High affinity binding of 3H rauwolscine and 3H RX781094 to α 2 adrenergic receptors and non-stereo selective sites in human and rabbit brain cortex membranes

Abstract

The radiolabeled antagonists 3H RX 781094 and 3H rauwolscine bind with high affinity to α 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 μM 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 μM (−)-epinephrine is used for the determinatlon of the non-specific binding, both radioligands bind specifically to α 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 B max = 113 ± 15 fmol/mg protein and K d = 22.8 ± 4.2 nM for 3H RX781094 and B max = 110 ± 17 fmol/mg protein and K d = 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 α 2 adrenergic receptors, i.e. phentolamine > yohimbine > prazosin for the antagonists and UK 14304 > p-aminoclonidine ⩾ (−)-epinephrine > (+)-epinephrine > isoproterenol for the agonists. Whereas the α 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 μM (−)-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-HT 1-receptor. The 3H RX 781094 binding displays low affinity for most α adrenergic ligands and do not correspond to β adrenergic, dopaminergic or serotonergic receptors.