INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

PANCREATIC DUCT EPITHELIAL cells (PDEC) secrete fluid and electrolytes. Together with acinar cells, which secrete digestive enzymes, they are the main components of pancreatic exocrine secretion. Because the pancreatic and the bile ducts share a common outflow in the duodenum, obstruction of the ampulla of Vater may cause bile to reflux backward into the pancreatic duct, exposing PDEC to bile acids. Indeed, in 1901, Opie suggested that refluxed bile is a factor in gallstone pancreatitis. Bile acids stimulate electrolyte secretion by epithelial cells from different portions of the gastrointestinal tract, such as the large bowel and the biliary tree (8, 9, 11, 12). However, to our knowledge, the secretory effects of bile acids on PDEC have not been evaluated thoroughly. Using cultured nontransformed dog PDEC that express many secretory functions characteristic of PDEC (1, 13, 20), such as mucin secretion (21), cAMP- and Ca²⁺-activated Cl channels (14), and Ca²⁺-activated K⁺ channels (16), we 1) examined whether bile acids activate Cl and K⁺ conductances, 2) identified the signaling pathway mediating this effect, 3) compared the secretory effects of different bile acids, 4) studied the effects of bile acids on monolayer transepithelial resistance (TER), and 5) compared the effects of bile acids on the apical or basolateral membrane of the PDEC.

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and reagents. Taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, charybdotoxin, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), sodium pyrophosphate, lactate, and tissue culture medium and supplements were from Sigma (St. Louis, MO). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was from Calbiochem (San Diego, CA), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was from Research Biochemicals International (Natick, MA). Na¹²⁵I (16 mCi/mg iodide) was from Amersham (Arlington Heights, IL), and ⁸⁶RbCl (4.66 mCi/mg ⁸⁶Rb⁺) was from NEN (Boston, MA).

PDEC culture. As previously described (21), PDEC isolated from the accessory pancreatic duct of a dog were cultured on Transwell or Snapwell inserts (Costar, Cambridge, MA) coated with 0.7 ml (for Transwell) or 0.15 ml (for Snapwell) of a 1:1 solution of Eagle's MEM-Vitrogen (Collagen, Palo Alto, CA) over a feeder layer of myofibroblasts and in Eagle's MEM supplemented with 10% FBS, 2 mM L-glutamine, 20 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml bovine insulin, 5 µg/ml human transferrin, and 5 ng/ml sodium selenite. These PDEC express a cAMP-activated Cl channel, corresponding to the cystic fibrosis transmembrane conductance regulator, a Ca²⁺-activated Cl channel (14), and Ca²⁺-activated K⁺ channels (16). Cells used in this report were between passages 9 and 30. Efflux studies, Ussing chamber recordings, TER measurements, and lactate dehydrogenase (LDH) assays used confluent PDEC on Transwell or Snapwell filters that were isolated from the myofibroblast feeder layer.

Efflux studies. Studies of the activation of Cl and K⁺ conductances through cellular ¹²⁵I and ⁸⁶Rb⁺ effluxes have been validated in T84 cells (23) and have been used extensively to characterize the Cl and K⁺ conductances on PDEC (14-19).

