Membrane interactions between secretion granules and plasmalemma in three exocrine glands

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Abstract
Three types of membrane interactions were studied in 3 exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland and guinea pig pancreas) by freeze-fracture and thin-section EM, exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all 3 glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. After injection of secretagogues, IMP were rapidly cleared from the areas of granule-plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrow necks and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Increasing the centrifugal force from 1600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. No such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in sections after centrifugation at 50 and 100,000 g for 10 min. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands, IMP-free blisters and vesicles were often observed, whereas in the pancreas they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. These blisters and vesicles probably arise artifactually during glutaraldehyde fixation. They were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO4. No increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.