Inhibition of epithelial chloride secretion by butyrate: role
of reduced adenylyl cyclase expression and activity
Silvia
Resta-Lenert,
Francis
Truong,
Kim E.
Barrett, and
Lars
Eckmann
Department of Medicine, University of California, San
Diego, School of Medicine, San Diego, California 92103
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ABSTRACT |
Butyrate and
other short-chain fatty acids (SCFAs) are found at high concentrations
in the colonic lumen and affect multiple epithelial cell functions. To
better understand how SCFAs regulate ion transport, we investigated the
effects of SCFAs on Cl
secretion in human colonic
epithelial cell line T84. Butyrate inhibited
Cl
secretory responses to prostaglandin E2,
forskolin, and cholera toxin. Other SCFAs were less effective or
inactive. Reduced secretion was associated with decreased synthesis of
the second messenger cAMP rather than increased degradation. Expression
and activity of adenylyl cyclase were decreased by butyrate, whereas
phosphodiesterase activity was unaffected and phosphodiesterase
inhibition did not reverse the effects of butyrate on Cl
secretion. Furthermore, butyrate decreased expression of the basolateral Na-K-2Cl cotransporter, indicating that it might modulate the secretory capacity of the cells. However, butyrate did not affect
secretory responses to the calcium-dependent secretagogue carbachol,
cAMP analogs, or uroguanylin, indicating that normal secretory
responses to adequate levels of second messengers in butyrate-treated
T84 cells are possible. These results show that butyrate
affects several aspects of epithelial Cl
secretion,
including second messenger generation and expression of key ion
transporters. However, these effects may not all be equally important
in determining Cl
secretion in response to
physiologically relevant secretagogues.
intestinal epithelial cells; short-chain fatty acids, ion
transport
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INTRODUCTION |
SHORT-CHAIN FATTY
ACIDS (SCFAs), including acetate, propionate, and butyrate, are
by-products of bacterial fermentation of dietary fiber, i.e.,
undigested starches and proteins, in the colonic lumen. SCFAs are the
predominant anions in the colon, with a total concentration exceeding
100 mM. Butyrate, the anion of the 4-carbon aliphatic carboxylic acid
butyric acid, is present in the colonic lumen at concentrations of
10-30 mM. Colonocytes show a marked preference, above glucose, for
butyrate as a source of energy, and 60-70% of the energy consumed
by the colonic epithelium is derived from this SCFA (35).
Impaired oxidation and/or decreased levels of butyrate may also play a
role in the pathogenesis of colonic inflammation (7, 40).
Local administration of butyrate has shown benefits in reducing colonic
inflammation and alleviates diarrhea associated with inflammatory bowel
disease, diversion colitis, and other intestinal inflammatory
conditions (21, 36, 37).
In vitro, butyrate has been shown to induce cell differentiation
in colonic and other epithelial cell lines (6, 10, 14, 17,
35), which is accompanied by alterations in gene expression via
both translational and transcriptional mechanisms (35). In
cultured colonic epithelial cells, butyrate increases the expression of
alkaline phosphatase, mucins, the polymeric immunoglobulin receptor,
and intercellular adhesion molecule-1 and modulates the expression and
function of electrolyte transporters (16, 27, 30).
Electroneutral absorption of NaCl is greatly stimulated by butyrate
(5), whereas chloride secretion appears to be inhibited (12). The effect of butyrate on chloride transport was
previously ascribed to downregulation of the Na-K-2Cl cotransporter
NKCC1, whereas the expression and function of the apical cystic
fibrosis transmembrane conductance regulator (CFTR) chloride channel
was not altered by the SCFA in vitro (30). Butyrate has
also been shown to alter ion transporter function [e.g., Na/H
exchanger (NHE)2] by directly altering the pH microclimate in the
apical region of enterocytes (19).
In this study, we undertook a more detailed analysis of the effect of
butyrate as a negative regulator of chloride secretion in the
T84 cell line. We hypothesized that the effect of this SCFA
on chloride secretion might be multifactorial and directed at other
cellular events beyond downregulation of NKCC1. We demonstrate that
butyrate has significant and irreversible inhibitory effects on
chloride secretion evoked by prostaglandin (PG)E2,
forskolin, and cholera toxin (CT). This can be ascribed predominantly
to reduced intracellular cAMP production secondary to downregulation of
the expression and activity of adenylyl cyclase (AC). NKCC1 expression
is also reduced. In addition, we show that butyrate does not have any
direct effect on phosphodiesterase (PDE) functions, because inhibition
of PDEs does not reverse the effects of the SCFA on chloride
secretion. Finally, the effects of butyrate on chloride
secretion do not extend to calcium-dependent stimulation of this
transport mechanism or to secretion evoked by the addition of
cell-permeant cAMP analogs or the cGMP-dependent agonist uroguanylin.
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MATERIALS AND METHODS |
Cell culture.
All studies were performed with T84 human colonic
epithelial cells. Cells were grown in 50% DMEM, 50% F12 medium,
supplemented with 5% heat-inactivated newborn calf serum, in an
atmosphere of 95% air-5% CO2 at 37°C. To obtain
polarized monolayers, cells not older than passage 22 were
seeded onto 0.6-cm2 Millicell HA filter inserts (Millipore,
Bedford, MA) and grown until 2-3 days after confluence.
Electrophysiological studies.
Vectorial ion transport was examined in modified Ussing chambers as
described previously (4). The mucosal and serosal baths contained Ringer buffered salt solution supplemented with glucose (in
mM: 115 NaCl, 25 NaHCO3, 2.4 K2HPO4, 0.4 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 10 glucose; adjusted to pH
7.4), which was gassed with 95% O2-5% CO2 at
37°C. The transepithelial voltage clamp connected to the Ussing
chambers provided continuous monitoring of short-circuit current
(Isc). Open-circuit potential difference was
measured every 1-5 min, and transepithelial resistance was
calculated using Ohm's law. After initial stabilization, baseline
measurements of Isc, potential difference, and
conductance were recorded for 10-15 min. Subsequently, agonists
were added to the serosal and mucosal baths to stimulate chloride
secretion and changes in electrophysiological parameters were recorded
at regular intervals.
cAMP and cGMP assays.
