Purification and structural determination of new vasoactive peptides and elucidation of their effects on the vascular wall cells

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In order to maintain homeostatic balance of the circulation system, the living body has a precisely and highly organized regulatory system, which is still far from the complete elucidation at the present stage. One of the reasons why we can understand this regulatory system is coming from the fact that there are still unidentified mechanisms which are regulated with unknown peptides, proteins or other compounds. In this project, we tried to identify a new vasoactive peptide and elucidate its physiological function.As a source of vasoactive peptides, we first examined the supernatant of cultured vascular endothelial cells. Although endothelin-1 was isolated from the conditioned media, most of other known biological active peptides were proteolytically cleaved and the ratio of the biologically active molecules was found to be relatively low as compared to the degraded products. In this study, therefore, we extracted the starting peptide fraction from pig brain (20kg) by the method previo … More usly established.As for the target cells, we set up a series of cultured endothelial cells and vascular smooth muscle cells of human, rat, pig and bovine origins. We prepared primary cultured cells of rat neonatal cardiomyocytes and cardiofibroblasts. In addition, renal tubular epithelial cells, glia cells and their related cells were also introduced for measurements of biological activity, as these cells elicited appropriate responses in the following assays in the commercially available cells. As for the measurements of biological activity, we evaluated the levels of second messengers, such as cAMP, cGMP and calcium, because the concentrations of these second messengers were expected to be altered by unknown vasoactive peptides. For this purpose, we set up the high-throughput measurement system for cAMP and calcium concentrations. Among the vascular wall cells, rat endothelial cells and vascular smooth muscle cells were shown to elicit high responses to known peptides. Thus, we used these cells for the screening.By using rat endothelial cells and vascular smooth muscle cells, we observed clear responses in the cAMP assay. Based on the stimulatory effects of cAMP levels, we purified more than 10 peptides from the extracts of pig brain. As we have noticed that most of the cAMP-increasing activity is derived from calcitonin gene-related peptide(CGRP), the immunoreactive CGRP level was also measured. Some of the peaks were free from CGRP immunoreactivity and eluted at the retention times distinct from that of CGRP on the reverse phase HPLC.However, structural analyses indicated that these were CGRP-related peptides that partially degraded, oxidized or modified. Vasoactive intestinal peptide(VIP) was also isolated by using this assay system. In the assay system using renal epithelial cells and glia cells, CGRP, VIP and pituitary adenylate cyclase-activating polypeptide and their oxidized or degraded peptides were isolated and identified. As we still have other candidates for unidentified vasoactive peptides in the present assay system, further purification and identification of these candidates are on going. Less