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 I2-imidazoline binding sites (Ki = 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 (Ki∼ 2–3 μM) against I2A-imidazoline sites in the rabbit cerebral cortex and I2B-imidazoline sites in the rat cerebral cortex labelled by [3H]2-(2-benzofuranyl)-2-imidazoline ([3H]2-BFI). However, preincubation (30 min at 25° 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 [3H]2-BFI to I2B-imidazoline sites. IBI (10–5 M to 10–3 M) also bound irreversibly to I2A-imidazoline sites in rabbit cerebral cortex but with a lesser efficacy (27% to 83% reduction of [3H]2-BFI binding). Saturation curves of [3H]2-BFI binding in the rat cerebral cortex indicated that preincubation with 10–6 M IBI reduced the total density (Bmax) without affecting the affinity (Kd) of I2B-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 I2B-imidazoline sites. These results demonstrate the ability of IBI to alkylate I2-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.