Apical Na+/H+ exchange near the base
of mouse colonic crypts
Jingsong
Chu*,
Shaoyou
Chu*, and
Marshall H.
Montrose
Department of Cellular and Integrative Physiology, Indiana
University School of Medicine, Indianapolis, Indiana 46202-5120
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ABSTRACT |
Colonic
crypts can absorb fluid, but the identity of the absorptive
transporters remains speculative. Near the crypt base, the epithelial
cells responsible for vectorial transport are relatively undifferentiated and often presumed to mediate only Cl
secretion. We have applied confocal microscopy in combination with an
extracellular fluid marker [Lucifer yellow (LY)] or a pH-sensitive
dye (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to
study mouse colonic crypt epithelial cells directly adjacent to the
crypt base within an intact mucosal sheet. Measurements of
intracellular pH report activation of colonocyte
Na+/H+ exchange in response to luminal or
serosal Na+. Studies with LY demonstrate the presence of a
paracellular fluid flux, but luminal Na+ does not activate
Na+/H+ exchange in the nonepithelial cells of
the lamina propria, and studies with LY suggest that the fluid bathing
colonocyte basolateral membranes is rapidly refreshed by serosal
perfusates. The apical Na+/H+ exchange in crypt
colonocytes is inhibited equivalently by luminal 20 µM
ethylisopropylamiloride and 20 µM HOE-694 but is not inhibited by
luminal 20 µM S-1611. Immunostaining reveals the presence of epitopes
from NHE1 and NHE2, but not NHE3, in epithelial cells near the base of
colonic crypts. Comparison of apical Na+/H+
exchange activity in the presence of Cl
with that in the
absence of Cl
(substitution by gluconate or nitrate)
revealed no evidence of the Cl
-dependent
Na+/H+ exchange that had been previously
reported as the sole apical Na+/H+ exchange
activity in the colonic crypt. Results suggest the presence of an
apical Na+/H+ exchanger near the base of crypts
with functional attributes similar to those of the cloned NHE2 isoform.
intracellular pH; NHE2; NHE3; NHE1; sodium absorption; epithelial
polarity; laser scanning confocal microscopy; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; Lucifer
yellow; immunofluorescence
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INTRODUCTION |
IN MAMMALIAN
COLON, intracellular pH (pHi) regulation and
electroneutral NaCl absorption are closely related functions. Apical Na+/H+ exchange is the luminal uptake mechanism
contributing to transcellular Na+ absorption (3,
38), and its activity consequently affects pHi in
the colonic epithelium (8, 29, 44, 51). Among the six
known Na+/H+ exchanger isoforms, only NHE1,
NHE2, and NHE3 have been found in the colonic epithelium on the basis
of mRNA expression and immunological detection (4, 5, 18).
NHE1 is ubiquitously expressed in mammalian cells to maintain pH
homeostasis of cells (57). In epithelial cells of the
intestine and colon, NHE1 is localized in the basolateral membrane
(4). In contrast, NHE2 and NHE3 have a more restricted
tissue distribution and are apical membrane proteins in intestinal
epithelia (4, 5, 26, 54, 55). Within the rat colon,
immunostaining detected NHE3 exclusively in the surface cells, whereas
NHE2 mRNA was detected predominantly on the surface cells with a
diminishing gradient that terminated in the upper third of the colonic
crypt (4, 5). In human colon, evidence has suggested that
NHE2 mRNA is present deeper in the crypts (18).
The physiological role of different
Na+/H+ exchangers is controversial. In
NHE3-knockout mice, basal fluid absorption by the intestine was
severely diminished (49), demonstrating that NHE3 plays at
least a permissive role in overall salt and water absorption. In
complementary experiments, NHE2-knockout mice have no obvious intestinal absorption defect (48). Aldosterone increases
abundance of NHE3, but not NHE2, in the rat proximal colon
(11). However, it is NHE2, and not NHE3, that is
responsible for the enhanced Na+ absorption in response to
a low-Na+ diet in chicken colon (17), and it
has been reported that rat colonic NHE2 and NHE3 are affected in
parallel by Na+ depletion (27). In rat
proximal colon, evidence suggests that NHE2 is the predominant
contributor to basal Na+ absorption (7),
although the role of NHE3 seems more dominant in isolated membrane
vesicles from the same tissue (11, 27). Adding to the
complexity, evidence suggests that rat colonic crypts may contain
apical Na+/H+ exchange activity that requires
extracellular Cl
(44, 45).
Cl
-dependent function has been proposed as evidence of a
new Cl-NHE isoform Na+/H+ exchanger in the
colon, but an intracellular Cl
dependence has recently
been shown to be a feature of conventional NHE isoforms as well
(1).
There is also a controversy about the colonic epithelial cell types
contributing to salt and water absorption. In contrast to the classic
view of crypts as secretory structures, recent studies have shown that
colonic crypts also contribute to fluid absorption (21,
52). Although crypt epithelial cells are less directly exposed
to the luminal contents than surface epithelial cells, they are an
abundant cell type of the colonic epithelium because of the density and
size of crypts in the tissue (23). Therefore, their
overall contribution to absorption could be substantial.
The major goal of this study was to question whether apical
Na+/H+ exchange was a feature of the cells near
the base of colonic crypts and, if so, to characterize that function
with respect to known NHE isoforms and the Cl
-dependent
NHE that have been reported. We have loaded isolated mouse colonic
mucosa with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester (BCECF-AM), a pH-sensitive dye, and measured activity of apical Na+/H+ exchange by
confocal microscopy while retaining normal epithelial architecture in
the mucosa. Results demonstrate that even cells in the base of colonic
crypts have the potential to contribute to Na+ absorption
and that known NHE isoforms are sufficient to explain results.
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MATERIALS AND METHODS |
Tissue preparation.
ICR mice (Harlan, Indianapolis, IN) were killed with halothane vapor
(Halocarbon Laboratories). The distal colon was excised, flushed with
saline, and stripped of muscle layers, as described previously
(12). Mucosal sheets were kept in DMEM (GIBCO BRL) on ice
and used within 3 h. For experiments, a microscope chamber allowed
mounting of mucosal sheets of muscle-stripped mouse colon, such that a
physiological saline could be superfused independently at the luminal
and serosal surfaces (12). The serosal surface of the
tissue was mounted facing the microscope objective lens (Zeiss C-Apo
×40) to facilitate study of crypt epithelium. Dye loading and
perfusion were performed on tissue mounted in the microscope chamber.
For pHi measurements, the solution contained 5 µM
BCECF-AM (Molecular Probes) in Na+ medium [in mM: 130 NaCl, 5 KCl, 1 MgSO4, 2 CaCl2, 1 (Na)PO4, 20 HEPES, 25 mannose, and 1 probenecid, titrated
to pH 7.4 with NaOH]. Incubation was at room temperature for 30 min.
After BCECF dye loading, the chamber was placed on the microscope stage
and continuously perfused.
Perfusate solutions.
