Investigative ophthalmology & visual science · 1994
Characterization of alpha 2-adrenoceptor binding sites in rabbit ciliary body membranes.
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Jin Y, Verstappen A, Yorio T
Аннотация
PURPOSE: This study sought to identify and characterize subtypes of alpha 2-adrenoceptors in rabbit ciliary body.
METHODS: Radioligand binding assays were performed with the alpha 2-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, Kd = 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 alpha 2-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 alpha 2-adrenoceptors were further studied by competition for [125I]PIC binding by subtype-selective compounds. [125I]PIC binding sites showed the pharmacologic characteristics of an alpha 2A-adrenoceptor (oxymetazoline > chlorpromazine >> prazosin), 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 microM 5'-guanylimidodiphosphate.
CONCLUSIONS: A large number of alpha 2-adrenoceptors are present in rabbit ciliary body. They are not the imidazoline-preferring receptor but are alpha 2-adrenergic-specific receptors of the alpha 2A subtype.
METHODS: Radioligand binding assays were performed with the alpha 2-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, Kd = 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 alpha 2-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 alpha 2-adrenoceptors were further studied by competition for [125I]PIC binding by subtype-selective compounds. [125I]PIC binding sites showed the pharmacologic characteristics of an alpha 2A-adrenoceptor (oxymetazoline > chlorpromazine >> prazosin), 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 microM 5'-guanylimidodiphosphate.
CONCLUSIONS: A large number of alpha 2-adrenoceptors are present in rabbit ciliary body. They are not the imidazoline-preferring receptor but are alpha 2-adrenergic-specific receptors of the alpha 2A subtype.