Labelling of I-2B-imidazoline receptors by [H-3]2-(2-benzofuranyl)-2-imidazoline (2-BFI) in rat brain and liver: Characterization, regulation and relation to monoamine oxidase enzymes

The novel selective imidazoline radioligand [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) was used to characterize and assess further the nature of I-2-imidazoline receptors in rat brain and liver. In the cerebral cortex, 2-BFI displayed high affinity (K-i= 9.8 nM) for a single class of [H-3]2-BFI binding sites. Other imidazoline/guanidine compounds (e.g. aganodine, cirazoline and idazoxan) displayed biphasic competition curves, indicating the existence of high (K-iH= 2.9-78 nM; R-H= 61-83%) and low (K-iL= 4.7-158 mu M) affinity sites. The pharmacological profile for [H-3]2-BFI binding (aganodine > cirazoline > 2-BFI >> clonidine > amiloride >> efaroxan) was typical of that for I-2-sites. This profile was almost identical to that obtained against [H-3]idazoxan (correlation between pK(i) values, r = 0.97) which indicated that the sites characterized with [H-3]2-BFI in brain corresponded to I-2-imidazoline receptors. The low affinity of amiloride against [H-3]2-BFI (K-i= 900 nM) further indicated that these brain I-2-sites belong to the I-2B-subtype. [H-3]2-BFI binding sites (B-max= 72 fmol/mg protein) in brain were differentially modulated by treatment (7 days) with cirazoline (up-regulation: 25%) and the MAO inhibitor phenelzine (down-regulation: 31%), indicating that these I-2-sites are regulated in vivo, as is the case for those labelled by [H-3]idazoxan. Chronic treatment with 2-phenylethylamine, a phenelzine metabolite and endogenous amine, did not alter the density of brain of I-2-imidazoline receptors labelled by [H-3]idazoxan. Preincubation of liver membranes with the MAO inhibitor clorgyline (10(-7) M) abolished the binding of [H-3]Ro 41-1049 (N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide) to MAO-A, but it did not alter the binding of [H-3]Ro 19-6327 (N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide) to MAO-B or that of [H-3]2-BFI to I-2-sites. At 10(-4) M it also abolished MAO-B sites, but a substantial proportion of I-2-sites (40%) remained intact. Preincubation of liver membranes at 60 degrees C also abolished MAO-A/B sites, whereas still 22% of I-2-sites remained. The results indicate that [H-3]2-BFI is a good tool for the identification of I-2-imidazoline receptors and suggest further that certain I-2-sites and MAO are different proteins.