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: 7802973
• 负责人: David I Yule
• 金额: $ 29.16万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7802973
• 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）：分泌刺激后的[Ca2+] i的升高在外分泌胰腺的消化酶分泌方面起着基本作用。 [Ca2+] I的增加是一个严格调节的事件，并且表现出特征性的时间和空间图案，这对于适当的外分泌功能绝对必需。值得注意的是，Ca2+信号的破坏是胰腺炎模型中的早期事件。这些Ca2+信号特性的主要决定因素是肌醇1,4,5-三磷酸受体（INSP3R）的性质。最近的一项研究报告说，这是2型和3型INSP3R小鼠的消融，这是最好的说明，这是由于该信号传导途径的失败以及消化酶的分泌而导致死亡。与INSP3R-1形成鲜明对比的是，关于INSP3R-2/3-显然对胰腺外分泌功能至关重要的特定生物物理和电生理特性的信息很少。因此，我们将定义这些受体的基本特性，从而研究其个人对腺泡细胞信号传导的贡献。将要解决的具体目的将通过细胞内ATP（AIM 2）和PKA磷酸化（AIM 3）调查Ca2+（AIM 1）对INSP3R-2和INSP3R-3的调节。生物物理数据将集成到INSP3R和外分泌功能的数学模型中。将使用全细胞斑块钳通过在质膜中利用INSP3R的表达来研究每个受体的单通道电生理特性，以孤立的一系列表达INSP3R-2或INSP3R-3和突变构建体的稳定细胞系的质膜表达。这些数据将通过研究在ER膜中通过对野生型或INSP3R-2无动物的分离胰腺核的制剂的核核片的研究来补充。 CA2+释放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。该提案将对控制CA2+信号传导的基本过程以及外分泌胰腺的正常功能提供详细的理解。研究的长期目标是由一个核心观念驱动的，即对正常功能的详细知识对于了解胰腺疾病状态中这些机制的破坏是绝对必要的，因此是为了最终开发新的疾病治疗策略。 公共卫生相关性：胰腺外分泌细胞的主要功能是分泌消化酶以消化食物。导致分泌的关键事件是细胞内部自由钙浓度的升高。在胰腺疾病状态等急性胰腺炎等疾病状态，正常的钙升高受到干扰。肌醇三磷酸受体（INSP3R）位于细胞的特定亚细胞区域，这在很大程度上是钙的增加的原因。最近的一项研究清楚地表明了INSP3R的重要性，该研究表明，由于没有分泌消化酶的分泌，因此在基因工程上缺乏胰腺中存在的INSP3R形式。但是，关于这些形式的INSP3R的详细信息很少。因此，在此提案中，我们将研究这些特定受体对胰腺功能至关重要的特定特性和调节。这些研究的长期目标是对这些过程提供一定程度的理解，这些过程最终将有助于设计胰腺疾病的新型治疗策略。