[H-3] 2-(2-BENZOFURANYL)-2-IMIDAZOLINE, A HIGHLY SELECTIVE RADIOLIGAND FOR I-2-IMIDAZOLINE RECEPTOR-BINDING SITES - STUDIES IN RABBIT KIDNEY MEMBRANES

2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characteris ed as a selective ligand for the I-2 type of imidazoline-receptor bind ing site(s) (I-2-RBS). The present studies determined the relative lev els of specific [H-3]2-BFI binding to membrane homogenates of brain an d kidney from rat, guinea pig and rabbit and identified the pharmacolo gical characteristics of [H-3]2-BFI binding sites in rabbit kidney mem branes. Rabbit kidney membranes had the highest relative density of sp ecific [H-3]2-BFI binding of all tissues studied (2000 fmol/mg protein ). Rabbit brain and guinea pig kidney had moderate levels of specific [H-3]2-BFI binding (350-500 fmol/mg protein), while rat kidney and gui nea pig and rat brain displayed much lower densities of binding (40-65 fmol/mg protein). Studies of [H-3]2-BFI binding kinetics in rabbit ki dney homogenates revealed binding to two distinct sites with K-d value s of 0.10 +/- 0.01 nmol/l and 1.00 +/- 0.36 nmol/l respectively. Equil ibrium saturation studies were also consistent with the presence of tw o binding sites - [H-3]2-BFI (0.01-20 nmol/l) bound to sites with affi nities of 0.10 +/- 0.01 nmol/l and 0.92 +/- 0.13 nmol/l and binding de nsities of 470 +/- 80 and 840 +/- 60 fmol/mg protein (n = 3), represen ting 36 and 64% respectively. Drug inhibition studies revealed that L- adrenaline; alpha(1)-adrenoceptor drugs (prazosin, L-phenylephrine) an d alpha(2)-adrenoceptor drugs (rauwolscine, methoxyidazoxan, oxyphenyl )-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3- (2H,4H)-isoquinolindione (AR C-239) had extremely low affinities for [H-3]2-BFI binding sites (IC50 greater than or equal to 10 mu mol/l). Putative I-1-RBS compounds, p- aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, in hibited [H-3]2-BFI binding to rabbit renal membranes with low to very low affinities (K-i values 3 to greater than or equal to 100 mu mol/l) , suggesting [H-3]2-BFI does not label I-1-RBS in rabbit kidney membra nes. I-2-RBS compounds - 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoli ne- potently inhibited [H-3]2-BFI binding (K-i values 0.37-1.6 nmol/l) , confirming the labelling of I-2-RBS. Inhibition of [H-3]2-BFI bindin g by certain compounds was consistent with their interaction with two binding site populations - for example (drug, K-i values) guanabenz, 0 .65 nmol/l and 0.17 mu mol/l and 26 mu mol/l rilmenidine, 150 nmol/l a nd 50 mu mol/l; and clonidine, 230 nmol/l and 70 mu mol/l. The high af finity of amiloride for a high proportion (85%) of the binding is cons istent with the presence of the I-2A-subtype of I-RBS in rabbit kidney . These results demonstrate that [H-3]2-BFI is a highly selective and high affinity radioligand for I-2-RBS which should be useful for the f urther characterisation of these sites in mammalian tissues.