From the Departments of Medicine and Physiology, Division of
Gastroenterology, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205, the 
Department of Medicine,
University of Arkansas for Medical Sciences, Little Rock, Arkansas
72205, and § Unité de Recherches sur la
Différenciation Cellulaire Intestinale, INSERM U178, 94807 Villejuif Cedex, France
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INTRODUCTION |
Of the five isoforms of mammalian Na+/H+
exchangers (NHEs)1 fully cloned to date,
the two characterized in most detail are NHE1 and NHE3. NHE1 is present
in nearly all mammalian cells and is involved in regulation of
cytoplasmic pH, volume, and perhaps cell proliferation (1, 2). In most
native mammalian epithelia, NHE1 is found in the basolateral membrane
domain. The other well characterized isoform, NHE3, has been found in
some segments of small and large intestine and renal tubules, where it
has been localized exclusively to the apical membrane domain (3-5).
NHE3 plays a role in net NaCl, HCO3, and probably
NH4 reabsorption in renal tubules and in neutral NaCl
absorption in the intestine. In dogs, NHE3 accounts for all basal ileal
Na+ absorption and the neurohormonal-induced increase
in ileal Na+ absorption that occurs after meals (6, 7). In
the rabbit ileal brush border (BB), NHE3 contributes approximately 50%
of the basal Na+/H+ exchange, the rest being
contributed by NHE2 (8).
Regulation of NHE1 is generally modulated by changes in
K'(H+) (i.e. the exchanger's
affinity for intracellular H+ ions) (9, 10). In contrast,
the activity of NHE3 is regulated mainly by changes in maximal velocity
(Vmax) of the exchanger. Growth factors, serum,
okadaic acid, and several calmodulin blockers all increase
Vmax of NHE3, whereas agents that increase
protein kinase C (PKC) activity (phorbol ester, carbachol) decrease
Vmax (11-13). Phorbol 12-myristate 13-acetate
(PMA) and/or PKC have been shown to inhibit endogenous as well as
transfected NHE3 activity in a variety of cells including rabbit
gallbladder epithelium (14), the opossum kidney (OK) cell line (15,
16), Chinese hamster ovary (AP-1) cell line (17, 18), and Chinese
hamster lung fibroblastic cell line PS120 (11, 13). Since the
inhibition occurs via a decrease in Vmax and is
observed within minutes, this type of control might theoretically be
achieved by a decrease in the number of active molecules at the BB (a
redistribution-dependent mechanism and/or rapid
degradation), by changes in the turnover number of individual exchanger
molecules, or both.
Recently, multiple plasma membrane transport proteins have been shown
to be regulated, at least in part, by cellular redistribution (endocytic retrieval from and/or exocytic insertion into the plasma membrane). Well characterized examples include glucose transporters GLUT1 and GLUT4 in adipocytes (19), the
K+,H+-ATPase pump in gastric parietal cells
(20), the water channel aquaporin 2 in the kidney (21), the renal
NaPi-2 cotransporter (22), the Na+/glucose cotransporter
SGLT1 (23), the renal Na+,K+-ATPase pump (24),
and the cystic fibrosis transmembrane conductance regulator (CFTR)
(25). Much less is known, however, about the mechanisms of short term
regulation of NHE3. The acute regulation, via growth factors and
protein kinases, may be important in rapid adjustments of changing
intraluminal sodium concentration in renal tubules and in altering
Na+ absorption in the intestine and the kidney. Some
evidence indicates that translocation from BB into the cytoplasm might
be involved in the short term inhibition of NHE3 in renal proximal
tubules, although the specific nature of the cytoplasmic vesicular
compartment containing the internalized NHE3 has not been defined (26,
27). Until now, no results have addressed the possible contribution of
the redistribution-based mechanism in the regulation of intestinal NHE3.
The results presented in this study indicate that acute inhibition of
NHE3 activity by PKC in the human colon adenocarcinoma cell line Caco-2
involves redistribution of exchanger molecules from the BB into a
subapical cytoplasmic compartment. A comparison of the effect of PMA on
NHE3 activity with the exchanger's redistribution suggests that
approximately half of the inhibitory effect of PMA is exerted via
retrieval of NHE3 molecules from the BB.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human colonic adenocarcinoma cell line Caco-2,
clone PF-11, was characterized previously (28) and was recently found
to express endogenous NHE3 (29). NHE3 is present exclusively at the BB,
and its activity peaks at 17-22 days postconfluency. At that time,
NHE3 constitutes 85-90% of total BB Na+/H+
exchange with only marginal contribution of NHE1. For all experiments described in this report, Caco-2 cells were plated at a density 5-8 × 104 cells/cm2 on a 0.45-µm pore
size, HD Falcon PET membranes (Becton Dickinson Labware, Franklin
Lakes, NJ) and grown in Dulbecco's modified Eagle's medium
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. For evaluation of
Na+/H+ exchange activity by fluorometry, cells
were grown on small fragments of polyethylene terephtalate (PET)
membranes glued over apertures in plastic coverslips
("filterslips") as described previously (30). In all experiments,
the monolayers were investigated at 17-22 days postconfluency.
Measurement of Na+/H+ Exchange
Rate--
For functional evaluation of apical (AP) and basolateral
(BL) Na+/H+ exchange in Caco-2 cells,
intracellular pH (pHi) was measured by a fluorometric method
based on cytosolic loading with a pH-sensitive fluorophore,
acetoxymethyl ester of 2',7'-bis(carboxyethyl)5-6-carboxyfluorescein (BCECF) (Molecular Probes, Eugene, OR), as described in detail elsewhere (30, 31). Briefly, monolayers grown on filterslips were
serum-starved for 8-12 h and incubated with BCECF-AM (5 µM) in the presence of 50 mM
NH4Cl (to promote subsequent intracellular acidification)
for 50 min at room temperature. Filterslips were then mounted in a
cuvette, which enabled separate AP and BL perfusion of the monolayers.
