1 Intestinal Disease Research Program and 2 Center for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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Growth
factors affect a variety of epithelial functions. We examined the
ability of TGF- to modulate epithelial ion transport and
permeability. Filter-grown monolayers of human colonic epithelia, T84
and HT-29 cells, were treated with TGF-
(0.1-100 ng/ml,
15 min-72 h) or infected with an adenoviral vector encoding
TGF-
(Ad-TGF
) for 144 h. Ion transport (i.e., short-circuit
current, Isc) and transepithelial resistance
(TER) were assessed in Ussing chambers. Neither recombinant TGF-
nor
Ad-TGF
infection affected baseline Isc;
however, exposure to
1 ng/ml TGF-
led to a significant (30-50%) reduction in the Isc responses to
forskolin, vasoactive intestinal peptide, and cholera toxin (agents
that evoke Cl
secretion via cAMP mobilization) and to the
cell-permeant dibutyryl cAMP. Pharmacological analysis of signaling
pathways revealed that the inhibition of cAMP-driven epithelial
Cl
secretion by TGF-
was blocked by pretreatment with
SB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors of
JNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF-
enhanced the barrier function of the treated monolayers by up to threefold as
assessed by TER; however, this event was temporally displaced from the
altered Isc response, being statistically
significant only at 72 h posttreatment. Thus, in addition to
TGF-
promotion of epithelial barrier function, we show that this
growth factor also reduces responsiveness to cAMP-dependent
secretagogues in a chronic manner and speculate that this serves as a
braking mechanism to limit secretory enteropathies.
short-circuit current; T84 epithelia; growth factor; transepithelial resistance
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INTRODUCTION |
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A NUMBER OF ESSENTIAL FUNCTIONS are provided by the epithelial lining of the gastrointestinal (GI) tract. In addition to nutrient digestion/absorption and immune surveillance roles, the gut epithelium also secretes electrolytes; it is this vectorial electrolyte secretion that establishes a driving force for directed water movement. Excess water in the lumen can lead to diarrhea (essentially a protective process), which if recurrent or prolonged may result in dehydration and even death. Given the association of GI epithelia with the enteric nervous system, neighboring enteroendocrine cells, and the luminal contents, it is not surprising that nervous and endocrine input as well as bacteria and/or their products constitute the major regulators of epithelial function. Recent literature, however, particularly from in vitro studies, has shown that immune mediators including cytokines can also modify epithelial function (24).
Growth factors are cytokines that have been shown to have both
mitogenic and nonmitogenic effects. Specifically, in terms of ion
transport, treatment of colonic epithelial cell lines with transforming
growth factor (TGF)- or epidermal growth factor (EGF) resulted in
decreased secretory responses to stimulants of chloride secretion
(6, 39). TGF-
is a multifunctional cytokine, the
bioactivity of which can be grouped into three main properties:
1) regulation of cell growth and proliferation,
2) immunomodulation, and 3) stimulation of wound
repair (epithelial restitution). Studies have shown that TGF-
is
upregulated in many diseases, including inflammatory bowel disease
(IBD) (2), an enteropathy often characterized by perturbed
water movement. Given that altered epithelial electrolyte transport can
lead to aberrant water balance in the gut, that other growth factors
have been shown to affect this process, and that TGF-
is upregulated during enteric disease, the primary aim of this study was to determine the effect of TGF-
on epithelial ion transport function.
Using monolayers of the human T84 or HT-29 colonic epithelial cell
lines as model epithelia, we have shown that exposure to TGF- leads
to a ~30% decrease in epithelial secretory responses to stimuli that
initiate cAMP-dependent Cl
secretion. This disruption of
normal electrolyte transport events is temporally distinct from the
ability of TGF-
to increase epithelial barrier function and can be
restored by treatment with SB-203580, an inhibitor of the p38 mitogen
activated protein kinase (MAPK) signaling pathway.
