MODULATION OF ARACHIDONATE CASCADE IN AIRWAY EPITHELIUM

吸入臭氧和高浓度氧气可引起 伴发呼吸道上皮细胞局灶性细胞坏死和塌陷 炎症细胞募集，粘膜水肿，粘液分泌增加 和呼吸道高反应性。气管上皮(TE)细胞能够 产生花生四烯酸(AA)的各种代谢物，并可能对 通过改变这些物质的轮廓和生产来暴露氧化性气体 强有力的促炎介质。呼吸道坏死脱屑 上皮细胞还可以导致神经肽的释放增加，如 外露传入神经的P物质与局部缓激肽的产生 从渗出的血浆中。P物质和缓激肽的受体是 存在于呼吸道上皮细胞上，这些介质也可能激活AA 这些细胞在呼吸道损伤部位的新陈代谢。在预赛中 研究中我们观察到了AA代谢物的变化 由体外臭氧暴露后的兔TE单层产生 高氧和P物质激活AA级联反应 缓激肽。 这些研究的目的是详细研究臭氧的影响。 高氧暴露对培养细胞产生AA代谢物的影响 兔和人TE细胞及其细胞内信号转导的研究 P物质和缓激肽激活脑内AA代谢的机制 呼吸道上皮细胞。兔和人TE细胞的单层培养 维持在允许内源基因表达的条件下 二十烷类新陈代谢将暴露于临床相关浓度 臭氧和高氧在允许体外暴露的系统中 细胞处于类似活体条件的气液界面。 AA代谢物光谱的定性和定量变化 TE细胞对氧化气体暴露和物质产生的反应 P和缓激肽将通过高压信号机制进行评估 将多肽受体偶联与激活AA代谢联系起来 通过测量细胞内游离钙的变化来研究 荧光指示剂Fura-2和评估G蛋白信号转导， 以及各机制对激活AA的相对贡献。 由选择性药物抑制或刺激所决定的级联反应 每条信号通路。

Inhalational exposure to ozone and high concentration of oxygen can induce focal cell necrosis and sloughing of airway epithelial cells accompanied by recruitment of inflammatory cells, mucosal edema, increased mucus secretion and airway hyperresponsiveness. Tracheal epithelial (TE) cells are capable of generating various metabolites of arachidonic acid (AA) and may respond to oxidant gas exposure by altering the profile and production of these potent proinflammatory mediators. Necrosis and desquamation of airway epithelium can also cause increased release of neuropeptides such as Substance P from exposed afferent nerves and local generation of bradykinin from extravasated plasma. Receptors for Substance P and bradykinin are present on airway epithelial cells and these mediators may also activate AA metabolism by these cells at sites of airway injury. In preliminary studies we have observed alterations in the profile of AA metabolites generated by rabbit TE monolayers following in vitro exposure to ozone and hyperoxia as well as activation of AA cascade by Substance P and bradykinin. The objective of these studies is to examine in detail the effects of ozone and hyperoxic exposure on the production of AA metabolites by cultured rabbit and human TE cells and to determine the intracellular signalling mechanisms by which Substance P and bradykinin activate AA metabolism in airway epithelial cells. Monolayer cultures of rabbit and human TE cells maintained under conditions which allow for expression of endogenous eicosanoid metabolism will be exposed to clinically relevant concentrations of ozone and hyperoxia in a system which allows for in vitro exposure of cells at a gas-fluid interface analogous to in vivo conditions. Qualitative and quantitative changes in the spectrum of AA metabolites generated by TE cells in response to oxidant gas exposure and to Substance P and bradykinin will be assessed by high pressure signalling mechanisms linking peptide receptor coupling to activation of aA metabolism will be studied by measuring changes in free cytosolic calcium using the fluorescent indicator fura-2 and assessing G protein signal transduction, and the relative contributions of each mechanisms to activation of AA cascade determined by selective pharmacologic inhibition or stimulation of each signal pathway.