[³H]-RS-15385-197, a selective and high affinity radioligand for α₂-receptors: Implications for receptor classification

1. RS-15385-197 is the most potent and selective a₂-adrenoceptor antagonist available. We have used [³H]-RS-15385-197 to define a₂-adrenoceptor subtypes. The binding of [³H]-RS-15385-197 to membranes of rat cerebral cortex, rat neonatal lung and human platelets was reversible, saturable and of high affinity. Saturation experiments indicated that [³H]-RS-15385-197 bound to single population of sites in all 3 tissues with high affinity (0.08-0.14 nM). The density of sites labelled by [³H]-RS-15385-197 was greater in the cortex (275 fmol mg^-1 protein) than in the neonate lung (174 fmol mg^-1 protein) and human platelet (170 fmol mg^-1 protein). The density of sites labelled with [³H]-RS-15385-197 in the cortex was significantly greater than that labelled with [³H]-yohimbine (121 fmol mg^-1 protein). 2. The selective a₂-adrenoceptor antagonists, idazoxan, yohimbine, rauwolscine and WY 26703 displaced [³H]-RS-15385-197 binding to rat cerebral cortex in a simple manner with Hill slopes close to unity. The affinities derived for these antagonists against [³H]-RS-15385-197 were similar to the values obtained for the displacement of [³H]-yohimbine indicating the a₂-adrenoceptor nature of the binding site. 3. a(2A)-Adrenoceptor selective compounds, oxymetazoline and BRL 44409, showed high affinity for [³H]RS-15385-197 binding in the human platelet and lower affinity in the neonatal lung, while the a(2B)-selective compounds, prazosin and imiloxan, showed high affinity for [³H]-RS-15385-197 binding in the neonate lung. This suggests that [³H]-RS-15385-197 labels both a(2A)- and a(2B)-adrenoceptor subtypes. 4. Prazosin and methysergide inhibited the binding of [³H]-RS-15385-197 in the rat cerebral cortex in a simple manner consistent with an interaction at a single site. Although oxymetazoline inhibited [³H]-RS-15385-197 with a Hill slope significantly different from unity, the slope was increased to unity in the presence of Gpp(NH)p, suggesting an agonist-like interaction. 5. The site labelled by [³H]-RS-15385-197 in the rat cortex shows high affinity for oxymetazoline and low affinity for prazosin which could be taken as evidence for classifying the site as an a(2A)-subtype. However, the affinities of yohimbine, rauwolscine and oxymetazoline at this site do not correspond to the population of sites in the human platelet. Yohimbine and rauwolscine were 20 fold selective for the platelet a(2A)-subtype, whereas phentolamine was 2-fold and imiloxan was 10 fold selective for the cortex subtype. Indeed although the site showed some similarities with the a(2A)-subtype, the highest degree of homology was observed between this site and the rat submaxillary gland and the RG20 clone, tentatively called the a(2D)-adrenoceptor subtype. We propose that the a₂-adrenoceptor in the rat cortex may therefore correspond to the putative a(2D)-subtype of the adrenoceptor.