The cuvette was inserted into the fluorometer (SPF 500C, SLM, Urbana,
IL) and initially superfused at both surfaces with Na+-free
buffer (containing 130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM tetramethylammonium
phosphate (TMA)2PO4, 25 mM glucose,
20 mM HEPES, pH 7.4). Then one surface of the monolayer was
exposed to Na+ buffer in which tetramethylammonium chloride
was replaced by 130 mM NaCl. Rates of
Na+-dependent intracellular alkalinization
(efflux of H+, in µM/s) were calculated for a
given pHi as the product of 
pH/T and buffering
capacity using the Enzfitter software (Biosoft Corp.) (31). The
buffering capacity of Caco-2 cells was evaluated in a separate series
of experiments, as described previously (32). To differentiate between
NHE1 and NHE3 activity at a given monolayer surface we used an
inhibitor of Na+/H+ exchange HOE694 (kindly
provided by Dr. H. Lang, Hoechst, Germany). The compound's
Ki for NHE3 is 650 µM, whereas its
Ki for NHE1 (also present in Caco-2 cells) is only
0.15 µM (33). Complete inhibition of NHE1 without any
effect on NHE3 activity is observed with HOE694 concentrations of
10-20 µM (33, 34). In all experiments, 20 µM HOE694 was present in both AP and BL superfusates.
Inhibition of NHE2 activity was unnecessary, since the PF-11 clone of
Caco-2 cells has no endogenous NHE2 (based on Northern analysis and
Western analysis using anti-NHE2 antibody (Ab) 597 (35).2
In experiments in which the effect of PMA on the
Na+/H+ exchange rate was evaluated, phorbol
ester (phorbol 12-myristate 13-acetate; 1 µM; Sigma) was
added for the last 20 min of incubation with BCECF and was then present
in all superfusion buffers (both AP and BL) at the same concentration.
In a separate set of experiments, cells were acutely exposed to 1 µM PMA for 5, 10, 20, and 40 min before measuring the
rate of intracellular alkalinization in the presence of apical
Na+ (130 mM). In all of the above experiments,
separate groups of monolayers were exposed to PMA in the presence of
the PKC inhibitor H7 (65 µM; Seikagaku Kogyo Co., Tokyo,
Japan), which was preceded by 20 min of preincubation with H7
alone.
Immunolocalization of NHE3 and BB Labeling--
For
immunolocalization of NHE3, Caco-2 monolayers were rinsed twice with
Dulbecco's PBS and fixed in 3% paraformaldehyde (Sigma) in PBS, pH
7.4, for 45 min. BB was labeled by incubating the monolayers with
FITC-conjugated lectin PHA-E (FITC/PHA; 50 µg/ml; Sigma) for 30 min.
Following extensive washing, the permeabilization and blocking of
nonspecific binding sites were performed in one step by incubating the
monolayers with PBS containing 0.075% saponin, 1% bovine serum
albumin (Goldmark Biologicals, Phillipsburg, NJ), and 15% normal goat
serum (Jackson Immunoresearch, West Grove, PA) (PBG buffer) for 45 min.
Monolayers were then incubated with anti-NHE3 polyclonal Ab 1380 (1:50
dilution) in PBG buffer for 1 h, followed by washing with PBS
containing 0.05% saponin and 1% bovine serum albumin. Anti-NHE3
polyclonal Ab 1380 has been previously shown to specifically recognize
rabbit NHE3 transfected into PS120 fibroblasts as well as endogenous
NHE3 in rabbit and human intestinal epithelia but not to cross-react
with NHE1 and NHE2 (5). For control of nonspecific staining, Ab 1380 was 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, West Grove,
PA) for 45 min, postfixed with 3% paraformaldehyde, and mounted in
50% glycerol in PBS containing 0.2% p-phenylendiamine as
an antiphotobleaching agent.
In a series of preliminary experiments, we tested the possibility that
incomplete penetration of FITC/PHA into the intermicrovillar domains of
the BB might affect the calculated intracellular limit of the BB. In
these experiments, we labeled the BB from within the cell by
fluorescently staining the actin cytoskeleton. Caco-2 monolayers were
fixed as described above, permeabilized, and incubated with
FITC-conjugated phalloidin (FITC/phalloidin; 0.6 µM;
Sigma) for 20 min at room temperature. Monolayers were rinsed twice
with PBS, and NHE3 was immunolabeled as described above. The rationale for this approach was based on the fact that nearly all of the actin
within the apical zone of polarized absorptive epithelial cells is
located within the microvilli, terminal web, and tight junctional
complexes.
All monolayers were examined in xy and xz planes
using a Zeiss LSM410 confocal fluorescence microscope as described
below.
Morphometric Confocal Analysis of Intracellular Distribution of
NHE3--
For morphometric confocal analysis, serum-starved Caco-2
monolayers were exposed to PMA (1 µM) or control medium
for 20 min at 37 °C. Cells were then cooled rapidly to 4 °C,
fixed with cold 3% paraformaldehyde, and stained as above. Using the
confocal microscope, serial optical sections were obtained parallel to the monolayer surface (xy plane) at 0.4-µm steps. FITC/PHA
and Cy3-labeled NHE3 were excited by separate lasers at 488 nm (argon laser) and 543 nm (HeNe laser), respectively. Emissions from FITC (505-530 nm) and Cy3 (580-660 nm) were collected sequentially (to
avoid detection of FITC in the Cy3 channel), and the images were stored
on an optical disc.
As discussed below, not all cells had adequate Cy3 fluorescence due to
heterogeneity of NHE3 expression. Therefore, only cells with
significant Cy3 signal (up to 70% of all cells; defined to be adequate
when exceeding the average background fluorescence intensity by more
than 4 times) were randomly chosen for analysis. Images of these cells
were analyzed using MetaMorph software (Universal Imaging Corp.) as
follows. Four separate areas of 5 × 5 pixels were randomly
selected at the surface of each analyzed cell, and pixel density in
these areas was determined in consecutive optical sections along the
apical-basal (xz) axis of the cell, separately for FITC and
Cy3. Obtained values were plotted against the optical section number.
