Vibrio cholerae ACE stimulates
Ca2+-dependent Cl
/HCO3
secretion in T84 cells in vitro
Michele
Trucksis1,2,
Timothy L.
Conn1,
Steven S.
Wasserman1, and
Cynthia L.
Sears3
1 Center for Vaccine Development, Department of Medicine,
University of Maryland School of Medicine, and 2 Medical
Service, Veterans Affairs Medical Center, Baltimore 21201; and
3 Divisions of Infectious Diseases and Gastroenterology,
Department of Medicine, Johns Hopkins School of Medicine,
Baltimore, Maryland 21205
 |
ABSTRACT |
ACE, accessory cholera enterotoxin, the third
enterotoxin in Vibrio cholerae, has been reported to
increase short-circuit current (Isc) in rabbit
ileum and to cause fluid secretion in ligated rabbit ileal loops. We
studied the ACE-induced change in Isc and
potential difference (PD) in T84 monolayers mounted in modified Ussing
chambers, an in vitro model of a Cl
secretory cell. ACE
added to the apical surface alone stimulated a rapid increase in
Isc and PD that was concentration dependent and
immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl
and HCO3
. ACE acted synergistically with the
Ca2+-dependent acetylcholine analog, carbachol, to
stimulate secretion in T84 monolayers. In contrast, the secretory
response to cAMP or cGMP agonists was not enhanced by ACE. The
ACE-stimulated secretion was dependent on extracellular and
intracellular Ca2+ but was not associated with an increase
in intracellular cyclic nucleotides. We conclude that the mechanism of
secretion by ACE involves Ca2+ as a second messenger and
that this toxin stimulates a novel Ca2+-dependent synergy.
bacterial toxin; second messenger; Ussing chamber; cholera; bacterial pathogenesis; accessory cholera enterotoxin
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INTRODUCTION |
COLONIZATION
OF THE SMALL INTESTINE by Vibrio cholerae causes
the potentially lethal disease cholera due to massive salt and water
secretion. The dehydrating diarrhea of cholera is attributed primarily
to the intestinal secretion stimulated by cholera toxin (CT)
(28). However, two other toxins of V. cholerae
that alter short-circuit current (Isc)
and/or resistance in Ussing chambers have been identified. These are
zonula occludens toxin (ZOT) (8, 10),
which acts by disrupting tight junctions, and accessory cholera
enterotoxin (ACE) (32).
We have previously reported the identification, cloning, and
purification of the ACE protein (31, 32). We
now report the investigation of the mechanism of action of ACE
utilizing the Cl
-secreting T84 epithelial cell line. This
cell line is derived from a human colonic carcinoma, resembles crypt
cells morphologically, and secretes Cl
in response to
secretagogues whose actions are mediated via cAMP-, cGMP-, or
Ca2+-related mechanisms (3). With the
utilization of T84 cell monolayers, we identified that ACE stimulates
anion secretion in T84 cells, and we showed that this is dependent on
the apical influx of extracellular Ca2+ and most likely
select intracellular Ca2+ pools. Furthermore, ACE exhibits
a novel synergy with the acetylcholine analog carbachol, but not with
cyclic nucleotide-dependent agonists including the heat-stable
enterotoxin type a (STa) of Escherichia coli and forskolin.
 |
METHODS |
Materials.
CT, E. coli heat-stable enterotoxin, carbachol,
collagen, bumetanide, 1,2-bis(2-aminophenoxy)
ethane- N,N,N',N'-tetraacetic acid (BAPTA)-AM, nifedipine,
verapamil,
-conotoxin GVIA, clotrimazole, dantrolene,
staurosporine, DIDS, nystatin, genistein, and thapsigargin were
obtained from Sigma Chemical. Forskolin was obtained from Calbiochem-Novabiochem (San Diego, CA).
Sample preparation for Ussing chambers.
Cultures of V. cholerae bacterial strains ACE
(CVD113, CT
, ZOT
, ACE
)
(11) and ACE+ [CVD113, (pCVD630,
ACE+)] (11, 32) were grown in L
broth at 37°C with shaking. Culture supernatants were prepared by
centrifugation followed by filtration through a 0.45-µm filter. The
filtered supernatant was then fractionated and concentrated 1,000-fold
using Pall Filtron Omega stir cells (Pall Filtron, Northborough, MA) to
obtain a 5,000-30,000 relative molecular weight
(Mr) fraction. The fraction was washed
and resuspended in PBS. The partially purified ACE+ and
ACE
supernatants were used for all experiments except
where noted. The concentration of ACE in the partially purified
supernatants was estimated at 4.5 × 10
7 M based on
a comparison of peak
Isc induced by the
partially purified supernatants, compared with the peak
Isc induced by using purified ACE toxin (see
Fig. 3B). Native purified ACE monomer (see
RESULTS) was used in a subset of 1-2
experiments of each type to confirm that purified ACE gave the same
results as the partially purified preparation. In addition, the
concentration-response experiments (see Fig. 3B) were
performed with native purified ACE monomer. All samples were stored at
20°C until tested in Ussing chambers.
