Article date: November 1991
By: Staffan Uhlén, Jarl E.S. Wikberg, in Volume 104, Issue 3, pages 657-664
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.
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.
(−)‐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.
[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.
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.
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.
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.
(−)‐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.
[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.
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.
DOI: 10.1111/j.1476-5381.1991.tb12485.x
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