Characterization of α₂-adrenoceptor binding sites in rabbit ciliary body membranes

Purpose. This study sought to identify and characterize subtypes of α₂- adrenoceptors in rabbit ciliary body. Methods. Radioligand binding assays were performed with the α₂-agonist ligand [¹²⁵I]-p-iodoclonidine ([¹²⁵I]PIC) and the antagonist ligand [³H]rauwolscine. Results. Both [¹²⁵I]PIC and [³H]rauwolscine bound to a single population of receptors in membrane preparations of rabbit ciliary body. The densities for the two ligands were the same (about 640 fmol/mg). However, the affinity of [¹²⁵I]PIC (dissociation constant, K(d) = 1.91 ± 0.24 nM) was about threefold higher than that of [³H]rauwolscine (dissociation constant = 6.79 ± 1.5 nM), and binding of [¹²⁵I]PIC exhibited guanine nucleotide sensitivity. Inhibition of [¹²⁵I]PIC binding by epinephrine, idazoxan, and amiloride was examined to differentiate between α₂-adrenoceptors and the imidazoline-preferring receptor. Epinephrine and idazoxan competed for all of the [¹²⁵I]PIC binding; relative potency was epinephrine > idazoxan >> amiloride. Subtypes of α₂-adrenoceptors were further studied by competition for [¹²⁵I]PIC binding by subtype-selective compounds. [¹²⁵I]PIC binding sites showed the pharmacologic characteristics of an α(2A)-adrenoceptor (oxymetazoline > chlorpromazine >> prazosine), and competition by oxymetazoline and chlorpromazine was best fit by a one-site model. Likewise, the relative potency of inhibition of [³H]rauwolscine binding was oxymetazoline > chlorpromazine. However, inhibition of [³H]rauwolscine- binding by oxymetazoline was better fit by a two-site model, which was converted to a one-site model in the presence of 100 μM 5'- guanylimidodiphosphate. Conclusions. A large number of α₂-adrenoceptors are present in rabbit ciliary body. They are not the imidazoline-preferring receptor but are α₂-adrenergic-specific receptors of the α(2A) subtype.