Volume 122, Issue 7, Pages 1941-1953 (June 2002) Transporter-mediated bile acid uptake causes Ca2+-dependent cell death in rat pancreatic acinar cells  Joo Young Kim, Kyung Hwan Kim, Jin Ah Lee, Wan Namkung, An–Qiang Sun, Meena Ananthanarayanan, Frederick J. Suchy, Dong Min Shin, Shmuel Muallem, Min Goo Lee  Gastroenterology  Volume 122, Issue 7, Pages 1941-1953 (June 2002) DOI: 10.1053/gast.2002.33617 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Activation of CCE by low bile acid concentrations in pancreatic acinar cells. (A) Effect of conjugated bile acids on [Ca2+]i was measured in cells loaded with Fura-2 and superfused with Ca2+-containing or Ca2+-free solutions. A bile acid mixture (BA) was used to simulate the in vivo situation. BA at 0.01% concentration corresponds to approximately 200 μmol/L of total bile acids. (B) Effect of BA on CCE was measured with the Mn2+-quench technique. CCE was compared in control cells and cells treated with 0.01% BA for 5 minutes. In the right panels, acinar cells were treated with 100 μmol/L of the SERCA pump inhibitor BHQ to maximally activate CCE. *P < 0.05. (C) Effect of BA on cell structure and membrane integrity was examined by electron microscopy. Acinar cells were treated with various BA concentrations for 20 minutes, fixed, and processed for obtaining electron microscopic images as detailed in the Materials and Methods section. Note that at BA concentrations at 0.5%, signs of membrane damage can be observed (arrows). In contrast, the 0.01% BA concentration used in Ca2+ influx measurement did not cause any membrane damage. (D) LDH release from cells treated with the indicated concentrations of BA. Gastroenterology 2002 122, 1941-1953DOI: (10.1053/gast.2002.33617) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Induction of inflammatory cell signals and cell death by low BA concentration. (A) Effect of BA on NF-κB/Rel activities was evaluated using the EMSA method. The Ca2+ dependence of NF-κB/Rel activation was tested by preloading the cells with the Ca2+ chelator, BAPTA-AM. The identities of individual DNA binding proteins were analyzed by super shift assays using specific antibodies against p50 and p65. (B) Effects of BA on the phosphorylation of JNK and ERK and their Ca2+ dependence were measured by immunoblotting using anti–phospho-JNK– or phospho-ERK–specific antibodies. (C) Effects of BAPTA pretreatment on BA-induced vacuolization. Cells were incubated with 0.01% BA for 20 minutes or 2 hours and processed for electron microscopy. (D) The effect of BA on cell survival was measured using a tetrazolium-based assay with primary cultures of pancreatic acinar cells. Note that chelation of intracellular Ca2+ completely inhibits acinar cell death induced by treatment with 0.01% BA for 15 hours. *P < 0.05. Gastroenterology 2002 122, 1941-1953DOI: (10.1053/gast.2002.33617) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Inhibition of SERCA pumps by bile acids. (A) Effect of BA on Ca2+ content in the intracellular store was measured using a low-affinity fluorescent probe, Mag-Fura-2. After dye loading and the BA treatment, acini were permeabilized, and the fluorescence of the remaining Mag-Fura-2 was measured before and after the addition of 1 μmol/L IP3. The left panel shows representative traces, and the right panel shows the average of 8 experiments with similar results. (B) SERCA activity was measured using Ca2+-clearing time after atropine treatment in carbachol-stimulated pancreatic acinar cells. An example of measurement in control cells is shown in the left panel. The effect of BA on total [Ca2+]i-clearing activity and on Ca2+ clearance by all mechanisms other than SERCA (BHQ treat) are summarized in the right panel. (C) Intracellular ATP concentration was measured in pancreatic acinar cells after treatment with the indicated BA concentrations. (D) The effect of BA on SERCA pump activities and SR Ca2+ release was analyzed in SR vesicles from rat skeletal muscles. SERCA pump activity was measured by 2 separate methods, ATP-dependent 45Ca2+ uptake (filled squares) and ATPase activity in the presence of 2 μmol/L A23187 (open squares, dotted line), as detailed in Materials and Methods. The Ca2+-releasing effect of BA (open circles) was measured by incubating 45Ca2+-preloaded SR vesicles with the indicated concentration of BA. *P < 0.05; **P < 0.01. Gastroenterology 2002 122, 1941-1953DOI: (10.1053/gast.2002.33617) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Na+-dependent bile acid transport in pancreatic acinar cells. (A) TC uptake was measured in isolated pancreatic acinar cells and hepatocytes using [3H]-TC. Na+ dependence of TC uptake in each cell type was estimated from incubating the cells in the presence and absence of Na+. The initial uptake rates of multiple experiments were summarized. (B) Expression of Na+-dependent bile transporters in the tail portion of the pancreas was analyzed by RT-PCR using 2 separate sets of primers. (C) Immunoblotting of Ntcp. (D) Immunostaining of Ntcp was performed using anti–GST-Ntcp antibodies and the relevant preimmune sera. Note that Ntcp showed luminal staining in pancreatic acinar cells (right), which was absent in preparations incubated with preimmune sera (left). M, molecular marker; Pan, pancreas; Liv, liver; Kid, kidney. **P < 0.01; *P < 0.05. Gastroenterology 2002 122, 1941-1953DOI: (10.1053/gast.2002.33617) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Na+-independent bile acid transport in pancreatic acinar cells. (A) The TC/HCO3− exchange activity, a representative function of Oatp1, was measured in pancreatic acinar cells by following the TC-induced decrease in pHi after HCO3− loading using the Cl−/ HCO3− exchange activity of the cell. (B) Expression of the Oatp isoforms was analyzed by RT-PCR. (C) Immunoblotting of Oatp1. The antibodies against Oatp1 detected an 80–85 KD protein in liver and pancreas. (D) Immunostaining of rat pancreas was performed with antibodies against Oatp1 and the corresponding blocking peptides, as indicated. Antibodies against Oatp1 stained both the basolateral (arrow) and the luminal membranes of pancreatic acinar cells (left). The basolateral staining was completely abolished by the blocking peptide, but the luminal staining was not (arrow, right). Gastroenterology 2002 122, 1941-1953DOI: (10.1053/gast.2002.33617) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 A model of bile-induced cell damage in rat pancreatic acinar cells. Bile acids can enter acinar cells through both the luminal Ntcp and basolateral Oatp. The transported bile acids inhibit SERCA and deplete intracellular Ca2+ stores. Store depletion increases Ca2+ influx through CCE. The sustained elevation in [Ca2+]i activates the inflammatory signals and causes cell death. Gastroenterology 2002 122, 1941-1953DOI: (10.1053/gast.2002.33617) Copyright © 2002 American Gastroenterological Association Terms and Conditions