Department of Medicine, Section of Digestive and Liver Diseases, University of Illinois and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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Enteric bacterial pathogens often increase
intestinal Cl secretion.
Enteropathogenic Escherichia coli
(EPEC) does not stimulate active ion secretion. In fact, EPEC infection
decreases net ion transport in response to classic secretagogues. This
has been presumed to reflect diminished
Cl
secretion. The aim of
this study was to investigate the influence of EPEC infection on
specific intestinal epithelial ion transport processes.
T84 cell monolayers infected with EPEC were used for these
studies. EPEC infection significantly decreased short-circuit current
(Isc) in
response to carbachol and forskolin, yet
125I efflux studies revealed no
difference in Cl
channel
activity. There was also no alteration in basolateral K+ channel or
Na+-K+-2Cl
cotransport activity. Furthermore, net
36Cl
flux was not decreased by EPEC. No alterations in either
K+ or
Na+ transport could be
demonstrated. Instead, removal of basolateral bicarbonate from
uninfected monolayers yielded an
Isc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished
Isc. These
studies suggest that the reduction in stimulated
Isc is not
secondary to diminished Cl
secretion. Alternatively, bicarbonate-dependent transport processes appear to be perturbed.
infectious diarrhea; enteric pathogens; bicarbonate
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INTRODUCTION |
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THE PARADIGM TYPICALLY associated with diarrhea that
results from infection by an enteric pathogen is one of
Cl secretion or, somewhat
less commonly, inhibition of Na+
and Cl
absorption. As more
effort has been invested into studying the interactions between
infectious agents and their target host cells, previously unrecognized
mechanisms of pathogenesis have been defined. For example, it is now
clear that several enteric bacterial pathogens and/or their
toxins alter tight junction permeability of intestinal epithelia (9).
Some, such as toxins A and B of Clostridium difficile, appear to induce this physiological function
by affecting the actin cytoskeleton and specific tight junction
proteins via inactivation of Rho (10, 11, 21). Others may directly
affect tight junction structure, such as zonula occludens toxin of
Vibrio cholera (8). Still others, such
as enteropathogenic Escherichia coli
(EPEC), alter tight junction permeability by activating specific signaling pathways (24). EPEC attachment to its host target cell
induces the phosphorylation of myosin light chain, thereby stimulating
contraction of the perijunctional actomyosin ring and opening tight
junctions (27). Perturbation of tight junction barrier function may
interfere with vectorial transport by allowing back diffusion and
equilibration of electrochemical gradients to occur. This isolated
alteration in intestinal epithelial physiology, however, is unlikely to
be sufficient to account for the resultant diarrhea associated with EPEC.
EPEC is a most interesting pathogen as it is neither invasive nor
toxigenic. Yet somehow by intimately attaching to its host cell, it
activates a number of signal transduction pathways that then presumably
translate into alterations in intestinal epithelial function, in turn
resulting in diarrhea. Interestingly, we and others have previously
reported (23, 26) that cultured intestinal epithelial T84 cell
monolayers infected with EPEC exhibit an attenuation in net ion
transport, measured as short-circuit current
(Isc), in
response to classic secretagogues such as forskolin and carbachol. This
observation, presumed to reflect decreased
Cl secretion, has not been
examined in detail. The aim of this study was to investigate the
effects of EPEC on intestinal epithelial transport processes with
specific attention to Cl
secretion. For these investigations, we have employed the
well-characterized human intestinal epithelial cell line, T84, and a
commonly used clinical isolate of EPEC, strain E2348/69.
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METHODS |
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Culture of T84 cells. T84 cells were kindly provided by Dr. Kim Barrett (University of California, San Diego, CA). Passages 25-40 were used for these studies. Cells were cultured as previously described (18) in a 1:1 mixture of Ham's F-12 and DMEM supplemented with 6% newborn calf serum.
Bacterial cultures. EPEC strain E2348/69, a gift from Dr. James Kaper (Center for Vaccine Development, University of Maryland, Baltimore, MD), was initially grown overnight in Luria-Bertani broth. On the day of experimentation, 30 µl of EPEC culture were transferred to 1 ml of serum- and antibiotic-free T84 cell medium and grown to early or midlog growth phase. T84 cell monolayers were then infected with 2 × 107 colony-forming units (cfu) of EPEC for 1 h. Nonadherent bacteria were then removed by washing, and incubation proceeded for another 1-2 h. EPEC strain UMD864, an espB deletion mutant, was provided by Dr. Michael Donnenberg (Section of Infectious Diseases, University of Maryland, Baltimore, MD).
