Low ph alters intracellular and extracellular communication in the acinar cell

低 pH 值改变腺泡细胞的细胞内和细胞外通讯

基本信息

批准号：
8675227
负责人：
Anamika Maragret Reed
金额：
$ 15.42万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2016-01-25
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8675227
关键词：
Acidosis Acids Acinar Cell Acinus organ component Acute Biliary Biological Assay Calcium Signaling Cell membrane Cells Cholecystokinin Clinical Communication Complications of Diabetes Mellitus Confocal Microscopy Connexins Data Degradation Pathway Development Development Plans Digestive System Disorders Disease Dyes Endoplasmic Reticulum Environment Enzyme Precursors Epithelium Faculty Fluorescence Recovery After Photobleaching Frequencies Gap Junctions Goals ITPR1 gene In Vitro Infection Injection of therapeutic agent Injury Inorganic Sulfates Inositol Knockout Mice Laboratories Life Link Measures Mediating Mentorship Modeling Molecular Mono-S Movement Organelles Pancreas Pancreatic duct Pancreatitis Pathogenesis Pathologic Pattern Physicians Physiological Principal Investigator Research Activity Research Personnel Research Training Risk Role Ryanodine Receptor Calcium Release Channel Scientist Signal Transduction Small Interfering RNA Structure Taurolithocholic Acid Testing Therapeutic Training Universities Unspecified or Sulfate Ion Sulfates acute pancreatitis base biomedical facility career development cell injury connexin 32 effective therapy extracellular in vivo in vivo Model inhibitor/antagonist intercellular communication knowledge base prevent receptor response skills tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): This proposal describes a five year training plan with the goal of developing the candidate into an independent investigator. The principal investigator will expand the scientific background and skills she has already attained through her research activities in the laboratory of Dr. Fred Gorelick, an expert in the molecular mechanisms of pancreatitis. By implementing a structured research, training, and career development plan under the mentorship of Dr. Gorelick and Dr. Michael Nathanson, an expert in calcium signaling in digestive epithelia, the candidate will gain the analytic framework, technical abilities, and knowledge base to succeed as an independent physician-scientist. Yale University and the Section of Digestive Diseases, with their rich network of diverse faculty and ample biomedical facilities, provide an ideal environment to pursue this plan. The pathogenesis of acute pancreatitis, a common and life-threatening disease, is incompletely understood. Clinical conditions characterized by acute acid loads predispose to the development of acute pancreatitis. Recently, the Gorelick lab has shown that low pH sensitizes to pancreatitis responses in vitro and in vivo. However, the mechanisms responsible for this sensitization are unknown. This proposal hypothesizes that low pH exerts its injurious effects by transforming intracellular calcium signaling and gap junctional intercellular communication in acinar cells from a physiologic pattern to one that causes pancreatitis. The specific aims of this proposal are to: 1) Determine the effects of low pH on intracellular calcium signaling in the acinar cell 2) Determine the effects of low pH on intercellular communication and 3) Determine whether acid-induced pancreatitis responses are mediated by pathologic calcium signals and/or changes in gap junctional intercellular communication. To establish the effects of low pH on calcium signals, isolated acinar cells will be examined through confocal microscopy. Functional aspects of gap junctional communication will be investigated by quantifying the degree of calcium signal synchrony and by examining dye transfer through gap junctions. Structural aspects of gap junctional intercellular communication will be examined by characterizing the degradation patterns of the predominant gap junction protein, connexin32. Finally, the role of calcium signals and gap junctional communication in acid-induced injury will be examined using in vitro and in vivo models of pancreatitis. Our preliminary data indicates that low pH decreases oscillation frequency and increases oscillation amplitude in isolated acini treated with physiologic concentrations of the cholecystokinin orthologue, cerulein. Additionally, low pH inhibits intercellular communication and leads to decreased levels of connexin32. Finally, we have found that inhibition of the ryanodine receptor, which mediates both the changes in calcium signaling and intercellular communication, reduces acid-induced zymogen activation and cellular injury in vitro. By establishing a link between harmful effects of acidemia and intracellular calcium signaling and intercellular gap junctional communication in the acinar cell, therapeutic strategies to treat or prevent acidosis-associated pancreatitis could be developed.

描述（由申请人提供）：该提案描述了一个五年培训计划，旨在将候选人发展成为一名独立研究者。首席研究员将扩大她通过在胰腺炎分子机制专家 Fred Gorelick 博士实验室的研究活动所获得的科学背景和技能。通过在消化上皮细胞钙信号传导专家 Gorelick 博士和 Michael Nathanson 博士的指导下实施结构化研究、培训和职业发展计划，候选人将获得成功所需的分析框架、技术能力和知识基础作为一名独立的医师科学家。耶鲁大学和消化疾病科拥有丰富的多元化教师网络和充足的生物医学设施，为实施这一计划提供了理想的环境。急性胰腺炎是一种常见且危及生命的疾病，其发病机制尚不完全清楚。以急性酸负荷为特征的临床状况容易发生急性胰腺炎。最近，Gorelick 实验室表明，低 pH 值在体外和体内对胰腺炎反应敏感。然而，造成这种敏化的机制尚不清楚。该提议假设，低 pH 值通过将腺泡细胞中的细胞内钙信号传导和间隙连接细胞间通讯从生理模式转变为导致胰腺炎的模式来发挥其有害作用。该提案的具体目标是：1) 确定低 pH 对腺泡细胞内钙信号传导的影响 2) 确定低 pH 对细胞间通讯的影响 3) 确定酸诱导的胰腺炎反应是否由病理介导钙信号和/或间隙连接细胞间通讯的变化。为了确定低 pH 对钙信号的影响，将通过共聚焦显微镜检查分离的腺泡细胞。将通过量化钙信号同步程度和检查通过间隙连接的染料转移来研究间隙连接通讯的功能方面。将通过表征主要间隙连接蛋白 connexin32 的降解模式来检查间隙连接细胞间通讯的结构方面。最后，将使用胰腺炎的体外和体内模型来检查钙信号和间隙连接通讯在酸诱导损伤中的作用。我们的初步数据表明，在用生理浓度的胆囊收缩素同源物雨蛙蛋白处理的分离腺泡中，低pH值会降低振荡频率并增加振荡幅度。此外，低 pH 值会抑制细胞间通讯并导致连接蛋白 32 水平降低。最后，我们发现抑制介导钙信号传导和细胞间通讯变化的兰尼碱受体可减少体外酸诱导的酶原激活和细胞损伤。通过建立酸血症的有害影响与腺泡细胞中的细胞内钙信号传导和细胞间间隙连接通讯之间的联系，可以开发治疗或预防酸中毒相关胰腺炎的治疗策略。