Perfusate solutions were based on the Na+ medium described
above. In Na+-free solutions, tetramethylammonium (TMA)
chloride replaced all NaCl mole for mole; in ammonium media, 25 mM
NH4Cl replaced equimolar NaCl or TMA chloride; and in
isobutyrate media, 130 mM sodium isobutyrate or TMA isobutyrate
replaced equimolar NaCl or TMA chloride. When perfusates with low
Na+ concentration were required, NaCl was substituted mole
for mole for TMA chloride in TMA medium to give defined Na+
concentrations. Two Cl
-free media were used: sodium
gluconate medium [in mM: 130 sodium gluconate, 5 potassium gluconate,
4 calcium gluconate, 1 MgSO4, 1 (Na)PO4, and 20 HEPES] and NaNO3 medium [in mM: 130 NaNO3, 5 KNO3, 2 Ca(NO3)2, 1 MgSO4, 1 (Na)PO4, and 20 HEPES]. When
necessary, Cl
-free media were made Na+ free
by equimolar substitution of the Na+ salt by 130 mM TMA
gluconate or TMA nitrate, respectively. To inhibit
Na+/H+ exchange activity, 20 µM
5-(N-ethyl-N-isopropyl)amiloride (EIPA; RBI),
S-1611, or HOE-694
[3-(methanesulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate; the two latter compounds were generous gifts from Dr.
H. J. Lang, Adventis Pharma Deutschland, Frankfurt/Main, Gemany]
was added to select media. All perfusate and drug solutions were
prepared fresh directly before use, and final perfusates were adjusted
to pH 7.4.
Confocal microscopy and image analysis.
Images were collected using a Zeiss LSM510 confocal microscope. To
measure BCECF fluorescence, excitation was alternated between 488- and
458-nm lines of an argon laser, with emission collected at >505 nm at
a single photomultiplier tube detector held at constant gain
and dark current during the experiment. The LSM510 confocal microscope
allowed rapid switching of excitation wavelength after each scan line
for excitation ratio imaging, so that <10 ms separated data collected
at the two wavelengths. Ratio images of fluorescence at 488 nm to
fluorescence at 458 nm were calculated after subtraction of background
images at each wavelength, and values from the entire cytosol of all
imaged epithelial cells within a crypt were averaged. To measure
pHi gradients, 1- to 2-µm2 regions in
subapical and subbasal areas of colonocytes were analyzed as previously
described (24, 34). In some experiments, tissue was not
loaded with BCECF but was superfused instead with Na+
medium containing 100 µM Lucifer yellow [CH lithium salt (LY); Molecular Probes]. The extracellular LY dye was imaged with 458-nm excitation and >505-nm emission. In each image, average LY
fluorescence (minus background) was recorded from crypt lumens, lateral
intercellular spaces (LIS), and lamina propria tissue adjacent to the
crypts. In BCECF and LY experiments, images were routinely collected at the same focal plane over time, with the confocal pinhole adjusted for
1.5-µm optical section thickness. The plane of focus was selected to
be directly adjacent to the base of crypts, such that the crypt lumen
was just visible and crypt epithelial cells within the image could be
visualized along their apical-to-basal pole. Background images were
collected from nontissue areas of the chamber. Post-data-acquisition image analysis was performed (MetaMorph, Universal Imaging) to analyze
results from four to six crypts per experiment.
pHi calibration.
To avoid problems with poor permeation of nigericin into tissue (data
not shown), intracellular dye calibration was performed with isolated
mouse colonocytes (28) using high-K+ medium
and nigericin (an artificial K+/H+ exchanger),
as described previously (36). Colonocytes were loaded with
5 µM BCECF-AM and superfused with 130 mM K+ and 10 µM
nigericin solution at pH 6-8 during imaging on the confocal
microscope. Excitation ratios of fluorescence at 488 nm to fluorescence
at 458 nm at different pH values were obtained (Fig.
1A) from confocal images. A
single-site concentration dependency equation was used to fit a
pHi calibration curve by nonlinear regression to data of
eight independent preparations (Prism, Graphpad Software; Fig.
1B). The calibration curve demonstrates a favorable dynamic
range of the ratio for pHi measurement and was used to calculate pHi of experimental data. After each experiment,
a solution containing 25 µM BCECF in pH 7 Na+ medium was
imaged on the confocal microscope stage as an internal standard to
normalize results of the nigericin calibration curve to daily settings
of the confocal microscope. All averaged results are presented as
means ± SE of separate experiments.

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Fig. 1.
Intracellular pH (pHi) calibration of mouse
colonocytes. Fresh isolated mouse distal colonocytes were loaded with 5 µM
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM in high-K+ medium with 10 µM nigericin. Cells
were superfused with high-K+ medium containing 10 µM
nigericin, and pH varied between 6 and 8 during imaging on the confocal
microscope stage. Background-corrected excitation ratio of fluorescence
at 488 nm to fluorescence at 458 nm was calculated from raw
fluorescence images. A: results from a representative
experiment showing time course of ratio change in response to changes
in medium pH. B: results from 8 experiments (means ± SE) are fit to the pH calibration curve used to determine
pHi values in subsequent experiments.
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Immunofluorescence staining.
Mouse colon was fixed with 2% paraformaldehyde in PBS through cardiac
perfusion of the mouse under thiobutabarbital (Inactin, 100 mg/kg)
anesthesia. The excised colon was further fixed for 2 h at 4°C
in the same fixative. Fixed tissue was rinsed with PBS twice and
transferred to 30% sucrose in PBS for 24-36 h at 4°C. The colon
was embedded with tissue-freezing medium (EMS). Cryosectioning was done
with a microtome cryostat (IEC) at
20°C, and 15- to 20-µm-thick
sections were collected on Polysine microscope slides (Erie
Scientific). Sections were treated sequentially with PBS (once for 5 min), washing buffer of PBS with 50 mM NH4Cl (twice for 10 min), blocking buffer of PBS with 50 mM NH4Cl, 2% BSA, and
0.05% saponin (once for 20 min), primary antibody in blocking buffer
(overnight at 4°C), washing buffer (4 times for 5 min), 10 µg/ml
secondary antibody (Alexa 488-labeled goat anti-rabbit IgG; Molecular
Probes) in blocking buffer (60 min at room temperature), washing buffer
(twice for 5 min), nuclear staining with 4 µg/ml propidium iodide in
PBS (once for 5 min), and washing buffer (twice for 5 min) and mounted
with Prolong Antifade Kit (Molecular Probes). The polyclonal antisera
against NHE1 (human NHE1 aa 631-746), NHE2 (rat NHE2 aa
676-813), and NHE3 (rat NHE3 aa 528-648) were produced in
rabbits (kindly provided by Drs. E. B. Chang and M. Musch,
University of Chicago). NHE1 and NHE3 antisera have been described
previously (4, 35). With the use of NHE2-transfected fibroblasts, NHE2 antiserum has been characterized as specific for an
84-kDa protein, competed by the immunizing peptide (M. Musch, personal
communication). Antisera to NHE1 and NHE3 were used with 1:100
dilutions and to NHE2 with 1:900 dilution. Preimmune serum from the
same rabbits producing antisera was used as control. Slides were imaged
on the confocal microscope. Samples were excited with 488 nm, and
emission was collected at 500-530 nm (Alexa) and 620-680 nm
(propidium iodide). Positive and control slides were imaged with
identical confocal settings.
