ACUTE ACTION OF CHORIOGONADOTROPIN ON LEYDIG TUMOR-CELLS - INDUCTION OF A HIGHER AFFINITY BENZODIAZEPINE-BINDING SITE RELATED TO STEROID-BIOSYNTHESIS

We previously demonstrated that the mitochondrial peripheral-type benz odiazepine receptor (PBR) is coupled to hormone-activated steroidogene sis by regulating the intramitochondrial cholesterol transport, the ra te-determining step of steroid biosynthesis. In the present study we e xamined whether PBR is the site of hormone action using the hCG-respon sive MA-10 mouse Leydig tumor cell line as a model system. Within 15 s ec of the addition of hCG to Leydig cells a 3-fold cAMP-dependent incr ease in PBR binding was observed. This rapid increase returned to basa l levels within 60 sec. No effect was observed after 1 min in the cont inued presence of hCG. Scatchard analysis revealed that in addition to the known high affinity (5.0 nM) benzodiazepine-binding site, a secon d, hormone-induced, higher affinity (0.2 nM) benzodiazepine-binding si te appeared. We then examined whether in such a short time frame stero id synthesis occurs. Fifteen-second incubation of MA-10 cells with the inhibitor of cholesterol metabolism aminoglutethimide together with h CG also resulted in an increased rate of pregnenolone formation by the ir isolated mitochondria that were washed and incubated in aminoglutet himide-free buffer. The dose response of benzodiazepine binding to hCG closely parallels the increase in steroid formation by the mitochondr ia of stimulated cells. Addition of the selective inhibitor of cAMP-de pendent protein kinase, H-89, completely blocked hormone-induced PBR b inding and steroid formation, whereas addition of the inactive analog H-85 was without any effect. The addition of flunitrazepam, a benzodia zepine previously shown to inhibit the trophic hormone action on stero idogenesis, completely abolished the hCG-induced rapid stimulation of steroid synthesis. These results demonstrate that in MA-10 cells, the most rapid effect described thus far of hCG and cAMP, is the transient induction of a higher affinity benzodiazepine-binding site, which occ urs concomitantly with an increase in the rate of steroid formation. T his, in turn, suggests that these hormones alter PBR to activate chole sterol delivery to the inner mitochondrial membrane and subsequent ste roid formation.