T84 cells, grown to confluence on Millicell HA filter
inserts or in six-well tissue culture plates, were washed with warmed Hanks' balanced salt solution (HBSS) and incubated with
PGE2, forskolin, or uroguanylin, as appropriate, at 37°C
for various times. Cells were then placed on ice, washed with ice-cold
HBSS containing 1 mM IBMX, and extracted with 500 µl/well ice-cold extraction buffer (67% ethanol, 33% HBSS, 1 mM IBMX) for 15 min. Cells were scraped from the well or filter, and the suspension was
mixed and centrifuged at 12,000 rpm for 10 min at 4°C. Pellets and
supernatants were dried separately under vacuum at room temperature. Dried pellets were resuspended in PBS, and protein was determined using
the Lowry method (Bio-Rad Laboratories, Hercules, CA). Dried supernatants were resuspended in PBS, and cyclic nucleotide levels were
assayed by enzyme immunoassay using commercially available kits (Cayman
Chemical, Ann Arbor, MI; R&D Systems, Minneapolis, MN).
Immunoprecipitation and immunoblot analysis.
Cell monolayers were grown on 0.45-cm2 Millicell
filter inserts, and whole cell lysates were prepared as described
previously (25). T84 monolayers were washed
and incubated in cold lysis buffer [PBS, 0.1% Tween 20, 10 µM
leupeptin , 10 µM aprotinin, 10 µM phenylmethylsulfonyl fluoride
(PMSF), 50 µM sodium vanadate] on ice for 30 min. Cell membranes
were isolated by centrifugation after lysis with hypotonic buffer (20 mM Tris base, 20 mM NaCl, 10 mM MgCl2, 1 mM vanadate, 1 µg/ml leupeptin, 100 µg/ml PMSF) as described previously
(25). The filters were then scraped, and cell lysates were
collected and centrifuged at 12,000 g for 10 min.
Supernatants were removed, and protein concentrations were determined.
The sample volumes were adjusted to provide equivalent protein
concentrations. For immunoprecipitation, cell lysates were supplemented
with additional PMSF (20 µM) and incubated overnight at 4°C with an
optimal amount of specific antibody [anti-NKCC1 NH2-terminal polyclonal antibody, kindly provided by Dr.
Christian Lytle, Univ. of California, Riverside, CA; anti-AC r-32
polyclonal antibody, Santa Cruz Biotechnology; or anti-CFTR
(COOH-terminal epitope) monoclonal antibody, Genzyme, Cambridge, MA].
The samples were then incubated with protein A agarose at 4°C for
1 h with constant mixing. The immunoprecipitates were collected by
centrifugation at 15,000 g for 2 min, and the pellets were
washed three times with lysis buffer. Samples were run on precast 7.5%
or 4.5-15% acrylamide gels, and proteins were transferred onto a
blotting membrane. After nonspecific binding sites were blocked with a PBS-Tween buffer containing nonfat dry milk, blots were incubated with
optimal dilutions of primary (anti-NKCC1, anti-CFTR, or anti-AC) and
secondary (horseradish peroxidase-labeled anti-mouse IgG or anti-rabbit
IgG, as appropriate) antibodies for 30-60 min. Membranes were washed in PBS-Tween, incubated with chemiluminescent reagents (Amersham ECL System) for 2-3 min, and exposed to X-ray film for optimal periods.
Assays of AC and PDE activities.
Cell membranes were prepared by nitrogen cavitation using
published protocols (24). Briefly, T84 cell
monolayers were washed with HBSS, scraped into homogenization buffer
(340 mM sucrose, 10 mM HEPES pH 7.4, 1 mM EDTA, 0.1 mM
MgCl2, 1 mM Na2ATP, 1.25 mM PMSF, 10 mg/ml
chymostatin), and centrifuged at 3,000 g for 5 min. The
pellets were resuspended in 1 ml of homogenization buffer, and the
suspensions were exposed to 200 psi of 100% nitrogen for 15 min at
4°C. After nitrogen cavitation, the homogenates were centrifuged at
3,000 g for 20 min to remove nuclei and cellular debris. The
supernatants containing the membranes were collected and used to
determine total protein content and enzymatic activities.
AC activity was determined as described by others (23)
with the following modifications. Aliquots (20-50 µg total
protein) of the membrane preparations were added to reaction buffer (50 mM Tris pH 7.5, 13 mM MgCl2, 2.6 mM EDTA, 25 mM
phosphocreatine, 1 mg/ml creatine phosphokinase, 0.25 mM IBMX, 1 mM
cAMP, 1 × 106 cpm [
-32P]ATP, and 0.5 mM ATP) in a total volume of 50 µl. The membrane suspension was
incubated for 15 min at 37°C with or without 100 µM forskolin. The
reaction was terminated by adding 100 µl of stop solution (2% SDS,
40 mM ATP, 1.4 mM cAMP). Approximately 104 counts of
[3H]cAMP were added to each reaction tube as a recovery
marker. A double column separation to separate ATP from cAMP was
performed according to Mittal (31). Each sample was added
to a 1-ml bed of Dowex AG50W-X4 resin and eluted with 3 ml of water
directly into scintillation vials to determine
[
-32P]ATP. The columns were then placed over 0.6-g
alumina columns and washed with 5 ml of water. Bound cAMP was eluted
from the alumina columns with 4 ml of 100 mM imidazole directly into
20-ml scintillation vials filled with 10 ml of scintillation fluid. Radioactivity was determined in a scintillation counter.
PDE activity was determined as described by Fuhrmann et al.
(15). Cell monolayers were washed twice with HBSS,
collected into lysis buffer (PBS containing 10 mM HEPES, 1 mM
MgCl2, 5 mM DTT, 10 µM trypsin inhibitor, 1 mM EGTA, 5 µM pepstatin, 10 µM leupeptin, 2 mM benzamidine), and sonicated on
ice for 5 s. Cell lysates (20-50 µg protein) were added to
the reaction buffer (60 mM Tris pH 7.4, 5 mM MgCl2, 1.25 mM
CaCl2, 100 µM calmodulin, 105 cpm
[3H]cAMP, and 0.5 µM cAMP) in a final volume of 200 µl. Approximately 104 cpm [
-32P]ATP were
added as a recovery marker. Reactions were incubated for 15 min at
37°C. Samples were then cooled on ice for 10 min, 50 µl of
5'-nucleotidase (2 mg/ml Crotalis atrox venom in 400 mM
Tris, pH 8.5) were added, and the reaction mixtures were incubated at
37°C for 15 min. Samples were added to 1-ml beds of DEAE A-25 Sephadex columns (prewashed with 100 mM Tris buffer, pH 7.2) and eluted
with 2 ml of distilled water directly into 20-ml scintillation vials.
