Regulation of intracellular pH gradients by identified Na/H
exchanger isoforms and a short-chain fatty acid
Tamas
Gonda,
Djikolngar
Maouyo,
Sharon E.
Rees, and
Marshall H.
Montrose
Departments of Medicine and Physiology, Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
Colonic luminal short-chain fatty acids (SCFA)
stimulate electroneutral sodium absorption via activation of apical
Na/H exchange. HT29-C1 cells were used previously to demonstrate that
transepithelial SCFA gradients selectively activate polarized Na/H
exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy
SNARF-1, respectively) are used to measure intracellular pH
(pHi) in HT29-C1 cells, to find
out which Na/H exchanger isoforms are expressed and if results are due
to pHi gradients. Inhibition of
Na/H exchange by HOE-694 identified
1) two inhibitory sites [50%
inhibitory dose (ID50) = 1.6 and
0.05 µM] in suspended cells and
2) one inhibitory site each in the
apical and basolateral membranes of filter-attached cells (apical
ID50 = 1.4 µM, basolateral
ID50 = 0.3 µM). RT-PCR detected
mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal
microscopy of filter-attached cells reported HOE-694-sensitive
pHi recovery in response to
luminal or serosal 130 mM propionate. Confocal analysis along the
apical-to-basal axis revealed that
1) luminal or serosal propionate
establishes transcellular pHi
gradients and 2) the predominant
site of pHi acidification and
pHi recovery is the apical portion
of cells. Luminal propionate produced a significantly greater
acidification of the apical vs. basal portion of the cell (compared
with serosal propionate), but no other dependence on the orientation of
the SCFA gradient was observed. Results provide direct evidence for a
subcellular response that assures robust activation of apical NHE2 and
dampening of basolateral NHE1 during
pHi regulation.
SNARF-1; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; laser
scanning confocal microscopy; epithelium; polarity; propionate; NHE1; NHE2; NHE3
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INTRODUCTION |
SHORT-CHAIN FATTY ACIDS (SCFAs) are produced in the
lumen of the large intestine by bacterial fermentation of the
nonabsorbed carbohydrate and protein that progresses into the colonic
lumen. There is a large transepithelial gradient of SCFAs across the colonic epithelium under physiological conditions because these monocarboxylates (e.g., acetate, propionate, and butyrate) accumulate to a steady-state level of >100 mM in the colonic lumen but are at
<0.5 mM in blood (13). Absorbed SCFAs stimulate salt and water
absorption, and their metabolism will generate 7-10% of body
energy reserves (3). The mechanism of colonic SCFA absorption is
controversial but is coincident with intracellular and extracellular pH
changes in the colonic mucosa (5, 7, 8, 10, 14, 16, 47, 48). Present
evidence suggests that nonionic and carrier-mediated SCFA transports
will directly contribute to the changes in pH (5, 9, 24, 31, 38, 42).
SCFAs stimulate electroneutral sodium absorption up to fivefold in
human colon (44), and the model of SCFA-stimulated sodium absorption is
tightly linked to the ability of SCFA fluxes to change pH. SCFAs
stimulate colonic sodium absorption via activation of apical Na/H
exchange. It is commonly accepted that SCFAs cause this activation by
changing pH, in simple models by the cellular acidification that occurs
when these weak acids are taken up by nonionic diffusion. It is known
that multiple isoforms of the NHE family of Na/H exchangers are
expressed in the colonic mucosa. These include the NHE2 and NHE3
isoforms that are expressed predominantly in epithelial cells as well
as the virtually ubiquitous NHE1 isoform that is found in the
basolateral membrane of epithelial cells (50, 51). In addition, both
apical and basolateral Na/H exchange activities have been measured in
colonocytes (17, 19, 20, 37, 39, 40). At this point, the specific
apical NHE isoform or isoforms that mediate electroneutral sodium
absorption in the colon have not been confirmed. Identification of
these isoforms is important because each has a characteristic response
to activation by protons and sodium (51), which will dictate tissue
response to the physiological stimulus of SCFA-induced pH change.
Because colonocytes can express apical and/or basolateral Na/H
exchangers, it has been unclear how SCFA-stimulated changes in pH could
mediate selective activation of apical vs. basolateral Na/H exchange to
facilitate efficient sodium absorption. Results from native tissue
suggest that this does happen because luminal but not serosal SCFAs can
stimulate sodium absorption (1, 21, 36). Previous experiments
demonstrated that SCFAs could preferentially activate either apical or
basolateral Na/H exchange in HT29-C1 cells (a cloned epithelial cell
line derived from a colon carcinoma) (46). This action required
transepithelial gradients of SCFAs and was not due to an allosteric
effect of SCFAs on the Na/H exchangers. In an apparent paradox, results
suggested that changes in pH were the mechanism underlying SCFA
effects, but measurements of intracellular pH
(pHi) could not resolve the
reason for differing effects of transepithelial SCFA gradients (46).
On the basis of these results in HT29-C1 cells, we hypothesized that
localized changes in pH near the plasma membrane were affecting local
activation of Na/H exchangers. Subsequent experiments in native tissue
demonstrated that changes in extracellular pH occurred near the crypt
epithelium and were part of SCFA effects that should contribute to
polarized activation of Na/H exchange (7). Such observations were
consistent with some, but not all, prior observations of a pH
microclimate at the colonic surface that was affected by SCFAs (25,
41). Recently, it has been proposed that
pHi microenvironments may also be
an important regulator of sodium absorption in colonic epithelium (4,
14, 15). This possibility is consistent with the prior observation of
pHi gradients in a variety of cell
types, using either optical or electrophysiological methods (22, 23,
34, 43). It is also consistent with all prior measurements of
pHi, which were averaged from the
entire cytosol and colonocyte could not resolve
pHi gradients (14, 15, 46).
The goals of the current work are to define the NHE isoforms that
mediate polarized Na/H exchange in the HT29-C1 model and to use
confocal microscopy to question whether transcellular
pHi gradients explain how SCFA
gradients selectively activate apical vs. basolateral Na/H exchange.
Confocal microscopy has the spatial resolution to resolve subcellular
events and is applicable to study of living cells (35). Results show
that multiple NHE isoforms are present and that subcellular
pHi heterogeneity can only
partially explain how SCFAs selectively activate polarized exchangers.
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METHODS |
Tissue culture.
