Dynamics of salivary secretion studied by confocal laser and scanning electron microscopy.

Plasma membrane events during secretion of the parotid and submandibular glands of humans and rats were observed in living cells by confocal microscopy and from the cytoplasmic side by scanning electron microscopy after removal of the cell organelles by the OsO4 maceration method. These new microscopic techniques revealed in acinar cells two distinct exocytosis-endocytosis coupling mechanisms elicited in response to different secretory stimuli. Beta-adrenergic stimulation with isoproterenol and its second messenger analog dibutyryl cyclic AMP evoked exocytosis after which fused granule membranes were individually removed from the luminal plasma membrane within several minutes. On the fused membrane area, small endocytotic vesicles about 100-150 nm in diameter were abundant. Muscarinic stimulation with carbachol also induced exocytosis, but in this case the fused granule membranes coalesced to form enlarged invaginations which stayed on the luminal membrane for more than 30 min. These invaginations, almost devoid of small endocytotic vesicles, were then pinched off from the luminal membrane and dispersed into the cytoplasm in the form of light microscopically detectable vesicles. After treatment with isoproterenol and carbachol, acinar cells exhibited different distributional changes of F-actin around the fused membrane area, suggesting the involvement of microfilaments in regulating the membrane events following exocytosis.