Electrophysiological studies.
A simplified apparatus for measuring electrophysiological parameters
previously described by Madara et al. (16) was used for these studies.
Isc in response
to carbachol (104 M) or
forskolin (10
6 M) was
determined. Resistance was calculated using Ohm's law (V = I/R,
where V is voltage,
I is current, and
R is resistance) by determining
voltage produced in response to the passage of 25 µA of current. All
experiments were performed in Ringer solution containing (in mM) 114 NaCl, 5 KCl, 1.65 Na2HPO4,
0.3 NaH2PO4, 25 NaHCO
3, 1.1 MgSO
4, 1.25 CaCl, and 5 glucose
unless indicated otherwise. For select experiments, specific ion-free
buffers were used. Na+-free buffer
contained (in mM) 116 choline chloride, 5 KCl, 1.65 K2HPO4,
0.3 KH2PO4,
25 choline HCO
3, 1.1 MgSO4, 1.25 CaCl2, and 10 glucose.
HEPES-phosphate-buffered Ringer solution (HPBR) was used for
HCO
3-free studies and contained the
following (in mM): 135 NaCl, 5 KCl, 3.33 NaH2PO4,
0.83 Na2HPO4, 1 CaCl2, 1 MgCl, 5 HEPES, and 10 glucose.
125I and 86Rb
efflux studies.
T84 cell monolayers were grown to confluence on collagen-coated
permeable supports (0.4 µm pore size Transwells; Costar,
Cambridge, MA). Control and EPEC-infected monolayers were loaded with 2 µCi of either 125I or
86Rb by incubating for 3 h.
Monolayers were then rapidly washed four times with HPBR, and a
previously described "sample/replace" technique (25) was used to
determine the rate constants of
125I and
86Rb efflux to estimate the
secretagogue-stimulated activation of Cl and
K+ channels, respectively. Before
addition of secretagogue, four baseline samples were obtained. Samples
were collected every 2 min following activation with secretagogue.
Residual intracellular radioactivity was determined following
extraction with 1 ml of 0.1 N NaOH. Samples were counted in a
scintillation counter. The efflux rate constant was calculated as
[ln(R2)
ln(R1)](t2
t1),
where R2 and
R1 are the percentages of
radioactivity remaining in the monolayer at times
t2 and
t1
(25).
86Rb uptake.
T84 monolayers grown on permeable supports were treated or not with
forskolin in the presence and absence of bumetanide (20 µM for 20 min). HPBR containing 2 µCi/ml of
86Rb was then added to the
basolateral reservoir of monolayers, and uptake was allowed to proceed
over 3 min. Termination of uptake was accomplished by immersing the
monolayers in ice-cold buffer composed of 100 mM
MgCl2 and 10 mM
Tris · HCl, pH 7.5. Radioactivity was extracted from
monolayers with 0.1 N NaOH and counted in a scintillation counter.
Protein concentration was determined by the Bradford assay.
Na+-K+-2Cl
cotransporter activity was defined as the bumetanide-sensitive portion
of 86Rb uptake.
Ussing chamber studies.
T84 cell monolayers were grown on
2-cm2 collagen-coated
polycarbonate filters glued onto lexan rings as previously described (2). Both uninfected control monolayers and EPEC-infected monolayers were used for these studies. Infected monolayers were incubated with
EPEC for 3 h before insertion into modified Ussing chambers. Resistances were matched to within 15% for all studies. Unidirectional fluxes of
36Cl
were performed utilizing either Ringer solution or
HCO
3-free HPBR. After a 20-min
equilibration period, 2 µCi/ml of
36Cl
(Amersham, Arlington Heights, IL) was added to either the mucosal or
serosal reservoir (5 ml each). Four baseline samples were collected before basal stimulation with forskolin. Samples (1 ml) were then collected from the "cold" reservoir at 2-min intervals for 12 min. Sample volume was replaced with the appropriate solution. At the
end of the experiment, 250 µl were collected from the "hot" reservoir so that flux rates could be calculated as previously described (11). Net Cl
flux
is calculated as the mucosal-to-serosal flux rate minus the
serosal-mucosal flux rate. Unidirectional fluxes of
22Na+
were also performed across control and EPEC-infected monolayers using
the methodology described for
36Cl
fluxes.
