Molecular and neurochemical evidence for the biosynthesis of dehydroepiandrosterone in the adult rat spinal cord
Molecular and neurochemical evidence for the biosynthesis of dehydroepiandrosterone in the adult rat spinal cord
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DOI：
10.1111/j.1471-4159.2005.03113.x
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发表时间：
2005-06
影响因子：
4.7
通讯作者：
Cherkaouia Kibaly;C. Patte-Mensah;A. Mensah-Nyagan
中科院分区：
文献类型：
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作者：
Cherkaouia Kibaly;C. Patte-Mensah;A. Mensah-Nyagan
各种研究表明，外源性脱氢表雄酮（DHEA）调节啮齿动物中枢神经系统的几种机制。由于成年啮齿动物腺体不分泌大量的DHEA，因此只有当DHEA由神经细胞产生时，其作为CNS的内源性调节剂的作用才有可能。因此，在过去的十年中，各种旨在证明成年啮齿动物CNS中关键DHEA合成酶细胞色素P450 c17（P450 c17）活性的研究均未成功。本研究结合分子、解剖、细胞和神经化学方法首次证实了P450 c17在成年大鼠脊髓中的存在及其生物活性。真实的实时RT-PCR显示P450 c17基因在所有SC节段中表达。免疫印迹分析允许识别一个特定的P450 c17蛋白在SC和免疫组织化学研究定位P450 c17在神经元和神经胶质细胞。结合HPLC和放射性类固醇检测的脉冲追踪实验表明，SC切片将[3 H]双烯醇酮转化为[3 H]DHEA，P450 c17抑制剂酮康唑显著降低了这种转化。动力学研究表明，积累的[3 H]DHEA新合成的SC片在孵育培养基中，其量下降缓慢。成年啮齿动物SC中活性P450 c17的第一个细胞图谱表明，脊髓神经网络中合成的内源性DHEA可能控制各种脊髓介导的活动。
Various studies have indicated that exogenous dehydroepiandrosterone (DHEA) modulates several mechanisms in the CNS of rodents. As adult rodent glands do not secrete significant amounts of DHEA, its role as endogenous modulator of the CNS remains possible only if DHEA is produced by nerve cells. Therefore, the last decade has been marked by diverse unsuccessful investigations aiming to demonstrate the activity of cytochrome P450c17 (P450c17), the key DHEA‐synthesizing enzyme, in adult rodent CNS. Here, we combined molecular, anatomical, cellular and neurochemical approaches to provide the first demonstration of the existence of P450c17 and bioactivity in adult rat spinal cord (SC). Real‐time RT‐PCR revealed P450c17 gene expression in all SC segments. Western blot analyses allowed identification of a specific P450c17 protein in the SC and immunohistochemical studies localized P450c17 in neurones and glial cells. Pulse‐chase experiments combined with HPLC and radioactive steroid detection showed that SC slices converted [3H]pregnenolone into [3H]DHEA, a conversion markedly reduced by ketoconazole, a P450c17 inhibitor. Kinetics studies revealed accumulation of [3H]DHEA newly synthesized by SC slices in the incubation medium as its amount declined slowly. This first cellular mapping of an active P450c17 in adult rodent SC suggests that endogenous DHEA synthesized in spinal neural networks may control various spinally‐mediated activities.