Synthesis, Intracellular Transport and Discharge of Exportable Proteins in the Pancreatic Acinar Cell and Other Cells

Abstract

SUMMARY After an outline description of pancreatic structure and function, and a more detailed account of acinar cell morphology, this review traces the pathway of amino acids as they are taken up by the acinar cell, incorporated into digestive hydrolases, transported through the cell and finally discharged from the cell, and considers the mechanisms by which these steps are controlled. At all stages comparisons are made with other secretory cells. The use of radioautography and cell‐fractionation techniques in determining this pathway in the pancreas are described. The route and kinetics of the process in pancreas are compared with those in other cells. Amino‐acid entry is by an active mechanism. However the intracellular pool of accumulated amino acids may not be used directly in protein synthesis. Selection of amino acids for incorporation into proteins may occur whilst they are associated with carrier systems within the plasma membrane. There is no convincing evidence that amino‐acid entry can be influenced by the pancreatic secretagogues, cholecystokin‐pancreazymin (CCK‐PZ) or acetylcholine. Secretory proteins are synthesized on ribosomes bound to the endoplasmic reticulum (ER) and the nascent proteins vectorially transferred across the ER membrane into the ER cisternae. All messenger RNA molecules which are templates for secretory proteins appear to possess an initial sequence of codons whose translation produces a ‘signal’ sequence of amino acids. This signal sequence somehow triggers attachment of the ribosomes to the ER, thereby automatically determining that the final translation product is destined for the ER cisternae. The effects of CCK‐PZ and acetylcholine on pancreatic protein synthesis are controversial. Whereas stimulation can be observed in vivo, this has not been convincingly demonstrated in vitro. I conclude that while CCK‐PZ and acetylcholine may accelerate protein synthesis, the physiological significance of this effect remains to be clarified. Long‐term stimulation can modify pancreatic enzyme synthesis and this, together with other factors, may be the means of dietary adaptation by the gland. Newly synthesized proteins travel from the ER cisternae via the peripheral Golgi components to the Golgi cisternae. Transport from ER to Golgi cisternae may occur by a vesicle shuttle service or by direct tubular connexions. Although sustained stimulation with CCK‐PZ analogues can accelerate this intracellular transport step, pancreatic secretagogues have not yet been shown to accelerate transport under physiological conditions. The Golgi complex has a number of functions including: glycosylation and, where appropriate, sulphation of glycoprotein and mucopolysaccharide components of the zymogen granules (ZG) and granule membranes; sequestration of divalent cations which bind to secretory proteins; the formation of condensing vacuoles (CV) from the inner Golgi cisternae. Aggregation of proteins occurs passively within CV so as to form osmotically inert complexes, thereby reducing internal osmotic activity and causing water to diffuse out. This condensation imparts a gel‐like consistency to the mature ZG so formed. Discharge of ZG occurs by a process of exocytosis involving fusion of the ZG membrane with the apical plasma membrane, release of the ZG contents, and retrieval of the ZG membrane from the plasma membrane by endocytotic mechanisms. The mechanisms responsible for migration of ZG towards the cell apex and for exocytosis remain unknown but may involve the participation of microtubules and/or microfilaments. Although there is a small, basal discharge of ZG at all times, stimulation with CCK‐PZ or acetylcholine greatly accelerates the process. The basic tenet of the secretory mechanism summarized above is that, following synthesis, secretory proteins are confined within an intracellular organelle at all times. This ‘segregation’ hypothesis has been challenged by the ‘equilibrium’ hypothesis in which secretory proteins are suggested to move across cellular membranes and are therefore at equilibrium within the various compartments of the cell. While many of the observations on which the equilibrium hypothesis are based are tenuous, some others cannot readily be explained by the segregation model. Proponents of the equilibrium hypothesis therefore suggest that preferential release of individual hydrolases from ZG occurs, followed by their separate transport across the apical cell membrane. The claims of this alternative model are discussed. In the final section are discussed the intracellular mechanisms by which CCK‐PZ and acetylcholine act on the acinar cell to cause discharge. The overall membrane perturbations brought about by CCK‐PZ and acetylcholine appear to be the same and include cell depolarization, and perhaps increased phospholipid turnover. Both events may be related to an altered membrane permeability to cations. CCK‐PZ, but not acetylcholine, will activate adenylate cyclase, but cyclic AMP does not appear to be involved in regulating enzyme discharge. Instead, Ca2+ is the major intracellular second messenger. However, rather than increase Ca2+ uptake into the cell, CCK‐PZ and acetylcholine appear to raise the intracellular Ca2+ concentration by causing release of Ca2+ from intracellular stores. The mechanism by which they do this, and the role of Ca2+ in the discharge process remain unknown.

Intracellular transport and storage of secretory proteins in relation to cytodifferentiation in neoplastic pancreatic acinar cells.

“…Analysis of the GEF cells following pulsechase electron microscope autoradiography established that these cells transport newly synthesized protein destined for export in a vectorial manner reminiscent of normal pancreatic acinar cells (2,4,20,27). The data show that the pulse-labeled protein in GEF cells moves successively from RER to the Golgi apparatus and from there to the post-Golgi vesicles (vacuoles or immature granules) and eventually to mature secretory granules as in a variety of other cells that store their secretory product (5,6,8,15). Transfer of label to the Golgi apparatus occurs very rapidly in these GEF ceils; by 10-min postpulse, ~26% of the total grains is associated with the Golgi apparatus, reaching a peak of-35% by 20-min postpulse.…”

“…These studies suggest that At 60 min, the stack of Golgi cisternae no longer contains appreciable quantity of the label, but the small vesicles in the inner face of the Golgi complex are labeled (arrows). Some label is seen associated with secretory granules (5). By 240 rain of chase incubation, the label is concentrated over secretory granules.…”

“…These studies have identified six successive steps in the secretory process namely, synthesis, segregation, intraceUular transport via the Golgi complex, concentration, intracellular storage of discharge (for detailed discussion of this process see references 18 and 27). The existence of such a secretory process in a variety of highly differentiated eucaryotic cell types is now well documented (5,6,8,15,18,27,47). Information regarding the acquisition of various steps of the secretory process during cytodifferentiation is, however, lacking.…”

The inhibitory effect of CR-1409 on amylase secretion and intracellular Ca2+ mobilization in rat pancreatic acini in vitro.

“…Stimulus-secretion coupling in pancreatic acinar cells involves an increase in the concentration of cytoplasmic free Cat +, and as a first step, the hormonereceptor binding evokes a rapid increase in the concentration of cytoplasmic Cat + (CASE, 1978;WILLIAMS, 1980;PETERSEN, 1982). In this study, we examined the changes in intracellular free Cat + in rat pancreatic acini induced by these secretagogues using a fluorescent Cat+-indicator, fura-2.…”

Protein Composition and Biogenesis of the Pancreatic Zymogen Granules

“…Stimulation of the acinar cells by secretagogues such as acetylcholine and cholecystokinin triggers fusion of ZG membrane with the apical plasma membrane and the release of digestive enzymes into the pancreatic ductal system. In the duodenum, trypsinogen is converted to trypsin by proteolytic cleavage via enterokinase and activated trypsin then proteolytically activates the other zymogen enzymes (8,55).…”