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 isoquinolinecarboxa- been proposed that glial cells can synthesize steroids de novo (6, 7). In fact, glial cells can convert cholesterol to pregnenolone, which then may be further metabolized to pregnenolone sulfate, 3f3-hydroxy-5-pregnene-20-one, 3/3-hydroxy-5-androstene-17-one, 3a-hydroxy-5a-pregnane-20-one, and 5a-pregnane-3a,21-diol-20-one, which positively or negatively modulate the GABA-gating of Cl-channels (4,8).Although detailed characterization of this biosynthetic apparatus in glial cells is far from complete, the cytochrome P450 side-chain-cleavage enzyme (P450scc), which participates in steroid biosynthesis by converting cholesterol to pregnenolone, has been identified immunocytochemically in primary glial cultures and in specific rat brain regions (7, 9). Two important findings prompted the present investigation: (i) the observations that in adrenal and testis receptors located on the outer membrane of mitochondria and which recognize benzodiazepines, isoquinolinecarboxamides, and imidazopyridines with high affinity-previously defined as peripheral-type benzodiazepine receptors-regulate intramitochondrial cholesterol transport, the rate-limiting step in steroidogenesis (10)(11)(12), and (ii) the demonstration that diazepam binding inhibitor (DBI), a polypeptide abundant in steroidogenic cells (1,13,14), can stimulate pregnenolone formation in adrenocortical and testicular Leydig cell mitochondria by an interaction with these mitochondrial receptors (15, 16). The present experiments show that rat glioma C6-2B cells and mitochondria prepared from these cells contain the P-45Scc enzyme and thereby convert cholesterol to pregnenolone. This enzymatic reaction can be activated by nanomolar amounts of DBI and the DBI fragment tetratriacontaneuropeptide [TTN,]. Since DBI, which is synthesized and processed in glial cells (17), competes against the binding of the mitochondrial receptor ligands 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) and 4'-chlorodiazepam (4'CD) with an affinity comparable to its potency in the steroidogenic effect, it appears to be the natural agonist for the receptor that controls steroidogenesis in glial cells. Therefore, we suggest that this glial mitochondrial receptor be termed MDR (mitochondrial DBI receptor). METHODSCell Culture. The C6-2B subclone of the rat glioma C6 cell line was obtained from Gary Brooker at this institute (18). These cells were adapted for growth in serum-fr...

Neurosteroids (steroids synthesized in the CNS) function by modulating neurotransmission. To establish an experimental model for investigation of neurosteroid synthesis and regulation, independent of blood‐borne steroids, we examined the steroidogenic activity of isolated rat retinas. We identified progesterone, pregnenolone, dehydroepiandrosterone, desoxycorticosterone, 3α,5α‐tetrahydrodesoxycorticosterone, 3α‐hydroxy‐5α‐dihydroprogesterone, 17‐hydroxyprogesterone, and 17‐hydroxypregnenolone together with their esterified forms. As pregnenolone is the precursor of all steroids, its formation was studied in detail as an index of a steroid‐synthesizing tissue. Pregnenolone was identified further by gas chromatography coupled to mass spectrometry, and its in vitro synthesis was inhibited by lovastatin, an inhibitor of mevalonolactone and cholesterol biosynthesis. We then examined pregnenolone synthesis in the presence of mevalonolactone as a precursor of sterol formation together with lovastatin, which reduces endogenous mevalonolactone synthesis, as well as with inhibitors of pregnenolone metabolism. The incorporation of mevalonolactone into pregnenolone and its sulfate ester was time‐ and concentration‐dependent and blocked by aminoglutethimide, a competitive inhibitor of cytochrome P450 side‐chain cleavage (P450scc) enzyme. Immunocytochemical studies with a specific antibody to P450scc revealed a primary localization of the enzyme at the retinal ganglion cell layer. A less pronounced immunostaining was also seen at cells of the inner nuclear layer. Compounds known to stimulate cyclic AMP content also stimulated pregnenolone formation by rat retinas. These results demonstrate that rat retinas synthesize steroids and, for the first time, they reveal the steroidogenic ability of neuronal cells. We propose rat retinas as an in vitro model system to study neurosteroidogenesis in the CNS.

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