PMC

RC exposure causes acinar cell: (1) activation of PLC; (2) generation of IP3 resulting in IP3R-induced Ca²⁺ release; (3) downstream activation of calcineurin; (4) translocation of NF-κB to the nucleus, leading to (5) acinar cell injury and pancreatitis. The inhibitory scheme is shown in red. PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacyglycerol.

(A) Schema for the administration of FK506 (1 mg/kg) relative to the infusion of RC. (B & F) Representative H&E sections from the pancreatic head. (C & F) Overall severity score (left), and serum amylase measurements (right). (n=5 animals per group). (D) Bioluminescence from the pancreas of mice that had received intraductal infusions of AAV6- NF-κB-luciferase. Quantification of the pancreatic NF-κB-bioluminescent signal over 36 hr (n=3 animals per group). (F) Gene expression for IL-6, GADD45B, and IL-1β from the pancreatic head (n=3). *, #, P<0.05 compared with NS sham and RC alone, respectively.

Mouse acinar cells were infected with Ad-NFAT-luciferase and stimulated with RC (A) for varying time periods (25% RC) or (B) with increasing concentrations for 5 hr. RC (9%)-induced NFAT-luciferase activity was prevented by (C) 30 min pre-treatment with the calcineurin inhibitors FK506 (24 μM) and cyclosporine (CsA; 16 μM) or (D) 30 min pre-treatment with the IP3R inhibitor 2-APB (100 μM) or the intracellular Ca²⁺ chelator BAPTA-AM (64 μM). (n=3). *, #, P<0.05, relative to the control or RC alone, respectively.

(A) Acinar cells were treated with increasing concentrations of RC for 6 hr, and propidium iodide uptake was measured. RC (12%)-induced acinar cell injury (6 hr incubation) was prevented by (B) U73122 (5 μM), 2-APB (100 μM), BAPTA (64 μM), or (C) the NF-κB inhibitor IKK-2 (20 μM). (D) Inhibition of calcineurin (FK506; 24 μM, CsA; 16 μM) or (E) genetic deletion of the regulatory calcineurin Aβ subunit (CnAβ). (F) ATP levels were measured from acinar cells treated with RC (12%) for 3.5 hr ± FK506. (n=3). *, #, P<0.05, relative to the control or RC alone, respectively.

Western blots from primary mouse acinar cells stimulated with (A) 20% RC or varying concentrations of RC for 15 min or 30 min and probed for phosphorylated IκBα or p65. Densitometry shown below for each blot. (B) Acinar-differentiated AR42J cells were infected overnight with Ad-NF-κB-luciferase and then incubated for 6 hr with increasing concentrations of RC. AR42J cells were exposed to RC (25%) for varying times (solid lines) then washed off with buffer (dashed lines) and incubated for a total of 6 hr. (C) NF-κB-luciferase activity was prevented in AR42J cells by pre-treatment with the U73122 (5 μM), 2-APB (100 μM), BAPTA (64 μM), (D) FK506 (24 μM), and CsA (16 μM). (n=3). *, #, P<0.05, relative to the control or RC alone, respectively. (E) AR42J cells were infected with increasing titers of an adenovirus carrying a constitutively active form of the catalytic calcineurin A subunit (Ad-ΔCn) or infected with Ad-EGFP (negative control). NFAT- and NF-κB-luciferase activity were measured. Ad-EGFP at amounts greater than 4×10⁵ infectious units (IFU) failed to increase luciferase levels. Correlation between NFAT- and NF-κB-luciferase activity in AR42J cells (R²=0.9185; P = 0.0002). (n=3). *, P<0.05, relative to Ad-EGFP alone.

(A) Schema for RC infusion into the distal common bile duct (arrows) using a perfusion pump (P, pancreas; D, duodenum). (B) Representative HE sections of the pancreatic head from NS sham control and RC-infused (at 20 μl/min, 100 μl volume) mice. A combination of intra-ductal RC along with an increased rate and volume induced greater histological severity and higher serum amylase levels. (n=5 animals per condition). *, P<0.05 relative to NS sham. (C) Mouse pancreatic acinar cells are loaded onto a custom-made perifusion chamber and imaged using a confocal microscope. (D) Phase contrast image and pseudo-colored images of acini loaded with the Ca²⁺ dye Fluo-4AM at baseline, during the initial rise in Ca²⁺ fluorescence, and peak fluorescence during perfusion with RC. An individual acinar cell is outlined in the dashed white line, and the red arrows indicate the progression of the Ca²⁺ signal from the apical to basal region of the cell. (E) Summation of whole cell tracings of Ca²⁺ flux with increasing concentrations of RC. (F) Amplitude and area under the curve measurements of the Ca²⁺ signal. Acinar cells were perfused with RC (25%) in the presence or absence of Ca²⁺ containing media (n=20–30 cells per condition). *, P<0.05 compared to 17% RC.