| The pivotal role of calcium (Ca2+) in regulating exocrine epithelial cell function under normal and pathophysiological conditions is well established; however, the molecular changes that occur in response to elevated Ca2+ are largely unknown. This dissertation is aimed at understanding how changes in cytosolic Ca2+ modulate protein secretion from digestive secretory cells. Ca2+-responsive heat-stable protein (CRHSP-28) is a key regulatory molecule in the secretory pathway of pancreatic acinar cells. This dissertation addresses the intracellular signaling mechanisms that regulate CRHSP-28 function following stimulation by gastrointestinal hormones and neurotransmitters. The first Specific Aim was designed to establish an immortalized cultured-cell model that could be used to characterize changes in CRHSP-28 phosphorylation and secretory function. The second Specific Aim tested the hypothesis that CRHSP-28 phosphorylation occurs under physiological conditions, and may be used a molecular indicator of Ca2+-signalling in vivo. In the third Specific Aim, studies were conducted to test if the Ca2+-calmodulin regulated protein kinase II (CaMKII) catalyzed CRHSP-28 phosphorylation in the cell and to determine the specific sites on CRHSP-28 that are phosphorylated in response to secretagogue-stimulation.; Findings establish that CRHSP-28 is present in a cultured colonic secretory epithelial cell line, T84. Phosphorylation of CRHSP-28 is regulated by G protein-coupled receptor activation and mediated exclusively through transient increases in cell Ca2+ concentrations. Additionally, data support that CRHSP-28 is regulated by a CaMKII-like enzyme in vivo, as substantiated by the following methods: (1) in vitro phosphorylation studies of wild type CRHSP-28 and a CaMKII-consensus-site CRHSP-28 phospho-mutant, (2) phosphopeptide mapping analysis of in situ- and in vitro phosphorylated CRHSP-28, and (3) the observation that loss of CaMKII localization at the subapical actin-cytoskeleton inhibited CRHSP-28 phosphorylation in response to cell stimulation. The cellular regulation of CRHSP-28 phosphorylation was clearly established as a physiologically relevant event as it was directly coupled to the consumption of a meal in fasted animals. Based on reports that CRHSP-28 interacts in a Ca2+-dependent manner with regulatory proteins within the endocytic-exocytic cycle, we propose that CRHSP-28 phosphorylation plays a key role in the vesicular trafficking of protein and phospholipids within exocrine epithelia. |
