Delineation of three pharmacological subtypes of α2‐adrenoceptor in the rat kidney

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Abstract
1 Simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked [3H]-RX821002 saturation curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves revealed that the drugs bound to three α2-adrenoceptor subtypes in the rat kidney with grossly differing selectivities. These α2-adrenoceptor subtypes were termed α2A, α2B1 and α2B2. The order of affinities for [3H]-RX821002 for the adrenoceptor sites was α2A > α2B1 > α2B2, the Kd s being 0.62 ± 0.05, 2.52 ± 0.11 and 6.74 ± 1.21 nm, respectively. The order of affinities for ARC 239 was α2B1 > α2B2 ≫ α2A with Kd s 4.78 ± 1.04, 28.8 ± 4.1 and 1460 ± 270 nm, respectively. For guanoxabenz the order of affinities was α2A > α2B1 ≫ α2B2 with Kd s 99.7 ± 15.1, 508 ± 135 and 25,400 ± 2400 nm, respectively. 2 Binding constants for 14 compounds for the three rat kidney α2-adrenoceptor subtypes were determined by the simultaneous computer modelling of plain and ARC 239- and guanoxabenz-masked drug competition curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239-masked guanoxabenz competition curves. Of the 14 compounds tested, oxymetazoline and guanfacine were found to bind with low affinities to both of the α2B1- and α2B2-adrenoceptors but with high affinity to the α2A-adrenoceptor. Prazosin instead bound with high affinities to the α2B1- and α2B2-adrenoceptors but with low affinity to the α2A-adrenoceptor. By contrast, guanoxabenz and ARC 239 delineated clearly between all the three α2-adrenoceptor subtypes. Notably the affinities of guanoxabenz for α2B1- and α2B2-adrenoceptors differed 72 fold and for α2A- and α2B2-adrenoceptors 380 fold. The selectivities of a number of other drugs were less marked but their Kd s were consistent with all three sites being α2-adrenoceptors. 3 (−)-Adrenaline and (−)-noradrenaline showed dissimilar order of affinities for the three α2-adrenoceptors. For (−)-adrenaline the order of affinities was α2B1 ≥ α2A > α2B2 and for (−)-noradrenaline α2B2 ≥ α2B1 > α2A. All three α2-adrenoceptors showed the expected stereoselective binding for adrenaline enantiomers, the (+)-form being 7–10 fold less potent than the (−)-form. 4 [3H]-yohimbine was also used as radioligand. The data with this ligand were fully compatible with the [3H]-RX821002 data. However, [3H]-yohimbine appeared to label only α2B1- and α2B2-adrenoceptors presumably because it had too low an affinity for α2A-adrenoceptors. 5 We conclude that three pharmacological subtypes of α2-adrenoceptors are labelled by [3H]-RX821002 in the rat kidney. Guanoxabenz and ARC 239 may be used in competition studies to delineate between these three α2-adrenoceptor subtypes. Moreoever, we here present a method allowing the determination of binding constants for an arbitrary drug to the three α2-adrenoceptor subtypes.