RELATIONSHIPS BETWEEN PROSTANOIDS AND CELLULAR INJURY

感染性(内毒素)休克被列为 死亡率。我们将检验细菌内毒素作用于 引起微血管长期改变的微循环 内皮细胞(MEC)花生四烯酸(AA)代谢。由此产生的 前列腺素E_2基础生成率增加可能是 免疫抑制和心血管生理学改变的观察 感染性休克状态。这项提议的主要目的是充分 描述内毒素与微血管内皮细胞相互作用的机制 启动AA新陈代谢。要使用的模型是主要的 来源的连续微血管内皮细胞的培养 兔或人的脂肪组织。初步研究将确定 人脐静脉内皮细胞中AA代谢的时程和产物形成谱 对内毒素的反应。将建立剂量-反应关系 对于由几种来源的内毒素引发的二十烷类化合物的形成 细菌的种类。放射性标记内毒素与整体的结合 MECS和TO分离的质膜碎片将通过 Scatchard分析计算内毒素受体数目和亲和力。 将使用电泳法分离和鉴定 微血管内皮细胞中存在内毒素受体。竞争研究使用 生物无活性内毒素将被用来进一步鉴定 内毒素与细胞膜的相互作用。更多的研究将 确定内毒素启动的亚细胞机制 二十烷类化合物的生物合成。研究将使用西区和 Northern印迹分析确定细菌内毒素是否影响 前列腺素的转录或翻译调控 负责二十烷类化合物的关键酶--过氧化内酯合成酶 综合。将进行实验以不可逆转地抑制 前列腺素内源性过氧化合酶和监测其回报率 酶活性作为该酶基因组表达的一个指标。 其他研究将确定磷脂酶A2的作用，这种酶可以 从膜磷脂中释放AA，以及脂肪酸的作用 为二十烷类化合物提供底物的脱酰/反应酶 系统。最后一组研究将调查可能的第二个 连接血浆内毒素相互作用的信使系统(S) 与花生四烯酸级联反应的机制有关 激活了。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。总而言之，这些研究将充分说明 细菌内毒素与微血管内皮细胞的相互作用 加强长期的二十烷类化合物生产。有人建议这一点 改变的二十烷类化合物的产生在改变的 内毒素休克的微血管和免疫学反应。一种知识 内毒素改变内皮细胞功能的机制 对设计有效的内毒素治疗方法很重要- 相关疾病状态，如儿童和老年感染性休克， 烧伤相关性败血症、外科败血症及相关败血症 患有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.