Parathyroid Cells exhibit an inverse regulation of parathyroid hormone (PTh) secretion by extracellular Ca2+ ([Ca2+]0) that is essential for Ca2+ homeostasis. The recently characterized CA2 sensing receptor (CaR) mediates [Ca2+]0 regulation of PTH secretion. The steps along the secretory pathway regulated by the CaR are not known, and the molecular events that mediate inhibition of PTH secretion are not well defined. A better understanding of the intracellular events that regulate PTh secretion will provide a framework to study abnormal states of PTh secretion that contribute to the pathophysiology of disorders of mineral ion metabolism. The studies outlined in this proposal will help define the steps along the secretory pathway from the trans Golgi network to the plasma membrane that are regulated by [Ca2+]0. Our specific goals examine, with a combination of biochemical and cell biological imaging techniques, the regulation of mature secretory granule release, the fate of immature vesicles containing PTH and the secretion of vesicles loaded with a marker of constitutive secretion. The regulatory role of CaR-regulated second messengers will be tested on each of these vesicle traffic events. We will also concentrate our efforts in developing markers to visualize the pathways of protein and membrane release as they are regulated by changes in [Ca2+]0. The research course outlined by this proposal gives me the opportunity to develop an expertise in parathyroid physiology as I receive critical training in molecular biology, analysis of signal transduction pathways and techniques in intracellular imaging that were not stressed during my.Ph.D. training. This extensive training is essential- to become a competitive independent investigator in parathyroid biology. It will also provide an adequate vehicle to enrich my current skills with the recent technical developments in the field of secretion and its regulation. Dr. Edward Brown's laboratory has an extensive experience in the field of parathyroid physiology, molecular biology and signal transduction pathways, making it an ideal setting to combine acquired skills with new techniques. My long term plan is to develop a research program devoted to the identification and characterization of molecular components involved in membrane traffic effected by CaR-regulated second messengers in parathyroid and other cells.