The curves obtained for Cy3 (representing distribution of NHE3) were
divided into the part overlapping the BB (defined by the distribution
of PHA-E; see below) and the part corresponding to the subapical
cytoplasmic compartment (SAC). SAC was defined as an apical cytoplasmic
area containing Cy3 fluorescent signal and located between the BB and
the background fluorescence of the cytoplasm. Finally, the total
surface area under the Cy3 curve corresponding to BB plus SAC was
calculated (after subtracting the background fluorescence), and the
results were expressed as SAC/(BB + SAC) × 100. The obtained values
represented the relative amount of immunolabeled NHE3 located in SAC as
a percentage of total apical NHE3 amount (BB + SAC) and were,
therefore, independent of differences in longitudinal BB dimensions and
intensity of Cy3 fluorescence among the cells examined.
The apical and intracellular BB limits were defined as the positions at
which the BB signal decreased most rapidly during scanning along the
apical-basal axis. This was determined using an algorithm to localize
the maximum values of the second derivative of the FITC fluorescent
signal (BB-bound lectin or, in some experiments, phalloidin-labeled
cortical actin) at consecutive optical sections, after smoothing data
with the Savitzky-Golay method to decrease random noise (Microcal
Origin version 4.1, Microcal Software). The maximum values of the
second derivative curve indicated the maximal changes in the slopes of
the FITC curve and were assumed to correspond to the apical and
intracellular BB limits.
Since the definition of intracellular limit of the BB was critical for
the subsequent calculations, we used another way to verify the results
of the derivative approach. The corresponding FITC/PHA and Cy3/NHE3
curves were normalized to 100% maximal pixel density, and the ratio of
FITC versus Cy3 was calculated for each optical section. The
point at which the Cy3 values diverged from the intracellular portion
of the FITC curve (ratio of Cy3 versus FITC significantly
higher than 1.0) was calculated for each cell. This approach determined
the point of separation of the NHE3-related signal within the cytoplasm
from the BB-defining signal.
Evaluation of Redistribution of NHE3 from BB to the Cytoplasm by
Reversible Surface Biotinylation--
Reversible surface biotinylation
was used to complement the data obtained by confocal analysis, since
the results of biotinylation do not rely on calculated limits of the
BB. A modification of the procedure described by Le Bivic et
al. (37) was used, with omission of the immunoprecipitation step.
Serum-starved Caco-2 monolayers grown on 25-mm Falcon culture inserts
were collected and cooled rapidly to 4 °C to inhibit endocytosis (an
abbreviated flow chart of the procedure is shown in Fig. 1). The entire
subsequent procedure was performed at 4 °C unless otherwise
indicated. The apical monolayer surface was rinsed with PBS and exposed
to Sulfo-NHS-SS-Biotin (0.5 mg/ml; Pierce) in borate buffer (10 mM H3BO3, 154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl2, pH 9.0)
for 1 h, followed by extensive washing. Next, groups of 4-6
monolayers were warmed to 37 °C and incubated in serum-free DMEM
containing PMA (1 µM; Fig. 1, group 3) or in DMEM only
(control group; Fig. 1, group 4) for 20 min. Separate groups were
incubated as above but in the presence of 65 µM H7 (also
present during the preceding hour of biotinylation). Monolayers were
cooled to 4 °C and exposed to 50 mM glutathione (free
acid; Sigma) in 75 mM NaCl supplemented with 10% fetal
bovine serum (pH 9.0) for 40 min, followed by quenching of the free
NHS-SS-Biotin with Tris buffer (20 mM Tris, 120 mM NaCl, pH 7.4) for 10 min. In one group of monolayers
studied to estimate the total initial amount of biotinylated NHE3 at
the BB (Fig. 1, group 1), cells were exposed to control solution
lacking glutathione. Also, a separate group was biotinylated and then
exposed to glutathione with omission of the step at 37 °C to
evaluate the efficiency of the stripping procedure (Fig. 1, group 5).
Finally, in a separate group of monolayers a possible loss of surface
biotinylated NHE3 during the 20 min of incubation at 37 °C was
evaluated (Fig. 1, group 2). If such a
loss was significant, it should be taken into account when calculating
the amount of NHE3 retrieved from BB during incubation at 37 °C.
These monolayers were biotinylated at 4 °C and incubated with 1 µM PMA at 37 °C for 20 min, and the cells were lysed
with omission of the glutathione stripping step. The calculated total
biotinylated NHE3 (BB + intracellular) was then compared with the
initial (i.e. prior to incubation at 37 °C) amount of
biotinylated NHE3 at the BB, using Western analysis as described
below.
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Fig. 1.
An abbreviated flow chart showing the major
steps of reversible cell surface biotinylation used to quantitate the
PKC-induced internalization of BB NHE3 in Caco-2 cells. A short
description of the rationale for each of the five experimental groups
(groups 1-5) is given under the group number. The entire procedure was
performed at 4 °C with the exception of endocytic steps in groups
2-4, which were performed at 37 °C.
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All cells were scraped in lysis buffer (60 mM HEPES, 150 mM NaCl, 3 mM KCl, 5 mM EDTA, 1%
Triton X-100, 3 µM aprotinin, 20 µM
phosphoramidone, 0.3 mM phenylmethylsulfonyl fluoride, and 10 µM leupeptin, pH 7.4), sonicated, and the lysates were
incubated for 30 min. Following centrifugation at 16,000 × g for 15 min, the lysates were incubated with avidin-agarose
beads (Pierce) for 60 min. The beads were separated by brief
centrifugation, and the supernatants were incubated with a second
aliquot of avidin-agarose for another hour (to recover any residual
biotinylated proteins). The agarose beads (fractions A1 and A2) were
washed with lysis buffer and boiled in Laemmli sample buffer for 10 min, followed by brief centrifugation. The biotinylated proteins in the
supernatants were separated by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose, and probed with anti-NHE3 Ab 1380 (1:500). The membrane preparation obtained from PS120 fibroblasts
transfected with NHE3 (11) was used as an internal standard. NHE3 bands were revealed by incubation of the membranes with horseradish peroxidase-conjugated donkey anti-rabbit IgG followed by
chemiluminescence detection (Western Blot Chemiluminescence Reagent
Plus, NEN Life Science Products).
