Slow sustained release from mast cells that is mediated by vesicle transport may be the most relevant mode of secretion in cell-mediated immunologic reactions and diseases of cell-mediated immunity (i.e., graft rejection, viral diseases, tumor rejection, contact allergy). In salivary gland secretion this mechanism could provide sustained baseline secretory products for normal use without release of a major bolus (e.g. as entire granules are released). Secretion by exocytosis is effected by a sequence of membrane fusion events that occurs after stimulation and results in the release of granule matrix proteins to the extracellular milieu. The mechanisms that underlie other secretory mechanisms (i.e., constitutive secretion, piecemeal degranulation) are less certain. Previously, a distinctive release reaction, termed """"""""piecemeal degranulation: has been described in guinea pig and human basophils and in human mast cells and eosinophilis. Piecemeal degranulation is differentiated by its morphology from the more widely recognized morphology of exocytosis that accompanies various regulated secretory processes. An increase in the number of cytoplasmic vesicles and the progressive emptying of cytoplasmic granules in place are the morphologic criteria that define piecemeal degranulation. In this proposal, I will proposal, I will rigorously examine the potential role of cytoplasmic vesicles as a key transport mechanism for piecemeal degranulation as well as extend documentation of this form of rapid secretion to rat mast cells, specialized secretory cells that contain pro- inflammatory mediators and cytokines, and to rat salivary gland acinar cells, as a more general example of secretion (i.e., exocrine secretory cells that contain digestive enzymes). Because the proposed processes to be studied (i.e., vesicular transport) are known to occur sufficiently fast that conventional chemical fixation for electron microscopy may fail to capture these events, the experimental approach will make use of a new method of microwave energy-assisted, ultrafast fixation recently developed and tested in our laboratory, ultrastructural immunocytochemistry, and computer assisted morphometry. Specificantly, the role of cytoplasmic vesicles in nontraditional secretory mechanisms will be evaluated as follows: 1) during stimulated release of granule associated histamine and chymase from rat peritoneal mast cells by various concentrations of two degranulation stimuli (antibody to IgE and compound 48/80). And 2) during constitutive and stimulated release of granule associated amylase, mucin glycoprotein, glutamine/glutamic acid- rich protein, and granule specific membrane proteins from dispersed acinar aggregates of rat parotid and submandibular glands by various concentrations of alpha and beta adrenergic stimuli.