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MATERIALS AND METHODS |
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Cell culture. The human colonic adenocarcinoma-derived T84 epithelial cell line was maintained in media containing equal volumes of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium, supplemented with 10% (vol/vol) fetal calf serum, 1.5% (vol/vol) HEPES, and 2% (vol/vol) penicillin-streptomycin (all from Life Technologies, Grand Island, NY) at 37°C, 5% CO2. The HT-29cl.19A cell line (HT-29; a kind gift from Dr. J. A. Groot, University of Amsterdam) was maintained in DMEM supplemented with 5% (vol/vol) fetal calf serum, 0.1% vol/vol L-glutamine, 2% (vol/vol) penicillin-streptomycin, and 5% (vol/vol) sodium bicarbonate. Cells were seeded onto semipermeable filter supports (0.4-µm pore size; Costar, Cambridge, MA) with a surface area of 1 cm2 (106 cells) for physiological assessment and grown to confluence as determined by transepithelial resistance (TER; minimum 6 days growth).
Physiological assessment studies.
Recombinant human TGF- (R&D Systems, Minneapolis, MN) was added to
the basolateral compartment of the semipermeable filter supports at
concentrations of 0.1, 1, 10, or 100 ng/ml. The basolateral surface of
T84 cells was exposed to TGF-
for 4, 8, 16, 24, and 72 h, at
which point cells were mounted in specialized Ussing chambers
(Precision Instrument Design, Tahoe City, CA) under voltage-clamped conditions as previously described (25), and short-circuit
current (Isc) responses to known stimuli of
Cl
secretion were measured. In other studies, T84
monolayers were exposed to TGF-
for 15 min, then rinsed twice in
fresh medium, and Isc responses examined 16 h later. Comparative studies examined Isc
responses to EGF (10 or 100 ng/ml; R&D Systems) after 16 h of
exposure. Acute experiments exposed T84 cells to TGF-
for 30 min (1 or 10 ng/ml) while mounted in the Ussing chambers, followed by
measurement of Isc responses. In all
experiments, baseline Isc was obtained after 10 min of equilibration. Stimulated Cl
secretion was induced
by forskolin (10
5 M), vasoactive intestinal peptide (VIP,
10
7 M), cholera toxin (10 µg/ml), and dibutyryl cAMP
(200 µM) (all from Sigma Chemical, St. Louis, MO), and the maximal
change in Isc (
Isc)
was recorded. These secretagogues were chosen because of their
known ability to elicit Cl
secretion via raising
intracellular cAMP (13, 23, 44). TER (in
/cm2) of the T84 monolayers was recorded with chopstick
electrodes and a voltmeter (Millipore, Bedford, MA) as a measure of
paracellular permeability.
Adenoviral infection.
Twenty-four hours after seeding (106 cells/ml on
semipermeable filter supports), T84 cells were infected with
replication-deficient adenovirus constructs encoding active TGF-
[Ad-TGF
(35)] at a multiplicity of infection (MOI) of
10, 20, or 50 virus particles/cell. Sixteen hours later, T84 cells were
rinsed twice with medium to remove any residual virus and cultured for
6 days, whereupon they were mounted in Ussing chambers for analysis of
secretory responsiveness to forskolin. Changes in barrier function were
monitored daily throughout the 6-day postinfection period by recording TER.
Pharmacological inhibition of intracellular signaling.
Physiological assessment experiments were conducted by using a single
dose (10 ng/ml) and time (16 h) of TGF- exposure to T84 monolayers.
T84 cells were pretreated with inhibitors of 1) p38 MAPK [1
h, 10 µM, SB-203580; Calbiochem, La Jolla, CA (18, 20,
33)]; 2) c-Jun NH2-terminal kinase (JNK)
[30 min, 10 µM, SP-600125; Calbiochem (17)];
3) extracellular signal-regulated kinase 1/2 (ERK1/2)
signaling, via inhibition of MEK, the enzyme upstream of ERK1/2 [1 h,
25 µM, PD-98059; Calbiochem (4, 8)]; or 4)
phosphatidylinositol 3' kinase (PI 3-K) [15 min, 20 µM, LY-294002;
Sigma Chemical (42)]. TGF-
was subsequently added to
the basal compartment of the culture well (inhibitor not washed out)
and, after a defined incubation time, the monolayers were mounted in
Ussing chambers and Isc responses to forskolin
were recorded. Pharmacological inhibition of the p38 MAPK signaling cascade in HT-29 cells was accomplished by pretreating monolayers with
SB-203580 for 30 min (0.1-50 µM), subsequently followed by TGF-
application (100 ng/ml, 24 h).