For quantitation of electroblotted NHE3, the fluorograms were scanned
on a Hewlett Packard Scan Jet 4C scanner, and the density of NHE3 bands
were analyzed using MetaMorph software and a threshold background
subtraction algorithm. Serial dilutions of the biotinylated BB
preparation were used to determine whether the analyzed NHE3 band
densities were within the linear portion of the concentration curve.
To estimate the degree of possible biotinylation of the cytosolic
proteins in damaged ("leaky") cells, cell lysate as well as
fractions containing biotinylated BB NHE3 or internalized, biotinylated
NHE3 were separated and electroblotted as described above.
Nitrocellulose was probed with a general anti-actin monoclonal antibody
(A-4700; Sigma), and actin bands were visualized by incubation with
donkey anti-mouse IgG followed by chemiluminescence detection. Since
actin is an abundant cytosolic protein present within BB, detection of
this protein in any of the biotinylated fractions would indicate
unintentional biotinylation of cytosolic proteins including NHE3. This
would cause an overestimation of the internalized fraction of the
exchanger in the subsequent calculations.
Statistical Analysis--
Numerical data are expressed as
means ± S.D., and the significance of difference between
experimental groups was analyzed by a two-tailed Student's
t test.
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RESULTS |
PKC Inhibits Endogenous NHE3 Activity in Caco-2 Cells--
In
control Caco-2 monolayers, the average Na+/H+
exchange rate in the presence of 130 mM Na+ (at
the AP surface) was 360 ± 55 µM/s (mean ± S.D.; at pHi 6.4) (Table I).
Preincubation of monolayers with 1 µM PMA inhibited the
NHE3 activity (Fig. 2 and Table I). The
maximal effect of PMA was noted at 10 min of exposure and did not
change significantly for up to 40 min of incubation with PMA (Fig. 2).
At all time points, preincubation of the cells with H7 (65 µM) resulted in a complete reversal of the PMA effect
(Fig. 2).
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Table I
Comparison of the effect of PMA on the relative distribution of NHE3
between BB and SAC obtained by confocal analysis with the NHE3 activity
evaluated by the BCECF fluorometric method
Caco-2 cells (17-19 days postconfluency) were exposed to PMA (1 µM) for 20 min in the absence or presence of protein
kinase C inhibitor H7 (65 µM). Intracellular distribution
of NHE3 was examined by confocal analysis (Confocal analysis), and NHE3
activity was evaluated by the fluorometric method
(Na+/H+ exchange). Averaged (mean ± S.D.) results
from three separate experiments are shown for each method.
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Fig. 2.
Effect of PMA exposure time on inhibition of
endogenous NHE3 in Caco-2 cells. Monolayers were exposed to PMA (1 µM) in the absence (solid bars) or presence of
protein kinase C inhibitor H7 (65 µM; shaded
bars) for the indicated periods of time before measuring NHE3
activity by the fluorometric method. Data represent percentage
inhibition of the rate of intracellular alkalinization as compared with
control cells and are expressed as mean ± S.D. of values obtained
from eight monolayers in two separate experiments.
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Since stable inhibition of NHE3 activity was observed between 10 and 40 min of cell exposure to PMA, we chose a 20-min incubation with PMA in
all subsequent experiments. Twenty minutes of exposure of the
monolayers to 1 µM PMA resulted in a significant
inhibition of the rate of intracellular alkalinization to 259 ± 29 µM/s (p < 0.01), i.e. by
approximately 28% of the control values (Table I). Preincubation of
the cells with H7 completely abolished the effects of PMA but had no
effect on NHE3 activity in control monolayers (Table I).
Endogenous NHE3 Is Present at the Brush Border as Well as in the
Subapical Cytoplasmic Compartment--
Between 17 and 22 days
postconfluency, approximately ~70% of all Caco-2 cells exhibited a
strong labeling of NHE3 at the apical surface. At that time, confocal
microscopy in the xy plane revealed heterogeneous expression
of NHE3 at the apical surface of adjacent cells (Fig.
3), which resembled the "mosaic"
pattern of distribution of several BB hydrolases described previously
(38, 39). In contrast to the monolayer as a whole, the distribution of
immunolabeled NHE3 throughout the apical surface of cells expressing
NHE3 was very homogenous, and no significant differences were found
among the NHE3 distribution patterns in any of the four 5 × 5-pixel areas analyzed in each cell.
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Fig. 3.
Indirect immunofluorescent micrograph of
apically expressed endogenous NHE3 in Caco-2 monolayers at 17 days
postconfluency. Cells were fixed with paraformaldehyde,
permeabilized, and incubated with anti-NHE3 Ab 1380, followed by
incubation with Cy3-conjugated goat anti-rabbit secondary antibody. The
image combines multiple images obtained in the xy plane by
confocal microscopy to present a 10-µm-thick optical section at the
brush border level. Note the uneven ("mosaic") expression of
immunoreactive NHE3 at the brush border of the adjacent cells.
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Analysis of the images in the xz plane revealed the presence
of NHE3 in a relatively wide apical zone of Caco-2 cells. To better
define the cellular distribution of NHE3, we labeled the BB with
FITC/PHA before the permeabilization and incubation of the monolayers
with anti-NHE3 antibody. As shown in Fig.
4A, the lectin labeled a
smaller apical region of the cells compared with NHE3-Ab (Fig.
4B). These results suggested the presence of NHE3 in both
the BB and a closely associated SAC (Fig. 4B).
