Labelling of I2B-imidazoline receptors by [3H]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 I₂-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 ³H]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 μM) affinity sites. The pharmacological profile for ³H]2-BFI binding (aganodine > cirazoline > 2-BFI >> clonidine > amiloride >> efaroxan) was typical of that for I₂-sites. This profile was almost identical to that obtained against ³H]idazoxan (correlation between pK _i values, r = 0.97) which indicated that the sites characterized with ³H]2-BFI in brain corresponded to I₂-imidazoline receptors. The low affinity of amiloride against ³H]2-BFI (K _i= 900 nM) further indicated that these brain I₂-sites belong to the I_2B-subtype. ³H]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 I₂-sites are regulated in vivo, as is the case for those labelled by ³H]idazoxan. Chronic treatment with 2-phenylethylamine, a phenelzine metabolite and endogenous amine, did not alter the density of brain of I₂-imidazoline receptors labelled by ³H]idazoxan. Preincubation of liver membranes with the MAO inhibitor clorgyline (10^-7 M) abolished the binding of ³H]Ro 41-1049 (N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide) to MAO-A, but it did not alter the binding of ³H]Ro 19-6327 (N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide) to MAO-B or that of ³H]2-BFI to I₂-sites. At 10^-4 M it also abolished MAO-B sites, but a substantial proportion of I₂-sites (40%) remained intact. Preincubation of liver membranes at 60 °C also abolished MAO-A/B sites, whereas still 22% of I₂-sites remained. The results indicate that ³H]2-BFI is a good tool for the identification of I₂-imidazoline receptors and suggest further that certain I₂-sites and MAO are different proteins. Received: 10 January 1997 / Accepted: 6 March 1997