Culture of functionally active human pituitary cells: investigation of gonadotropin regulation

A method for culturing cells from human pituitaries obtained at autopsy for studies of the secretion of gonadotropins and other hormones is described. The EC50 of GnRH-stimulated LH secretion from human pituitary cells was 1.25 nmol/L, similar to that found with rat pituitary cells (2.45 nmol/L). The GnRH agonist (D-Ala6, N alpha MeLeu7, Pro9-NEt)GnRH was 10-fold more active in stimulating LH release from both human and rat pituitary cells (EC50, 0.21 and 0.35 nmol/L, respectively). The GnRH antagonists (Ac-D-Nal(2)1,D-alpha-Me-4-ClPhe2,D-3-Pal3,D-Arg6,D-Ala10 )GnRH and (D-pGlu1,D-Phe2,D-Trp3,6)GnRH both inhibited 3 nmol/L GnRH-stimulated LH release from human pituitary cells (IC50, 2.40 and 78.6 nmol/L, respectively) with potencies similar to those observed in the rat (IC50, 0.68 and 33.0 nmol/L, respectively). Stimulation of the human pituitary cells with 100 nmol/L GnRH for 44 min resulted in biphasic release of LH and FSH. The initial phase occurred during the first 4 min of stimulation, and the second protracted plateau phase continued for at least the ensuing 40 min. The kinetics of LH and FSH release were identical, with no evidence of differential release at any stage. GnRH-stimulated LH and FSH secretion was Ca2+ dependent. The presence of 3 mmol/L EGTA prevented the gonadotropin response to GnRH, and the Ca2+ ionophore A23187 stimulated release of both LH and FSH. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate also stimulated gonadotropin secretion, indicating that activation of protein kinase-C is able to elicit LH release. GnRH desensitized the cells to subsequent stimulation with GnRH. After initial 2-h incubation with 0.1-100 nmol/L GnRH, LH release during a second 2-h incubation with the same doses was reduced by 50-100%. The reduction in FSH ranged from 63-92%. Depletion of gonadotropin pools may contribute to desensitization, since total LH and FSH cell content decreased 48% and 49%, respectively, after 100 nmol/L GnRH stimulation for 4 h. We conclude that functionally active cells can be cultured from human pituitaries obtained postmortem and that the mechanism of GnRH action and the relative potencies of GnRH analogs closely parallel those in the rat.