Statistical analysis.
Data are presented as means ± SE. Student's
t-test was used to compare data.
Significance was defined as P 0.05.
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RESULTS |
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Effect of EPEC on baseline and secretagogue-stimulated
Isc.
Confluent T84 monolayers (0.33 cm2) were infected with 2 × 107 midlog growth phase
EPEC, and Isc was
measured at 15- to 30-min intervals for up to 6 h. At no point in time
did EPEC stimulate the
Isc above
baseline (data not shown). To determine whether EPEC infection
influenced the
Isc response to
secretagogues, uninfected and infected monolayers were challenged with
either the Ca2+-mediated
secretagogue carbachol (104
M) or the cAMP-mediated secretagogue forskolin
(10
6 M). As shown in Fig.
1, EPEC infection
significantly decreased Isc in response
to both carbachol and forskolin compared with uninfected controls.
Although EPEC has been previously demonstrated to increase tight
junction permeability, that attenuation in
Isc could not be
attributed to alterations in resistance was shown in two ways. First,
the changes in secretagogue-induced
Isc could be
demonstrated before the EPEC-associated decrease in resistance occurred
(Fig. 2). Second, the EPEC mutant UMD864, a
deletion of the espB gene necessary
for the activation of several host signaling pathways, does not alter
transepithelial resistance (+20 ± 8 vs.
6 ± 5% change
in resistance for UMD864 vs. control), yet it still causes the observed
attenuation in forskolin-stimulated Isc (90 ± 4 vs. 62 ± 4 µA/cm2 for
control and UMD864, respectively). Sterile bacterial supernatants had
no effect on secretagogue-stimulated
Isc (data not
shown), suggesting that a soluble factor is not responsible.
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Diminished Cl secretion does not
account for the decrease in secretagogue-induced
Isc.
T84 monolayers have been widely used as a model cell line in which to
study Cl
secretion (1).
Because EPEC has been shown to diminish the Isc response in
T84 monolayers challenged with secretagogues, it has been presumed that
decreased Cl
secretion is
responsible. This hypothesis, however, has not been directly tested.
Electrogenic Cl
secretion
requires the coordinated activity of several ion transporters. These
include the apical Cl
channel and basolateral
Na+-K+-ATPase,
Na+-K+-2Cl
cotransporter, and K+ channels.
Our approach, therefore, was to systematically evaluate the effect of
EPEC on the transport components that contribute to apical
Cl
secretion.
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Neither stimulation of apical
K+ secretion nor
inhibition of
Na+ absorption
accounts for diminished Isc
in EPEC-infected monolayers.
Because diminished Cl
secretion did not explain the attenuated
Isc response in
EPEC-infected monolayers, alternative explanations were explored.
First, the possibility that apical
K+ secretion might be stimulated,
thus blunting measured
Isc, was examined. To test this possibility, apical
86Rb efflux studies were
performed. No difference was seen, however, between uninfected and
infected monolayers (0.001 ± 0.0001 vs. 0.001 ± 0.0003 efflux
rate constants for control and EPEC-infected monolayers;
n = 8;
P = 0.1). Second, the role of altered
Na+ transport was explored in
three ways. First, if inhibition of Na+ absorption were the cause of
blunted Isc, then
removal of Na+ from the apical
reservoir should increase stimulated
Isc back to the
level seen in uninfected control monolayers. Removal of apical
Na+ had no effect on
Isc. Second,
apical Na+ absorption in the
intestine occurs, in part, via the
Na+/H+
exchanger 3 (NHE3). To inhibit NHE3, amiloride (100 µM) was added to
the apical reservoir of monolayers before stimulation with secretagogues. The
Isc response in
EPEC-infected monolayers was not altered by amiloride pretreatment.