Radioactivity was determined in a scintillation counter.
Materials.
Unless otherwise indicated, all chemicals were obtained from Sigma (St.
Louis, MO). Purified cholera toxin (no. C3012) was obtained from Sigma
and stored at 4°C before use.
Data analysis.
Data are expressed as means ± SE. Differences between groups were
analyzed by ANOVA or Student's t-test, as appropriate. The statistical analysis comprised comparisons between butyrate and time-matched controls. P values of <0.05 were taken to
indicate statistically significant differences.
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RESULTS |
Butyrate treatment inhibits PGE2-stimulated chloride
secretion in T84 colonic epithelial cells.
Monolayers of polarized T84 cells were treated with
butyrate at various concentrations (1-50 mM) and for various times
(1-72 h), washed, and mounted in Ussing chambers to assess
baseline and stimulated chloride secretion. Changes in
Isc in T84 cells are wholly
representative of chloride secretion (3). T84
monolayers treated for up to 72 h with up to 50 mM butyrate showed
no changes in transepithelial resistance (data not shown), indicating
that these conditions had no effect on the viability or barrier
integrity of the epithelial monolayers. Furthermore, baseline
Isc was not different between butyrate-treated
and control cells (data not shown), indicating that butyrate itself had
no secretagogue activity.
We then tested whether butyrate treatment modulates chloride secretory
responses by intestinal epithelial cells after stimulation with known
agonists of this response, as suggested by previous studies (12,
30). Stimulation of control T84 monolayers with the
potent secretagogue PGE2 strongly increased
Isc, and this response was inhibited in
butyrate-treated cells in a concentration-dependent manner (Fig.
1A). A significant decrease in
PGE2-stimulated Isc was observed as
early as 3 h after butyrate addition with all doses tested, and
maximal inhibition occurred after 48-72 h of butyrate treatment
(Fig. 1B). Significant inhibition of
PGE2-stimulated Isc was seen after
addition of as little as 1 mM butyrate for 48 h, and maximal
inhibition occurred at 50 mM (Fig. 1). The presence of butyrate for
only 1 h at the beginning of the culture period, followed by
further incubation without butyrate, had no significant effect on
chloride secretion at 3 h at low concentrations of butyrate (1 and
5 mM), but even these concentrations were sufficient to significantly
inhibit the PGE2-induced secretory response at 24-48 h
after butyrate treatment using this washout design (Fig.
2). Thus, although continuous incubation
with butyrate exerted a stronger inhibitory effect than transient
exposure (cf. Fig. 1B), butyrate appeared to initiate its
effects quite rapidly and irreversibly, although the functional
consequences required a longer period to become fully apparent. To
determine whether the effects of butyrate required its metabolism, we
examined whether the poorly metabolized derivative isobutyrate was also
able to inhibit chloride secretion. This fatty acid had no effect at
1-10 mM concentrations and only a modest effect at the 50 mM dose
at some of the time points examined (Fig.
3). In contrast, propionate and acetate inhibited PGE2-stimulated chloride secretion at low
concentrations, although even these SCFAs had inhibitory effects that
were much less pronounced than those evoked by butyrate (Fig. 3). A 1-h incubation of T84 cells with isobutyrate, acetate, and
propionate, followed by incubation in medium alone, did not result in
significant inhibition of chloride secretion (Fig. 2).

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Fig. 1.
Butyrate inhibits chloride secretion stimulated by
prostaglandin (PG)E2 in T84 cell monolayers.
A: T84 cells were treated for 48 h with
butyrate at 1 ( ), 5 ( ), 10 ( ), or 50 ( ) mM or were left untreated
as controls ( ). Monolayers were mounted in Ussing
chambers, and chloride secretion (short-circuit current,
Isc) was measured after addition of
PGE2 (3 µM) to both sides of the monolayers. A time
course of Isc responses after PGE2
addition is shown. B: peak Isc
responses induced by PGE2 stimulation after various times
of butyrate treatment. The bars represent treatment with butyrate at 1, 5, 10, and 50 mM. Data in B are expressed as % of the
values obtained in PGE2-stimulated control cells not
treated with butyrate. Significant inhibitory effects of butyrate vs.
time-matched controls: *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA). Values are means ± SE; n = 9. In A, all doses of butyrate
had significant inhibitory effects, but asterisks are omitted for
clarity.
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Fig. 2.
Short-term incubation with butyrate, but not other
short-chain fatty acids (SCFAs), has a progressive inhibitory effect on
chloride secretion in T84 cells. T84 cells were
treated for 1 h with SCFAs at 1 ( ), 5 ( ), 10 ( ), or 50 ( ) mM
or were left untreated as controls ( ) and then
incubated in medium without SCFAs for the indicated times. The peak
Isc after PGE2 stimulation was
then determined and is expressed as % of the value obtained in
PGE2-stimulated control cells not treated with SCFAs.
A-D: effects of butyrate, isobutyrate, propionate, and
acetate, respectively. Data in all panels are means ± SE;
n = 6. Significant inhibitory effects of butyrate vs.
time-matched controls: ** P < 0.01, ***P < 0.001 (ANOVA). In the case of overlapping
symbols, the same level of significance (or lack thereof) applies to
all; where error bars are not shown, they are obscured by the symbol.
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Fig. 3.
SCFAs other than butyrate have little effect on chloride secretion
stimulated by PGE2 in T84 cell monolayers.
T84 cells were treated for various times with SCFAs at
concentrations of 1, 5, 10, and 50 mM. Peak
Isc after subsequent PGE2
stimulation was determined and is expressed as % of the value obtained
in PGE2-stimulated control cells not treated with SCFAs.
A-C: effect of isobutyrate, propionate, and acetate,
respectively. Data in all panels are means ± SE;
n = 6. Significant inhibitory effects of the SCFA
shown: *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA).
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Butyrate treatment downregulates expression of NKCC1 in
T84 cells.
