Pancreatic Function: G-Protein Mediated Ca2+ Signaling

描述（由申请人提供）：促分泌剂刺激后[Ca2+]i升高在胰脏外分泌的消化酶分泌中起基础作用。[Ca2+]i的增加是一个严格调控的事件，并表现出一定的时间和空间模式，这是适当的外分泌功能所绝对需要的。值得注意的是，Ca2+信号的破坏发生在胰腺炎模型的早期事件。这些Ca2+信号特征的主要决定因素是通过肌醇1,4,5-三磷酸受体（InsP3R）的特性。最近的一项研究最好地说明了这一点，该研究报道了2型和3型InsP3R小鼠消融导致死亡，因为该信号通路失败，从而导致消化酶分泌失败。与InsP3R-1形成鲜明对比的是，关于InsP3R-2/3的特定生物物理和电生理特性的信息很少，而这些特性显然对胰腺外分泌功能至关重要。因此，我们将定义这些受体的基本特性，从而研究它们对腺泡细胞信号传导的个体贡献。具体目标将研究Ca2+（目标1）、细胞内ATP（目标2）和PKA磷酸化（目标3）对InsP3R-2和InsP3R-3的调节。生物物理数据将被整合到InsP3R和外分泌功能的数学模型中。每个受体的单通道电生理特性将使用全细胞膜片钳，通过在分离表达InsP3R-2或InsP3R-3和突变构建的一系列稳定细胞系的质膜中利用InsP3R的表达来研究。这些数据将通过对野生型或无InsP3R-2动物的离体胰腺细胞核进行核补片来研究内质膜中表达的天然InsP3R的特性来补充。Ca2+释放将监测使用单向通量测定渗透细胞和数字成像完整的细胞。该提案将提供一个详细的了解控制Ca2+信号的基本过程，从而正常功能的外分泌胰腺。研究的长期目标是由核心思想驱动的，即对正常功能的详细了解对于理解这些机制如何在胰腺疾病状态中被破坏是绝对必要的，因此对于疾病治疗的新策略的最终发展是必要的。

DESCRIPTION (provided by applicant): 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.