BETA-1-ADRENERGIC AND DOPAMINE (D1)-RECEPTORS COUPLED TO ADENYLYL CYCLASE ACTIVATION IN GT1 GONADOTROPIN-RELEASING-HORMONE NEUROSECRETORY-CELLS

Binding sites labeled by the beta-adrenergic receptor radioligand (-)-[I-125]iodocyanopindolol ([I-125]ICYP) and the selective D1-subtype dopamine (DA) receptor radioligand (+)-[I-125]SCH 23982 were identified on immortalized hypothalamic GnRH neurons (GT1-7 cell lines). Saturation analyses in crude particulate suspensions of GT1 cells described high affinity and low capacity binding sites for [I-125]ICYP (K(d), 41 pm; binding capacity, 25 fmol/mg protein) and [I-125]SCH 23982 (K(d), 320 pm; binding capacity, 23 fmol/mg protein). These binding sites were further characterized in competition assays using a variety of agonists and antagonists selective for either beta-adrenergic or DA receptor subtypes. The pharmacological profiles of [I-125]ICYP and [I-125]SCH 23982 binding obtained from these studies indicated that the radioligands were labeling beta1-adrenergic and D1-dopaminergic receptor sites, respectively. Northern blot analyses of purified GT1 cell mRNA documented the expression of D1-dopaminergic and beta1-adrenergic receptor mRNAs. Beta2-Adrenergic receptor mRNA was not identified. All three transcripts were detected in mouse brain mRNA. Both beta1-adrenergic and D1-receptors were discovered to be positively coupled to adenylyl cyclase. DA and the beta-adrenergic agonist isoproterenol each provoked a rapid and marked stimulation of adenylyl cyclase activity in GT1 cell membrane suspensions. Subtype-selective beta-adrenergic and DA receptor antagonists were used to inhibit isoproterenol- and DA-stimulated adenylyl cyclase activities. Their relative potencies indicated that the isoproterenol stimulation was mediated via the beta1-adrenergic receptor. The DA-stimulated adenylyl cyclase activity was mediated via the D1-DA receptor. These studies have identified functional beta1-adrenergic and D1-dopaminergic receptors positively coupled to adenylyl cyclase on GT1 GnRH neurosecretory cells. The existence of these receptors suggests that the noradrenergic and dopaminergic regulation of gonadotropin secretion may be mediated at least in part via direct synapses on GnRH neurons.