Abstract
Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.
Highlights
Acute pancreatitis is a complex inflammatory disease of the pancreas that is of uncertain pathogenesis
As had been observed following infusion of sodium taurocholate, retrograde ductal infusion of taurolithocholic acid 3-sulfate disodium salt (TLCS) (50 l, 3 mM (i.e. 0.18%)) resulted in hyperamylasemia, pancreatic edema, and extensive acinar cell injury/ necrosis, but Protease-activated receptor-2 (PAR2) deletion markedly reduced the extent of each of these features of pancreatic injury. In contrast to these observations noted in the bile salt and secretagogue-induced models of pancreatitis, no change in the mortality rate of choline deficient diet-induced pancreatitis was associated with genetic deletion of PAR2
These observations replicate the pattern of response observed. Both Namkung et al [6] and our group [7] have shown that the severity of pancreatic injury in caerulein-induced pancreatitis is reduced in rats or mice that have been pretreated with the PAR2-activating peptide SLIGRL
Summary
Experimental Animals—All experiments were performed using non-sex-selected wild type C57/Bl/6 mice (20 –30 g) purchased from Charles River Laboratories (Wilmington, MA) and 20 –30-g PAR2Ϫ/Ϫ mice, of either sex, that had been bred from founder C57/Bl/6 knock-out animals kindly donated by Dr P. Diet-induced pancreatitis was elicited by feeding female mice (13–15 g) a choline-deficient 0.5% ethionine-supplemented diet (3 g diet/24 h) for 72 h preceded by a 24-h period of fasting as described previously [12]. They were given regular diet ad libitum and observed for an additional 3 days. Measurement of Calcium Transients within Single Acinar Cells—Freshly prepared small pancreatic acini (1–10 cells per cluster) were pre-loaded with 1 M Fura-2/AM in the presence of 0.0005% Pluronic F-127 for 10 min at room temperature followed by extensive washing, and calcium transients in single cells within the acinar cluster were monitored using a Nikon Eclipse TEU 2000 inverted microscope as described previously [13]. A p value of Յ0.0083, which is the Bonferroni adjusted p-value to control the experimentwise alpha at 0.05 when there are six comparisons, was considered to indicate significant difference
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