Volume 126, Issue 7, Pages 1844-1859 (June 2004) Protease-activated receptor 2 exerts local protection and mediates some systemic complications in acute pancreatitis  Wan Namkung, Wonsun Han, Xiang Luo, Shmuel Muallem, Kyung Hee Cho, Kyung Hwan Kim, Min Goo Lee  Gastroenterology  Volume 126, Issue 7, Pages 1844-1859 (June 2004) DOI: 10.1053/j.gastro.2004.03.019

Figure 1 Expression of protease-activated receptors in the pancreas. (A) Isoform-specific RT-PCR was performed in isolated rat pancreatic acini. Messenger RNA samples from brain tissues were used as positive controls. All reactions were performed with the same amount of RNA (0.05 μg per reaction). (B) Proteins (100 μg) extracted from isolated pancreatic acini or CAPAN-1 cells were blotted with antibodies against PAR isoforms. M, base pair marker. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 2 Activation of Ca2+ signaling and the mitogen-activated protein kinase by PAR2 agonists in pancreatic acinar cells. (A-D) [Ca2+]i was measured in pancreatic acinar cells stimulated with the indicated PAR agonists. (E) The effect of PAR2 stimulation on the phosphorylation of ERK was measured by immunoblotting with anti-phospho-ERK antibodies. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 3 Effects of PAR2 activation on cell survival and HCO3− transport in pancreatic cells. (A) Cell survival was measured in primary cultures of pancreatic acini stimulated with the indicated PAR2-AP concentration by using a tetrazolium-based [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. (B) Effects of PAR2 activation on acinar cell death induced by low and high concentrations of bile acids. (C) The activity of luminal Cl−/HCO3− exchange was measured in monolayers of CAPAN-1 cells that originated from pancreatic ducts. A high concentration of K+ (100 mmol/L) was used to block Cl− transport by electrogenic or K+-coupled pathways. Basolateral application of PAR2-AP (100 μmol/L) stimulated luminal Cl−/HCO3− exchange. ∗P < 0.05. LM, luminal membrane; BLM, basolateral membrane. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 4 Effects of PAR2 activation on edema and cell damage in the cerulein-induced pancreatitis model. (A) Treatment protocols. PAR2-AP and cerulein were administered by intraperitoneal injections. Control rats received an equal volume (1 mL/kg) of saline. (B) Changes in pancreas wet weight relative to body weight. (C) Light microscopic images of pancreatic tissues stained with H&E. Well-preserved acinar and islet structures are observed in control and PAR2-AP-stimulated pancreata. Pancreas sections from cerulein-injected animals show interstitial edema, intracellular vacuoles, and dead cell debris (arrowheads). Note that pretreatment with PAR2-AP greatly decreased the cerulein-induced pathologic changes (right panel). (D) Electron microscopy (EM) images. Well-preserved intracellular structures are observed in control and PAR2-AP-stimulated pancreata. A canalicular structure can be seen in the PAR2-AP-treated section. Cerulein-treated samples show interstitial edema between the basolateral membranes (i) and intracellular vacuoles in the luminal area (v). Pretreatment with PAR2-AP decreased edema and vacuole formation (right panel). ∗∗P < 0.01. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 5 Measurement of trypsin levels in serum of rats with taurocholate-induced acute pancreatitis. Trypsin activity was measured in blood samples collected from the portal vein of control rats and of rats with taurocholate-induced pancreatitis by using a BzArgNap-based fluorometric assay. (A) Active trypsin concentration was measured in the absence of enterokinase treatment. Induction of pancreatitis (6 hours) significantly increased serum trypsin activity, and the value was not statistically different from that measured in serum collected from rats infused with exogenous trypsin 2 mg/kg. (B) Total trypsinogen concentration was estimated after treatment of blood samples with enterokinase (10 U per reaction; 45 minutes). ∗P < 0.05; ∗∗P < 0.01. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 6 Effects of PAR2 activation on monocytic THP-1 cell functions. (A and B) Effect of PAR2 stimulation on [Ca2+]i in THP-1 cells. Trypsin 30 nmol/L sufficiently increased [Ca2+]i (A). Repeated PAR2 stimulation desensitized the PAR2-activated Ca2+ signaling (B). (C) The effects of PAR2 stimulation on NF-κB activity were measured with the electrophoretic mobility shift assay method. Similar results were obtained in 3 separate experiments. (D) Semiquantitative measurement of IL-8 mRNA levels with RT-PCR. PAR2 stimulation increased IL-8 mRNA levels. (E) Measurements of IL-8 release by enzyme-linked immunosorbent assay. Stimulation with PAR2-AP (100 μmol/L) induced IL-8 release in THP-1 cells that was maximal after 12 hours of incubation. M, base pair marker. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 7 Effects of PAR2 activation on endothelial HUVEC cell functions. (A and B) Stimulation of PAR2 increased [Ca2+]i in HUVEC cells. Trypsin 30 nmol/L sufficiently increased [Ca2+]i (A). Repeated stimulation of PAR2 indicated that PAR2 did not undergo significant desensitization in HUVEC cells (B). (C and D) Generation of peroxynitrite, the combined product of nitric oxide and reactive oxygen species, was monitored with H2DCF fluorescence. (C) Representative confocal images of H2DCF fluorescence of control cells and cells treated with NG-nitro-l-arginine methyl ester (l-NAME) stimulated with PAR2-AP 100 μmol/L. (D) Traces of H2DCF fluorescence obtained in 4 separate experiments. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 8 Effects of PAR2 activation on arterial blood pressure. Arterial blood pressure was recorded on a polygraph through a cannula connected to the common carotid artery while PAR2-AP or trypsin was infused through the jugular vein. (A-C) Effects of trypsin infusion on arterial blood pressure. (A and B) Representative traces; (C) summary of results obtained with 6 control rats and 8 rats with taurocholate-induced pancreatitis. Infusion of trypsin evoked an instantaneous decrease in arterial blood pressure (BP), particularly in diastolic pressure. Note that the decrease in blood pressure induced by exogenous trypsin was much smaller in rats with acute pancreatitis. (D) Treatment of rats with PAR2-AP 1 mg/kg evoked changes in blood pressure similar to those measured with trypsin 2 mg/kg. ∗P < 0.05; ∗∗P < 0.01 vs. control. Systol, systolic blood pressure; diastol, diastolic blood pressure. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)

Figure 9 Maintained vasodilative responsiveness of rats with taurocholate-induced pancreatitis. (A) The effect of trypsin on arterial pressure was measured after treatment with 20 mg/kg of the serine protease inhibitor camostat. (B) A summary of 6 experiments with camostat pretreatment. The control values were taken from Figure 8C. (C) The vasodilator effect of carbachol (Cch) was measured in control rats and in rats with taurocholate-induced pancreatitis. (D) A summary of the response to Cch stimulation (n = 6 for each group). ∗P < 0.05; ∗∗P < 0.01 vs. control. BP, blood pressure; systol, systolic blood pressure; diastol, diastolic blood pressure. Gastroenterology 2004 126, 1844-1859DOI: (10.1053/j.gastro.2004.03.019)