In vitro effects of crude extracts of Parkia biglobosa (Mimosaceae), Stereospermum kunthianum (Bignoniaceae) and Biophytum petersianum (Oxalidaceae) on corticosteroid secretion in rat.

Previous studies conducted in guinea pig, rat and rabbit have revealed that crude extracts from Parkia biglobosa, Stereospermum kunthianum and Biophytum petersianum exert hypotensive and/or hypoglycemic activities. Since corticosteroids are involved in the control of arterial blood pressure and glycemia, we have investigated the possible effects of these plant extracts on rat adrenal tissue in vitro. Short-term administration of crude semi-ethanolic extracts of P. biglobosa and S. kunthianum to perifused rat adrenal tissue did not induce any significant changes in corticosteroid output. Conversely, the B. petersianum extract caused a dose-dependent increase in corticosterone and aldosterone secretion. Repeated infusions or prolonged administration of B. petersianum extract did not produce any apparent attenuation of the steroid response. Altogether, these data indicate that a semi-ethanolic extract of B. petersianum dose-dependently stimulates corticosterone and aldosterone secretion in rat without any desensitization phenomenon.

The octadecaneuropeptide ODN stimulates neurosteroid biosynthesis through activation of central-type benzodiazepine receptors.

Neurosteroids may play a major role in the regulation of various neurophysiological and behavioural processes. However, while the biochemical pathways involved in the synthesis of neuroactive steroids in the central nervous system are now elucidated, the mechanisms controlling the activity of neurosteroid-producing cells remain almost completely unknown. In the present study, we have investigated the effect of the octadecaneuropeptide (ODN), an endogenous ligand of benzodiazepine receptors, in the control of steroid biosynthesis in the frog hypothalamus. Glial cells containing ODN-like immunoreactivity were found to send their thick processes in the close vicinity of neurones expressing the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase. Exposure of frog hypothalamic explants to graded concentrations of ODN (10(-10)-10(-5) M) produced a dose-dependent increase in the conversion of tritiated pregnenolone into various radioactive steroids, including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone and dihydrotestosterone. The ODN-induced stimulation of neurosteroid biosynthesis was mimicked by the central-type benzodiazepine receptor (CBR) inverse agonists methyl beta-carboline-3-carboxylate (beta-CCM) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The stimulatory effects of ODN, beta-CCM and DMCM on steroid formation was markedly reduced by the CBR antagonist flumazenil. The ODN-evoked stimulation of neurosteroid production was also significantly attenuated by GABA. Collectively, these data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3 beta-hydroxysteroid dehydrogenase-containing neurones, stimulates the biosynthesis of neurosteroids through activation of central-type benzodiazepines receptors.

gamma-Aminobutyric acid, acting through gamma -aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus.

Most of the actions of neurosteroids on the central nervous system are mediated through allosteric modulation of the gamma-aminobutyric acid type A (GABA(A)) receptor, but a direct effect of GABA on the regulation of neurosteroid biosynthesis has never been investigated. In the present report, we have attempted to determine whether 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-containing neurons, which secrete neurosteroids in the frog hypothalamus, also express the GABA(A) receptor, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants. Double immunohistochemical labeling revealed that most 3beta-HSD-positive neurons also contain GABA(A) receptor alpha(3) and beta(2)/beta(3) subunit-like immunoreactivities. Pulse-chase experiments showed that GABA inhibited in a dose-dependent manner the conversion of tritiated pregnenolone into radioactive steroids, including 17-hydroxy-pregnenolone, progesterone, 17-hydroxy-progesterone, dehydroepiandrosterone, and dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA(A) receptor agonist muscimol but was not affected by the GABA(B) receptor agonist baclofen. The selective GABA(A) receptor antagonists bicuculline and SR95531 reversed the inhibitory effect of GABA on neurosteroid formation. The present results indicate that steroid-producing neurons of the frog hypothalamus express the GABA(A) receptor alpha(3) and beta(2)/beta(3) subunits. Our data also demonstrate that GABA, acting on GABA(A) receptors at the hypothalamic level, inhibits the activity of several key steroidogenic enzymes, including 3beta-HSD and cytochrome P450(C17) (17alpha-hydroxylase).

In vivo evidence for the production of sulfated steroids in the frog brain.

