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

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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.