Statistical and data analysis. Data are normalized to time-matched controls (i.e., percentage of control response) and are presented as means ± SE. Data were analyzed using one-way ANOVA, and P < 0.05 was accepted as the level of statistical significance.
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RESULTS |
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TGF- reduces Isc responses to cAMP secretagogues.
Treatment with TGF-
(0.1-100 ng/ml, 4-72 h) did not
significantly affect baseline Isc compared with
naive time-matched controls [0.17 ± 0.17 vs. 0.33 ± 0.33 µA/cm2 for control and TGF-
-treated (10 ng/ml, 16h),
respectively, n = 6 monolayers from a representative
experiment]. Addition of TGF-
to the Ussing chamber for 30 min
(0.1, 1, 10 ng/ml) had no effect on epithelial baseline
Isc or
Isc evoked by
forskolin (data not shown). Treatment with TGF-
for 16 h did,
however, cause a statistically significant decrease in
Isc responses to forskolin; this was evident
with
1 ng/ml (data not shown). Figure 1
shows that T84 cells exposed to TGF-
(10 ng/ml) for 16 or 24 h
demonstrated a statistically significant decrease in forskolin-induced Isc responses of ~30% compared with controls
(P < 0.0001). This reduced responsiveness to forskolin
was further enhanced by 72 h post-TGF-
treatment, being on
average only 50% of the magnitude of the response observed in
time-matched control monolayers (P < 0.05 compared
with 16 and 24 h of TGF-
exposure). After 72 h of TGF-
treatment, there was a significant increase in TER (1,405 ± 254
/cm2 compared with control at 846 ± 204
/cm2, P < 0.05, n = 9-12 monolayers) that was not observed with shorter TGF-
exposure periods. In "washout" experiments, cells exposed to
TGF-
(10 ng/ml) for 16 h and subsequently rinsed free of
TGF-
also displayed a similar decrease in Isc
responses to forskolin at 72 h posttreatment (40.6 ± 1.7%
of control response, P < 0.0001, n = 3 monolayers), comparable to those seen after 72 h of persistent TGF-
exposure (49.3 ± 7.5% of control responses,
P < 0.0001, n = 9 monolayers).
This washout treatment also resulted in increased TER (1,598 ± 48
/cm2 vs. controls at 847 ± 20
/cm2,
P < 0.001, n = 3). T84 cells exposed
to TGF-
for 16 h at the highest dose used (i.e., 100 ng/ml)
displayed a ~30% decrease in forskolin-induced increases in
Isc, which was not statistically different from
the inhibition of Isc observed with lower doses of cytokine (i.e., 10 ng/ml). Furthermore, additional washout experiments revealed that exposure to TGF-
for 15 min was sufficient to induce diminished responsiveness to forskolin upon examination 16 h later (untreated controls 37.3 ± 4 vs. TGF-
-treated
cells 17 ± 2.1 µA/cm2. P < 0.001, n = 6-7 monolayers), in this case a 55% reduction in
Isc.
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Exposure to TGF-1 delivered via adenoviral gene transfer causes
decreased forskolin responsiveness.
We sought to determine what effect chronic exposure to TGF-
would
have on epithelial function using adenoviral gene transfer of the
biologically active form of TGF-
. Cell infectivity was verified by
use of adenoviral vectors encoding marker genes (Fig. 3A). Cells infected with the
highest dose of virus (50 MOI) displayed no significant decrease in
viability, as determined by the trypan blue exclusion technique (data
not shown), and exhibited increased TGF-
production compared with
uninfected controls (e.g., 912.2 ± 202.4 pg/ml TGF-
in
supernatants collected 72 h after infection with Ad-TGF
vs.
217.9 ± 8.5 pg/ml in supernatants of naive controls, P < 0.05, n = 3). Assessment of
Isc responses to forskolin 6 days post-Ad-TGF
infection revealed a statistically significant decrease in secretory
responsiveness compared with naive controls and T84 cells infected with
Ad-delete or Ad-latent TGF
(Fig. 3B). In addition,
Ad-TGF
infection resulted in a statistically significant increase in
TER by the end of the 6-day (144 h) postinfection period (Fig.