Ussing chamber studies. Confluent monolayers of PDEC cultured on Snapwell inserts were mounted in modified Ussing chambers with an aperture area of 1.0 cm². Three different configurations were studied. For studies of net electrogenic transepithelial ion transport, both sides of the monolayer were bathed in Ringer solution (in mM: 115 NaCl, 1.2 CaCl₂, 1 MgCl₂, 0.4 KH₂PO₄, 2.4 K₂HPO₄, 25 NaHCO₃, and 10 glucose), warmed to 37°C with a circulating water jacket, and gently mixed and aerated with a constant inflow of 95% O₂-5% CO₂. For studies of apical Cl conductance, the basolateral surface of the PDEC was permeabilized by adding 0.20 mg/ml nystatin to the serosal compartment 15 min before the addition of TDCA. A serosal-to-luminal Cl gradient (intracellular-to-extracellular gradient across the apical membrane) was generated by adding, to the serosal compartment, a buffer consisting of 135 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mM K₂HPO₄, 0.6 mM KH₂PO₄, 10 mM HEPES, and 10 mM glucose, titrated to pH 7.4 with NaOH, and, to the luminal compartment, a buffer of 135 mM sodium gluconate, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mM K₂HPO₄, 0.6 mM KH₂PO₄, 10 mM HEPES, and 10 mM glucose, titrated to pH 7.4 with NaOH. For studies of basolateral K⁺ conductance, the apical membrane was permeabilized with nystatin (0.20 mg/ml, 15 min) and a luminal-to-serosal K⁺ gradient (intra-to-extracellular gradient across the basolateral membrane) generated using a luminal buffer of 10 mM NaCl, 1.25 mM CaCl₂, 1 mM MgCl₂, 118 mM potassium gluconate, 10 mM HEPES, and 10 mM glucose, titrated to pH 7.4 with NaOH, and a serosal buffer of 10 mM NaCl, 1.25 mM CaCl₂, 1 mM MgCl₂, 4 mM potassium gluconate, 114 mM N-methyl-D-glucamine, 10 mM HEPES, and 10 mM glucose, titrated to pH 7.4 with acetic acid.

Measurement of TER. Confluent PDEC monolayers cultured on Snapwell inserts were replenished with fresh culture medium and exposed, either apically or serosally, to different concentrations of TDCA for either 2 min, 5 min, 10 min, or 24 h. TER of the monolayers was measured at the beginning of the experiment, after TDCA exposure (or after 10 min for the control and 24 h treatments), after 15 min, and after 24 h, using an epithelial voltohmmeter (EVOM apparatus) from WPI (Sarasota, FL). TER measurements were calibrated using a Transwell membrane coated with Vitrogen but devoid of PDEC. For each monolayer, TER measurements were normalized to the value obtained at the beginning of the experiment.

LDH activity. To assess for cellular injury, LDH release from PDEC exposed to bile acids was monitored. The cultured PDEC and supporting filter were excised from the Transwell inserts and exposed for 5 min to 1 ml of efflux buffer containing different concentrations of bile acids. LDH released in the medium was assayed according to method of Amador et al. (3): 1.4 ml reaction buffer (77.5 mM lactic acid, 5.25 mM -nicotinamide adenine dinucleotide, and 0.05 M sodium pyrophosphate, pH 8.8) was mixed with 100 µl supernatant, and absorption at 340 nm was measured using a 160U ultraviolet spectrophotometer (Shimadzu). The absorption change after a lag time of 1 min was determined.

Statistics. All observations were based on at least three experiments. Unless specified otherwise, results were expressed as means and SE, and statistical significance was determined with the unpaired Student's t-tests or ANOVA (concentration-dependence studies) using the Statview 512+ (Abacus Concepts, Calabasas, CA) or InStat 3 (GraphPad Software) software.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

Iodide efflux studies. The possible activation of Cl conductances on PDEC by TDCA was evaluated by monitoring cellular ¹²⁵I efflux. As shown in Fig. 1A, TDCA, at a concentration of 1 mM, stimulated a robust increase in ¹²⁵I efflux to a peak rate coefficient of 0.726 ± 0.072 min¹ 30 s after addition of the conjugated bile acid (vs. 0.155 ± 0. 0.011 min¹ for control treatment, P < 0.01, n = 3). A much smaller increase was obtained with 500 µM TDCA with a peak rate coefficient of 0.224 ± 0.026 min¹ 45 s after TDCA addition (vs. 0.134 ± 0.014 min¹ for control treatment, P < 0.05, n = 3). At a concentration of 100 µM, TDCA failed to elicit an increased efflux different from control.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of taurodeoxycholic acid (TDCA) on 125I efflux. 125I efflux from pancreatic duct epithelial cells (PDEC) was determined as detailed in MATERIALS AND METHODS, and the efflux rate coefficient was calculated and shown. In A-D, means and SE from 3 experiments are shown. A: after 1 min of baseline determination, TDCA was added at the different final concentrations shown. NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid. B: after 1 min of baseline determination, 1 mM TDCA was added in the absence () or presence () of 500 µM NPPB. C: after 1 min of baseline determination, 1 mM TDCA was added in the absence () or presence () of 500 µM DIDS. D: after 1 min of baseline determination, 1 mM TDCA was added to cells pretreated with 50 µM BAPTA-AM () or untreated control cells ().

