Autoradiographic Comparison of [3H]-Clonidine Binding to Non-Adrenergic Sites and α2-Adrenergic Receptors in Human Brain

Abstract

Clonidine is a partial agonist at brain α2-adrenoceptors (α2AR), but also has high affinity (KD = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and α2AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 μM norepinephrine (NE; α2AR agonist) or 12.5 μM methoxy-idazoxan (MIDX; selective α2AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 μM cirazoline. Levels of [3H]-clonidine binding to α2AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r2 = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: α2AR (>4:1). Quantitative transepts drawn across hippocampal images revealed α2AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus—areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I1 and I2 subtypes. The rank-order of affinities were cirazoline > harmane > BDF6143 > idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 ± 12 nM). In conclusion: 1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from α2AR; and 2) the affinities of these sites was distinct from α1AR, α2AR, I1 or I2 sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.