The role of calcium in gonadotropin releasing hormone-induced luteinizing hormone secretion from the bovine pituitary

Typescript (photocopy).The hypothesis was tested that GnRH acts to release LH by increasing calcium uptake by the gonadotroph which in turn stimulates calcium-calmodulin activity and results in LH release from bovine pituitary cells as it does in the rat. Pituitary glands of calves (4-10 months of age) were enzymatically dispersed (0.2% collagenase) and grown for 5 days to confluency in multiwell plates (3x10^5/well). Cells treated with GnRH (10-1000 ng/ml in Hank's Balanced Salt Solution), Ca++ ionophore A23187 (2.5-10 μM), and ouabain (0.1-1 μM) all produced significant releases of LH. Extracellular Ca++ as low as 46 μM permitted GnRH-induced LH release in a pronounced "all or none" fashion, while thorough washing of the cells with 0.5 mM EGTA in Ca++-free media prevented the action of GnRH. GnRH (100 ng/ml) caused a rapid efflux (15 seconds) of ^45Ca++. Both GnRH-stimulated 45Ca efflux and LH release could be partially blocked by verapamil (10^-5 M). GnRH-induced LH release could also be blocked by nifedipine (10^-7-10^-5 M) and tetrodotoxin (10^-9 10^-7 M), although these agents did not affect 45Ca efflux. The calmodulin antagonists calmidazolium (10^-9-10^-8M) and W7 (10^-6-10^-5 M) were found to block GnRH induced LH release, as well as LH release induced by theophylline (1 mM), KC1 (25 mM), PGE2 (1 μM) and estradiol (25 ng/ml). These data indicated that: (1) calcium is required for GnRH action, but extracellular Ca++ does not regulate LH release; (2) GnRH elevates intracellular Ca++ by opening both voltage sensitive and receptor mediated Ca++ channels; (3) activation of calmodulin is one mechanism involved in GnRH-induced LH release