HT29-C1 cells obtained from D. Louvard (Paris, France) were used
between 10-19 passages after original subcloning from the parental
HT29-18 line. HT29-C1 cells are stably differentiated when grown in
DMEM containing 25 mM glucose, as described earlier (32, 45, 46). In
some experiments, cells grown on plastic flasks for 1 wk were suspended
using 0.005% trypsin and then exposed to 7 mg ovomucoid trypsin
inhibitor (Sigma) and dispersed into a single cell suspension for study
by fluorometry (45). For substrate-attached cell experiments, HT29-C1
cells were grown for 3-7 days (1-2 days postconfluency) on
permeant membrane filters (Anotec, Whatman) that were attached to
plastic frames (Ref. 46 or as slightly modified by D. Maouyo, S. Chu,
and M. H. Montrose, unpublished observations, for confocal microscopy).
Fluorometric measurement of pHi.
Either suspended cells or confluent cell monolayers on filters were
studied. Cells were incubated for 45 min at 37°C with 2 µM
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM
(BCECF-AM; Molecular Probes) in "NaCl medium" [containing,
in mM, 130 NaCl, 5 KCl, 20 HEPES, 25 mannose, 1 (Na)PO4, 2 CaCl2, and 1 MgSO4, titrated to pH 7.4].
After dye was loaded, suspended cells were centrifuged 10 s at
2,000 g, resuspended in the absence of
dye, and then placed in a stirred fluorometer cuvette thermostated at
37°C (45). After filter-grown cells were dye loaded, the filter was
mounted in a fluorometry chamber that allowed independent control of
apical and basolateral superfusion (30). BCECF fluorescence was
measured in an SLM 500C spectrofluorometer at 520- to 540-nm emission
in response to alternating excitation of 500 and 440 nm. The
fluorescence excitation ratio (500 to 440 nm) was calibrated vs.
pHi as previously described (30,
45, 46).
Confocal microscopy measurement of
pHi.
Confluent cells on filters were incubated with 10 µM carboxy
SNARF-1-AM (Molecular Probes) for 45 min at room temperature. The
filters were then mounted in a microscopy chamber placed on the stage
of a Zeiss LSM410 confocal microscope and continuously superfused (6)
during imaging with a ×40 C-Apo water-immersion objective.
Intracellular SNARF-1 was excited with a 488-nm argon laser light, and
two fluorescent emissions were collected simultaneously at 550-600
nm and 620-680 nm. During experiments, cells in the monolayer were
scanned along the apical-to-basal
(xz-plane) axis using eight line
averages (4 s per image), and an image was collected once per minute.
Results were analyzed postacquisition using Metamorph software
(Universal Imaging), Microsoft Excel, and GraphPad Prism. Background-corrected emission ratios (620-680 nm to 550-600
nm) were used to estimate pHi,
based on daily calibration of SNARF-1-free acid on the microscope stage
(6). Thresholding of raw fluorescence images was used to exclude
regions of low fluorescence during analysis. Image analysis was used to
estimate pHi either from the
entire cell or from five subcellular regions distributed equally along
the apical-to-basal axis. Subcellular regions were 4-µm-diameter circles (79 pixels each) that were equally spaced 1-2 µm apart to span the entire apical-to-basal axis of single cells. An example of
region placement is shown in Fig. 6A.
Superfusate solutions.
All superfusates were based on standard NaCl medium. In SCFA medium,
130 mM chloride was replaced with equimolar propionate. All sodium was
replaced with equimolar tetramethylammonium (TMA) in sodium-free
TMA-chloride medium. All media were isosomotic (Wescor 5500 osmometer),
and all solutions were titrated to pH 7.4. HOE-694 (a generous gift of
Dr. H. Lang at Hoescht Marion Roussel) was solubilized in NaCl medium.
For the experiments with SNARF-1, all perfusion solutions contained 1 mM probenecid to inhibit dye loss.
PCR.
As described previously, total RNA was isolated from confluent flasks
of HT29-C1 cells using guanidinium thiocyanate and centrifuging the
cell lysate through a CsCl cushion (32). First-strand cDNA was
transcribed with Moloney murine leukemia virus-RT (GIBCO
BRL) using random hexanucleotide primers (Pharmacia). Unless noted, cDNA templates were subjected to 30 cycles of amplification with recombinant Taq DNA polymerase (Perkin
Elmer Cetus) and an annealing temperature of 56°C, and then an
aliquot (3 µl) of reaction product was used as template to initiate a
second 30-cycle PCR with the same primers. NHE1 primers were
5'-AAGTGTCTGATAGCTGGC-3' and
5'-TGCTCCGCATCATGATGC-3'. NHE2 primers were
5'-AAACATGCCATAGAGATGGC-3' and
5'-CCACCTCATTCTTCCATTC-3'. NHE3 required two rounds of PCR
with nested primers. The first round used
5'-GAGAGAAAATGTCAGCGC-3' and
5'-GCAGGAAGGAGTCCACG-3'. Three microliters of this PCR
product were used as template for the second round of NHE3
amplification. The nested primers for the second 30 cycles of
amplification were 5'-AGGACATGGTCACGCACC-3' and
5'-GGAGAGTAGGGAATCTGC-3', with a 58°C annealing
temperature. Caco-2 RNA was a neighborly gift from C. H. C. Yun.
Statistics.
Averaged results are presented as means ± SE. Comparison between
two groups was performed by unpaired two-tailed
t-test, and comparisons among multiple
groups were performed by one-way ANOVA with the Bonferroni multiple
comparison test (Prism software, GraphPad). Differences of
P < 0.05 were considered
significant. When multiple comparisons are discussed, only the most
conservative level of significance is cited to simplify presentation.
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RESULTS |
Evidence for multiple NHE isoforms in HT29-C1 cells.
We had previously observed both apical and basolateral Na/H exchange in
HT29-C1 cells (46), but the molecular identity of these transporters
has not been established. To find out whether multiple members of the
NHE gene family of Na/H exchangers play a role in
pHi regulation of HT29-C1 cells,
we examined effects of the NHE inhibitor HOE-694 on suspended cells.
HOE-694 has widely different potency for inhibition of NHE1, NHE2, or
NHE3 (12). Suspended cells provided a system in which all plasma
membrane proteins were equally available to the extracellular
environment, albeit in the absence of cellular polarity. As performed
previously (45, 46), cells were loaded with a pH-sensitive fluorescent dye (BCECF), and Na/H exchange was measured as the sodium-dependent recovery of pHi from an acid load
induced by transient exposure to
NH4Cl.
