Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1

Abstract

Background & aims: The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G-protein-coupled, cell surface bile acid receptor.

Methods: Acute pancreatitis was induced in wild-type and Gpbar1(-/-) mice by either retrograde ductal infusion of taurolithocholic acid-3-sulfate (TLCS) or supramaximal secretagogue stimulation with caerulein. In vitro experiments were performed in which acini obtained from wild-type and Gpbar1(-/-) mice were exposed to either submicellar concentrations of TLCS (200-500 microM) or a supramaximally stimulating concentration of caerulein (10 nM).

Results: Gpbar1 is expressed at the apical pole of acinar cells and its genetic deletion is associated with reduced hyperamylasemia, edema, inflammation, and acinar cell injury in TLCS-induced, but not caerulein-induced, pancreatitis. In vitro, genetic deletion of Gpbar1 is associated with markedly reduced generation of pathological calcium transients, intracellular activation of digestive zymogens, and cell injury when these responses are induced by exposure to TLCS, but not when they are induced by exposure to caerulein.

Conclusions: Gpbar1 may play a critical role in the evolution of bile-acid-induced pancreatitis by coupling exposure to bile acids with generation of pathological intracellular calcium transients, intra-acinar cell zymogen activation, and acinar cell injury. Acute biliary pancreatitis may be a "receptor-mediated" disease and interventions that interfere with Gpbar1 function might prove beneficial in the treatment and/or prevention of biliary acute pancreatitis.

Figure 1. Gpbar1 is expressed in the mouse pancreas

Gpbar1 expression in the pancreas of wild type and Gpbar1^−/− mice was evaluated by rt-PCR (Panel A), and immunofluorescence microscopy (Panel B) as described in the text. Note intense anti-Gpbar1 antibody fluorescence at the apical pole of wild type acinar cells which is adjacent but not identically localized to the sites occupied by f-actin (Panel B, overlay) and absence of Gpbar1 expression in Gpbar1^−/− pancreas (Panels A, B).

FIGURE 2. Genetic deletion of Gpbar1 reduces the severity of TLCS- but not caerulein-induced pancreatitis

Acute pancreatitis was induced, in wild type and Gpbar1^−/− mice either by retrograde ductal infusion with 3 mM TLCS (Panel A) or supramaximal stimulation with repeated administration of 50 μg/kg/injection of caerulein (Panel B) as described in the text. Twenty-four hours after the start of pancreatitis-induction, the animals were sacrificed and pancreatitis severity was evaluated as described in the text. Solid bars report results obtained using wild type mice while open bars report results from Gpbar1^−/− animals. Asterisks denote significant differences in bracketed groups in which results from wild type and Gpbar1^−/− mice are compared. NS = not significant.

Figure 3. Genetic deletion of Gpbar1 alters frequency with which pathological calcium transients are observed after exposure of cells to TLCS but not after exposure of cells to a supramaximally stimulating concentration of caerulein

Small pancreatic acini from wild type and Gpbar1^−/− mice were exposed to 200 μM and 500 μM TLCS or to 10 nM caerulein and calcium transients were monitored in individual acinar cells as described in the text. Panel A shows tracings that are representative of the 4 patterns as described in the text. Long arrows denote time of TLCS addition while short arrows denote time of caerulein addition. This latter addition was used to confirm maintenance of cell viability and functionality throughout the experiment. Panel B reports frequency with which each response pattern was observed after exposure of wild type or Gpbar1 ^−/− acini to either TLCS or caerulein. Asterisks denote significant differences (p<0.01) when Gpbar1^−/− acini are compared to wild type acini. NS = not significant.

Figure 4. Genetic deletion of Gpbar1 prevents intra-acinar cell activation of trypsinogen and chymotrypsinogen after exposure to TLCS but not after exposure to caerulein

Large pancreatic acini from wild type (solid black columns) and Gpbar1^−/− (open columns) mice were exposed to either 500 μM TLCS or 10 nM caerulein. Trypsinogen and chymotrypsinogen activation 15 and 30 min later, respectively, was quantitated as described in the text. Asterisks denote significant differences (p<0.05) between bracketed groups. NS = not significant.

Figure 5. Genetic deletion of Gpbar1 prevents acinar cell injury induced by TLCS but not caerulein

Large pancreatic acini from wild type (wt) and Gpbar1^−/− mice were exposed to either 500 μM TLCS or 10 nM caerulein. LDH leakage from the acini was quantitated as described in the text. Results reflect net LDH leakage, expressed as a percent of total LDH content, after subtracting leakage noted in the absence of both TLCS and caerulein. That subtracted value was 11.6 +/− 0.9% and 12.8 +/− 0.6 % of total LDH content for wild type and Gpbar1^−/− acini, respectively. Asterisks denote significant differences when results obtained with Gpbar1^−/− mice are compared to those obtained with wild type acini.

Figure 6. TLCS- and caerulein-induced calcium-dependent acinar cell discharge of amylase is not altered by genetic deletion of Gpbar1

Large pancreatic acini from wild type (left plots) and Gpbar1^−/− (right plots) mice were exposed to varying concentrations of either TLCS (upper plots) or caerulein (lower plots). Others (hatched columns) were pre-loaded with BAPTA and similarly treated. Amylase discharge, over 15 min, was measured as described in the text. Asterisks denote significant differences when acini pre-loaded with BAPTA are compared with those not pre-loaded with BAPTA or when acini exposed to TLCS were compared to those not exposed to TLCS.