 µg/ml) and 10% fetal bovine
serum, in a 10% CO2-humidified incubator at 37°C. The same batch of serum was used in
all experiments. In some experiments, cells were plated on the Falcon
polyethylene terephtalate (PET) membranes covered with human laminin (1 µg/cm2; Sigma Chemical, St.
Louis, MO).
For fluorometry, cells were grown as polarized monolayers on small
pieces of Falcon PET membranes glued over an aperture in rectangular
plastic coverslips ("filterslips") as described previously (27).
Filterslips with attached cells were kept in standard 12-well culture
plates, and culture medium was replaced every other day.
Measurement of
Na+/H+
exchange rate.
A fluorometric method based on pH-sensitive fluorophore
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl
ester (BCECF-AM; Molecular Probes, Eugene, OR) was used for functional characterization of
Na+/H+
exchange separately at the apical (AP) and BL surfaces of the monolayers, as described in detail previously (27, 48). Briefly, monolayers grown on filterslips and serum deprived for 8-12 h were
exposed to BCECF-AM (5 µM) and
NH4Cl (40 mM, to promote
subsequent intracellular acidification) in
Na+ medium [in mM, 131 NaCl,
5 KCl, 2 CaCl2, 1 MgSO4, 0.8 Na2H(PO4), 0.2 NaH2(PO4),
25 glucose, 20 HEPES, pH 7.4] for 50 min at room temperature.
Filterslips were then mounted in a cuvette, placed in the fluorometer
(SPF 500C; SLM, Urbana, IL), and perfused at both monolayer surfaces
with tetramethylammonium (TMA) medium (identical to
Na+ medium except that
Na+ salts were replaced by TMA
salts) to allow for rapid intracellular acidification. The
TMA medium was subsequently replaced with
Na+ medium at one monolayer
surface. Changes in pHi were
monitored by alternating the excitation wavelengths between 440 and 500 nm and collecting the emission signal at 530 nm. Rates of
Na+-dependent intracellular
alkalinization (efflux of H+, in
µM/s) were calculated for a given
pHi (within the linear phase of
the initial rate of intracellular alkalinization, as the product of
pH/
T and buffering capacity) using Enzfitter software (Biosoft)
(48). The buffering capacity of Caco-2 cells was
determined separately, as described elsewhere (6, 48). The monolayers'
integrity was systematically tested by adding 1 mM amiloride
[which, at this concentration, inhibits both NHE1 and NHE3 (8,
32)] to the AP superfusate (TMA medium) in the presence of 131 mM
Na+ in the BL superfusate, and
comparing the rate of intracellular alkalinization with that observed
in the absence of amiloride. In these experiments, <3% of tested
monolayers exhibited inhibition of BL
Na+/H+
exchange, which suggested a leak of amiloride from AP to BL side and,
therefore, a break in the integrity of the monolayer.
To differentiate among NHE1, NHE2, and NHE3 activity at a given
monolayer surface, we used two known
Na+/H+
exchange inhibitors: HOE-694 and amiloride. HOE-694 is a
benzoylguanidine derivative initially characterized using PS120
fibroblasts transfected with various NHE isoforms (8). In
these cells, the compound's inhibitor constant for NHE3 was 650 µM,
whereas its inhibitor constant for NHE1 was only 0.15 µM. In our
hands (see RESULTS) complete inhibition of endogenous NHE1
in Caco-2 cells without any effect on NHE3 activity was observed with a
HOE-694 concentration of 20 µM. In the presence of 131 mM Na+, the
IC50 for NHE2 transfected into
PS120 fibroblasts was 20.5 µM (see below). We used 1 mM amiloride
(instead of HOE-694) for inhibition of NHE3, inasmuch as we were not
able to solubilize HOE-694 in Na+
buffer at a concentration higher than 1 mM (which was required for
complete inhibition of NHE3). The NHE1 activity was calculated by
subtraction of the exchange rate observed at a given
pHi in presence of 20 µM HOE-694
from the total exchange rate observed at a chosen monolayer surface in
the presence of 131 mM Na+. The
Na+/H+
activity observed in the presence of 20 µM HOE-694 was due to NHE3
only, and it was predictably inhibited by 1 mM amiloride. NHE2 was not
expressed in any of the Caco-2 clones examined (see below).
In experiments with EGF (200 ng/ml) and phorbol 12-myristate 13-acetate
(PMA, 1 µM) (Sigma Chemical), the tested substances were present in
Na+ medium during the final 20 min
of incubation with BCECF, and then in both AP and BL media during the
entire perfusion. In some experiments, separate groups of monolayers
were exposed to PMA in the presence of the PKC inhibitor
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 65 µM; Seikagaku
Kogyo, Tokyo, Japan), which was preceded by 20 min of preincubation
with H-7 alone.
Inhibition of AP and BL
Na+/H+
exchange activity by amiloride and HOE-694.
Examination of the response kinetics of
Na+/H+
exchange to amiloride and HOE-694 was performed separately at the AP
and BL surfaces of Caco-2 monolayers by using the BCECF fluorometric
method as previously described (48). Caco-2 monolayers at
17 postconfluent days (PCD) were superfused at the chosen monolayer
surface with Na+ medium without or
with various concentrations of the inhibitors. The opposite surface of
the monolayer was perfused with TMA medium. Na+/H+
exchange kinetics were then calculated using Enzfitter software, and
the rates were compared at the same
pHi within the linear portion of
the kinetic curves. The dose-response curves were obtained using a
computer-enhanced curve-fitting algorithm (Microcal Origin, Microcal Software).
Inhibitory effects of HOE-694 on NHE1 and NHE2 in PS120 fibroblasts
were evaluated using PS120 cells transfected with cDNA encoding for
NHE1 and NHE2, respectively (41, 42). The rate of intracellular
alkalinization in the presence of 131 mM
Na+ and different concentrations
of HOE-694 was examined as previously described for Caco-2 cells, with
the exception that PS120 cells were cultured on glass coverslips and
were therefore superfused only at the cell surface.
Localization of intracellular NHE1 and NHE3 by indirect
immunofluorescence.
Labeling of intracellular NHE1 and NHE3 was performed as described in
detail previously (15). Briefly, Caco-2 monolayers grown on PET
membranes were rinsed with PBS and fixed with 3% paraformaldehyde in
PBS, pH 7.4, for 45 min. The permeabilization and blocking of
nonspecific binding sites were performed in one step by incubating the
monolayers with PBS containing 0.075% saponin, 1% BSA (Goldmark
Biologicals, Phillipsburg, NJ), and 15% normal goat serum (buffer PBG;
Jackson Immunoresearch, West Grove, PA) for 45 min. Monolayers were
then incubated with anti-NHE3 polyclonal antibody (Ab) 1380 (1:50
dilution) or with anti-NHE1 Ab 1950 (1:50 dilution) in buffer PBG for 1 h, followed by washing with PBS containing 0.05% saponin and 1% BSA.
Both antibodies were raised in rabbits and have been shown previously
(by immunohistochemistry and Western analysis) to specifically
recognize respective exchanger molecules expressed in PS120 fibroblasts
as well as endogenous exchangers in rabbit and human intestinal
epithelia and not to cross-react with each other or with NHE2 (14, 41).
For control of nonspecific binding, the antisera were substituted with
preimmune rabbit serum at 1:50 dilution. Monolayers were subsequently
incubated with Cy3-conjugated goat anti-rabbit IgG (1:300 dilution;
Jackson ImmunoResearch) for 45 min, postfixed with 3%
paraformaldehyde, and mounted in 50% glycerol in PBS containing 0.2%
p-phenylenediamine as an
anti-photobleaching agent. Intracellular localization of the
immunolabeled exchanger molecules was performed in XY and XZ planes using Zeiss laser confocal microscope LSM 410.
In some experiments, we evaluated the effect of PMA and EGF on the BB
length. Monolayers were exposed to PMA or EGF and fixed as previously
described. The BB was visualized by labeling with FITC-conjugated
Phaseolus vulgaris lectin phytohemagglutinin-E (PHA-E, 50 µg/ml; Sigma Chemicals, St. Louis, MO) for 30 min, and the BB length
was calculated using MetaMorph software as described previously
(15).
Examination of membrane targeting of NHE1 and NHE3 by cell surface
biotinylation.