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RESULTS |
As described in MATERIALS AND METHODS,
muscle-stripped colonic mucosa was mounted in a microscope chamber and
examined by confocal microscopy. Our goal was to examine polarized
Na+/H+ exchange function in crypt epithelial
cells, but interpreting those experiments required characterization of
how well the luminal and serosal perfusates were separated in the
perfusion chamber. Specifically, we needed to test whether the
epithelium sustained a barrier between luminal and serosal compartments
and whether the LIS between adjacent crypt epithelial cells was a site
of restricted access to the serosal perfusates. For these general questions, we superfused tissue with the membrane-impermeant
fluorescent dye LY in the luminal or serosal compartment and imaged LY
fluorescence by confocal microscopy every 10 s while we focused
near the base of colonic crypts.
When LY was added to the serosal compartment, LY fluorescence rapidly
equilibrated into LIS [half time (t1/2) = 14 ± 3 (SE) s, n = 6 experiments] and the lamina
propria tissue between crypts (t1/2 = 20 ± 0 s). Some LY bound to collagen fibers in the lamina propria and could not be washed out, but in the majority of lamina propria regions, LY fluorescence was rapidly reversible on removal of
LY from the perfusate and washed out with rapid kinetics
(t1/2 = 30 ± 3 s). LY also
appeared in crypt lumens (t1/2 = 16 ± 2 s), demonstrating a transepithelial leak of the dye. As shown in
Fig. 2, all fluorescence in LIS and crypt
lumens was rapidly and completely reversible on removal of serosal LY
(t1/2 = 11 ± 2 and 13 ± 2 s, respectively). The levels of steady-state fluorescence in the presence of serosal LY are shown in Table
1, with results normalized to the
(reversible) fluorescence in lamina propria. Luminal fluorescence was
10% of fluorescence in the lamina propria. Because the LIS are
physically smaller than optical resolution (i.e., any imaged pixel
includes regions containing LIS and non-LIS regions), fewer dye
molecules are available to report fluorescence, and so LIS are dim,
even when equilibrated with LY concentrations in the lamina propria
[also observed previously with carboxyseminaphthorhodofluor (SNARF)-1
fluorescence] (12). In addition to demonstrating
transepithelial leak of LY, results suggest that the rate of fluid
entry and exit at the LIS and surrounding lamina propria tissue is
rapid and indistinguishable.

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Fig. 2.
Transmucosal permeability of extracellular Lucifer yellow
(LY) dye. Tissue in the microscope chamber was superfused with
Na+ medium with or without 100 µM LY. Confocal images of
tissue were collected over time while tissue was continuously
superfused. Superfusates were independently controlled at the luminal
(L) or serosal (S) surfaces. Results were compiled separately from the
same images for dye accumulation in the crypt lumens and the lateral
intercellular spaces (LIS) between adjacent crypt colonocytes. Raw
fluorescence values were collected from a representative time course
during polarized presentation of LY to the colonic mucosa. Results are
representative of 6 preparations.
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When added to the luminal compartment, LY slowly equilibrated in the
lumen (t1/2 = 122 ± 12 s,
n = 6 experiments; Fig. 2). This is consistent with
previous measurements of SNARF-1 fluorescence (13) and
shows that fluid in the luminal perfusates only slowly migrates down
the crypt lumen to undergo mixing. Even after luminal fluorescence
attained steady-state levels, Table 1 shows that negligible
fluorescence (i.e., not significantly different from zero in one-sample
t-test, P > 0.2) was detected in the LIS
and lamina propria (2 and 4% of luminal fluorescence, respectively). Given the known transepithelial leak of LY, defined by the serosal application of dye in the same experiments, results suggest that rapid
mixing in the serosal compartment leads to effective control of
extracellular fluid composition in this space, despite known transepithelial leakage from the lumen. Conversely, serosally added
components will have a more substantial effect to alter composition of
the fluid in the crypt lumen because of relatively slow mixing in the
luminal compartment. Thus, for studies of polarized functions (e.g.,
Na+/H+ exchange) activated by extracellular
factors (e.g., Na+) in perfusates, results encourage
luminal application of those factors to yield the tightest conclusions
about the membrane localization of effects.
Visualization of BCECF-loaded colonic mucosa during superfusion.
As described in MATERIALS AND METHODS,
muscle-stripped colonic mucosa was loaded with BCECF-AM and then
continuously superfused with dye-free medium. Figure
3 shows confocal fluorescence images of
BCECF-loaded colonic mucosa during superfusion, imaged at 488-nm excitation and >505-nm emission. BCECF loads into crypt epithelial cells as well as nonepithelial cells in the lamina propria surrounding crypts. Because the mucosa was oriented in the chamber with serosal surface adjacent to the objective lens, we could image crypt epithelial cells clearly. The series of images shows the transition between imaging the base of crypts (Fig. 3A) and the crypt opening
(Fig. 3C) as focus is advanced in the microscope. In all
subsequent experiments, results are reported from the region directly
above the base of the crypt, at the point where multiple crypt lumens were visible and individual epithelial cells were aligned along their
apical-basal axis in a single focal plane of the image. Because of
tissue motion in the dually perfused chamber, the plane of focus could
shift along the crypt-surface axis of the crypt during an experiment.
The crypt base is used as a landmark to reposition the focal plane as
needed during an experiment.

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Fig. 3.
Confocal images of BCECF-AM-loaded mouse colonic mucosa.
Smooth muscle-stripped mouse distal colonic mucosa was mounted into a
microscope chamber, loaded with BCECF-AM, and superfused with
Na+ medium. Mucosa was imaged with a Zeiss LSM510 confocal
microscope at 488-nm excitation and >500-nm emission. Large, circular
multicellular structures are colonic crypts imaged in cross section.
Series of images were taken at different focal planes at the base of
crypts (i.e., nearest the objective lens; A), focused 5 µm
deeper into tissue to the midpoint of the basal cells (B),
and focused another 5 µm into tissue to reach the crypt lumen
(C). Arrowhead points to crypt epithelium, and arrow points
to crypt lumen.
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Polarized activation of
Na+/H+
exchangers.
With the use of confocal microscopy, images of BCECF-loaded tissue were
collected every 1 min, and results were analyzed from crypt colonocytes
in the field of view. To activate Na+/H+
exchange, we used media without added bicarbonate/CO2 and
acidified the tissue by transient exposure to a weak base (25 mM
NH4Cl; ammonia medium) in luminal and serosal superfusates.
