Definition and functional polarity of the regulated secretory pathway in AR42J cells, a pancreatic acinar cell line.

摘要

I have examined hormonally stimulated protein secretion in a rat pancreatic acinar cell line, AR42J. These cells assume a more fully differentiated phenotype after treatment with the synthetic steroid hormone dexamethasone. Dexamethasone treatment induces an increase in the amount of protein synthesized and secreted, the amount of protein synthetic apparatus, and the number of membrane bounded secretory granules present within the cytoplasm. Although receptors for cholecystokinin are present in untreated AR42J cells, only dexamethasone treated AR42J cells are sensitive to stimulation by cholecystokinin, as measured by an enhanced rate of amylase secretion in the presence of this hormone.; When grown on suspended filters coated with basement membrane components (laminin and collagen type IV), AR42J cells form layers, consisting of approximately 30% morphologically polarized cells. In spite of this, secretion of amylase from these layers in response to cholecystokinin stimulation occurs predominantly into the apical compartment. Basolateral secretion may be due to heterogeneity in cell polarity, and further characterization of the polarity of secretion from AR42J cells should be undertaken using a subcloned line.; The regulated secretory pathway is defined by (a) secretion of protein(s) in response to hormonal stimulus, (b) presence of the secreted protein(s) within secretory granules, and (c) a relatively long half life of the secreted protein(s) within the cell. I have shown that dexamethasone treatment induces the formation of a regulated secretory pathway in AR42J cells. I then developed a pulse labeling protocol which allowed for distinction between stimulated exocytosis of prestored protein (represented by amylase), and stimulated exocytosis of newly synthesized proteins, which can only occur if these proteins enter the secretory granules. Examination of cholecystokinin stimulated secretion from dexamethasone induced cells pretreated with various physiologic perturbants, allowed me to determine the role, if any, of the cytoskeleton, continuous protein synthesis, an acidic intracellular compartment, normal ATP levels, physiologic temperature, and GTP-binding proteins in routing of newly synthesized proteins to the secretory granules. Only artificial raising of intracompartmental pH (in the presence of either NH{dollar}sb4{dollar}Cl or monensin) inhibited transfer of newly synthesized proteins from the late Golgi into the secretory granules.