ALCOHOL-INDUCED MODULATION OF CYTOKINES AND NITRIC-OXIDE IN RAT LUNG

酒精中毒会增加肺部感染的发生率。 该机制 未知，但可能由乙醇、肿瘤 坏死因子（TNF）、皮质酮和一氧化氮（NO）。 我们 假设乙醇诱导肺血管中NO的变化 内皮细胞（EC）、中性粒细胞（PMN）和肺泡巨噬细胞（AM） 部分介导其对大鼠肺宿主防御的抑制， 急性和慢性酒精中毒模型。 具体问题是 回答是（一）：乙醇上调EC生产的NO？免费 将测量单细胞内和细胞外NO和NO2 肺动脉（PA）和肺静脉（PV）EC的电流测量 酒精大鼠（AR）和非酒精等热量饮食（NAI）大鼠。 将在基础条件下和刺激期间测量NO， TNF、内毒素（LPS）、PMN、缓激肽（BK）和A23187。 组成NO 合成酶（NOS）I和诱导型NOS II活性将在 通过定量14 C-瓜氨酸和NO2的形成来测定NAI和AR的EC 14 C-精氨酸。 我们将确定乙醇是否上调NOS 通过测量NOS I和II的mRNA的基因表达或翻译 在NAI和AR的PA和PV中使用c-ERT PCR。 的性别差异 将使用雄性和雌性大鼠（第2天）测试乙醇对NO的影响 发情期）（n-5/gp/性别）。 (b)：EC NO的上调是否抑制PMN 粘附EC？从AR和NAI获得的PMN、PA和PV将被共同分析。 在37 ℃下于Ussing室中孵育。 中性粒细胞的粘附和迁移 使用组织学和51 Gr标记技术进行评估。 EC和PMN NO合成、酶活性、mRNA和超氧化物（SOX）的变化 将评价通过细胞色素C还原酶活性测定的结果。(c)： 乙醇可下调AM的吞噬功能，并募集PMN NO，降低吞噬功能 和细菌的杀灭AR和NAI将使用克雷伯菌治疗 测定肺炎或LPS以及AM和PMN的细菌杀伤， 吞噬作用以及如上所述的NO和SOX系统的变化。 NO在肺宿主防御中的作用将通过使用 AR和NAI用NO系统的调节剂预处理。 (d)： 乙醇通过下调TNF α来下调NOS I和II， 上调皮质类固醇？ AT和NAI将被给予外源性TNF， LPS、TNF受体拮抗剂或RU 38486。 PMN粘附，AM和PMN 如上所述测定细菌杀伤、吞噬作用、TNF和NO。 cAMP、腺苷、Ca ~（2+）和细胞内Fe ~（3+）在此过程中的作用 会得到考验 该项目将确定新的方法， 和治疗乙醇诱导的肺宿主防御机制抑制。

Alcoholism increases the incidence of lung infection. The mechanism is unknown but may result from an interaction between ethanol, tumor necrosis factor (TNF), corticosterone and nitric oxide (NO). We hypothesize that ethanol-induced changes in NO in lung vascular endothelium (EC), neutrophils (PMN) and alveolar macrophages (AM) mediate, in part, its suppression of pulmonary host defense in a rat model of acute and chronic alcoholism. The specific questions to be answered are (a): Does ethanol upregulate EC production of NO? Free single cell intra- and extracellular NO and NO2 will be measured amperiometrically in the EC of pulmonary artery (PA) and vein (PV) of alcoholic rats (AR) and rats on a non-alcoholic, isocaloric diet (NAI). NO will be measured under basal conditions and during stimulation with TNF, endotoxin (LPS), PMN, bradykinin (BK) and A23187. Constitutive NO synthase (NOS) I and inducible NOS II activity will be assessed in the EC of NAI and AR by quantifying the formation of 14C-citrulline and NO2 from 14C-arginine with HPLC. We will define if ethanol upregulates NOS gene expression or translation by measurement of mRNA for NOS I and II using c-ERT PCR in PA and PV from NAI and AR. Sex differences in ethanol's effect on NO will be tested using male and female rats (day 2 estrous) (n-5/gp/sex). (b): Does up-regulation of EC NO suppress PMN adhesion to EC? PMN, PA and PV obtained from AR and NAI will be co- incubated in Ussing chambers at 37C. Adhesion and migration of PMNs will be assessed using histologic and 51Gr-labelling techniques. EC and PMN changes in NO synthesis, enzyme activity and mRNA and superoxide (SOX) assayed by cytochrome c reductase activity will be evaluated. (c): Does ethanol down-regulate AM and recruited PMN NO to decrease phagocytosis and bacterial killing? AR and NAI will be treated with Klebsiella pneumonia or LPS and AM and PMN assayed for bacterial killing, phagocytosis and changes in the NO and SOX systems as described above. The role of NO in pulmonary host defense will be critically tested using AR and NAI pretreated with modulators of the NO system. (d): Does ethanol deregulated NOS I and II by down-regulating TNFalpha and upregulating corticosteroids?. AT and NAI will be given exogenous TNF, LPS, TNF receptor antagonist or RU38486. PMN adhesion, AM and PMN bacterial killing, phagocytosis, TNF and NO assayed as described above. The role of cAMP, adenosine, Ca2+ and intracellular Fe3 in this process will be tested. This project will define new approaches for preventing and treating ethanol-induced suppression of lung host-defense mechanisms.