Hormone Regulation of [Ca2+] in Pancreatic Acinar Cells

胰腺腺泡细胞中[Ca2]的激素调节

• 批准号: 7905573
• 负责人: Shmuel Muallem
• 金额: $ 7.68万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-05 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7905573
• 关键词: Acinar Cell; Address; Affect; Agonist; Alcohols; Amylases; Apoptosis; Bile Acids; Binding; Caerulein; Caspase; Cell Death; Cell membrane; Cell model; Cell physiology; Cells; Cellular Stress; Charge; Complex; Disease; Dyes; Electrostatics; Environment; Enzymes; Esters; Exocytosis; Functional disorder; Gene Expression; Genes; Goals; Hormones; In Vitro; Individual; Inflammation; Ions; Isotonic Exercise; Knockout Mice; Knowledge; Lactate Dehydrogenase; Liquid substance; Measures; Mediating; Modeling; Molecular; Mus; Neurotransmitters; Nodal; Pancreas; Pancreatitis; Patients; Physiological; Process; Protein Biosynthesis; Regulation; Reporter; Role; STIM1 gene; Second Messenger Systems; Signal Transduction; Stress; System; Time; Toxin; acute pancreatitis; cell injury; extracellular; hormone regulation; palmitoleic acid; public health relevance; receptor; response; scaffold; second messenger; sensor

DESCRIPTION (provided by applicant): The chief function of pancreatic acinar cells is the secretion of digestive enzymes and a small amount of isotonic fluid. Both functions are regulated by hormones and neurotransmitters that use Ca2+ as the second messenger. At the same time, aberrant Ca2+ signaling is the nodal point in all forms of pancreatitis. The hypothesis of this proposal is that specifically aberrant Ca2+ influx by store-operated Ca2+ channels (SOCs) is responsible for pancreatitis. Therefore, understanding regulation and function of Ca2+ influx channels is critical for understanding acinar cell function and dysfunction in the context of pancreatitis. Acinar cells have several Ca2+ influx channels, TRPC1, TRPC3, TRPC4, TRPC6 and Orai1, all of which are gated by the ER Ca2+ sensor STIM1. TRPCs are also gated by the scaffold Homer1 that inhibits their activity. Considering the involvement of SOCs in pancreatitis, the overall goal of this proposal is to understand gating mechanism of the pancreatic SOCs by STIM1 and Homer1 and their role in pancreatitis. This will be achieved in four aims: Aim 1: Decipher the molecular mechanism of TRPCs gating by STIM1 and Homer1. Homer1 interacts with a PPXF motif to keep the channels in a close state. STIM1 opens the TRPCs by interacting with two conserved residues that are only 4 aresidues upstream of the Homer1 binding motif. We propose to determine a) the gating mechanism of TRPCs by STIM1; b) how Homer1 and STIM1 function in tandem to gate the TRPCs. Aim 2: Determine molecular mechanism of Orai1 gating by STIM1. Agonist-stimulated pancreatic Ca2+ influx is mediated by a combination of TRPCs and Orai1 that are differentially gated by STIM1. We discovered that the STIM1(234-535) fragment is sufficient for full activation of Orai1. Hence we will: a) determine the minimal STIM1 domain that gates Orai1; b) how STIM1 opens Orai1; c) what is the relationship between the NATIVE TRPCs and NATIVE Orai1. Aim 3: The role of TRPCs, Orai1 and STIM1 in physiological and pathological acinar cells Ca2+ signaling and exocytosis will be determined by characterizing Ca2+ signaling and exocytosis in acinar cells from TRPC1-/-, TRPC3-/-, TRPC6-/-, Orai1-/- and STIM1-/- mice. Aim 4: The role of TRPC channels, Orai1, STIM1 will be studies in vitro-induced pancreatitis and measure cellular reporters of cell stress and cell damage. Then the KO mice will be used to evaluate the role of the Ca2+ influx channels in careluin and bile acid models of pancreatitis. PUBLIC HEALTH RELEVANCE: The pancreas secretes digestive enzymes and fluid in response to stimulation by hormones and neurotransmitters. The stimulants tell the pancreatic cells how to do so by changing the concentration of Ca2+ ions within the cells. Most of the Ca2+ enters the cells from the extracellular environment. When this process is aberrant, the digestive enzymes remain trapped within the cells and the cells dye and the patient develop the disease called pancreatitis. The aims of this proposal are to understand how Ca2+ ions enter the cells and how the aberrant Ca2+ entry causes the pancreatitis. This will be achieved by studying Ca2+ entry into the cells in model cell systems and in the cells of the pancreas of mice from which the genes that are responsible for the entry of Ca2+ into the cells have been deleted.