For detection of endogenous NHE1 and NHE3 by surface biotinylation, 25 mm PET inserts containing Caco-2 monolayers were collected at various
times between 3 and 22 PCD, rinsed with ice-cold PBS, and exposed at
either AP or BL surface to sulfo-NHS-biotin (0.5 mg/ml; Pierce,
Rockford, IL) in borate buffer (in mM, 154 NaCl, 7.2 KCl, 1.8 CaCl2, 10 H3BO3,
pH 9.0) for 1 h, followed by extensive washing. Free
biotin was quenched with Tris buffer (120 mM NaCl, 20 mM Tris, pH 7.4)
for 10 min, and cells were scraped in lysis buffer (150 mM NaCl, 3 mM
KCl, 5 mM EDTA, 1% Triton X-100, 3 µM aprotinin, 20 µM
phosphoramidone, 0.3 mM phenylmethylsulfonyl fluoride, 10 µM
leupeptin, 60 mM HEPES, pH 7.4). After brief sonication the lysates were incubated with rocking for 30 min and spun at 16,000 g for 15 min. The lysates were then
incubated with avidin-agarose beads (Pierce, Rockford, IL) for 60 min
and spun briefly. The entire procedure was performed at 4°C. The
agarose beads with biotinylated proteins were washed extensively with
lysis buffer and boiled in Laemmli sample buffer for 10 min, followed
by brief centrifugation. The biotinylated proteins in the supernatant
were separated by SDS-PAGE, transferred to nitrocellulose, and probed with either anti-NHE3 Ab 1380 or anti-NHE1 Ab 1950 (both diluted 1:500). Membrane preparations obtained from PS120 fibroblasts transfected with NHE3 or NHE1 cDNA, respectively, were used as an
internal standard. Protein bands were revealed by incubation of
membranes with horseradish peroxidase-conjugated donkey anti-rabbit IgG
followed by chemiluminescence detection (Western Blot Chemiluminescence Reagent Plus, NEN Life Science Products, Boston, MA).
SDS-PAGE and Western blotting.
For Western analysis of total cellular NHE3, monolayers were collected
at 3, 11, or 22 PCD and rinsed with ice-cold BSA, and cells were
scraped into lysis buffer as described for the biotinylation procedure.
The lysates were sonicated briefly, spun at 16,000 g for 15 min, and
boiled in Laemmli buffer for 10 min. The lysate proteins were separated
by SDS-PAGE as previously described. Semiquantitative evaluation of the
relative abundance of NHE3 in biotinylated BB preparations as well as
in the whole cell lysates was performed as described previously (15).
For detection of NHE2, total cell lysates obtained from Caco-2
monolayers at 5 PCD were separated by SDS-PAGE and transferred to
nitrocellulose as previously described. The protein blots were probed
with anti-NHE2 polyclonal Ab 597 at 1:1,000 dilution. This antibody was
shown previously not to cross-react with rabbit NHE3 and NHE1 (41) and
to specifically recognize human NHE2 (14).
Statistical analysis.
Numerical data are expressed as means ± SD, and the
significance of difference between experimental groups was analyzed by two-tailed Student's t-test.
 |
RESULTS |
General characteristics of the studied Caco-2 clones.
Once confluence was reached (4-5 days), all three clones studied
exhibited morphological heterogeneity, as described previously (44). At
the level of phase contrast microscopy, islands of well-demarcated
polygonal cells were interspersed with areas covered with more
irregular, larger cells. Formation of domes was observed within
2-3 PCD in monolayers grown on solid substrate. The ATCC and PF-11
clones were more heterogeneous and produced significantly more domes
than the TC-7 clone (not shown). Moreover, TC-7 cells had a higher
mitotic index than the other two clones (doubling time of ~24 and 36 h, respectively), consistent with previous reports (7). In the PF-11
clone, the average cell density at confluency was ~3.1 × 105/cm2,
and gradually increased to ~7.2 × 105/cm2
at 3-4 PCD, after which time the cell density remained relatively stable for up to 30 PCD. Similar results were observed in TC-7 and ATCC clones.
Gradual development of polarized
Na+/H+
exchange activity in Caco-2 monolayers.
Polarized expression of NHE1 and NHE3 activities was investigated
starting as soon as the formation of the occluding junctions was
completed, e.g., between 48 and 72 h postconfluency (38). A
representative pattern of
Na+-dependent intracellular
alkalinization at 3 PCD is shown in Fig. 1A.
Exposure of BL monolayer surface to
Na+ medium resulted in a rapid
intracellular alkalinization, with the average
H+ efflux rate being 733 ± 83 µM/s (n = 8) within the linear
portion of the alkalinization curve. The
pHi reached plateau at 7.12 ± 0.08 within 12-14 min (not shown). This BL
Na+/H+
exchange was completely suppressed by 20 µM HOE-694, indicating that
it was entirely due to the activity of NHE1. Significant Na+/H+
exchange activity was also noted when the monolayers were perfused with
Na+ medium at the AP surface.
However, only ~67% of this activity was suppressed by 20 µM
HOE-694, the residual activity requiring 1 mM amiloride for complete
inhibition. These data suggest the presence of activities of both NHE1
and NHE3 (~67 and 33% of NHE1 and NHE3, respectively) at the AP
surface of the early postconfluent monolayers (Fig.
1A and Table
1). Lack of detectable NHE2 in these cells
(Fig. 2) speaks against the possibility of
significant contribution of this isoform to the observed "amiloride
sensitive" AP and/or BL
Na+/H+
exchange at this early postconfluent period. With increasing time
postconfluency, a progressively higher activity of NHE3 was observed at
the AP surface of the Caco-2 cell monolayers, and this was paralleled
by a significant decrease of NHE1 activity at this membrane domain. At
17 PCD, NHE3 contributed ~93% to the total AP
Na+/H+
exchange, the residual activity being represented by NHE1 (Fig. 1B). As was the case with early
postconfluent monolayers, the entire BL
Na+/H+
exchange at 17 PCD was due to activity of NHE1 (Fig.
1B). The pattern of gradual
development of the polarized functional expression of NHE3 and NHE1 in
clone PF-11 is summarized in Fig. 3. During 22 PCD examined, the total AP
Na+/H+
exchange rate increased from 284 ± 30 µM
H+/s at 3 PCD to 468 ± 32 µM
H+/s at 22 PCD
(n = 8, P < 0.01). This increase was due to
a rapid increase in the NHE3 activity (from 93 ± 16 to 450 ± 72 µM H+/s,
n = 8, P < 0.01), and it was associated
with a significant decrease in NHE1 activity at the AP surface (from
192 ± 15 to 18 ± 6 µM
H+/s 3 and 22 PCD, respectively,
n = 8, P < 0.01). The total BL exchange
rate increased rapidly between days 3 and 7 postconfluency (from 733 ± 83 to 1,261 ± 150 µM H+/s,
n = 8, P < 0.01) and remained stable
thereafter.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Patterns of Na+-dependent
intracellular alkalinization in Caco-2 cells (clone PF-11) examined at
3 (A) and 17 (B) postconfluent days (PCD). Cells
on filterslips were loaded with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein,
acidified with NH4Cl, and
superfused in the fluorometer with
Na+ medium at either an apical (Na
in AP) or basolateral (Na in BL) monolayer surface. Separate groups of
monolayers were superfused at AP surface with
Na+ medium containing either 20 µM HOE-694 (Na/HOE in AP) or 1 mM amiloride (Na/Amil in AP). In all
experiments, opposite monolayer surface was superfused with
tetramethylammonium (TMA) medium. Arrows labeled Na, onset of exposure
of monolayers to sodium at respective surface. Arrows labeled HOE,
onset of addition of 20 µM HOE-694 to the BL compartment.
Representative tracings of intracellular alkalinization observed in 4 separate monolayers are shown in each panel.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Comparison of total apical Na+/H+
exchange and relative contribution of NHE3 to this exchange in 3 Caco-2 cell clones
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2.
Western analysis of total cell lysate obtained at 5 PCD from wild-type
Caco-2 clone PF-11 (Caco-2) or PF-11 clone transfected with NHE2 cDNA
(Caco-2 E2). Whole cell lysates were prepared as described in
MATERIALS AND METHODS and were
separated using SDS-PAGE. Blotted proteins were probed with anti-NHE2
antibody (Ab) 597 (at 1:1,000 dilution). Note the presence of two
protein bands of molecular mass of ~75 kDa and 85 kDa in Caco-2 E2
lane, consistent with the presence of NHE2 protein in transfected
Caco-2 cells and a complete lack of a corresponding band in wild-type
Caco-2 cells. Material separated in each lane was obtained from
comparable number of cells. Similar to findings in clone PF-11, no NHE2
was detected in clones ATCC and TC-7 (data not shown).
