[Ca2+]i and Secretory Dynamics in Parotid Acinar Cells

描述（由申请人提供）：唾液的液体和蛋白质组分的促分泌素刺激中的关键事件是细胞溶质游离钙浓度[Ca 2 +] i的增加。[Ca 2 +]i的这种增加具有特定的时间和空间特征，这对于适当激活对唾液分泌至关重要的效应物是重要的。乙酰胆碱和大多数其他促分泌素以促代谢的方式起作用，刺激1，4，5-三磷酸肌醇（InsP 3）的产生和细胞内Ca 2+的释放。在唾液腺泡细胞中，Ca 2+信号也可以通过在三磷酸腺苷（ATP）激活P2 X类Ca 2+渗透性离子通道之后的替代的、很大程度上独立的亲离子过程产生。后一种机制将被预测为导致Ca 2+信号和效应器激活具有不同的空间和时间特征。使用当代的光学技术，如数字成像，全内反射显微镜和共聚焦显微镜结合转基因动物模型和特定的药理学，我们将扩大在孤立的唾液腺腺泡中产生的发现，以确定通过代谢刺激，特别是通过P2 X4嘌呤能受体（P2 X4 R）在器官型腮腺切片制备（目的1）的钙信号传导事件。作为P2 X4 R激活的结果的Ca 2+信号在蛋白激酶A（PKA）激活后显著增强。由于PKA被激活时液体分泌也增强，这可能是生理相关的情况。在具体的目标2中，我们提出定义这一事件背后的分子机制。在具体目标3中，主要目标还将是评估通过P2 X4受体（P2 X4 R）的离子型嘌呤能信号传导（单独或在特定药理学操作之后）是否是流体和胞吐分泌机制的有效激活剂。这些研究旨在促进我们对导致液体分泌的一系列复杂的受调节分子事件的理解，并可能最终导致基于利用剩余功能性腺泡组织的生理学来设计用于治疗口干症的治疗策略。唾液对口腔健康至关重要。它对口腔粘膜的水合作用至关重要;它提供润滑，开始营养消化，并为口腔和上消化道提供抗菌和机械保护。唾液流量减少（口干症）极大地影响患者的生活质量。唾液的液体和蛋白质组分的促分泌素刺激中的关键事件是细胞溶质游离钙浓度[Ca 2 +] i的增加。这些研究旨在促进我们对导致[Ca 2 +]i增加并因此导致液体分泌增加的一系列复杂的受调节分子事件的理解。最终，这些数据的目的是导致设计的治疗策略，用于治疗口干症的基础上利用剩余的功能腺泡组织的生理。

DESCRIPTION (provided by applicant): A key event in the secretagogue stimulation of both the fluid and protein component of saliva is an increase in the cytosolic free calcium concentration [Ca2+].i This increase in [Ca2+]i has specific temporal and spatial characteristics which are important for appropriately activating effectors pivotal for the secretion of saliva. Acetylcholine and the majority of other secretagogues act in a metabotropic manner stimulating the production of inositol 1,4,5-trisphosphate (InsP3) and the release of intracellular Ca2+. In salivary acinar cells, Ca2+ signals can also be generated by an alternative, largely independent ionotropic process following adenosine triphosphate (ATP) activation of the P2X class of Ca2+ permeable ion channels. The latter mechanism would be predicted to result in Ca2+ signals and effector activation with distinct spatial and temporal characteristics. Using contempory optical techniques, such as digital imaging, total internal reflection microscopy and confocal microscopy combined with transgenic animal models and specific pharmacology we will expand on findings generated in isolated salivary gland acini to define Ca2+ signaling events stimulated through both metabotropic and in particular through the P2X4 purinergic receptor (P2X4R) in an organotypic parotid gland slice preparation (aim 1). Ca2+ signals as a result of P2X4R activation are profoundly enhanced following protein kinase A (PKA) activation. Since fluid secretion is also enhanced when PKA is activated this may be the physiologically relevant situation. In specific aim 2 we propose to define the molecular mechanism underlying this event. In specific aim 3 a primary goal will also be to assess whether ionotropic purinergic signaling through P2X4 receptors (P2X4R), either alone, or following specific pharmacological manipulation is an effective activator of the fluid and exocytotic secretion mechanism. These studies are designed to advance our understanding of the complex series of regulated molecular events which result in fluid secretion and may ultimately lead to the design of therapeutic strategies for the treatment of xerostomia based on exploiting the physiology of remaining functional acinar tissue. Saliva is vital for oral health. It is essential for the hydration of the oral mucosa; it provides lubrication, begins nutrient digestion and imparts antimicrobial and mechanical protection for the mouth and upper gastrointestinal tract. Reduced flow of saliva (xerostomia) greatly impacts the quality of life of sufferers. A key event in the secretagogue stimulation of both the fluid and protein component of saliva is an increase in the cytosolic free calcium concentration [Ca2+].i These studies are designed to advance our understanding of the complex series of regulated molecular events which result in the increase in [Ca2+]i and hence fluid secretion. Ultimately these data are designed to lead to the design of therapeutic strategies for the treatment of xerostomia based on exploiting the physiology of remaining functional acinar tissue.