The non-benzodiazepine anxiolytic drug etifoxine causes a rapid, receptor-independent stimulation of neurosteroid biosynthesis.

Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.

Influence of 17beta-estradiol on the synthesis of reduced neurosteroids in the brain (in vivo) and in glioma cells (in vitro): possible relevance to mental disorders in women.

Brain neurosteroids modulate gamma-aminobutyric acid type A (GABAA) receptor activity, thereby playing a role in mood disorders. Alterations in 17beta-estradiol (E2) and progesterone (P) are also known to play a significant role in psychopathology in women. The aim of the present study was to evaluate the synthesis of dihydroprogesterone (DHP), tetrahydroprogesterone (THP), and the activity of 5alpha-reductase (5alphaR) which regulates the reduction of P to DHP on exposure to supraphysiological levels of E2 in vitro (C6 glioma cells) and in vivo (mouse brain). The results showed that supraphysiological levels of E2 induced a decrease in the accumulation of both neurosteroids, probably by decreasing the activity of 5alphaR. We hypothesize that the high levels of E2 in pregnancy attenuate the increase in the conversion of P to THP in the brain and that the ratio of E2/P modulates the sedative effect of THP. This process may be relevant to psychopathological disorders that are ascribed to drastic alterations in estrogen levels, such as premenstrual syndrome, pregnancy-related mental disorders, and postpartum "blues".

The direct luteotropic effect of tokishakuyakusan in rats.

The effect of Tokishakuyakusan (TS) on the corpus luteum function in pseudopregnant rats was examined in vivo. On day 4 of pseudopregnancy (PSP), induced by cervical stimulation, TS (20 micrograms) stimulated the progesterone secretion rate (PSR) in the ovarian venous plasma. There was also a significant increase in the rate of progesterone to 20 alpha-OH-progesterone. However, on day 8 of PSP, there was no apparent change in PSR in the ovarian venous plasma after the administration of TS. These data suggest that the sensitivity to TS of the corpus luteum varies according to its age.

Influence of electrical stimulation of the hypothalamus on ovarian steroidogenesis in hypophysectomized and adrenalectomized rats.

The effects of electrical stimulation of the lateral hypothalamic area (LHA), periventricular arcuate nucleus (ARC) and ventromedial hypothalamic nucleus (VMH) on the rates of 14C transfer from 14C-1 acetate into ovarian steroids in ovarian slices of hypophysectomized and adrenalectomized (H-A) rats were investigated. The 14C transfer rates into estrogen and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) were decreased by LHA stimulation. The stimulation of the ARC and VMH increased the rates of 14C transfer into estrogen, progesterone and 20 alpha-OH-P. From these results, it might be suggested that these hypothalamic structures were involved in the regulation of ovarian steroidogenesis without participation of the pituitary and adrenal.

Forskolin and phosphodiesterase inhibitors stimulate rat granulosa cell differentiation.

The effect of forskolin (an adenyl cyclase activator) and 1-methyl-3-isobutylxanthine (MIX, a phosphodiesterase inhibitor) on granulosa cell steroidogenesis and LH receptor formation was studied in vitro. Granulosa cells from immature hypophysectomized, estrogen-treated rats were cultured for 2-3 days in androstenedione-supplemented media in the absence or presence of FSH or forskolin (10(-7)-10(-4) M). Some cultures were also treated with forskolin with or without MIX (0.125-1.0 mM) or theophylline (1.25-10 mM). Forskolin (3 X 10(-6)-10(-4) M) stimulated the production of estrogen, progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) and cAMP in a dose-related manner to levels similar to or higher than that elicited by FSH alone. Similarly, forskolin and FSH both increased LH/hCG receptor content in cultured granulosa cells, although forskolin was only 50% as effective as FSH. Treatment with MIX alone increased basal levels of cAMP, accompanied by elevations of estrogen and progestin biosynthesis without affecting LH/hCG receptor content. In contrast, theophylline treatment only increased cAMP and progestin accumulation. Furthermore, MIX potentiated the stimulatory effects of forskolin and FSH on cAMP and progestin production. In contrast, MIX inhibited FSH- and forskolin-stimulated estrogen production. Thus, activation of adenyl cyclase and inhibition of cAMP breakdown in the cultured rat granulosa cells enhance steroidogenesis and LH receptor formation, reinforcing the concept that cAMP is a (but may not be the only) second messenger in the hormonal regulation of granulosa cell differentiation.