Characterization of alpha 2A-adrenergic receptors in GT1 neurosecretory cells

In this paper, we report the endogenous expression of functional alpha 2-adrenergic receptors (alpha 2-ARs) in the immortalized hypothalamic cell line, GT1. Membranes from GT1 cells exhibited high-affinity binding for the specific alpha 2-AR radioligand [3H]RX821002 (Kd = 0.2 +/- 0.03 nM, Bmax = 29.5 +/- 2.1 fmol/mg protein, n = 3). The Ki values for the adrenergic ligands, oxymetazoline (1.6 +/- 0.3 nM, n = 3) and prazosin (287 +/- 89 mM, n = 3), were consistent with the pharmacological properties of the A subtype of alpha 2-AR (alpha 2A-AR). The presence of mRNA encoding the alpha 2A-AR in GT1 cell extracts was confirmed by Northern blot analysis. alpha 2-ARs in GT1 cells were found to be coupled to the inhibition of adenylyl cyclase through the pertussis toxin-sensitive class of heterotrimeric G-proteins. A maximal dose of the alpha 2-AR agonist UK 14304 inhibited forskolin-stimulated cAMP production in GT1 cells by 53% (EC50 = 316 nM). Double labeling of rodent brain sections with antibodies specific for GnRH and the alpha 2A-AR indicated a large proportion of neurons labeled with the GnRH antibody also contained alpha 2A-AR-like immunoreactivity. In both GT1 cells and GnRH-immunopositive neurons in brain, clusters of alpha 2A-AR-like immunoreactivity were associated with cell bodies and occasionally with neuritic processes. Punctate alpha 2A-AR-like immunoreactivity was localized to intracellular compartments in GT1 cells as determined by confocal microscopy. Whole cell radioligand binding techniques were used to show that at least one third of the alpha 2-AR population in GT1 cells was intracellular. In view of these data, GT1 cells may serve as a representative system in which to study the biology of alpha 2A-ARs in relation to neuronal and neurosecretory functions.