Immunoreactive NHE3 was not detected at the basolateral membrane domain
in any of the monolayers examined between 3 and 22 days
postconfluency.
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Fig. 4.
Indirect immunofluorescence micrograph of
apically expressed endogenous NHE3 in Caco-2 cells at 17 days
postconfluency. Following exposure of the monolayers to 1 µM PMA for 20 min, cells were fixed, incubated with
FITC-conjugated PHA-E lectin, permeabilized, and incubated with
anti-NHE3 Ab 1380 and goat anti-rabbit secondary antibody. Panels
A and B show the same section of the monolayer
reconstructed in the xz (vertical) plane. Panel A
shows the BB labeled with FITC/PHA before the permeabilization step.
The image was obtained at emission wavelength of 520 nm. Panel
B shows intracellular localization of NHE3 labeled with anti-NHE3
Ab 1380 and Cy3-conjugated secondary antibody. Panel C shows
a representative image of the control monolayer in which anti-NHE3 Ab
1380 was replaced by preimmune rabbit serum. Images B and
C were obtained at an emission wavelength of 570 nm. Note
the distribution of NHE3 overlapping the area of BB and extending into
the subapical cytoplasmic compartment. Vertical lines marked
MH indicate monolayer height.
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In all of the cells examined, a speckled pattern of weak Cy3
fluorescence was observed within the cytoplasm below the 4-6-µm-wide zone of SAC. For the purpose of confocal analysis, this staining was
considered to represent background fluorescence, since it did not
differ significantly from the pattern observed in control monolayers in
which Ab 1380 was replaced by preimmune rabbit serum (Fig.
4C).
Quantitative Comparison of BB and NHE3 Staining
Patterns--
Assuming that lectin labeled exclusively the BB,
analyses were developed to assign the edges of the BB detected by this
probe. Analysis of derivative curves obtained from 30 cells in which the BB was labeled with FITC/PHA (both control and exposed to PMA)
revealed remarkable similarity of the slopes, with the apical BB limit
defined at 30 ± 5%, and the intracellular BB limit at 50 ± 7% of the maximal amplitude of BB fluorescence (Fig.
5). These values were used to define the
BB limits in all subsequent calculations. Since the maximal pixel
density of FITC/PHA was normalized to 100% for the calculations of BB
limits, differences in the density of the BB fluorescence observed
among the cells examined did not affect the calculated BB limits, the
latter depending on the slope of the distribution curves rather than on
their amplitudes.
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Fig. 5.
Algorithm used to define the apical and
intracellular BB limits for confocal morphometric analysis. Caco-2
cells were fixed, and the BB was labeled with FITC-conjugated lectin
PHA-E before the membrane permeabilization step. Serial optical
sections were obtained by confocal microscopy, and the pixel density in
a chosen 5 × 5-pixel area was quantified in consecutive sections
separated by 0.4 µm. Panel A shows the second derivative
tracing obtained from the FITC curve, as indicated by the
schematic at the top. Panel B shows a
representative curve obtained by plotting the relative pixel density of
FITC/PHA (expressed as percentage of maximal pixel density)
versus the optical section number and represents the
distribution of the FITC signal. In the representative example shown,
the apical and cytoplasmic BB limits were 35.8 and 47.8% of maximal
pixel density, respectively.
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A similar general pattern of distribution of the BB marker was observed
in cells in which the BB actin was labeled with FITC/phalloidin (Fig.
6). In these cells, the apical and
intracellular BB limits, obtained by the derivative method, were not
significantly different from those obtained using FITC/PHA (34 ± 5% for apical limit and 46 ± 7% for intracellular limit
(n = 20)). The slight shift of the intracytoplasmic BB
limit observed in the FITC/phalloidin method might result from labeling
of actin within the terminal web. However, this difference was not
statistically significant.
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Fig. 6.
Results of labeling of the BB of Caco-2 cells
with FITC-conjugated phalloidin. Caco-2 monolayers were fixed,
permeabilized with saponin, and incubated with FITC/phalloidin for 20 min. Cells were examined in both the xy and xz
planes by confocal microscopy, and distribution of the FITC/phalloidin
(representing labeled actin) was analyzed in serial optical sections
obtained parallel to the monolayer surface at 0.4-µm intervals. The
upper panel shows a representative image of the monolayer
obtained in the xz plane. Note the labeling of the cortical
actin in the area of the BB. The vertical line marked
MH indicates monolayer height. The lower panel
shows a representative distribution of the relative pixel density along
the apical-cytoplasmic axis of the BB (  ). Maximal pixel
density was normalized to equal 100%. The dotted line
represents the numerical values of the second derivative of the pixel
distribution. The points of maximal values of the second derivative
correspond to maximal deflections of the slopes of the density tracing
and are assumed to represent apical and intracellular limits of the BB.
In the example shown, the deflections were localized at 29.0 and 47.8%
(arrows) of maximal pixel density.
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The rationale for the derivative method was significantly supported by
the observation that, in all cells examined, the BB-marking FITC curve
diverged from the NHE3-marking Cy3 curve at the upper boundary of the
intracellular portion of the distribution curves (Fig.
7). Analysis of patterns obtained from 40 cells in which the BB was labeled with FITC/PHA revealed that the
intracellular BB limit defined by the derivative method was also the
"point of divergence" (47 ± 6 versus 50 ± 7% of maximal pixel density for the point of divergence
versus derivative method, respectively (not significant)).
This validated the derivative approach as a means to estimate a
physiologically relevant edge of the BB.
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Fig. 7.
Morphometric confocal analysis of the
subcellular distribution of endogenous NHE3 in Caco-2 cells. Data
were obtained from a single, representative cell in a control
(upper panel) and PMA-treated (lower panel)
monolayer, respectively. Curves drawn with a solid line
represent the distribution of pixel density of Cy3 (NHE3), and those
drawn with a dotted line represent the distribution of
FITC-conjugated PHA-E (bound to brush border) along the longitudinal
(apical-basal) cell axis. The areas limited by the vertical
dotted lines represent the BB and SAC, as depicted by the
schematics in each panel. In the two cells
examined, the relative amounts of NHE3 in SAC expressed as a percentage
of the total (BB + SAC) amount were 17.5% in the control cell
(upper panel), and 30.1% in the PMA-treated cell
(lower panel).
