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

描述(申请人提供)：唾液和蛋白质成分的促分泌剂刺激中的一个关键事件是细胞内游离钙浓度[Ca~(2+)]i的增加。这种[Ca~(2+)]i的增加具有特定的时间和空间特征，这对于适当地激活唾液分泌的关键效应器是重要的。乙酰胆碱和大多数其他促分泌剂以代谢性方式刺激肌醇1，4，5-三磷酸(InsP3)的产生和细胞内钙的释放。在唾液腺泡细胞中，在三磷酸腺苷(ATP)激活P2X类钙离子通透离子通道后，也可以通过另一种基本上独立的离子亲电过程产生钙信号。后一种机制预计会导致钙信号和效应器的激活，具有明显的时空特征。利用现代光学技术，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药理学，我们将扩展在分离的唾液腺腺泡中产生的发现，以确定通过代谢性，特别是通过器官型腮腺切片制剂中的P2X4嘌呤能受体(P2X4R)刺激的钙信号事件(目标1)。在蛋白激酶A(PKA)激活后，由于P2X4R激活而导致的钙信号显著增强。因为当PKA被激活时，体液分泌也会增加，这可能是生理上的相关情况。在具体目标2中，我们建议定义这一事件背后的分子机制。在具体目标3中，一个主要目标也将是评估通过P2X4受体(P2X4R)的离子亲和性嘌呤能信号是否是液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调控分子事件的理解，并最终可能导致基于利用剩余功能性腺泡组织的生理学来设计治疗口干症的策略。唾液对口腔健康至关重要。它对口腔粘膜的水合是必不可少的；它提供润滑，开始营养消化，并为口腔和上胃肠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。唾液和唾液中蛋白质组分的促分泌作用的一个关键事件是细胞内游离钙浓度的增加。这些研究旨在促进我们对一系列复杂的调节分子事件的理解，这些分子事件导致[Ca~(2+)]i增加，从而导致液体分泌。最终，这些数据被设计成基于利用剩余功能性腺泡组织的生理学来设计治疗口干症的治疗策略。

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.