Vibrio cholerae ACE stimulates Ca2+-dependent Clminus /HCO3minus 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, omega -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 Delta Isc induced by the partially purified supernatants, compared with the peak Delta 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 Omega /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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (20K):
[in this window]
[in a new window]
 
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. open circle , V. cholerae ACE- culture supernatant; , V. cholerae ACE+ culture supernatant, apical addition; black-triangle, 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. open circle , V. cholerae ACE- culture supernatant; , V. cholerae ACE+ culture supernatant, apical addition; black-lozenge , 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. open circle , V. cholerae ACE- culture supernatant; , V. cholerae ACE+ culture supernatant, apical addition, n = 4.

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).


View larger version (14K):
[in this window]
[in a new window]
 
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.

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).


View larger version (13K):
[in this window]
[in a new window]
 
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 (Delta ) 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.

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).


View larger version (12K):
[in this window]
[in a new window]
 
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).

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).


View larger version (12K):
[in this window]
[in a new window]
 
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).

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Cyclic nucleotide concentration in T84 monolayers after addition of secretagogues

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 omega -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.


View larger version (21K):
[in this window]
[in a new window]
 
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 omega -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; black-down-triangle , 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).

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).


View larger version (16K):
[in this window]
[in a new window]
 
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); diamond , predicted additive effect. Genistein had no effect on Isc response to ACE (ACE + genistein observed vs. predicted, P = 0.3, n = 4).

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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8.   A: effect of ACE and carbachol alone or simultaneous addition of both on Isc response by T84 monolayers. open circle , carbachol alone (10-4 M) added to basolateral bath; , ACE alone; , simultaneous addition of ACE + carbachol; diamond , 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. open circle , 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).

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).


View larger version (10K):
[in this window]
[in a new window]
 
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).

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.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 10.   Effect of clotrimazole pretreatment 30 min before ACE addition on Isc response by T84 monolayers. open circle , 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 omega -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 omega -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, CTXPhi (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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Devor, DC, Singh AK, Gerlach AC, Frizzell RA, and Bridges RJ. Inhibition of intestinal Cl- secretion by clotrimazole: direct effect on basolateral membrane K+ channels. Am J Physiol Cell Physiol 273: C531-C540, 1997[Abstract/Free Full Text].

2.   Dharmsathaphorn, K, Cohn J, and Beuerlein G. Multiple calcium-mediated effector mechanisms regulate chloride secretory responses in T84-cells. Am J Physiol Cell Physiol 256: C1224-C1230, 1989[Abstract/Free Full Text].

3.   Dharmsathaphorn, K, and Madara JL. Methods in Enzymology: Biomembranes, edited by Fleischer S, and Fleischer B.. San Diego, CA: Academic, 1990, p. 354-389.

4.   Dharmsathaphorn, K, Mandel KG, Masui H, and McRoberts JA. Vasoactive intestinal polypeptide-induced chloride secretion by a colonic epithelial cell line. J Clin Invest 75: 462-471, 1985[ISI][Medline].

5.   Dharmsathaphorn, K, and Pandol SJ. Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. J Clin Invest 77: 348-354, 1986[ISI][Medline].

6.   Dreyfus, LA, Harville B, Howard DE, Shaban R, Beatty DM, and Morris SJ. Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB). Proc Natl Acad Sci USA 90: 3202-3206, 1993[Abstract].

7.   Fabbri, A, Falzano L, Frank C, Donelli G, Matarrese P, Raimondi F, Fasano A, and Fiorentini C. Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells. Infect Immun 67: 1139-1148, 1999[Abstract/Free Full Text].

8.   Fasano, A, Baudry B, Pumplin DW, Wasserman SS, Tall BD, Ketley JM, and Kaper JB. Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions. Proc Natl Acad Sci USA 88: 5242-5246, 1991[Abstract].

9.   Fasano, A, Hokama Y, Russell R, and Morris JG. Diarrhea in ciguatera fish poisoning: preliminary evaluation of pathophysiological mechanisms. Gastroenterology 100: 471-476, 1991[ISI][Medline].

10.   Fasano, A, and Uzzau S. Modulation of intestinal tight junctions by Zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J Clin Invest 99: 1158-1164, 1997[Abstract/Free Full Text].

11.   Fiore, AE, Michalski JM, Russell RG, Sears CL, and Kaper JB. Cloning, characterization, and chromosomal mapping of a phospholipase (lecithinase) produced by Vibrio cholerae. Infect Immun 65: 3112-3117, 1997[Abstract].

12.   Guba, M, Kuhn M, Forssmann W-G, Classen M, Gregor M, and Seidler U. Guanylin strongly stimulates rat duodenal HCO3- secretion: proposed mechanism and comparison with other secretagogues. Gastroenterology 111: 1558-1568, 1996[ISI][Medline].

13.   Hecht, G, and Koutsouris A. Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl- secretion. Am J Physiol Gastrointest Liver Physiol 276: G781-G788, 1999[Abstract/Free Full Text].

14.   Hogan, DL, Crombie DL, Isenberg JI, Svendsen P, De Muckadell OBS, and Ainsworth MA. CFTR mediates cAMP- and Ca2+-activated duodenal epithelial HCO3- secretion. Am J Physiol Gastrointest Liver Physiol 272: G872-G878, 1997[Abstract/Free Full Text].

15.   Huott, PA, Liu W, McRoberts JA, Giannella RA, and Dharmsathaphorn K. Mechanism of action of Escherichia coli heat stable enterotoxin in a human colonic cell line. J Clin Invest 82: 514-523, 1988[ISI][Medline].