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PKC Causes Partial Redistribution of NHE3 from BB to SAC--
The
effect of PMA on the intracellular distribution of NHE3 was examined
utilizing two independent but complementary methods: confocal
morphometric analysis and reversible cell surface biotinylation. By
confocal analysis, 18.7 ± 2.8% (mean ± S.D.) of total (BB + SAC) NHE3 was localized in SAC of the control cells (Table I and Fig.
7). Preincubation of the cells with PMA resulted in a significant
increase of the SAC fraction to 29.2 ± 3.5% (p < 0.01), which corresponded to a relative decrease of the BB content
of NHE3 by 12.9% (calculated as ((100 
SACC) (100 SACPMA))/BBC × 100, where
SACC, SACPMA, and BBC represent the
relative abundance of NHE3 in SAC of control cells, SAC of PMA-treated
cells, and BB of control cells, respectively) (Table I and Fig. 7).
Pretreatment with H7 resulted in a complete inhibition of the
PMA-dependent redistribution of NHE3, whereas no
significant effect of H7 alone was observed in control monolayers
(Table I). Moreover, PMA treatment did not significantly change the
width of the BB (Table II). In preliminary experiments, Caco-2 cells were incubated in 50 mM NH4Cl with or without PMA and then briefly
exposed to 130 mM Na+ before fixation, as
described under "Experimental Procedures" for studies of NHE3
activity. This procedure, performed to acidify the cell interior prior
to fixation, did not result in any significant change in distribution
of NHE3 in control or PMA-treated cells (data not shown) and,
therefore, was not performed in subsequent experiments.
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Table II
Comparison of calculated BB dimension and relative amounts of NHE3 in
SAC (presented as percentage of BB + SAC) obtained by marking the
BB of Caco-2 cells with FITC-labeled lectin PHA-E or by labeling of the
BB cortical actin cytoskeleton with FITC-phalloidin
Caco-2 cells (17-19 days postconfluency) were incubated in control
medium or were exposed to 1 µM PMA for 20 min. The
intracellular distribution of NHE3 was examined by confocal analysis.
The dimension of the BB in the apical-cytoplasmic axis was calculated
for each method using the derivative approach. Averaged (mean ± S.D.) results from two (BB dimension) or three (NHE3 distribution)
experiments are shown. No significant difference was observed between
values within each column.
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Possible incomplete penetration of FITC/PHA into the intermicrovillar
domains of the BB was not a significant factor affecting the calculated
distribution of NHE3 by confocal morphometric analysis. As shown in
Table II, neither the BB dimensions nor the calculated NHE3
distribution were significantly different when either FITC/PHA (labeling the BB from outside the cell) or FITC/phalloidin (labeling the cortical actin cytoskeleton within the BB) were used to mark the BB
of Caco-2 cells. Thus, labeling the BB of Caco-2 cells with
FITC/phalloidin is comparable with the FITC/PHA method. However, possible alterations of the amounts of F-actin versus
G-actin caused by protein kinase agonists and/or growth factors makes the first method a less optimal approach to label the BB.
The data obtained by confocal analysis were confirmed by the results of
reversible surface biotinylation. In control monolayers, 12.9 ± 3.7% (mean ± S.D.; calculated after subtraction of the poststripping background) of the total initial BB content of
biotinylated NHE3 was found within the cytoplasm after 20 min of
incubation at 37 °C, the results most probably representing the net
effect of apical membrane recycling (Fig.
8). Incubation of the cells with PMA for
20 min at 37 °C resulted in an internalized fraction of biotinylated
NHE3 constituting 28.1 ± 6.1% of the initial BB amount, which
was a significant increase versus the absence of PMA
(p < 0.05).
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Fig. 8.
Demonstration of PKC-induced redistribution
of NHE3 from brush border into the cytoplasm of Caco-2 cells by cell
surface biotinylation. The cell surface was biotinylated with
NHS-SS-Biotin at 4 °C before exposure to PMA (1 µM) or
control medium at 37 °C in the absence or presence of PKC inhibitor
H7 (65 µM). Following surface biotin stripping and
incubation with avidin-agarose beads, biotinylated material was
separated by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and probed with anti-NHE3 Ab 1380. The initial amount
of NHE3 at the brush border is compared with that found in the
following experimental groups: monolayers with surface biotin stripped
with glutathione after initial biotinylation (STRIP);
monolayers biotinylated, exposed to PMA at 37 °C, and then stripped
(PMA); and biotinylated monolayers warmed to 37 °C for 20 min in control medium and then stripped (CONTR). Material
separated in each lane was obtained from a comparable number of cells.
The upper panel shows a fluorogram obtained from one
representative experiment. Material extracted after the first and
second incubation with avidin-agarose is shown in lanes A1
and A2, respectively. Internal standard (membrane
preparation from NHE3-transfected PS120 cells) is in the
lane marked PS. The photomicrograph shown is a
composite picture obtained from the same immunoblot from which certain
lanes, irrelevant for the presented data, were removed. Also, NHE3
bands obtained from experiments carried out in the presence of H7 are
not shown for clarity. The lower panel shows relative
density of NHE3 bands (A1 and A2 combined) obtained from densitometric
analysis of the fluorograms and represent means ± S.D. from three
separate experiments. Data are presented as a percentage of the initial
amount of biotinylated NHE3 at the BB. Solid bars and
shaded bars show the relative density of NHE3 bands obtained
from experiments carried out in the absence and presence of H7,
respectively. Abbreviations of the experimental groups are the same as
for the upper panel. a, significantly different
(p < 0.05) from STRIP; b, significantly
different (p < 0.05) from CONTR.
