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Cloning of cDNA encoding a receptor to the glutamate derivative acromelic acid and the involvement in the pain induction system

编码谷氨酸衍生物丙烯酸受体的 cDNA 的克隆及其参与疼痛诱导系统

基本信息

批准号：
15590283
负责人：
NISHIZAWA Mikio
金额：
$ 2.3万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Acromelic acid (ACRO), a kainate analogue isolated from a poisonous mushroom Clitocybe acromelalga, produced tactile pain (mechanical allodynia), when intrathecally (i.t.) injected to mice. Induction of allodynia by prostaglandin (PG) F2alpha, as well as ACRO, was selectively lost one week after i.t. injection of a sublethal dose of ACRO, although no neuronal damage except slight gliosis was observed in the lower spinal cord. To isolate cDNA involving in allodynia, we established the method for i.t. administration of antisense oligonucleotides through tubing to mice. We identified an antisense oligonucleotide targeting FP mRNA, which caused disappearance of PGF2alpha-induced allodynia and decrease of FP mRNA in the spinal cord. PGF2alpha rapidly increased [Ca^<2+>]i of the cells in the deeper layer of the dorsal horn, when the spinal cord slices were prepared from the saline- administered mice. When the FP antisense oligonucleotide was administered, the population of PGF2alpha-responsi … More ve cells in the slices reduced, and PGF2alpha-induced [Ca^<2+>]i increase of these cells diminished. These data suggested that there are the FP-expressing cells involved in PGF2alpha-induced allodynia in the dorsal horn. As a biochemical probe for an ACRO receptor, we designed and synthesized a novel ACRO analog possessing an azido-functionalized phenyl group. Although the analog exerted a biological activity equivalent to ACRO, specific bands could not be identified by photoaffinity labeling experiments. To isolate candidates for an ACRO receptor, we screened a subtraction cDNA library using the spinal cord of the mice to which ACRO or saline was injected. Positive clones expressed specifically in spinal cord were isolated, and then the full-length cDNAs were isolated. Antisense oligonucleotides targeting these cDNAs were injected to mice by the above-mentioned method, and several suppressed the PGF2alpha-induced allodynia. Some cDNAs showed interaction with FP and other receptors, suggesting the involvement to the ACRO receptor. Less

Acromelic Acid (ACRO) 是一种从有毒蘑菇 Clitocybe acromelalga 中分离出来的红藻氨酸类似物，当鞘内注射给小鼠时，会产生触觉疼痛（机械性异常性疼痛）。前列腺素 (PG) F2α 以及 ACRO 诱导的异常性疼痛在 1 周后选择性消失。注射亚致死剂量的 ACRO，尽管在下脊髓中除了轻微的神经胶质增生外没有观察到神经元损伤。为了分离与异常性疼痛有关的cDNA，我们建立了它的方法。通过管道向小鼠施用反义寡核苷酸。我们鉴定了一种针对 FP mRNA 的反义寡核苷酸，它导致 PGF2α 诱导的异常性疼痛消失，并减少脊髓中 FP mRNA。当从给予盐水的小鼠制备脊髓切片时，PGF2α迅速增加背角深层细胞的[Ca 2+ ] i 。当施用 FP 反义寡核苷酸时，切片中 PGF2α 反应细胞的数量减少，并且 PGF2α 诱导的这些细胞的 [Ca^2+]i 增加减少。这些数据表明，表达 FP 的细胞参与了 PGF2α 诱导的背角异常性疼痛。作为 ACRO 受体的生化探针，我们设计并合成了一种具有叠氮基功能化苯基的新型 ACRO 类似物。尽管该类似物具有与 ACRO 相当的生物活性，但光亲和标记实验无法识别特定条带。为了分离 ACRO 受体的候选者，我们使用注射了 ACRO 或盐水的小鼠的脊髓筛选了消减 cDNA 文库。分离在脊髓中特异表达的阳性克隆，然后分离全长cDNA。通过上述方法将针对这些 cDNA 的反义寡核苷酸注射到小鼠体内，其中几种可以抑制 PGF2α 诱导的异常性疼痛。一些 cDNA 显示与 FP 和其他受体相互作用，表明与 ACRO 受体有关。较少的