MODULATION OF ARACHIDONATE CASCADE IN AIRWAY EPITHELIUM

吸入臭氧和高浓度氧气可导致 局部细胞坏死和气道上皮细胞脱落， 炎症细胞募集、粘膜水肿、粘液分泌增加 和气道高反应性。 气管上皮（TE）细胞能够 花生四烯酸（AA）的各种代谢产物，并可能响应 氧化剂气体暴露通过改变这些的轮廓和生产 强效促炎介质。 气道坏死和脱屑 上皮细胞也可引起神经肽的释放增加， 暴露传入神经的P物质与缓激肽的产生 从外渗的血浆中 P物质和缓激肽的受体是 存在于气道上皮细胞上，这些介质也可以激活AA 这些细胞在气道损伤部位的代谢。 初步 研究中，我们观察到AA代谢产物的变化 在体外暴露于臭氧后由兔TE单层产生， 高氧以及由P物质激活AA级联， 缓激肽 这些研究的目的是详细检查臭氧的影响 和高氧暴露对培养的 兔和人TE细胞，并确定细胞内信号传导 P物质和缓激肽激活AA代谢的机制 气道上皮细胞。 兔和人TE细胞的单层培养物 在允许表达内源性 类花生酸代谢将暴露于临床相关浓度 臭氧和高氧的系统，允许在体外暴露的 细胞在类似于体内条件的气体-流体界面处。 AA代谢产物谱的定性和定量变化 由TE电池响应于氧化剂气体暴露和物质 P和缓激肽将通过高压信号机制进行评估 将肽受体偶联与aA代谢的激活联系起来， 通过测量游离胞浆钙的变化， 荧光指示剂fura-2和评估G蛋白信号转导， 以及每种机制对AA激活的相对贡献 通过选择性药理学抑制或刺激 每一个信号通路

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.