Ryanodine Receptor in Pancreatitis

描述（由申请人提供）：急性胰腺炎是一种常见的危及生命的胰腺疾病。异常的腺泡细胞Ca2+信号在引发这种疾病中起关键作用。我们之前已经表明，基底区异常升高的Ca2+信号与病理性腺泡内蛋白酶激活有关，这是胰腺炎发展的早期和关键事件。这种Ca2+信号是由内质网（ER） Ca2+通道，即ryanodine受体（RyR）介导的。在本提案中，我们研究了调节腺泡细胞中这种病理性RyR Ca2+释放的机制。我们已经证明，在腺泡细胞中增加cAMP会导致RyR磷酸化、RyR Ca2+释放和蛋白酶激活增强1,2。在初步工作中，我们证明了酒精，胰腺炎的主要原因，触发camp介导的RyR磷酸化以及RyR Ca2+释放和增强蛋白酶激活。因此，我们假设腺泡内蛋白酶激活和胰腺炎，特别是由酒精暴露引起的胰腺炎，是由病理激活的磷酸化RyRs释放的腺泡细胞Ca2+触发的。在这项提议中，我们将使用新的遗传和药理学工具的组合，在酒精存在或不存在的情况下，追求以下具体目标：(1)检查RyR磷酸化是否导致分离的腺泡细胞中RyR Ca2+释放(2)研究RyR磷酸化和RyR Ca2+释放是否易导致分离的腺泡细胞蛋白酶激活(3)确定RyR磷酸化和RyR Ca2+释放是否易导致体内蛋白酶激活和胰腺炎。磷酸化对RyR位点的影响被认为介导PKA依赖的RyR Ca2+释放，将直接使用先前产生的转基因小鼠进行测试，这些小鼠在RyR PKA磷酸化位点中含有模仿磷酸化或耐磷酸化突变。预计这些关于RyR在病理性蛋白酶激活和胰腺炎中的作用的研究将(1)提高对胰腺炎中异常Ca2+信号的理解，(2)提供酒精和RyR Ca2+释放之间的新联系，这可能对酒精在其他多个器官系统中的并发症的机制有更广泛的影响，(3)提出针对胰腺中RyR的治疗策略。公共卫生相关性：急性胰腺炎是一种危及生命和痛苦的胰腺疾病，始于自身消化酶的自动消化。酒精性疾病是导致这种疾病的主要原因。胰腺内的钙信号是这种病理性消化所必需的，因此我们的研究重点是了解钙通过一种称为ryanodine受体的蛋白钙通道异常释放在自动消化和由此导致的胰腺炎发展中的作用，特别是对酒精暴露。

DESCRIPTION (provided by applicant): Acute pancreatitis is a common, life-threatening disorder of the pancreas. Abnormal acinar cell Ca2+ signals play a crucial role in initiating this disease. We have previously shown that abnormally elevated Ca2+ signals in the basal region are associated with pathologic intra-acinar protease activation, an early and critical event in the development of pancreatitis. This Ca2+ signal is mediated by an endoplasmic reticulum (ER) Ca2+ channel, the ryanodine receptor (RyR). In this proposal, we examine mechanisms that regulate this pathologic RyR Ca2+ release in the acinar cell. We have shown that increasing cAMP in acinar cells causes RyR phosphorylation, RyR Ca2+ release, and enhanced protease activation1, 2. In preliminary work, we demonstrate that alcohol, a leading cause of pancreatitis, triggers cAMP-mediated RyR phosphorylation as well as RyR Ca2+ release and enhanced protease activation. Therefore, we hypothesize that intra-acinar protease activation and pancreatitis, particularly that induced by alcohol exposure, are triggered by release of acinar cell Ca2+ from pathologically activated, phosphorylated RyRs. In this proposal, using a combination of novel genetic and pharmacologic tools, we will, in the presence or absence of alcohol, pursue the following Specific Aims: (1) Examine whether RyR phosphorylation causes RyR Ca2+ release in isolated acini (2) Study whether RyR phosphorylation and RyR Ca2+ release predispose to protease activation in isolated acini (3) Determine whether RyR phosphorylation and RyR Ca2+ release predispose to protease activation and pancreatitis in vivo. The effects of phosphorylation on RyR sites thought to mediate PKA-dependent RyR Ca2+ release will be directly tested using previously generated transgenic mice that harbor phospho-mimetic or phospho-resistant mutations in the RyR PKA phosphorylation site. It is anticipated that these studies on the role of the RyR in pathologic protease activation and pancreatitis will (1) lead to improved understanding of aberrant Ca2+ signaling in pancreatitis, (2) provide a novel link between alcohol and RyR Ca2+ release that may have broader implications for mechanisms contributing to alcohol's complications in multiple other organ systems, and (3) suggest treatment strategies that target the RyR in the pancreas. PUBLIC HEALTH RELEVANCE: Acute pancreatitis is a life-threatening and painful disease of the pancreas that starts with auto-digestion by its own digestive enzymes. Alcoholic disease is a leading cause of this disease. Calcium signals within the pancreas are required for this pathologic digestion, and thus our studies are focused on understanding the role of abnormal release of calcium through a protein calcium channel called the ryanodine receptor in the development of auto-digestion and resultant pancreatitis, in particular vis-`-vis alcohol exposure.