Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

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.

Cyclic AMP accelerates calcium waves in pancreatic acinar cells.

Cytosolic Ca(2+) (Ca(i)(2+)) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca(2+) wave generated by the Ca(2+)-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca(2+) channel opening by affecting either cAMP-dependent protein kinase (PKA) or exchange protein directly activated by cAMP (Epac). Isolated pancreatic acinar cells from rats were stimulated with carbachol (1 muM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca(i)(2+) was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 muM)-stimulated Ca(2+) wave was 8.63 +/- 0.68 microm/s; it increased to 19.66 +/- 2.22 microm/s (*P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 microM). The Ca(2+) rise time after carbachol stimulation was reduced in both regions but to a greater degree in the basal. Lag time and maximal Ca(2+) elevation were not significantly affected by cAMP. The effect of cAMP on Ca(2+) waves also did not appear to depend on extracellular Ca(2+). However, the ryanodine receptor (RyR) inhibitor dantrolene (100 microM) reduced the cAMP-enhancement of wave speed. It was also reduced by the PKA inhibitor PKI (1 microM). 8-(4-chloro-phenylthio)-2'-O-Me-cAMP, a specific agonist of Epac, caused a similar increase as 8-Br-cAMP or VIP. These data suggest that cAMP accelerates the speed of the Ca(2+) wave in pancreatic acinar cells. A likely target of this modulation is the RyR, and these effects are mediated independently by PKA and Epac pathways.

Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin.

Aberrant cytosolic Ca(2+) flux in pancreatic acinar cells is critical to the pathological pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of Ca(2+)-activated protein phosphatase 2B (or calcineurin) in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiological caerulein (100 nM) with or without the calcineurin inhibitors FK506 or cell-permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation, and the percent amylase secretion was used as a measure of enzyme secretion. Cytosolic Ca(2+) changes were recorded in acinar cells loaded with the intermediate Ca(2+)-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 microM FK506 or 10 microM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca(2+) after caerulein stimulation. These findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation but not enzyme secretion or the initial caerulein-induced cytosolic Ca(2+) signal.

Cyclic AMP-dependent protein kinase and Epac mediate cyclic AMP responses in pancreatic acini.

The pancreatic acinar cell has several phenotypic responses to cAMP agonists. At physiological concentrations of the muscarinic agonist carbachol (1 microM) or the CCK analog caerulein (100 pM), ligands that increase cytosolic Ca(2+), cAMP acts synergistically to enhance secretion. Supraphysiological concentrations of carbachol (1 mM) or caerulein (100 nM) suppress secretion and cause intracellular zymogen activation; cAMP enhances both zymogen activation and reverses the suppression of secretion. In addition to stimulating cAMP-dependent protein kinase (PKA), recent studies using cAMP analogs that lack a PKA response have shown that cAMP can also act through the cAMP-binding protein, Epac (exchange protein directly activated by cyclic AMP). The roles of PKA and Epac in cAMP responses were examined in isolated pancreatic acini. The activation of both cAMP-dependent pathways or the selective activation of Epac was found to enhance amylase secretion induced by physiological and supraphysiological concentrations of the muscarinic agonist carbachol. Similarly, activation of both PKA or the specific activation of Epac enhanced carbachol-induced activation of trypsinogen and chymotrypsinogen. Disorganization of the apical actin cytoskeleton has been linked to the decreased secretion observed with supraphysiological concentrations of carbachol and caerulein. Although stimulation of PKA and Epac or Epac alone could largely overcome the decreased secretion observed with either supraphysiological carbachol or caerulein, stimulation of cAMP pathways did not reduce the disorganization of the apical cytoskeleton. These studies demonstrate that PKA and Epac pathways are coupled to both secretion and zymogen activation in the pancreatic acinar cell.

The ryanodine receptor mediates early zymogen activation in pancreatitis.

Acute pancreatitis is characterized by the pathologic activation of zymogens within pancreatic acinar cells. The process requires a rise in cytosolic Ca(2+) from undefined intracellular stores. We hypothesized that zymogen activation is mediated by ryanodine receptor (RYR)-regulated Ca(2+) release, because early zymogen activation takes place in a supranuclear compartment that overlaps in distribution with the RYR. Ca(2+) signals in the basolateral, but not apical, region of acinar cells observed during supraphysiologic agonist stimulation were dependent on RYR Ca(2+) release. Inhibition of RYR or depletion of RYR-sensitive Ca(2+) pools each reduced pathologic zymogen activation in isolated acinar cells, but neither treatment affected amylase secretion. Inhibition of RYR also inhibited zymogen activation in vivo. We propose that Ca(2+) release from the RYR mediates zymogen activation but not enzyme secretion. The findings imply a role for the RYR in acute pancreatitis.

Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions.

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.