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In monolayers incubated with PMA at 37 °C with omission of the
glutathione stripping step, the total (BB + intracellular) amount of
biotinylated NHE3 was 96 ± 8% of that found initially at the BB
(n = 3, NS). This indicated that no significant amount of the initially biotinylated NHE3 was lost from the BB during the
20-min incubation with PMA and allowed us to calculate the PMA-induced
decrease of the BB content of NHE3 to be 15.2% (calculated as
((TOTPMA TOTSTRIP) (TOTCONTR TOTSTRIP)), where TOTPMA, TOTCONTR, and TOTSTRIP represent total amounts
of biotinylated NHE3 in monolayers treated with PMA at 37 °C and
stripped, control monolayers warmed to 37 °C and stripped, and
control monolayers that were stripped without warming, respectively).
Incubation with H7 abolished the PMA-induced increase in the
intracellular pool of NHE3, whereas no significant effect of H7 was
observed in monolayers incubated at 37 °C in control medium (Fig. 8,
bottom panel).
Control experiments were carried out to rule out two potentially
confounding factors in the biotinylation experiments, namely the
possibility that a significant amount of residual biotin was left at
the BB after the glutathione stripping step and/or that cytosolic
proteins in damaged cells were initially biotinylated and then
contributed to the pool assumed to represent endocytosed, biotinylated
surface molecules. As evident from the data shown in Fig. 8, the amount
of biotin left at the BB after stripping step was very low (3.3 ± 1.9% of the initial BB amount) and, most importantly, significantly
lower than the cytoplasmic, internalized pool. To investigate the
second possible source of error mentioned above, the biotinylated
material was separated on SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose, and Western analysis was performed with
anti-actin monoclonal antibody. As expected, a protein band
corresponding to actin standard preparation was found in the cell
lysate obtained from biotinylated Caco-2 monolayers and not incubated
with avidin-agarose beads. In contrast, no detectable actin was found
in the fractions recovered from the Avidin beads, indicating that the
intracellular biotinylated NHE3 is derived only from the biotinylated
pool of the surface molecules (Fig.
9).
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Fig. 9.
Degree of contamination with actin (used as a
marker of cytosolic proteins) of various preparations obtained from
cell surface biotinylation of Caco-2 cells. Monolayers were
surface-biotinylated and exposed to PMA (1 µM) at
37 °C for 20 min. Following surface biotin stripping and incubation
of cell lysates with avidin-agarose, the biotinylated material was
separated by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and probed with anti-actin antibody. Standard actin is
shown in lane 1 (ACTIN). The cell lysate obtained
after surface biotinylation is shown in lane 2 (CELL
LYSATE). The fraction of cell lysate bound to avidin-agarose is
shown in lane 3 (BIOTINYL. BB), and the material
obtained after exposure to PMA, stripping with glutathione, and
incubation with avidin-agarose is shown in lane 4 (BIOTINYL. SAC). The absence of any detectable actin in
lanes 3 and 4 suggests a negligible amount of
biotinylated cytosolic proteins in these two preparations. Material
separated on lanes 2-4 was obtained from a comparable
number of cells.
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DISCUSSION |
In this study, we have shown that the PMA-induced acute inhibition
of endogenous NHE3 in Caco-2 cells is mediated by PKC and that it
involves redistribution of the exchanger molecules from the BB to SAC.
This conclusion is drawn from the results of two complementary methods:
a novel morphometric method based on semiquantitative analysis of
intracellular distribution of immunoreactive NHE3 in Caco-2 cells by
confocal microscopy and reversible cell surface biotinylation. Since
PMA caused approximately 28% decrease in NHE3 activity but only
13-15% decrease in the BB amount of the exchanger, we further
conclude that the internalization of the BB NHE3 molecules is
responsible for approximately 50% of the observed PMA effect. Other
mechanisms, probably involving decrease in the turnover number of
residual BB NHE3 molecules, are likely to account for the remainder of
the inhibition.
Until now, quantitative evaluation of internalization of plasma
membrane proteins was most commonly performed by analysis of cellular
vesicular fractions obtained by density gradient centrifugation and a
counter-current phase partitioning technique (26, 27, 40). The major
limitation of this approach results from difficulties in isolating well
defined and homogenous populations of vesicles that represent specific
intracellular membranous compartments. Moreover, recognition of
internalized molecules is partially based on their functional
properties, which could be altered following endocytosis. In contrast,
confocal analysis does not depend on the activity of the internalized
molecules. Moreover, the analysis can be performed on a small number of
cells in native as well as cultured epithelia, as opposed to a
relatively large quantity of material needed for biochemical analyses.
Finally, confocal analysis combines semiquantitation of the
internalized molecules with respect to their BB content with their
intracellular localization, although the latter may require
colocalization studies with markers of specific intracellular
compartments. In this report, we did not define the exact nature of
SAC. However, we have reasons to believe that it represents the early
endosomal compartment and not the trans-Golgi network (see below).
One aspect of confocal analysis requires comment. The obtained
numerical values reflecting the abundance of NHE3 in SAC are relative,
and they depend on the definition of the BB limits. However, due to the
limits of confocal resolution and a convoluted shape of the physical
edges of the BB, the observed curves were bell-shaped, with upper and
lower limits difficult to localize by visual inspection (Fig. 5). To
resolve this problem, we developed an algorithm based on the assumption
that the BB limits correspond to points at which the BB marker changes
most steeply. Three lines of evidence support this rationale. First,
comparison of multiple BB-defining curves resulted in a remarkably
similar location of the apical and intracellular points of maximal
deflections relative to the maximal pixel density of every tracing.