Simultaneous with ammonium exposure, Na+ was removed from
the luminal perfusate (substitution with TMA; TMA medium) to allow
adequate time to wash Na+ from this compartment. On removal
of ammonia medium, colonocyte pHi acidified rapidly (Fig.
4A). During this treatment,
Na+ was removed from the serosal medium to halt all
Na+/H+ exchange activity. Na+ was
then selectively returned to the luminal or serosal perfusate to
activate apical or basolateral Na+/H+ exchange,
respectively. As shown in Fig. 4A, colonocyte
pHi alkalinized after cells were exposed to luminal
Na+. To activate NHE activity, we added luminal 140 mM
Na+, an Na+ concentration much higher than the
Michaelis-Menten constant of NHEs for Na+ (55, 57,
59) . In this case, full activation of apical NHE will occur
before the crypt lumen is equilibrated with the perfusate
Na+ concentration. On the basis of mixing kinetics in the
crypt lumen, we predict that even 2 min after addition of luminal
Na+, the luminal Na+ concentration is 70 mM,
which should maximally stimulate the transporter. This is reasonably
well matched with our rate of data collection (every 1 min) and should
allow reliable measures of transport activity to be collected.
Subsequent serosal Na+ addition elicited a more rapid
pHi recovery to return pHi to normal resting
level. A second round of NH4Cl exposure, acidification, and
pHi recovery showed that the pHi recovery was
robust and reproducible in our experimental system. Rates of
Na+-dependent pHi recovery were calculated for
both rounds of acidification by using the lowest common pHi
value from the two recoveries as the starting value for rate
calculation: a way to account for the known pHi sensitivity
of Na+/H+ exchange activity and accommodate a
data set in which the level of acidification cannot be perfectly
controlled. No significant difference between the first and second
acidification was detected in the rates of pHi recovery in
response to luminal Na+ (Fig. 4B) calculated
from 4 min of pHi recovery from the lowest common
pHi. Similar calculations were performed for results from the subsequent addition of serosal Na+ after subtraction of
the rate of apical Na+/H+ exchange directly
before addition of serosal Na+ (to estimate rates of
basolateral Na+/H+ exchange). There was no
significant difference (P = 0.25) in basolateral
Na+/H+ exchange after the first vs. second
acidification (0.25 ± 0.03 and 0.21 ± 0.04 pH/min,
respectively, n = 6). These absolute values calculated
from (2-3 min) pHi recovery after serosal
Na+ should be viewed with reservation, because linear rates
of pHi change were sometimes poorly resolved due to rapid
pHi recoveries.

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Fig. 4.
Luminal and serosal Na+ activate pHi
recovery in colonocytes. Values are means ± SE from 4 experiments. A: time course of cellular pHi
response in colonic crypt epithelial cells acidified by
NH4Cl prepulse and Na+-free superfusion.
Luminal Na+ addition activated a pHi recovery.
Subsequent serosal Na+ addition accelerated pHi
rise toward normal resting level. Data show 2 rounds of acidification
and subsequent pHi recovery. TMA, tetramethylammonium.
B: pHi recovery rates of initial 4 min after
luminal Na+ addition from 1st and 2nd rounds were
calculated starting at lowest common pHi between the 2 rounds of acidification.
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Results tentatively suggest the presence of apical and basolateral
Na+/H+ exchange but required further
validation, because transepithelial Na+ leakage may be
different from transepithelial LY leakage. Therefore, we asked whether
luminal Na+ was able to stimulate pHi recovery
in crypt epithelial cells and cells in the lamina propria close to the
crypts. Because cells in the lamina propria are not in direct contact
with the luminal compartment, this is a stringent test of whether
sufficient Na+ from the lumen infiltrates the tissue to
activate the (presumptive NHE1) Na+/H+
exchanger on these cells. As shown qualitatively in the sequence of
ratio images of fluorescence at 488 nm to fluorescence at 458 nm of
Fig. 5, A-C, all cells in
the colonic mucosa acidified after ammonium prepulse (Fig.
5A). Crypt epithelial cells, but not cells in the lamina
propria, alkalinize after addition of 140 mM luminal Na+
(Fig. 5B). Subsequent addition of 140 mM serosal
Na+ confirmed that the lamina propria cells were able to
activate Na+/H+ exchange (Fig. 5C).
These results are substantiated by direct comparison of the time course
of BCECF response in the two cell types (Fig. 5D). Results
in Fig. 5D are plotted as fluorescence ratio, since we lack
pH calibration data for the lamina propria cells. Results document that
luminal Na+ cannot increase Na+ concentration
in the lamina propria sufficiently to activate Na+/H+ exchange.

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Fig. 5.
Polarized Na+ addition: comparison of pHi
response between epithelial and nonepithelial cells in the colonic
mucosa. Experiments were performed as described in Fig. 4 legend.
A-C: pseudocolor images of intracellular BCECF ratio
(488 nm/458 nm), with qualitative correspondence to pH change indicated
in the color bar. A: cells in mucosa were acidified by
NH4Cl prepulse and kept in Na+-free TMA medium.
Crypt colonocytes (arrows) and nonepithelial cells in the lamina
propria (arrowheads) acidified. B: luminal Na+
addition caused pHi recovery of crypt colonocytes but not
lamina propria cells. Note lamina propria cell at top left, directly
adjacent to colonic crypt base. C: subsequent serosal
Na+ addition caused further pHi rise in
colonocytes and initiated pHi recovery in lamina propria
cells. D: direct comparison of BCECF fluorescence ratio time
course simultaneously recorded from crypt epithelial cells and lamina
propria cells in the same experiment. Results are representative of 4 experiments.
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This observation was refined by questioning how much Na+
would have been required in the serosal tissue to activate
Na+/H+ exchange on colonocytes or lamina
propria cells. For this purpose, we added different concentrations of
Na+ to the serosal perfusate and monitored pHi
recovery in both cell types. Results (Fig.
6) show that 1 mM Na+ did not
stimulate measurable pHi recovery, 3 mM Na+
induced a slow but detectable pHi recovery, and
10 mM
Na+ activated significant pHi recovery.
Importantly, the pHi recovery of epithelial and lamina
propria cells was equally sensitive to low concentrations of added
serosal Na+, and even low Na+ concentrations
caused a simultaneous and prompt activation of Na+/H+ exchange in both cell types. Results
suggested no evidence of a diffusion barrier to Na+ or
sequestering of Na+ at the colonocyte basolateral membranes
containing Na+/H+ exchangers, and to explain
the lack of pHi recovery by lamina propria cells in this
condition, the direct addition of luminal 140 mM Na+ must
have resulted in <3 mM Na+ in the lamina propria tissue.
On the basis of this estimate, the serosal tissue accumulation of
Na+ is maximally 2% of the added luminal Na+
concentration, a percentage similar to that directly measured for LY
fluorescence (4%) after luminal LY addition.

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Fig. 6.
pHi response to different serosal
Na+ concentrations: comparison between epithelial and
nonepithelial cells in the colonic mucosa. BCECF fluorescence ratio
time course simultaneously recorded from crypt epithelial cells and
lamina propria cells was directly compared in the same experiment.
