Exocytotic release of hormones and neurotransmitters is central to intercellular communication and synaptic transmission. The process is fundamental to the normal function of the endocrine, cardiovascular and nervous systems. In the previous grant period we made fundamental advances in defining the steps involved in the pathway of Ca2+-activated exocytosis using intact and digitonin permeabilized bovine chromaffin cells. An important part of the competing renewal is the extension of the investigations by implementation of two new systems that we have developed for studying secretion. They allow us to manipulate more directly than heretofore possible components of the secretory pathway. We have developed a novel transient transfection system in bovine chromaffin cells (primary, non-dividing cells) to study the role of specific proteins in the secretory pathway. A key part of the approach is to coexpress with a protein of interest, human growth hormone (GH), which serves as a reporter for the regulated secretory pathway in the small fraction of cells that are transfected. We also found that chromaffin granule membranes injected into frog oocytes undergo exocytosis. This reconstitution technique allows us for the first time to manipulate the secretory vesicle apart from the cell to explore components of the secretory vesicle necessary for exocytosis. These new systems will allows us to ask more precise questions concerning the biochemical mechanisms underlying the pathway. The development of both these systems allows us to use functional assays to study chromaffin granule as well as other cellular protein components in exocytosis. The following topics will be investigated: 1) The role of the GTP-binding protein rab3a in regulated exocytosis in adrenal chromaffin cells. 2) The role in exocytosis of synaptotagmin (p65), an integral membrane protein of secretory vesicles. 3) The role of polyphosphoinositides (PPIs) in exocytosis. 4) The identification of protein components on the chromaffin granule membrane important in the exocytotic response in frog oocytes.