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
Gradual development of polarized functional expression of endogenous
NHE1 and NHE3 in Caco-2 monolayers (clone PF-11) examined at 3, 7, 11, 17, and 22 PCD. Results are presented as rates of intracellular
alkalinization [H+ efflux
rate in µM/s, at intracellular pH
(pHi) 6.40] in the
presence of 131 mM Na+ at either
AP or BL surface, as described in MATERIALS AND
METHODS. Calculation of relative participation of NHE1
and NHE3 in AP
Na+/H+
exchange activity was based on effects of 20 µM HOE-694 and 1 mM
amiloride on exchange rate (see MATERIALS AND
METHODS). The 4 consecutive bars (from left to right)
in each group represent: 1) total AP
Na+/H+
exchange activity (Total AP), 2) AP
NHE3 activity (NHE3 AP), 3) AP NHE1
activity (NHE1 AP), and 4) BL NHE1
activity (NHE1 BL). Data represent means ± SD from 8 monolayers per
condition in 2 separate experiments.
|
|
Data presented in Figs. 1 and 2 were obtained from clone PF-11 of
Caco-2 cells. The overall NHE3 activity was only slightly and not
significantly lower in the ATCC clone. Moreover, the pattern of gradual
development of the AP NHE3 activity associated with rapidly decreasing
activity of NHE1 at this membrane domain was similar in both clones
(Table 1). In contrast, significantly lower overall AP
Na+/H+
exchange rate as well as a significantly smaller contribution of NHE3
to the total AP
Na+/H+
exchange was observed in clone TC-7 (Table 1). Because cell number did
not change significantly after 3-4 PCD in any of the clones
examined, these data suggest significant differences in the patterns of
development of endogenous NHE3 activity between clones PF-11/ATCC and
clone TC-7. On the other hand, the total BL
Na+/H+
exchange rate observed in TC-7 cells was only slightly, and not significantly, lower than that observed in PF-11 and ATCC monolayers at
the respective postconfluency periods, and was accomplished entirely by
NHE1 (data not shown).
Sensitivity of AP and BL
Na+/H+
exchange activity to HOE-694 and amiloride.
Evaluation of the dose-response kinetics of AP and BL
Na+/H+
exchange for HOE-694 and amiloride was performed using clone PF-11 at
17 PCD, i.e., at the time of stable polarized expression of NHE1 and
NHE3. The dose-response curve obtained for HOE-694 applied basolaterally suggested the presence of a single isoform of NHE, with
IC50 equaling 0.13 µM (Fig.
4, top).
In contrast, the curve obtained when the inhibitor was applied at the
AP surface of Caco-2 monolayers suggested the presence of two NHE
isoforms, with IC50 of the
"HOE-694-sensitive" component being ~0.11 µM and the
IC50 for the
"HOE-694-resistant" component being 655 µM. Moreover, the
HOE-694-sensitive component was apparently responsible for only ~9%
of the overall AP
Na+/H+
exchange. These values are consistent with the predominant contribution of NHE3, and only a small contribution of NHE1 to the total AP Na+/H+
exchange in this Caco-2 clone at 17 PCD. The purely sigmoid shapes of
the dose-response curves obtained for the HOE-694-sensitive Na+/H+
exchange at the AP and the BL monolayer surfaces speaks against the
presence of NHE2 activity in these monolayers. Computer-enhanced analysis of both curves (Fig. 4,
top) suggested the presence of only
one exchanger with IC50 of ~12
µM. This was similar to the IC50
obtained from PS120 cells transfected with NHE1 cDNA (0.25 µM, Fig.
5). Because the
IC50 for NHE2 was 20.5 µM (Fig.
5, PS120 cells), contribution of NHE2 to the AP and/or BL
Na+/H+
exchange in Caco-2 cells would result in a bimodal shape of the respective segments of the dose-response curves.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Patterns of inhibition of AP ( ) and BL ( )
Na+/H+
exchange in Caco-2 monolayers (clone PF-11, 17 PCD) exposed to various
concentrations of HOE-694 (top) or
amiloride (bottom). Monolayers on
filterslips were acidified and then superfused in a fluorometer in the
presence of 131 mM Na+ with
indicated concentrations of respective inhibitor at either AP or BL
surface. Opposite monolayer surface was superfused with TMA medium.
Magnitude of inhibition is presented as percent of
H+ efflux rate observed in the
absence of inhibitors, and was calculated from rate of intracellular
alkalinization at pHi 6.40. Rescaled segments of curves obtained from AP surfaces at concentrations
of HOE-694 (top) and amiloride
(bottom) of 0-10 µM are shown
in the insets. Each data point represents mean from total of 4 monolayers from 2 separate experiments.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Patterns of inhibition of
Na+/H+
exchange in PS120 fibroblasts transfected with cDNA encoding for NHE1
( ) or NHE2 ( ) and exposed to various concentrations of HOE-694.
Confluent monolayers on glass coverslips were acidified and then
superfused in a fluorometer in the presence of 131 mM
Na+ with indicated concentrations
of HOE-694. Magnitude of inhibition is presented as percent of control
H+ efflux rate observed in the
absence of inhibitors and was calculated from the rate of intracellular
alkalinization at pHi 6.40. Each
data point represents mean from total of 3 monolayers from 2 separate
experiments.
|
|
The dose-response patterns obtained for amiloride are shown in Fig. 4,
bottom. Similar to the patterns shown
in Fig. 4, top for HOE-694, they
suggest that the BL
Na+/H+
exchange was entirely due to NHE1 activity
(IC50 2.1 µM), and that both
NHE3 (~92% of overall AP activity,
IC50 115 µM) and NHE1 (~8% of
overall AP activity, IC50 2.1 µM) were present at the AP surface of these monolayers.
Subcellular localization of NHE1 and NHE3 by immunofluorescence.
NHE3 was found at the AP surface of a small number of Caco-2 cells
(clone PF-11) shortly after postconfluence, and the number of
positively labeled cells increased progressively with time, from
~15% positive cells seen at 3 PCD to ~52% at 11 PCD and 85% at
22 PCD (Table 2). Similar results were
obtained with ATCC monolayers, whereas only very small numbers of
NHE3-positive cells were found in TC-7 cells, regardless of the
postconfluent culture period (Table 2).
View this table:
[in this window]
[in a new window]
|
Table 2.
Effect of laminin on relative number of cells expressing brush-border
NHE3 and on apical NHE3 activity in 3 Caco-2 cell clones analyzed at 3, 11, and 22 PCD
|
|
Because the extracellular matrix and in particular laminin appear to
affect the functional maturation of Caco-2 cells (45), we tested the
effect of coating the permeable culture substrate with exogenous
laminin on the expression of NHE3 in the three clones of Caco-2 cells.
In PF-11 and ATCC clones the presence of laminin resulted in a
significant increase in the number of NHE3 expressing cells at 3 and 11 PCD, with no significant effect of laminin seen at 22 PCD (Table 2).
The effect of laminin on the number of NHE3-positive cells was
paralleled by the NHE3 activity of the monolayers. In contrast, no
significant effect of laminin on NHE3 expression was observed in TC-7
clone at any postconfluent period examined (Table 2).
The representative pattern of expression of surface NHE3 labeled by
indirect immunofluorescence in Caco-2 (PF-11) monolayer at 17 PCD is
shown in Fig. 6. The labeled cells were
aggregated in irregular islands, interspersed with areas of cells that
exhibited a much weaker fluorescent signal. With use of confocal
microscopy, two distinctive morphological patterns of labeled BB could
be recognized: 1) a relatively
smooth and uniform "standard" pattern and
2) a "flower" pattern, with
labeling aggregated into small clusters on the surface of a single
cell. These patterns were consistent with the description of
immunolabeling of some BB hydrolases (sucrase-isomaltase, alkaline
phosphatase, dipeptidylpeptidase IV, lactase) in Caco-2 cells in
earlier reports (44, 46). Reconstruction of the images in the vertical
(XZ) plane revealed that the immunolabeling was exclusively
present in the AP cell area corresponding to the BB and a narrow zone
of subapical cytoplasm (Fig. 6B). No
significant labeling was observed in the areas corresponding to the BL
surface.

