Delivery ofcholesterol to inner mitochondrial membranes is rate-limiting for steroidogenesis in the zona fasciculata of adrenal cortex. A protein that stimulates this process was isolated to homogeneity from bovine adrenal tissue. This protein's primary structure has been determined in its entirety by a combination of automated Edman microsequencing, fast-atom bombardment mass spectrometry (FAB-MS). The sequence was identical to that previously reported for bovine brain endozepine, except that it lacks the last two residues, -Gly-Ile, at the C terminus. To our knowledge, isolation of an endozepine-related protein from a tissue other than brain has not been reported previously. Endozepine competes with benzodiazepines for saturable binding sites in synaptosomes and in mitochondria of specific peripheral tissues. Previous reports have localized the adrenal benzodiazepine receptor to the outer mitochondrial membrane. In this report, we show that the prototypic benzoiazepine, diazepam, effects a stimulation of adrenal mitochondrial cholesterol delivery similar to that observed for endozepine. The effective diazepam concentration was consistent with that previously shown to displace a high-affinity ligand of the mitochondrial benzodiazepine receptor. The action of diazepam in adrenal mitochondria suggests that the mediation of corticotropininduced steroidogenesis may be the physiological function of the peripheral-type benzodiazepine receptor. These studies provide new insights into the previously unknown function of peripheral benzodiazepine receptors and should allow new investigations into the stimulation of steroidogenesis by endozepines and benzodiazepines in the brain and in certain peripheral tissues.

We have purified to homogeneity a protein from bovine adrenal (fasciculata) cells that is capable of: 1) stimulating synthesis of pregnenolone (in a concentration-dependent fashion) by mitochondria when added with exogenous cholesterol; 2) increasing the concentration of cholesterol in outer and inner membrane when added to mitochondria with exogenous cholesterol; 3) increasing the rate of transport of cholesterol from outer membrane to inner membrane when the two membranes are incubated together in an aqueous buffer and subsequently separated by centrifugation; and 4) increasing the proportion of molecules of C27 side-chain cleavage P-450 that are bound to the substrate cholesterol. The protein is homogeneous according to two-dimensional polyacrylamide gels, shows a molecular weight of 8200 and is therefore referred to as 8.2 K. It is proposed that 8.2 K may be involved in the regulation of steroid synthesis in bovine fasciculata cells, possibly by stimulating entry of cholesterol into the outer membrane and by altering the intramitochondrial distribution of this substrate.

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.

Bovine but not rat fasciculata cells show a concentration-dependent stimulation of the production of corticosteroids by addition of external Ca2+ to the incubation medium. Both cell types respond to ACTH in a concentration-dependent manner. Increasing concentrations of K+ (0-20 mM) cause increased production of corticosteroids and accelerated influx of Ca2+ in bovine fasciculata cells but no change in either of these two parameters in rat fasciculata cells. Two inhibitors of Ca2+ channels (nifedipine and PY108-068) inhibit both the production of steroids by unstimulated bovine cells and the stimulation produced by three agents (ACTH, (Bu)2 cAMP, and K+). Half-maximal inhibition of these responses was produced in each case by submicromolar or low micromolar concentrations of the inhibitors. These inhibitors are without effect in rat fasciculata cells. One Ca2(+)-channel agonist (BAY K8644) stimulated synthesis of steroids by bovine cells and potentiated the response to ACTH. The agonist acts in the low micromolar range but was without effect on rat cells or on the responses of steroid synthesis by these cells to either ACTH or dibutyryl cAMP. Moreover, bovine fasciculata cells show specific binding sites for [+]PN 200-110, a specific ligand for 1,4-dihydropyridine receptors associated with voltage-dependent Ca2+ channels [dissociation constant (Kd), 14.3 nM; maximum number of binding sites (Bmax), 0.52 pmol/10(6) cells]. Rat cells show no specific binding of PN 200-110. We conclude that bovine fasciculata cells possess voltage-dependent Ca2+ channels which are involved in the regulation of steroid synthesis in these cells and that rat fasciculata cells are without such channels.