Expression of mitochondrial benzodiazepine receptor and its putative endogenous ligand diazepam binding inhibitor in cultured primary astrocytes and C-6 cells: relation to cell growth.

A recognition site for benzodiazepines structurally different from that linked to the gamma-aminobutyric acid receptor subtype A or the "central type" benzodiazepine receptor has been located mainly in the outer membranes of mitochondria and designated mitochondrial benzodiazepine receptor (MBR). A putative endogenous ligand for MBR is the peptide, diazepam binding inhibitor (DBI), which inhibits benzodiazepine ligand binding in mitochondrial membranes. In this study, DBI- and MBR-like immunoreactivities (LI) were examined at the cellular and ultrastructural levels, and their changes during cell growth were followed in rat primary cerebellar astroglial and C-6 cell cultures. During the early stages of the cultures (7-14 days in vitro), MBR and DBI were expressed virtually in all astrocytes and C-6 cells of the cultures. The highest MBR/DBI immunoreactivity was observed in dividing cells. When the astrocytes had formed a confluent layer (21 days in vitro), MBR staining intensity was significantly decreased. Electron microscopic analysis demonstrated an even distribution of DBI-LI throughout the cytoplasm, while MBR-LI was mainly observed in a close association with the outer mitochondrial membranes. However, dividing cells also displayed strong MBR immunoreactivity in endoplasmic reticulum, nuclear membranes, and centrioles. Treatment of the confluent cultures with MBR ligands PK 11195 and Ro 5-4864 at nanomolar concentrations increased the density of MBR-LI and the progesterone content in the medium 2-3-fold over the basal levels. These results demonstrate a close association between DBI and mitochondrial benzodiazepine receptors and lend support to the theory that they have a possible role in the regulation of steroid production. The relation of MBR and DBI expression to cell growth and division suggests a novel role for these elements in the regulation of important intracellular events.