Amiloride also blocks Na+
channels; therefore, taken together, these data indicate that neither
inhibition of Na+ absorption by
NHE3 nor inhibition of Na+
channels accounts for the attenuation in
Isc associated
with EPEC infection. Finally, that altered
Na+ transport was not responsible
for the observed diminution in Isc was shown by
performing unidirectional
22Na+
fluxes (Table 1). There was minimal movement of
Na+ in response to cAMP-mediated
secretagogues as has been demonstrated previously (5). These data also
confirm the integrity of the tight junctions, dispelling the
possibility that enhanced paracellular permeability might account for
some of the difference in
Isc between control and EPEC-infected monolayers.
EPEC infection alters HCO3
transport.
Because investigations regarding the effect of EPEC on each of the
major ions transported by intestinal epithelium revealed no significant
differences, a role for HCO
3 was
considered. HCO
3 transport by the
basolateral Na+-HCO
3
cotransporter is electrogenic; therefore, the impact of removal of
basolateral HCO
3 on forskolin-induced
Isc was examined
in control and EPEC-infected monolayers. As shown in Fig.
5, removal of basolateral
HCO
3 decreases
Isc in
EPEC-infected monolayers to a much greater extent than it does in
controls. These findings suggest that a basolateral, HCO
3-dependent process contributes
significantly to
Isc generated by
EPEC-infected monolayers.
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EPEC-induced changes in
Isc are not driven by
mucosal acidification.
In this model, the apical surface of T84 cells is infected by EPEC.
Under these conditions, the medium in the apical reservoir becomes
acidified over time. HCO3 secretion via CFTR has been demonstrated to be stimulated by mucosal
acidification (13). When
Isc measurements
are taken using our model, however, the original medium is removed and
replaced with Ringer solution (pH 7.4) before challenge with
secretagogues. To confirm that apical acidification is not the factor
driving the altered
Isc response,
additional experiments were performed. First, acidified culture medium
from infected T84 cells was removed, filtered, and added to new
monolayers for 3 h. These new monolayers were than challenged with
either forskolin or carbachol. There was no significant difference in
the Isc response
between control and supernatant-treated monolayers stimulated with
forskolin (94 ± 4 vs. 86 ± 11 µA/cm2 for control and infected
monolayers; n = 8;
P = 0.1) or carbachol (61 ± 4 vs.
56 ± 11 µA/cm2 for control
and EPEC-infected monolayers, respectively;
n = 8, P = 0.5).
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DISCUSSION |
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The pathophysiology underlying EPEC-associated diarrhea appears to be much more complex than initially realized. The originally described morphological lesion, which included microvillus degeneration, seemed initially to provide a logical explanation for the resultant diarrhea. That is, it was presumed that the loss of crucial microvillar brush-border enzymes resulted in malabsorption and osmotic diarrhea. Subsequent data resulting from several different avenues of investigation, however, suggest that this is not the case. For example, human volunteer studies have shown that the onset of diarrhea following the ingestion of EPEC occurs as early as 4 h (6), too short a time for the sequence of attachment, effacement, and malabsorption to occur. Also, EPEC adheres as microcolonies, not individual bacteria, thus infecting only a portion (~50%) of enterocytes (14). As such, a substantial number of unaffected absorptive cells remain. Furthermore, only those microvilli in close proximity to adherent EPEC are altered, leaving some normal-appearing microvilli on the infected enterocyte. Last, although EPEC infection undoubtedly accelerates the normal process of vesicular shedding of microvillar digestive enzymes (2), there is evidence to suggest that the enterocyte may respond by a compensatory increase in the synthesis of such enzymes (7). These data, in addition to more recent studies that examine in detail the impact of EPEC infection on intestinal epithelial cell function, suggest that the pathophysiology of EPEC-associated diarrhea is quite complex and cannot be simply attributed to loss of brush-border enzymes.
The elucidation of the pathophysiology underlying EPEC-associated diarrhea is further complicated by the finding that EPEC does not induce active ion secretion. In an early study by Moon et al. (20), it was reported that infection of ligated ileal loops by EPEC in both pigs and rabbits did not induce fluid accumulation despite the presence of the attaching and effacing lesion. Interestingly, infection was found more frequently and was characterized as being more extensive in the large vs. the small intestine. Colonic loop experiments were not performed in this study.
More recently, attention has shifted to examining the effects of EPEC
specifically on its host target cell, the intestinal epithelial cell.
