DETERMINATION OF DNA SEQUENCE OF NEUROPEPTIDE Y Y3-RECEPTOR

为了探讨神经源性肺水肿的发病机制，我们采用了三个实验：（1）克隆神经肽YY_3受体并测定其DNA序列;（2）血管内皮细胞的参与;（3）中枢神经系统的作用：我们已报道神经肽Y是一种神经递质，由交感神经末梢释放，在纤维蛋白诱导的肺水肿（一种神经源性肺水肿模型）的发生过程中可能增加肺血管通透性。因此，它扰乱了一代新药。我们提取mR* 并创建cDNA。将该cDNA连接到ZAP表达载体上，建立cDNA文库。用噬菌体感染大肠杆菌DNA，噬菌体携带cDNA。孵化后，E. coli在琼脂平板上成功形成噬斑。用抗NPY抗体和抗PYY抗体处理印迹的硝酸纤维素膜。通过彩色化可视化，c ...更多信息 其次，我们利用大鼠主动脉内皮细胞，使细胞在琼脂平板中存活。现在研究内皮细胞，以检测具有渗透性分子如荧光分子结合白蛋白的血管渗透性。同时，我们还研究了血管内皮细胞生长因子（VEGF）的发育，它使血管床对大分子白蛋白具有通透性。静脉麻醉药、氯胺酮、异丙酚、硫喷妥钠、神经递质、神经肽Y、去甲肾上腺素和 * 乙酰胆碱等生物活性物质。氯胺酮和硫喷妥钠具有增加血管内皮细胞释放VEGF的活性，我们已经报道了延髓中NO的释放促进了神经源性肺水肿的发展。* 由于单侧左迷走神经切断2周后NO合成酶增加，我们检查了纤维蛋白诱导的肺水肿，获得了NO抑制水肿发展的结果。然而，血管通透性的增加在2周时间点达到最大值，但在4周时增加更多。目前，我们正在研究1-谷氨酸（一种兴奋性神经递质）对纤维蛋白诱导的肺水肿的影响。少

In order to investigate a mechanism involved in the development of neurogenic pulmonary edema, we utilized three experiments: (1) cloning of neuropeptide Y Y3-receptor and determination of its DNA sequenc* (2) involvement of vascular endothelial cells; (3) action of central nervous system.We have reported that neuropeptide Y is a neurotransmitter, released from the sympathetic nerve terminal, which might increa* the pulmonary vascular permeability in the development of fibrin-induced pulmonary edema, a model of neurogenic pulmonary edema* So far, DNA sequence of NPY Y3-receptor has not been elucidated. Therefore it disturbs a generation of new drug. We extracted mR* and created cDNA. The cDNA was ligated to ZAP Express Vector, creating cDNA library. E.coli DNA was infected by phage which h* the cDNA. After incubation, E. coli successfully made plaques on agar plate. Blotted nitrocellulose membrane was treated with ant* NPY antibody and anti PYY antibody. Visualized with colorization, c … More loning is now under study.Secondly, we utilized rat aortic endothelial cells, making the cells alive in agar plates. Endothelial cells are now investigated f* detecting the vascular permeability with permeating molecules such as fluorescent molecule-binding albumin. At the same time, we * also studying the development of vascular endothelial cell growth factor (VEGF) which makes the vascular bed permeable to large * molecule, albumin. Venous anesthetics, ketamine, propofol, and thiopental, neurotransmitters, neuropeptide Y, norepinephrine and * acetylcholine, and other bioactive substances. Ketamine and thiopental have an activity to increase the release ofVEGF from the * vascular endothelial cells.We already reported that NO release in the medulla oblongata has enhanced the development of neurogenic pulmonary edema. * Since unilateral left-vagotomy increase the NO synthase 2 weeks later, we have examined the fibrin-induced pulmonary edema, obtain * the results demonstrating the inhibitory action of NO on the edema development. However, the increase in vascular permeability was * maximum at 2 weeks time point, but rather increased more at 4 weeks. Now we are studying the effects of 1-glutamate, an excitable * neurotransmitter, on the fibrin-induced pulmonary edema. Less