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.