Low ph alters intracellular and extracellular communication in the acinar cell

描述（由申请人提供）：该提案描述了一个五年的培训计划，目标是将候选人发展成为一名独立的调查员。首席研究员将扩展她在胰腺炎分子机制专家Fred Gorelick博士实验室的研究活动中已经获得的科学背景和技能。在Gorelick博士和消化上皮钙信号专家Michael Nathanson博士的指导下，通过实施结构化的研究、培训和职业发展计划，候选人将获得分析框架、技术能力和知识基础，以成功成为一名独立的内科科学家。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，为实现这一计划提供了理想的环境。急性胰腺炎是一种常见的危及生命的疾病，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易导致急性胰腺炎的发展。最近，Gorelick实验室已经证明，低pH值对体外和体内胰腺炎反应敏感。然而，这种致敏的机制尚不清楚。本研究假设低pH通过将腺泡细胞内钙信号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损伤作用。本研究的具体目的是：1)确定低pH对腺泡细胞内钙信号的影响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性钙信号和/或间隙连接细胞间通讯的改变介导。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。通过定量钙信号同步程度和检查通过间隙连接的染料转移，将研究间隙连接通信的功能方面。通过表征主要的间隙连接蛋白connexin32的降解模式，将研究间隙连接细胞间通讯的结构方面。最后，我们将通过体外和体内胰腺炎模型来研究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，低pH值降低了分离的腺泡在生理浓度的胆囊收缩素同源物蓝蛋白处理下的振荡频率，增加了振荡幅度。此外，低pH抑制细胞间通讯并导致连接蛋白水平降低32。最后，我们发现，抑制ryanodine受体（介导钙信号和细胞间通讯的变化）可以减少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细胞内钙信号和细胞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性胰腺炎的治疗策略。

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.