SUPPRESSION OF FOLLICLE-STIMULATING-HORMONE BY THE GONADAL- AND NEUROSTEROID 3-ALPHA-HYDROXY-4-PREGNEN-20-ONE INVOLVES ACTIONS AT THE LEVELOF THE GONADOTROPE MEMBRANE CALCIUM CHANNEL/

We have previously shown that the gonadal- and neurosteroid 3 alpha-hy droxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells. We undertook exploration of the mechanis ms of this suppression by examining the possible sites of 3 alpha HP a ction in isolated anterior pituitary cells of rats. The specific objec tive of this study was to determine if 3 alpha HP suppresses FSH by ac tion at the level of the gonadotrope membrane and/or calcium channels. Pituitary cells from adult randomly cycling female rats were precultu red for 72 h and then treated for 4 h with 10 nM GnRH and 0.1 nM 3 alp ha HP with or without Ca2+ channel agonists or antagonists. In other e xperiments, cells were treated with BSA-conjugated 3 alpha HP, progest erone, or 3 beta HP (the stereoisomer of 3 alpha HP). Levels of FSH we re determined by RIA in media and cells. GnRH-stimulated FSH release a nd the total FSH (released plus cellular) were significantly suppresse d by 3(alpha Hp. The Ca2+ ionophore A23187 induced FSH release and 3 a lpha HP significantly suppressed both released and total FSH in its pr esence. In combination with a high dose (100 mu M) of the dihydropyrid ine-sensitive Ca2+ channel antagonist nifedipine, 3 alpha HP suppresse d FSH secretion to a greater extent than the antagonist alone. Cellula r content of FSH was also decreased by nifedipine (100 mu M) and was f urther suppressed in the presence of 3 alpha HP. The phenylalkylamine- sensitive Ca2+ channel antagonist methoxyverapamil (D600) suppressed G nRH-induced FSH release, and 3 alpha HP significantly potentiated the suppression. Released and cellular FSH were increased by the dihydropy ridine-sensitive agonist BAYK 8644, whereas 0.1 nM 3 alpha HP suppress ed this agonist-induced FSH to a greater extent than the maximum dose (100 mu M) of nifedipine. In order to test for direct action at the le vel of the gonadotrope membrane, 3 alpha HP was conjugated to BSA (3 a lpha HP-BSA) and administered to cultured pituitary cells. The 3 alpha HP-BSA conjugate (but not progesterone-BSA or 3 beta HP-BSA) signific antly 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 s uppress FSH release (and possibly synthesis) through direct action at the level of the gonadotrope membrane.