FUNCTIONAL AND MORPHOLOGICAL CHARACTERIZATION OF 4 CELL-LINES DERIVEDFROM GH(3) CELLS STABLY TRANSFECTED WITH GONADOTROPIN-RELEASING-HORMONE RECEPTOR COMPLEMENTARY DEOXYRIBONUCLEIC-ACID

Four cell lines, stably transfected with rat GnRH receptor complementa ry DNA, have been prepared from the lactotropic GH(3) cell line. All f our lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, charact eristic of high protein synthetic activity, although secretory granule s are virtually absent; the rough endoplasmic reticulum (rER) cisterna e were short and straight. Instances were observed in which the ER rea ches to the plasma membrane, suggesting a possible nongranular secreto ry route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depend ing on the cell line) after exposure to 10 nM GnRH; receptors then rec overed (2-7 h). This pattern is reminiscent of the GnRH receptor in th e primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer(6)-des-Gly(10).Pro(9)-ethy lamide-GnRH), an event that was inhibited by all three major classes o f Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines , and diltiazem); in contrast, GnRH-stimulated LH release from pituita ry-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, althoug h the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 mu g/ml Buserelin than to 1 mu g/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.