These studies have yielded interesting information. In contrast to the
usual paradigm of bacteria-induced
Cl secretion, EPEC has been
shown herein and by others (23) to not activate this process. In fact,
net ion secretion stimulated by classic secretagogues is blunted in
EPEC-infected monolayers. The results presented here suggest that such
attenuation is a global phenomenon in that both
Ca2+- and cAMP-stimulated
Isc values are
decreased. Philpott et al. (23), however, demonstrated attenuated
Isc only when
stimulated with forskolin, not carbachol. This scenario is reminiscent
of what occurs when the actin cytoskeleton is stabilized with agents such as phalloidin (19). In this case, diminished
Isc in response to forskolin is attributable to inhibition of the basolateral Na+-K+-2Cl
cotransporter. It was, therefore, hypothesized that the effect of EPEC
on forskolin-stimulated
Isc, which was
presumed to reflect diminished
Cl
secretion, may be linked
to this same mechanism. These investigators, however, did not examine
the effect of EPEC on Cl
secretion specifically but only examined
Isc, a
measurement of net ion transport. Furthermore, EPEC has not been shown
to alter basal actin, a component demonstrated to regulate
Na+-K+-2Cl
cotransporter activity.
The data presented here show that the diminution in stimulated
Isc caused by
infection with EPEC is, surprisingly, not due to diminished
Cl secretion. This was
demonstrated by showing that each specific transporter participating in
apical Cl
secretion,
including apical Cl
channels and the basolateral
Na+-K+-2Cl
cotransporter, was not altered by EPEC. In addition,
Cl
flux studies performed
in Ussing chambers confirmed that net Cl
secretion was identical
in control and infected monolayers. It should be noted, however, that a
recent publication reported that EPEC infection of Caco-2 monolayers
induced a rapid but transient increase in
Isc, a portion
(~25%) of which was Cl
dependent (4). The peak change in
Isc was seen at
10-min postinfection; however, a model of synchronized infection was
employed whereby bacteria were centrifuged onto the cell surface.
Obviously, the differences in the model employed, both cell line and
method of infection, could account for the variability in results.
Our studies also suggest that altered Na+ absorption does not account for the diminished Isc seen in EPEC-infected monolayers. Neither removal of Na+ from the apical buffer nor treatment with amiloride, which inhibits NHE3 as well as Na+ channels, altered the attenuated Isc response. In addition, Na+ flux studies showed no difference in Na+ transport.
It is becoming increasingly apparent that CFTR serves not only as a
conductor for the active secretion of
Cl but also for
HCO
3 (3, 12, 13). The role of CFTR in
HCO
3 secretion is, however, more complex. Much like Cl
secretion, the secretion of HCO
3
requires the orchestration of several transporters, including an anion
channel, apical
Cl
/HCO
3
exchanger, and basolateral uptake by the Na+-HCO
3
cotransporter. Intracellular HCO
3 may
also be generated through the action of carbonic anhydrase. The effect
of cAMP and Ca2+ stimulation on
these various components involved in
HCO
3 secretion is just beginning to be investigated.
The contribution of HCO3 to
secretagogue-stimulated
Isc in T84 cells
has not been addressed. Our studies suggest, contrary to popular dogma,
that HCO
3 may be responsible for up to
40% of forskolin-stimulated
Isc, the
difference between calculated and measured
Isc shown herein. In reviewing the data published as a part of the initial
characterization of the transport properties of T84 cells (5), the
measured Isc was
20% greater than that predicted from the net secretion of
Cl
. This unexplained
Isc may be
attributable to HCO
3 secretion. Our
Cl
flux studies performed
in the absence of HCO
3 showed that
both predicted and measured
Isc dropped
significantly. This does not necessarily imply that
HCO
3 secretion directly accounts for
nearly one-half of the generated
Isc. Instead, it
has been proposed that the activities of CFTR and the apical Cl
/HCO
3
exchanger are linked (3). One offered explanation of this interaction
is that the
Cl
/HCO
3
exchanger serves to recycle
Cl
through the apical
membrane (3). If
Cl
/HCO
3
activity is interrupted, either by direct inhibition or removal of
HCO
3, then there may be a decrease in
net Cl
secretion as we
observed in our flux studies. Under these conditions, measured and
predicted Isc
corresponded perfectly.
