RELATIONSHIPS BETWEEN PROSTANOIDS AND CELLULAR INJURY

脓毒性（内毒素）休克是一个重要的原因， mortality. 我们将检验细菌内毒素作用于 微循环引起微血管的长期改变 内皮细胞（MEC）花生四烯酸（AA）代谢。 所得 基础前列腺素E2生成速率的增加可能是 免疫抑制和心血管生理学的改变， 败血性休克状态 这项建议的主要目的是充分 表征内毒素与MEC相互作用的机制， 启动AA新生代谢。 要使用的模型是主要的 连续培养微血管内皮细胞， 兔子或人的脂肪组织。 初步研究将描述 在MEC中AA代谢的时间过程和产物形成概况 对内毒素的反应 将建立剂量-反应关系 对于由源自几种细菌的内毒素引发的类二十烷酸形成， 细菌的种类。 放射性标记内毒素与完整 MECS和分离的质膜碎片将通过 Scatchard分析计算内毒素受体数量和亲和力。 电泳技术将用于分离和表征 内毒素受体存在于MEC中。 竞争研究使用 将制备无生物活性的内毒素，以进一步表征 内毒素与细胞膜的相互作用。 其他研究将 确定内毒素启动的亚细胞机制 类花生酸生物合成 研究将使用西方和 北方印迹分析，以确定细菌内毒素是否影响 前列腺素的转录或翻译调节 内过氧化物合成酶，负责类花生酸的关键酶 合成. 将进行实验以不可逆地抑制 前列腺素内过氧化物合酶和监测的回报率 酶活性作为酶的基因组表达的指标。 其他研究将确定磷脂酶A2的作用， 从膜磷脂中释放AA，脂肪酸 脱酰/再酰化酶为类花生酸提供底物 系统 最后一组研究将调查可能的第二次 连接血浆中内毒素相互作用的信使系统 膜的机制，其中花生四烯酸级联是 激活 这些系统包括蛋白激酶C、鸟嘌呤核苷酸 结合蛋白，磷脂酰肌醇循环，酪氨酸激酶，和 环核苷酸 总之，这些研究将充分表征 细菌内毒素和MEC之间的相互作用，导致 增强长期类花生酸的产生。 有人建议， 改变的类二十烷酸产生在改变的 内毒素休克的微血管和免疫反应。 知识 内毒素改变内皮细胞功能的机制是 对于设计有效的内毒素治疗方法很重要- 相关的疾病状态如小儿和老年感染性休克， 烧伤相关败血症、外科败血症和相关败血症 获得性免疫缺陷综合症

Septic (endotoxic) shock is advancing in rank as a significant cause of mortality. We will test the hypothesis that bacterial endotoxins act at the microcirculation to induce long-term alterations in microvascular endothelial cell (MEC) arachidonic acid (AA) metabolism. The resultant enhanced rate of basal prostaglandin E2 production may be responsible for the immunodepression and altered cardiovascular physiology observed in the septic shock state. The primary aim of this proposal is to fully characterize the mechanism by which endotoxins interact with MECs to initiate de novo AA metabolism. The model to be used is a primary culture of continuous, microvascular endothelial cells derived from rabbit or human adipose tissue. Initial studies will characterize the time course and product formation profile of AA metabolism in MECs in response to endotoxin. Dose-response relationships will be established for eicosanoid formation initiated by endotoxins derived from several species of bacteria. The binding of radio-labeled endotoxin to intact MECS and to isolated plasma membrane fragments will be analyzed by Scatchard analysis to calculate endotoxin receptor number and affinity. Electrophoretic techniques will be used to isolate and characterize endotoxin receptors present in MECs. Competition studies using biologically inactive endotoxins will be made to further characterize the endotoxin interaction with the cell membrane. Additional studies will identify the subcellular mechanism by which endotoxins initiate eicosanoid biosynthesis. Studies will be carried out using Western and Northern blot analysis to determine whether bacterial endotoxins affect the transcriptional or translational regulation of prostaglandin endoperoxide synthase, the key enzyme responsible for eicosanoid synthesis. Experiments will be done to irreversibly inhibit prostaglandin endoperoxide synthase and monitor the rate of return of enzymatic activity as an index of genomic expression of the enzyme. Other studies will determine the role of phospholipase A2, an enzyme that releases AA from membrane phospholipids, and the role of fatty acid deacylation/reacylation enzymes in providing substrate for the eicosanoid system. A final group of studies will investigate the possible second messenger system(s) that link the interaction of endotoxins at the plasma membrane to the mechanisms by which the arachidonic acid cascade is activated. These systems include protein kinase C, guanine nucleotide binding proteins, the phosphatidylinositol cycle, tyrosine kinase, and cyclic nucleotides. In summary, these studies will fully characterize the interaction between bacterial endotoxins and MECs that results in enhance, long-term eicosanoid production. It is suggested that this altered eicosanoid production plays a significant role in the altered microvascular and immunologic responses to endotoxic shock. A knowledge of the mechanisms by which endotoxin alters endothelial cell function is important to the design of effective therapeutic approaches to endotoxin- associated disease states such as pediatric and geriatric septic shock, burn-associated septicemia, surgical sepsis and septicemia associated with the acquired immune deficiency syndrome.