Synthesis, metabolism and levels of the neuroactive steroid, 3 α-hydroxy-4-pregnen-20-one (3 αHP), in rat pituitaries

Abstract

The neuroactive steroid, 3 α-hydroxy-4-pregnen-20-one (3 αHP), is a metabolite of progesterone and a precursor of 3 α-hydroxy-5 α-pregnan-20-one (5 αP3 α; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA A receptor complex, 3 αHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca 2+-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3 αHP, 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 αHP 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 3H- or 14C-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 αHP along with 5 αP3 α, 5 α-pregnane-3,20-dione, 20 α-hydroxy-5 α-pregnan-3-one, 3 β-hydroxy-5 α-pregnan-20-one, 5 α-pregnane-3 α( β), 20 α-diols, 20 α-hydroxy-4-pregnen-3-one, and 4-pregnene-3 α( β), 20 α-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3 α-hydroxysteroid oxidoreductase (3 α-HSO), 20 α-HSO, 3 β-HSO, and 5 α-reductase. The activities of 5 α-reductase and 3 α-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 αHP had been formed. The accumulation of 3 αHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7- 3H]3 αHP showed that pituitary cells convert about 29% and 8% of the 3 αHP to progesterone and 5 αP3 α, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3 αHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3 αHP 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 αHP 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 αHP, from progesterone, and in turn to metabolize 3 αHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3 αHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3 αHP may be (in part, or in whole) via paracrine or autocrine mechanisms.