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View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of different bile acids on 125I efflux. 125I efflux from PDEC was determined as detailed in MATERIALS AND METHODS, and the efflux rate coefficient was calculated and shown. In A and B, means and SE from 3 experiments are shown. A: after 1 min of baseline determination, 1 mM of deoxycholic acid conjugated to either taurine () or glycine () was added. B: after 1 min of baseline determination, 1 mM of either deoxycholic acid (), chenodeoxycholic acid (), or cholic acid (), all conjugated to taurine, was added.

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⁸⁶Rb+ efflux studies. As previously reported, these PDEC also express Ca²⁺-activated K⁺ conductances that can be studied through measurements of ⁸⁶Rb⁺ efflux (16). Because the findings above suggested that TDCA caused a [Ca²⁺]_i increase to stimulate Ca²⁺-activated Cl channels, the effect of this [Ca²⁺]_i increase on Ca²⁺-activated K⁺ channels was also evaluated. As shown in Fig. 3A, 1 mM TDCA also stimulated an increased ⁸⁶Rb⁺ efflux in a concentration-dependent manner. Unlike the ¹²⁵I efflux, the ⁸⁶Rb⁺ efflux was sustained over time. Indeed, 4 min after the addition of TDCA, the ⁸⁶Rb⁺ efflux rate coefficients were at stable values of 0.045 ± 0.007, 0.088 ± 0.008, and 0.145 ± 0.007 min¹ for control, 0.75 mM, and 1 mM TDCA (P < 0.01 for 1 mM vs. control and for 1 mM vs. 0.75 mM; P < 0.05 for 0.75 mM vs. control, n = 3), respectively. This effect was also inhibited by 100 nM charybdotoxin, an inhibitor of Ca²⁺-activated K⁺ channels, from a efflux rate coefficient of 0.164 ± 0.009 to 0.049 ± 0.009 min¹ 4 min after the addition of 1 mM TDCA (P < 0.01, n = 3; Fig. 3B).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of TDCA on 86Rb+ efflux. 86Rb+ efflux from PDEC was determined as detailed in MATERIALS AND METHODS, and the efflux rate coefficient was calculated and shown. In A and B, means and SE from 3 experiments are shown. A: after 1 min of baseline determination, TDCA was added at the different final concentrations shown. B: after 1 min of baseline determination, 1 mM TDCA was added in the absence () or presence () of 100 nM charybdotoxin.

Ussing chamber studies. As previously reported, the cultured dog PDEC exhibit tight junctions, generating the high transepithelial monolayer electrical resistance necessary for studies in Ussing chambers (16, 17, 19). They are also polarized with apical and basolateral membranes that may differ in their ability to mediate the secretory effects of bile acids. We therefore compared the effects of TDCA added to either the luminal or serosal compartment of Ussing chambers.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of TDCA on net electrogenic ion transport by PDEC monolayers. Monolayers of confluent PDEC were mounted in Ussing chambers, as described in MATERIALS AND METHODS, and the short-circuit current (Isc) was measured at 10-s intervals. After ~5 min of baseline determination, TDCA was added at the different final concentrations shown. The Isc increases shown (calculated by subtracting the Isc recorded just before the addition of TDCA) were obtained from monolayers cultured and studied at the same time. A: TDCA was added to the serosal compartment in contact with the basolateral membrane of the PDEC. These tracings are representative of 3 experiments, and the average peak Isc increases (highest Isc within 10 min of TDCA addition  Isc just before addition of TDCA) are shown in the inset [significant differences from 0 (), 0.2 (#), 0.4 (@), and 0.6 (&) mM, P < 0.05 by ANOVA]. B: TDCA was added to the luminal compartment in contact with the apical membrane of the PDEC. These tracings are representative of 5 experiments, and the average peak Isc increases (highest Isc within 10 min of TDCA addition  Isc just before addition of TDCA) are shown in the inset [significant differences from 0 (), 1 (#), 1.25 (@), 1.5 (&), and 1.75 (*) mM, P < 0.05 by ANOVA].

