JOURNAL OF SURGICAL RESEARCH 74, 43–46 (1998)ARTICLE NO. JR975202

Acute Effects of Bile Acids on the Pancreatic Duct Epithelium in Vitro

Carlos Alvarez, M.D.,* Alessio Fasano, M.D.,† and Barbara L. Bass, M.D.*

*Surgical Service, Baltimore VAMC, Baltimore, Maryland 21201; Department of Surgery, University of Maryland; and†Department of Pediatrics, University of Maryland, Baltimore, Maryland

Presented at the Annual Symposium of the Association of Veterans Administration Surgeons, Louisville, Kentucky, May 5–7, 1997

possibility, that bile refluxes into and damages the pan-Background. Acute pancreatitis is associated with creas, has received extensive attention [3–7]. Injection

passage of gallstones, although the mechanism(s) link- of bile through the pancreatic duct causes damage toing the two processes remains undefined. Bile reflux the epithelial cells lining the ducts at the same timeinto the pancreatic duct could play a role but the ex- that it produces parenchymal injury. Thus, it has beenperimental conditions often employed to induce pan- postulated that the duct epithelium may serve as acreatitis rarely develop clinically. Here we examined protective barrier in a manner analogous to the gastro-whether low concentrations of bile affect ductal elec- duodenal mucosa [8, 9].trophysiology as an indirect measure of ductal epithe- We have previously shown that the duct epitheliumlial integrity and function in vitro. is highly sensitive to bile acids in vitro, at concentra-Methods. The main duct was dissected out of freshly

tions much lower than normally found in biliary andharvested bovine pancreata, cut into 1- 1 2-cm sec-duodenal fluids, and lower than those typically used totions, placed in tissue culture for 48–72 h, then placedstudy experimental pancreatitis in vivo [10]. The timein Ussing chambers. Changes in tissue resistance (Rt )course employed in that series of experiments exposedand short-circuit current (Isc) were monitored. The re-the duct epithelium to bile acid for 48 h, also an un-sponses to forskolin and bile (taurodeoxycholic acid,likely clinical scenario. We thus were interested in ex-TDCA) were examined separately and together.amining the more physiologically relevant effect ofResults. Forskolin (10 mM) produced a decrease inacute bile acid exposure on the duct epithelium. Thethe Isc without a significant change in Rt , suggesting aUssing chamber, which has been used extensively tosecretory response, followed by a return to baseline.

TDCA caused a similarly reversible decrease in the Isc study secretion in the gastrointestinal tract, has alsoat low doses, but a persistent drop at higher concentra- been used to monitor acute changes in epithelial integ-tions. A concurrent drop in Rt was noted at all TDCA rity and the technology has recently been applied toconcentrations, the duration of which correlated with the pancreatic duct epithelium [11–14]. Thus, it ap-dosage and degree of histological damage. Prior expo- peared as an excellent tool to define the effect of bilesure to low (0.5 mM) doses of TDCA significantly acids on the pancreatic duct epithelium isolated fromblunted the response to subsequent forskolin chal- other components of the exocrine pancreas.lenge.

Conclusions. Acute exposure to TDCA in vitro causesepithelial damage at levels lower than those normally METHODSused to induce experimental pancreatitis. At the lowerconcentrations, Rt returns to baseline rapidly, sug- Explant harvest and culture. Fresh whole bovine pancreas glandsgesting recovery (restitution) from epithelial damage were obtained from a local beef processing plant and transported on

ice in 2% antibiotic antimycotic solution in phosphate-buffered salinebut with a persistent loss of the response to forskolin.(PBS-AA). Using sterile technique, the proximal 5–6 cm of the mainReflux of minute amounts of bile into the pancreaticpancreatic duct was dissected free from the surrounding parenchymaduct could play a significant role in the pathogenesisand removed. The duct was then cut open lengthwise, divided intoof gallstone pancreatitis by uncoupling the normal1- 1 2-cm segments avoiding secondary branches and extensively

stimulus-secretion apparatus of the ductal system and washed in cold PBS-AA. The segments of ducts (explants) were thenbreaking down the epithelial barrier. q 1998 Academic Press placed individually on gelatin (Gelfoam, Upjohn) ‘‘boats’’ in plastic

