High affinity binding of 3H rauwolscine and 3H RX781094 to α2 adrenergic receptors and non-stereo selective sites in human and rabbit brain cortex membranes

The radiolabeled antagonists 3H RX 781094 and 3H rauwolscine bind with high affinity to alpha 2 adrenergic receptors as well as to non-receptor sites in human and rabbit brain cortex membranes. These non-receptor sites form an important contaminant of the specific binding when non-specific binding is determined in the presence of 10 microM phentolamine or more. While phentolamine is no suitable ligand to discriminate both sites, (-)-epinephrine displays a sufficient affinity ratio to separate radioligand binding to these sites. When 1 microM (-)-epinephrine is used for the determination of the non-specific binding, both radioligands bind specifically to alpha 2 receptors. Under these conditions, 3H rauwolscine and 3H RX 781094 bind to the same amount of non-cooperative sites; binding isotherms for human brain are Bmax = 113 +/- 15 fmol/mg protein and Kd = 22.8 +/- 4.2 nM for 3H RX781094 and Bmax = 110 +/- 17 fmol/mg protein and Kd = 4.7 +/- 2.5 nM for 3H rauwolscine. Competition binding experiments show, for both radioligands and in both species, the typical pharmacological potency order of alpha 2 adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin for the antagonists and UK 14304 greater than p-aminoclonidine greater than or equal to (-)-epinephrine greater than (+)-epinephrine greater than isoproterenol for the agonists. Whereas the alpha 2 receptor sites display high affinity and stereoselectivity towards (-)-epinephrine and (+)-epinephrine, the non-receptor sites bind both epinephrine isomers with equal low affinity. Specific binding of both radioligands to these sites can be determined when total binding is performed in the presence of 1 microM (-)-epinephrine and non-specific binding the presence of 1 mM phentolamine. 3H rauwolscine binding to the non-stereoselective sites can be displaced with high affinity by 5-HT, suggesting binding to a 5-HT1-receptor. The 3H RX 781094 binding displays low affinity for most alpha adrenergic ligands and do not correspond to beta adrenergic, dopaminergic or serotonergic receptors.