calcimycin and 3-hydroxypregn-4-en-20-one

We have previously shown that the gonadal and neurosteroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), can selectively suppress gonadotrophin-releasing hormone (GnRH) induced follicle-stimulating hormone (FSH) release from static cultures of anterior pituitary cells during a 4-h incubation period. The actions appeared to be at the level of the gonadotroph membrane and the cell signaling pathway involving Ca2+ and protein kinase C (PKC). In order to investigate further if the effects of 3 alpha HP on FSH release are generated by nongenomic mechanisms, we monitored the short-term effects of 3 alpha HP using dispersed anterior pituitary cells in a low dead-volume perifusion system with short (< or = 5 min) exposures to the steroid. Pulses of GnRH (10(-8) or 10(-7) M) lasting 2-5 min resulted in marked peaks of FSH release, and the variation in FSH amounts released from the cells in a particular column were minimal if the interval between successive GnRH pulses was at least 3-4 h. A 5-min pulse of 3 alpha HP (10(-9) M) administered simultaneously with the GnRH pulse suppressed GnRH-induced FSH release. On the other hand, similar treatment with the stereoisomer 3 beta-hydroxy-4-pregnen-20-one (3 beta HP), had no effect, but progesterone and estradiol pulses augmented the GnRH-induced FSH release. Pretreatment of cells with a 5-min pulse of 3 alpha HP, at 120, 60, or 30 min prior to a GnRH pulse suppressed the GnRH-induced FSH release. The suppression of GnRH-induced FSH release by 3 alpha HP was only partial if the start of the 3 alpha HP pulse occurred 0.5 or 1.0 min after the start of the GnRH pulse, and no suppression occurred if the start of the 3 alpha HP pulse was delayed by 2-5 min. The FSH release elicited by 5-min pulses of the Ca2+ ionophore A23187, the Ca2+ agonist BAY K8644, the PKC activator phorbol 12-myristate 13-acetate (PMA), or phospholipase C (PLC) was suppressed by simultaneous pulses of 3 alpha HP. The suppression of FSH release by 3 alpha HP appeared to be stereospecific, since no suppression was observed with 5 alpha-pregnane-3,20-dione (5 alpha P) or 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha P3 alpha). In separate experiments, cells were treated with pulses of BSA conjugates of 3 alpha HP, 3 beta HP, or progesterone; the 3 alpha HP-BSA, but not the 3 beta HP-BSA or the progesterone-BSA, suppressed the GnRH-induced release of FSH. The results of this study provide the first evidence that 3 alpha HP exerts immediate (nongenomic)

Topics: 20-alpha-Dihydroprogesterone; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcimycin; Calcium Channel Agonists; Cells, Cultured; Estradiol; Female; Follicle Stimulating Hormone; GABA Modulators; Gonadotropin-Releasing Hormone; Ionophores; Perfusion; Pituitary Gland, Anterior; Pregnanolone; Progesterone; Rats; Rats, Sprague-Dawley; Serum Albumin; Tetradecanoylphorbol Acetate; Time Factors; Type C Phospholipases

We have previously shown that the gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells. We undertook exploration of the mechanisms of this suppression by examining the possible sites of 3 alpha HP action in isolated anterior pituitary cells of rats. The specific objective of this study was to determine if 3 alpha HP suppresses FSH by action at the level of the gonadotrope membrane and/or calcium channels. Pituitary cells from adult randomly cycling female rats were precultured for 72 h and then treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with or without Ca2+ channel agonists or antagonist. In other experiments, cells were treated with BSA-conjugated 3 alpha HP, progesterone, or 3 beta HP (the stereoisomer of 3 alpha HP). Levels of FSH were determined by RIA in media and cells. GnRH-stimulated FSH release and the total FSH (released plus cellular) were significantly suppressed by 3 alpha HP. The Ca2+ ionophore A23187 induced FSH release and 3 alpha HP significantly suppressed both released and total FSH in its presence. In combination with a high dose (100 microM) of the dihydropyridine-sensitive Ca2+ channel antagonist nifedipine, 3 alpha HP suppressed FSH secretion to a greater extent than the antagonist alone. Cellular content of FSH was also decreased by nifedipine (100 microM) and was further suppressed in the presence of 3 alpha HP. The phenylalkylamine-sensitive Ca2+ channel antagonist methoxyverapamil (D600) suppressed GnRH-induced FSH release, and 3 alpha HP significantly potentiated the suppression. Released and cellular FSH were increased by the dihydropyridine-sensitive agonist BAYK 8644, whereas 0.1 nM 3 alpha HP suppressed this agonist-induced FSH to a greater extent than the maximum dose (100 microM) of nifedipine. In order to test for direct action at the level of the gonadotrope membrane, 3 alpha HP was conjugated to BSA (3 alpha HP-BSA) and administered to cultured pituitary cells. The 3 alpha HP-BSA conjugate (but not progesterone-BSA or 3 beta HP-BSA) significantly suppressed release of FSH. The results of the study suggest that 3 alpha HP may be interacting with the Ca2+ channel component of the GnRH signal transduction mechanism; in addition, 3 alpha HP may also suppress FSH release (and possibly synthesis) through direct action at the level of the gonadotrope membrane.

Topics: 20-alpha-Dihydroprogesterone; Animals; Calcimycin; Calcium Channel Blockers; Calcium Channels; Cell Membrane; Female; Follicle Stimulating Hormone; Gonadotropin-Releasing Hormone; Luteinizing Hormone; Pituitary Gland, Anterior; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine

The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP [Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) but not 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20 alpha-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20 alpha-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH. We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 20-alpha-Dihydroprogesterone; Animals; Bucladesine; Calcimycin; Cells, Cultured; Female; Follicle Stimulating Hormone; Granulosa Cells; Pregnenolone; Progesterone; Rats