We examined the effects of varying concentrations of HOE-694 added
during the pHi recovery from an
acid load. Drug inhibition was calculated as the percent change in the
linear rate of pHi recovery after
vs. before HOE-694 addition. Results of individual runs are shown in
Fig. 1; nonlinear least squares curves fit
to the data assumed either one or two binding sites for HOE-694. Statistical comparison of the two fits (Prism software, GraphPad) indicated that the two-site model was a better fit to the data (P < 0.005), which suggested that at
least two kinetically distinct transporters were responsible for
pHi regulation. On the basis of
prior work showing that NHE3 had an HOE-694
ID50 = 650 µM (12), the complete
inhibition of Na/H exchange by 10 µM HOE-694 suggested that NHE3 was
unlikely to contribute to pHi
regulation.

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Fig. 1.
HOE-694 concentration dependency for inhibition of Na/H exchange.
Intracellular pH (pHi) was
measured in suspended HT29-C1 cells using
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
as described in METHODS. Na/H exchange
was quantified as the sodium-dependent recovery from an acid load
induced by 30 mM NH4Cl prepulse
(45). Linear rates of pHi recovery
were compared before vs. immediately after addition of indicated
concentrations of HOE-694. Extent of inhibition was calculated as the
percent decrease in the rate of
pHi recovery by the drug, and each
point represents a single determination with results compiled from 3 experiments. Results were fit by 2 equations: a 1-binding site model
(dotted line) and a 2-binding site competition model (solid line). Data
deviated from the 1-site model (P < 0.05) but not from the 2-site model (P = 0.37). The 2-site model was a significantly better fit
(P = 0.005), even accounting for the
larger number of fit variables compared with the 1-site model (Prism
software, GraphPad). Inset key
presents the best-fit parameters derived from both models.
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Experiments were performed to determine whether kinetically distinct
Na/H exchangers segregated to the apical vs. basolateral membranes.
Confluent HT29-C1 cell monolayers on filters were studied in a
fluorometry chamber, which allowed independent control of luminal and
serosal superfusion (bathing the apical and basolateral membranes,
respectively). Cells were acidified by
NH4 prepulse, and then sodium was
added unilaterally to initiate pHi
recovery. During pHi recovery,
varying concentrations of HOE-694 were superfused to the same cell
surface to measure drug sensitivity. Figure
2 shows results from a single experiment,
qualitatively demonstrating differences between sensitivity of Na/H
exchange to 1 µM HOE-694 added at either the apical or basolateral
membrane. Figure 3 compiles results from
five experiments to quantify the inhibitory potency of HOE-694. Curve
fitting of results at the apical or basolateral membrane gave the best
fit with only a single HOE-694 inhibitor site vs. a two-site model,
suggesting that only one Na/H exchange was kinetically resolved at
either membrane domain. The dotted line in Fig. 3 is the best fit of
the basolateral data set to a two-site model having the HOE-694
affinities defined from suspended cells. This special case of the
two-site model is clearly a poor fit. Results suggest the presence of
two Na/H exchangers in HT29-C1 cells: one at the apical membrane having
a low-affinity HOE-694 binding site and one at the basolateral membrane
with a higher-affinity HOE-694 binding. The low-affinity value for
suspended cells is indistinguishable from the apical membrane value and
is similar to the ID50 reported
for NHE2 (12). The high-affinity site value for suspended cells differs
by sixfold from the basolateral membrane value, but both most closely
resemble the ID50 reported for
NHE1 (12).

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Fig. 2.
Differing potency of HOE-694 against apical and basolateral Na/H
exchange. pHi was measured in
filter-grown HT29-C1 cells using BCECF and a conventional fluorometer
as described in METHODS. Results from
a single monolayer during 2 separate recoveries from an acid load
induced by 30 mM NH4Cl prepulse
are shown. After cells were equilibrated in tetramethylammonium
(TMA)-chloride medium (TMA), NaCl medium (Na) was added to either the
luminal or serosal superfusate to selectively activate apical or
basolateral Na/H exchange, respectively. At the indicated times, 1 µM
HOE-694 was added to the same side of the monolayer as the sodium. As
shown, apical Na/H exchange was more resistant to inhibition by HOE-694
than basolateral Na/H exchange.
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Fig. 3.
HOE-694 concentration dependency for inhibition of polarized Na/H
exchangers. Results were compiled from 5 experiments such as that shown
in Fig. 2, using different concentrations of HOE-694 to separately
quantify inhibition of apical ( ) or basolateral ( ) Na/H exchange.
Inhibition of Na/H exchange was measured as described in Fig. 1. Each
data point is mean of 3-5 determinations. Results were fit by a
1-site model (solid line) but would not converge with a 2-site model
having full freedom to determine 50% inhibitory dose
(ID50) values (because only 1 site was needed to fit the curve; the other site was indeterminate). If
ID50 values were fixed at 0.05 and
1.6 µM (values determined from suspended cells) and the 2-site model
was applied to the basolateral data set, best-fit parameters ascribed
52% of transport to the high-affinity site, although the curve fit
(dotted line) was inferior. Key shows the best-fit parameters derived
from the 1-site model. These and all averaged results are presented as
means ± SE.
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To complement these functional analyses, PCR was used to detect the
presence of mRNA for specific NHE isoforms. Primers were designed to
amplify the most divergent portion of the isoforms, the COOH-terminal
region (51). As shown in Fig. 4, RT-PCR
detected the presence of NHE1 and NHE2 but not NHE3. The identity of
amplified fragments was confirmed by digestion with rare-cutting (6-bp
recognition) restriction enzymes predicted from published human NHE
sequences. The NHE2 restriction sites were commonly predicted from the
human NHE2 sequence of Ramaswamy et al. (18) (GenBank no. S83549) and
Ghishan et al. (11) (GenBank no. 81591). The significance of negative
NHE3 results in HT29-C1 was confirmed by positive amplification of NHE3
from Caco-2 cells, another human colon carcinoma line that expresses
NHE3 several weeks postconfluency (29). Both kinetic and molecular
analyses support the lack of NHE3 and the presence of basolateral NHE1
and apical NHE2 in HT29-C1 cells.

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Fig. 4.
RT-PCR analysis of NHE isoforms present in HT29-C1 cells.
Isoform-specific primers were used to amplify COOH-terminal sequences
from NHE1, NHE2, and NHE3 mRNA. PCR products were run on agarose gels
and visualized with ethidium bromide. Restriction enzymes confirmed the
identity of the amplified fragments.
A: cDNA from HT29-C1 was used to
amplify fragments of NHE1 or NHE2, as indicated. Predicted 621-bp NHE1
amplification product was observed (lane
1) and had the predicted restriction sites for
Ban I (lane
2) and Apa I
(lane 3). Similarly, 517-bp NHE2
amplification product (lane 4) had
the predicted restriction sites for
Hind III (5' 340-bp fragment and
3' 177-bp fragment) (lane 5)
and Tth III (5' 162-bp fragment
and 3' 355-bp fragment) (lane
6). B: NHE3 primers
were used to amplify cDNA from Caco-2 or HT29-C1 cDNA as indicated.