3C), which was first apparent 72 h postinfection (Fig.
3D). Similar to the experiments with recombinant TGF-
, HT-29 cells infected with Ad-TGF
(50 MOI) displayed reduced
Isc responses to forskolin 6 days postinfection
(66 ± 18.7 compared with controls at 118.7 ± 23.7 µA/cm2, P < 0.05, n = 3).
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Inhibition of p38 MAPK, but not JNK, ERK1/2 MAPK, or PI 3-K,
reduced the TGF- effect on cAMP-mediated Isc.
Pretreatment of HT-29 cells with SB-203580 (
1 µM), a potent
inhibitor of p38 MAPK activity, 30 min before TGF-
application completely restored the Isc responses to
forskolin to control values (Fig. 4),
whereas similarly treated T84 monolayers resulted in a significant, but
only partial, improvement in Isc responses to
forskolin (Table 2). Other signaling
pathways in T84 epithelial cells did not appear to contribute to the
diminished Isc, because pretreatment with the
JNK inhibitor (SP-600125), ERK1/2 pathway inhibitor (PD-98059), and the
PI 3-K inhibitor (LY-294002) did not inhibit the observed effect of
TGF-
on ion transport; in fact, exposure to PD-98059 or LY-294002
alone caused a significant decrease in Isc
responses to forskolin ~16 h later (Table 2).
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DISCUSSION |
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TGF- is a multifunctional peptide that affects cell
proliferation and differentiation and has immunosuppressive properties. Using human enteric epithelial cell lines, we have shown that 1) exposure to TGF-
significantly decreases cAMP-driven
Cl
secretion; 2) this effect can be blocked by
an inhibitor of p38 MAPK but not by pharmacological inhibitors of JNK,
ERK1/2 MAPK, or PI 3-K activity; and 3) the increased
barrier function caused by TGF-
is temporally distinct (i.e.,
delayed) from the altered ion transport characteristics of the treated epithelium.
Ion transport is an important element of gut homeostasis. As the
driving force for water movement, it facilitates surface hydration,
which, if dysregulated, can result in debilitating diarrhea or
constipation. TGF- had no effect on tonic epithelial ion transport
(i.e., baseline Isc) but significantly reduced
secretory responses to three cAMP-dependent secretagogues that operate
via different mechanisms. This effect likely lies downstream of cAMP generation, because TGF-
-treated cells stimulated with dibutyryl cAMP displayed similarly reduced Isc events. The
responses to forskolin, VIP, cholera toxin, and dibutyryl cAMP were all
reduced by a similar magnitude (i.e., ~30%), suggesting that
although TGF-
can dampen cAMP-driven Cl
secretion, the
pathophysiology that would be associated with total blockade of
Cl
secretion is avoided. The inhibition of secretory
responsiveness required a minimum of 1 ng/ml TGF-
and was
statistically significant 16 h posttreatment; however, constant
exposure to TGF-
was not essential, because a 15-min exposure
resulted in reduced
Isc to forskolin 16 h later. Higher doses of TGF-
did not affect the time required to
observe a reduced
Isc to forskolin, and they
did not affect the magnitude of the response. This lack of a dose
response has been noted for TGF-
inhibition of bradykinin-induced
Isc in HCA-7 colonocytes, although in this
instance the Isc responses were reduced by
6 h posttreatment (6).
The effects of recombinant TGF- were reproduced in epithelia
infected with adenovirus encoding the gene for the active form of
TGF-
; diminished
Isc to forskolin were
apparent 144 h postinfection (i.e., end of the experiment).
However, unlike other growth factors, such as EGF and IGF, which induce
acute effects on epithelial
Isc (39,
11), TGF-
when added to T84 monolayers in Ussing chambers for
30 min had no effect on subsequent forskolin-evoked
Isc. Thus TGF-
can be added to the list of
cytokines that directly modulate epithelial ion transport (reviewed in
Ref. 24). In addition, the effect of TGF-
is set apart
from those of other growth factors that rapidly affect ion transport;
rather, the TGF-
effect is reminiscent of IFN-
or IL-4 diminution
of epithelial secretory responsiveness, which typically requires
24 h or longer to become apparent (1, 10).