Cell culture and filter preparation.
T84 cells were grown in a 1:1 mixture of DMEM and Ham's F-12 nutrient
supplement with 29 mM NaHCO3, 20 mM HEPES, 50 U/ml
penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum. T84
cells were plated onto collagen-coated Transwell polycarbonate inserts
(Corning Costar, Acton, MA) at a density of 7 × 104
cells/cm2. Transepithelial resistances attained stable
levels (>1,000
/cm2) after 12 days.
Ussing chamber voltage-clamp transport studies.
Transepithelial transport studies were carried out across T84 confluent
monolayers in a simplified apparatus for measuring electrophysiological
parameters (surface area 1.0 cm2) designed for study of
filter-grown cells previously described by Madara et al.
(23). Isc and open-circuit PD
measurements were carried out in culture media (except where noted to
be in Ringer or Ca2+-, HCO3
-, or
Cl
-free Ringer) using Ag-AgCl and calomel electrodes via
4% agar bridges made with Ringer buffer. The electrodes were connected to an automatic voltage clamp (DVC 1000; World Precision Instruments, New Haven, CT). The PD was recorded under open-circuit conditions every
10 min (or at shorter intervals as displayed in RESULTS), and then the voltage was clamped and the Isc was
recorded (19, 20). Resistance of the
monolayer was calculated from the Isc and
open-circuit PD according to Ohm's law. Ringer solution contained (in
mM) 140 Na+, 25 HCO3
, 5.2 K+,
1.2 Ca2+, 1.2 Mg2+, 119.8 Cl
, 0.4 H2PO4
, 2.4 HPO42
, 10 glucose, and 5 HEPES, pH 7.4. For the Cl
-free Ringer, the
NaCl was replaced by sodium isethionate, and the CaCl2 and
MgCl2 were replaced by CaSO4 and
MgSO4 at the same molarities. For the
HCO3
-free Ringer, the NaHCO3 was replaced
by sodium isethionate. In experiments where the effect of
Ca2+ on ACE secretory activity was examined,
Ca2+-free Ringer (9) with the following
composition was used (in mM): 140 Na+, 25 HCO3
, 5.2 K+, 1.0 Mg2+, 117 Cl
, 0.4 H2PO4
, 2.4 HPO42
, 10 glucose, 5 HEPES, and 1.0 EGTA, pH 7.4. The
intracellular Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) was loaded into the cells during a
1-h preincubation period in Ringer solution at the desired concentration.
Purification of native ACE toxin.
Culture supernatant of wild-type V. cholerae strain E7946
was fractionated using Pall Filtron Omega stir cells and a Mini Prep
Cell (Bio-Rad Laboratories) as reported previously (31). Both the monomer and dimer forms of the ACE toxin were purified separately, as previously reported, and each yielded a single band with
silver stain (data not shown).
Measurement of cyclic nucleotides.
T84 cells grown on Costar inserts were treated with ACE, forskolin,
V. cholerae CT, E. coli STa, and
carbachol as described in the text. Intracellular cAMP was extracted
with ice-cold 50% ethanol-50% Ringer solution (vol/vol). Extraction
for cGMP measurements was performed with ice-cold 67% ethanol-33%
Ringer solution (vol/vol). The cell extracts were frozen at
20°C
until assayed by cyclic nucleotide enzyme immunoassay (EIA, cAMP, and
cGMP) system (Amersham Life Science) according to the manufacturer's instructions.
Statistical analysis.
The effects of various treatments were analyzed by repeated-measures
analysis of variance (ANOVA) where the dependent variable was PD or
Isc, the independent variable was the treatment
group (treated vs. control), and with time 0 as a covariate.
Each of these analyses were tested for a group effect (i.e., mean
difference in PD between treatment groups) and a group × time
interaction (differential change in PD over time in the 2 groups). Data
shown are means ± SE. Statistical hypotheses were evaluated at
the 5% level.
 |
RESULTS |
ACE stimulates a reversible increase in Isc and PD in
T84 cell monolayers.
The addition of ACE to the apical (Fig.
1, A and
B) or apical plus basolateral bathing solution of T84 cell
monolayers caused increases in Isc and PD as
measured in modified Ussing chambers. Basolateral addition alone of ACE
had no effect (Fig. 1A, peak Isc,
basolateral addition vs. negative control, P = 0.3).
Maximal response was reached by 20 min after the addition of ACE, and the effect persisted for at least 2 h. The peak
Isc and PD values for supernatants of an
ACE+ V. cholerae strain compared with an
ACE
V. cholerae strain (negative control) were
11.8 ± 2.4 µAmp/cm2 vs. 0.8 ± 1.1 µAmp/cm2 (Isc, P = 0.006, Fig. 1A) and
18.5 ± 2.2 mV vs. 0.6 ± 0.2 mV (PD, P < 0.001, Fig. 1B). The increase
in Isc and PD of an ACE+ V. cholerae strain compared with an ACE
V. cholerae strain was significant throughout a 2-h time course. Twenty minutes after the addition of apical ACE (but not basolateral), resistance of monolayers dropped ~20% (P = 0.08) and then
returned to baseline by 60 min (Fig. 1C). Subsequent studies
of ACE were performed with apical addition alone.