View larger version (80K):
[in this window]
[in a new window]
|
Fig. 6.
Representative images of Caco-2 monolayer (clone PF-11, 17 PCD)
immunolabeled with anti-NHE3 Ab 1380. A: combines multiple images obtained
in XY plane by confocal microscopy to present a 15-µm
thick optical section at brush-border (BB) level. Note uneven
expression of immunoreactive NHE3 at surface of adjacent cells and the
presence of 2 distinctive patterns of the BB labeling, namely smooth
"standard" pattern (S) and "flower" pattern (F).
B: reconstruction of monolayer
obtained in vertical (XZ) plane. Note predominant
immunolocalization of NHE3 at the BB and adjacent, narrow zone of
subapical cytoplasm. C: reconstruction
in XZ plane of image of control monolayer incubated with
preimmune serum instead of Ab 1380. Dashed lines, surface of permeable
culture support. Bars correspond to 20 µm.
|
|
Because Ab 1380 was used at a relatively low dilution, the possibility
of nonspecific binding had to be considered. Three pieces of evidence
speak against this possibility. First, no significant binding was
observed when preimmune serum was used instead of Ab 1380 (Fig.
6C). Second, in late postconfluent
monolayers of TC-7 cells in which BB was fully and uniformly developed
(as judged by labeling with PHA-E, data not shown) virtually no
immunolabeling of NHE3 was observed, and this corresponded to the very
low AP NHE3 activity observed in these cells (Table 1). Finally, the same Ab 1380 was shown to specifically detect NHE3 in PS120 cells as
well as at the BB of native human and rabbit intestinal epithelia (14).
In contrast to NHE3, immunolabeling of NHE1 in Caco-2 monolayers with
anti-NHE1 Ab 1950 revealed a "chicken wire" pattern suggesting BL
localization of the exchanger. This was confirmed by images
reconstructed in the XZ plane (Fig.
7). In early postconfluent monolayers (3 PCD), the NHE1 labeling was present predominantly in the BL areas, but
some weak labeling was also occasionally observed at the BB (not
shown). These findings were consistent with the predominantly BL
localization of NHE1 activity in fully matured Caco-2 monolayers and
with a presence of this exchanger also at the BB, especially in the
early postconfluent period.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 7.
Indirect immunofluorescent labeling of endogenous NHE1 in Caco-2 cells
(clone PF-11) examined at 17 PCD. A:
pattern of labeling obtained by confocal fluorescent microscopy in
XY plane at approximately midpoint of monolayer height (1 µm optical section). Note "chicken wire" labeling pattern
suggesting BL localization of exchanger, which is confirmed by
reconstruction in XZ plane shown in
B. Bars correspond to 20 µm. Dotted
line, surface of permeable culture support.
|
|
Subcellular localization of NHE1 and NHE3 and quantitation of NHE3
by surface biotinylation and Western analysis.
Surface biotinylation experiments were performed to confirm the pattern
of NHE3 and NHE1 intracellular localization suggested by microscopic
studies. In Caco-2 monolayers at 17 PCD (clone PF-11), a prominent
protein band of molecular mass, ~85 kDa, was observed in the
immunoblots of cell lysates obtained from monolayers biotinylated at
the AP surface (Fig. 8,
top). This band
corresponded to the 85-kDa band observed in the immunoblots obtained
from the lysates of PS120 fibroblasts transfected with rabbit NHE3. In contrast, no detectable biotinylated protein of molecular mass ~85
kDa was detected in the Caco-2 lysates obtained from monolayers biotinylated at the BL surface (Fig. 8,
top). These data confirmed the
exclusive AP targeting of endogenous NHE3 in Caco-2 cells. On the other
hand, performance of a similar procedure but with use of anti-NHE1 Ab
1950 resulted in detection at the BL monolayer surface of significant
amounts of a protein of molecular mass ~110 kDa, which corresponded
to similar band obtained from PS120 cells transfected with NHE1 cDNA.
(Fig. 8, bottom). However, only a
trace of this protein was detected in cell lysates obtained from
monolayers biotinylated at the AP surface. Similar results were
obtained from clones ATCC and TC-7 (data not shown). These findings
confirmed a predominantly BL targeting of NHE1 in the examined Caco-2
clones and also excluded the possibility that NHE3 was not detected at
the BL membrane domain due to poor accessibility of this domain to
biotin.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 8.
Western analysis of biotinylated membrane proteins from Caco-2
monolayers (clone PF-11, 17 PCD) and PS120 fibroblasts. Cell surface
biotinylation was performed as described in MATERIALS
AND METHODS. Biotinylated membrane proteins were run in
consecutive lanes as follows. Top:
lane 1, preparation obtained from
PS120 fibroblasts transfected with rabbit NHE3 (PS120 E3);
lane 2, AP surface biotinylated
proteins from Caco-2 monolayers (Caco-2 AP); lane
3, BL surface biotinylated proteins from Caco-2
monolayers (Caco-2 BL); lane 4,
biotinylated membrane proteins from wild-type PS120 fibroblasts lacking
any NHE isoform (PS120). Blot was probed with anti-NHE3 Ab 1830. Note
prominent band of molecular mass ~85 kDa in lane
2, corresponding to similar band in
lane 1, and representing endogenous
NHE3 biotinylated at AP surface of Caco-2 monolayers. Also, note
absence of corresponding bands in lanes
3 and 4.
Bottom: lane
1, membrane proteins from PS120 cells transfected with
NHE1 cDNA (PS120 E1); lane 2, BL
surface biotinylated proteins from Caco-2 monolayers (Caco-2 BL);
lane 3, AP surface biotinylated
proteins from Caco-2 monolayers (Caco-2 AP); lane
4, biotinylated membrane proteins from wild-type PS120
fibroblasts (PS120). Blot was probed with anti-NHE1 Ab 1950. Note
prominent band of molecular mass ~110 kDa in lane
2, corresponding to similar band in
lane 1, and representing endogenous
NHE1 biotinylated at BL surface of Caco-2 monolayers. Also, note
absence of corresponding bands in lanes
3 and 4. Material
separated in each lane in respective panel was obtained from comparable
number of cells.
|
|
The relative abundance of BB NHE3, as measured by cell surface
biotinylation (and normalized by the number of cells) steadily increased during postconfluent maturation (Fig.
9). At 3 PCD, surface NHE3 constituted 19%
and at 11 PCD it was 56% of the total amount of NHE3 (100%) found at
22 PCD (Fig. 9, A and
C). Interestingly, the pattern of
increase of the total cellular NHE3 amount closely followed the pattern
observed for the amount of NHE3 at the BB (Fig. 9,
B and
C). Similar results were obtained
from the ATCC clone, whereas virtually no NHE3 protein was detected in
either biotinylated preparations or in the whole cell lysates obtained from TC-7 cells (data not shown). Because only ~18% of the total cellular NHE3 is present in the cytoplasm of mature Caco-2 cells (clone
PF-11) (15), these data suggest that most of the total cellular NHE3
was targeted to BB of Caco-2 cells (PF-11 and ATCC) regardless of the
length of the postconfluent culture period.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
Relative abundance of NHE3 protein at BB and in whole cell lysates
obtained from Caco-2 monolayers at 3, 11, and 22 PCD. Biotinylated AP
surface protein preparations or whole cell lysates obtained from PF-11
clone cultured for indicated periods were subjected to Western
analysis, and bands corresponding to NHE3 were quantitated by
densitometry as described in MATERIALS AND
METHODS. A:
representative pattern of NHE3 abundance in biotinylated preparations.
B: representative pattern obtained
from whole cell lysates. Material separated in each lane of
A and
B was obtained from similar number of
cells (differences in cell number among lanes were 5% of mean cell
number). C: comparison of patterns of
increase of the surface (open bars) and the whole cell (shaded bars)
amounts of NHE3, expressed as percent of respective amounts found at 22 PCD (shown as 100% for each group). Data in
C are means ± SD from 3 independent experiments. Data shown by line tracing ( ) represent
average NHE3 activity measured in parallel monolayers at respective
postconfluent periods and are presented as percent of activity observed
at 22 PCD.
|
|
Regulation of activity of endogenous NHE3 by PMA and EGF.
