Evidence that neurosteroids are potent modulators of the action of GABA at GABAA receptors has prompted the investigation of the mechanism that controls brain neurosteroid synthesis by glial cell mitochondria in vivo. In vitro studies suggest that the interaction of the diazepam binding inhibitor (DBI)--a polypeptide that is abundant in steroidogenic cells--with glial mitochondrial DBI receptors (MDRs) is a crucial step in the physiological regulation of neurosteroid biosynthesis. MDRs bind 4'-chlorodiazepam (4'-CD), N,N-di-n-hexyl-2-(4-fluorophenyl)-indol-3-acetamide (FGIN-1-27), and the isoquinoline carboxamide PK 11195 with high affinity, and these ligands have been used to investigate whether the stimulation of glial MDRs increases brain pregnenolone production in vivo. Adrenalectomized and castrated (A-C) male rats (to eliminate peripheral sources of pregnenolone) were pretreated with trilostane (to prevent pregnenolone metabolism to progesterone), and the pregnenolone content in brain regions dissected after fixation with a 0.8-s exposure to microwave irradiation focused to the head was determined by HPLC followed by specific radioimmunoassay. The forebrain and cerebellum of A-C rats contained 4-7 ng of pregnenolone/g of tissue, and the olfactory bulb contained 10-14 ng/g. These concentrations of brain pregnenolone are only 30-40% lower than those of sham-operated rats. In contrast, the plasma pregnenolone content of sham-operated rats was 2-3 ng/ml, but it was only 0.15-0.20 ng/ml in the plasma of A-C rats. In A-C rats, treatment with the MDR ligands 4'-CD and FGIN-1-27 increased the pregnenolone content in the brain but failed to change the plasma or peripheral tissue content of this steroid. The effect of 4'-CD on brain pregnenolone content was maximal (70-100% increase) at the dose of 18 mumol/kg, 5-10 min after intravenous injection. The effect of oral administration of FGIN-1-27 on brain pregnenolone content was maximal (80-150% increase) at doses of 400-800 mumol/kg and peaked at approximately 1 h. That this effect of FGIN-1-27 was mediated by the MDR was documented by pretreatment with the MDR partial agonist PK 11195 (100 mumol/kg, i.p.). PK 11195 did not affect basal brain pregnenolone content but prevented the accumulation of brain pregnenolone induced by FGIN-1-27. FGIN-1-27 and 4'-CD failed to increase the brain concentration of dehydroepiandrosterone in A-C rats. These data suggest that glial cell MDRs play a role in neurosteroid biosynthesis in vivo.

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Metabolites of [3H]progesterone were studied in slices prepared from different brain regions of male rat, mouse, and monkey. The major metabolites were 5 alpha-dihydroprogesterone (5 alpha-DHP) and 3 alpha,5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THP) in rat brain slices, 5 alpha-DHP and 20 alpha-dihydroprogesterone (20 alpha-DHP) in mouse brain slices, and 20 alpha-DHP in monkey brain slices. In rat olfactory bulb slices, 5 alpha-DHP represented 25.2 +/- 3.3% of total radioactivity and 3 alpha,5 alpha-THP 17.5 +/- 2.8%, whereas in rat medulla oblongata slices, 5 alpha-DHP was 31.3 +/- 3.5% and 3 alpha,5 alpha-THP 5.4 +/- 1.5% of total radioactivity. In slices from other rat brain regions, both metabolites represented 12-20% of total radioactivity. The highest metabolite content in mouse brain was also detected in olfactory bulb slices, where 5 alpha-DHP represented 16.6 +/- 4.6% and 20 alpha-DHP 9.5 +/- 2.3% of total radioactivity. In cortical and corpus callosum slices of monkey brain, 26.8 +/- 4.4% and 2.4 +/- 0.5% of total radioactivity, respectively, were converted to 20 alpha-DHP, and less than 3% of total radioactivity could be attributed to any of the other metabolites detected. The 3 alpha, 5 alpha-THP content in both rat and monkey brain was below 1 nM, but increased in rat brain to 6.7 +/- 2.5 nM after electroshock. Endogenous 3 alpha,5 alpha-THP might play an important role in the regulation of rat behavior through the modulation of GABA action on the GABAA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

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