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Fig. 1.
A: time course of short-circuit current
(Isc) response to Vibrio cholerae
accessory cholera enterotoxin (ACE)+ and
ACE culture supernatants by T84 monolayers in the Ussing
chamber. , V. cholerae ACE
culture supernatant; , V. cholerae ACE+
culture supernatant, apical addition; , V. cholerae
ACE+ culture supernatant, basolateral addition. V. cholerae ACE+ culture supernatant, apical addition vs.
V. cholerae ACE culture supernatant,
P = 0.006, n = 4. B: time
course of potential difference (PD) response to V. cholerae
ACE+ and ACE culture supernatants by T84
monolayers in the Ussing chamber. , V. cholerae
ACE culture supernatant; , V. cholerae ACE+ culture supernatant, apical addition;
, V. cholerae ACE+ culture supernatant,
basolateral addition. V. cholerae ACE+ culture
supernatant, apical addition vs. V. cholerae
ACE culture supernatant, P < 0.001, n = 4. C: time course of resistance
(R) response to V. cholerae ACE+ and
ACE culture supernatants by T84 monolayers in the Ussing
chamber. , V. cholerae ACE
culture supernatant; , V. cholerae ACE+
culture supernatant, apical addition, n = 4.
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To determine whether the increase in the monolayer's electrical
parameters was reversible, the bathing media was replaced at different
time points with ACE-free media after ACE stimulated increases in
Isc and PD. The increase in
Isc and PD induced by ACE was immediately
reversible whether removed after 4 (Fig.
2), 60, or 120 min (data not shown).

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Fig. 2.
Effect of the removal of toxin on ACE-induced
Isc response by T84 monolayers;
n = 3. Similar results were obtained with ACE removal
at 60 and 120 min.
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Purified ACE protein stimulates concentration-dependent increases
in Isc and PD as seen with partially purified
ACE+ culture supernatants.
The ACE protein was purified from a wild-type V. cholerae
strain E7946. As we previously reported (31), the
predominant form of the ACE toxin produced in V. cholerae
had a molecular weight of 18,000 representing an ACE dimer. A second
protein of molecular weight 9,000 consistent with a monomer form of ACE
was also present. When these proteins were analyzed on T84 cells, the
monomer form of ACE produced a concentration-dependent increase in
Isc compared with the negative control (Fig.
3B). The
threshold concentration of purified ACE that induced a significant
increase in Isc was ~10
8 M (36 nM; P = 0.008) with a maximal effect at
~10
7 M (900 nM; Fig. 3B). Because of
limitations in the availability of purified ACE (31), we
were unable to stimulate the monolayers with a high enough
concentration of ACE to clearly saturate the Isc
response and thus were unable to calculate the half-maximal stimulatory
concentration. The time-to-peak Isc was
concentration dependent, because increasing the concentration of toxin
shifted the peak Isc to an earlier time (Fig.
3C). The time-dependent PD, Isc, and
resistance responses to purified ACE were similar to those observed
with the partially purified culture supernatant (see Fig. 1, data not
shown). The dimer form of ACE demonstrated less activity (Fig.
3A, ACE dimer vs. negative control, P
0.3 at 20, 30, and 60 min; P
0.7 at 10 min
and 90-240 min).

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Fig. 3.
A: time course of
Isc response to purified ACE monomer, dimer, and
ACE culture supernatant by T84 monolayers in the Ussing
chamber, n = 4. B: concentration response
with ACE toxin. Peak change ( ) in Isc
stimulated by purified ACE toxin in T84 monolayers. The threshold
concentration of ACE that stimulated a significant increase in
Isc was 36 nM (P = 0.008 vs.
negative control), and a maximal increase in Isc
was 900 nM ACE (P = 0.02). Results are means of number
of experiments, as indicated in parentheses. Similar results were
obtained when PD was analyzed (data not shown). C: time
course of Isc response to purified ACE monomer
at 2 concentrations, single experiment, illustrating the earlier peak
Isc response with increasing monomer
concentration.
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ACE-stimulated secretion is equally dependent on
Cl
or HCO3
ions.
It has previously been shown that the loop diuretic, bumetanide,
inhibits the basolaterally localized
Na+-K+-2Cl
cotransport system in
the T84 cell line (4). This transport pathway serves as
the principal Cl
-uptake pathway, and its inhibition by
bumetanide results in a reversal or inhibition of Cl
secretion mediated by cyclic nucleotides or Ca2+.
Therefore, bumetanide was used to test the involvement of this cotransport pathway in the Cl
secretory process activated
by ACE. As is the case for prostaglandin E1-
(37) and STa-induced (15)
Cl
secretion, pretreatment (30 min) of T84 cell
monolayers with bumetanide (10
4 M) substantially
(~60%) inhibited the action of ACE (Fig.
