Heterogeneity of the specific imidazoline binding of [3H]idazoxan in the human cerebral cortex

The aim of the present study was to verify whether [3H]idazoxan can be considered as a highly selective ligand for imidazoline preferring receptors (IPR). In human frontal cortex membrane preparations [3H]idazoxan at a low concentration (2 nM) only labelled imidazoline sensitive, catecholamine insensitive sites. Binding was of high affinity, saturable and stereospecific. The rank order of potency of different compounds able to inhibit this binding was cirazoline > (+/-)-idazoxan > guanoxan > (-)-idazoxan > tolazoline > UK-14304 > clonidine. Amiloride, imidazol-4-acetic acid and histamine had no significant affinity for IPR labelled by [3H]idazoxan. [3H]idazoxan bound to 2 different sites (KD1 = 1 nM and KD2 = 16.4 nM). Clonidine behaved as a non competitive, non allosteric inhibitor of [3H]idazoxan binding. Both [3H]idazoxan binding sites were equally affected by clonidine. In membrane preparations obtained from the Nucleus Reticularis Lateralis region (NRL) of the brainstem, [3H]idazoxan binding was similar to that in cortical membranes, particularly with regard to specificity and kinetics. However, in the NRL region binding sites were 4-5 times more numerous than in the frontal cortex. Non linear analyses of saturation data obtained with NRL membrane preparations were compatible with both a one site and a two sites model. No significant effects of 1 mM MgCl2 alone or with 100 microM Gpp(NH)p were observed on either [3H]idazoxan binding or the competition with clonidine or rilmenidine. As in the cortical membrane, clonidine was a non competitive inhibitor of [3H]idazoxan binding to membranes from the NRL region. In conclusion, we show that when a low concentration is used, [3H]idazoxan binding to human brain involves sites almost completely insensitive to catecholamines and specific for imidazolines or related compounds. This binding involves two distinct sites. We also report that [3H]idazoxan imidazoline binding sites are not coupled with a G protein. Because of the non competitive interaction between clonidine and [3H]idazoxan for the binding sites of the latter, we are unable to conclude that the binding sites of the two drugs are identical. However, the non competitive, non allosteric interaction suggests a complex model of multiple binding sites.