The long-term objective of the proposed research is to develop a mechanistic understanding of exocytosis as it relates to the specific interactions among biological membranes and their dynamic flow in cells specialized for synthesizing and sequestering macromolecules destined for export. Secretion granules, their limiting membranes, and plasma membranes have been obtained as purified fractions (by centrifugation) from rat parotid salivary plands. In attempts to establish an in vitro analog of exocytosis each type of membrane will be mixed (as a prospective fusion partner) with secretion granules under a variety of conditions postulated to approximate the cytosolic environment in stimulated cells. For plasma membrane-granule mixing, amylase assay will be used to detect a membrane-dependent release of granule contents as a way to diagnose possible fusion events (subject to rigorous subsequent confirmation). In the case of granule membrane-granule mixing, the reduction of a nitroxide spin label internalized into membrane visicles by a spin label reducing agent known to be located in the content of intact granules will be monitored by electron paramagnetic resonance spectroscopy as a means to follow prospective fusion kinetically. The establishment of a bonafide fusion assay is expected to provide the means to systematically explore the mechanism of membrane fusion and to identify approaches applicable to the study of exocytosis in vivo. The interactions of known effector molecules (Ca2+, ATP, and cyclic nucleotides) with the surfaces of intact secretion granules will also be examined with the same intent. Studies of rat peritoneal mast cells have identified in vivo an enhanced incorporation of fatty acid into phosphatidylcholine that is correlated both in time and extent with histamine release. Its presumed coupling to the preceding action of phospholipase A2 will be tested, and localization by autoradiography of the cellular site of incorporation in relation to the sites of granule discharge will be attempted. This cell type is considered to be unusually favorable for examining exocytosis and related metabolic events; its study is expected to guide both future in vivo and in vitro investigations. Although no pathology directly involving exocytosis has been identified, the universality of this process among eukaryotic cells justifies it investigation as related to health sciences.