4). Bumetanide, by itself, had no effect
on Isc or PD. Bumetanide also reversed the
action of ACE when added after ACE had elicited a response (Fig. 4).
Similarly, ouabain (250 µM), which inhibits the
Na+-K+-ATPase necessary for active
transepithelial Cl
secretion, inhibited and reversed
ACE-induced Isc when added to the basolateral
reservoir (data not shown).

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Fig. 4.
Effect of bumetanide on ACE-induced
Isc response by T84 monolayers;
n = 3. Bumetanide (100 µM, added to the basolateral
reservoir) substantially inhibited the activity of ACE
(P = 0.003).
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The inhibition of Cl
secretion by bumetanide, described
above, suggests that Na+, Cl
, and possibly
K+ are required for the Cl
-uptake step in
ACE's action and that this process is localized to the basolateral
membrane of the T84 cells. To verify the involvement of
Cl
and/or HCO3
in the ACE-stimulated
increase in Isc/PD, ion replacement studies were
performed. Of note, the peak of ACE activity was ~60-80% inhibited when the Ringer solution was replaced by
Cl
-free Ringer (Fig. 5,
P = 0.03) or when replaced by
HCO3
-free Ringer (Fig. 5). When both ions were
removed from the Ringer solution, there was complete inhibition of
ACE-induced current (Fig. 5, P = 0.005).

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Fig. 5.
Time course of Isc response to
V. cholerae ACE+ culture supernatants by T84
monolayers in the Ussing chamber in Ringer buffer vs.
Cl -free, HCO3 -free, or
Cl -/HCO3 -free Ringer buffer. Of note,
removal of Cl or HCO3 individually
reduced ACE-stimulated Isc in a nearly
equivalent manner (P = 0.03), whereas ACE-stimulated
Isc was abrogated by removal of both anions
(P = 0.005; n = 5).
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The effect of ACE on second messengers.
To explore further the mechanism of action of ACE, we measured the
effect of the enterotoxin on cellular cAMP and cGMP. ACE and carbachol
had no significant effect on cellular cGMP or cAMP, whereas
STa increased cGMP but had no effect on cAMP and forskolin, and V. cholerae CT increased cAMP but had no effect
on cGMP (Table 1).
To examine the role of Ca2+ as a second messenger, ion
replacement studies were performed with Ringer solution replaced by
Ca2+-free Ringer in the apical reservoir 30 min before the
addition of the ACE toxin. ACE was added at a near maximal
concentration (90-900 nM). The basolateral reservoir retained
normal Ringer solution, which is required to maintain tight junction
integrity (33). The peak action of ACE was ~65%
inhibited when the apical Ringer solution was replaced by
Ca2+-free Ringer (Fig.
6A, P = 0.04).
The resistance of the monolayers was unchanged compared with the
resistance of parallel controls in normal Ringer solution. Furthermore,
pretreatment of T84 monolayers with the Ca2+ channel
blocker nifedipine inhibited the ACE-induced Isc
response (Fig. 6B, P = 0.001). In contrast,
pretreatment with the Ca2+ channel blockers
-conotoxin
and verapamil had no significant effect on the ACE-induced
Isc response (Fig. 6B). Together
these results suggest that the apical influx of extracellular
Ca2+ is required for the ACE effect on
Isc.

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Fig. 6.
Ca2+-dependence of ACE-induced
Isc. A: time course of
Isc response to V. cholerae
ACE+ culture supernatants by T84 monolayers in the
Ussing chamber in Ringer buffer vs. Ca2+-free Ringer
buffer; n = 5. ACE activity was significantly inhibited
in Ca2+-free Ringer buffer (P = 0.04).
Ca2+ was removed from the apical reservoir only to preserve
tight junction integrity (33). B: effect of
Ca2+ channel blockers, nifedipine, verapamil, and
-conotoxin, on ACE-induced Isc response.
Ca2+ channel blockers were added to the apical bath 30 min
before the addition of ACE. Only nifedipine significantly inhibited
ACE-induced Isc (P = 0.001;
n = 3). C: effect of
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) on ACE-induced Isc response.
Ca2+ chelation via BAPTA nearly ablated ACE activity
without altering monolayer resistance (n = 5).
D: effect of dantrolene on ACE-induced
Isc response. Dantrolene, an intracellular
Ca2+ antagonist, significantly reduced ACE-induced
Isc (P = 0.002;
n = 4). E: effect of ACE and thapsigargin
(Thaps) alone or thapsigargin pretreatment 2 h before ACE addition
on ACE-induced Isc response by T84 monolayers.
, thapsigargin (300 nM) alone to basolateral bath; , ACE
alone; , thapsigargin (300 nM) pretreatment in
basolateral bath followed by addition of ACE 2 h later;
, predicted additive effect. Thapsigargin, which
stimulates an increase in Isc or PD by
discharging endoplasmic reticulum Ca2+ stores
(34), potentiated ACE-stimulated
Isc (P < 0.001, n = 5).
