Characterization and autoradiographical localization of non‐adrenoceptor idazoxan binding sites in the rat brain (1992)

1. In rat whole brain homogenates, saturation analysis revealed that both [3H]-idazoxan and [3H]-RX821002, a selective alpha 2-adrenoceptor ligand, bound with high affinity to an apparent single population of sites. However, the Bmax for [3H]-idazoxan was significantly (P less than 0.01) greater than that for [3H]-RX821002. 2. In competition studies, (-)-adrenaline displaced 3 nM [3H]-idazoxan binding with an affinity consistent with [3H]-idazoxan labelling alpha 2-adrenoceptors. However, this displacement was incomplete since 23.68 +/- 1.11% of specific [3H]-idazoxan binding remained in the presence of an excess concentration (100 microM) of (-)-adrenaline. In contrast, unlabelled idazoxan promoted a complete displacement of [3H]-idazoxan binding with a Hill slope close to unity and an affinity comparable with its KD determined in saturation studies. 3. Displacement of [3H]-idazoxan binding by the alpha 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 greater than RX811059 greater than yohimbine) is similar to that determined against the binding of [3H]-RX821002 to rat brain, suggesting that these components reflect the inhibition of [3H]-idazoxan binding to alpha 2-adrenoceptors.The remaining component in each displacement curve exhibiting low affinity towards these antagonists is attributable to the displacement of [3H]-idazoxin from a non-adrenoceptor idazoxan binding site (NAIBS) since a comparable amount of [3H]-idazoxan binding was not displaced by an excess concentration of (-)-adrenaline.5. The displacement of [3H]-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 Ki of RX801023 for the remainder of the sites labelled was similar to its Ki versus [3H]-RX821002, indicating that this drug displays improved affinity and NAIBS/z2-adrenoceptor selectivity compared with idazoxan.6. In autoradiographical studies, the distribution of 5 nM [3H]-idazoxan binding to sections of rat whole brain was consistent with that reported from previous studies and resembled the distribution ofM2-adrenoceptors. However, when sections of brain were coincubated with concentrations of alpha2-adrenoceptor agonists or antagonists predicted to saturate alpha2-adrenoceptors, there remained distinct areas of binding corresponding to discrete brain nuclei. This remaining binding was however displaced by unlabelled idazoxan (3 microM) or RX801023 (3 microM) indicative of the labelling of NAIBS.7. Quantitative autoradiography of NAIBS revealed several brain nuclei which contained higher densities of these sites than alpha2-adrenoceptors, notably the area postrema, interpeduncular nucleus,arcuate nucleus, ependyma and pineal gland.