Investigations on the effects of PMA (1 µM) or EGF (200 ng/ml) on
endogenous NHE3 in Caco-2 cells were conducted using clone PF-11. A
representative pattern of the effect of PMA on the initial rate of
Na+-dependent intracellular
alkalinization in monolayers at 17 PCD is shown in Fig.
10A and
is consistent with our previous report (15). In the experiment shown,
PMA inhibited the control H+
efflux rate by 27% (from 380 to 273 µM
H+/s at
pHi 6.40). In three separate
experiments, the average inhibition of NHE3 by PMA was 28 ± 5% of
control value (Fig. 11). This effect was
completely abolished by the PKC inhibitor H-7 (Fig. 11), suggesting the
involvement of the PKC signal transduction pathway in the observed
PMA-mediated inhibition of NHE3 activity. H-7 alone did not have any
significant effect on the control
Na+/H+
exchange rate. Similar magnitude of PMA-induced inhibition of NHE3 was
observed in early postconfluent PF-11 monolayers, although the overall
activity of NHE3 was much lower at that time. At 7 PCD PMA (1 µM)
inhibited NHE3 activity by 25 ± 3% (means ± SD, n = 8 monolayers), whereas exposure to
PMA in the presence of H-7 resulted in a nonsignificant change of
activity compared with control. Twenty minutes of exposure of PF-11
cells (17 PCD) to 1 µM PMA did not result in a significant change in
the BB length [3.8 ± 0.5 µm in control cells vs. 3.5 ± 0.4 (SD) µm in PMA-treated cells, n = 30 cells from 5 monolayers for each condition].

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 10.
Inhibition by phorbol 12-myristate 13-acetate (PMA,
A) and stimulation by epidermal
growth factor (EGF, B) of activity
of endogenous NHE3 in Caco-2 cells (clone PF-11, 17 PCD). Monolayers on
filterslips were preincubated with PMA (1 µM) or EGF (200 ng/ml) for
20 min, and the Na+-dependent rate
of intracellular alkalinization was evaluated using fluorometric
method, as described in MATERIALS AND
METHODS. Data show means ± SD of rates observed in
9 monolayers in 3 separate experiments for each treatment. Dotted
lines, least-square linear fit curves for control condition (CTR) and
for monolayers exposed to PMA or monolayers exposed to EGF. Arrows,
onset of exposure of the AP surface of monolayers to 131 mM
Na+.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 11.
Cumulative results of effects of PMA and EGF on rate of
Na+-dependent intracellular
alkalinization in Caco-2 monolayers (clone PF-11, 17 PCD). Rate of
intracellular alkalinization in presence of 131 mM
Na+ was examined after
preincubation of monolayers with control medium (CTR) or with PMA (1 µM) or EGF (200 ng/ml), as described in the legend for Fig. 10.
Separate sets of monolayers were preincubated (20 min) with 65 µM H-7
(+H7) before perfusion with CTR medium or with PMA. Data are expressed
as percent of control H+ efflux
rate observed at pHi 6.40 and are
means ± SD from total of 9 monolayers in 3 separate experiments.
* Significantly different (P < 0.05) from respective bracketed bar.
|
|
On the other hand, the presence of EGF in the AP and BL superfusates
resulted in a small but significant increase of the initial rate of
intracellular alkalinization. As shown in Fig.
10B, EGF stimulated the exchange rate
by ~19% over control (from 326 to 385 µM
H+/s at
pHi 6.40). The average stimulation
observed in three separate experiments was 18 ± 6% over control
(Fig. 11). Similarly small but significant stimulation of NHE3 activity
by EGF was observed at 7 PCD (21 ± 5% over control, means ± SD, n = 8 monolayers). Exposure to EGF
resulted in a small but significant increase in the length of the BB
[4.2 ± 0.6 µm in control cells vs. 5.5 ± 0.6 (SD) µm in EGF-treated cells, 17 PCD,
P < 0.01, n = 30 cells from 5 monolayers for
each condition].
 |
DISCUSSION |
In this report we present data indicating that NHE3 is endogenously
expressed exclusively at the BB of the human colonic adenocarcinoma cells Caco-2. The magnitude of the AP
Na+/H+
exchange due to NHE3 activity increased gradually between 3 and 22 PCD
in clones PF-11 and ATCC of Caco-2 cells, the increase being a result
of progressively increasing number of NHE3-expressing cells. This was
in contrast with clone TC-7, in which NHE3 contributed only marginally
to the total AP
Na+/H+
exchange up to 22 PCD examined. In all three clones, the housekeeping isoform NHE1 was present predominantly at the BL membrane domain and,
to much lesser degree, also at the AP domain. The AP expression of NHE1
decreased dramatically during the postconfluent differentiation of
clones PF-11 and ATCC, and it contributed only 4-7% to the total
BB exchange activity in the mature monolayers. In TC-7 clone, however,
NHE1 was responsible for the majority of the AP
Na+/H+
exchange, even in mature monolayers. Neither clone expressed, in our
hands, detectable amounts of NHE2. Stimulation of the PKC signal
transduction pathway by PMA resulted in an inhibition of NHE3 activity
in clones expressing significant amounts of NHE3 at the BB (ATCC and
PF-11), whereas exposure to EGF resulted in small but significant
stimulation of the exchanger's activity.
Our studies demonstrate a gradual development of the functional
expression of endogenous NHE3 at the BB of Caco-2 cells, which increased almost fivefold during 22 days postconfluency. This pattern
closely resembled the previously reported gradual development of
expression of sucrase-isomaltase, lactase, aminopeptidase N, and
alkaline phosphatase at the BB of Caco-2 cells (46). Two major
mechanistic scenarios could explain the phenomenon of time-dependent increase in morphological and functional expression of NHE3 in the
examined Caco-2 clones. In the first scenario, all the cells gradually,
and in parallel, differentiate into a mature, polarized phenotype. In
such a situation, the majority of the cells in the immediate
postconfluent period would exhibit a poorly differentiated phenotype
with little "per cell" expression of NHE3. In the second scenario, the gradual increase in the AP NHE3 activity of the cultured
population as a whole would occur by a progressively increasing number
of cells expressing the exchanger at the BB. Data presented in this
communication support the second possibility. Indeed, with increasing
time postconfluency, we observed an increasing amount of BB NHE3
(biotinylation experiments), which paralleled an increasing amount of
total cellular NHE3 and an increase in NHE3 activity at the BB, but
which was not accompanied by any significant increase in the cell
number (Fig. 9). Thus it seems that once Caco-2 cells were capable of
expressing NHE3, both the targeting and regulatory mechanisms were
fully operational, and it was the increasing number of the
phenotypically differentiated cells which was solely responsible for
the time-related increase in the monolayers' NHE3 activity and NHE3
content. Similar conclusions were drawn by Vachon et al. (46) to
explain the time-dependent increase in expression of BB hydrolases in
Caco-2 cells. The authors suggested that the gradual increase of
activity of hydrolases reflected the changing "transient mosaic
pattern" of expression, resulting from a steadily increasing number
of functionally differentiated cells in the cultured population. Some
evidence suggests that this transient mosaic pattern results from an
uncoordinated pattern of expression of genes involved in some aspects
of differentiation of Caco-2 cells, one result of which might be a
mosaic deposition of heterotrimeric laminin (a basement membrane
component essential for terminal differentiation of enterocytes) (45).
The latter hypothesis is supported by our observation that the presence
of exogenous laminin significantly accelerated the development of NHE3
expression in PF-11 and ATCC clones (Table 2).
The comparison of NHE3 expression with that of BB hydrolases, and
especially sucrase-isomaltase, should be done cautiously, however.
Although expression of sucrase-isomaltase was reported to be
severalfold higher in late TC-7 clone than in early PF-11 clone (7),
the opposite was observed by us in respect to NHE3 expression. Clearly,
many factors, including amount and composition of the deposited
extracellular matrix, differential gene expression in various clones,
and even the passage number of the same clone, are involved in the in
vitro differentiation of Caco-2 cells. Therefore, in this study the
term "differentiation" has been replaced by the term
"maturation" whenever we discussed the phenomenon of
time-dependent increase in the overall expression of NHE3 by Caco-2
monolayers. However, this in vitro phenomenon may, indeed, reflect the
changes actually occurring during enterocyte differentiation along the
crypt-villus axis. A mosaic pattern of expression of BB hydrolases and
some other BB proteins has been described during development of the gut
in rats and humans (3, 39), as well as in certain pathological
conditions in humans (25, 29). Moreover, NHE3 was found predominantly
in the BB of villus and upper crypt epithelial cells and only in small
amounts in the lower crypt cells of jejunum and ileum in humans, thus
supporting the notion that the gradually increasing expression of some
BB enzymes and transport proteins parallels some aspects of terminal enterocyte differentiation.
