An elevation in the [Ca2+]i following secretagogue stimulation plays a fundamental role in underlying digestive enzyme secretion from the exocrine pancreas. The increase in [Ca2+]i is a tightly regulated event and exhibits characteristic temporal and spatial patterning which is absolutely required for appropriate exocrine function. Of note, disruption of Ca2+ signaling occurs as an early event in models of pancreatitis. A major determinant of the characteristics of these Ca2+ signals is through the properties of inositol 1,4,5-trisphosphate receptors (InsP3R). This is best illustrated by a recent study reporting that ablation in mice of the type-2 and type 3 InsP3R resulted in death because of failure of this signaling pathway and thus of digestive enzyme secretion. In stark contrast to the InsP3R-1, little information is available regarding the specific biophysical and electrophysiological properties of InsP3R-2/3- obviously crucial to pancreatic exocrine function. We will therefore define the fundamental properties of these receptors and thus investigate their individual contributions to acinar cell signaling.
The specific aims that will be addressed will investigate the regulation of InsP3R-2 and InsP3R-3 by Ca2+ (aim 1) by intracellular ATP (aim 2) and following PKA phosphorylation (aim 3). The biophysical data will be integrated into mathematical models of InsP3R and exocrine function. The single-channel electrophysiological properties of each receptor will be studied using whole-cell patch-clamp by exploiting the expression of InsP3R in the plasma membrane of a series of stable cell lines expressing InsP3R-2 or InsP3R-3 and mutant constructs in isolation. These data will be complemented by study of the properties of native InsP3R expressed in the ER membrane by on-nucleus patching of preparations of isolated pancreatic nuclei from wild-type or InsP3R-2 null animals. Ca2+ release will be monitored using a unidirectional flux assay in permeabilized cells and by digital imaging in intact cells. The proposal will provide a detailed understanding of the fundamental processes controlling Ca2+ signaling and thus normal function of the exocrine pancreas. The long term goal of the studies is driven by the central idea that a detailed knowledge of normal function is absolutely necessary to understanding how these mechanisms are disrupted in pancreatic disease states and thus for the ultimate development of novel strategies for the treatment of disease. Public Health Relevance: The primary function of the exocrine cells of the pancreas is to secrete digestive enzymes to digest food following a meal. The key event which results in the secretion is an elevation in the free calcium concentration inside the cell. In disease states of the pancreas like acute pancreatitis, the normal calcium elevation is disrupted. The inositol trisphosphate receptor (InsP3R) localized to specific subcellular regions of the cell is largely responsible for this increase in calcium. The importance of the InsP3R is clearly shown by a recent study which demonstrated that mice genetically engineered to lack the forms of InsP3R present in the pancreas died because no digestive enzymes were secreted. Very little detailed information is available however regarding these forms of the InsP3R. In this proposal we will therefore investigate the specific properties and regulation of these particular receptors vitally important for pancreatic function. The long term goal of these studies is to provide a level of understanding of these processes that will ultimately aid in the design of novel therapeutic strategies for the treatment of pancreatic disease.
|Wagner 2nd, Larry E; Yule, David I (2012) Differential regulation of the InsP? receptor type-1 and -2 single channel properties by InsP?, Ca²? and ATP. J Physiol 590:3245-59|
|Won, Jong Hak; Zhang, Yu; Ji, Baoan et al. (2011) Phenotypic changes in mouse pancreatic stellate cell Ca2+ signaling events following activation in culture and in a disease model of pancreatitis. Mol Biol Cell 22:421-36|
|Park, Keigan M; Yule, David I; Bowers, William J (2010) Impaired TNF-alpha control of IP3R-mediated Ca2+ release in Alzheimer's disease mouse neurons. Cell Signal 22:519-26|
|Betzenhauser, Matthew J; Wagner 2nd, Larry E; Park, Hyung Seo et al. (2009) ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs. J Biol Chem 284:16156-63|
|Betzenhauser, Matthew J; Fike, Jenna L; Wagner 2nd, Larry E et al. (2009) Protein kinase A increases type-2 inositol 1,4,5-trisphosphate receptor activity by phosphorylation of serine 937. J Biol Chem 284:25116-25|
|Betzenhauser, Matthew J; Wagner 2nd, Larry E; Iwai, Miwako et al. (2008) ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action. J Biol Chem 283:21579-87|
|Park, Hyung Seo; Betzenhauser, Matthew J; Won, Jong Hak et al. (2008) The type 2 inositol (1,4,5)-trisphosphate (InsP3) receptor determines the sensitivity of InsP3-induced Ca2+ release to ATP in pancreatic acinar cells. J Biol Chem 283:26081-8|
|Betzenhauser, Matthew J; Wagner 2nd, Larry E; Won, Jong Hak et al. (2008) Studying isoform-specific inositol 1,4,5-trisphosphate receptor function and regulation. Methods 46:177-82|
|Brown, David A; Yule, David I (2007) Protein kinase C regulation of P2X3 receptors is unlikely to involve direct receptor phosphorylation. Biochim Biophys Acta 1773:166-75|
|Won, Jong Hak; Cottrell, William J; Foster, Thomas H et al. (2007) Ca2+ release dynamics in parotid and pancreatic exocrine acinar cells evoked by spatially limited flash photolysis. Am J Physiol Gastrointest Liver Physiol 293:G1166-77|
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