It is well established that sulfated neurosteroids are potent regulators of neuronal activity but the biosynthesis of sulfate esters of steroids in the central nervous system (CNS) has received little attention. In particular, the localization of hydroxysteroid sulfotransferase (HST), the enzyme which is responsible for the formation of sulfated steroids, has never been determined in the brain. We took advantage of the availability of an antiserum raised against rat liver HST to investigate the distribution of this enzyme in the CNS of the frog Rana ridibunda. Two populations of HST-positive neurons were localized in the anterior preoptic area and the magnocellular nucleus of the hypothalamus. Numerous HST-immunoreactive fibers were visualized throughout the telencephalon and the diencephalon. Reversed-phase high performance liquid chromatography (HPLC) analysis of frog telencephalon and hypothalamus extracts combined with radioimmunoasssay (RIA) detection showed the presence of substantial amounts of DHEAS-immunoreactive material which coeluted with synthetic DHEAS. The concentrations of DHEAS detected in the telencephalon and hypothalamus were respectively eight and five times higher than in the serum. The present study demonstrates the occurrence of HST-immunoreactive material in neurons of the frog telencephalon and diencephalon. This report also provides evidence for the presence of HST bioactivity, in vivo, in the frog brain.

The endozepine triakontatetraneuropeptide diazepam-binding inhibitor [17-50] stimulates neurosteroid biosynthesis in the frog hypothalamus.

Neurons and glial cells are capable of synthesizing various bioactive steroids, but the neuronal mechanisms controlling neurosteroid-secreting cells are poorly understood. In the present study, we have investigated the possible effect of an endogenous ligand of benzodiazepine receptors, the triakontatetraneuropeptide [17-50] (TTN), on steroid biosynthesis in the frog hypothalamus. Immunohistochemical studies revealed that most hypothalamic neurons expressing 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase also contained peripheral-type benzodiazepine receptor-like immunoreactivity. Confocal laser scanning microscopic analysis revealed that the peripheral-type benzodiazepine receptor-immunoreactive material was located both in the cytoplasm and at the periphery of the cell bodies. By using the pulse-chase technique, TTN was found to stimulate the conversion of [3H]pregnenolone into various steroids, including 17-hydroxypregnenolone, 5 alpha-dihydrotestosterone and 17-hydroxyprogesterone, in a dose-dependent manner. The peripheral-type benzodiazepine receptor agonist Ro5-4864 mimicked the stimulatory effect of TTN on the formation of neurosteroids. The peripheral-type benzodiazepine receptor antagonist PK11195 significantly reduced the effect of TTN on neurosteroid synthesis, while the central-type benzodiazepine receptor antagonist flumazenil did not affect the formation of neurosteroids evoked by TTN. These data indicate that TTN stimulates the biosynthesis of 3-keto-17 alpha-hydroxysteroids in frog hypothalamic neurons through activation of peripheral-type benzodiazepine receptors likely located at the plasma membrane level.

Localization of 17beta-hydroxysteroid dehydrogenase and characterization of testosterone in the brain of the male frog.

Several enzymes involved in the formation of steroids of the pregnene and pregnane series have been identified in the brain, but the biosynthesis of testosterone has never been reported in the central nervous system. In the present study, we have investigated the distribution and bioactivity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) (EC 1.1.1.62; a key enzyme that is required for the formation of testosterone and estradiol) in the brain of the male frog Rana ridibunda. By using an antiserum against human type I placental 17beta-HSD, immunoreactivity was localized in a discrete group of ependymal glial cells bordering the telencephalic ventricles. HPLC analysis of telencephalon and hypothalamus extracts combined with testosterone radioimmunoassay revealed the existence of two peaks coeluting with testosterone and 5alpha-dihydrotestosterone. After HPLC purification, testosterone was identified by gas chromatography/mass spectrometry. Incubation of telencephalon slices with [3H]pregnenolone resulted in the formation of metabolites which coeluted with progesterone, 17alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone. The newly synthesized steroid comigrating with testosterone was selectively immunodetected by using testosterone antibodies. These data indicate that 17beta-HSD is expressed in a subpopulation of gliocytes in the frog telencephalon and that telencephalic cells are capable of synthesizing various androgens, including dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone.