Selective Activation Of Cannabimoid Receptor Subtype2（CB2R） Eliminates Agonist-induced Ca²⁺ Oscillations In Pancreatic Acinar Cells:a Novel Cellular Mechanism For Acute Pancreatitis Therapeutics

Backgoroud:Acute pancreatitis is an inflammatory disease of the pancreas, which has several causes and symptoms and requires immediate medical attention. It occurs when pancreatic pro-enzymes (especially trypsin) secreted from acinar cells that digest food are activated in the pancreas instead of the small intestine and consequently cause autodigestion. In clinical practice, there are still no efficient drugs that specifically treat acute pancreatitis. Emerging lines of evidence demonstrate that the primary event initiating the process of acute pancreatitis is the excessive release of Ca2+from intracellular stores. These studies provide a promising therapeutic strategy that the blockade of Ca2+release-activated Ca2+currents in pancreatic acinar cells may provide protection against overload of intracellular Ca2+, protease activation and necrosis, which are the major triggers of acute pancreatitis. The cannabinoid receptor type2,(CB2R) is a G protein-coupled receptor from the cannabinoid receptor family that in humans is encoded by the CNR2gene. CB2R agonists can be useful for treatment of inflammation and pain. Therefore, it is possible that the activation of CB2Rs modulates intracellular Ca2+signals, and treating acute pancreatitis.Methods:(1) Single pancreatic cell dissociationSingle pancreatic cells were dissociated as previous reports. In short, adult wild-type mouse was anesthetized with isoflurane, the pancreatic glands was removed quickly and injected with collagenase (150-200U/ml) before it was placed in a small tube full of the same collagenase. Then the tube was put in a shaker for digestion (20-25min,37℃). At the end of collagenase digestion, the tube was shaked with the hand for30seconds and the cell suspension was transferred to another tube, and the cells were then washed three times gently using standard external solution. Thereafter, a150μl aliquot of suspension was added into a35-mm culture dish containing standard external solution. The isolated cells were usually adhered to the bottom within15min, and these cells were used within3h. All experiments were performed at room temperature21＋1℃(2) Patch-clamp whole-cell recording and perforated patch recordingCa2+-activated Cl-currents were recorded using conventional whole-cell recordings or perforated patch recording to monitor intracellular Ca2+signal oscillations as previously reported. To prepare perforated-patch recording, perforating drug (amphotericin B) was dissolved in the standard pipette solution (150-200μg/ml). When a tight seal (>1GΩ.) was formed between the electrode tip and the cell surface, the perforated process starts, and it usually takes1-5min to form the perforated whole-cel configuration. The hold potential (VH) for voltage-clamp recording was-30mV. Transmembrane currents were recorded with a patch-clamp amplifier.(3) Immunocytochemistry:Immunohistochemistry (IHC) AssaysCB2antibodies (CB2-Abs):mCB2-Ab (1:250) purchased from Alomone (ACR-002, Jerusalem, Israel), which recognizes the intracellular3rd loop, was used to identify CB2R protein expression in isolated pancreatic acinar cells. The secondary antibodies was goat anti-rabbit Alexa488for CB2receptors (1:500) in5%bovine serum albumin and0.5%Triton X-100phosphate buffer for2hr at room temperature. Cells were then washed, mounted, and cover slipped. Fluorescent images were taken with a fluorescence microscope (Nikon Eclipse80i) or confocal microscope (Nikon Eclipse Cl)(4) Real-time PCRReal-time PCR was used to test the expression of CBR mRNA on pancratic acinar cells. The probes and primers were purchased from Applied Biosystems (Foster City, CA, USA). The qRT-PCR reactions were performed using a QIAGEN OneStep(?) RT-PCR kit (Category number:210212, QIAGEN Inc., Valencia, CA, USA). (5) Confocal imagingConfocal imaging was used to test Calcium oscillaition on pancreatic acinar cells which were incubated with dye Fluo-4for20min. The Ca2+signals were collected by a continuous two-dimensional scanning (XYT) style.(6) Experimental animalsWild-type mice were grouped into control group, model group and GW group, blood, lung and panceas were collected after mice was injected with drugs for24hours. Blood was used to test AMS, lung was used to test MPO. Part of pancreas was used to test pathogegesis, and another part was used to test calcium oscillation with Confocal imaging. Both CB1R KO and CB2R KO were also used to confirm the effects of GW that is mediated via CB2Rs.