The inhibition of the PGE2-induced secretory response in
butyrate-treated T84 cells is consistent with the previous
finding that secretory responses to forskolin are attenuated in
butyrate-treated T84 cells (30), because both
PGE2 and forskolin exert their effects on chloride
secretion via increases in cellular cAMP levels. Consistent with the
previous studies, we also found inhibition of forskolin-stimulated
chloride secretion in T84 cells treated with butyrate (Fig.
4) but not in those treated with
isobutyrate (not shown). Previous studies had also shown that butyrate
treatment decreased epithelial expression of one of the crucial ion
transporters, NKCC1, required for regulated chloride secretion in
intestinal epithelia, suggesting that this event was limiting for
forskolin-induced chloride secretory responses (30). We
also observed decreased levels of NKCC1 in T84 cells at 24 and 48 h of butyrate treatment, although no significant decrease
in expression was seen at 6 h either in whole cell lysates or in a
membrane fraction (Fig. 5). The latter
finding suggests that decreased NKCC1 expression in butyrate-treated
T84 cells cannot be the sole mechanism underlying the
decrease in chloride secretory response to PGE2, as this
was already significantly inhibited after 3 h of butyrate
treatment (Fig. 1B). Exposure of T84 cells to
isobutyrate, acetate, and propionate had no significant effect on NKCC1
expression (data not shown).

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Fig. 4.
Butyrate inhibits forskolin-induced chloride secretion in
T84 cells. T84 cells were treated for 48 h
(A) or the indicated times (B) with butyrate at 1 ( ), 5 ( ), 10 ( ), or 50 ( ) mM or were left untreated as controls
( ). Monolayers were mounted in Ussing chambers, and
Isc was determined after addition of forskolin
(3 µM) to both sides of the monolayers. A: time course of
Isc responses induced by forskolin addition.
B: Isc responses induced by
forskolin stimulation after various times of butyrate treatment,
expressed as % of the value obtained in forskolin-stimulated
time-matched control cells not treated with butyrate. Data are
means ± SE; n = 6. Significant inhibitory effects
of butyrate: **P < 0.01, ***P < 0.001 (ANOVA). In the case of overlapping symbols, the same level of
significance (or lack thereof) applies to all; where error bars are not
shown they are obscured by the symbol.
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Fig. 5.
Effect of butyrate on Na-K-2Cl cotransporter NKCC1
expression in T84 cells. T84 cells were treated
with 10 mM butyrate, or left untreated, for the indicated times. NKCC1
was immunoprecipitated from cell membrane fractions (A) or
cell lysates (B) and detected by immunoblot analysis. A
representative blot is shown at the top of each panel.
Results of densitometric analysis of 4 separate experiments (means ± SE; expressed in arbitrary units, a.u.) are shown at the
bottom: filled bars, control cells; open bars,
butyrate-treated cells. ***Significant difference (P < 0.01, ANOVA) vs. controls not treated with butyrate.
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Butyrate inhibits effect of CT in T84 cells.
Chloride secretory responses in epithelial cells are known to be
mediated by calcium or cyclic nucleotides. cAMP production by AC in
T84 cells can be activated by different mechanisms: by agonists that interact directly with AC (e.g., forskolin), by agonists
binding to G protein-coupled receptors (PGE2), or by agonists that interact with stimulatory G proteins (CT) or by uncoupling of inhibitory G proteins (Gi or Go).
The common result is an increased level of cAMP production by AC. To
examine possible effects of butyrate at various points in this
signaling pathway, we investigated responses to CT. CT is an
arginine-specific ADP-ribosyl transferase that irreversibly inhibits
Gs protein GTPase activity, thereby promoting accumulation
of cAMP with subsequent sustained activation of chloride secretion by
intestinal epithelial cells (13, 29). CT (30 pM)
was added to monolayers for 2 h; the cells were then mounted in
Ussing chambers, and Isc response was monitored.
Butyrate significantly inhibited the effect of CT (30 pM, 2 h,
37°C) on chloride secretion by T84 cells (Fig.
6). These data are therefore consistent
with the hypothesis that butyrate inhibits cAMP-mediated chloride
secretory responses by modulating cyclic nucleotide generation.

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Fig. 6.
Butyrate inhibits cholera toxin-induced chloride
secretion in T84 cells. T84 cells were treated
for the indicated times with butyrate at 10 mM ( ) or
were left untreated as controls ( ). For the last 2 h before mounting in Ussing chambers, the cells were treated with
cholera toxin (30 pM). Maximal chloride secretory responses to cholera
toxin were then determined by monitoring Isc for
an additional 90 min. Data are means ± SE; n = 4. ***Significant inhibitory effects of butyrate vs. time-matched
controls (P < 0.001, ANOVA).
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Butyrate treatment has no effect on chloride secretory responses to
carbachol or uroguanylin.
Because NKCC1 expression was substantially inhibited in T84
cells after 24-48 h of butyrate treatment and this ion transporter is required for vectorial chloride secretion in response to multiple agonists (3), we reasoned that secretory responses to
physiological secretagogues acting via mechanisms independent of cAMP
might also be inhibited in butyrate-treated T84 cells.
However, this was not the case, because peak chloride secretion in
response to the Ca2+-dependent agonist carbachol was not
significantly different in SCFA-treated vs. control cells, even at the
highest concentration of butyrate or other SCFAs tested (Fig.
7). Butyrate treatment also failed to
significantly inhibit responses to the cGMP-dependent secretagogue
uroguanylin (Fig. 8). These data show
that butyrate treatment does not cause a general reduction in
epithelial secretory responses but appears to affect cAMP-mediated
secretory responses selectively, such as those observed after
PGE2, forskolin, or CT stimulation. They further suggest
that decreased NKCC1 expression is at most only partially responsible
for the attenuation of PGE2-induced chloride secretion in
butyrate-treated T84 epithelia.

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Fig. 7.
Butyrate and other SCFAs have no effect on
calcium-mediated chloride secretion in T84 cells.
A: T84 cells were treated for 48 h with
butyrate at 1 ( ), 5 ( ), 10 ( ), or 50 ( ) mM or were left untreated
as controls ( ). Monolayers were mounted in Ussing
chambers, and Isc was determined after addition
of carbachol (100 µM) to the basolateral side of the monolayers.
