Multiple Cholinergic Signaling Pathways in Pituitary Gonadotrophs

Abstract

Acetylcholine (ACh) has been established as a paracrine factor in the anterior pituitary gland, but the receptors mediating ACh action and the cell types bearing these receptors have not been identified. Our results showed that the expression of the nicotinic subunits mRNAs followed the order beta 2 > beta 1 = alpha 9 > alpha 4 in cultured rat pituitary cells. The expression of the subunits in immortalized L beta T2 mouse gonadotrophs followed the order beta 2 > alpha 4 = alpha 1. M4 > M3 muscarinic receptor mRNA were also identified in pituitary and L beta T2 cells. The treatment of cultured pituitary cells with GnRH down-regulated the expression of alpha 9 and alpha 4 mRNAs, without affecting the expression of M3 and M4 receptor mRNAs, and ACh did not alter the expression of GnRH receptor mRNA. We also performed double immunostaining to show the expression of beta 2-subunit and M4 receptor proteins in gonadotrophs. Functional nicotinic channels capable of generating an inward current, facilitation of electrical activity, and Ca2+ influx were identified in single gonadotrophs and L beta T2 cells. In both cell types, the M3 receptor-mediated, phospholipase C-dependent Ca2+ mobilization activated an outward apamin-sensitive K+ current and caused hyperpolarization. The activation of M4 receptors by ACh inhibited cAMP production and GnRH-induced LH release in a pertussis toxin-sensitive manner. We concluded that multiple cholinergic receptors are expressed in gonadotrophs and that the main secretory action of ACh is inhibitory through M4 receptor-mediated down-regulation of cAMP production. The expression of nicotinic receptors in vitro compensates for the lack of regular GnRH stimulation of gonadotrophs. (Endocrinology 154: 421-433, 2013)