Tissue was exposed to indicated Na+ concentration in the
serosal perfusate while images were collected every 30 s.
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In all colonocyte measurements (Figs. 4-6), the
pHi response of entire crypts was averaged. To test the
possibility of intercellular heterogeneity of
Na+/H+ exchange among the ring of crypt
epithelial cells at this focal plane of the crypt, we measured
pHi of individual cells during Na+-dependent
pHi recovery. With the use of raw fluorescence images, in
which individual cell boundaries can be defined (Fig. 3), four to six
4.5-µm-diameter regions were positioned in individual cells of each
crypt to record pHi from the same cells in the subsequently derived ratio images. Results in Fig. 7
show the average magnitude of pHi recovery from a single
experiment. Every cell near the crypt base activated
Na+/H+ exchange activity in response to luminal
and serosal Na+. Identical results were obtained in four
experiments, suggesting that the majority of colonocytes at the crypt
base have similar Na+/H+ exchange activity. On
the basis of the surprising suggestion of apical
Na+/H+ exchange at the base of colonic crypts,
we tested different Na+/H+ exchange inhibitors
to learn whether known NHE isoforms were responsible for the putative
apical Na+/H+ exchange activity.

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Fig. 7.
Homogeneity of pHi response among crypt
colonocytes. pHi from 5 randomly selected colonic
epithelial cells in each crypt shown in Fig. 5, A-C, is
quantified. Each analyzed colonocyte responded with pHi
alkalinization to luminal (Lum) and serosal (Ser) Na+
presentation of 140 mM Na+. Values are means ± SD;
n = 20 cells. **P < 0.001.
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|
Apical
Na+/H+
exchanger is inhibited by EIPA.
We first tested EIPA, an inhibitor that blocks a wide spectrum of NHE
isoforms, including NHE1, NHE2, and NHE3 (39, 59). The
experimental protocol described for Fig. 4 was used, but 20 µM EIPA
was added to luminal perfusates to inhibit apical
Na+/H+ exchange activity in the second round of
acidification. Results in Fig.
8A qualitatively show that
EIPA inhibited pHi recovery elicited by luminal
Na+. The pHi recovery rate was calculated as
described for Fig. 4, and results in Fig. 8B show that
luminal EIPA inhibited the response to luminal Na+ by
79 ± 3.9% (n = 4). We also tested the effect of
basolateral EIPA on Na+/H+ exchange activity of
crypt colonocytes. Results (Fig. 9)
showed that basolateral 20 µM EIPA inhibited the
Na+/H+ exchange activated by luminal
Na+, a result requiring EIPA and/or Na+ leakage
across the epithelium. As noted earlier, serosal addition of substances
leads to greater problems defining sidedness of action. Because
basolateral Na+/H+ exchange was also strongly
inhibited by EIPA (Fig. 9), we could test for the reversibility after
removal of EIPA. As shown in Fig. 9, EIPA inhibition was not readily
reversible during 15 min after EIPA removal from perfusates. Given
evidence that EIPA is at best slowly reversible, results in Fig. 8 can
be evaluated for the inhibition of basolateral NHE as well as the
putative apical transporter. In that experimental series, rates of
pHi recovery after addition of serosal Na+
(0.31 ± 0.02 pH/min) were significantly less after exposure to EIPA (0.22 ± 0.01 pH/min, n = 4, P < 0.05), although the 28% decrease was only
slightly greater than the nonsignificant 20% decrease reported in the
absence of drug and clearly distinct from the 79% inhibition in
response to luminal Na+ activation. Results suggest that
the combination of luminal EIPA and luminal Na+ is an
effective pairing for selective analysis of apical
Na+/H+ exchange and, combined with earlier
results, support the presence of distinct apical and basolateral
Na+/H+ exchangers in colonocytes.

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Fig. 8.
Luminal ethylisopropylamiloride (EIPA) inhibits colonocyte
Na+/H+ exchange activity in response to
luminal Na+. Values are means ± SE from 4 experiments. A: after a 1st round of NH4Cl
prepulse, luminal Na+-dependent pHi recovery
was activated. After a 2nd round of acidification, 20 µM EIPA was
added to the luminal superfusion before luminal Na+
addition. B: pHi recovery rates of initial 4 min
after luminal Na+ addition from 1st and 2nd rounds were
calculated starting at lowest common pHi between the 2 rounds of acidification. **P < 0.001 vs.
control.
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Fig. 9.
Serosal EIPA inhibits colonocyte pHi recovery
in response to luminal and serosal Na+ and is poorly
reversible. Values are means ± SE from 3 experiments.
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|
Differential effect of isoform-specific NHE inhibitors.
To identify candidate NHE isoforms on the apical membrane of crypt
epithelium, we compared two NHE inhibitors. To inhibit NHE3, we used
S-1611, a compound that has relatively high specificity for inhibition
of NHE3 vs. NHE2 (50). Conversely, we used
HOE-694, which is relatively specific for inhibition of NHE2 vs. NHE3
(15). Figure
10A shows qualitatively
that, in the presence of luminal 20 µM S-1611, the pHi
recovery stimulated by luminal Na+ was similar to control,
a result confirmed by quantification in Fig. 10B. In
contrast, luminal HOE-694 inhibited the pHi recovery stimulated by luminal Na+ by 89 ± 2%
(n = 4; Fig. 11).
Similar to results with serosal EIPA, serosal 20 µM HOE-694 caused
inhibition of apical and basolateral NHE (data not shown), suggesting
that the drug was also leaky across the epithelial layer. Results are
most consistent with NHE2, but not NHE3, as a candidate for the apical
Na+/H+ exchanger near the base of colonic
crypts.

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Fig. 10.
Luminal S-1611 does not inhibit colonocyte
Na+/H+ exchange activity in response to luminal
Na+. Protocol described in Fig. 8 legend was used to
compare results in the absence with results in the presence of 20 µM
luminal S-1611. Values are means ± SE from 4 experiments.
A: time course of pHi response in the absence or
presence of 20 µM luminal S-1611. B: pHi
recovery rates of initial 4 min after luminal Na+ addition
from 1st and 2nd rounds were calculated starting at lowest common
pHi between the 2 rounds of acidification.
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Fig. 11.
Luminal HOE-694 inhibits colonocyte Na+/H+
exchange activity in response to luminal Na+. Protocol
described in Fig. 8 legend was used to compare results in the absence
with results in the presence of 20 µM luminal HOE-694. Values are
means ± SE from 4 experiments. A: time course of
pHi response in the absence or presence of 20 µM luminal
HOE-694. B: pHi recovery rates of initial 4 min
after luminal Na+ addition from 1st and 2nd rounds were
calculated starting at lowest common pHi between the 2 rounds of acidification. *P = 0.01 vs.
control.
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Short-chain fatty acids.
