Peter Thorn
Department of Pharmacology, University of Cambridge, UK

https://doi.org/10.36866/pn.60.20

Pancreatic acinar cells have long been used as a model system to study the mechanisms that underlie polarized secretion. The determination of the pathways of synthesis of digestive enzymes (the major secretory product) in the endoplasmic reticulum, and then their passage through the Golgi and condensing vacuoles into the secretory granules, was the basis for the Nobel Prize in Physiology given, as part of a joint award, to GE Palade in 1974. Palade’s work showed that the final stage of secretion, the release of secretory products to the outside, depends on the fusion of secretory granule membrane with the apical plasma membrane. The year before this Nobel award, it was demonstrated that the trigger for this digestive enzyme release was a rise in calcium (Matthews et al. 1973).

Modern imaging techniques now make it possible to observe these final steps of granule fusion with the plasma membrane, the process of exocytosis. Perhaps the most interesting findings indicate that secretion in these cells may follow processes somewhat distinct from those found in neurones and endothelial cells. Firstly, it appears as if almost all aspects of exocytosis are slow. Secondly, there is evidence that granule membrane retrieval, endocytosis, may follow a distinct pathway.

Looking at the temporal aspects of exocytosis, the exocytotic response in acinar cells follows a calcium rise with a delay of seconds (Ito et al. 1997) compared with millisecond delays seen in neurones. After fusion of the granule and apical plasma membrane, granule contents are lost relatively rapidly (~10 s, Thorn & Parker, 2005) but the granule stays at the plasma membrane (Nemoto et al. 2001), with direct evidence that the fusion pore stays open for many minutes (Thorn et al. 2004).

Looking at endocytosis, in principle, the granule membrane could collapse into the plasma membrane and then be retrieved or it could be that the whole granule is recaptured. In the latter case we are left with a basic problem – how could it be refilled with its proteinaceous contents? Recent experiments suggest a third possibility. We have shown that the fusion pore apparatus forms a barrier to the movement of lipids even as the granule disappears, a time when we presume endocytosis is taking place. This has led to our proposal that granule membrane recovery (endocytosis) may proceed in a piecemeal fashion (Fig.1) with small pieces of membrane gradually recovered from the original granule and then reconstituted in the Golgi into a new granule ready for another round of exocytosis (Thorn et al. 2004). For a cell, this method of endocytosis would provide an efficient mechanism of recycling intact granule membrane without the need to re-sort the membrane constituents.

Recycling of granule components is supported by early experiments showing that the lifetime of granule membrane proteins is much longer than the lifetime of the proteins that make up the granule contents (Jamieson & Palade, 1971). In addition, the model of piecemeal endocytosis is specifically supported by freeze-fracture experiments in salivary gland acinar cells, where differences in intramembrane particle density between the apical plasma membrane (high density) and the granule membrane (low density) are maintained even after exocytosis and apparently during endocytosis (De Camilli et al. 1976).

However, unfortunately there is no corresponding data for pancreatic acinar cells since the intramembrane particle densities, prior to exocytosis, are the same in the plasma membrane and granule membrane. Further, recent evidence apparently contradicts the idea that the fusion pore blocks interchange between the granule and the plasma membrane and suggests that syntaxin 2, a plasma membrane protein thought to be important in exocytosis, may get into the granule membrane after exocytosis (Pickett et al. 2005). Clearly, more work is needed to understand the endocytotic process in epithelial cells.

The specific proteins that might regulate the slow kinetics of epithelial cell exocytosis and regulate the process of endocytosis are not known. Proteins known to control exocytosis in other cells, have been identified on the apical plasma membrane and on the secretory granule, but the specific proteins involved have yet to be identified; even the calcium sensor itself remains unknown. Imaging the processes of exocytosis and endocytosis in epithelial cells and defining the proteins involved will certainly be an exciting challenge for the future.

References

De Camilli P, Peluchetti D & Meldolesi J (1976). Dynamic changes of the luminal plasmalemma in stimulated parotid acinar cells. A freeze­-fracture study. J Cell Biol 70, 59-74.

Ito K, Miyashita Y & Kasai H (1997). Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells. EMBO J 16, 242-251.

Jamieson JD & Palade GE (1971). Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells. J Cell Biol 50, 135-158.

Matthews EK, Petersen OH & Williams JA (1973). Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase release. J Physiol 234, 689-701.

Nemoto T, Kimura R, Ito K, Tachikawa A, Miyashita Y, Iino M & Kasai H (2001). Sequential-replenishment mechanism of exocytosis in pancreatic acini. Nature Cell Biol 3, 253-258.

Pickett JA, Thorn P & Edwardson JM (2005). The plasma membrane Q-SNARE syntaxin 2 enters the zymogen granule membrane during exocytosis in the pancreatic acinar cell. J Biol Chem 280, 1506-1511.

Thorn P, Fogarty KE & Parker I (2004). Zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity. Proc Natl Acad Sci 101, 6774-6779.

Thorn P & Parker I (2005). Two phases of zymogen granule lifetime in mouse pancreas: ghost granules linger after exocytosis of contents. J Physiol 563, 433-442.