Upon identifying that TGF- exerts an effect on epithelial ion
transport, a number of mechanistic issues arose. One option was to
investigate the physiological changes mediated by TGF-
, such as the
possible change in amount, location, or activity of the apical
Cl
channel, CFTR. Indeed, growth factor regulation of
specific ion channels (3, 34) and cytokine modulation of
CFTR gene expression have been reported (7, 9, 30).
Alternatively, the TGF-
signaling pathway leading to the decreased
secretory response could be addressed. Data from many cell types show
that TGF-
binding to its surface receptor causes mobilization of
classic MAPKs and a unique series of molecules designated SMADs, and
these signaling cascades regulate specific aspects of the biological effects of TGF-
(29, 37, 41, 46). Little is known,
however, of the TGF-
signaling pathways in epithelia in general
(36), with a distinct lack of data relevant to enteric
epithelial cells.
Using a pharmacological approach, we found that the TGF- effects on
ion transport were reduced in two cell lines by pretreatment with the
established inhibitor of the p38 MAPK pathway, SB-203580. This is the
first time p38 MAPK has been implicated in governing a TGF-
function
in gut epithelia, and these findings are complementary to a recent
report showing that hyperosmolar stress-induced reduction in colonic
CFTR mRNA was blocked by inhibition of p38 MAPK activity (5). Whereas low-dose SB-203580 (i.e., 1 µM) completely
restored the TGF-
-induced diminished ion transport events in HT-29
cells, higher doses of the pharmacological inhibitor only partially
corrected the defect in T84 epithelia. This finding indicates cell
line-specific differences and, in the case of T84 cells, suggests
either reduced sensitivity to SB-203580 or that other signaling
pathways participate in the TGF-
effect. Thus we adopted the same
pharmacological approach to assess the putative involvement of other
signaling molecules in TGF-
modulation of ion transport. JNK is
likely activated in tandem with p38 MAPK in response to TGF-
(22, 43) and can affect the
Na+-K+-2Cl
transporter
(19). Interference with the activity of this transporter would impact on apical Cl
secretion events. Furthermore,
high-dose SB-203580 (i.e., 10-20 µM) may inhibit JNK activity
(45), yet use of a reputedly specific inhibitor of JNK
activity did not ameliorate the TGF-
-induced diminution of T84
Isc to forskolin. ERK, the third member of the MAPK family, is mobilized in a variety of cell types by TGF-
(14, 15, 28): pharmacological inhibition of MEK, the
enzyme upstream of ERK, did not affect the decreased
Isc induced by TGF-
. Finally, use of an
established inhibitor of PI 3-K activity excluded the involvement of
this ubiquitous signaling molecule in the TGF-
-induced perturbation
of cAMP-driven Cl
secretion. The latter observation
contrasts with the diminished carbachol-induced increases in
Isc in T84 cells caused by EGF that are PI 3-K
sensitive (40). In this context, we observed that TGF-
did not consistently alter carbachol-elicited increases in
Isc in T84 or HT-29 epithelia (personal observation).
Exposure to the MEK and PI 3-K inhibitors alone reduced T84
Isc to forskolin (Table 2), suggesting that
longer inhibition of ubiquitous signaling pathways may affect multiple
energy-dependent processes such as epithelial ion transport. The
inhibition of forskolin-induced
Isc by
LY-294002 appears to contradict the work of Dickson et al.
(12). The discrepancy may be due to the fact that in the
latter study, the epithelium was exposed to LY-294002 in Ussing
chambers for
30 min and Isc responses were
immediately assessed. Indeed, when those investigators inhibited PI 3-K
via wortmannin for 24 h, they observed an ~50% decrease in
forskolin-stimulated Isc, which is compatible
with the data in the current study.
Agent specificity must be considered in any pharmacological study.