Short-chain fatty acids (SCFAs) are major anions of the colonic
lumen and stimulate apical Na+/H+ exchange to
promote colonic Na+ absorption (2, 20, 47). We
asked whether luminal SCFA activates the HOE-694-sensitive
Na+/H+ exchanger in crypt colonocytes. The
pHi of crypt epithelial cells was monitored during exposure
to luminal 130 mM isobutyrate in the absence and presence of luminal 20 µM HOE-694. Results in Fig. 12 show
that isobutyrate acidified crypt epithelial cells and activated an
HOE-694-sensitive Na+/H+ exchanger. HOE-694
inhibited 76 ± 2% (n = 4) of apical
Na+/H+ exchange activated by the SCFA,
suggesting that ammonium prepulse and SCFA activate a similar or
identical transporter.

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Fig. 12.
Luminal HOE-694 inhibits isobutyrate-activated colonocyte
Na+/ H+ exchange activity. Values are
means ± SE from 4 experiments. A: luminal
TMA-isobutyrate (T-B) medium acidified crypt epithelial cells, and
subsequent switch to sodium isobutyrate (Na-B) medium initiated
pHi recovery. Apical Na+/H+
exchange in the absence and presence of 20 µM apical HOE-694 is
compared using the protocol described in Fig. 8 legend. B:
pHi recovery rates of initial 4 min after luminal
Na+ addition from 1st and 2nd rounds were calculated
starting at lowest common pHi between the 2 rounds of
acidification. *P < 0.01 vs. control.
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Testing Cl
dependence of crypt
apical
Na+/H+
exchange.
Recent studies have reported that all apical
Na+/H+ exchange is dependent on extracellular
Cl
in crypt epithelial cells from rat distal colon
(43-45). To test for Cl
dependence of
apical Na+/H+ exchange in crypt epithelial
cells from mouse distal colon, we measured apical
Na+/H+ exchange with and without
Cl
(Fig. 13A).
The mucosa was first exposed to 25 mM ammonium in Na+-free
solution. Subsequent removal of ammonium caused rapid cellular acidification. Simultaneously, Cl
was removed from the
luminal and serosal superfusion (gluconate substitution) for 8-10
min. Returning luminal Na+ activated pHi
recovery in the absence of Cl
, and subsequent luminal
Cl
addition did not accelerate the pHi
recovery rate. To test for effects of different anion substitution,
Cl
was replaced by gluconate (Fig. 13A) or
nitrate. Control experiments with the continuous presence of
Cl
were used for comparison with Cl
-free
conditions. Results from four experiments using each condition are
shown in Fig. 13B, which shows that
Na+/H+ exchange activity is similar in the
presence of Cl
, gluconate, or nitrate. Results indicate
that any apical Na+/H+ exchange in mouse distal
colonic crypt epithelium is independent of extracellular
Cl
.

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Fig. 13.
Crypt colonocyte Na+/H+ exchange is not
dependent on extracellular Cl . A:
representative time course experiment showing crypt colonocytes
acidified by ammonium prepulse and then exposed to Na+-free
and Cl -free TMA-gluconate (TMA-G) medium. After
pHi acidified to a steady state, luminal Na+
was returned to luminal Cl -free superfusion [sodium
gluconate (Na-G) medium] to activate pHi recovery. Further
addition of Cl to the luminal superfusion did not
accelerate pHi recovery. B: compiled results
comparing pHi recovery rates of initial 4 min mediated by
return of luminal Na+ in Cl -free conditions
(NaCl replaced by sodium gluconate or NaNO3) with rates in
the presence of Cl throughout the experiment (i.e., using
conventional TMA medium and Na+ medium). Rates were
calculated from the same starting pHi. Results under the 3 conditions were not significantly different (P > 0.05, n = 4 experiments in each condition).
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Testing for pHi gradient in crypt colonocytes.
Using polarized HT29-C1 cells, we had observed more extreme
pHi changes in the subapical cytoplasm than in the region
adjacent to the basal pole of the cell. This led to pHi
gradients being observed in HT29-C1 cells in response to luminal SCFA
exposure or NH4Cl prepulse (24, 34). Because
the apical-basal axis of the epithelial cells was directly imaged in
our native tissue experiments, we asked whether a similar
pHi gradient could be observed in mouse crypt colonocytes
under comparable conditions. We analyzed small (1-2
µm2) regions of cytoplasm and compared the
pHi reported at the subapical and subbasal domains of crypt
epithelial cells. In Fig. 14, these subcellular pHi values in the resting state (exposed to
Na+ medium), in the presence of luminal SCFA (absence of
Na+ in all superfusates), and after NH4Cl
prepulse (also in the absence of Na+ in all superfusates)
are compared. As shown in Fig. 14, pHi was not
significantly different between the two subcellular sites under any of
the tested conditions.

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Fig. 14.
Testing for pHi gradients in crypt
colonocytes. Measurements from subcellular regions were made in cells
exposed to Na+ medium (resting pH, n = 8)
or in Na+-free conditions during exposure to luminal
isobutyrate (+isobutyrate, n = 3) or
Na+-free conditions after transient exposure to
NH4Cl medium (post NH4, n = 5).
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Immunostaining of NHE isoforms.
Figure 15 shows immunofluorescent
staining of mouse distal colon with polyclonal antisera raised against
the divergent COOH-terminal sequences of NHE1, NHE2, and NHE3. With the
use of nuclear staining to mark cellular structures, results show that
NHE1 was expressed on the basolateral membrane of epithelial cells
along the crypt-surface axis (Fig. 15, a and b).
In contrast, NHE3 was expressed on surface but not crypt epithelial
cells (Fig. 15, g and h), and the majority of
NHE3 was in the apical or subapical domain of the surface cells. The
expression of NHE2 in the colon was more complex: it was observed at
the apical region of the surface epithelial cells (Fig. 15d) and also in crypt epithelial cells (Fig. 15, d and
e). NHE2 stained the apical membrane of cells near the base
of the crypts but, similar to NHE3, also displayed cytosolic staining.
For all antisera, preimmune serum confirmed specificity of the signal.
Results suggest that NHE2, or another NHE isoform with similar
epitopes, is a candidate for the apical exchanger near the base of
colonic crypts.

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Fig. 15.
Immunofluorescent localization of NHEs in mouse distal
colon. Red, nuclear staining with propidium iodide; green, NHE
immunofluorescence (see MATERIALS AND METHODS
for staining protocols). *, Crypt lumen; arrow, surface epithelium.
Images are for NHE1 (a-c), NHE2
(d-f), and NHE3 (g-i). Images
a, d, and g exposed crypt-to-surface view of the
colonic epithelium. Images b, e, and h are cross
sections of crypts near the crypt base. Images c, f, and
i are controls using the same dilution of preimmune sera as
the primary antisera. Images are representative of results from 3 animals. Scale bars, 20 µm.