Despite use of nested PCR and attempts with several primer pairs, we
could not observe a clean PCR product. However, cDNA from Caco-2 cells
demonstrated the most prominent band at the predicted 549 bp size
(lane 1, marked by arrow) that was cut
by Sal I to generate the predicted
fragments (160 and 389 bp) (lane 2,
marked by *) and by Nco I to generate
the correct fragments (264 and 285 bp) (lane
3, marked by >). In contrast, the PCR fragments
amplified from HT29-C1 (lane 4) did
not give the predicted Nco I fragments
(lane 5).
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Regulation of pHi gradients by SCFAs and
Na/H exchange.
Heterogeneity of pHi might explain
why luminal-to-serosal transepithelial gradients of SCFAs are required
to observe selective stimulation of apical Na/H exchange (4, 14, 15,
46). Therefore, confocal microscopy was used to resolve
pHi at a subcellular level.
Because luminal SCFA preferentially activates apical Na/H exchange
activity, and serosal SCFA preferentially activates basolateral Na/H
exchange (46), we compared these two conditions as those predicted to
yield the most widely different
pHi gradients.
We first tested the sensitivity of confocal microscopy to report
pHi recovery of the entire cell
cytosol, when imaging along the apical-to-basal pole using the SNARF-1
fluorophore. As shown in the abbreviated time course of Fig.
5A, cells
acidified after exposure to serosal 130 mM sodium propionate at
time zero and mounted a
pHi recovery over the ensuing 14 min in the continuous presence of the SCFAs. Figure
5B shows that the
pHi recovery is inhibited by
serosal addition of 10 µM HOE-694 or bilateral removal of sodium from
the medium. Figure 5C shows
pHi recovery in response to
luminal 130 mM sodium propionate added at time
zero, which examined the same time course as in Fig.
5A. Figure
5D shows that the response to luminal
propionate is inhibited by luminal addition of 10 µM HOE-694 or
bilateral removal of sodium. These results validate the sensitivity of
confocal microscopy to detect activation and inhibition of apical or
basolateral Na/H exchange in response to SCFA gradients. Results after
HOE-694 addition also suggest apical Na/H exchange is responsible for
the majority of total Na/H exchange following stimulation by luminal
SCFA, and, conversely, basolateral Na/H exchange predominates after
serosal SCFA. Both results are consistent with prior observations that
the orientation of the SCFA gradient determines the preferential
activation of polarized Na/H exchangers (46).

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Fig. 5.
Response to short-chain fatty acids (SCFA) reported in whole cell
confocal analyses. Confocal microscopy was used to
image cell monolayers loaded with carboxy SNARF-1, as described in
METHODS. Cells were directly imaged
along the apical-to-basal axis
(xz-images) during superfusion with
different solutions, and pHi was
estimated from the fluorescence of the entire visualized cytosol.
Results are presented for 4 time points: 2 min before SCFA addition
while cells were in NaCl medium ( 2 min), at the nadir
pHi directly following SCFA
addition (defined as time 0), and 7 and 14 min after time 0 in the
continued presence of SCFA. A: cells
were exposed to serosal 130 mM sodium propionate while NaCl medium was
maintained in the luminal superfusate
[n (no. of experiments) = 8]. As shown, confocal microscopy detects
pHi recovery in the presence of
SCFA. B: cells were exposed to serosal
130 mM TMA-propionate in combination with luminal TMA-chloride ( ,
n = 3) or serosal sodium propionate
plus 10 µM HOE-694 in combination with luminal NaCl medium ( ,
n = 5).
C: cells were exposed to luminal 130 mM sodium propionate while NaCl medium was maintained in the serosal
superfusate (n = 6).
D: cells were exposed to luminal 130 mM TMA-propionate in combination with serosal TMA-chloride ( ,
n = 3) or luminal sodium propionate
plus 10 µM HOE-694 in combination with serosal NaCl medium ( ,
n = 4). As shown, either removal of
sodium or addition of HOE-694 blocked
pHi recovery.
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We next questioned whether differences in
pHi could be resolved at different
regions of the cell along the apical-to-basal axis. Figure
6A shows a
SNARF-1 fluorescence image of a cell monolayer directly imaged along
the apical-to-basal axis during superfusion with luminal sodium
propionate. Superimposed on the image are five regions (each 10 pixels
in diameter) that indicate the size and approximate spacing of
subcellular domains that were independently analyzed to assess the
response along the apical-to-basal axis. Analysis of the five regions
was performed at each time point for four or five cells in each
monolayer. Figure 6B shows a SNARF-1 ratio image calculated from the cells in Fig.
6A. As shown qualitatively from the
pseudocolor scale, the ratio image reports that the apical regions of
cells in the monolayer were more acidic than the basal regions. To
investigate whether results were influenced by optical artifacts,
control experiments questioned whether ratio images of 0.1 mM carboxy
SNARF-1-free acid in solution reported constant pH
1) at the interface between the dye
solution and a glass coverslip and
2) as a function of focal depth into
the dye solution. No pH measurements at the different locations in the
dye solution were significantly different
(P > 0.7), suggesting that optical artifacts are unlikely to be responsible for
pHi differences that are reported
at different depths of the cell. These results confirmed our previous
results when using a different confocal microscope (6).

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Fig. 6.
Direct confocal imaging of polarized HT29-C1 cells along the
apical-to-basal axis. Filter-grown HT29-C1 cells were imaged during
superfusion with luminal sodium propionate and serosal NaCl medium, at
the time point reporting the nadir
pHi in whole cell analyses (i.e.,
time 0 in Fig.
5A).
A: confocal fluorescence image of
SNARF-1-loaded cells. The xz-plane
image is an average of the fluorescence intensities from simultaneously
collected 550- to 600-nm and 620- to 680-nm images. Apical and basal
boundaries of the cell monolayer are indicated by arrows. Superimposed
on the image are 5 circular regions that have the size and placement
typically used for subcellular analyses of
pHi.
B: pseudocolor ratio image derived
from data in A. Color bar defines the
correspondence of color to pHi. As
shown, the apical pole of cells is relatively acidic compared with the
basal pole.