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To further confirm that the ACE effect is mediated by Ca2+,
we employed the intracellular Ca2+ chelator BAPTA. When T84
cell monolayers were preloaded with 50 µM BAPTA, there was a near
ablation of ACE-induced Isc (Fig. 6C,
P < 0.001). In addition, pretreatment of T84
monolayers with dantrolene, an intracellular Ca2+
antagonist, inhibited the ACE-induced Isc
response (Fig. 6D, P = 0.002). However, when
monolayers were pretreated with thapsigargin (300 nM), a naturally
occurring sesquiterpene lactone that induces a rapid increase in the
concentration of cytosolic-free Ca2+ by direct discharge of
intracellular stores (30), there was a potentiation of
ACE-induced Isc (thapsigargin alone, mean peak Isc = 10.2 ± 4.3; ACE alone, mean
peak Isc = 14.0 ± 3.5; thapsigargin followed by ACE, mean peak Isc = 81.6 ± 7.1; predicted additive effect, mean peak
Isc = 24.6 ± 3.0; P < 0.001, Fig. 6E). Together these results suggest that
ACE's activity is dependent on both intra- and extracellular
Ca2+ and that ACE and thapsigargin act via different
intracellular Ca2+ pools.
To further examine the signaling pathways involved in the ACE-induced
Isc, we utilized the broad spectrum inhibitor of
protein kinases, staurosporine (100 nM), and the tyrosine kinase
inhibitor genistein (100 µM). Staurosporine inhibited 45% of peak
ACE-induced Isc (Fig.
7A), whereas genistein had no
effect on ACE-induced Cl
/HCO3
secretion
(Fig. 7B).

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Fig. 7.
Effect of kinase inhibitors on ACE-induced
Isc response. A: effect of protein
kinase inhibitor, staurosporine, on ACE-induced
Isc response. , staurosporine
pretreatment to basolateral bath followed by ACE 15 min later.
Staurosporine inhibits 45% of the peak Isc
response to ACE (P = 0.02; n = 4).
B: effect of genistein, a tyrosine kinase inhibitor, on
ACE-induced Isc response. , ACE alone;
, ACE treatment followed by genistein added to
basolateral bath 20 min later; , genistein alone to basolateral bath
(100 µM); , predicted additive effect. Genistein had no effect on
Isc response to ACE (ACE + genistein
observed vs. predicted, P = 0.3, n = 4).
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The Cl
secretory responses of T84 monolayers to ACE
plus agonists acting via Ca2+ (carbachol) or cyclic
nucleotides (STa, forskolin).
For these experiments, agonists were added at a concentration that
stimulated a maximal Isc response when added
individually (10
4 M carbachol, 4.4 × 10
7 M STa, 1 × 10
5 M
forskolin). ACE was utilized at a near-maximal concentration of 5 × 10
7 M. As previously reported (5), the
Cl
secretory response to carbachol (added to the
basolateral membrane) was rapid and transient, with a peak
Isc of 9.8 ± 1.1 µAmp/cm2 at
4 min and a return nearly to baseline by 10 min (Fig.
8A). In contrast, ACE alone
stimulates a rapid but persistent increase in
Isc with a peak of 10.5 ± 0.9 µAmp/cm2 at 4 min (Fig. 8A). Simultaneous
addition of ACE and carbachol (Fig. 8A) or serial addition
of ACE then carbachol (data not shown) resulted in a synergistic
response that was apparent by 4 min, with a peak
Isc of 71.0 ± 6.0 µAmp/cm2
and that persisted for at least 30 min (predicted additive effect, 20.0 ± 1.3 µAmp/cm2; P < 0.001). However, if carbachol is added before ACE, i.e., T84
monolayers are pretreated with carbachol (10
4 M,
basolateral membrane) 10 min before the addition of ACE, the ACE-induced Cl
secretion is not augmented (Fig.
8B) nor blocked. Of note, pretreatment of T84 monolayers
with carbachol 10 min before the addition of thapsigargin blocked
thapsigargin-induced Cl
secretion as previously reported
(Fig. 8B) (17). This, combined with the data
presented in Fig. 6, again suggests that the mechanism of ACE- and
thapsigargin-induced Isc is through activation
of different Ca2+ pools.

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Fig. 8.
A: effect of ACE and carbachol alone or
simultaneous addition of both on Isc response by
T84 monolayers. , carbachol alone (10 4 M)
added to basolateral bath; , ACE alone; ,
simultaneous addition of ACE + carbachol; , predicted additive
effect. Treatment of T84 monolayers simultaneously with ACE and
carbachol stimulated a synergistic increase in
Isc (P < 0.001, n = 3), which persisted at least 30 min. B:
effect of serial addition of both carbachol and ACE or carbachol and
thapsigargin on Isc response by T84 monolayers.
