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

Acromelic acid （ACRO）是一种从毒蘑菇Clitocybe acromelalga中分离出来的盐酸盐类似物，当鞘内注射给小鼠时，会产生触觉痛（机械性异常痛）。前列腺素（PG） F2alpha和ACRO诱导的异常性疼痛在注射亚致死剂量ACRO一周后选择性消失，尽管除了在脊髓下段观察到轻微胶质细胞增生外，未见神经元损伤。为了分离与异常性痛觉有关的cDNA，我们建立了通过管道给药小鼠的方法。我们发现了一个针对FP mRNA的反义寡核苷酸，该核苷酸导致pgf2alpha诱导的异常性疼痛消失和脊髓中FP mRNA的减少。在给药小鼠脊髓切片中，PGF2alpha迅速增加背角深层细胞[Ca^<2+>]i。当给药FP反义寡核苷酸时，pgf2α -反应的细胞数量减少，pgf2α -诱导的[Ca^<2+>]i增加的细胞数量减少。这些数据表明，在pgf2alpha诱导的背角异常性疼痛中有表达fp的细胞参与。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团功能化苯基的新型ACRO类似物。虽然该类似物具有与ACRO相当的生物活性，但无法通过光亲和标记实验识别特异性条带。为了分离候选的ACRO受体，我们使用注射了ACRO或生理盐水的小鼠脊髓筛选了一个减法cDNA文库。分离脊髓特异性表达阳性克隆，分离全长cdna。通过上述方法将靶向这些cdna的反义寡核苷酸注射到小鼠体内，其中一些抑制了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，提示与ACRO受体有关。少

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