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Quantitative analysis of subcellular events from a single monolayer is
shown in Fig. 7. Figure
7A shows results analyzed for the
entire cytosol during a time course experiment compared with results in
Fig. 7B, which analyzed five
subcellular regions along the apical-to-basal axis (results averaged
from 4 cells in the monolayer). As shown, exposure to serosal SCFA
caused a prominent acidification of the apical regions of the cell
(qualitatively similar to the response to luminal SCFA shown in Fig.
7B) in either the presence or
absence of sodium. Results in Fig. 7B
also suggest that sodium-dependent
pHi recovery occurs prominently in
the most apical region of the cell and that
pHi gradients can be generated and
sustained in the absence of Na/H exchange.

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Fig. 7.
Comparison of cellular response to serosal SCFA reported for whole cell
and subcellular regions along the apical-to-basal axis. Results are
from a time course experiment that imaged a filter-grown HT29-C1
monolayer with a confocal microscope. Cells were directly imaged along
the apical-to-basal axis while the response to either serosal sodium
propionate (NaProp) or TMA-propionate (TMAProp) was evaluated.
A: measurement of
pHi reported from whole cell
analyses. B: measurement of
pHi from 5 subcellular regions
spanning the apical-to-basal dimension of the cells. Size and spacing
of regions were as depicted in Fig.
6A, and results from each region are
reported separately (key identifies most apical, near-apical, mid-cell,
near-basal, and most basal from top to
bottom, respectively). Subcellular
analysis was performed for 5 cells in the monolayer, and average values
of pHi are
reported.
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To substantiate these conclusions, results were compiled from multiple
experiments that compared the subcellular
pHi values before, during, and
after exposure to serosal SCFA. Results in Fig.
8A are
from six separate experiments, with five subcellular compartments
analyzed in at least four cells for each time point in each experiment.
Three time points were examined during serosal SCFA exposure.
Time zero is defined as the nadir
pHi (as determined from whole cell
analyses) directly after SCFA addition. The
pHi was also reported after an
additional 7 or 14 min of SCFA exposure past time
zero. Statistical comparisons (one-way ANOVA with
Bonferroni multiple comparison tests) were first made to determine
which individual regions significantly changed
pHi during the experiment. As
predicted from inspection of the results, each cellular region acidified due to serosal sodium propionate addition at
time zero (P < 0.05 for each region, comparing
before vs. 0 min). During the 14 min of SCFA exposure, only the most
apical region underwent significant
pHi recovery alkalinization
(P < 0.01, comparing 0 vs. 14 min,
and P < 0.05, comparing 0 vs. 7 min), although all regions reported increasing values of
pHi. All regions alkalinized upon
SCFA removal (P < 0.001 for each
region comparing 14 min vs. after) and the
pHi after SCFA removal was higher
than the starting pHi
(P < 0.001 for each region,
comparing before vs. after). This is consistent with our previous
observations of a pHi overshoot
following pHi recovery by Na/H
exchange during SCFA exposure of isolated mouse colonocytes (8).
Statistical comparisons also questioned which regions within the cell
were different at any given time point in Fig.
8A. Before SCFA addition, cells were exposed to NaCl medium and were at resting
pHi. Under these resting conditions ("before"), the most apical region was significantly acidic compared with the three most basal regions of the cell (P < 0.01), but no other differences
were significant among regions. During serosal SCFA exposure, the most
apical region was more acidic than all other portions of the cell
(P < 0.01), and the adjacent
near-apical region was also more acidic than the most basal portion of
the cell (P < 0.05). Thus the apical
portions of the cells act as a distinct subcellular compartment that is the site of the majority of pHi
recovery. After SCFA removal, there was no difference among pH values
in intracellular regions.

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Fig. 8.
Analysis of subcellular pHi
following serosal SCFA addition. Results from confocal time course
experiments were compiled to quantitatively compare the subcellular
response to serosal sodium propionate in the presence vs. absence of
active Na/H exchange. Multiple time points were analyzed. Before SCFA
addition ("before"), cells were exposed to NaCl medium in both
luminal and serosal superfusates. SCFA medium was then added only to
the serosal medium. During SCFA exposure,
pHi was analyzed at the nadir
pHi determined from whole cell
experiments (defined as time 0) and
at 7 and 14 min past time 0. When SCFA
medium was removed (14-24 min after SCFA addition), it was
replaced with NaCl medium and pHi
was evaluated 2-4 min later ("after"). Size and spacing of
subcellular regions were as depicted in Fig.
6A, and results from each region are
reported separately for each time point (key identifies most apical,
near-apical, mid-cell, near-basal, and most basal, from
top to
bottom, respectively). Subcellular
analysis was performed for 5 cells in each monolayer at each time
point, and average values of pHi
are reported. A: cells previously
equilibrated in NaCl medium (i.e., at resting
pHi) were exposed to serosal 130 mM sodium propionate. Results are compiled from 6 experiments.
B: cells were previously exposed to
conditions described in A and so were
not at resting pHi at the before
time point. Cells were exposed to serosal 130 mM sodium propionate plus
serosal 10 µM HOE-694 at time 0.
Results are compiled from 4 experiments.
|
|
In prior experiments, we had shown that transepithelial gradients of
SCFAs lead to selective activation of polarized Na/H exchangers.
Specifically, serosal propionate preferentially activated basolateral,
but not apical, Na/H exchange (46). To determine whether basolateral
Na/H exchange was affecting the
pHi gradient, similar experiments
were performed in the presence of serosal 10 µM HOE-694, to fully
inhibit basolateral Na/H exchange. Figure 8B compiles results from multiple
experiments (n = 4) that compared subcellular pHi values before,
during, and after exposure to serosal SCFA plus 10 µM HOE-694. In
this data set, cells had previously been exposed to serosal sodium
propionate alone; therefore, the before results in Fig.
8B are similar to the "after" condition in Fig.
8A. In the presence of HOE-694, all
cellular regions acidified when SCFA was added and alkalinized when
SCFA was removed (P < 0.05), but no
pHi recovery trend was noted in
any region. Qualitatively similar results were observed when cells were
acidified by serosal TMA-propionate to inactivate Na/H exchange (data
not shown). In the presence of HOE-694, the most apical region was more
acidic than other regions of the cell (P < 0.05)
at all time points during SCFA exposure. When Fig.
8A is compared with Fig.
8B, a similar subcellular
pHi gradient is noted in both the
presence and absence of HOE-694, suggesting that
1) the presence of the SCFA, not the activity of Na/H exchange, was responsible for generating the pHi gradient and
2) the apical acid/base equivalents
do not equilibrate with the rest of the cell in the absence of
pHi regulation.