, carbachol (10 4 M) added to basolateral
bath at time 0, followed by ACE at 15 min; , ACE
alone at 15 min; , carbachol (10 4 M) added to
basolateral bath at time 0, followed by thapsigargin (1 µM) to basolateral bath at 15 min; , thapsigargin (1 µM) alone added to basolateral bath at 15 min. Pretreatment with
carbachol had no effect on ACE-induced Isc,
whereas pretreatment with carbachol inhibited the
Isc response to thapsigargin (P < 0.08, n = 3) as previously reported
(17).
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In contrast, the Cl
secretory responses of T84 monolayers
to E. coli STa, or forskolin, cGMP, and cAMP
agonists, respectively, are not enhanced by ACE. Simultaneous addition
of ACE and STa or ACE and forskolin produced an additive
response with a peak Isc of 32 ± 3.8 µAmp/cm2, predicted additive effect, 20.0 ± 5.5 µAmp/cm2 (ACE + STa, actual vs.
predicted, P = 0.14, n = 3) and a peak Isc of 38.9 ± 5.7 µAmp/cm2,
predicted additive effect, 54.3 ± 6.8 µAmp/cm2
(ACE + forskolin, actual vs. predicted, P = 0.15, n = 3). This lack of synergy between a Ca2+
agonist (ACE) and cyclic nucleotide agonist (STa or
forskolin) was unexpected, because Ca2+- and cyclic
nucleotide-dependent agonists normally show synergy. We performed
control experiments examining the secretory responses of T84 monolayers
to serial addition of E. coli STa followed by carbachol and response of monolayers to forskolin followed by carbachol. As expected, these agonists demonstrated the reported synergy of Ca2+- and cyclic nucleotide-dependent
agonists (21, 22, 36) (STa followed by carbachol, mean peak
Isc = 45.0 µAmps/cm2 vs.
predicted additive effect, mean peak Isc = 14.5 ± 0.5, P < 0.001, n = 2;
forskolin followed by carbachol, mean peak
Isc = 69.3 ± 2.4 µAmps/cm2 vs. predicted additive effect, mean peak
Isc = 49.0 ± 2.9, P = 0.005, n = 3).
ACE-stimulated Cl
/HCO3
secretion is
partially dependent on a DIDS-sensitive apical Cl
channel.
The apical membrane of polarized T84 cells contains two distinct
Cl
channels, differentiated by their sensitivity to DIDS
and anion selectivity (24). The DIDS-insensitive channel
that is activated by cAMP agonists presumably represents cystic
fibrosis transmembrane conductance regulator (CFTR). Both cAMP and
Ca2+ agonists activate the DIDS-sensitive Cl
channel. To determine which of the two channels is activated by ACE, we
treated T84 monolayers with 500 µM DIDS on the apical membrane.
DIDS-treated monolayers showed a 50% inhibition of the ACE-induced
Isc response (Fig.
9). This is in contrast to 100% inhibition of thapsigargin-induced Isc response
and 40% inhibition of forskolin-induced Isc
response by DIDS reported previously (24).

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Fig. 9.
Effect of ACE and DIDS alone or DIDS pretreatment 30 min
before ACE addition on Isc response by T84
monolayers. , DIDS (500 µM) added to apical bath alone;
, DIDS pretreatment to apical bath followed by addition
of ACE 30 min later. DIDS inhibited peak ACE-stimulated
Isc by 50% (P < 0.001, n = 7).
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ACE-stimulated secretion is inhibited by clotrimazole.
To ascertain the involvement of the basolateral membrane in
ACE-stimulated secretion, we evaluated the effects of clotrimazole on
ACE-mediated Cl
/HCO3
secretion. In
previously reported studies, clotrimazole was identified as an
inhibitor of both basolateral membrane K+ channels,
KCa and KcAMP (1).
Clotrimazole-treated monolayers showed a 92% reduction in
ACE-stimulated Isc compared with control monolayers (Fig. 10). This is similar
to the 91-94% inhibition of cyclic nucleotide agonist-dependent
Cl
secretion by clotrimazole (26) and the
84% inhibition of carbachol-dependent secretion (25).
These results indicate that ACE, like other cyclic nucleotide and
Ca2+-mediated agonists, depends on basolateral
K+ efflux pathways.

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|
Fig. 10.
Effect of clotrimazole pretreatment 30 min before ACE
addition on Isc response by T84 monolayers.
, clotrimazole (30 µM) added to apical and
basolateral baths; , Clotrimazole pretreatment followed
by addition of ACE 30 min later. Clotrimazole inhibition experiments
were performed with monolayers in Ringer buffer. Clotrimazole inhibited
ACE-stimulated Isc by 92% (P < 0.001, n = 4).
|
|
 |
DISCUSSION |
This report indicates that the V. cholerae enterotoxin
ACE is a Ca2+-dependent agonist that acts at the apical
membrane of polarized intestinal epithelial cells (T84 model) to
stimulate anion secretion, consistent with prior reports of its
secretory activity in animal models (32). The mechanism by
which ACE stimulates Isc and PD in T84
monolayers appears to involve both an influx of extracellular Ca2+ across the apical membrane of the cells as well as
intracellular Ca2+ stores. Selective inhibition with the
Ca2+ channel blocker nifedipine, but not the
Ca2+ channel blocker
-conotoxin (Fig. 6B),
suggests that an L-type voltage-gated Ca2+ channel is
involved in the ACE response rather than an N-type Ca2+
channel (inhibited by
-conotoxin). Of note, although thapsigargin potentiated the response of T84 cells to ACE, dantrolene and BAPTA-AM inhibited the activity of ACE. These data are consistent with the
hypothesis that the action of ACE on T84 cells is dependent on
extracellular and select (but as yet undefined) intracellular Ca2+ stores.