Although accepted as a specific p38 MAPK inhibitor, SB-203580 has
recently been shown to block the activity of ALK5, a kinase that
phosphorylates SMAD3 (21). Thus involvement of SMAD3 in TGF- inhibition of
Isc to forskolin is a
possibility. We have been unable to convincingly and consistently show
increased amounts of phosphorylated p38 MAPK in nuclear or whole cell
extracts of serum-starved TGF-
-treated epithelial cells, or elevated
p38 MAPK activity (data not shown). This may be due to 1)
the relatively high constitutive phospho-p38 MAPK found in control
extracts; 2) the possibility that there may in fact be only
very subtle increases in activated p38 MAPK, because TGF-
causes
only a partial ablation of the Isc response; or
3) the TGF-
effect on ion transport is via a specific p38
MAPK isoform (i.e.,
,
,
, or
), the increases of which are
masked by constitutive expression of the other isoforms. Indeed, in
light of the data from Laping et al. (21), a finding of
increased phospho-p38 MAPK in TGF-
-treated epithelia would be
suggestive, but not unequivocal proof, of involvement of this enzyme in
the altered ion transport. Thus, although we have ruled out JNK, ERK,
and PI 3-K, definitive statements regarding the intracellular signals
that mediate the TGF-
effect on enteric epithelial ion transport
require the development of p38 MAPK and SMAD3 knockout (and preferably
inducible knockout) cell lines suitable for the analysis of vectorial
ion transport.
Recombinant or adenovirally delivered TGF- was shown to increase
TER, an accepted index of the paracellular permeability pathway. This
finding is in accordance with studies where TGF-
was shown to
increase epithelial barrier function and/or preserve barrier integrity
compromised by inflammatory cytokines (26, 31, 32).
Intriguingly, we found that the effects of TGF-
on barrier function
were temporally distinct from those on ion transport: TER was, unlike
Isc, unaltered by 16-24 h post-TGF-
treatment. This divergence in the timing of TGF-
effects on two of
the primary roles of the enteric epithelium implies utilization of
different or additional signaling pathways in the modulation of
epithelial barrier and ion transport. Research directed toward understanding the structural basis for the TGF-
-induced increased TER, the signaling events underlying this process, and definition of
the mechanism(s) responsible for the temporal separation of enhancement
of the epithelial barrier and diminished cAMP-driven Cl
secretion are required. As noted, TGF-
can antagonize
IFN-
-mediated disruption of the barrier function of T84 monolayers
(32). Because IFN-
can reduce TER within 24 h of
exposure and TGF-
-induced increases in TER are not apparent at this
time (i.e., 24 h), it is suggested that the TGF-
inhibition of
IFN-
-induced decreases in TER occurs via a distinct mechanism, that
is, inhibition of the IFN-
signaling cascade. The reciprocal event
(i.e., IFN-
interference of TGF-
SMAD signaling) has been shown
(38).
In conclusion, irrespective of the intracellular signaling mechanism,
exposure of model gut epithelia to TGF- not only led to the expected
increase in TER (i.e., at 72 h posttreatment) but also resulted in
diminished responsiveness to cAMP-dependent secretagogues 16 h
posttreatment. Prolonged exposure to TGF-
(using adenoviral vectors
encoding the gene for the active form of TGF-
) maintained the
Cl
secretion abnormality. The temporal separation of
TGF-
modulation of epithelial ion transport and barrier functions
adds to our appreciation of the complexity and spectrum of biological
activities coordinated by this pleiotropic cytokine. Finally, we
speculate that in addition to its ability to maintain or enhance
epithelial barrier function, TGF-
may reduce pathophysiological
complications resulting from excess water movement into the gut lumen
by limiting cAMP-driven Cl
secretion.
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ACKNOWLEDGEMENTS |
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We thank J. Lu and I. Gunawan for technical assistance.
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FOOTNOTES |
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K. Howe has been a recipient of an Ontario Graduate Scholarship in Science and Technology (2000) and a Natural Sciences and Engineering Research Council Studentship (2001-2003). D. M. McKay is a Canadian Institutes of Health Research (CIHR) Scholar (1998-2003). This work was funded by CIHR Grant MT-13421 (to D. M. McKay).
Address for reprint requests and other correspondence: D. M. McKay, Intestinal Disease Research Program, McMaster Univ., HSC-3N5C, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: mckayd{at}mcmaster.ca).
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.
August 14, 2002;10.1152/ajpcell.00414.2001
Received 31 July 2001; accepted in final form 1 August 2002.
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