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。其机制是 未知，但可能是乙醇、肿瘤 血清肿瘤坏死因子(TNF)、皮质酮、一氧化氮。我们 乙醇诱导肺血管一氧化氮变化的假说 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作用 急性和慢性酒精中毒模型。具体问题是 答案是(A)：乙醇是否上调了EC产生的NO？免费 将测量单个细胞内和细胞外的NO和NO2 肺动脉(PA)和静脉(PV)内皮细胞的计量学研究 酒精大鼠(AR)和非酒精等卡路里饮食(NAI)大鼠。 NO将在基础条件下和在刺激期间进行测量 肿瘤坏死因子、内毒素、中性粒细胞、缓激肽和A23187。本体式NO 合酶(NOS)I和诱导型NOS II的活性将在 ~(14)C-瓜氨酸和NO_2生成定量测定NAI和AR的EC 用高效液相色谱法从14C-精氨酸中分离得到。我们将确定乙醇是否会上调一氧化氮合酶 通过测量一氧化氮合酶I和II的mRNA表达或翻译 应用c-ERT聚合酶链式反应(c-ERT)从NAI和AR获得PA和PV。中国的性别差异 乙醇对NO的影响将在雄性和雌性大鼠(第2天)身上进行测试 动情期)(n-5/gp/性别)。(B)：上调EC是否会抑制PMN 对EC的粘合？从AR和NAI获得的PMN、PA和PV将共同 在37℃的Ussing小室中孵化。中性粒细胞的黏附和迁移将 使用组织学和51Gr标记技术进行评估。EC和PMN 一氧化氮合成、酶活性及信使核糖核酸和超氧化物歧化酶的变化 用细胞色素C还原酶活性测定法进行评价。(C)：是否 乙醇下调AM和募集PMN-NO以减少吞噬功能 那细菌杀戮呢？AR和NAI将接受克雷伯氏菌治疗 肺炎或内毒素、AM和PMN进行细菌杀灭试验， 吞噬作用和上述NO和SOX系统的变化。 NO在肺宿主防御中的作用将通过 AR和NAI用NO系统的调节剂进行预处理。(D)：是否 乙醇通过下调肿瘤坏死因子α和一氧化氮合酶抑制一氧化氮合酶的表达 皮质类固醇激素的增加？AT和NAI将被给予外源性肿瘤坏死因子， 脂多糖、肿瘤坏死因子受体拮抗剂或RU38486。中性粒细胞黏附、AM和PMN 杀菌、吞噬、肿瘤坏死因子和一氧化氮如上所述测定。 CAMP、腺苷、钙离子和胞内Fe3在此过程中的作用 将会受到考验。该项目将定义新的预防方法 以及治疗乙醇诱导的肺宿主防御机制的抑制。

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.