Pancreatic Function: G-Protein Mediated Ca2+ Signaling

胰腺功能：G 蛋白介导的 Ca2 信号转导

• 批准号: 7905591
• 负责人: David I Yule
• 金额: $ 9.97万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7905591
• 关键词: Ablation; Acinar Cell; Address; Animals; Biological Assay; Calcium; Cell Nucleus; Cell membrane; Cells; Cessation of life; Characteristics; Complement; Cyclic AMP-Dependent Protein Kinases; Data; Development; Disease; Enzymes; Event; Exhibits; Exocrine pancreas; Exocrine pancreatic insufficiency; Exocytosis; Failure; Food; GTP-Binding Proteins; Genetically Engineered Mouse; Goals; Hormonal; Individual; Inositol; Knockout Mice; Knowledge; Mediating; Membrane; Metabolic; Modeling; Monitor; Mus; Neurons; Pancreas; Pancreatic Diseases; Pancreatic Exocrine Secretion; Pancreatitis; Pattern; Phosphorylation; Physiology; Play; Preparation; Process; Property; Proteins; Regulation; Reporting; Role; Series; Shapes; Signal Pathway; Signal Transduction; Site; Stimulus; System; Testing; Tissues; Weaning; Weight Gain; acute pancreatitis; design; digital imaging; mathematical model; mutant; novel strategies; novel therapeutics; patch clamp; public health relevance; receptor; receptor binding; receptor function; relating to nervous system; stable cell line; treatment strategy

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.

描述（由申请人提供）：促分泌素刺激后 [Ca2+]i 的升高在外分泌胰腺的潜在消化酶分泌中起着重要作用。 [Ca2+]i 的增加是一个严格调控的事件，并表现出特征性的时间和空间模式，这对于适当的外分泌功能是绝对必需的。值得注意的是，Ca2+信号传导的破坏是胰腺炎模型中的早期事件。这些 Ca2+ 信号特征的主要决定因素是肌醇 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，该研究报告称，2 型和 3 型 InsP3R 的消融导致小鼠死亡，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学特性的信息很少，这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。将要解决的具体目标将研究 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。将通过利用一系列表达 InsP3R-2 或 InsP3R-3 的稳定细胞系和分离的突变体构建体的质膜中 InsP3R 的表达，使用全细胞膜片钳研究每种受体的单通道电生理特性。这些数据将通过对来自野生型或 InsP3R-2 无效动物的分离胰腺细胞核制剂进行核上修补来研究 ER 膜中表达的天然 InsP3R 的特性来补充。将使用透化细胞中的单向通量测定和完整细胞中的数字成像来监测 Ca2+ 释放。该提案将详细了解控制 Ca2+ 信号传导的基本过程，从而控制外分泌胰腺的正常功能。这些研究的长期目标是由以下中心思想驱动的：对正常功能的详细了解对于理解这些机制在胰腺疾病状态下如何被破坏是绝对必要的，从而最终开发出治疗疾病的新策略。 公共健康相关性：胰腺外分泌细胞的主要功能是分泌消化酶来消化餐后的食物。导致分泌的关键事件是细胞内游离钙浓度的升高。在急性胰腺炎等胰腺疾病状态下，正常的钙升高被破坏。位于细胞特定亚细胞区域的肌醇三磷酸受体（InsP3R）是钙含量增加的主要原因。最近的一项研究清楚地表明了 InsP3R 的重要性，该研究表明，经过基因改造而缺乏胰腺中 InsP3R 形式的小鼠会因不分泌消化酶而死亡。然而，关于 InsP3R 的这些形式的详细信息非常少。因此，在本提案中，我们将研究这些对胰腺功能至关重要的特定受体的具体特性和调节。这些研究的长期目标是提供对这些过程的一定程度的理解，最终有助于设计治疗胰腺疾病的新治疗策略。