Wang Wei, Ji Ping, Riopelle Richard J, Dow Kimberly E
Department of Pediatrics, Kingston General Hospital, Ontario, Canada.
J Neurochem. 2002 Jan;80(2):287-94. doi: 10.1046/j.0022-3042.2001.00687.x.
Corticotropin-releasing hormone (CRH), known as a key regulator of the hypothalamic-pituitary-adrenal axis response to stress, elicits its biological effects by binding to two membrane receptors (CRH-R1 and CRH-R2). The present studies examined the presence of functional expression of CRH receptors in cultured microglia of rat. CRH-R1 mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and receptor chemical cross-linking assay in cultured microglia. CRH-R2 mRNA was undetectable by RT-PCR. The radioligand binding analysis using [125I]Tyr-rat/human CRH revealed a high affinity binding site (Kd of 1.2 nm and Bmax of 84 fmol/mg of protein). Competition studies using CRH and related peptides indicated kinetic and pharmacological characteristics consistent with the CRH-R1 receptor subtype. Receptor chemical cross-linking assay demonstrated a single band of CRH receptor with a molecular weight of -77 kDa, which was inhibited in the presence of excess unlabeled rat/human CRH in a dose-dependent manner and inhibited by a CRH receptor antagonist astressin. Functional coupled cAMP production in cultured microglia was stimulated by exogenous addition of CRH and related peptides in a dose-dependent manner and blocked by astressin. Our findings suggest the functional expression of CRH-R1 receptor in rat microglia, indicating an important mechanism of interaction between immune and neuroendocrine systems in brain physiological and pathological conditions.
促肾上腺皮质激素释放激素（CRH）是下丘脑 - 垂体 - 肾上腺轴对应激反应的关键调节因子，它通过与两种膜受体（CRH - R1和CRH - R2）结合来发挥其生物学效应。本研究检测了大鼠培养的小胶质细胞中CRH受体功能表达的情况。通过逆转录聚合酶链反应（RT - PCR）、蛋白质印迹法和受体化学交联分析在培养的小胶质细胞中检测到了CRH - R1 mRNA和蛋白质。RT - PCR未检测到CRH - R2 mRNA。使用[125I]Tyr - 大鼠/人CRH进行的放射性配体结合分析显示存在一个高亲和力结合位点（解离常数Kd为1.2 nM，最大结合量Bmax为84 fmol/mg蛋白质）。使用CRH和相关肽进行的竞争研究表明其动力学和药理学特性与CRH - R1受体亚型一致。受体化学交联分析显示有一条分子量约为77 kDa的CRH受体条带，在存在过量未标记的大鼠/人CRH时，其呈剂量依赖性受到抑制，并且被CRH受体拮抗剂阿斯特辛抑制。外源性添加CRH和相关肽以剂量依赖性方式刺激培养的小胶质细胞中功能性偶联的环磷酸腺苷（cAMP）产生，并被阿斯特辛阻断。我们的研究结果表明大鼠小胶质细胞中存在CRH - R1受体的功能表达，这表明在脑生理和病理状态下免疫和神经内分泌系统之间相互作用的一个重要机制。