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of TDCA on apical Cl channels. Monolayers of confluent PDEC, cultured and studied at the same time, were mounted in Ussing chambers, in the presence of a 135 mM serosal-to-luminal Cl gradient, and basolaterally permeabilized with 0.2 mg/ml nystatin for 15 min, as described in MATERIALS AND METHODS. A: monolayer pretreated for 30 min with 50 µM BAPTA-AM. B: monolayer pretreated for 30 min with 500 µM DIDS. After ~5 min of baseline determination, 1 mM TDCA was added to the serosal compartment. The Isc was measured at 10-s intervals, and the Isc increases were calculated by subtracting the Isc recorded just before TDCA addition. The tracings displayed are representative of 3 experiments, and the average peak Isc increases (highest Isc within 10 min of TDCA addition  Isc just before addition of TDCA) from these experiments are shown in the insets. *Significant difference between Isc increases in the presence or absence of either BAPTA-AM or DIDS (P < 0.05 by unpaired 1-tailed t-test).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of TDCA on basolateral K+ channels. Monolayers of confluent PDEC, cultured and studied at the same time, were mounted in Ussing chambers in the presence of a 114 mM luminal-to-serosal K+ gradient and apically permeabilized with 0.2 mg/ml nystatin for 15 min as described in MATERIALS AND METHODS. A: monolayer pretreated for 30 min with 50 µM BAPTA-AM. B: monolayer pretreated for 5 min with 100 nM charybdotoxin. After ~5 min of baseline determination, 1 mM TDCA was added to the serosal compartment. The Isc was measured at 10-s intervals, and the Isc increases were calculated by subtracting the Isc recorded just before TDCA addition. The tracings displayed are representative of 3 experiments, and the average peak Isc increases (highest Isc within 10 min of TDCA addition  Isc just before addition of TDCA) from these experiments are shown in the inset. *Significant difference between Isc increases in the presence and absence of either BAPTA-AM or charybdotoxin (P < 0.05 by unpaired 2-tailed t-test).

Measurements of TER. TER is another electrophysiological property of PDEC monolayers that may be affected by bile acids. Figure 7 shows that the effects of TDCA on monolayer TER also differ according to the side of exposure. From the serosal compartment (Fig. 7A), transient treatments with 1 mM TDCA (2, 5, and 10 min) only produced temporary TER decreases of up to 12 ± 2% (n = 6), resolving when TDCA was removed (15-min time point). However, 24 h after treatment, TER was only 40-45% of the initial value. Continuous exposure to 1 mM TDCA resulted in a marked TER decrease of 95% after 24 h. When PDEC were exposed serosally to 2 mM TDCA, the resulting decrease in TER increased with time. Indeed, after only 10 min of TDCA exposure, the TER was 22 ± 3% of its original value; after 24 h, this value decreased to 5%.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effects of TDCA on transepithelial resistance (TER) of PDEC monolayers. PDEC monolayers were either untreated (control) or treated with 1, 2, 3, or 4 mM TDCA added to either the serosal (A) or luminal (B) compartment for 2 min, 5 min, 10 min, or 24 h. Monolayer TERs were measured, as described in MATERIALS AND METHODS, before treatment, at the end of treatment (or at 10 min for the control and 24-h exposures), at 15 min, and at 24 h. The mean ± SE of the TERs, normalized to the initial value before treatment, are shown (n = 6).  Statistical difference from 100% (P < 0.05, t-test, 5 degrees of freedom). For the serosal exposure, the initial TERs were, respectively, 706 ± 13, 604 ± 9, 592 ± 19, 653 ± 24, 796 ± 29, 621 ± 55, 711 ± 67, 693 ± 94, and 757 ± 109  · cm2 for the control, 1 mM/2 min, 1 mM/5 min, 1 mM/10 min, 1 mM/24 h, 2 mM/2 min, 2 mM/5 min, 2 mM/10 min, and 2 mM/24 h treatments. For the luminal exposure, the initial TERs were, respectively, 839 ± 35, 861 ± 58, 849 ± 67, 807 ± 43, 860 ± 44, 929 ± 90, 904 ± 112, 929 ± 142, 917 ± 130, 901 ± 119, 875 ± 145, 943 ± 149, and 907 ± 160  · cm2 for the control, 2 mM/2 min, 2 mM/5 min, 2 mM/10 min, 2 mM/24 h, 3 mM/2 min, 3 mM/5 min, 3 mM/10 min, 3 mM/24 h, 4 mM/2 min, 4 mM/5 min, 4 mM/10 min, and 4 mM/24 h treatments.

