Ca2+ and secretory dynamics in salivary acinar cells

描述(申请人提供)：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。唾液流量减少会导致口干(口干症)，并极大地影响患者的生活质量。唾液腺功能低下的一个主要原因是干燥综合征(SS)，这是一种相对常见的影响唾液腺和泪腺的自身免疫性外分泌疾病。SS会导致口干和眼干(干眼症)的逐渐恶化，因为随着淋巴细胞的渗透，重要的腺体组织最终会被破坏。值得注意的是，在已建立的动物模型中 在SS中，如IL14a B细胞“敲入”小鼠或C57BL/6.NOD-Aec1Aec2小鼠，信号事件和液体流量在任何腺体组织被破坏的证据之前显著减少。此外，SS患者经常保留形态完整的组织，但矛盾的是，显然对刺激是不敏感的。有了这些信息，这个项目将检验这一假设，即刺激-分泌耦合缺陷是疾病早期唾液形成减少的原因，随着疾病的进展，剩余的功能组织也会减少。该提案将利用SS的小鼠模型来探索随着疾病的发展导致唾液和泪腺分泌液体和蛋白质的途径中的潜在缺陷。在小鼠模型中的重要发现将通过对SS患者的唇腺活检进行类似的实验来验证。在特定的目标1，将进行实验，以确定为什么细胞内钙信号，液体分泌的主要刺激因素在SS小鼠模型中受到影响。实验将确定SS中第二信使的产生是否受到影响。腺小叶的多光子成像和高速广野成像，以及离体腺泡的局部闪光光解，将质疑SS中负责钙释放、钙内流或钙清除的机制是否发生了变化。在具体目标2中，将进行实验以阐明为什么SS模型中蛋白质的胞吐作用减弱，以及液体分泌所必需的离子通道的功能是否改变。液体相标记物的多光子成像和细胞膜表面积的电容测量将用于监测胞吐作用。我们将确定SS中的信号或胞吐机制是否被破坏。最后，我们将评估钙激活的Cl-和K+通道在SS中的功能是否发生改变。这些研究旨在提供关于SS刺激-分泌耦合过程中发生的功能变化的基本信息。这个项目的最终目标是利用这些知识来设计和测试基于SS中剩余功能分泌组织的生理学基础上的拯救和维持分泌物的策略。

DESCRIPTION (provided by applicant): 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 results in "dry mouth" (xerostomia) and greatly impacts the quality of life of sufferers. A major cause of salivary gland hypofunction is Sjogrens syndrome (SS), a relatively common autoimmune exocrinopathy affecting the salivary and lacrimal glands. SS results in progressively worsening dry mouth and dry eye (xerophthalmia) as significant glandular tissue is ultimately destroyed following lymphocyte infiltration. Significantly, in established animal models of SS, such as the IL14a B cell "knock-in" mouse, or the C57BL/6.NOD-Aec1Aec2 mouse, signaling events and fluid flow are markedly decreased prior to any evidence of destruction of glandular tissue. Moreover, morphologically intact tissue is often retained in SS patients, but paradoxically, is apparently refractory to stimulation. Given this information, this project will tst the hypothesis that defects in stimulus-secretion coupling are responsible for decreased saliva formation early in the disease and in remaining functional tissue as the disease progresses. The proposal will utilize mouse models of SS to probe the underlying defects in pathways leading to fluid and protein secretion from salivary and lacrimal glands as the disease develops. Important findings in mouse models will be validated by performing similar experiments in labial gland biopsies from SS patients. In specific aim 1, experiments will be performed to ascertain why intracellular Ca2+ signaling, the primary stimulus for fluid secretion is compromised in SS mouse models. Experiments will determine whether the production of second-messengers is compromised in SS. Multi-photon imaging in gland lobules together with high-speed wide-field imaging, combined with focal flash photolysis in isolated acini, will question if the machinery responsible for Ca2+ release, Ca2+ influx or Ca2+ clearance is altered in SS. In specific aim 2, experiments will be performed to elucidate why the exocytosis of protein is attenuated in SS models and if the function of ion channels necessary for fluid secretion is altered. Multi-photon imaging of fluid phase markers together with capacitance measurements of cell membrane surface area will be used to monitor exocytosis. We will determine whether signaling, or the exocytotic machinery in SS is disrupted. Finally, we will evaluate if the function of Ca2+- activated Cl- and K+ channels are altered in SS. These studies are designed to provide fundamental information regarding the functional changes occurring in stimulus-secretion coupling in SS. The ultimate goal of this project is to use this knowledge to rationale design and test strategies for the rescue and maintenance of secretion based on exploiting the physiology of remaining functional secretory tissue in SS.