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

为了维持体内循环系统的稳态平衡，生物体具有精确的、高度组织化的调节系统，这在现阶段还远未完全阐明。我们能够理解这种调节系统的原因之一是，仍然存在未知的机制，这些机制由未知的肽、蛋白质或其他化合物调节。在本项目中，我们试图鉴定一种新的血管活性肽并阐明其生理功能。作为血管活性肽的来源，我们首先检测了培养的血管内皮细胞的上清液。虽然内皮素-1是从条件培养基中分离出来的，但大多数其他已知的生物活性肽被蛋白水解裂解，并且发现与降解产物相比，生物活性分子的比例相对较低。因此，在本研究中，我们用上述方法从猪脑（20 kg）中提取起始肽级分， ...更多信息 在靶细胞方面，我们建立了一系列培养的人、大鼠、猪和牛的内皮细胞和血管平滑肌细胞。我们制备了原代培养的新生大鼠心肌细胞和心脏成纤维细胞。此外，还引入肾小管上皮细胞、神经胶质细胞及其相关细胞用于测量生物活性，因为这些细胞在以下测定中在市售细胞中引起适当的反应。至于生物活性的测量，我们评估了第二信使的水平，如cAMP、cGMP和钙，因为这些第二信使的浓度预计会被未知的血管活性肽改变。为此，我们建立了cAMP和钙离子浓度的高通量测量系统。在血管壁细胞中，大鼠内皮细胞和血管平滑肌细胞显示出对已知肽的高反应。因此，我们使用这些细胞进行筛选。通过使用大鼠内皮细胞和血管平滑肌细胞，我们在cAMP测定中观察到明确的反应。基于对cAMP水平的刺激作用，我们从猪脑提取物中纯化了10多个肽。由于我们已经注意到大部分cAMP增加活性来自降钙素基因相关肽（CGRP），因此还测量了免疫反应性CGRP水平。一些峰没有CGRP免疫反应性，在反相HPLC上洗脱的保留时间与CGRP的保留时间不同，然而，结构分析表明这些是部分降解、氧化或修饰的CGRP相关肽。利用该系统还分离了血管活性肠肽（VIP）。在使用肾上皮细胞和神经胶质细胞的测定系统中，分离并鉴定了CGRP、VIP和垂体腺苷酸环化酶激活多肽及其氧化或降解肽。由于我们在目前的检测系统中仍然有其他候选的未鉴定的血管活性肽，这些候选肽的进一步纯化和鉴定正在进行中。少

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