Our observations that ACE acts synergistically with the
Ca2+-dependent agonist carbachol, but not with cyclic
nucleotide-dependent agonists in T84 cells, is novel.
Ca2+-Ca2+-dependent synergy has not been
previously described and lends support to the concept that regional,
but interactive, pools of Ca2+ may be stimulated by
agonists. Conversely, prior reports have routinely identified
short-lived synergy of Ca2+- and cyclic
nucleotide-dependent agonists in T84 monolayers, whereas in the current
report, no such synergy was identified between ACE and cyclic
nucleotide agonists. The mechanism of synergy between
Ca2+-dependent and cyclic nucleotide-dependent agonists has
been postulated as due to a complementary increase in the driving force
for Cl
secretion due to the action of Ca2+ on
basolateral membrane K+ channels, combined with increased
apical membrane Cl
conductance due to the phosphorylation
of CFTR by cyclic nucleotide-dependent protein kinases. The lack of
synergy observed in response to ACE and cyclic nucleotide-dependent
agonists suggested to us initially that ACE, unlike carbachol, may not
modify K+ efflux across the basolateral membrane of T84
monolayers. However, our experiments using the inhibitor clotrimazole
(Fig. 10) suggest that ACE-induced secretion is dependent on
K+ efflux across the basolateral membrane of T84 cells.
Thus we hypothesize that apical influx of Ca2+ stimulated
by ACE may trigger mechanisms altering the transport properties of
apical anion channels (also supported by our experiments using DIDS,
Fig. 9) and that synergy with carbachol may result in part from a
synergistic or additive effect of ACE and carbachol on the same or
distinct basolateral K+ channels, respectively. Testing
this hypothesis will require both direct measurements of intracellular
Ca2+ and specific transport/ion efflux studies after
treatment of T84 cells with ACE.
The calcium dependence of ACE's activity is notable, as few enteric
toxins to date have been clearly defined to act in a
Ca2+-dependent manner. The best-studied examples are the
second heat-stable enterotoxin of E. coli (STb)
that act via a G protein-linked Ca2+ channel to stimulate
influx of Ca2+ from extracellular stores (6)
and the thermostabile direct hemolysin (TDH) of Vibrio
parahemolyticus that also stimulates influx of extracellular
Ca2+ (7). Similar to ACE, both of these toxins
act at the apical membrane of intestinal epithelial cells. However,
unlike ACE, which stimulates a prolonged increase in
Isc/PD, STb and TDH stimulate considerably briefer increases in Isc/PD in
animal tissues (15-40 min). In contrast,
Ca2+-dependent neurohumoral agonists (e.g., carbachol,
histamine, and serotonin) act at the basolateral membrane of intestinal
epithelial cells and stimulate only very brief increases in
Isc and PD. The rapid termination of the
Isc response to these agonists is attributed to
the production of the negative regulator,
D-myo-inositol 3,4,5,6-tetrakisphosphate (IP4), influx of extracellular Ca2+, activation
of protein kinase C, and/or a tyrosine kinase-dependent signaling
pathway (18). Our data suggest that ACE may modify the
cellular production of IP4 or prevent activation of one of the other known inhibitory pathways to produce the protracted Isc/PD response to ACE that we observed (Fig.
1). Inhibition by ACE of IP4 production (or another
inhibitory pathway) stimulated by carbachol would also be predicted to
contribute to the synergistic response of ACE and carbachol (Fig.
8A). Our data that demonstrate that the synergistic response
to ACE is ablated when carbachol is added 10 min before ACE (Fig.
8B) further suggest that ACE is insensitive to the
inhibitory action of IP4. This is in contrast to
thapsigargin, which shows a marked inhibition of the
Isc/PD response after carbachol pretreatment
(Fig. 8B and Ref. 17). Finally, our finding that a protein
kinase inhibitor (staurosporine) blocks ACE-induced
Isc, whereas a tyrosine kinase inhibitor
(genistein) has no effect, suggests that activation of protein kinase C
or Ca2+-calmodulin-dependent kinases may be essential to
ACE's activity. The observed diversity in the site of activity of
Ca2+-dependent agonists (2), the apparent
Ca2+ stores involved, and the magnitude and time course of
the identified physiological responses suggest that further studies to
better delineate the mechanisms and localization of Ca2+
responses in intestinal epithelial cells are warranted.
