Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.

Abstract

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...

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