Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.
Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.
复制标题
DOI：
10.1073/pnas.89.11.5113
复制
发表时间：
1992-06
影响因子：
11.1
通讯作者：
V. Papadopoulos;Patrizia GUARNERIt;Karl E. KRUEGERt;Alessandro GUIDOTTIt;Erminio COSTAt
中科院分区：
文献类型：
--
作者：
V. Papadopoulos;Patrizia GUARNERIt;Karl E. KRUEGERt;Alessandro GUIDOTTIt;Erminio COSTAt
C6-2B胶质瘤细胞系富含与苯二氮卓类、咪唑并吡啶和异喹啉羧胺类化合物(以前称为外周型苯二氮卓类受体)高亲和力的线粒体受体，以此为模型，研究多肽安定结合抑制物(DBI)及其加工产物在线粒体受体调节的类固醇合成中的意义。在C6-2B胶质瘤细胞中，DBI及其天然存在的片段被发现以较高的浓度存在，能与特异性的异喹啉甲酰胺或4‘-氯安定以高亲和力与线粒体识别位点竞争，并刺激线粒体孕烯醇酮的形成。这些数据表明，这种类型的细胞可能同时表达受体和假定的激动剂配体来调节类固醇的生成。因此，我们建议将这种线粒体受体命名为MDR(线粒体DBI受体)，以表明其在类固醇生物合成中对DBI的反应性。在目前的工作中，我们证明了C6-2B细胞的线粒体通过氨基戊二酰亚胺阻断的机制将(22R)-22-羟基胆固醇转化为孕烯醇酮。免疫印迹证实C6-2B细胞线粒体中存在较高水平的细胞色素P-450胆固醇侧链裂解酶。此外，与MDR的18 kDa线粒体多肽亚单位相关的异喹啉羧胺结合位点在C6-2B胶质瘤细胞线粒体中含量丰富(Bmax约为30pmol/mg蛋白质)，并与类固醇生物合成的调节有关。具有纳摩尔浓度的天然多肽DBI及其天然加工产物四核苷酸神经肽[DBI-(17-50)]的MDR的占据增加了孕烯醇酮的形成。氯硝西潘和十八肽[DBI-(33-50)]对γ-氨基丁酸A型受体有较高的亲和力，但对MDR的亲和力较低，对孕烯醇酮的合成没有促进作用。这些发现为C6-2B细胞表现出与外周内分泌器官相似的显着类固醇生成活性提供了证据，并提示MDRs和DBI参与神经胶质细胞类固醇生成的调节。
The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high concentrations in C6-2B glioma cells, to compete against specific isoquinolinecarboxamide or 4'-chlorodiazepam binding to mitochondrial recognition sites with high affinity, and to stimulate mitochondrial pregnenolone formation. These data suggest that this cell type may express both the receptor and the putative agonist ligand to regulate steroidogenesis. Therefore, we propose to term this mitochondrial receptor MDR (mitochondrial DBI receptor) to indicate its responsiveness to DBI in steroid biosynthesis. In the present work, we show that mitochondria of C6-2B cells convert (22R)-22-hydroxycholesterol to pregnenolone by a mechanism blocked by aminoglutethimide. Immunoblotting confirmed the presence of relatively high levels of cytochrome P-450 cholesterol side-chain-cleavage enzyme in C6-2B cell mitochondria. Furthermore, isoquinolinecarboxamide binding sites associated with the 18-kDa mitochondrial polypeptide subunit of the MDR are abundant in C6-2B glioma cell mitochondria (Bmax approximately 30 pmol/mg protein) and are coupled to the regulation of steroid biosynthesis. Occupancy of MDRs with nanomolar concentrations of the naturally occurring polypeptide, DBI, as well as its naturally occurring processing product tetratriacontaneuropeptide [DBI-(17-50)] increases pregnenolone formation. Clonazepam and octadecaneuropeptide [DBI-(33-50)], which exhibit a higher affinity for gamma-aminobutyric acid type A receptors but a low affinity for MDR, were ineffective in stimulating pregnenolone synthesis. These findings provide evidence that C6-2B cells exhibit a significant steroidogenic activity which resembles that found in peripheral endocrine organs and they suggest that MDRs and DBI are involved in the regulation of glial cell steroidogenesis.