A: time course of Isc after carbachol
addition. B: peak Isc induced by
carbachol stimulation after various times of butyrate treatment as
indicated in A or after treatment with 50 mM isobutyrate
( ), propionate ( ), or acetate
( ).
Data are means ± SE; n = 6. No significant
differences were observed between SCFA-treated and control cells under
any of the conditions tested, as assessed by ANOVA.
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Fig. 8.
Butyrate and other SCFAs have no effect on chloride
secretory responses of T84 cells to uroguanylin.
T84 cell monolayers were treated for various time periods
(6-48 h) with butyrate at 1 ( ), 5 ( ), 10 ( ), or 50 ( ) mM
or left untreated as controls ( ) or were treated with
50 mM of isobutyrate ( ), propionate ( ),
or acetate
( ).
Cells were then stimulated with 1 µM of uroguanylin and peak
Isc responses were determined. Data are
means ± SE; n = 4. No significant differences
were observed between SCFA-treated and control cells under any of the
conditions tested, as assessed by ANOVA.
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Butyrate treatment of T84 cells does not alter
secretory responses to cell-permeant cAMP.
The decreased secretory response to PGE2 in
butyrate-treated T84 cells could be due to reduced second
messenger generation (i.e., lower cAMP levels after PGE2,
forskolin, or CT stimulation), reduced secretory responses in the face
of normal second messenger generation (i.e., a reduced capacity of the
chloride secretory mechanism to respond to normal cAMP levels), or
both. To test whether reduced responsiveness to cAMP plays a role, we
stimulated T84 cells with three cell-permeant analogs of
cAMP, dibutyryl cAMP-AM, dibutyryl cAMP, and 8-bromo-cAMP (8-BrcAMP).
This approach bypasses the cellular signaling events required for CT-,
forskolin-, or PGE2-stimulated cAMP generation and
selectively tests whether the signaling pathways and ion transport
processes downstream of cAMP generation are affected in
butyrate-treated cells. Chloride secretion induced by dibutyryl cAMP-AM
in butyrate-treated T84 cell monolayers was not
significantly different from that in untreated control cells, even if
cells were treated with butyrate for up to 72 h (Fig.
9A). Similarly, chloride
secretion stimulated by dibutyryl cAMP or 8-BrcAMP was not markedly
affected by butyrate treatment (Fig. 9, B and C).
Interestingly, at a low concentration (0.1 mM) and early time points,
dibutyryl cAMP-stimulated chloride secretion was slightly inhibited in
the presence of butyrate, but this was a transient effect rather than
the progressive inhibition seen with PGE2. Responses to
8-BrcAMP were not altered at any time (Fig. 9, B and
C). These data indicate that butyrate-treated T84 cells can secrete chloride normally in the presence of
adequate levels of intracellular cAMP, suggesting that butyrate
treatment of T84 cells has relatively little effect on the
signaling pathways and ion transport processes downstream of cAMP
generation. Consistent with this conclusion, and as shown by others
(30), expression of the apically located cAMP-dependent
chloride channel CFTR was not altered in butyrate-treated compared with
control cells, as assessed by immunoblot analysis (data not shown).

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Fig. 9.
Butyrate has no effect on the chloride secretory response
of T84 cells to cell-permeant cAMP analogs while inhibiting
responses to PGE2. A: T84 cell
monolayers were treated for the indicated times with 10 mM
butyrate( , ) or left untreated as
controls ( , ). Peak
Isc was determined after addition of 3 µM
PGE2 (as a positive control; ,
) or 3 µM dibutyryl cAMP-AM ( ,
) to both sides of the monolayers. B and
C: peak Isc induced by dibutyryl cAMP (1 mM;
B) or 8-bromo-cAMP (0.3 mM; C) stimulation after
various times of butyrate treatment. Results are means ± SE;
n = 4. Significant differences: *P < 0.05, ***P < 0.001 (ANOVA) vs. controls stimulated
with the same agonist but not treated with butyrate.
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Butyrate treatment decreases agonist-stimulated cAMP levels in
T84 cells.
The data described above indicated that butyrate had little effect on
responses to the second messenger cAMP, which raised the possibility
that the generation of this second messenger in response to the binding
of PGE2 to its receptor is altered in butyrate-treated
T84 cells. To test this, we determined cAMP levels after
PGE2 stimulation in butyrate-treated and control
T84 cells. As shown in Fig.
10, butyrate treatment resulted in
reduced cAMP levels in response to PGE2 stimulation, with
this effect being concentration dependent. The inhibitory effect on
cAMP levels was observed within 3 h of butyrate treatment,
although PGE2-stimulated cAMP levels continued to decrease
further over time (Fig. 10A). Significant inhibition of
PGE2-stimulated cAMP production was observed even at the
lowest concentration of butyrate tested (1 mM). These results indicate
that butyrate treatment of T84 cells caused a decrease in
second messenger generation in response to PGE2
stimulation, and this inhibition occurred with a time course that
parallels the inhibitory effects of butyrate on epithelial chloride
responses (compare Fig. 10 with Fig. 1B). Figure
10B shows cAMP production after PGE2 stimulation
in cells that had been treated for only 1 h with butyrate (10 mM)
and then incubated in fresh medium for up to 48 h before
PGE2 addition. This experimental design resulted in a
significant reduction in cAMP, although with delayed kinetics compared
with continuous incubation. These findings were comparable to the
studies of chloride secretion reported above (Fig. 2). Similarly,
forskolin stimulation of T84 cells, i.e., using a direct
agonist of AC, produced results comparable to those obtained with PG
stimulation, both in terms of chloride secretion (Fig. 5) and cAMP
production (data not shown).

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Fig. 10.
Effect of butyrate on PGE2-stimulated cAMP
levels. A: T84 cells were treated for the
indicated times with butyrate at 1 ( ), 5 ( ), 10 ( ), or 50 ( ) mM
or were left untreated as controls ( ). Cells were then
stimulated for 15 min with 10 µM PGE2, and cell extracts
were assayed for cAMP. B: experiments similar to those shown
in A, with the exception that the cells were treated with
butyrate (10 mM) for 1 h only and then incubated with fresh medium
for the times indicated before PGE2 stimulation. Results
are means ± SE; n = 4. Significant inhibitory
effects of butyrate: *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA). Data are means ± SE;
n = 6. Basal levels of cAMP were similar in control and
butyrate-treated cells (200 ± 50 vs. 220 ± 50 pmol/mg
protein; n = 4, P = not significant).