Key Words: pancreatic ducts; bile acids; epithelial tissue culture dishes. Waymouth’s culture media (Sigma) containing5% fetal bovine serum (GIBCO), 2% antibiotic antimycotic (GIBCO),cell; cell culture.and 0.1 mg/ml soybean trypsin inhibitor (Sigma) was added and theexplants were incubated at 377C for 48 to 72 h in humidified 95%O2/5% CO2. The medium was replaced every 24 h. The 48-h minimumperiod of recovery was prompted by significant duct epithelial abnor-The close relationship between passage of a gallstonemalities and occasional cell sloughing noted in the freshly harvestedand the development of acute pancreatitis has been tissue, probably the result of ischemia and trauma from handling and

known for nearly 100 years [1], yet the mechanisms dissection. By 48 h, the normal epithelial appearance had returned inmost explants.underlying this association remain obscure [2]. One

43 0022-4804/98 $25.00Copyright q 1998 by Academic Press

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44 JOURNAL OF SURGICAL RESEARCH: VOL. 74, NO. 1, JANUARY 1998

the absence of any intervention, the preparation re-mained stable for up to 1 h (n Å 6). Addition of 1 mMTDCA to the apical surface caused a brief decrease inthe Isc , which did not achieve statistical significancecompared to the controls (n Å 8). There was no changein the Rt at this TDCA dose. In contrast, adding 5 mMTDCA to the apical side lead to a prompt and substan-tial decline in both the Rt and the Isc (n Å 6). Thisresponse is persistent beyond 3 h (data not shown).

Histological examination of the explants after bile ex-posure, as shown in Fig. 3, demonstrated that, comparedto control specimens (Fig. 3A) which maintained a cuboi-dal appearance typical of the duct epithelium, 1 mMTDCA (Fig. 3C) led to flattening of the epithelial cellsFIG. 1. Effect of 10 mM forskolin on pancreatic duct explant epi-

thelial Rt and Isc . Note drop in the Isc without an associated drop in but no significant cell loss, so that the integrity of thethe Rt , suggesting secretion of anions into the luminal space (*P õ epithelium was maintained. On the other hand, exposure0.05 vs t0). of the apical surface of the explants to 5 mM TDCA (Fig.

3D) produced extensive abnormalities in the epithelialUssing studies. The explants were mounted on Ussing chambersappearance, including considerable cell sloughing.with an exposed membrane diameter of 0.67 cm2 (World Precision

Instruments). Both sides of the chamber were filled with standard As expected, with exposure to 0.5 mM TDCA thereRinger’s solution (in mM: 53 NaCl, 5 KCl, 30.5 Na2SO4, 30.5 manni- was less histological evidence of epithelial injury thantol, 30.5 Na2HPO4, 1.69 NaH2PO4, 0.3 Na2HPO4, 1.25 CaCl2, 25 with higher doses (Fig. 3B). Nevertheless, at this doseNaHCO3, and 10 D-glucose) at pH 7.4, warmed at 377C, and bubbled

there was a short-lived decrease in both the Rt and thecontinuously with 95% O2/5% CO2. The fluid resistance was calcu-Isc , the latter closely resembling the response of thelated prior to tissue mounting and corrected for during the remainder

of each experiment. The transepithelial open-circuit potential differ- same explants to 10 mM forskolin (Fig. 4). Despite re-ence (V0) was monitored continuously under current clamp condi- turn to baseline values and a nearly normal histologicaltions. A 100 mA/cm2 current was passed at various time points and appearance, the tissue remained insensitive to for-the resulting change in potential difference (V100) was used to calcu-

skolin stimulation, even at double the earlier dose.late the electrical resistance of the tissue (Rt) from the relationship,Rt Å (V100 0 V0)/I. The short-circuit current was determined fromthe equation, Isc Å V0/Rt , as the amount of external current required DISCUSSIONto neutralize V0 .

Experimental conditions. The V0 and baseline Rt achieved stableAlthough the factors associated with the develop-values after 30 min in the chambers. At that point, experimental

conditions were established. The adenyl cyclase activator forskolin ment of acute pancreatitis are well-established, little(Sigma) was dissolved in DMSO and taurodeoxycholic acid (TDCA, progress has been made in understanding the determi-Sigma) was dissolved in Ringer’s solution. Both agents were added nants of disease development, of an individual’s suscep-in 10- 20-mL aliquots of their concentrated stock solutions to their

tibility, and of the typically variable course of the dis-respective 10-ml chamber, forskolin to the basolateral and TDCAease. Although pancreatitis associated with gallstonesto the apical. For examination of the interaction between forskolin

stimulation and TDCA exposure, forskolin (10 mM) was added to thebasolateral chamber, the response was observed for 20 min, and thenboth chambers were rinsed and fresh Ringer’s was added. After asecond 30-min period of stabilization, 0.5 mM TDCA was added tothe apical side, followed at 20 min by a second dose of forskolin (20mM) added to the basolateral chamber. The explants were then fixedin 4% paraformaldehyde overnight, paraffin-embedded, cut andstained with hematoxylin and eosin for microscopic evaluation ofepithelial damage.