Regarding the effect of EPEC infection on stimulated net ion transport,
our studies clearly demonstrate that diminished
Cl secretion does not
account for the observed difference in
Isc. Examination
of the influence of EPEC on the transport of other major ions, namely
Na+ and
K+, failed to reveal any change.
That diminished transepithelial resistance was not responsible for the
measured changes in active ion transport was shown in two ways. First,
alterations in secretagogue-stimulated Isc were
demonstrated before changes in permeability, as shown in Fig. 2.
Second, if altered paracellular permeability were responsible, the flux
rate of Na+ would have increased.
Furthermore, a mutant strain of EPEC known to not decrease epithelial
resistance still induces alterations in
Isc.
How, then, might the EPEC-induced diminution in
Isc be explained?
In that the degree of decrease in net ion transport is quite substantial and alterations in the transport of none of the major ions,
including Cl,
Na+, and
K+, appear to account for this
change, HCO
3 is a likely candidate.
When HCO
3 is removed from uninfected
control monolayers, secretagogue-stimulated
Isc decreases significantly, ranging from 20 to 40%. In this case,
Isc approximates that seen in EPEC-infected monolayers. We, therefore, propose that EPEC
is altering the transport of HCO
3. Whether this change is being effected at the apical or basolateral membrane cannot be distinguished at this point. Also, the complex interaction that appears to exist between CFTR and the
Cl
/HCO
3
exchanger makes the regulation of HCO
3 secretion difficult to unravel. Clarke and Harline (3) suggest that, in
the duodenal epithelia, the relative contribution of CFTR as a
conductor of HCO
3 and as a
"facilitator" of apical
Cl
/HCO
3
exchange likely changes in response to cues from the luminal
environment. In addition, the activities of the basolateral
Na+-HCO
3
cotransporter and intracellular carbonic anhydrase must be considered.
To reconcile the specific mechanisms by which EPEC interferes with
HCO
3 transport, many additional
studies will be required. EPEC-infected intestinal epithelial cells
should, however, provide an interesting model for deciphering the
complex pathways that regulate HCO
3 secretion.
Although these studies enhance our understanding of the effects of EPEC infection on active ion transport, they do not fit the usual paradigm for diarrhea that occurs in response to infection by enteric pathogens. It has been shown that with time EPEC increases the permeability of tight junctions by inducing the phosphorylation of myosin light chain (27). This event may contribute to the production of diarrhea, but it is likely that other factors are involved as well.
In vivo, EPEC infection induces the recruitment of inflammatory cells, primarily neutrophils, not only into the lamina propria but also across intact crypts (20). Activated neutrophils produce 5'-AMP, which is converted by epithelial cell 5'-ectonucleotidase to adenosine, a potent secretagogue (17). In our in vitro model, EPEC infection also attenuates Isc in response to adenosine (unpublished observation). In vivo, however, EPEC attaches primarily to surface epithelial cells and less often to crypt cells, causing severe villous atrophy and crypt hyperplasia (22). Neutrophils tend to accumulate in intestinal crypts where it would be possible for uninfected crypt cells in the in vivo situation to demonstrate a full secretory response to neutrophil-derived adenosine.
In summary, the studies presented herein confirm that EPEC infection of
intestinal epithelial cells diminishes net ion transport, as measured
by Isc in
response to classic secretagogues. Unexpectedly, our results clearly
show that this decrease in net ion transport does not represent
diminished Cl secretion as
one might predict. Instead, it appears that EPEC alters
HCO
3 secretion and possibly perturbs other HCO
3-dependent transport
processes. Additional studies are needed to further define these
transport changes.
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ACKNOWLEDGEMENTS |
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We are grateful to our colleagues, Drs. Ramaswamy and Rao, for insightful suggestions regarding these studies.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50694 and the Department of Veterans Affairs (Merit Award) to G. Hecht.
This work was presented in preliminary form at the annual meeting of the American Gastroenterological Association held May 16-20, 1998, in New Orleans, LA.
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 and other correspondence: G. Hecht, Univ. of Illinois, Dept. of Medicine, Digestive and Liver Diseases (M/C 787), 840 South Wood St., CSB Rm. 704, Chicago, IL 60612 (E-mail: gahecht{at}uic.edu).
Received 6 July 1998; accepted in final form 3 December 1998.
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