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 |
DISCUSSION |
This study introduces the use of BCECF, the most commonly used
fluorescent pHi indicator, for ratiometric measurements of living native tissue by confocal microscopy. Recent advances in commercial instrumentation allow rapid switching between appropriate excitation wavelengths (458 and 488 nm) for the use of excitation ratio
imaging while minimizing concern about motion artifacts in living
specimens. Using the method in conjunction with a chamber that allows
separate superfusion of the luminal and serosal compartments of
isolated colonic tissue (12), we have applied the method to analyze functional properties of the Na+/H+
exchangers expressed in the crypt epithelium.
Experiments analyzed the fidelity of the preparation for selective
presentation of substances to the luminal vs. serosal
compartments and the apical vs. basolateral membrane of the
colonocytes. In the serosal compartment, Na+ and the
structurally unrelated LY dye had rapid access to nonepithelial cells
in the lamina propria and basolateral membrane of colonocytes. No
evidence was found for slow or restricted mixing of the serosal perfusate in the LIS between colonocytes compared with the directly adjacent lamina propria tissue. Others and we previously observed that
pH in the LIS of cultured epithelia and colonocytes can be distinct
from that in the serosal tissue and is relatively constant in response
to changes in acid/base conditions (9, 12, 25, 34). This
was shown to be due to fixed proton buffers on LIS membranes, rather
than altered ion diffusion in the LIS (19, 25, 33, 58).
This model is supported by our observation of rapid fluid exchange
between the lamina propria and the LIS. Our results suggest that LIS
Na+ concentration can be rapidly altered, even by low
concentrations of added Na+, suggesting lack of substantial
Na+ binding or buffering in LIS. Of importance for studies
in which Na+ is added to luminal or serosal perfusates,
results demonstrated that the basolateral membrane of colonocytes was
exposed to a fluid that was rapidly and efficiently refreshed by
serosal perfusates.
In contrast, multiple results suggested that the crypt lumen has only
slow access to luminal perfusates and is thereby subject to greater
influence from leakage of fluid and substances from the serosal
compartment. The most direct demonstration is that luminal
equilibration of an extracellular marker (LY) added to the luminal
perfusate required sixfold more time than serosal equilibration of the
same marker added to the serosal perfusate. We also observed a marked
asymmetry when the transepithelial LY leakage between the bulk fluid
environments of the crypt lumen and the lamina propria was compared as
the orientation of LY transepithelial gradients was reversed. In
response to serosal LY, 10% of the dye concentration appeared in the
crypt lumen. The response to luminal LY addition was significantly less
(P = 0.026) dye accumulation in the lamina propria, at
only 4% (and this value was not significantly different from zero).
This suggests that the extracellular fluid in the serosal compartment
of the colonic mucosa is more rapidly and efficiently controlled by the
perfusate than the extracellular fluid in the luminal compartment.
Experiments unequivocally demonstrated limited leakage of an
extracellular fluid marker (LY, 450 g/mol) between the serosal and
luminal compartments. Additionally, experiments showed that NHE
inhibitors (EIPA and HOE-694) and/or Na+ could also
traverse the mucosa. Our results place limits on how leakage of
Na+ across the mucosa could confound the conclusion that
apical Na+/H+ exchange mediates the response to
luminal Na+. On the basis of our resolution of
pHi recovery in lamina propria cells (Fig. 6), the amount
of luminal 140 mM Na+ reaching the serosal tissue adjacent
to colonocytes must be <3 mM. To explain the rapid rate of colonocyte
Na+/H+ exchange activated by luminal
Na+ as due to erroneous presentation of Na+ at
basolateral membranes, the LIS/basal membranes would have to be exposed
to a much higher Na+ concentration:
10 mM Na+
at basolateral membranes. This more than threefold Na+
gradient over 10-30 µm is clearly incompatible with results
being due to 1) gross mixing of the two perfusates via a
poorly built microscope chamber or 2) transepithelial
Na+ absorption at the surface epithelium adding
Na+ to the serosal tissue around crypts. The only tenable
explanations are those in which Na+ appears at the
basolateral surface by flux directly across the crypt epithelium. One
possibility is a substantial transcellular Na+ flux that
loads the LIS/basal extracellular region with Na+. This
Na+ absorptive function requires a robust apical
Na+ uptake route in the crypt base cells to continually
fuel Na+ loading in these well-mixed extracellular spaces,
and the apical route would have to be EIPA and HOE-694 insensitive and
a transport reaction other than Na+/H+
exchange. The only other apical transport mechanism contributing to
mammalian Na+ absorption is Na+ channels, and
evidence suggests that these are solely expressed at the surface
epithelium (31). The second possibility is a paracellular
Na+ flux locally activating LIS
Na+/H+ exchangers. Because of the localized
entry of Na+ across tight junctions and rapid fluid mixing
in the LIS, a gradient of decreasing Na+/H+
exchange activity along the apical-basal axis of the LIS is predicted. No gradient of pH or Na+ concentration has been observed
along the apical-basal axis of the LIS (9, 10, 25, 32). In
contrast, our observations would require a steep Na+
gradient characterized by <3 mM Na+ near the basal pole of
colonocytes and an average 10 mM Na+ along the length of
the LIS. Although such a gradient may seem unlikely, LY leakage ensures
some paracellular Na+ flux. Results suggest that the crypt
colonocyte tight junctions must be relatively impermeable to
Na+, since transepithelial accumulation of the 450 g/mol LY
is greater (4% on the basis of results in Table 1) than the maximal
accumulation of Na+ that is predicted from study of lamina
propria cells (<2% on the basis of results in Fig. 6). In our
analysis of the apical Na+/H+ exchange, we have
optimized conditions for selectively detecting apical transport by
adding Na+ and inhibitors only to the luminal compartment.
In addition to the studies described above that evaluate the influence
and accessibility of perfusates to the spaces surrounding colonocytes,
the suitability of this approach is suggested by experiments with EIPA.
Luminal application of EIPA produces only slight inhibition of
basolateral NHE (presumptive NHE1) compared with apical NHE, despite
using an EIPA concentration (20 µM) that is ~1,000-fold greater
than the inhibition constant (Ki) of NHE1 for
the inhibitor (39, 59) and 100-fold greater than the
IC50 in the presence of 145 mM Na+
(42). For all the reasons cited above, we believe that our analysis has resolved an Na+/H+ exchanger
localized to the apical membrane of crypt colonocytes.
Absorptive transporter in relatively undifferentiated colonic crypt
epithelium.