In the case of overlapping symbols, the same level of significance
applies to all.
|
|
To test the specificity of the effect of butyrate on cyclic nucleotide
generation, we stimulated T84 cells with uroguanylin (1 µM, 30 min). This agonist stimulated an increase in cGMP levels from
10 ± 1 to 48 ± 5 pmol/mg protein in control T84
cells (means ± SE; n = 3, P < 0.01). In T84 cells treated for 48 h with 10 mM
butyrate, uroguanylin stimulation increased cGMP levels from 8 ± 1 to 42 ± 5 pmol/mg protein (means ± SE; n = 3, P < 0.01). These data show that butyrate
treatment had no significant effect on cGMP levels after uroguanylin
stimulation, suggesting that the butyrate effects on cAMP generation
are specific and do not extend to other cyclic nucleotides.
Effects of butyrate on epithelial cAMP levels and chloride
secretion are not reversed by PDE inhibition.
Reduced cAMP levels after PGE2 stimulation of
butyrate-treated T84 monolayers could be the result of
decreased cAMP generation, increased cAMP degradation, or both. To test
whether increased degradation contributes to decreased cAMP levels, we
inhibited cAMP degradation in butyrate-treated T84 cells
with the broadly specific PDE inhibitor IBMX and evaluated the effects
on cAMP levels and chloride secretion in response to PGE2
or forskolin. As shown in Fig. 11,
addition of IBMX to butyrate-treated T84 cells had
relatively little effect on the butyrate-induced inhibition of
PGE2-stimulated cAMP levels when tested after 48 h of
butyrate treatment. Consistent with this finding, IBMX did not reverse the inhibitory effect of butyrate on PGE2-stimulated
chloride secretion after 48-72 h of butyrate treatment (Fig.
12). At earlier times of butyrate
treatment, IBMX increased PGE2-stimulated chloride secretory responses in butyrate-treated cells, as might be predicted on
the basis of its ability to sustain PGE2-stimulated cAMP
generation. This effect occurred to a similar extent in control cells,
suggesting that IBMX did not specifically reverse the butyrate-induced
inhibition of chloride secretion. Furthermore, IBMX treatment of
T84 monolayers also failed to reverse the inhibitory effect
of butyrate on forskolin-induced chloride secretion
(
Isc at 24 h in µA/cm2:
17.4 ± 1.8 vs. 57.3 ± 2.2, butyrate vs. control after IBMX
treatment, respectively; n = 6, P < 0.001). Together these data suggest that increased cAMP degradation is
not responsible for the decrease in cAMP levels and chloride secretion
after PGE2 or forskolin stimulation of butyrate-treated
T84 cells.

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Fig. 11.
Inhibition of phosphodiesterase activity does not
reverse the inhibitory effect of butyrate on
PGE2-stimulated cAMP levels. T84 cells treated
with 10 mM butyrate for 48 h ( , )
or untreated controls ( , ) were
incubated with 0.5 mM IBMX ( , ) or
buffer alone ( , ) for 30 min, after
which time cells were stimulated with PGE2 (10 µM) for
the times indicated on the x-axis. Cell extracts were
prepared, and cAMP levels were determined. Results are means ± SE; n = 4. ***Significant differences
(P < 0.001, ANOVA) vs. respective time-matched
controls not treated with butyrate.
|
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Fig. 12.
Inhibition of phosphodiesterase activity does not
reverse the inhibitory effect of butyrate on
PGE2-stimulated chloride secretion. T84 cells
treated with 10 mM butyrate ( , ), or
left untreated as controls ( , ), for
the indicated times were incubated with 0.5 mM IBMX ( ,
), or buffer alone ( , )
for 60 min, after which time monolayers were mounted in Ussing
chambers. Peak Isc was then determined after
the addition of 3 µM PGE2. Data are means ± SE;
n = 6. ***Significant differences (P < 0.001, ANOVA) vs. respective time-matched controls not treated with
butyrate. In the case of overlapping symbols, the same level of
significance applies to all.
|
|
To confirm these findings, we determined PDE activity directly in cell
lysates from control and butyrate-treated T84 cells. Control cells had an IBMX-inhibitable PDE activity of 11.7 ± 0.7 pmol cAMP · min
1 · mg
protein
1 (means ± SE; n = 4). In
comparison, T84 cells treated with 10 mM butyrate for
48 h showed 83.5 ± 12.0% of the IBMX-inhibitable PDE
activity of controls analyzed in parallel (means ± SE;
n = 4, P = not significant by
Student's t-test). Determination of PDE activities in
lysates from cells stimulated for 10 min with PGE2 before
the lysates were prepared yielded similar results (data not shown).
These data indicate that butyrate-treated T84 cells
exhibited no increase in PDE activity, giving further support to the
conclusion that the reduction in PGE2-stimulated cAMP
levels in butyrate-treated cells is not caused by increased cellular cAMP degradation.
Butyrate treatment downregulates activity and expression of
epithelial ACs.
Because cAMP degradation was not affected by butyrate treatment of
T84 cells, we next investigated the possibility that cAMP synthesis was inhibited under these conditions. Membranes were prepared
from control T84 cells and cells treated with 10 mM
butyrate for 48 h and were assayed for AC activity after forskolin
stimulation. Membranes from control cells showed 92 ± 20 pmol
cAMP · min
1 · mg protein
1
of forskolin-inducible AC activity (means ± SE; n = 4). In contrast, membranes from T84 cells treated with 10 mM butyrate for 48 h showed only 66.3 ± 3.5% of the
inducible AC activity of controls (means ± SE; n = 4, P < 0.01 by Student's t-test). These
results indicate that butyrate treatment of T84 cells
caused a significant decrease in AC activity, which can provide an
explanation for the decrease in cAMP generation after PGE2
stimulation in these cells.
Decreased AC activity could be related to changes in expression,
posttranslational events, or both. To assess expression of ACs in
T84 cells, we determined protein levels of total ACs by immunoblot analysis using an antibody that recognizes all AC isoforms. This approach was chosen because the enzyme activity data were obtained
by stimulating membranes with forskolin, a pharmacological activator of
the majority of AC isoforms (22). Cells treated with
butyrate showed a significant decrease in total AC expression relative
to controls, both in total cell lysates (Fig.