Figure 9 presents an analogous compilation of
subcellular pHi values following
exposure to luminal sodium propionate in either the absence (Fig.
9A; n = 6) or presence (Fig. 9B;
n = 3) of 10 µM HOE-694. In response
to luminal sodium propionate (Fig.
9A), all regions except the most
basal were significantly acidified by SCFA addition and alkalinized
after SCFA removal (P < 0.05). Within cells, there was no significant difference among the regional pHi values either before or after
SCFA exposure. However, at all time points during SCFA exposure, the
most apical region was acidic compared with all other regions
(P < 0.05), and the near-apical region was also acidic compared with the most basal region
(P < 0.05). Overall, results were
qualitatively similar to those following serosal sodium propionate and
suggested that the apical portion of the cell was the most responsive
site of pHi change elicited by
either luminal or serosal SCFA exposure. The results also confirm prior
results showing that apical Na/H exchange is predominately activated in
response to luminal SCFA (46).

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Fig. 9.
Analysis of subcellular pHi
following luminal SCFA addition. Results from confocal time course
experiments were compiled to quantitatively compare the subcellular
response to luminal sodium propionate in the presence vs. absence of
active Na/H exchange. Before SCFA addition (before), cells were exposed
to NaCl medium in both luminal and serosal superfusates. SCFA medium
was then added only to the luminal medium. During SCFA exposure,
pHi was analyzed at the nadir
pHi determined from whole cell
experiments (defined as time 0) and
at 7 and 14 min past time 0. When SCFA
medium was removed (14-26 min after SCFA addition), it was
replaced with NaCl medium and pHi
evaluated 2-4 min later (after). Size and spacing of subcellular
regions were as depicted in Fig. 6A,
and results from each region are reported separately for each time
point (key identifies most apical, near-apical, mid-cell, near-basal,
and most basal from top to
bottom, respectively). Subcellular
analysis was performed for 4 or 5 cells in each monolayer at each time
point, and average values of pHi
are reported. A: cells previously
equilibrated in NaCl medium (i.e., at resting
pHi) were exposed to luminal
130 mM sodium propionate. Results are compiled from 6 experiments. B: cells
were exposed to luminal 130 mM sodium propionate plus luminal 10 µM
HOE-694. Results are compiled from 3 experiments.
|
|
Comparison of transcellular pHi gradients
caused by apical or basolateral SCFA.
On the basis of the differential activation of polarized Na/H
exchangers by luminal vs. serosal SCFAs (46), we had previously predicted that exposure to luminal SCFA might produce a different subcellular heterogeneity of pHi
from exposure to serosal SCFA. To formally test this prediction, we
compared the extent of intracellular acidification caused by either
luminal or serosal SCFA at each of the five subcellular regions.
Results in Fig. 10 were normalized to the
pHi change observed at the most
basal portion of the cell, to facilitate comparisons between
conditions. Both luminal and serosal SCFA produced greater
acidification at the apical vs. basolateral pole of the cell
(P < 0.001). However, as indicated in Fig. 10, luminal SCFA caused a greater
pHi acidification than serosal
SCFA in the entire apical half of the cell
(P < 0.05).

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Fig. 10.
Comparison of transcellular acidification caused by luminal or serosal
SCFA. We compared the magnitude of
pHi change
( pHi) elicited immediately on
SCFA addition to either luminal or serosal medium.
pHi was quantified for each
subcellular region individually as the difference between "0 min"
and "before" pHi values from
experiments such as those in Figs. 8 and 9. Subcellular heterogeneity
of acidification was similar whether caused by sodium propionate in the
absence or presence of HOE-694 or from exposure to TMA-propionate (data
not shown); therefore, these conditions were compiled together. Values
in each monolayer were normalized to the acidification observed at the
most basal region of the cell to facilitate transcellular comparisons
between conditions. Before normalization, average acidification
observed in the most basal region was 0.40 ± 0.05 pH units (13 experiments) when exposed to serosal SCFA and 0.25 ± 0.05 pH units
(9 experiments) when exposed to luminal SCFA.
* P < 0.05, ** P < 0.01 when luminal vs.
serosal responses at the same subcellular site are compared.
|
|
Because luminal SCFA predominantly activates apical Na/H exchange
(NHE2), and serosal SCFA predominantly activates basolateral Na/H
exchange (NHE1) (46), it was also of interest to ask if the
pHi recovery in response to these
two stimuli was different across the cell. Therefore, we compared the
extent of pHi recovery (Na/H
exchange) during 14 min of either luminal or serosal SCFA exposure at
each of the five subcellular regions. Results are shown in Fig.
11. Both luminal and serosal SCFA
elicited a pHi recovery of similar
magnitude at the apical region of the cell, which was larger in both
cases than that observed at the basal region
(P < 0.05). In the basal half of the
cell, serosal SCFA elicited a pHi
recovery that was consistently greater than that seen during luminal
SCFA, but this did not attain statistical significance except for one
cell region (indicated in Fig. 11). Qualitatively similar results were
observed when results were examined after 7 min of
pHi recovery (data not shown).
Results suggest that subcellular sites of
pHi recovery are similar, although potentially not identical, when comparing between luminal and serosal
SCFA exposure.

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Fig. 11.
Comparison of transcellular pHi
recovery during exposure to luminal or serosal SCFA. We compared the
magnitude of pHi change
( pHi) elicited during 14 min
of sodium propionate exposure at either luminal or serosal surface.
pHi was quantified for each
subcellular region individually as the difference between "14
min" and 0 min pHi values from
experiments such as those in Figs. 8A
and 9A. Results are compiled from
multiple experiments during exposure to serosal SCFA (6 experiments),
or luminal SCFA (6 experiments).
** P = 0.018 when luminal vs.
serosal responses at the same subcellular site are
compared.
|
|
 |
DISCUSSION |
Similar to colonic tissue (17, 19, 20, 37, 39, 40), the colon carcinoma
cell line HT29-C1 has both apical and basolateral Na/H exchange
activity (46). We previously reported that the polarized Na/H
exchangers in HT29-C1 cells could be activated by SCFAs and that
flipping the orientation of a transepithelial SCFA gradient
preferentially activated either apical or basolateral Na/H exchange
(46). Physiologically (luminal-to-serosal) oriented SCFA gradients
preferentially activated apical Na/H exchange, in keeping with
physiological expectations. These results parallel observations in
native tissue (1, 14, 15, 21, 36, 47) and suggest that local activation
of Na/H exchangers might be explained by microdomains of pH near the
polarized plasma membranes [either within (4) or outside (7)
cells]. Our goal in this study was to address two questions
raised by these observations. First, we sought to identify the Na/H
exchanger isoforms that mediated the response to SCFAs in the HT29-C1
model. Second, we sought to test the prediction that
pHi gradients may play a role in
selective activation of the polarized exchangers of HT29-C1 cells.
