Characterization of alpha 2-adrenergic receptors of calf retina membranes by [ 3H]-rauwolscine and [ 3H]-RX 781094 binding

Abstract

Alpha 2-adrenergic receptors were identified in calf retina membranes by binding of the radiolabelled antagonists [ 3H]-RX 781094 and [ 3H]-rauwolscine. When 10 μM phentolamine was used to determine the non-specific binding, both radioligands labelled a single class of non-cooperative sites: B max =1051 ± 252 fmol/mg protein, K d = 5.1 ± 1.5 nM for [ 3H]-RX 78104 and B max = 1167 ± 449 fmol/mg protein, K d = 21.0 ± 4.1 nM for [ 3H]-rauwolscine. Competition binding experiments showed the typical pharmacological potency order of alpha 2-adrenergic receptors, i.e. phentolamine > yohimbine > prazosin. Agonist competition binding curves revealed the presence of two receptor populations, having respectively high affinity (70% of the total receptor population) and low affinity for agonists, but with the same affinity for the antagonists. The high affinity sites could be converted into low affinity sites by guanine nucleotides. The non-specific binding of [ 3H]-RX 781094 was the same if 0.1 mM (−)-epinephrine was used instead of phentolamine. In contrast, the non-specific binding of [ 3H]-rauwolscine was markedly lower with (−)-epinephrine than with phentolamine. Under this condition, the Scatchard plot of [ 3H]-rauwolscine saturation binding was curvilinear, indicating the presence of low affinity sites for the radioligand in addition to alpha 2-adrenergic receptors. Competition binding experiments revealed that these low affinity sites were distinct from adrenergic receptors: the catecholamine agonists (−)- and (−)-epinephrine, (−)-norepinephrine, (−)-isoproterenol and dopamine competed with similar K i values (μM range) whereas clonidine did not interact. Furthermore, these sites bound reserpine and the alpha 2-adrenergic antagonists yohimbine and rauwolscine but not phentolamine.