Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.
Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.
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DOI：
10.4049/jimmunol.147.10.3482
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发表时间：
1991-11
影响因子：
4.4
通讯作者：
J. Mcgillis;S. Humphreys;S. Reid
中科院分区：
文献类型：
--
作者：
J. Mcgillis;S. Humphreys;S. Reid
降钙素基因相关肽（CGRP）是一种存在于周围神经元中的血管活性神经肽，在局部炎症部位释放。在这些研究中，特异性高亲和力腺苷酸环化酶连接的CGRP受体在大鼠淋巴细胞上被表征。通过放射配体结合分析CGRP受体的分布、亲和力和特异性。125I-[His10]CGRP与大鼠淋巴细胞结合迅速，在22℃下20 ~ 30 min达到平衡，并依赖于细胞浓度。CGRP受体在纯化T淋巴细胞和B淋巴细胞上的解离常数Kd分别为0.807 +/- 0.168 nM和0.387 +/- 0.072 nM，密度分别为774 +/- 387和747 +/- 244个结合位点/细胞。竞争结合研究表明，大鼠CGRP抑制125I-[His10]CGRP与淋巴细胞结合的亲和力最高（Ki = 0.192 +/- 0.073），其次是人CGRP和CGRP受体拮抗剂CGRP8-37。125I-[His10]CGRP与大鼠淋巴细胞的结合不受神经肽物质P、降钙素或神经肽y的抑制。淋巴细胞CGRP受体蛋白通过二琥珀酰亚酸共价交联125I-[His10]CGRP与其受体通过亲和标记鉴定。SDS-PAGE分析显示特异性标记的CGRP结合蛋白分子量为74.5和220 kDa。同时还观察到第三条没有穿透凝胶的高分子质量蛋白带。在功能研究中，CGRP刺激cAMP快速持续增加，ED50约为8 pM。在比较异丙肾上腺素（β 2-肾上腺素能激动剂）和CGRP最佳浓度的实验中，异丙肾上腺素治疗后30分钟细胞内cAMP升高恢复到基础水平，而CGRP治疗后60分钟cAMP仍升高。对CGRP的反应是特异性的，可以被CGRP8-37完全阻断。T淋巴细胞和B淋巴细胞上存在高亲和功能的CGRP受体，这为CGRP在调节淋巴细胞功能中的调节作用提供了证据。
Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.