Pancreatic Function: G-Protein Mediated Ca2+ Signaling

描述（由申请人提供）：促分泌素刺激后[Ca 2 +]i升高在胰腺外分泌的潜在消化酶分泌中起重要作用。[Ca 2 +]i的增加是一个严格调节的事件，并表现出特征性的时间和空间模式，这是绝对需要适当的外分泌功能。值得注意的是，Ca 2+信号传导的中断在胰腺炎模型中作为早期事件发生。这些Ca 2+信号的特征的主要决定因素是通过肌醇1，4，5-三磷酸受体（InsP 3R）的性质。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InsP 3R在小鼠中的消融导致死亡，因为这种信号传导途径失败，因此消化酶分泌失败。与InsP 3R-1形成鲜明对比的是，关于InsP 3R-2/3的特定生物物理学和电生理学特性的信息很少-InsP 3R-2/3对胰腺外分泌功能显然至关重要。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单独贡献。将解决的具体目标将研究Ca 2+（目的1）、细胞内ATP（目的2）和PKA磷酸化（目的3）对InsP 3R-2和InsP 3R-3的调节。生物物理数据将被整合到InsP 3R和外分泌功能的数学模型中。将使用全细胞膜片钳通过利用InsP 3R在一系列表达InsP 3R-2或InsP 3R-3的稳定细胞系的质膜中的表达以及分离的突变体构建体来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InsP 3R-2无效动物的分离的胰腺细胞核的制备物的核上修补来研究ER膜中表达的天然InsP 3R的性质来补充。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Ca 2+释放。该提案将提供控制Ca 2+信号传导的基本过程的详细了解，从而外分泌胰腺的正常功能。这些研究的长期目标是由中心思想驱动的，即对正常功能的详细了解对于了解这些机制在胰腺疾病状态下如何被破坏以及最终开发治疗疾病的新策略是绝对必要的。 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，以便在餐后消化食物。导致分泌的关键事件是细胞内游离钙浓度的升高。在胰腺的疾病状态下，如急性胰腺炎，正常的钙升高被破坏。位于细胞特定亚细胞区域的三磷酸肌醇受体（InsP 3R）主要负责钙的增加。最近的一项研究清楚地表明了InsP 3R的重要性，该研究表明，基因工程改造缺乏胰腺中存在的InsP 3R形式的小鼠死亡，因为没有分泌消化酶。然而，关于InsP 3R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特定特性和调节。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。

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.