The long-term goal of this project is to describe the molecular events that mediate the inhibition of parathyroid hormone (PTH) secretion following calcium-sensing receptor (CaR) activation in parathyroid cells. Inhibition of secretion occurs despite a concomitant CaR-mediated rise in intracellular calcium, a step normally associated with stimulation of secretory granule exocytosis in other endocrine cells. Acute elevations in extracellular calcium cause a rapid (< 1 min) inhibition of PTH secretion, suggesting that late stages of the secretory pathway, possibly involving the priming, docking or fusion of secretory granules with the plasma membrane, are the likely targets for inhibition in parathyroid cells. The overall hypothesis for this research proposal is that parathyroid-specific factor(s), regulated by CaR activation, inhibit the normal stimulus-secretion coupling associated with exocytosis of secretory granules in parathyroid cells. An experimental approach based on the ability to reconstitute in vitro the inhibitory effect of parathyroid-derived factors on the late stages of secretion in a permeabilized cell will be used to achieve the following specific aims: 1) To isolate and identify parathyroid-specific factors that mediate The inhibition of secretory granule exocytosis. Various biochemical separation methods will be applied to identify the factor(s) present in a crude parathyroid extract shown to block the exocytosis of secretory granules. 2) To determine the role of these parathyroid-specific factors in the inhibition of secretory vesicle exocytosis in both the regulated and constitutive pathway. Specifically, the role of these factors in preventing the priming of secretory vesicles for exocytosis will be explored, including the possibility that the cytoskeleton is a potential target for these parathyroid-derived factors. This research effort will help define regulatory mechanisms that inhibit intracellular membrane traffic in secretory cells and identify molecular targets for the treatment of medical disorders with abnormal hormone secretion.
