In Vitro Stimulation of Enzyme Secretion and the Synthesis of Microsomal Membranes in the Pancreas of the Guinea Pig

Abstract

Several mechanisms have been suggested to explain how secretory cells remove from the plasmalemma the excess membrane resulting from the insertion of granule membrane during exocytosis : intact patches of membrane may be internalized and then reutilized within the cell ; alternatively these membranes may be either disassembled to subunits or degraded . In the latter case new membranes should be synthetized at other sites of the cell, probably in the rough-surfaced endoplasmic reticulum (RER) and the Golgi complex . In the present research, membrane subfractions were obtained from rough microsomes (derived from fragmented and resealed RER cisternae) and from smooth microsomes (primarily contributed by Golgi stacks and vesicles) of the guinea pig pancreas by incubation at 4°C for 4 hr in 0 .0005 M puromycin at high ionic strength followed by mild (pH 7 .8) alkaline extraction with 0 .2 M NaHCO3 . Such treatments release the majority of nonmembrane components of both microsomal fractions (i .e., contained secretory enzymes, ribosomes, and absorbed proteins of the cell sap) and allow the membranes to be recovered by centrifugation . The effect of in vitro stimulation of enzyme secretion (brought about in pancreas slices by 0 .0001 M carbamoyl choline) on the rate of synthesis of the phospholipid (PLP) and protein of these membranes was then investigated . In agreement with previous data, we observed that in stimulated slices the synthesis of microsomal PLP was greatly increased . In contrast, the synthesis of microsomal membrane proteins was unchanged . These results suggest that exocytosis is not coupled with an increased rate of synthesis of complete ER and Golgi membranes and are, therefore, consistent with the view that excess plasma membrane is preserved and reutilized, either as discrete membrane patches or as membrane macromolecules, throughout the secretory cycle . 6It is now firmly established that, in the acinar cells of the pancreas, stimulation of secretion ultimately results in the discharge of digestive enzymes stored within zymogen granules by a process akin to exocytosis (1-5) . Such a process consists of fusion of the limiting membrane of the granule with the apical portion of the plasmalemma and therefore results, at least temporarily, in the incorporation of the first membrane into the latter. The mechanism of removal of excess membrane from the acinar lumen is still unknown . As proposed by Palade (1), intact patches of membranes may be internalized from the cell surface and reutilized in the packaging of new zymogen granules . Alternatively these membranes may be degraded as suggested originally by Fawcett (6), new membranes being synthesized at other sites in the cell .The results of a series of papers by the Hokins

Composition of Cellular Membranes in the Pancreas of the Guinea Pig

“…3 and 4E) and in the KCI-puromycin-released subfraction (containing ribosomes, secretory, and adsorbed proteins (8)) (Figs . 3 and 4D) and absent from the subfractions containing either secretory proteins (NaHCO 3-extracted) (8) (Figs . 3 and 4C) or membranes (8) (Figs .…”

“…3 and 4D) and absent from the subfractions containing either secretory proteins (NaHCO 3-extracted) (8) (Figs . 3 and 4C) or membranes (8) (Figs . 3 and 4B) .…”

“…Such treatment releases the bulk of secretory proteins and absorbed cell sap proteins (7,8) ; however, -50% of the RNA is recovered in these membrane subfractions (7) .…”

“…The purification was achieved by removing from both microsome fractions and ZG the nonmembrane components while leaving the structure of the membranes apparently unaffected (7,8) . Since the purification of membrane fractions is a necessary prerequisite to the work presented in this paper, we have summarized in Table I filaments associated with the terminal web .…”

“…In previous papers we have reported that the membranes involved in the process, i .e ., the membranes of rough microsomes (RM) (=RER), of smooth microsomes (SM) (primarily Golgi vesicles and cisternae), of ZG, and the plasma membranes (PM) can be isolated as highly purified fractions from homogenates of guinea pig pancreas (7,8) . The comparative study on the lipid composition (9) and enzymic activities (10) of these fractions led us to the conclusion that considerable differences exist among the various types of membranes, thus suggesting that they do not mix randomly with one another during the transport of secretory products .…”