NHE isoforms in HT29-C1 cells.
A combination of kinetic and molecular approaches was used to identify
which members of the NHE gene family of Na/H exchangers were present in
HT29-C1 cells. The Na/H exchange inhibitor HOE-694 is known to have
widely different potencies against NHE1
(ID50 = 0.16 µM), NHE2
(ID50 = 5 µM), and NHE3
(ID50 = 650 µM) (12). In
suspended or polarized HT29-C1 cells, all Na/H exchange was inhibited
by 10 µM HOE-694, suggesting NHE3 was not functionally important.
This was consistent with the lack of NHE3 mRNA observed in HT29-C1
cells by RT-PCR. Inhibition kinetics identified two distinct
HOE-694-sensitive components of Na/H exchange in suspended HT29-C1
cells, which appeared to segregate into the single kinetic components
identified in the apical or basolateral membrane of polarized cells. At
a minimum, our work defined the presence of functionally different Na/H
exchangers at the two membrane domains of HT29-C1 cells and identified
a concentration of HOE-694 (10 µM) that is able to fully inhibit
either transporter.
Results allowed us to provisionally assign specific NHE isoforms as the
apical and basolateral exchangers of HT29-C1 cells. On the basis of
results with HOE-694, the "low-affinity"
ID50 value from suspended cells
(1.6 µM) was indistinguishable from the value observed at the apical
membrane (1.4 µM) and was most closely aligned with known properties
of NHE2. In support of the suggestion that NHE2 is the apical Na/H
exchanger, NHE2 mRNA was readily detected from HT29-C1 cells. The
assignment of the basolateral transporter was less clear because the
"high-affinity" ID50 from suspended cells (0.05 µM) was sixfold lower than the
ID50 value at the basolateral
membrane (0.3 µM). Together, these values bracket the previously
reported ID50 value for NHE1 (0.16 µM), and NHE1 mRNA was detected in HT29-C1 cells. The lower affinity
at the basolateral membrane could potentially be explained by a mixture of NHE1 and NHE2 in that membrane [NHE2 is a basolateral membrane protein in kidney medulla (49)] or a mixture of NHE1 with another untested isoform having low HOE-694 affinity. However, neither possibility is likely because the basolateral membrane only reports a
single HOE-694 binding site and results are not well fit by any mixture
of ID50 values in a two-site model
(see Fig. 3). Instead, our working hypothesis is that either restricted
access or altered environmental conditions (e.g., pH) at the
basolateral surface of polarized cells decrease HOE-694 binding to NHE1
and lower the apparent affinity.
Mammalian colon expresses NHE1, NHE2, and NHE3 (18, 52). NHE1 is
accepted as a virtually ubiquitous protein, expressed in the
basolateral membrane of epithelial cells. It has previously been
suggested that 25-55% of NHE1 is expressed in the apical membranes of another HT29 clone (HT29-19A) and in Caco-2 cells (33). Our results suggest a higher fidelity of membrane protein sorting
in the HT29-C1 clone under our experimental conditions. NHE2 and NHE3
have been identified as apical exchangers in colonic tissue (2, 18,
26), but it is unclear which isoform(s) contributes to sodium
absorption. The current work establishes HT29-C1 as a simplified
colonic model system for exploring activation of only a single apical isoform.
Visualizing subcellular pHi
gradients.
To test for the presence of pHi
gradients that may affect activation of NHE1 and NHE2, we applied
confocal microscopy to study filter-attached epithelial cells while
independently controlling the composition of superfusates bathing the
apical and basolateral membrane domains. The focal axis of the
microscope was the same as the apical-to-basal axis of the epithelial
cells. Given the numerical aperture of the microscope objective (1.2),
the refractive index of the water immersion (1.33), and the Stoke's
shift of SNARF-1, we should theoretically be able to resolve 0.8 µm
along the focal axis using the 488-nm laser for excitation (35). In our
work, subcellular measurements reported
pHi from 4-µm-diameter regions
that were separated by 1-2 µm, well within the capability of our
instrument to spatially resolve differences among regions.
There is a concern that the reported subcellular heterogeneity could be
explained wholly by optical and/or chemical artifacts. As
discussed in RESULTS, measurements of
dye in solution under identical conditions have discounted gross
optical artifacts (e.g., edge effects, inaccuracy of confocal
measurements as a function of focal depth) that could skew results (6).
It should also be noted that confocal microscopy faithfully reported
pHi recovery in whole cell
analyses (Fig. 5), supporting their general validity for measuring
pHi. Furthermore, depending on
experimental conditions, the disparate
pHi results could be reported from
multiple cellular regions or alternatively could be completely absent.
Thus subcellular pHi was
biologically responsive: a property not predicted for an optical artifact.
At the next level, artifact could result from varying chemical
properties of SNARF-1 dye at different intracellular regions of the
cell. If pHi gradients were purely
artifactual, this would require postulating either that SNARF-1
fluorescence is more sensitive to
pHi changes at the apical region
and/or SNARF-1 has an acid-shifted pKa at the apical
regions. However, if a fixed relationship existed between the
properties of apical vs. basal dye, then (with the small pH ranges
encountered in these experiments) the apical response should always be
proportional to the basal response. Contrary to this prediction,
results show that the ratio of basal to apical response is not always
constant (Figs. 10 and 11). In addition, both postulates would have to
be invoked simultaneously to explain how the cell can display
transcellular heterogeneity under some, but not all, conditions (Figs.
8 and 9). On the basis of these points, it is unlikely that results are
wholly due to subcellular variability in SNARF-1 response, and we
therefore assume that subcellular
pHi gradients exist and can be
regulated by SCFA exposure and Na/H exchange activity. This is
consistent with the observation by other investigators of
pHi gradients in multiple cell
types, as reported using both optical and electrophysiological methods (22, 23, 34, 43).
Generating and regulating transcellular
pHi gradients.
