Oncogenic Ras requires activation for pathological levels of activity

Abstract

Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA Background: Previous studies have shown that the small G-protein Rab27A regulates the intracellular trafficking of secretory granules in various cell types, including islet beta cells. However, its role in mouse pancreatic acini is poorly understood. We utilized ashen mice, which lack Rab27A to investigate the function of Rab27A in pancreatic acinar cells. Aim: To investigate the effects of Rab27A deficiency on pancreatic acinar cell physiology and function. Methods: Isolated pancreatic acini were prepared from ashen or wildtype (WT) mouse pancreases by collagenase digestion and CCKor carbachol-induced amylase secretion was measured. Expression of Rab27A, Rab27B, Rab3D and digestive enzymes were measured by western blot. GTP-bound states of Rab27B and Rab3D in ashen or WT mouse acini were assessed by GST-pulldown assay. Localization of Rab27B and Rab3D in ashen and WT pancreases was shown by immunofluorescence. Results: Expression of Rab27A was detected in isolated WT mouse pancreatic acinar cells by western blot and RT-PCR. Expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. The GTP-bound states of Rab27B and Rab3D in ashen and WT mouse acini also remained constant. Acini from ashenmice showed lower basal level of amylase release but no obvious change in the entire dose response curves of CCKor carbachol-stimulated amylase release. Localization of Rab27B, Rab3D and amylase by immunohistochemistry was not obviously changed in acinar cells from both mice. Localization of Rab27A could not be determined using available antibody. Conclusions: Rab27A is present in mouse pancreatic acinar cells but not involved in the final secretory steps of zymogen granule secretion.