Isolation and identification of the enzyme synthesising Up4A from endothelial cells and isolation, identification, and characterization of further "endothelial-derived vasoconstrictive factors" (EDCF)

内皮不仅是血液和血管壁之间的机械屏障，而且是具有多种调节功能的内分泌器官。通过鉴定一氧化氮和内皮素作为血管调节因子，我们对内皮介导的血管调节的认识发生了革命性的变化。在文献中有几个迹象表明进一步的内皮源性血管收缩因子（EDCF）。在我们自己的实验中，我们从受刺激的人内皮细胞的上清液中分离出四磷酸尿苷腺苷作为另一种高效的EDCF。接下来的问题是内皮细胞是如何产生Up4A的。初步实验表明，固定化内皮细胞含有一种酶，该酶能够从ADP和UDP合成Up4A。因此，分离和鉴定合成Up4A的酶是本课题的主要目的。进一步的实验表明，除了Up4A，人类内皮细胞还会分泌更多未知的edcf，这些edcf也属于核苷酸类。由于培养的内皮细胞只分泌少量的edcf，我们还不能在初步实验中分离和鉴定这些化合物。因此，在提案的第二部分，我们计划分离，识别，量化和表征这些仍然未知的edcf。在这些化合物被鉴定后，除非它们是市售的，否则它们必须被合成，并且对血管调节的影响必须通过在申请人组中建立的那些生物测定来详细表征。在计划项目的第三部分，我们将使用分子生物学技术检查Up4A和其他额外的edcf的信号转导，这些edcf将在本项目中被确定。

The endothelium not only acts as a mechanical barrier between blood and vessel walls, but is also an endocrine organ with multiple regulatory functions. By the identification of NO and endothelin as vasoregulatory factors our knowledge of endothelial-mediated vascular regulation was revolutionized. In literature there are several indications of further ¿endothelialderived vasoconstrictive factors (EDCF). In our own experiments we isolated uridine adenosine tetraphosphate as a further highly effective EDCF from supernatants of stimulated human endothelial cells. Next the question arose how Up4A is being produced in endothelial cells. First preliminary experiments showed that immobilized endothelial cells contain an enzyme, which is capable of synthesizing Up4A from ADP and UDP. Therefore it is the primary aim of the project to isolate and identify the enzyme synthesizing Up4A. Further experiments showed that human endothelial cells secrete further still unknown EDCFs beyond Up4A, which also belong to the class of nucleotides. Since cultivated endothelial cells secrete only small absolute amounts of these EDCFs, we have not yet been able to isolate and identify these compounds in our preliminary experiments. Therefore, in the second part of the proposal we plan to isolate, identify, quantify, and characterize these still unknown EDCFs. After these compounds have been identified, they will have to be synthesized unless they are commercially available, and the effects on vascular regulation will have to be characterized in detail by those bioassays, which are established in the applicant s group. In a third part of the proposed project we will examine the signal transduction of Up4A and of other, additional EDCFs, which will have been identified in this project, using molecular biology techniques.