Interaction of GnRH with Anterior Pituitary. III. Role of Divalent Cations, Microtubules and Microfilaments in the GnRH Activated Gonadotroph1

Abstract

The role of divalent cations, microtubules and microfilaments in mediating secretion of LH from GnRH stimulated gonadotrophs was examined in ovine anterior pituitary cells cultured in suspension. GnRH did not stimulate release of LH in suspensions of acutely dispersed cells. Release of LH in response to GnRH was reestablished with time of culture; maximal responsiveness occurred after 18 h of culture. Conversely, release of cAMP in response to GnRH was increased 6-fold in acutely dispersed cells. GnRH specific release of cAMP from cells immediately after dissociation did not differ from GnRH specific release from cells cultured for 18 h. Culture of anterior pituitary cells with actinomycin D or cycloheximide reduced total LH (media plus cells) following 18 h of culture. GnRH specific release of LH, but not cAMP, was depressed relative to the 18 h control from cells cultured for 18 h with inhibitors of transcription or translation. In a calcium-free environment, GnRH did not stimulate release of LH. Upon readdition of calcium, however, release of LH from gonadotrophs stimulated by GnRH was elevated above basal levels with maximal release occurring at concentrations of extracellular calcium of 1 mM and greater. Release of LH induced by 73.5 mM K⁺ was also calcium dependent. High K⁺ stimulated release of LH above basal release at 0.5 mM calcium. At no concentration of calcium did the release of LH induced by 73.5 mM K⁺ approach the release stimulated by 20 nM GnRH. Release of LH induced by 73.5 mM K⁺ and 20 nM GnRH was not additive. Increasing concentrations of magnesium (0-16 mM) had no effect on specific release of LH stimulated by 200 nM GnRH, but increased the GnRH specific release of cAMP 2.21-fold. Conversely, increasing concentrations of barium (0-4 mM) increased GnRH specific release of LH 2.8-fold but depressed specific release of cAMP by 67%.