13) and in isolated membranes (data not
shown). A significant decrease in AC expression was first observed
after 3 h of butyrate treatment, consistent with the timing of the
effect of butyrate on chloride secretion. AC expression reached a
minimum by 24-48 h of butyrate treatment, with AC levels in
butyrate-treated cells being only 15-20% of the levels in control
cells as determined by densitometric analysis of the immunoblots (Fig.
13). These data suggest that decreased AC expression is responsible for
decreased AC activity in butyrate-treated T84 cells. In
keeping with this conclusion, isobutyrate, acetate, and propionate had
no significant effect on AC expression (Fig.
14). Moreover, when cells were
incubated with butyrate for only 1 h and incubation was continued
in fresh medium, AC levels still declined, albeit to a lesser extent
than seen with continuous butyrate exposure (Fig.
15). Under these conditions, a
significant decrease in AC expression was only seen at 24 and 48 h
(Fig. 15).

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Fig. 13.
Decreased adenylyl cyclase (AC) expression in butyrate-treated
T84 cells. T84 cells were treated with 10 mM
butyrate for the indicated times or left untreated as controls. Total
ACs were immunoprecipitated from cell lysates and detected by
immunoblot analysis. A purified AC protein mixture was used as a
positive control. A representative blot is shown at top.
Below this, another blot shows that AC levels do not change over time
in untreated cells. Results of densitometric analysis of 6 separate
experiments (means ± SE) are shown at bottom. Filled
bars, control cells; open bars, butyrate-treated cells. Significant
differences vs. time-matched controls not treated with butyrate
(** P < 0.01, *** P < 0.001, ANOVA).
|
|

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Fig. 14.
Isobutyrate, propionate, and acetate have no significant
effect on AC expression in T84 cells. T84 cells
were treated with 10 mM SCFAs (isobutyrate, acetate, or propionate) for
the indicated times or left untreated as controls. Total ACs were
immunoprecipitated from cell lysates and detected by immunoblot
analysis. A purified AC (Purif AC) protein mixture was used as a
positive control (pos contr). A representative blot is shown at
top; results of densitometric analysis of 6 separate
experiments (means ± SE) are shown at bottom.
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Fig. 15.
One-hour exposure to butyrate inhibits AC expression in
T84 cells, but with delayed kinetics. T84 cells
were treated with 10 mM butyrate for 1 h and then incubated with
fresh medium or left untreated as controls. Total ACs were
immunoprecipitated from cell lysates and detected by immunoblot
analysis. A purified AC (Pur AC) protein mixture was used as a positive
control. A representative blot is shown at top. Below this,
another blot shows that AC levels do not change over time in untreated
control cells. Results of densitometric analysis of 6 separate
experiments (means ± SE) are shown at bottom. Filled
bars, controls; open bars, butyrate-treated cells. **Significant
differences vs. time-matched controls not treated with butyrate
(P < 0.01, ANOVA).
|
|
 |
DISCUSSION |
This study demonstrates that butyrate inhibits cAMP-mediated
chloride secretory responses in T84 cells in a
concentration- and time-dependent manner. The inhibitory effect of
butyrate was first observed within 3 h and was paralleled by an
inhibition of cAMP accumulation after PGE2 or forskolin
stimulation and decreased AC expression. These findings suggest that
decreased AC activity early after butyrate treatment of T84
cells is responsible for decreased cAMP production after
PGE2 or forskolin stimulation, leading to a decreased
chloride secretory response. In addition to the initial inhibitory
effects, butyrate caused a more sustained inhibition of cAMP-mediated
chloride secretory responses, which required 24-48 h to become
fully apparent.
Besides inhibition of AC activity and cAMP generation, the later
inhibitory effects of butyrate were paralleled by decreased NKCC1
expression. The latter finding is consistent with previous observations
in HT-29 colonic epithelial cells (30). These studies showed that butyrate inhibition of forskolin-activated chloride secretion was accompanied by reductions in mRNA and protein expression of the basolateral Na-K-2Cl transporter NKCC1, which is responsible for
mediating basolateral uptake of chloride into epithelial cells (30). Although it is not clear whether the levels of NKCC1
or cAMP are limiting for chloride secretion in intestinal epithelia, it
is possible that the effects of butyrate on both NKCC1 protein expression and generation of the second messenger cAMP produce a
synergistic effect in inhibiting chloride secretion. On the other hand,
our data show that the inhibitory effect of butyrate on chloride
secretion does not extend to calcium- or cGMP-dependent secretory
mechanisms or to secretion evoked by cell-permeant cAMP analogs. This
suggests that the epithelial chloride secretory capacity in response to
adequate levels of second messengers is not affected by butyrate,
indicating that NKCC1 levels may not be limiting for the chloride
secretory response under these conditions. Data showing an acute
inhibitory effect of butyrate on preexisting chloride secretion
(12, 36) also support a nonlimiting role of NKCC1 in the
mechanism of action of SCFAs on chloride secretion. Furthermore, other
effects of butyrate in modulating cellular ion transport have recently
been described by Musch and colleagues (33) and others
(5, 19, 36), underscoring the effects of this SCFA on
sodium-hydrogen exchange and sodium-chloride absorption. By altering
sodium and chloride absorption, in addition to effects on chloride
secretion, the net effect of butyrate in vivo would be potentiated.
Moreover, this study supports the evidence for unaltered CFTR
expression in the presence of butyrate in intestinal epithelial cells
(30). These data differ from the findings of Loffing et
al. (28), which showed increased expression of CFTR after
6-day exposure to 5 mM butyrate or phenylbutyrate in Calu-3 airway
epithelial cells. Organ-specific features, as well as the use of a
different model (nystatin-permeabilized epithelial airway cells) and
experimental design (6-day exposure to SCFAs), may explain the lack of
concurrence with our observations.
Although our data align with previous findings by Matthews et al.
(30), they differ from some of the conclusions proposed by
Dagher et al. (12). Differences in the model (rat colon) and experimental design (acute exposure to SCFAs for both rat colon and
nystatin-permeabilized T84 cells) may account for the discordant results, especially in relation to the interaction of
butyrate with basolateral transport mechanisms and second messenger production. Our findings with CT are also internally consistent with
our data and agree with previous work (2, 36).
