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

• 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
• 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

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（申请人提供）：该提案描述了一项为期五年的培训计划，其目的是将候选人发展为独立的研究者。首席研究人员将通过她在胰腺炎分子机制专家弗雷德·戈雷利克（Fred Gorelick）的研究活动中扩大她已经获得的科学背景和技能。通过在Gorelick博士的指导下实施一项结构化的研究，培训和职业发展计划，而Divestive上皮中的钙信号专家Michael Nathanson博士将获得分析框架，技术能力和知识基础，以成功成为独立的医师科学家。耶鲁大学和消化系统疾病部分，拥有丰富的各种教师和充足的生物医学设施网络，为追求这一计划提供了理想的环境。急性胰腺炎的发病机理是一种常见和威胁生命的疾病，尚不完全了解。以急性酸负荷为特征的临床状况易于发育。最近，Gorelick Lab表明，低pH值对体外和体内的胰腺炎反应敏感。但是，造成这种敏化的机制尚不清楚。该提议假设低pH值通过转化细胞内钙信号传导和间隙连接腺细胞的间隙细胞间通信从生理模式变为引起胰腺炎的一种。该提案的具体目的是：1）确定低pH值对腺泡细胞中细胞内钙信号传导的影响2）确定低pH值对细胞间通信的影响； 3）确定酸诱导的胰腺炎反应是否由病理钙信号和/或间隙连接细胞间的细胞内界面细胞间通信介导。为了确定低pH值对钙信号的影响，将通过共聚焦显微镜检查分离的腺泡细胞。差距连接通信的功能方面将通过量化钙信号同步的程度并通过间隙连接检查染料转移来研究。间隙连接间间通信的结构方面将通过表征主要间隙连接蛋白的降解模式（Connexin32）来检查。最后，将使用体外和体内胰腺炎模型检查钙信号和间隙连接在酸诱导的损伤中的作用。我们的初步数据表明，低pH值降低了振荡频率，并增加了用胆囊基蛋白直系同源物（Cerulein）生理浓度处理的分离的acini中的振荡振幅。此外，低pH值抑制细胞间通信，并导致连接率降低。最后，我们发现抑制ryanodine受体介导了钙信号传导和细胞间通信的变化，可减少酸诱导的酶原激活和体外细胞损伤。通过建立酸血症和细胞内钙信号传导的有害作用与腺泡细胞中细胞间间隙连接通信之间的联系，可以开发治疗或预防与酸中毒相关的胰腺炎的治疗策略。