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

AcroMelic acid(ACRO)是从有毒蘑菇Clitcybe acromelalga中分离出来的一种海人藻酸类似物，当鞘内注射(I.T.)时，会产生触觉疼痛(机械性超敏)。注射给小鼠。注射后一周，前列腺素(PG)F2pha和ACRO诱导的痛觉超敏反应选择性消失。注射亚致死剂量的ACRO，但在脊髓下部未观察到神经元损伤，仅有轻微的胶质细胞增生。为了分离与痛觉异常有关的基因，我们建立了I.T.小鼠经管内注射反义寡核苷酸。我们发现了一种针对FP mRNA的反义寡核苷酸，它能导致PGF2α诱导的痛觉异常消失，并导致脊髓中FP mRNA的减少。用生理盐水制备小鼠脊髓切片后，PGF2α可使脊髓背角深层细胞[Ca~(2+)]i迅速升高。当给予FP反义寡核苷酸时，PGF2α反应的…群体脑片内VE细胞减少，PGF2α诱导的[Ca^&lt；2+&gt；]i升高作用减弱。这些结果提示，在背角存在着参与PGF2α引起的痛觉过敏的FP表达细胞。作为ACRO受体的生化探针，我们设计并合成了一种新型的具有叠氮功能化苯基的ACRO类似物。虽然该类似物具有相当于ACRO的生物活性，但通过光亲和标记实验无法识别特定的条带。为了分离ACRO受体的候选基因，我们使用注射了ACRO或生理盐水的小鼠的脊髓筛选了一个消减cDNA文库。分离脊髓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，并有几种反义寡核苷酸抑制了PGF2pha诱导的痛觉过敏。一些cDNA与FP和其他受体相互作用，提示ACRO受体参与其中。较少

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