Evidence for the existence of two forms of α(2A)-adrenoceptors in the rat

The α(2A)-adrenoceptors in rat spleen, kidney, spinal cord and cerebral cortex were studied using [(3)H]-RX821002 radioligand binding. In the spleen, spinal cord and cerebral cortex, the ligand bound to saturable sites with a K (d) of about 1 nmol/l and capacities of 134, 240 and 290 fmol/mg protein, respectively. Computer modelling competition curves for 39 drugs, including those for α(2A)-, α(2B)- or α(2C)-adrenoceptor selective drugs, indicated that the sites labelled by [(3)H]-RX821002 in the spleen consisted of a single population of α(2A)-adrenoceptors. However, the competition curves for guanoxabenz were definitely biphasic and resolved into two site fits, indicating that guanoxabenz was binding to both high affinity (K (d) = 35 nmol/1) and low affinity (K (d) = 8900 nmol/1) α(2A)-adrenoceptor sites in the proportions 57% and 43%, respectively. The K (d) (S)for a number of α(2)-adrenoceptor subtype selective drugs, measured in competition with [(3)H]-RX821002 in cerebral cortex and spinal cord, were highly correlated with those obtained in the spleen indicating their α(2A)-adrenoceptor nature. However, by contrast to the results with the spleen, the guanoxabenz competition curves for the spinal cord and cerebral cortex were monophasic and resolved only into one site fits, the K (d) of guanoxabenz being about 4000 nmol/l for both tissues. Drug K (d) (S)for kidney α(2A)-adrenoceptors were also determined using [(3)H]-RX821002. For nearly all drugs tested, the K (d) (S)were highly correlated with those found for the α(2A)-adrenoceptors in the other rat tissues. However, for guanoxabenz, the data indicated that it competed with [(3)H]-RX821002 at a single α(2A)-adrenoceptor site with a K (d) of 39 nmol/1. When the rat α(2A)-adrenoceptor gene RG20 was transiently expressed in COS-7 cells and its ligand binding properties probed using [(3)H]-RX821002, the drug K (d) (S)obtained were also highly correlated with those found for the α(2A)-adrenoceptors in the spleen, cerebral cortex, spinal cord and kidney of the rat. For the RG20 encoded receptor, the guanoxabenz competition curves were steep and monophasic and modelled best into one site fits, with the Kd of guanoxabenz being 5200 nmol/1. It is suggested that guanoxabenz can differentiate between two forms of α(2A)-adrenoceptors in the rat: α(2A1) and α(2A2). The α(2A1)-form is present in the spleen and kidney where it shows a high apparent affinity for guanoxabenz. The α(2A2)-form shows a low apparent affinity for guanoxabenz and is present in the spleen, cerebal cortex and spinal cord. The α(2A2)-form of the rat α(2)-adrenoceptor appears to be encoded by the RG20 gene. The α(2A), and α(2A2)-adrenoceptor forms do not represent high and low affinity receptor forms for agonists because assays included EDTA, Gpp(NH)p and Na(+), which eliminated the high affinity receptors for agonists.