Secretion from mammalian cells is classified as being constitutive (occurring continuously) or regulated (exocytosis occurs in response to neurohormonal stimulation). The pancreatic acinar cell has been a paradigm for studying regulated exocytosis and recent data from our laboratory indicate that the process involves novel members of the rab3 family of small MrGTP-binding proteins based on functional and immunocytochemical studies. Exocytosis from the acinar cell is accompanied by compensatory endocytosis of excess membrane from the apical plasmalemma; the fate of internalized (recycled) membrane is not known.
The aim of our project is to examine the biogenesis of regulated secretion in the developing rat pancreas. One to 2 days prior to birth, the following cytodifferentiation, acinar cells do not couple hormonal stimulation with exocytosis but are capable of constitutive secretion. Immediately following birth, stimulus-secretion coupling occurs. Data from our laboratory indicate that maturation of regulated exocytosis may be due to expression of small Mr GTP binding proteins (?rab3-like proteins), their effectors or modulators. This system should allow us to dissect the factors involved in conversion of the acinar cell from a constitutive secreting state (analogous to yeast which possesses only constitutive secretion) to a regulated system.
Specific aims are (1) to characterize biochemically the developmental appearance of membrane proteins including members of the rab family that are implicated in regulated exocytosis (rab3) and coupled endocytosis (rab4); (2) to assess biochemically effectors and regulators of G-protein cycles such as GTPases (GAP), GNRP's (guanine nucleotide releasing proteins and GDI's (guanine nucleotide dissociation inhibitors) during maturation of regulated secretion; (3) to use immunocytochemistry to determine the rate of appearance of putative G proteins and other membrane-associated proteins in acquisition of regulated secretion; to use confocal and video microscopy to examine development of coupled exo- and endocytosis in living cells whose exocytic (zymogen granule) and endocytic membranes have been labeled with fluorescent probes and (4) carry out functional studies in which candidate rab proteins, peptides from their effector domains, and antibodies against them and other proteins implicated in coupled exo-endocytosis will be introduced into permeabilized developing acinar cells. The latter approach may complement/bypass the developmental defect in neonatal glands. Future studies will examine pancreatic acinar cell tumors whose secretory refractivity to stimulants may parallel fetal maturation of stimulus-secretion coupling observed in the developing pancreas.
Showing the most recent 10 out of 167 publications
