Protease activation during in vivo pancreatitis is dependent on calcineurin activation

Abstract

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Fig. 1.

Schema for in vivo Ca²⁺/calmodulin-dependent calcineurin (PP2B) inhibition using FK506 (1 mg/kg) and pancreatitis induction using caerulein (Caer; 50 μg/kg).

Fig. 2.

Pancreatic PP2B activity can be reduced with FK506 administration. Mice received intraperitoneal injections of FK506 (1 mg/kg) as described in methods and PP2B activity was assayed. Activity from kidney was used as a positive control (n = 3). NS, normal saline. *P < 0.05 compared with the saline-treated group.

Fig. 3.

FK506 treatment reduces pancreatic trypsin activity in vivo. Trypsin activity was assayed from pancreatic homogenates 1 (A) and 8 h (B) after the first caerulein dose (n = 5 animals per group). *, #P < 0.005 compared with saline-treated and caerulein-alone groups, respectively. Values are expressed as means ± SE and are normalized to a maximum value.

Fig. 4.

FK506 treatment reduces serum amylase and IL-6 elevations observed during pancreatitis. Caerulein hyperstimulation caused a time-dependent increase in serum amylase and IL-6. FK506 administration reduced levels as early as 4 h (n = 5 animals per group). *P < 0.005; #P < 0.05 compared with saline-treated and caerulein-alone groups, respectively. SE shown for IL-6. Serum values from FK506-only treated animals did not rise above baseline at 1, 4, or 8 h after caerulein stimulation (shaded bar; n = 3). Representative images at 1 h demonstrate edema and vacuolization (arrow).

Fig. 5.

FK506 treatment reduces histological severity of pancreatitis. A: representative hematoxylin and eosin sections of pancreas from saline- (top left) or caerulein-treated animals over an 8-h time course. B: histological scoring of pancreatitis severity with or without FK506 administration (n = 5 animals per group). #P < 0.05, P < 0.001, P < 0.005 compared with caerulein-alone groups at 1, 4, and 8 h, respectively.

Fig. 6.

Analysis of histological parameters shows that FK506 treatment causes a reduction in severity early in the course of pancreatitis. A score of 0–3 was given on the basis of the degree of edema, inflammation, apoptosis, and vacuole formation at 1 (A), 4 (B), and 8 h (C) after the first caerulein dose (n = 5 animals per group). #P < 0.005 compared with caerulein-alone groups. All parameters were minimally increased for saline controls or FK506-alone animals. Representative images demonstrate edema and vacuolization (arrow).

Fig. 7.

The FK506 dosing regimen does not affect pancreatic heat shock protein 70 (HSP70) expression. A: pancreatic tissue was isolated for Western blot analysis from mice receiving hourly injections of normal saline or FK506 (1 mg/kg) after 1 and 8 h of treatment. Representative blot demonstrates no increase in HSP70 expression with FK506 treatment. B: quantification of HSP70 expression, normalized to actin (n = 3).