描述（申请人提供）：胰腺腺泡细胞的主要功能是分泌消化酶和少量等渗液。这两种功能均由使用 Ca2+ 作为第二信使的激素和神经递质调节。同时，异常的 Ca2+ 信号传导是所有形式胰腺炎的关键。该提议的假设是，钙库操纵的 Ca2+ 通道 (SOC) 异常的 Ca2+ 流入是胰腺炎的原因。因此，了解 Ca2+ 流入通道的调节和功能对于了解胰腺炎中腺泡细胞的功能和功能障碍至关重要。腺泡细胞有多个 Ca2+ 流入通道：TRPC1、TRPC3、TRPC4、TRPC6 和 Orai1，所有这些通道均由 ER Ca2+ 传感器 STIM1 门控。 TRPC 还受到抑制其活性的支架 Homer1 的门控。考虑到SOCs参与胰腺炎，本提案的总体目标是了解STIM1和Homer1对胰腺SOCs的门控机制及其在胰腺炎中的作用。这将通过四个目标来实现： 目标 1：破译 STIM1 和 Homer1 门控 TRPC 的分子机制。 Homer1 与 PPXF 基序相互作用，使通道保持关闭状态。 STIM1 通过与 Homer1 结合基序上游仅 4 个残基的两个保守残基相互作用来打开 TRPC。我们建议通过 STIM1 确定 a) TRPC 的门控机制； b) Homer1 和 STIM1 如何协同作用来门控 TRPC。目标 2：确定 STIM1 门控 Orai1 的分子机制。激动剂刺激的胰腺 Ca2+ 内流是由 TRPC 和 Orai1 的组合介导的，而 TRPC 和 Orai1 则由 STIM1 进行差异门控。我们发现 STIM1(234-535) 片段足以完全激活 Orai1。因此，我们将： a) 确定门控 Orai1 的最小 STIM1 域； b) STIM1如何打开Orai1； c) NATIVE TRPC 和 NATIVE Orai1 之间的关系是什么。目标 3：通过表征 TRPC1-/-、TRPC3-/-、TRPC6-/-、Orai1-/- 和 STIM1-/- 小鼠腺泡细胞中的 Ca2+ 信号传导和胞吐作用，确定 TRPC、Orai1 和 STIM1 在生理和病理性腺泡细胞 Ca2+ 信号传导和胞吐作用中的作用。目标 4：TRPC 通道、Orai1、STIM1 的作用将在体外诱导的胰腺炎中进行研究，并测量细胞应激和细胞损伤的细胞报告基因。然后使用 KO 小鼠评估 Ca2+ 流入通道在胰腺炎卡鲁因和胆汁酸模型中的作用。公共卫生相关性：胰腺响应激素和神经递质的刺激而分泌消化酶和液体。兴奋剂通过改变细胞内 Ca2+ 离子的浓度来告诉胰腺细胞如何做到这一点。大部分Ca2+从细胞外环境进入细胞。当这个过程异常时，消化酶仍然被困在细胞内，细胞会染色，患者就会患上胰腺炎。该提案的目的是了解 Ca2+ 离子如何进入细胞以及异常的 Ca2+ 进入如何导致胰腺炎。这将通过研究 Ca2+ 进入模型细胞系统和小鼠胰腺细胞中的细胞来实现，其中负责 Ca2+ 进入细胞的基因已被删除。