The pattern of development of functional expression of NHE1 at the BL
membrane domain reported here did not parallel that of NHE3. The
expression of NHE1 was maximal at ~5-7 PCD, shortly after the
monolayers reached a maximal cell density. Therefore, the expression of
NHE1 during Caco-2 in vitro maturation seems to be regulated
differently from that of NHE3, and NHE1 is rather ubiquitously
expressed in these cells already at the early stages of postconfluent
growth. This conclusion is supported by the observation that
immunoreactive NHE1 was detected at the BL membrane domain of all cells
as early as 3 PCD (data not shown). NHE1 was also targeted to AP
membrane domain, although both the absolute and relative amount of this
isoform at the BB dramatically decreased during postconfluent
maturation. It is unlikely that the BB amiloride-sensitive Na+/H+
exchange activity was contaminated with, or represented by, an endogenous NHE2, because no NHE2 protein was detected in these monolayers by Western analysis (Fig. 2). Although NHE1 has not been
detected in the BB of native intestinal epithelia examined so far, such
"mistargeting" of other basolaterally targeted proteins in in
vitro epithelial cell models as well as in human kidney epithelium
(polycystic kidney disease) has been described (11, 20, 32, 49).
Nevertheless, in mature Caco-2 cells only ~4% of total NHE1 activity
was localized at the BB. This is in contrast with a much higher
proportion of apically targeted NHE1 found in Caco-2 cells as well as
in Madin-Darby canine kidney (MDCK) and HT-29 by Noel et al.
(32). The reason for this discrepancy is not clear.
In contrast to NHE1, both functional and morphological expression of
NHE3 was exclusively AP in all three Caco-2 clones examined. This
exclusively AP functional expression of NHE3 paralleled the subcellular
distribution of the exchanger molecules, as indicated by the results of
immunofluorescent studies as well as cell surface biotinylation.
Although we never found NHE3 at the BL membrane domain of the three
Caco-2 clones studied, the magnitude of the AP expression of this
isoform was significantly smaller in TC-7 cells than in the other two
clones. This observation suggests differential regulation of expression
of BB proteins among various Caco-2 clones. Our data emphasize the need
for a careful characterization of a studied Caco-2 clone with respect
to the expression of the protein(s) of interest. Full expression of
these proteins may require relatively long postconfluent growing time
and, additionally, the magnitude of expression may differ significantly
among clones. It is important to notice that the significant
differences in expression of NHE3 between clone TC-7 and the other two
clones examined occurred in otherwise morphologically
well-differentiated cells (7). Moreover, although TC-7 cells expressed
significantly less NHE3 than the PF-11 clone, they were previously
reported to express more sucrase-isomaltase and dipeptidylpeptidase IV (7). Despite these differences in the magnitude of NHE3 expression among the clones, NHE3 has not been targeted to the BL membrane domain
in any clone and at any time postconfluency, a finding consistent with
results of in vivo studies, in which NHE3 was exclusively found at the
BB of ileal and colonic epithelium in human, rat, and rabbit (5, 14).
The presence of NHE3 at the BB of Caco-2 cells has been reported by us
previously using morphological and biochemical approaches (15). Also,
the activity of an endogenous Na+/H+
exchanger with NHE3 characteristics at the AP surface of Caco-2 cells
has been suggested by results reported earlier by Osypiw et al. (34).
Using a fluorometric method, the authors observed the activity of a
relatively amiloride-resistant
(IC50 287 µM) Na+/H+
exchanger at the BB of Caco-2 cells, which most probably represented NHE3. The authors also observed a
Na+/H+
exchanger at the BL membrane domain of Caco-2 cells, which probably represented NHE1, although no details concerning activity of this exchanger at low amiloride concentrations was reported. In contrast, no
evidence of
Na+/H+
exchange at the BB of Caco-2 monolayers (ATCC clone) was reported by
Watson and colleagues (48) from our laboratory. These authors described
the presence of an amiloride-sensitive
Na+/H+
exchanger (NHE1) at the BL membrane domain of Caco-2 monolayers, but
they did not observe any significant
Na+/H+
exchange at the AP domain. One possible explanation for this discrepancy is that these studies were conducted at an early
postconfluent period, when relatively little endogenous NHE3 activity
is present at the BB. More recently, McSwine and colleagues (26)
characterized a clonal line of Caco-2 cells (C2/bbe), which also lacked
endogenous NHE3 expression. These data suggest that a subpopulation of
cells with no or with a very small capacity for NHE3 expression did exist in the originally derived Caco-2 adenocarcinoma cell line and
that therefore some clones obtained by dilutional ("single cell")
cloning may remain incapable of expressing a significant quantity of
the exchanger. This hypothesis is supported by our observation that
~10% of all cells in the PF-11 clone and ~17% of all cells in the
ATCC clone did not express NHE3 for up to 30 PCD, and that very low
levels of NHE3 protein and activity were found in some single cell
clones obtained in our laboratory from the PF-11 cell clone (data not
shown). Finally, although the presence of exogenous laminin
significantly accelerated development of NHE3 expression in PF-11 and
ATCC clones, laminin was not capable of inducing NHE3 expression in all
the cells in the cultured populations. Moreover, laminin had no effect
on NHE3 expression in TC-7 cells. These findings would suggest an early
block in NHE3 expression in some cells, even within clonal populations,
a block that could not be reversed simply by increasing duration of
culture or by the presence of exogenous laminin.
The activity of endogenous NHE3 in Caco-2 cells was inhibited by PMA,
and it was stimulated by EGF. We believe that the observed inhibitory
effect of PMA on NHE3 was due to stimulation of PKC activity, because
it was abolished by H-7, a PKC inhibitor. We recently reported that
~50% of the PKC-induced inhibition of NHE3 in Caco-2 cells is due to
redistribution of the exchanger molecules from the BB into the
subapical cytoplasmic compartment (15). Magnitude of the NHE3
inhibition (~28%) resembles that reported for other cell types,
including OK cells, PS120 fibroblasts, gallbladder epithelial cells,
and Caco-2 cells transfected with NHE3 cDNA (2, 13, 21, 26, 40). On the
other hand, the stimulatory effect of EGF, although statistically
significant, was of lower magnitude than that reported for fibroblast
growth factor in PS120 fibroblasts (22, 51). One possible reason for
the relatively small stimulatory effect of EGF could be the autocrine
downregulation of the EGF receptors by transforming growth factor-
, a growth factor reported to be secreted by Caco-2 cells
(1). Such a downregulation of EGF receptors was shown
previously in type 2 pneumocytes (30), and one preliminary report
suggests a similar phenomenon occurring during postconfluent
differentiation of Caco-2 cells (19). We attempted to answer this
question by pretreatment of PF-11 Caco-2 monolayers with suramin (1 mM), the polyanionic compound that complexes many growth factors and
thus may prevent downregulation of the growth factor receptors (30).
However, this approach did not result in a significant increase of the stimulatory effect of EGF on NHE3 activity (data not shown).
Importantly, the observed inhibitory effect of PMA and stimulatory
effect of EGF on NHE3 activity in Caco-2 cells resembled those
described for native ileal
Na+-absorbing epithelial cells. In
the rabbit ileum, carbachol was shown to inhibit neutral NaCl
absorption, which was associated with an asymmetric increase of PKC
activity at the BB of ileal epithelium, translocation of phospholipase
C (PLC)-
to the BB, and activation of AP, but not BL,
phosphatidylinositol 4,5-bisphosphate-PLC activity (16).
