Ryanodine Receptor in Pancreatitis

描述(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。腺泡细胞钙信号异常在本病的发病机制中起着至关重要的作用。我们以前已经证明，基底区异常升高的钙信号与病理性腺泡内蛋白水解酶的激活有关，这是胰腺炎发展过程中的早期和关键事件。这种钙信号是由内质网(ER)钙通道--兰尼定受体(RyR)介导的。在这项建议中，我们研究了腺泡细胞中这种病理性RyR钙释放的调节机制。我们已经证明，腺泡细胞中cAMP的增加会导致RyR磷酸化，RyR钙释放，并增强蛋白酶激活1，2。在初步工作中，我们证明了酒精是胰腺炎的主要原因，触发了cAMP介导的RyR磷酸化以及RyR钙释放和增强的蛋白酶激活。因此，我们假设腺泡内蛋白水解酶的激活和胰腺炎，特别是酒精暴露引起的胰腺炎，是由病理激活的、磷酸化的RyRs释放的腺泡细胞钙触发的。在这项建议中，我们将结合新的遗传学和药理学工具，在酒精存在或不存在的情况下，追求以下特定目的：(1)检测RyR磷酸化是否导致分离的腺泡中RyR钙释放(2)研究RyR磷酸化和RyR钙释放是否易于在分离的腺泡中激活蛋白水解酶(3)确定RyR磷酸化和RyR钙释放是否在体内易导致蛋白酶激活和胰腺炎。被认为介导依赖PKA的RyR钙释放的RyR位点上的磷酸化的影响将通过先前产生的在RyR PKA磷酸化位点上存在磷酸模拟突变或磷酸抗性突变的转基因小鼠来直接测试。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)促进对胰腺炎中异常的钙信号的理解，(2)提供酒精和RyR钙释放之间的新的联系，这可能对酒精在多个其他器官系统中引起并发症的机制具有更广泛的影响，以及(3)建议针对胰腺中的RyR的治疗策略。与公共卫生相关：急性胰腺炎是一种威胁生命的胰腺疼痛疾病，始于自身消化酶的自动消化。酒精性疾病是这种疾病的主要原因。这种病理性消化需要胰腺内的钙信号，因此我们的研究集中在了解通过一种称为兰尼定受体的蛋白钙通道异常释放钙在自身消化和所致胰腺炎的发展中的作用，特别是与酒精暴露有关的作用。

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.