Moreover, these values were not significantly different when the BB was
marked by staining intracellular actin, which speaks against artifacts resulting from possible incomplete penetration of the FITC/PHA into the
intermicrovillar domains of the BB. Second, an alternative method of
defining the lower BB limit (based on the location of the "point of
divergence") yielded results virtually identical to those obtained by
the second derivative approach. Finally, values of BB/SAC distribution
of NHE3 obtained from confocal analysis closely resembled those
obtained from surface biotinylation experiments, the latter method not
relying on the definition of the BB limits. Interestingly, the BB/SAC
distribution of NHE3 in control Caco-2 cells obtained by confocal
analysis (81% at the BB versus 19% in the cytoplasm)
closely resembles that reported for rat proximal tubule NHE3 (75% at
the BB versus 25% in the cytoplasm, respectively), the
latter obtained by a differential gradient centrifugation method
(40).
In contrast to our data obtained from Caco-2 cells, only ~27% of the
total cellular NHE3 content was found at the plasma membrane of
NHE3-transfected PS120 fibroblasts (as quantified by standard surface
biotinylation technique) (13). It is not clear whether the
significantly higher plasma membrane/cytoplasm ratio of NHE3 abundance
in epithelial cells versus in nonepithelial PS120 cells results from an overexpression of NHE3 in the transfected cells in
comparison with endogenous levels in Caco-2 cells or is due to cell
type-specific differences in the exchanger's intracellular processing
and plasma membrane recycling mechanism.
In this report, we have shown that PMA significantly inhibited the
activity of NHE3 in Caco-2 cells and that maximum inhibition was
observed as early as 10 min. The fact that the PKC blocker H7 inhibited
the PMA effects indicates that the effects were mediated via increased
activity of PKC. Both PMA and/or PKC have been shown to inhibit NHE3
activity in a variety of cell types, with the magnitude of the effects
similar to that described here. Thus, in rabbit gallbladder epithelium
PMA inhibited NHE3-dependent sodium transport by
approximately 30%, an effect reversed by the PKC inhibitor calphostin
C (14). In opossum kidney cells, PMA inhibited the apical NHE3 by
approximately 50-65% (15, 16), and similar effects were reported for
NHE3 transfected into AP-1 cells (17). In NHE3-transfected PS120
fibroblasts, PMA decreased the Vmax of the
exchanger by approximately 64% of control, an effect partially blocked
by PKC inhibitor H7 (10). None of these reports, however, addressed the
possibility that retrieval of NHE3 from the plasma membrane could be
involved in the observed, PKC-mediated inhibition of the exchanger.
Initial evidence that endocytic retrieval of NHE3 may occur during the
inhibition of the exchanger was reported by Hensley et al.
in 1989 (26). In rat kidney proximal tubule epithelium, PTH inhibited
BB Na+/H+ exchange that was accompanied by an
increase of the exchanger activity in an intracellular population of
vesicles, thus suggesting internalization of the BB NHE3. Although PTH
may stimulate both PKC- and PKA-associated signal transduction pathways
in renal epithelium, it is believed that at physiological conditions it acts via stimulation of PKC (16). More recently, inhibition of renal
NHE3 by both endocytic retrieval and decrease in turnover number has
been reported in acutely hypertensive rats, although the nature of the
second messenger(s) involved in this process was not investigated
(27).
The results of confocal analysis presented here demonstrate a
PKC-dependent redistribution of NHE3 from BB to SAC in
Caco-2 cells. Two major processes that could lead to such
redistribution are the stimulation of endocytic retrieval of NHE3 from
the BB, and/or inhibition of the exocytic insertion of the exchanger
into the BB. By complementing confocal analysis with cell surface
biotinylation, we were able to rule out two additional mechanisms that
theoretically could be involved, namely increased degradation of NHE3
at the BB and increased delivery of NHE3 to SAC from the trans-Golgi network, since neither mechanism per se would result in the
increased amount of biotinylated NHE3 in the cytoplasm. Moreover, a
remarkable similarity of the PMA-induced increase of NHE3 amount in the
intracellular pool of the exchanger obtained by both methods suggests
that degradation of the exchanger and/or delivery of newly synthesized
molecules to the intracellular pool do not play any significant role
during the 20 min of exposure to PMA. Recently, PKC has been reported to generally stimulate apical, but not basolateral, endocytosis in
Caco-2 cells (41, 42). Moreover, the degree of stimulation was
remarkably similar to that reported here. Also, apical endocytosis of
NHE3 was observed during rapid inhibition of the exchanger in kidney
proximal tubule epithelium in acutely hypertensive rats (27). Based on
these data, we suggest that endocytic retrieval was, at least in part,
responsible for the PMA-induced increase in the intracellular pool of
NHE3 in Caco-2 cells reported here.
The degree of PMA-induced redistribution of NHE3 does not fully explain
the overall inhibitory effect of PMA on the exchanger's activity,
however. The calculated decrease of NHE3 amount at the BB was ~14%,
whereas the observed inhibition of the NHE3 activity was ~28%.
Theoretically, three mechanisms could explain this discrepancy: 1) a
PMA-stimulated increase in NHE3 degradation at the BB; 2) a selective
retrieval from the BB of NHE3 molecules with the highest Na+/H+ exchange activity; and 3) PMA-induced
changes in turnover number of the residual NHE3 molecules at the BB.
Results of biotinylation experiments presented in this report argue
against the first of the possibilities mentioned above. The second
mechanism is an interesting possibility. However, such a selective
retrieval of NHE3 from plasma membrane of any cell type has yet to be
demonstrated. Thus, our working hypothesis is that PKC inhibits NHE3 in
Caco-2 cells by both stimulating translocation of the exchanger
molecules from BB into SAC as well and changing the turnover number of
the residual BB molecules. Simultaneous occurrence of both mechanisms has been suggested for multiple plasma membrane transport proteins including retinal taurine transporter (43), glucose transporters GLUT1
and GLUT4 (44), and Na+,K+-ATPase (24, 27). It
will be important to further define the relative contribution of each
of these two mechanisms in the acute regulation of NHE3 in both
epithelial and nonepithelial cells.
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 the HOE694 compound.
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Abstract
Introduction
