ASTROCYTES AND NEUROSTEROIDS - METABOLISM OF PREGNENOLONE AND DEHYDROEPIANDROSTERONE - REGULATION BY CELL-DENSITY

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).