We know little about the minimum requirements to sustain
pHi at distinct values in one
subcellular region vs. another. Subcellular heterogeneity is difficult
to detect when cells are equilibrated in a conventional NaCl medium
(significant in Fig. 9A before but not
in Fig. 8A before), so at a minimum
there is not yet compelling evidence for the maintenance of subcellular
pHi heterogeneity in the absence
of SCFA stimuli. In the presence of 130 mM SCFA, transcellular
pHi gradients are rapidly
established in 1-2 min, in either the absence or presence of
active pHi-regulatory mechanisms. Because the apical domain acidifies most in response to either apical
or basolateral SCFA, it seems likely that this effect requires predominantly the mere presence of SCFA rather than
1) the vectorial energy in the
transepithelial SCFA gradient or 2)
a transcellular gradient of SCFA acting as a variable proton buffer. In
these experiments, propionate is used as a surrogate for total SCFA concentration (in the 100-150 mM range). This is probably a
reasonable assumption, since equimolar amounts of the major SCFAs
(C2-C5 aliphatic monocarboxylates) produce similar activation of
Na/H exchange in HT29-C1 cells [assayed as amiloride-sensitive
swelling (45)]. Furthermore, lower SCFA concentrations are known
to produce transient acidification in colonic tissue (14) and isolated colonocytes (8, 16, 47).
During pHi regulation by NHE1 or
NHE2, pHi recovery is observed in
the apical compartment, implying that protons leave this space (or
hydroxyl anions enter it). Results in Fig. 11 suggest that NHE1 does a
marginally better job than NHE2 at clearing protons from the basal half
of the cell, but there is no other evidence to suggest whether the net
proton fluxes that predominate in each region are those that go across
the plasma membrane or that mix with other regions.
The simplest explanation for these results would be a gradient of fixed
proton buffers in cytosol, with greater buffering capacity in the basal
half of the cell (27, 28). This would also require the assumption that
proton diffusion is not at equilibrium within cells, which is supported
by the observation of pHi
gradients by other investigators in other cell types (22, 23, 34, 43).
In this model, addition of a bolus of acid or base to the cytosol could
produce subcellular changes in pHi
proportional to the buffering capacity in each region. This would
explain how luminal and serosal SCFA could both cause predominantly
subapical pHi acidification and
how NHE2 and NHE1 could both cause
pHi recovery predominantly in the
subapical domains. However, strict proportionality of transcellular
pHi changes was not observed when
the acidification caused by luminal vs. serosal SCFA was compared (Fig.
10). Therefore, gradients of fixed proton buffers cannot explain all
observations. We predicted that transepithelial SCFA gradients would
cause polarized changes in pHi
(46), and this effect could conceivably have imposed a second force
affecting subcellular pHi that was
additive with effects of fixed buffer gradients. Further experiments
will be needed to test these possibilities.
The apical region was the most interesting from a physiological point
of view. It can be defined functionally as a compartment that
1) exists in the apical quarter of
the cell, 2) is accessible to SCFAs
and/or protons that flux across either apical or basolateral membranes, and 3) is the predominant
site of pHi acidification and
pHi recovery activated by SCFAs.
Some of these properties were correctly predicted by Dahger et al. (14,
15) when examining SCFA and CO2
stimulation of sodium absorption and have been summarized in a recent
review (4). We do not have enough information to physically define the
apical subcellular compartment. Because adjacent regions in the apical
half of the cells can also display significant
pHi differences from the basal
portions of the cell, there do not appear to be sharp boundaries to the
pHi microdomain. This makes it
less likely that the apical compartment is a previously unrecognized
organelle or a structure associated only with the brush-border
membrane. Although SNARF-1 dye will sometimes display punctate
fluorescence in subapical areas, above the usual homogeneous cytosolic
dye fluorescence, the ratio values reported from these "bright"
areas are not different from surrounding areas (data not shown). It
should also be noted that because the apical region reports the
expected pHi recovery from an acid
load, the basal regions may actually be the sites at which unusual
behavior is being manifest. In other words, it may be muting of the
basal response, rather than amplification of the apical response, that is the essential event in defining unusual transcellular behavior.
Do transcellular pHi gradients
explain selective activation of Na/H exchangers by SCFA
gradients?
Our experiments were designed to compare two transepithelial SCFA
gradient conditions that elicited a dramatic alteration in polarized
Na/H exchange activation in prior work (46). Based on a model of
transepithelial nonionic diffusion, we predicted that luminal SCFA
would cause mainly acidification of the apical domains of the cell and
serosal SCFA would cause mainly acidification of the basolateral
domains. This prediction was only partially fulfilled. In support of
the original prediction, luminal SCFA was shown to acidify the apical
domain more selectively than serosal SCFA (Fig. 10). However, the model
did not predict that both luminal and serosal SCFA would predominantly
cause acidification in the apical regions of the cell.
Results affect our working model of SCFA-stimulated Na/H exchange in
HT29-C1 cells. The highly reactive apical domain will act as an
amplifier of apical NHE2 activity when the cell is acidified. Physiological (apical-to-basolateral) SCFA gradients will also help to
enhance selective activation of apical NHE2 by preferential acidification of this subcellular domain adjacent to the membrane. For
these reasons, the subcellular response is ideally poised to activate
apical exchanger(s) and make sodium absorption exquisitely sensitive to
pHi regulation (4, 14, 15). In
contrast, the basal cell domains are relatively alkaline and resistant
to pHi perturbation, features that
should dampen activation of basal NHE1 under all tested conditions.
Transcellular pHi results do not
explain how serosal SCFA can preferentially activate basolateral NHE1,
since this condition still results in striking subapical acidification.
With our current resolution of subcellular events, we can only
speculate that the activation of NHE1 in the lateral membranes will be
intermediate between responses near the apical and basal poles. In
summary, pHi can be viewed as an
amplifier of cellular Na/H exchange in HT29-C1 cells, with subcellular
pHi heterogeneity playing a major
role in assuring robust activation of apical NHE2 during physiological
stimulation by SCFAs. However, the ability to flip SCFA gradients and
predominately activate NHE1 requires that other actions of SCFA
gradients [e.g., changes in extracellular pH; (7)] play a
dominant role in controlling activation of NHE1 activity and/or
inactivation of NHE2. Extrapolation of these conclusions to native
tissue must be made with caution, until a similar analysis can be
performed to appraise the presence and importance of
pHi gradients in the colonic
epithelium of animals.
 |
ACKNOWLEDGEMENTS |
Caco-2 mRNA was the generous gift of Dr. C. H. C. Yun. We gratefully
acknowledge critical reading of the manuscript by Dr. Chahrzad
Montrose-Rafizadeh.
 |
FOOTNOTES |
This work was supported by a Johns Hopkins University Provost's
Undergraduate Award to T. Gonda and National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-42457.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. H. Montrose, Indiana Univ., Med Sci
307, 635 Barnhill Drive, Indianapolis, IN 46202.
Received 26 January 1998; accepted in final form 26 September
1998.
 |
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