SUPPRESSION IN GONADOTROPES OF GONADOTROPIN-RELEASING HORMONE-STIMULATED FOLLICLE-STIMULATING-HORMONE RELEASE BY THE GONADAL- AND NEUROSTEROID 3-ALPHA-HYDROXY-4-PREGNEN-20-ONE INVOLVES CYTOSOLIC CALCIUM

The gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alph a HP) suppresses FSH release in cultures of anterior pituitary cells. In a previous report, we showed that this suppression is achieved at l east in part by an interaction at the plasma membrane level. We undert ook to examine the possible interaction of 3 alpha HP at the level of intracellular Ca2+. Anterior pituitary cells from adult randomly cycli ng female rats were treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with Or without protein kinase C activator (SC10), antagonist (H-7 ), intracellular Ca2+ chelator (TMB-8), and intracellular Ca2+ mobiliz er (glutamate), and with or without EGTA and Ca2+ in,the medium. FSH c ontent in media and cells was determined by RIA. The protein kinase C (PKC) activator, SC10, increased basal levels of secreted FSH. 3 alpha HP suppressed (P < 0.05) SC10-stimulated basal FSH release. The PKC i nhibitor, H7, decreased GnRH-induced FSH release; FSH was further supp ressed (P < 0.05) by 3 alpha HP in the presence of H7. These results w ere interpreted to indicate that 3 alpha HP may act in part at the lev el of PKC and also at another site(s). The intracellular Ca2+ chelator , TMB-8, suppressed released and cellular GnRH-stimulated FSH to the s ame extent as 3 alpha HP; FSH was not further decreased by 3(alpha HP in the presence of TMB-8. 3 alpha HP suppressed glutamate-stimulated F SH release in Ca2+-free medium (P < 0.01). Moreover, GnRH-induced rele ase of FSH was suppressed to the same degree by 10(-10) M 3 alpha HP a s by 10(-4) M EGTA. In pituitary cell suspensions, the GnRH-induced [C a2+]i elevations were significantly (P < 0.05) attenuated by 3 alpha H P. From these and previous results, a model is proposed for the action of 3 alpha HP. The model suggests that 3 alpha HP may interact with g onadotropes at the level of the PKC cell signaling pathway and intrace llular Ca2+ mobilization, in addition to the plasma membrane/calcium c hannel. The interaction effects a decrease in intracellular Ca2+, lead ing to decreases in FSH release from those pituitary gonadotropes that are responsible for FSH. The consistent decrease in total FSH (releas ed plus cellular content) by 3 alpha HP suggests that this neurosteroi d may also suppress FSH synthesis.