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 hypothalam ic cell line, GT1. Membranes from GT1 cells exhibited high-affinity bi nding for the specific alpha(2)-AR radioligand [H-3]RX821002 (K-d = 0. 2 +/- 0.03 nM, B-max = 29.5 +/- 2.1 fmol/mg protein, n = 3). The K-i v alues for the adrenergic ligands, oxymetazoline (1.6 +/- 0.3 nM, n = 3 ) and prazosin (287 +/- 89 nM, n = 3), were consistent with the pharma cological properties of the A subtype of alpha(2)-AR (alpha(2A)-AR). T he 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 f ound to be coupled to the inhibition of adenylyl cyclase through the p ertussis toxin-sensitive class of heterotrimeric G-proteins. A maximal dose of the alpha(2)-AR agonist UK 14304 inhibited forskolin-stimulat ed cAMP production in GT1 cells by 53% (EC(50) = 316 nM). Double label ing 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 b oth GT1 cells and GnRH-immunopositive neurons in brain, clusters of al pha(2A)-AR-like immunoreactivity were associated with cell bodies and occasionally with neuritic processes. Punctate alpha(2A)-AR-like immun oreactivity was localized to intracellular compartments in GT1 cells a s determined by confocal microscopy. Whole cell radioligand binding te chniques 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 biolo gy of alpha(2A)-ARs in relation to neuronal and neurosecretory functio ns.