Cellular LDH release. Because bile acids are detergents, the toxicity of these bile acids against PDEC was also evaluated by monitoring LDH release from cells exposed to bile acids. As shown in Fig. 8, after treatment with 0.5 and 1 mM TDCA for 5 min, minimal LDH was released in the supernatant, similar to the control treatment (LDH activity in arbitrary units; control: 120 ± 20.8, 1 mM TDCA: 96.7 ± 28.5, n = 3). A significant increase in LDH was released in the supernatant with 2 mM TDCA; with 5 mM TDCA, the amount of LDH released was equivalent to the amount released with 1 mM Triton X-100, used as positive control (LDH activity: 2 mM TDCA, 893 ± 91; 5 mM TDCA, 8,803 ± 2,112; and Triton X-100, 7,316 ± 1,839).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Lactate dehydrogenase (LDH) leakage from PDEC treated with TDCA. PDEC were treated with different concentrations of TDCA or with 1% Triton X-100, and the LDH released into the supernatant was assayed as described in MATERIALS AND METHODS. The means and SE from 3 determinations are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

PDEC mediate the secretion of fluid and electrolytes (mainly bicarbonate), one of the two main functions of the exocrine pancreas. Because these cells are exposed to refluxed bile acids after ampullary obstruction and because bile acids stimulate secretion from colonic and biliary epithelia, we examined the secretory effects of bile acids on PDEC. Nontransformed, well-differentiated, and polarized cultured dog PDEC served as the model since they were used successfully to characterize the secretory effects of histamine (acting via H₁ receptors), UTP and ATP (via P2Y₂ and P2Y₁₁ receptors), and trypsin (via proteinase-activated receptor-2; see Refs. 15, 17, 18, and 19). In addition, these PDEC are derived from the main pancreatic duct of the dog, the first pancreatic structure exposed to refluxed bile.

In this model, TDCA activated both Cl and K⁺ conductances. Indeed, it stimulated an increased ¹²⁵I efflux, inhibited by the Cl channel blockers NPPB or DIDS, and a ⁸⁶Rb⁺ efflux inhibited by the K⁺ channel blocker charybdotoxin. When PDEC were examined in Ussing chambers, TDCA also stimulated an I_sc increase from basolaterally permeabilized cells subject to a serosal-to-luminal Cl gradient and from apically permeabilized cells subject to a luminal-to-serosal K⁺ gradient. These I_sc increases were also inhibited, respectively, by the Cl channel inhibitors NPPB and DIDS and the K⁺ channel inhibitor charybdotoxin. As previously reported, these efflux and I_sc responses are characteristic of those mediated by the apical Ca²⁺-activated Cl channels and basolateral Ca²⁺-activated K⁺ channels (14, 16). Inhibition by BAPTA-AM, a cell-permeant Ca²⁺ chelator, of TDCA-stimulated ¹²⁵I efflux and I_sc increases from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl gradient and apically permeabilized PDEC subject to a luminal-to-serosal K⁺ gradient further verifies that activation of the Cl and K⁺ channels is mediated through an increase in [Ca²⁺]_i. A toxic effect of TDCA at the concentrations tested was excluded by the absence of increased LDH leakage after exposure to 1 mM TDCA. These findings are consistent with the activation, through increased [Ca²⁺]_i, by similar concentrations of bile acids of K⁺ and/or Cl conductances on colonic T84 cells and biliary cells and the stimulation by tauroursodeoxycholic acid of exocytosis in hepatocytes (6, 8, 9, 22).

