Isothiocyanatobenzyl imidazoline is an alkylating agent for I2-imidazoline binding sites in rat and rabbit tissues

Isothiocyanatobenzyl imidazoline (IBI), the 4'-NCS analogue of tolazoline, has been used to alkylate several receptor sites in rabbit iris muscles. Because of the high affinity of tolazoline for the I-2-imidazoline binding sites (K-i = 16-130 nM), this study was designed to assess whether IBI is also an alkylating agent for these sites. In competition studies, IBI displayed moderate affinity (K-i similar to 2-3 mu M) against I-2A-imidazoline sites in the rabbit cerebral cortex and I-2B-imidazoline sites in the rat cerebral cortex labelled by [H-3]2-(2-benzofuranyl)-2-imidazoline ([H-3]2-BFI). However, preincubation (30 min at 25 degrees C) of rat cortical and liver membranes with IBI (10(-7) M to 10(-3) M), followed by extensive washing, markedly decreased (17% to 96%) the specific binding of [H-3]2-BFI to I-2B-imidazoline sites. IBI (10(-5) M to 10(-3) M) also bound irreversibly to I-2A-imidazoline sites in rabbit cerebral cortex but with a lesser efficacy (27% to 83% reduction of [H-3]2-BFI binding). Saturation curves of [H-3]2-BFI binding in the rat cerebral cortex indicated that preincubation with 10(-6) M IBI reduced the total density (B-max) without affecting the affinity (K-d) of I-2B-imidazoline sites for IBI. Acute treatments (6 h) with IBI (10 and 30 mg/kg, i.p.) also dose-dependently reduced (26% and 41%; respectively) the total density of I-2B-imidazoline sites. These results demonstrate the ability of IBI to alkylate I2B-imidazoline binding sites in vitro and in vivo and provide evidence for the use of IBI as a new tool for the study of the functional implications of imidazoline binding sites.