This translocation of PKC to the BB and an increase in the kinase
activity strongly support the hypothesis that, at least in the
intestine, the PKC signal transduction pathway is involved in the
inhibitory regulation of the NHE3. On the other hand, EGF has been
shown to stimulate NaCl absorption and NHE3 activity in native ileal
epithelium, a process requiring an increased activity of
phosphotidylinositol 3-kinase (PI3K) (17). A similar dependence of EGF
stimulation of NHE3 on PI3K activity has also been reported for Caco-2
cells transfected with rabbit NHE3 (17). However, the molecular
mechanism of the acute stimulation of NHE3 by EGF in intestinal
epithelial cells remains unclear. Because the stimulation is caused by
changes in Vmax
(21), increased number of NHE3 molecules within the BB membrane is a
highly probable mechanism. This in turn could result from increase rate
of exocytic insertion of NHE3 molecules into the BB, from decreased
rate of endocytic retrieval of the exchanger from the BB, or both. The mechanism involving a rapid subcellular redistribution of NHE3 is
suggested by the results published by Hardin and colleagues (12). The
authors observed a significant increase in BB height and surface area
after acute exposure of rabbit jejunal epithelium to EGF, which
suggested a rapid, EGF-induced, cytoskeletal rearrangement and/or
exocytic insertion of preformed membranes into the BB. In our hands,
exposure to EGF resulted in a small but significant increase in the BB
height in Caco-2 cells, an observation that would support the
redistribution hypothesis. It is not clear, however, why we did not
observe a decrease in the BB height after exposure of the cells to PMA,
in which case endocytic retrieval is known to take place (15). One
possibility is that the changes in surface area of the BB after
exposure to PMA might be too small to be detected just by measuring the
monolayer height using optical microscopy. Alternatively, surface area
might not change following exposure to PMA even with removal of BB
membrane if compensatory changes in the BB cytoskeleton occur, as has
been reported previously (28, 37, 43).
In conclusion, our data demonstrate that the PF-11 and ATCC clones of
Caco-2 cells represent a promising "physiological" model for
studying the development and regulation of endogenous NHE3 in the
intestinal epithelium. In mature monolayers, both the polarized AP
expression of NHE3 as well as regulation of the exchanger by PKC and
receptor tyrosine kinase signal transduction pathways closely resemble
the behavior of endogenous NHE3 in the ileal and colonic epithelia. The
fact that Caco-2 cells are derived from human colonic epithelium adds
value to this in vitro model, because studies on native human
intestinal epithelium are limited for many reasons.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Ann Hubbard for advice and critique during the
development of the project. We also thank Dr. Shaoyou Chu and Greg
Martin for expert advice and help with the confocal microscopy and
immunostaining, and Dr. Hans-Jochen Lang from Hoechst for providing us
with HOE-694 compound.
 |
FOOTNOTES |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grants RO1-DK-26523, PO1-DK-44484, R29-DK-43778, T32-DK-07632, and K08-DK-02557; the Meyerhoff Digestive Diseases Center; and the Hopkins Center for Epithelial Disorders.
Part of this work was presented at Digestive Disease Week (Washington,
DC, May 10-16, 1997), and was published in abstract form
(Gastroenterology 112: A372, 1997).
Present addresses: M. H. Montrose, Dept. of Physiology, Univ. of
Indiana School of Medicine, Indianapolis, IN 46202-5120; F. Sanchez de
Medina, Dept. of Pharmacology, Univ. of Granada, 18071 Granada, Spain.
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: M. Donowitz,
Johns Hopkins Univ., School of Medicine, Division of Gastroenterology,
918 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205 (E-mail:
mdonowit{at}welchlink.welch.jhu.edu).
Received 13 November 1998; accepted in final form 15 April 1999.
 |
REFERENCES |
1.
Anzano, M. A.,
D. Rieman,
W. Prichett,
D. F. Bowen-Pope,
and
R. Greig.
Growth factor production by human colon carcinoma cell lines.
Cancer Res.
49:
2898-2904,
1989[Abstract].
2.
Azarani, A.,
D. Goltzman,
and
J. Orlowski.
Parathyroid hormone and parathyroid hormone-related peptide inhibit the apical Na+/H+ exchanger NHE-3 isoform in renal cells (OK) via a dual signaling cascade involving protein kinase A and C.
J. Biol. Chem.
270:
20004-20010,
1995[Abstract/Free Full Text].
3.
Beaulieu, J. F.,
P. H. Vachon,
and
S. Chartrand.
Immunolocalization of extracellular matrix components during organogenesis in the human small intestine.
Anat. Embryol. (Berl.)
183:
363-369,
1991[Medline].
4.
Biemesderfer, D.,
P. A. Rutherford,
T. Nagy,
J. H. Pizzonia,
A. K. Abu-Alfa,
and
P. S. Aronson.
Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney.
Am. J. Physiol.
273 (Renal Physiol. 42):
F289-F299,
1997[Abstract/Free Full Text].
5.
Bookstein, C.,
A. M. DePaoli,
Y. Xie,
P. Niu,
M. W. Musch,
M. C. Rao,
and
E. B. Chang.
Na+/H+ exchangers, NHE-1 and NHE-3, of rat intestine. Expression and localization.
J. Clin. Invest.
93:
106-113,
1994[Medline].
6.
Boyarsky, G.,
M. B. Ganz,
R. B. Sterzel,
and
W. F. Boron.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3.
Am. J. Physiol.
255 (Cell Physiol. 24):
C844-C856,
1988[Abstract/Free Full Text].
7.
Chantret, I.,
A. Rodolosse,
A. Barbat,
E. Dussaulx,
E. Brot-Laroche,
A. Zweibaum,
and
M. Rousset.
Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation.
J. Cell Sci.
107:
213-225,
1994[Abstract/Free Full Text].
8.
Counillon, L.,
W. Scholz,
H. J. Lang,
and
J. Pouyssegur.
Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties.
Mol. Pharmacol.
44:
1041-1045,
1993[Abstract].
9.
Donowitz, M.,
S. Levine,
C. Yun,
S. Brant,
S. Nath,
J. Yip,
S. Hoogerwerf,
J. Pouyssegur,
and
C. Tse.
Molecular studies of members of the mammalian Na/H exchanger gene family.
In: Molecular Biology of Membrane Transport Disorders, edited by S. G. Schultz,
T. E. Andreoli,
A. M. Brown,
D. M. Fambrough,
J. F. Hoffman,
and M. J. Welsh. New York: Plenum, 1996, p. 259-275.
10.
Donowitz, M.,
and
M. J. Welsh.
Regulation of mammalian small intestinal electrolyte secretion.
In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1987, p. 1351-1388.
11.
Hammerton, R. W.,
K. A. Krzeminski,
R. W. Mays,
T. A. Ryan,
D. A. Wollner,
and
W. J. Nelson.
Mechanism for regulating cell surface distribution of Na+,K(+)-ATPase in polarized epithelial cells (see comments).
Science
254:
847-850,
1991[Medline].
12.
Hardin, J. A.,
A. Buret,
J. B. Meddings,
and
D. G. Gall.
Effect of epidermal growth factor on enterocyte brush-border surface area.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G312-G318,
1993[Abstract/Free Full Text].
13.
Helmle-Kolb, C.,
M. H. Montrose,
G. Stange,
and
H. Murer.
Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters.
Pflügers Arch.
415:
461-470,
1990[Medline].
14.
Hoogerwerf, W. A.,
S. C. Tsao,
O. Devuyst,
S. A. Levine,
C. H. C. Yun,
J. W. Yip,
M. E. Cohen,
P. D. Wilson,
A. J. Lazenby,
C. M. Tse,
and
M. Donowitz.
NHE2 and NHE3 are human and rabbit intestinal brush-border proteins.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G29-G41,
1996[Abstract/Free Full Text].
15.
Janecki, A. J.,
M. H. Montrose,
P. Zimniak,
A. Zweibaum,
C. M. Tse,
S. Khurana,
and
M. Donowitz.
Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger.
J. Biol. Chem.
273:
8790-8798,
1998[Abstract/Free Full Text].
16.
Khurana, S.,
S. Kreydiyyeh,
A. Aronzon,
W. A. Hoogerwerf,
S. G. Rhee,
M. Donowitz,
and
M. E. Cohen.
Asymmetric signal transduction in polarized ileal Na(+)-absorbing cells: carbachol activates brush-border but not basolateral-membrane PIP2-PLC and translocates PLC-
1 only to the brush border.
Biochem. J.
313:
509-518,
1996[Medline].
17.
Khurana, S.,
S. K. Nath,
S. A. Levine,
J. M. Bowser,
C. M. Tse,
M. E. Cohen,
and
M. Donowitz.
Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na+/H+ exchange.
J. Biol. Chem.
271:
9919-9927,
1996[Abstract/Free Full Text].
18.
Khurana, S.,
C. M. Tse,
J. Teng,
and
M. Donowitz.
Ileal brush border actin-binding protein villin associates with the brush border sodium-hydrogen exchanger NHE3 (Abstract).
