Poster Presentation 6th Australian Health and Medical Research Congress 2012


Roya Safi 1 , Natasha Behrendorff 1 , Peter Thorn 1
  1. School of Biomedical Science, University of Queensland, Brisbane, QLD, Australia

Here we use live-cell methods to determine the importance of a protein, VAMP8, in the in vitro supramaximal stimulation model of acute pancreatitis.

VAMP8 is a soluble NSF attachment protein receptor (SNARE) found on intracellular membranes and plays a selective role in compound exocytosis during normal secretion in pancreatic acinar cells. In addition, VAMP8-/- mice show less inflammation and less tissue damage in models of acute pancreatitis.

Here we use live-cell imaging to measure vacuolisation; an early marker of acute pancreatitis. Supramaximal concentrations of agonist, applied to freshly isolated fragments of pancreatic tissue almost immediately (within seconds) promoted fusion of zymogen granules. This response was rapidly followed (within minutes) by the apparent fusion of granules with each other and the formation of vacuoles in the cell. Immunofluorescence showed these vacuoles contain digestive enzymes and are therefore likely to be similar to those observed in vivo in acute pancreatitis. In contrast to the extensive vacuolisation seen in wild-type animals (10.8+/- 2.1% mean+/-SEM of total cell area occupied by vacuoles), pancreatic fragments from VAMP8-/- animals showed almost no vacuolisation (2.00+/-0.3% of cell area) after supramaximal agonist treatment (Student’s t test p<0.01).

We next tested levels of activated enzyme; another marker of disease. In wild type pancreatic fragments enzyme activation was only observed after supramaximal stimulation. In contrast in VAMP8-/- pancreatic fragments enzyme was activated by 27 fold (compared to WT) even when un-stimulated. Western blot showed the VAMP8-/- pancreas had 13.5 times more chymotrpysinogen than wild type suggesting that the elevations in activated enzyme are due to the high levels of stored enzyme known in the VAMP8-/- pancreas.

We conclude that VAMP8 is important in the pathogenesis of vacuole formation in the supramaximal model of acute pancreatitis. Our new data, coupled with the published work on VAMP8-/- mice, suggest enzyme activation is not directly correlated with disease severity.