Our data suggest that ACE alters both Cl
and
HCO3
transport (Fig. 5, ion replacement studies) in
T84 monolayers via apical channel(s). Based on the DIDS-inhibition
studies, ACE appears to stimulate anion secretion by activating a
DIDS-sensitive, Ca2+-activated Cl
channel
(Fig. 9). In addition, ACE may also activate a DIDS-insensitive Cl
channel, most likely the CFTR. The ACE-stimulated
Isc also relies on the
Na+-K+-ATPase,
Na+-K+-2Cl
cotransporter, and
K+ channels (clotrimazole inhibition experiments) on the
basolateral cell membrane presumably to generate the Cl
gradient, which drives secretion when the apical channels are activated. Intriguingly, removal of either Cl
or
HCO3
caused nearly equivalent decreases in the
Isc/PD induced by ACE. Transport of
HCO3
by T84 cells has not been well defined. A recent
review of data published as part of the initial characterization of the
transport properties of T84 cells revealed that the measured
Isc was 20% that predicted from the net
secretion of Cl
, suggesting that under basal conditions,
T84 cells actively transport HCO3
(4,
13). This same report suggested that infection of T84 monolayers by enteropathogenic E. coli (EPEC) decreased net
transport by T84 monolayers by perturbing
HCO3
-dependent transport pathways (13).
In addition, transepithelial HCO3
transport is
reported to be insensitive to bumetanide, and bumetanide substantially,
but incompletely, inhibited the ACE-induced increase in
Isc (Fig. 4) similar to prior results with, for
example, genistein (27) and E. coli
STa (15). Together our results and recently reported data suggest that ACE most likely modifies both
Cl
- and HCO3
-dependent transport
mechanisms in T84 monolayers. Possible mechanisms that account for
these observations are that treatment of T84 monolayers with ACE
modifies the anion selectivity of CFTR [enabling it to conduct
HCO3
consistent with recent reports in the duodenal
epithelium (12, 14, 16,
29)] and/or the activity of an apical membrane
Cl
/HCO3
exchanger (or its linkage to
the activity of CFTR via, for example, recycling of Cl
through the apical membrane). Further studies of ACE will help define
both the transport changes stimulated by ACE and may assist in
evaluating the mechanisms by which T84 cells transport Cl
and HCO3
. Our observations, combined with the recent
observations regarding the effect of EPEC on ion transport in T84
monolayers (13), strongly suggest that future studies of
stimulated secretion in T84 cells consider the role of
HCO3
in the observed responses.
The ACE toxin is encoded in the V. cholerae chromosomal core
region, which encodes the filamentous bacteriophage, CTX
(35). Two of the open-reading frames in this region,
orfU and zot, are required for production of
phage particles (35). The role of ACE in the bacteriophage
has not been determined experimentally. We had previously shown that
the ACE toxin, cloned as a separate open-reading frame (that is,
without any of the other open-reading frames required for phage
production), had the activity of a classic enterotoxin in an in vivo
model (rabbit ileal loops) and in an in vitro model (rabbit Ussing
chambers) (32). Additionally, we were able to express the
ACE gene in yeast, and the ACE toxin produced had activity
in the rabbit Ussing chamber (31) and on monolayers of T84
cells (unpublished results). Furthermore, as shown in this paper, ACE
can be purified from the culture supernatants of wild-type V. cholerae, indicating that wild-type V. cholerae secretes biologically active ACE. Thus together, these data and the experimental results contained in this manuscript indicate that
ACE, like CT, is secreted by V. cholerae independent of
phage production and has physiological activity. Its role in the
pathogenesis of cholera has not been determined with the use of
isogenic mutants in human volunteers. However, the rapidity and potency
by which ACE increases Isc in T84 monolayers
suggests that ACE may contribute to an early phase of intestinal
secretion in V. cholerae infection before the onset of the
secretion stimulated by the more slowly acting cholera toxin.
These studies identify T84 cells as an excellent model for further
studies to delineate the cellular mechanism of action of ACE. The novel
physiology seen secondary to the ACE toxin makes this bacterial toxin
useful for understanding Ca2+-dependent signal transduction
in epithelial cells and may help clarify mechanisms of Cl
and HCO3
secretion. The suggestion that the ACE toxin
may act through the second messenger, Ca2+, and appears to
synergize with carbachol that also acts through the second messenger
Ca2+ makes this toxin a unique example of a secretagogue
exhibiting Ca2+-Ca2+ synergy.
 |
ACKNOWLEDGEMENTS |
We thank Alessio Fasano, James Nataro, and James Kaper for critical
reading of the manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Allergy and Infectious
Diseases Grant AI-35717 (M. Trucksis) and National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-45496 (C. L. Sears).
Address for reprint requests and other correspondence: M. Trucksis, Center for Vaccine Development, Univ. of Maryland School of
Medicine, 685 W. Baltimore St., Baltimore, MD 21201 (E-mail: mtrucksi{at}medicine.umaryland.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. §1734 solely to indicate this fact.
Received 19 July 1999; accepted in final form 14 March 2000.
 |
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