[3H]2-(2-Benzofuranyl)-2-imidazoline, a highly selective radioligand for I2-imidazoline receptor binding sites Studies in rabbit kidney membranes

Abstract

2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characterised as a selective ligand for the I₂-type of imidazoline-receptor binding site(s) (I₂-RBS). The present studies determined the relative levels of specific [³H]2-BFI binding to membrane homogenates of brain and kidney from rat, guinea pig and rabbit and identified the pharmacological characteristics of [³H]2-BFI binding sites in rabbit kidney membranes. Rabbit kidney membranes had the highest relative density of specific [³H]2-BFI binding of all tissues studied (2000 fmol/mg protein). Rabbit brain and guinea pig kidney had moderate levels of specific [³H]2-BFI binding (350–500 fmol/mg protein), while rat kidney and guinea pig and rat brain displayed much lower densities of binding (40–65 fmol/mg protein).

Studies of [³H]2-BFI binding kinetics in rabbit kidney homogenates revealed binding to two distinct sites with K _d values of 0.10 ± 0.01 nmol/l and 1.00 ± 0.36 nmol/l respectively. Equilibrium saturation studies were also consistent with the presence of two binding sites – [³H]2-BFI (0.01–20 nmol/l) bound to sites with affinities of 0.10 ± 0.01 nmol/l and 0.92 ± 0.13 nmol/l and binding densities of 470 ± 80 and 840 ± 60 fmol/mg protein (n=3), representing 36 and 64% respectively. Drug inhibition studies revealed that l-adrenaline; α₁-adrenoceptor drugs (prazosin, l-phenylephrine) and α₂-adrenoceptor drugs (rauwolscine, methoxyidazoxan, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) had extremely low affinities for [³H]2-BFI binding sites (IC₅₀ ≥ 10 μmol/l). Putative I₁-RBS compounds, p-aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, inhibited [³H]2-BFI binding to rabbit renal membranes with low to very low affinities (K _i values 3 to ≥100 μmol/l), suggesting [³H]2-BFI does not label I₁-RBS in rabbit kidney membranes. I₂-RBS compounds – 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoline – potently inhibited [³H]2-BFI binding (K _i values 0.37–1.6 nmol/l), confirming the labelling of I₂-RBS. Inhibition of [³H]2-BFI binding 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 μmol/l; naphazoline, 0.94 nmol/l and 2.8 μmol/l; amiloride, 76 nmol/l and 26 μmol/l rilmenidine, 150 nmol/l and 50 μmol/l; and clonidine, 230 nmol/l and 70 μmol/l. The high affinity of amiloride for a high proportion (85%) of the binding is consistent with the presence of the I_2A-subtype of I-RBS in rabbit kidney.

These results demonstrate that [³H]2-BFI is a highly selective and high affinity radioligand for I₂-RBS which should be useful for the further characterisation of these sites in mammalian tissues.