Gastroenterology
114:
G1579,
1998.
19.
Kuwada, S. K.,
X. F. Li,
and
H. S. Wiley.
The regulation of epidermal growth factor receptor expression in polarized intestinal epithelial cells (Abstract).
Gastroenterology
112:
A1165,
1997.
20.
Kuwahara, M.,
S. Sasaki,
S. Uchida,
E. J. Cragoe, Jr.,
and
F. Marumo.
Different development of apical and basolateral Na-H exchangers in LLC-PK1 renal epithelial cells: characterization by inhibitors and antisense oligonucleotide.
Biochim. Biophys. Acta
13:
132-138,
1994.
21.
Levine, S. A.,
M. H. 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].
22.
Levine, S. A.,
S. K. Nath,
C. H. Yun,
J. W. Yip,
M. Montrose,
M. Donowitz,
and
C. M. Tse.
Separate C-terminal domains of the epithelial specific brush border Na+/H+ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth factors.
J. Biol. Chem.
270:
13716-13725,
1995[Abstract/Free Full Text].
23.
Maher, M. M.,
J. D. Gontarek,
R. S. Bess,
M. Donowitz,
and
C. J. Yeo.
The Na+/H+ exchange isoform NHE3 regulates basal canine ileal Na+ absorption in vivo.
Gastroenterology
112:
174-183,
1997[Medline].
24.
Maher, M. M.,
J. D. Gontarek,
R. E. Jimenez,
and
C. J. Yeo.
The role of brush border Na+/H+ exchange in canine ileal absorption.
Dig. Dis. Sci.
41:
651-659,
1996[Medline].
25.
Maiuri, L.,
V. Raia,
J. Potter,
D. Swallow,
M. W. Ho,
R. Fiocca,
G. Finzi,
M. Cornaggia,
C. Capella,
and
A. Quaroni.
Mosaic pattern of lactase expression by villous enterocytes in human adult-type hypolactasia.
Gastroenterology
100:
359-369,
1991[Medline].
26.
McSwine, R. L.,
M. W. Musch,
C. Bookstein,
Y. Xie,
M. Rao,
and
E. B. Chang.
Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe.
Am. J. Physiol.
275 (Cell Physiol. 44):
C693-C701,
1998[Abstract].
27.
Montrose, M. H.,
and
H. Murer.
Measurement of intracellular pH in single LLC-PK1 cells: recovery from an acid load via basolateral Na/H exchange.
J. Biol. Chem.
97:
63-78,
1987.
28.
Myat, M. M.,
S. Anderson,
L. A. Allen,
and
A. Aderem.
MARCKS regulates membrane ruffling and cell spreading.
Curr. Biol.
7:
611-614,
1997[Medline].
29.
Nichols, B. L.,
F. Carrazza,
V. N. Nichols,
M. Putman,
P. Johnston,
M. Rodrigues,
A. Quaroni,
and
M. Shiner.
Mosaic expression of brush-border enzymes in infants with chronic diarrhea and malnutrition.
J. Pediatr. Gastroenterol. Nutr.
14:
371-379,
1992[Medline].
30.
Nici, L.,
M. Medina,
and
A. R. Frackelton.
The epidermal growth factor receptor network in type 2 pneumocytes exposed to hyperoxia in vitro.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L242-L250,
1996[Abstract/Free Full Text].
31.
Noel, J.,
and
J. Pouyssegur.
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].
32.
Noel, J.,
D. Roux,
and
J. Pouyssegur.
Differential localization of Na/H exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines.
J. Cell Sci.
109:
929-939,
1996[Abstract/Free Full Text].
33.
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].
34.
Osypiw, J. C.,
D. Gleeson,
R. W. Lobley,
P. W. Pemberton,
and
R. F. McMahon.
Acid-base transport systems in a polarized human intestinal cell monolayer: Caco-2.
Exp. Physiol.
79:
723-739,
1994[Abstract].
35.
Paillard, M.
Na+/H+ exchanger subtypes in the renal tubule: function and regulation in physiology and disease.
Exp. Nephrol.
5:
277-284,
1997[Medline].
36.
Park, K.,
J. A. Olschowka,
L. A. Richardson,
C. Bookstein,
E. B. Chang,
and
J. E. Melvin.
Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells.
Am. J. Physiol.
276 (Gastrointest. Liver Physiol. 39):
G470-G478,
1999[Abstract/Free Full Text].
37.
Perez, M.,
A. Barber,
and
F. Ponz.
Modulation of intestinal paracellular permeability by intracellular mediators and cytoskeleton.
Can. J. Physiol. Pharmacol.
75:
287-292,
1997[Medline].
38.
Pinto, M.,
S. Robine-Leon,
M. Appay,
M. Kedinger,
N. Triadou,
E. Dussaulx,
B. Lacroix,
P. Simon-Assman,
K. Haffen,
J. Fogh,
and
A. Zweibaum.
Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture.
Biol. Cell.
47:
323-330,
1983.
39.
Rubin, D. C.
Spatial analysis of transcriptional activation in fetal rat jejunal and ileal gut epithelium.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G853-G863,
1992[Abstract/Free Full Text].
40.
Silviani, V.,
V. Colombani,
L. Heyries,
A. Gerolami,
G. Cartouzou,
and
C. Marteau.
Role of the NHE3 isoform of the Na+/H+ exchanger in sodium absorption by the rabbit gallbladder.
Pflügers Arch.
432:
791-796,
1996[Medline].
41.
Tse, C. M.,
S. A. Levine,
C. H. Yun,
S. Khurana,
and
M. Donowitz.
Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein.
Biochemistry
33:
12954-12961,
1994[Medline].
42.
Tse, C. M.,
A. I. Ma,
V. W. Yang,
A. J. Watson,
S. Levine,
M. H. Montrose,
J. Potter,
C. Sardet,
J. Pouyssegur,
and
M. Donowitz.
Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.
EMBO J.
10:
1957-1967,
1991[Abstract].
43.
Vaaraniemi, J.,
V. Huotari,
V. P. Lehto,
and
S. Eskelinen.
Effect of PMA on the integrity of the membrane skeleton and morphology of epithelial MDCK cells is dependent on the activity of amiloride-sensitive ion transporters and membrane potential.
Eur. J. Cell Biol.
74:
262-272,
1997[Medline].
44.
Vachon, P. H.,
and
J. F. Beaulieu.
Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line.
Gastroenterology
103:
414-423,
1992[Medline].
45.
Vachon, P. H.,
and
J. F. Beaulieu.
Extracellular heterotrimeric laminin promotes differentiation in human enterocytes.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G857-G867,
1995[Abstract/Free Full Text].
46.
Vachon, P. H.,
N. Perreault,
P. Magny,
and
J. F. Beaulieu.
Uncoordinated, transient mosaic patterns of intestinal hydrolase expression in differentiating human enterocytes.
J. Cell. Physiol.
166:
198-207,
1996[Medline].
47.
Wakabayashi, S.,
M. Shigekawa,
and
J. Pouyssegur.
Molecular physiology of vertebrate Na/H exchangers.
Physiol. Rev.
77:
51-74,
1997[Abstract/Free Full Text].
48.
Watson, A. J. M.,
S. Levine,
M. Donowitz,
and
M. H. Montrose.
Kinetics and regulation of polarized Na+/H+ exchanger from Caco-2 cells, a human intestinal cell line.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G229-G239,
1991[Abstract/Free Full Text].
49.
Wilson, P. D.,
A. C. Sherwood,
K. Palla,
J. Du,
R. Watson,
and
J. T. Norman.
Reversed polarity of Na(+)-K(+)-ATPase: mislocation to apical plasma membranes in polycystic kidney disease epithelia.
Am. J. Physiol.
260 (Renal Fluid Electrolyte Physiol. 29):
F420-F430,
1991[Abstract/Free Full Text].
50.
Yeo, C. J.,
K. Barry,
J. D. Gontarek,
and
M. Donowitz.
Na+/H+ exchange mediates meal-stimulated ileal absorption.
Surgery
116:
388-394,
1994[Medline].
51.
Yip, J. W.,
W. H. Ko,
G. Viberti,
R. L. Huganir,
M. Donowitz,
and
C. M. Tse.
Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger.
J. Biol. Chem.
272:
18473-18480,
1997[Abstract/Free Full Text].
Am J Physiol Gastroint Liver Physiol 277(2):G292-G305
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society