Data analysis. Results are reported as percentage change({SEM) in Rt and Isc calculated from the change in each parameterfrom baseline values at the time of each intervention. Comparisonsbetween groups were made with analysis of variance and withingroups by paired t test (Prizm 2.01, GraphPad Software, Inc.). Statis-tical significance was accepted at P õ 0.05.

RESULTS

Baseline mean V0 was 00.66 { 0.13 mV, Rt 31.3 {2.7 V/cm2 and Isc was 025.3 { 8.0 mA/cm2. Explantswith baseline Rt lower than 10 V/cm2 were discarded,since these values accurately predicted loss of the epi-

FIG. 2. Effect of increasing doses of TDCA on pancreatic ductthelial lining as demonstrated by histology. Forskolinexplant epithelial Rt and Isc . Note steep and persistent drop in thecaused a further reduction in the Isc , which peaked at Rt with 5 mM TDCA, associated with a drop in the Isc , suggesting

18 min at 66 { 44%, without a significant change in epithelial barrier loss confirmed by histology (Fig. 3, *P õ 0.01 vsboth control and 1 mM TDCA, # P õ 0.01 vs control).the Rt (Fig. 1, n Å 13). Figure 2 demonstrates that in

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45ALVAREZ, FASANO, AND BASS: BILE INJURY IN THE PANCREATIC DUCT EPITHELIUM

ithelial damage result [3–8]. Nevertheless, detractorshave pointed out correctly that the conditions necessaryto demonstrate reflux or cause pancreatic injury (highpressures and bile concentrations, infected bile, activatedpancreatic enzymes, etc.) are somewhat artificial [3].

In our earlier series of experiments we showed thatexposure to concentrations of TDCA as low as 0.05 mMfor 48 h was able to cause noticeable damage to theduct epithelium in vitro [10]. This suggested that it isnot necessary to have massive degree of bile reflux toinjure the duct epithelium. Here we have documentedthat higher doses (5 mM) of bile acid are neverthelessnecessary to cause irreversible breakdown in the epi-thelial barrier as shown by concomitant loss of both Rt

and the Isc . However, at a lower dose, 1 mM TDCA, theelectrophysiological parameters suffer only brief de-clines before returning to normal. This correlates withepithelial cell flattening observed on histology whichwe believe represents spreading of the cells to maintainepithelial integrity (Fig. 3C). This phenomenon ofrapid reepithelialization of damaged mucosal surfaces,termed restitution, has been well documented in othergastrointestinal tissues [13, 16, 17]. If restitution doesoccur in the pancreatic duct, pharmacologic interven-tions to optimize the process could benefit to patientswith acute pancreatitis as severe disease is often asso-ciated with breakdown in ductal integrity [18–20].

Much of the focus on the ductal epithelium with re-gards to acute pancreatitis has fallen on its barrierfunction. Alternatively, the pancreatic ducts may bethought of simply as a conduit for the inactive digestiveenzymes produced by acinar cells. The discovery thatcystic fibrosis, which often leads to pancreatic exocrineinsufficiency and fibrosis, is caused by mutations in agene which codes for a duct cell chloride channel pro-

FIG. 3. Histological (H&E) sections of representative sections ofexplants after removal from the Ussing chamber. (All 1100.) (A)Control conditions, standard Ringer’s solution. Note normal cuboidalappearance of duct cells lining the epithelium. (B) Explant exposedto 0.5 mM TDCA. No significant evidence of epithelial injury. (C)Explant exposed to 1 mM TDCA. Note flattening of the cells but noloss of epithelial continuity. (D) Explant treated with 5 mM TDCA.Observe extensive loss of the epithelial cell lining, associated withpersistent decline in both Rt and Isc in the chambers.

has been convincingly shown to be caused by passageof a stone through the ampulla of Vater, considerabledisagreement exists as to whether obstruction of thepancreatic duct or reflux of bile into the duct is respon-sible for the development of pancreatitis [2, 15].

There is considerable evidence to support the refluxhypothesis. Reflux from the biliary to the pancreatic tree

FIG. 4. Effect of bile exposure on Rt and Isc of pancreatic ductcan be demonstrated in the majority of patients withexplant epithelium. Note brief drop in Rt after bile exposure and lossgallstone pancreatitis by antegrade cholangiography. of the Isc response to forskolin after explants are treated with 0.5

When bile is introduced into the pancreatic duct under mM TDCA (*P õ 0.01 vs 10 mM forskolin, #P õ 0.05 vs 10 mMforskolin).experimental conditions, both pancreatitis and ductal ep-

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