Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis

Abstract

Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2(-/-) mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca(2+) concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.

Fig. 1.

P2pal-18S inhibits proteinase-activated receptor-2 (PAR2)-induced generation of pathological intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients in acinar cells. A: representative examples of the 3 [Ca²⁺]_i response patterns (no response, oscillatory response, and peak-plateau response) that can occur in freshly isolated pancreatic acinar cells that have been loaded with fura 2 and then exposed to various concentrations of the PAR2-activating peptide SLIGRL-NH₂. Arrows point to the time SLIGRL-NH₂ was added. B: P2pal-18S, but not a similar but inactive pepducin, reduces oscillatory responses triggered by exposure to low SLIGRL-NH₂ concentration ([SLIGRL-NH₂]). Freshly isolated, fura 2-loaded acini were preincubated with P2pal-18S (17 μM), P2pal-18GF (17 μM), or vehicle, and, 30 min later, they were exposed to 30 μM SLIGRL-NH₂. C: P2pal-18S reduces both oscillatory and peak-plateau responses triggered by high [SLIGRL-NH₂]. Conditions are as described in B, but [SLIGRL-NH₂] was 100 μM. [Ca²⁺]_i levels were monitored as described in the text. Data shown represent pooled results obtained from 3 or more independent experiments.

Fig. 2.

P2pal-18S inhibits SLIGRL, but not caerulein, or taurolithocholic acid 3-sulfate disodium salt (TLCS)-induced secretion of amylase from pancreatic acini. Freshly isolated pancreatic acini were preincubated with or without 17 μM P2pal-18S for 10 min. Caerulein (0.1 nM), TLCS (200 μM), or SLIGRL-NH₂ (100 or 1,000 μM) was then added, and the incubation was continued for an additional 30 min. The amylase in the supernatant was measured and is expressed as a percent of total. Results are averages of 4 experiments, each performed in duplicate. *P < 0.05.

Fig. 3.

P2pal-18S reduces bile acid-induced Ca²⁺ transients in acinar cells. A: P2pal-18S reduces peak-plateau responses triggered by exposure to 200 μM TLCS. B: P2pal-18S reduces peak-plateau responses triggered by exposure to 0.3% sodium taurocholate (NaT). C: in acini harvested from PAR2^−/− mice, P2pal-18S does not alter peak-plateau responses triggered by exposure to TLCS. Conditions are as described in the legend for Fig. 1B, but the indicated bile acid was used in place of [SLIGRL-NH₂]. [Ca²⁺]_i levels were monitored as described in the text. Data shown represent pooled results obtained from 3 or more independent experiments.

Fig. 4.

P2pal-18S worsens caerulein-induced pancreatitis. Mice were treated with either vehicle or P2pal-18S (10 mg/kg, sc). Later (30 min) they received the first of 12 hourly injections of caerulein. They were killed 12 h after the last caerulein injection. Photomicrographs are representative of hematoxylin and eosin (H&E)-stained samples obtained from animals in each group. Bar in photomicrograph equals 200 μm. Serum amylase activity, pancreatic edema (i.e., water content), pancreatic inflammation (i.e., myeloperoxidase activity), and acinar cell necrosis were quantitated as described in the text. Results shown represent mean ± SD values obtained from 5 mice in each group. *P < 0.05 when mice given P2pal-18S were compared with mice receiving vehicle.

Fig. 5.

P2pal-18S ameliorates NaT-induced pancreatitis. Mice were treated with the vehicle or P2pal-18S (10 mg/kg, sc). Later (30 min), pancreatitis was induced by retrograde ductal infusion of 2% NaT as described in the text. Mice were killed 20 h after the start of duct infusion, and pancreatitis severity was quantitated as described in the legend for Fig. 4. Photomicrographs are representative of H&E-stained samples obtained from animals in each group, and the bar equals 200 μm. Results obtained from vehicle-treated mice were compared with those obtained from P2pal-18S-treated mice. Results shown are mean ± SD values for 5 animals in each group. *P < 0.05.

Fig. 6.

P2pal-18S ameliorates TLCS-induced pancreatitis. Mice were treated with the vehicle or P2pal-18S (10 mg/kg, sc). Later (30 min), pancreatitis was induced by retrograde ductal infusion of 10 mM TLCS as described in the text. Mice were killed 20 h after the start of duct infusion, and pancreatitis severity was quantitated as described in the legend for Fig. 4. Photomicrographs are representative of H&E-stained samples obtained from animals in each group, and the bar equals 200 μm. Results obtained from vehicle-treated mice were compared with those obtained from P2pal-18S-treated mice. Results shown are mean ± SD values for 5 animals in each group. *P < 0.05.

Fig. 7.

P2pal-18S, administered before or shortly after pancreatitis induction, reduces pancreatitis severity. Biliary pancreatitis was induced by retrograde ductal infusion of 10 mM TLCS, whereas control animals received ductal infusion of saline and no TLCS. P2pal-18S (10 mg/kg, sc) was given 30 min before or either 2 or 5 h after bile acid infusion, whereas control animals received only pretreatment with vehicle. Pancreatic acinar cell injury/necrosis was quantitated morphometrically as the percent of acinar cells that morphologically appeared to be either dead or injured. Note that there is a significant reduction in cell injury in the pancreas from mice that were pretreated or treated 2 h after the pancreatic duct infusion, but there was no significant reduction in the cell injury in mice that were treated 5 h after pancreatic duct infusion. *P < 0.05 compared with the vehicle-treated group.

Fig. 8.

P2pal-18S protects isolated acinar cells from bile acid-induced, in vitro injury/death. Freshly isolated pancreatic acini were prepared as described in the text. P2pal-18S (17 μM) was added to the medium, and, 30 min later, either TLCS (200 or 500 μM) or NaT (0.2%) was added. The incubation was continued for an additional 3 h (TLCS) or 20 min (NaT). Propidium iodide and Hoechst 33342 were then added, and cell injury was quantitated as described in the text. Results shown are the averages of 3 independent experiments and reflect net percent bile acid-induced cell injury, i.e., after subtracting the amount of cell injury noted in cells incubated in the absence of bile acids. *P < 0.05 compared with injury noted in the absence of P2pal-18S treatment.

Fig. 9.

P2pal-18S does not reduce in vitro intracellular trypsinogen activation triggered by either caerulein or TLCS. Freshly isolated pancreatic acini were preincubated with or without 17 μM P2pal-18S for 30 min. Caerulein (10 or 100 nM), TLCS (200 or 500 μM), or buffer alone was then added, and the incubation was continued for an additional 30 min. The acini were then homogenized, and trypsin activity in the homogenate was quantitated as described in the text. Results shown are the averages of 3 experiments, each performed in duplicate. Results obtained for samples preincubated with or without P2pal-18S were compared, and no significant difference was noted.

Fig. 10.

P2pal-18S does not reduce in vivo bile salt-induced protease activation. Mice were pretreated with the vehicle or P2pal-18S (1 μg/kg, sc). Later (30 min), pancreatitis was induced by retrograde ductal infusion of TLCS as described in the text. After the end of the ductal infusion (5 min), the pancreas was removed, and trypsin and chymotrypsin activity was determined by means of a fluorogenic substrate. Results shown are the averages of 5 animals in each group. No difference was seen in the trypsin or chymotrypsin activity between the pretreated and untreated group.