Synthesis, metabolism and levels of the neuroactive steroid, 3 alpha-hydroxy-1-pregnen-20-one (3 alpha HP), in rat pituitaries

The neuroactive steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), is a metabolite of progesterone and a precursor of 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha P3 alpha; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA(A) receptor complex, 3 alpha HP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca2+-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3 alpha HP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3 alpha HP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with H-3- or C-14-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3 alpha HP along with 5 alpha P3 alpha, 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one, 3 beta-hydroxy-5 alpha-pregnan-20-one, 5 alpha-pregnane-3 alpha(beta), 20 alpha-diols, 20 alpha-hydroxy-4-pregnen-3-one, and 4-pregnene-3 alpha(beta), 20 alpha-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSO), 20 alpha-HSO, 3 beta-HSO, and 5 alpha-reductase. The activities of 5 alpha-reductase and 3 alpha-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3 alpha HP had been formed. The accumulation of 3 alpha HP increased approximately Linearly with the time of incubation. Metabolism studies using [1,2,6,7-H-3]3 alpha HP showed that pituitary cells convert about 29% and 8% of the 3 alpha HP to progesterone and 5 alpha P3 alpha, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3 alpha HP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3 alpha HP were about 2-3-fold greater than those of progesterone in the same pituitaries. In older (80-100 days old) cycling rats, the levels of 3 alpha HP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3 alpha HP, from progesterone, and in turn to metabolize 3 alpha HP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3 alpha HP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3 alpha HP may be (in part, or in whole) via paracrine or autocrine mechanisms. (C) 1997 Elsevier Science B.V.