Further Evidence for the Existence of Two Forms of α2B‐Adrenoceptors in Rat

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Abstract
Analysis of saturation isotherms of the novel α2‐adrenoceptor antagonist radioligand [3H]‐MK 912 revealed that the ligand labelled a homogenous population of α2B‐adrenoceptors in the neonatal rat lung with a Kdof 0.77 nM and a Bmaxof 231 fmol/mg protein. In rat kidney, combined saturation and competition experiments, using [3H]‐MK 912 and the α2A‐adrenoceptor selective drug guanfacine, revealed that ~ 81% of the sites labelled by [3H]‐MK 912 were α2B‐adrenoceptors and ~ 19% α2A‐adrenoceptors; the Kds of [3H]‐MK 912 being. 1.1 and 2.0 nM and the Bmax134 and 33 fmol/mg protein, respectively. The kidney α2B‐adrenoceptors were studied separately by using ~ 1.5 nM [3H]‐MK 912 in the presence of 0.32 μM guanfacine, the latter which blocked ligand binding to α2A‐adrenoceptors completely. Analysis of drug competition curves obtained during these conditions revealed that 18 out of 20 different agonists and antagonists yielded steep and uniphasic competion curves which modelled best into one site fits. However, both guanoxabenz and LT 11 appeared to inhibit [3H]‐MK 912 binding at two sites; the Kds of guanoxabenz differing ~ 120‐fold and that of LT 11 differing ~ 35‐fold for the two sites. Moreover, the addition of mutual fixed concentrations of either 20 μM guanoxabenz or 20 μM LT 11 completely prevented the binding of the other compound to its high affinity site, indicating that both compounds labelled the same site with the high affinity. The analysis indicated that 29% of the sites were of high and 71% of low affinity. However, in the neonatal rat lung guanoxabenz and LT 11 (as well as 15 other compounds) yielded competition curves which modelled only into one site fits. The Kds obtained in the lung correlated well with the Kds obtained in the kidney for α2B‐adrenoceptors; for guanoxabenz and LT 11 the values from the lung were close to those determined in the kidney for the low affinity site for guanoxabenz and LT 11. Moreover, when the rat RNG α2B‐adrenoceptor was expressed in COS‐7 cells and its binding properties tested using [3H]‐MK 912 binding, guanoxabenz, LT 11 as well as a number of other drugs inhibited the ligand binding at a single α2‐adrenoceptor site; the drug Kds being practically the same as those found for the neonatal rat lung. It is suggested that rat α2B‐adrenoceptors may exist in two forms: α2B1and α2B2. The α2B1‐form, which shows high affinity for guanoxabenz and LT 11, is present in the adult rat kidney. The α2B2‐form, which shows low affinity for guanoxabenz and LT 11, is present in the neonatal rat lung as well as in the adult rat kidney and it is encoded by the RNG gene.

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