EX-VIVO REGULATION OF ADRENAL-CORTICAL CELL STEROID AND PROTEIN-SYNTHESIS, IN RESPONSE TO ADRENOCORTICOTROPIC HORMONE STIMULATION, BY THE GINKGO-BILOBA EXTRACT EGB-761 AND ISOLATED GINKGOLIDE-B

We previously demonstrated that repeated treatment of rats with the st andardized extract of Ginkgo biloba leaves, EGb 761, and its bioactive component ginkgolide B (GKB), specifically reduces the ligand binding , and protein and messenger RNA expression of the adrenal mitochondria l peripheral benzodiazepine receptor (PER), a key element in the regul ation of cholesterol transport, resulting in decreased circulating cor ticosterone levels. Adrenocortical cells were isolated from rats treat ed with EGb 761 or GKB and cultured for 2 and 12 days. The effect of A CTH on normal and metabolically labeled cells was examined. Corticoste rone levels were measured by RIA, and protein synthesis was analyzed b y two-dimensional gel electrophoresis. Ex vivo treatment with EGb 761 and GKB resulted, respectively, in 50% and 80% reductions of ACTH-stim ulated corticosterone production by adrenocortical cells cultured for 2 days compared with that by cells isolated from saline-treated rats. Two-dimensional gel electrophoresis analysis revealed that in cells fr om both control and drug-treated animals, ACTH induced the synthesis, at the same level, of a 29-kDa and pi 6.4-6.7 protein identified as th e adrenal steroidogenic acute regulatory protein (StAR). In addition, treatment with EGb 761 and GKB specifically altered the synthesis of s even proteins, including inhibition of synthesis of a 17-kDa, identifi ed as PER. After 12 days in culture, ACTH-stimulated adrenocortical ce ll steroid synthesis was maintained, and it was identical among the ce lls isolated from animals treated with GKB or saline. Under the same c onditions, the expression of PER was recovered, whereas no effect of A CTH on the 29-kDa and 6.4-6.7 pi protein (StAR) or other protein synth esis could be seen. A comparative analysis of the effects of GKB and E Gb 761 on adrenocortical steroidogenesis and protein synthesis identif ied, in addition to the 17-kDa PER, target proteins of 32 kDa (pi 6.7) and 40 kDa (pi 5.7-6.0) as potential mediators of the effect of EGb 7 61 and GKB on ACTH-stimulated glucocorticoid synthesis. In conclusion, these results 1) validate and extend our previous in vivo findings on the effect of EGb 761 and GKB on ACTH-stimulated adrenocortical stero idogenesis, 2) demonstrate the specificity and reversibility of EGb 76 1 and GKB treatment, 3) question the role of the 29-kDa, 6.4-6.7 pi pr otein (mature StAR) as the sole mediator of ACTH-stimulated steroid pr oduction, and 4) demonstrate the obligatory role of PER in hormone-reg ulated steroidogenesis.