Characterization and autoradiographical localization of non‐adrenoceptor idazoxan binding sites in the rat brain

1 In rat whole brain homogenates, saturation analysis revealed that both [ 3 H]‐idazoxan and [ 3 H]‐RX821002, a selective α 2 ‐adrenoceptor ligand, bound with high affinity to an apparent single population of sites. However, the B max for [ 3 H]‐idazoxan was significantly ( P < 0.01) greater than that for [ 3 H]‐RX821002. 2 In competition studies, (−)‐adrenaline displaced 3 n m [ 3 H]‐idazoxan binding with an affinity consistent with [ 3 H]‐idazoxan labelling α 2 ‐adrenoceptors. However, this displacement was incomplete since 23.68 ± 1.11% of specific [ 3 H]‐idazoxan binding remained in the presence of an excess concentration (100 μ m ) of (−)‐adrenaline. In contrast, unlabelled idazoxan promoted a complete displacement of [ 3 H]‐idazoxan binding with a Hill slope close to unity and an affinity comparable with its K D determined in saturation studies. 3 Displacement of [ 3 H]‐idazoxan binding by the α 2 ‐adrenoceptor antagonists yohimbine, RX821002 (2‐(2‐methoxy‐1,4‐benzodioxan‐2‐yl)‐2‐imidazoline) and RX811059 (2‐(2‐ethoxy‐1,4‐benzodioxan‐2‐yl)‐2‐imidazoline) was more complex, with Hill slopes considerably less than unity, and best described by a two‐site model of interaction comprising a high and low affinity component. The proportion of sites with high affinity for each antagonist was similar (60–80%). 4 The rank order of antagonist potency for the high affinity component in each displacement curve (RX821002 > RX811059 > yohimbine) is similar to that determined against the binding of [ 3 H]‐RX821002 to rat brain, suggesting that these components reflect the inhibition of [ 3 H]‐idazoxan binding to α 2 ‐adrenoceptors. The remaining component in each displacement curve exhibiting low affinity towards these antagonists is attributable to the displacement of [ 3 H]‐idazoxin from a non‐adrenoceptor idazoxan binding site (NAIBS) since a comparable amount of [ 3 H]‐idazoxan binding was not displaced by an excess concentration of (−)‐adrenaline. 5 The displacement of [ 3 H]‐idazoxan binding by RX801023 (6‐fluoro‐(2‐(1,4‐benzodioxan‐2‐yl)‐2‐imidazoline) was also best described by a model assuming a two site interaction with 20.07 ± 3.11% of the sites labelled displaying high affinity for RX801023. The K i of RX801023 for the remainder of the sites labelled was similar to its K i versus [ 3 H]‐RX821002, indicating that this drug displays improved affinity and NAIBS/α 2 ‐adrenoceptor selectivity compared with idazoxan. 6 In autoradiographical studies, the distribution of 5 n m [ 3 H]‐idazoxan binding to sections of rat whole brain was consistent with that reported from previous studies and resembled the distribution of α 2 ‐adrenoceptors. However, when sections of brain were coincubated with concentrations of α 2 ‐adrenoceptor agonists or antagonists predicted to saturate α 2 ‐adrenoceptors, there remained distinct areas of binding corresponding to discrete brain nuclei. This remaining binding was however displaced by unlabelled idazoxan (3 μ m ) or RX801023 (3 μ m ) indicative of the labelling of NAIBS. 7 Quantitative autoradiography of NAIBS revealed several brain nuclei which contained higher densities of these sites than α 2 ‐adrenoceptors, notably the area postrema, interpeduncular nucleus, arcuate nucleus, ependyma and pineal gland.