ASTROCYTES AND NEUROSTEROIDS - METABOLISM OF PREGNENOLONE AND DEHYDROEPIANDROSTERONE - REGULATION BY CELL-DENSITY
ASTROCYTES AND NEUROSTEROIDS - METABOLISM OF PREGNENOLONE AND DEHYDROEPIANDROSTERONE - REGULATION BY CELL-DENSITY
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DOI：
10.1083/jcb.121.1.135
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发表时间：
1993-04-01
影响因子：
7.8
通讯作者：
LEGOASCOGNE, C
中科院分区：
文献类型：
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作者：
AKWA, Y;SANANES, N;LEGOASCOGNE, C
此前已证明大鼠中枢神经系统 (CNS) 合成孕烯醇酮 (PREG) 并将其转化为黄体酮 (PROG) 和 7α-羟基-PREG (7α-OH PREG)。星形胶质细胞参与中枢神经系统功能的调节，可能参与神经类固醇的代谢。使用选择性培养条件从胎鼠前脑中获得纯化的 1 型星形胶质细胞，并通过特异性抗体（GFAP+、A2B5-）的免疫染色进行鉴定。将它们以低、中或高密度（分别为 2.5-5 x 10(5)、1-2 x 10(6) 或 4-8 x 10(6) 细胞/培养皿）铺板并维持 21 d。然后将它们与 C-14-PREG 和 C-14-DHEA 一起孵育 24 小时，并分析从细胞和培养基中提取的类固醇。大多数放射性衍生物被释放到培养介质中。主要观察到两条代谢途径。 PREG和DHEA分别被氧化为PROG和雄烯二酮（ADIONE）[3β-羟基类固醇脱氢酶，DELTA5→4 3-酮类固醇异构酶（3β-HSD）活性]，并分别转化为7α-OH PREG和7α-OH DHEA（7α-羟化酶活性）。低密度铺板后，PROG 和 ADIONE 的形成约为孵育放射性的 10%，是 7α-羟基化代谢物的十倍。相反，在高密度铺板后，形成低水平的PROG和ADIONE，而向7α-OH PREG或7α-OH DHEA的转化率大于或等于50%。每个细胞表达的结果表明，在高细胞密度下，3β-HSD 活性几乎完全受到抑制，而 7α-羟基化则保持或增加。类固醇代谢模式与测量时的细胞密度有关，而不是与细胞的早期定型有关：当原代培养物以高密度（8 x 10（6）细胞/培养皿）铺板，然后在多次稀释（3倍、9倍或27倍）后传代培养时，3β-HSD活性仅在低密度下恢复。此外，当离心 5 x 10(5) 个细胞并将所得簇铺板时，3β-HSD 活性降低，而类固醇 7α-羟基化增强。这意味着细胞密度本身，但细胞数量和扩散因子都不参与类固醇代谢的调节。我们得出的结论是，培养物中的星形胶质细胞代谢 PREG 和 DHEA，并且代谢转化以及相关的酶活性取决于细胞间的接触。因此，星形胶质细胞可以将类固醇递送至中枢神经系统中的其他细胞类型。形成的代谢物的性质取决于细胞聚集的状态，并且可能涉及生理和/或病理状况（中枢神经系统损伤的修复、星形细胞肿瘤）。
The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7alpha-hydroxy-PREG (7alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with C-14-PREG and C-14-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3beta-hydroxysteroid-dehydrogenase, DELTA5-->4 3-ketosteroid-isomerase (3beta-HSD) activity], and converted to 7alpha-OH PREG and 7alpha-OH DHEA, respectively (7alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7alpha-OH PREG or 7alpha-OH DHEA was greater-than-or-equal-to 50%. The results expressed per cell indicated that the 3beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3beta-HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3beta-HSD activity was decreased, whereas steroid 7alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts. Thus, astrocytes can deliver steroids to other cell types in the CNS. The nature of the metabolites formed depends on the state of cell aggregation and may be involved in physiological and/or pathological conditions (repair of CNS injury, astrocytic tumors).