Amino acid transporters and enzymes involved in glutathione synthesis are altered in pancreatic acinar cells during acute pancreatitis

s / Pancreatology 15 (2015) S1eS141 S4 activation appears very early in subcellular fractions, that are enriched with zymogen granules and dense lysosomal vesicles but lacking in the autophagosomal marker LC3B-II. Recently we found that zymogens are initial activated as early as 30min caerulein hyperstimulation in dense lysosomal vesicles independent of Atg5-related autophagy. Aims: Here we analyzed effects of autophagy modulation in terms of zymogen activation and its subcellular distribution in relation to lysosomal protease cathepsin B in caerulein hyperstimulated murine pancreas. Materials & methods: NMRI mice were pre-treated with autophagy inducers/inhibitors rapamycin, bafilomycin A1, 3-methyladenine (3-MA) or vinblastine by i.p. injection for up to 27 hours. After 300160 min caerulein hyperstimulation pancreata were homogenized and subcellular fractionated in 46 fractions after Percoll gradient centrifugation. Distribution of enzyme activities and marker proteins were determined by colorand fluorometric measurement or by Western blotting. Results: Whether blocking of autophagosome formation by 3-MA nor inhibition of autophagosome-lysosome fusion by Bafilomycin (VATPase-inhibitor) or autophagy induction by rapamycin affected pancreatic chymotrypsinogen activation in caerulein hyperstimulated mice. However inhibition of microtubule formation and associated blocking of autophagosome fusion with lysosomes by vinblastine decreased significantly chymotrypsinogen activation in caerulein hyperstimulated mice. Conclusion: The early zymogen activation occurs in a dense lysosomal compartment in an ATG5-independent process which requires microtubules formation. Our results give evidence that the activation compartment seems to be generated by fusion events of dense granular vesicles with lysosomes or autolysosomes.