16.   Joo, NS, London RM, Kim HD, Forte LR, and Clarke LL. Regulation of intestinal Cl- and HCO3- secretion by uroguanylin. Am J Physiol Gastrointest Liver Physiol 274: G633-G644, 1998[Abstract/Free Full Text].

17.   Kachintorn, U, Vajanaphanich M, Barrett KE, and Traynor-Kaplan AE. Elevation of inositol tetrakisphosphate parallels inhibition of Ca2+-dependent Cl- secretion in T84 cells. Am J Physiol Cell Physiol 264: C671-C676, 1993[Abstract/Free Full Text].

18.   Keely, SJ, Uribe JM, and Barrett KE. Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. J Biol Chem 273: 27111-27117, 1998[Abstract/Free Full Text].

19.   Lencer, WI, de Almeida JB, Moe S, Stow JL, Ausiello DA, and Madara JL. Entry of cholera toxin into polarized human intestinal epithelial cells: identification of an early brefeldin A sensitive event required for A1-peptide generation. J Clin Invest 92: 2941-2951, 1993[ISI][Medline].

20.   Lencer, WI, Strohmeier G, Moe S, Carlson SL, Constable CT, and Madara JL. Signal transduction by cholera toxin: processing in vesicular compartments does not require acidification. Am J Physiol Gastrointest Liver Physiol 269: G548-G557, 1995[Abstract/Free Full Text].

21.   Levine, SA, Donowitz M, Sharp GWG, Crane JK, and Weikel CS. Synergism in chloride secretion in T84 cells is dependent on the order and timing of addition of E. coli heat-stable enterotoxin (STa) and carbachol. Am J Physiol Gastrointest Liver Physiol 261: G592-G601, 1991[Abstract/Free Full Text].

22.   Levine, SA, Donowitz M, Watson AJM, Sharp GWG, Crane JK, and Weikel CS. Characterization of the synergistic interaction of Escherichia coli heat-stable toxin and carbachol. Am J Physiol Gastrointest Liver Physiol 261: G592-G601, 1991[Abstract/Free Full Text].

23.   Madara, JL, Colgan S, Nusrat A, Delp C, and Parkos C. A simple approach to measurement of electrical parameters of cultured epithelial monolayers: use in assessing neutrophil-epithelial interactions. J Tiss Cult Meth 14: 209-216, 1992.

24.   Merlin, D, Jiang L, Strohmeier GR, Nusrat A, Alper SL, Lencer WI, and Madara JL. Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells. Am J Physiol Cell Physiol 275: C484-C495, 1998[Abstract/Free Full Text].

25.   Rufo, PA, Jiang L, Moe SJ, Brugnara C, Alper SL, and Lencer WI. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells. J Clin Invest 98: 2066-2075, 1996[Abstract/Free Full Text].

26.   Rufo, PA, Merlin D, Riegler M, Ferguson-Maltzman MH, Dickinson BL, Brugnara C, Alper SL, and Lencer WI. The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances-demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera. J Clin Invest 100: 3111-3120, 1997[Abstract/Free Full Text].

27.   Sears, CL, Firoozmand F, Mellander A, Chambers FG, Eromar IG, Bot AGM, Scholte B, de Jonge HR, and Donowitz M. Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers. Am J Physiol Gastrointest Liver Physiol 269: G874-G882, 1995[Abstract/Free Full Text].

28.   Sears, CL, and Kaper JB. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev 60: 167-215, 1996[Free Full Text].

29.   Seidler, U, Blumenstein I, Kretz A, Viellard-Baron D, Rossmann H, Colledge WH, Evans M, Ratcliff R, and Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3- secretion. J Physiol (Lond) 505: 411-423, 1997[Abstract].

30.   Thastrup, O, Cullen PJ, Drobak BK, Hanley MR, and Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87: 2466-2470, 1990[Abstract].

31.   Trucksis, M, Conn TL, Fasano A, and Kaper JB. Production of Vibrio cholerae accessory cholera enterotoxin (ACE) in the yeast Pichia pastoris. Infect Immun 65: 4984-4988, 1997[Abstract].

32.   Trucksis, M, Galen JE, Michalski J, Fasano A, and Kaper JB. Accessory cholera enterotoxin (ACE), the third toxin of a Vibrio cholerae virulence cassette. Proc Natl Acad Sci USA 90: 5267-5271, 1993[Abstract].

33.   Unno, N, Baba S, and Fink MP. Cytosolic ionized Ca2+ modulates chemical hypoxia-induced hyperpermeability in intestinal epithelial monolayers. Am J Physiol Gastrointest Liver Physiol 274: G700-G708, 1998[Abstract/Free Full Text].

34.   Uribe, JM, Gelbmann CM, Traynor-Kaplan AE, and Barrett KE. Epidermal growth factor inhibits Ca2+-dependent Cl- transport in T84 human colonic epithelial cells. Am J Physiol Cell Physiol 271: C914-C922, 1996[Abstract/Free Full Text].

35.   Waldor, MK, and Mekalanos JJ. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272: 1910-1914, 1996[Abstract].

36.   Warhurst, G, Fogg KE, Higgs NB, Tonge A, and Grundy J. Ca(2+)-mobilizing agonists potentiate forskolin- and VIP-stimulated cAMP production in human colonic cell line, HT29-cl. 19A: role of [Ca2+]i and protein kinase C. Cell Calcium 15: 162-174, 1994[ISI][Medline].

37.   Weymer, A, Huott P, Liu W, McRoberts JA, and Dharmsathaphorn K. Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. J Clin Invest 76: 1828-1836, 1985[ISI][Medline].


Am J Physiol Cell Physiol 279(3):C567-C577
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society