Furthermore, our data on the lack of effect of butyrate on
cGMP-mediated chloride secretion and cGMP production are in agreement
with other studies (11, 38) showing that butyrate does not
interfere with cGMP production in response to guanylin or uroguanylin
or in response to E. coli heat-stable endotoxin
(STa) in T84 cells, although those authors also
showed that cGMP distribution between intracellular and extracellular
compartments is altered by the SCFA. Differences between our work and
that of Dagher et al. (12), as well as a recent study by
Charney et al. (8), which reported an inhibition of
STa-induced chloride secretion and cGMP production by
butyrate, may be ascribed to the model and experimental design (acute
SCFA exposure) used by Charney for chloride secretion and cGMP
determination (rat colon). Moreover, in intact tissues, cGMP may be
produced in other cell types in addition to the epithelium and thereby evoke secondary effects on chloride secretion via the release of other
mediators. The complexity of cell-cell interactions that regulate
intestinal ion transport underscores the utility of a reductionist
model such as the T84 cell line.
Butyrate treatment of T84 cells decreased the total
activity and protein expression of ACs, suggesting that the
downregulation of AC protein expression after butyrate is important for
the decrease in cAMP production. Furthermore, AC expression was
reduced, albeit with delayed kinetics, even in cells that had only a
brief exposure to butyrate. In cardiac myocytes, changes in the
expression of AC, rather than in G protein-coupled receptors or G
proteins, have a greater influence on agonist-induced cAMP levels
(18). Consistent with our findings regarding AC expression
in T84 cells, butyrate inhibited total AC activity by
40-50% in GH1 pituitary cells (2), indicating that
the effects of butyrate on AC activity may be conserved among different
cell types. The observation that butyrate decreases total AC protein
levels in T84 cells suggests that the expression of one or
several of the more abundant AC isoforms is affected by butyrate.
Little is currently known about the expression of specific AC isoforms
in intestinal epithelial cells, although AC isoform expression has been
well characterized in other systems. For example, brain cells express
all of the AC isoforms (AC1-9) (9, 20, 26), whereas
AC expression in other organs is limited to specific isoforms
(26, 39).
We observed no increase in total PDE activity after butyrate treatment
of T84 cells, suggesting that the decrease in cAMP levels
by butyrate is mostly regulated by pathways that affect cAMP synthesis
rather than its degradation. Consistent with these findings, butyrate
also failed to affect PDE activity in mastocytoma cell lines, in which
butyrate also induces cell differentiation (32). However,
butyrate treatment increased PDE activity in GH1 pituitary cells
(2), indicating that butyrate may have varying effects on
total PDE activity in different cell types. The effect of butyrate on
total PDE activity in GH1 cells suggests that the SCFA affects
expression and/or activity of one or several PDE isoforms in that
system. In fact, in a recent study by Zaharia et al. (41),
acute treatment of rat colon and human colonic biopsy samples with 50 mM butyrate or other SCFAs led to an inhibition of cGMP-PDE activity,
whereas cAMP-PDE activity was stimulated. Although our observations
show no increase of PDE activity induced by 1- to 48-h exposure to
butyrate in T84 cells, we cannot exclude the possibility
that butyrate affects the expression and/or activity of specific PDE
isoforms in T84 cells without altering total PDE activity
in these cells and that early effects on cAMP levels could be related
in part to modulation of specific PDE isoforms. This mechanism could
explain the small inhibitory effect of butyrate that we observed with
one cell-permeant form of cAMP (Fig. 9B). In fact, chloride
secretion stimulated by dibutyryl cAMP at a lower concentration of 0.1 mM was significantly inhibited by butyrate (10 mM), an effect that was
not reversed by addition of the PDE inhibitor IBMX (data not shown).
Nevertheless, chloride secretory responses evoked by maximally
effective concentrations of dibutyryl cAMP (1 mM) or the PDE-resistant
analog 8-BrcAMP, were not affected by butyrate in any major way.
Butyrate induces growth arrest and cell differentiation in cultured
colonic epithelial cell lines (10, 17), which has led to
its wide use to evoke an in vitro model of epithelial cell differentiation. Intestinal epithelial cell differentiation, which occurs in vivo during the migration of enterocytes from the crypts to
the surface in the colon or from the crypts to the villi in the small
intestine, is characterized by distinct molecular and phenotypical
changes. These include increased expression of membrane hydrolases,
such as sucrases and alkaline phosphatases (17), and
changes in second messenger signaling (e.g., cAMP) (1). Our findings show that decreased PGE2 signaling and reduced
AC expression and activity are likely to be part of the differentiation program of intestinal epithelial cells. Conversely, these data suggest
that the state of differentiation of intestinal epithelial cells is
important in the regulation of cAMP signaling in these cells, which may
be relevant pathophysiologically. For example, mucosal inflammation is
associated with epithelial damage, leading to increased epithelial
regeneration that is characterized by an increase in the number of
undifferentiated crypt cells (34). Under these conditions,
the ratio of undifferentiated to differentiated epithelial cells is
increased, which, on the basis of our present observations, would be
predicted to be accompanied by an increased overall epithelial
responsiveness to cAMP-mediated chloride secretion. Because fluid
secretion can be considered a protective host function, the increased
secretory capacity of the regenerating intestinal epithelium may help
to reduce exposure of the epithelium to microbial and noxious agents,
thus promoting healing. On the other hand, these responses may
contribute to the adverse outcome of secretory diarrhea.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Paul Insel for helpful advice, Dr. Martin
Kagnoff for laboratory support for parts of these studies, and Dr.
Christian Lytle for the generous gift of the NKCC1 antibody. We also
thank Glenda Wheeler for secretarial assistance.
 |
FOOTNOTES |
These studies were supported by National Institutes of Health (NIH)
Grants DK-28305 and DK-35108 (Unit 5) to K. E. Barrett, an NIH
supplemental award to S. Resta-Lenert, and a Research Grant from the
Crohn's and Colitis Foundation of America to L. Eckmann.
Portions of this work have been published in abstract form
(Gastroenterology 116: 3813, 1999).
Address for reprint requests and other correspondence: S. Resta-Lenert, UCSD Medical Center 8414, 200 West Arbor Drive, San Diego, CA 92103 (E-mail: srestalenert{at}ucsd.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.
Received 21 September 2000; accepted in final form 15 August 2001.
 |
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