Studies on CGRP receptor subtypes and their specific intracellular signaling pathways in the periodontal tissue

本论文在前人研究的基础上，尝试制备了CGRP的一些片段，并对这些新的多肽进行了生物活性测定。1）CGRP片段的产生和筛选-除了市售的人CGRP_<1-37>、CGRP_<8-37>、CGRP_<19-37>、CGRP_<22-37>和[Cys（Acm）2，7]-CGRP之外，我们还产生了CGRP_<1-12>、反向-CGRP_<1-8>和反向-CGRP_<1-14>。经cAMP生成量及其它指标筛选，发现CGRP_<8-37>，CGRP_<22-37>和[Cys（Acm）^&lt;2，7&gt;]-CGRP具有明显的激动或拮抗作用。2)生物活性CGRP片段对细胞内信号通路的影响-在成骨细胞MG 63中，CGRP<8-37>有效地拮抗CGRP<1-37>在cAMP产生中的作用。CGRP_<22-37>1也有较弱的拮抗作用。[Cys（Acm）^&lt;2，7&gt;]-CGRP显示中等拮抗和弱激动作用。在牙龈成纤维细胞Gin-1或口腔上皮SCC 25细胞中获得了类似的结果。3)生物活性CGRP片段的结合亲和力-氟-CGRP以浓度依赖性方式结合其受体，并且其结合被CGRP-特异性取代<8-37>，但不清楚地被[Cys（Acm）^&lt;2，7&gt;]-CGRP取代。4)CGRP片段对细胞增殖的影响-这里产生的任何CGRP片段都不能可重复地影响增殖，但只有CGRP_<1-37>微弱地刺激Gln-1增殖。5)CGRP片段对细胞凋亡的影响-任何CGRP片段在本文使用的任何细胞类型中均不明显诱导凋亡。6)CGRP受体亚型在牙周组织中的表达-CRLR和RAMP-I蛋白的表达使用那些特异性抗体在牙周组织中或在本文使用的任何细胞类型中检测。

Based on the published data about 3-D configuration of CGRP, we have attempted to produce some CGRP fragments and performed bioassay with those new peptides. However, we could not particularly find any bioactive peptides.1) Production of CGRP fragments and screening - In addition to commercially available human CGRP_<1-37>, CGRP_<8-37>, CGRP_<19-37>, CGRP_<22-37> and [Cys(Acm)^<2,7>]-CGRP, we have produced CGRP_<1-12>, reverse-CGRP_<1-8> and reverse-CGRP_<1-14>. On screening with cAMP production and other indeces, we have found that CGRP_<8-37>, CGRP_<22-37> and [Cys(Acm)^<2,7>]-CGRP have substantial agonistic or antagonistic action. 2) Effects of bioactive CGRP fragments on intracelllar signaling pathways - In osteoblastic MG63 cells, CGRP_<8-37> potently anta gonized CGRP_<1-37> action in cAMP production. CGRP_<22-37>, also had a weak antagonistic action. [Cys(Acm)^<2,7>]-CGRP showed both a medium antagonistic and a weak agonistic action. Similar results were obtained in either gingival fibroblastic Gin-1 or oral epithelial SCC25 cells. 3) Binding affinity of bioactive CGRP fragments - Fluoro-CGRP bound to its receptors in a concentration-dependent fashion, and its binding was specifically replaced with CGRP_<8-37>, but not clearly with [Cys(Acm)^<2,7>]-CGRP. 4) Effects of CGRP fragments on cell proliferation - Any CGRP fragments produced here failed to reproducibly influence the proliferation, but only CGRP_<1-37> weakly stimulated Gin-1 proliferation. 5) Effects of CGRP fragments on cell apoptosis - Any CGRP fragments did not apparently induce apoptosis in any cell types used here. 6) Expression of CGRP receptor subtypes in periodontal tissues - Expression of both CRLR and RAMP-I proteins were detected using those specific antibodies either in periodontal tissues or in any cell types used here.