Novel Molecular Insights into Stimulus-secretion Coupling in the Adrenal Medulla

Abstract

The adrenal medulla is an important branch of the sympathetic nervous system. Chromaffin cells, which serve as the secretory units of the medulla, receive direct sympathetic innervation via the splanchnic nerves. Secretion from the medulla is primarily caused by two neurotransmitters, acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP), released from splanchnic nerve terminals. ACh-stimulated secretion is associated with basal sympathetic tone; on the other hand, secretion stimulated by PACAP is associated with heightened sympathetic tone and the fight-or-flight response. The overarching goal of this dissertation was to test the hypothesis that the different intracellular mechanisms activated by ACh and PACAP regulate chromaffin cell fusion characteristics. The experiments described in this thesis sought to further characterize the intracellular signaling pathways of PACAP stimulated secretion and the mechanisms underlying regulation of granule cell secretion in adrenomedullary chromaffin cells by the calcium sensing proteins, synaptotagmins. Specifically, these studies used TIRF microscopy and primary mouse chromaffin cells from transgenic mouse lines to determine differences in fusion characteristics and identify intracellular proteins involved in exocytosis. Fluorescent cargo proteins and calcium indicators were utilized to monitor dense core granule fusion and intracellular calcium levels after stimulation with endogenous secretagogues, ACh and PACAP. The synaptotagmins syt-1 and syt-7 are the two major calcium sensors for exocytosis in adrenal chromaffin cells and are important in triggering calcium mediated exocytosis in chromaffin cells. Though the importance of synaptotagmins in the adrenomedullary system is appreciated, there are questions remaining as to the localization and function in mediated discharge of dense core granule cargos in response to physiological stimulation. To assess the subcellular localization of synaptotagmins in chromaffin cells, we used immunocytochemistry and subcellular fractionation, which showed that syt-7 is distributed in organelles, including dense core granules. TIRF imaging demonstrated that syt-7 -/- cells stimulated by ACh had lower probability of release and rapid fusion kinetics compared to WT chromaffin cells. The use of a reconstituted system employing cell-derived granules expressing either syt-1 or syt-7 allowed us to observe kinetic properties and calcium sensitivities of the synaptotagmins. In these studies, syt-7 had a greater calcium sensitivity and slowed the rate at which cargos are released compared to syt-1. Altogether, this study demonstrated that the high calcium sensitivity of syt-7 and its effects on fusion pore expansion are necessary for chromaffin cells to secrete normally to cholinergic stimulation. Chapter III of this thesis centered on determining the intracellular signaling components of PACAP-stimulated secretion. It is known that PACAP elicits secretion from chromaffin cells by binding to PAC1 receptors and activating a Gαs-coupled signaling pathway. However, the specific role of proteins downstream of Gαs in causing exocytosis are not well understood. This study was undertaken to fill this gap in our understanding. Using a combination of genetic animal models and TIRF-based imaging of secretion, it is shown that cells lacking phospholipase C epsilon (PLCε) – a protein whose activity is stimulated by the small GTPase Rap – exhibit neither PACAP-stimulated increases in intracellular Ca2+ nor exocytosis. These data establish a novel role for PLCε in a signaling cascade that couples PAC1 receptor activation to chromaffin cell secretion. Overall, the work presented in this thesis suggests that the differing intracellular signaling mechanisms between ACh and PACAP stimulation regulate fusion characteristics from chromaffin cells. Additionally, that synaptotagmins endow granules with distinct fusion properties.