PDEC polarization and the presence of monolayer resistance allowed us to examine the polarity of TDCA effects. At 1 mM, only serosal, but not luminal, TDCA stimulated an I_sc increase from intact monolayers; a higher concentration (2 mM) was required to produce a luminal effect. In T84 cells, <1 mM TDCA also produced only a serosal effect, and 1 mM TDCA was required for luminal action. It was postulated that the higher luminal concentrations of bile acids disrupted T84 cell monolayer integrity, allowing bile acids to reach the basolateral membrane and stimulate secretion (9). In this report, it is possible that TDCA interacts directly with the apical membrane of PDEC to stimulate secretion. Indeed, 2 mM luminal TDCA decreased monolayer TER by only 15-20%, which may be inadequate for rapid TDCA leakage in the serosal compartment. Furthermore, the onsets of action after serosal and luminal TDCA addition were similar, without the delay expected for transepithelial leakage.

The secretory effects of the different bile acid types were examined. Because biliary bile acids are also conjugated to glycine, we demonstrated that 1 mM glycodeoxycholic acid also stimulated an increase in ¹²⁵I efflux, albeit of smaller amplitude. The effects of cholic acid and chenodeoxycholic acid, which, together with deoxycholic acid, account for 90% of biliary bile acids, were also evaluated. Conjugated to taurine and tested at 1 mM, chenodeoxycholic acid and deoxycholic acid were equivalent in their secretory response; in contrast, cholic acid did not produce a significant response. Of note, taurocholic acid also did not activate conductances in T84 cells (8). Because taurocholic acid, like TDCA, is a strong detergent, its lack of activity further argues against the secretory effect of TDCA resulting from a nonspecific detergent action. Structural differences may account for these different effects as follows: both chenodeoxycholic acid and deoxycholic acid are dihydroxylated (at positions 3 and 7 for chenodeoxycholic acid and at positions 3 and 12 for deoxycholic acid), whereas cholic acid is trihydroxylated (at positions 3, 7, and 12).

The effects of TDCA on PDEC TER are consistent with the previous findings that bile acids at concentrations >1 mM increased the permeability of bovine pancreatic duct explants (2) and of the main pancreatic duct in different animal models of bile-induced pancreatitis (4, 5, 10). Again, higher luminal TDCA concentrations were also necessary to affect monolayer TER. Marked immediate and delayed decreases in TER followed transient treatment with 2 mM serosal TDCA and with 4 mM luminal TDCA. Of note, transient exposure to 1 mM serosal TDCA caused delayed TER decreases after 24 h, suggesting that serosal bile acids produce long-lasting effects. On the other hand, transient exposure to 2-3 mM luminal TDCA only caused a transient TER decreases, with full recovery after 24 h. The PDEC used in this study are well differentiated and polarized, with apical and basolateral membranes differentially exhibiting receptors for ATP, UTP, histamine, and trypsin (15, 17, 18, 19). The differential effects of luminal and serosal TDCA most likely reflect different interaction with apical and basolateral membranes.

Because serum concentrations of bile acids are in the micromolar range, it is unlikely that systemic bile acids will cause any serosal effect on PDEC under physiological conditions. On the other hand, our findings may be relevant to gallstone pancreatitis in which refluxed bile may yield conjugated bile acid concentrations in pancreatic juice in excess of 2 mM. The stimulation of secretion by subtoxic concentrations of TDCA may be a defense mechanism for diluting and washing off refluxed bile acids before critically toxic concentrations are reached. The relative resistance of the apical membrane to the toxic effects of TDCA further illustrates the importance of the barrier function of the pancreatic ductal mucosa.

In summary, we have shown that dihydroxy bile acids activate Cl and K⁺ conductances on PDEC and at higher concentrations impair epithelial barrier function. These effects will be relevant to conditions associated with bile reflux and disruption of the pancreatic ductal epithelial barrier.