In mouse colonic crypts from distal colon, epithelial cells near the
base of colonic crypts are proliferating and are only separated by a
few cell divisions from the stem cells positioned at the crypt base
(6, 41). Although our experiments are clearly not studying
the stem cells, the expression of more differentiated functions (such
as absorptive transporters) is believed to start nearer the colonic
surface. With the use of isolated, microperfused crypts from rat distal
colon, it has been observed that crypts (in the absence of secretory
stimuli) absorb fluid in an Na+-dependent manner and
express an (Cl
-dependent) apical
Na+/H+ exchange (21, 43-45,
52). In these experiments, the need to cannulate the upper
portion of the crypt and puncture/cannulate the crypt base allowed
analysis in the midregion of the crypt. In contrast, our measurements
preserve normal epithelial architecture while allowing study of
colonocytes directly adjacent to the crypt base. In this site, we also
observe apical Na+/H+ exchange and report that
this transport can be activated by physiological luminal stimuli such
as Na+ and SCFAs. These results strongly suggest that even
cells near the base of the crypt express an absorptive-type transporter
and, for this reason, have at least the potential to contribute to Na+ absorptive function. Given the high abundance of the
Cl
secretory channel cystic fibrosis transmembrane
conductance regulator in the lower crypt (56) and the
observation that all cells near the crypt base express apical
Na+/H+ exchange (Fig. 7), we speculate that
individual crypt epithelial cells in the colon may mediate
Na+ absorption and Cl
secretion.
Candidate NHEs at the crypt base.
Recent studies on isolated, microperfused rat distal colonic crypts and
apical membrane vesicles have shown a Cl
-dependent
Na+/H+ exchange activity
(43-45). Using protocols designed to closely parallel
those applied previously to resolve Cl-NHE (44, 45), we
were unable to demonstrate any Cl
dependence of apical
Na+/H+ exchange in mouse distal colonic crypts.
We compared a relatively permeant (nitrate) and impermeant (gluconate)
anion substitution with no difference in results. The discrepancy
between our results and previous studies may be due to species
differences (rat vs. mouse) or site of analysis (crypt base vs.
midcrypt region). It should be noted that known NHE isoforms can be
Cl
dependent in some cases due to an (incompletely
understood) effect of intracellular Cl
(1).
However, our results clearly demonstrate that the Cl-NHE as previously
analyzed in rat distal colonocytes is not a ubiquitous function of
colonic crypt epithelia.
Known NHE isoforms are sufficient to explain our observations in crypt
colonocytes. The basolateral exchanger identified by function is
presumed to be NHE1 (4), a conjecture made more likely by
demonstration of the presence of NHE1 in the basolateral membrane of
epithelial cells near the crypt base (Fig. 15). However, our main focus
was to discriminate among potential NHE isoforms at the apical
membrane. Experiments were designed to discriminate among the three
cloned NHE isoforms (NHE1, NHE2, and NHE3) expressed in small intestine
and colon (4, 5, 18). It was important to include NHE1 in
the functional analysis because of reports that NHE1 can be expressed
apically in some cultured intestinal epithelia (40) and
because of the general lack of information about cells near the crypt base.
Our experimental design used luminal addition of inhibitors with
differential activity against NHE isoforms, using the absence of
serosal Na+ to block activity of basolateral
Na+/H+ exchange. EIPA, able to inhibit all
three NHEs at the applied concentration of 20 µM
(Ki = 0.02-2.4 µM) (39, 42,
59), blocked >75% of apical Na+/H+
exchange activity. A similar level of inhibition was observed with
addition of 20 µM HOE-694, which was predicted to inhibit NHE1 and
NHE2 (Ki = 0.16-5 µM) but not NHE3
(Ki = 650 µM) (15). No
inhibition was observed with 20 µM S-1611, which was predicted to
inhibit NHE1 and NHE3 (Ki = 0.7-5
µM) but not NHE-2 (Ki = 89 µM)
(50). These functional studies suggested that NHE1 and
NHE3 do not contribute to apical Na+/H+
exchange near the crypt base. In addition, they suggested that ~75%
of the response to luminal Na+ may be due to NHE2. Results
were supported by immunostaining, which suggested that epithelial cells
near the crypt base did not express NHE3 but did express NHE2-like epitopes.
Our results cannot unequivocally assign NHE2 as the apical
Na+/H+ exchanger in the crypt. Other
undiscovered NHE isoforms may have a pharmacological profile and an
epitope signature similar to NHE2. However, it seems clear that the
crypt apical NHE will have properties closely related to those of NHE2.
Similar conclusions were made about HT29-C1 cells, where NHE2 and NHE1
(but not NHE3) are expressed, and use of HOE-694 also suggested that
NHE2 was apical and NHE1 was basolateral (24).
Interestingly, the HT29-C1 cells also express cystic fibrosis
transmembrane conductance regulator (37), as shown for
colonic epithelial cells near the base of the crypt. Together, results
suggest a close functional correlation between the cells near the crypt
base and HT29-C1 cells.
In native tissue and HT29-C1 cells, physiological gradients of SCFAs
cause selective activation of Na+/H+ exchange
in the apical membrane (16, 34, 46, 51). It has been
proposed that heterogeneity of intracellular and extracellular pH may
explain why apical exchange activity is activated in preference to
basolateral exchange activity (16, 34, 46, 51). In support
of this model, qualitatively similar changes in extracellular pH were
observed around HT29-C1 cells and crypt colonocytes exposed to
transepithelial SCFA gradients (13, 14, 22, 34). HT29-C1 cells also reported a pHi gradient in which the cytoplasm
near the apical membrane was significantly more acidic than the
cytoplasm near the basal pole of the cell (24, 34). It has
been observed that mouse small intestinal enterocytes can demonstrate
cytosolic pHi gradients (53). Therefore, we
analyzed whether pHi gradients were measurable in crypt
colonocytes under the tested conditions. Because cytosolic
pHi gradients were not observed, we assume that if any
pHi heterogeneity exists in colonocytes under these
conditions, it must be below our level of spatial resolution. In the
future, use of near-membrane pH probes might be helpful, inasmuch as
they have resolved pH microdomains near the apical membrane of
colonocytes exposed to SCFAs (22, 30). Nevertheless, there
seems a clear divergence between the observations made in HT29-C1 cells
and crypt colonocytes with respect to pHi gradients that
can be detected with cytosolic pH indicators.
In summary, several unexpected findings have come from this first
report of polarized epithelial cell function from cells adjacent to the
colonic crypt base. First, apical Na+/H+
exchange function is detected even among the relatively
undifferentiated cells in this region of the mouse crypt. Second, this
apical Na+/H+ exchanger has functional
attributes of NHE2, although the presence of NHE2 mRNA was not detected
in this region of the rat crypt (5). Our transport and
immunostaining results more closely mirror a report from the human
colon, where NHE2 mRNA was detected deeper in crypts (18).
Third, there is no evidence of the Cl-NHE that has been reported as the
sole apical Na+/H+ exchange activity in the rat
colonic crypt (43-45). Results suggest that the
colonic crypt has even broader expression of Na+ absorptive
transporters than was previously recognized and that our understanding
of cellular function in the crypt epithelium remains incomplete.
 |
FOOTNOTES |
*
J. Chu and S. Chu contributed equally to this work.
Address for reprint requests and other correspondence: M. Montrose, Med Sci 307H, 635 Barnhill Dr., Indianapolis, IN 46202-5120 (E: mmontros{at}iupui.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.01380.2000
Received 15 December 2000; accepted in final form 12 March 2002.
 |
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