Bitter and sweet taste receptor physiology in airway ciliated cells

项目摘要 气道细胞纤毛中的T2 R苦味受体检测细菌酰基高丝氨酸内酯 和喹诺酮类。这些T2 R的激活增加一氧化氮（NO）的产生，以增加 纤毛跳动并杀死细菌。因此，T2 R在气道先天免疫中起重要作用。 降低纤毛T2 Rs功能的多态性与革兰氏阴性菌增加相关 上呼吸道感染、慢性鼻窦炎易感性和较差的手术结果 鼻窦手术后靶向T2 R或增强它们的活性可以是互补的或互补的。 替代抗生素。增强气道或其他组织中内源性T2 R介导的免疫 器官可能是一个有用的抗病原体的战略，在面对日益普遍的 抗寄生虫的“超级细菌”NO既能杀菌，又能杀灭病毒。 T2 R还激活其他组织和免疫细胞（如巨噬细胞）中的NO产生， 其中T2 R到NO信号传导调节吞噬作用。T2 R激活内皮（e）形式的 一氧化氮合酶（eNOS）在初级气道细胞和巨噬细胞。该途径 在许多细胞类型中似乎是T2 R信号传导的标志，并调节多种 生理过程。了解T2 R如何向eNOS发出信号对于了解 口外味觉受体信号在许多组织中。我们假设T2 R到eNOS的信号传导 需要通过T2 R下游激活的激酶磷酸化eNOS。激酶 与许多其他GPCR相比，T2 R激活的途径非常未被探索。我们也 假设这种激酶磷酸化被HSP 90增强，HSP 90是一种重要的细胞因子， 参与NOS功能以及一些GPCR信号传导的伴侣蛋白。 在这个提议中，我们将研究T2 R介导的NOS磷酸化， 从手术残留物中分离的原代鼻上皮细胞中NO产生的结果 材料并在空气-液体界面处生长（目标1）。我们将使用NO生产的活细胞成像 和生物化学结合， 已知激活eNOS的途径。我们还将使用充分表征的药理学HSP 90 抑制剂，以表征HSP 90是否调节T2 R或eNOS定位于气道纤毛或功能 （钙信号或NO产生）。

PROJECT SUMMARY T2R bitter taste receptors in airway cell cilia detect bacterial acyl-homoserine lactones and quinolones. Activation of these T2Rs increases nitric oxide (NO) production to increase ciliary beating and kill bacteria. T2Rs thus play an important role in airway innate immunity. Polymorphisms that reduce the function of cilia T2Rs correlate with increased gram negative upper respiratory infections, susceptibility to chronic rhinosinusitis, and worse surgical outcomes after sinus surgery. Targeting T2Rs or enhancing their activity may be a complementary or alternative to antibiotics. Boosting endogenous T2R-mediated immunity in the airway or other organs may be a useful anti-pathogen strategy in the face of the increasing prevalence of antibiotic-resistant “superbugs.” NO can be bactericidal and also can inactivate viruses. T2Rs also activate NO production in other tissues and immune cells like macrophages, where T2R to NO signaling regulates phagocytosis. T2Rs activate the endothelial (e) form of nitric oxide synthase (eNOS) in both primary airway cells and macrophages. This pathway appears to be a hallmark of T2R signaling in many cell types and regulates multiple physiological processes. Understanding how T2Rs signal to eNOS is critical for understanding extraoral taste receptor signaling in many tissues. We hypothesize that T2R to eNOS signaling requires phosphorylation of eNOS by kinases activated downstream of the T2Rs. Kinase pathways activated by T2Rs are very unexplored compared with many other GPCRs. We also hypothesize that this kinase phosphorylation is enhanced by HSP90, an important cellular chaperone protein involved in NOS function as well as some GPCR signaling. In this proposal, we will investigate T2R-mediated phosphorylation of NOS and consequences for NO production in primary nasal epithelial cells isolated from residual surgical material and grown at air-liquid interface (Aim 1). We will use live cell imaging of NO production and biochemistry combined with well-characterized pharmacological inhibitors of kinase pathways known to activate eNOS. We will also use well characterized pharmacological HSP90 inhibitors to characterize if HSP90 regulates T2R or eNOS localization to airway cilia or function (calcium signaling or NO production).