(7)StatisticsFor patch-clamp data, the net charge of Cl" current (current area divided by cell capacitance) was normalized and the nomarlized current net charges were compared before, during and after CB2R ligands. If data come from the same cell, paired-sample t-test was used to analyze datas; if the multiple droups of data are compared (e.g., different concentrations of GW), one-way ANOVA was used. Results(1) Effects of CB receptor agonists on agonist-induced Ca2+oscillationsBath-application of10nM ACh induced Ca2+oscillations measured by Ca2+-dependent Cl-current using patch-clamp whole-cell recording. In the continuous presence of10nM ACh, exposure of10μM GW reduced the Ca2+oscillations, which was reversible after washout of GW. The similar effect was also observed in100nM ACh-induced Ca2+oscillations using confocal Ca2+imaging.(2) GW eliminated ACh-induced Ca2+oscillations in a concentration dependent mannerIncrease od GW concentrations induced more inhibition of Ca2+oscillations. Statistic analysis showed that the normalized net charges of Ca2+oscillation were reduced to88+15%(n=6, p>0.05),51+4.4%(p<0.01, n=8) and12+1.1%of control after exposure to GW1,10and100μM..(3) GW eliminated ACh-induced Ca2+oscillations through CB2RsThe results demonstrated that co-application of AM630(0.1μM) and GW (10μM) abolished inhibitory effect of GW on10nM ACh-induced Ca2+oscillations, while AM630alone (0.1μM) did not significantly affect these Ca2+oscillations. Further, we found that10μM GW-induced inhibition in30nM ACh-induced Ca2+oscillations were absent in CB2R, but not CB1R, KO mice Finally, we showed that the normalized,10nM ACh-induced Ca2+oscillation signals were no significant change by bath-application of CB1R agonist (10μM ACEA).(4) GW inhibited ACh-induced Ca2+oscillations through CB2Rs on cell surfaceWe added IP3(30μM) into pipette solution, and when the conventional whole-cell recording was formed, IP3was infused into the recorded cell and induced Ca2+oscillations. Under this condition (bypass muscurinic receptor), we examined the effects of GW on IP3-induced oscillations and did not find the inhibitory effect of GW on the Ca2+oscilltions induced by IP3. Then, we added GW (100μM) into recording electrode, but we did not find any detectable inhibition of internal GW on ACHh-induced oscillation. Finally, we intracellularly applied CB2R blocker (AM6301μM), and found that internal AM630failed to prevent bath-application of GW-induced enhancement of Ca2+oscillations.(5) Acute pancreatitis inducer, L-arginine potentiated ACh-induced Ca2+oscillationsIn single dissociated pancreatic acinar cells from normal mice, bath-application of L-arginine for10min remarkably enhanced ACh-induced Ca2+oscillations, and this effect was not reversible after washout for10min. In an in vivo model, the mice were treated with L-arginine, and ACh-induced Ca2+oscillations were measured in dissociated pancreatic acinar cells using confocal Ca2+imaging after L-arginine injection for24hours. Results showed that the average of intracellar Ca2+oscillations was significantly enhanced compared to the saline-treated mice at the same time points.(6)GW prevented both L-arginine-enhanced Ca2+oscillations and acinar cell pathogegesisA co-application of L-arginine with GW (10μM) abolished L-argininr-induced enhancement of Ca2+oscillations on single pancreatic acinar cells.In an in vivo experiment, pre-injection of GW prevented L-arginine-induced enhancement of Ca2+oscillations. In addition, in a L-arginine-induced acute pancreatitis mouse model, pre-treatment of GW eliminated AMS and MPO.(7) ImmunocytochemistryThe CB2are indeed expressed on isolated pancreatic acinar cells.(8)Expression of CB receptor mRNACB2is highly expressed in mouse acinar cells, almost as high as beta-actin. CB1is not in acinar cellsConclusion:· Both CB2R mRNA and protein are expressed in isolated mouse pancreatic acinar cells.· Selective activation of CB2Rs using GW inhibits ACh-induced Ca2+oscillations in a concentration-dependent manner, which is bloched by CB2R antagonist or absent in CB2R KO mice.· Functional CB2Rs are expressed on the surface, but not cytosolic aer of acinar cells.· Acute pancreatitis inducer, L-arginine, remarkably enhances ACh-induced Ca2+oscillations· CB2R agonist inhibits the enhanced effects of L-atginine on ACh-induced Ca2+oscillations,which demostrates that activtion of CB2R is a novel cellular mechanism for acute pancreatitis therapeutics.