An approach to analysis of radiolabeled ligand interactions with specific receptors

Abstract

The aim of the study was to reveal general characteristics of the ligand-receptor interaction in the binding and displacement of radiolabeled ligands. The binding and displacement of DL-[3H]propranolol hydrochloride ([3H]propranolol) and L-[propyl-2,3, -3H]dihydroalprenolol ([3H]dihydroalprenolol), beta-adrenoceptor antagonists, were studied with isolated rat red blood cells, their membranes and ghosts. The binding of [3H]dihydroalprenolol and L-quinuclidinyl-[phenyl-4-3H]-benzylate ([3H]quinuclidinyl benzylate), a muscarinic acetylcholine receptor antagonist, was studied with cerebral cortex membranes. The ligand-receptor interaction corresponded to that for a model of two pools of receptors in the same effector system, with the binding of two ligand molecules to the receptors and was described by the following equation: b=[(B(m1)A(2))/(K(d1)(2)+A(2))]+[(B(m2)A(2))/(K(d2)(2)+A(2))]. The parameters of [3H]propranolol binding to beta-adrenoceptors were as follows: K(d1)=0.74 nM, K(d2)=14.40 nM, B(m1)=24 unit/cell, and B(m2)=263 unit/cell for native red blood cells from rats; K(d1)=0.70 nM, K(d2)=19.59 nM, B(m1)=9 fmol/mg protein, and B(m2)=39 fmol/mg protein for blood ghosts. The parameters of [3H]quinuclidinyl benzylate binding to muscarinic acetylcholine receptors of cerebral cortex membrane were as follows: K(d1)=0.43 nM, K(d2)=2.83 nM, B(m1)=712 fmol/mg, B(m2)=677 fmol/mg. The analysis of the equilibrium binding and displacement of [3H]propranolol and [3H]dihydroalprenolol at beta-adrenoceptors of membranes, ghosts and native red cells of rats, [3H]dihydroalprenolol at beta-adrenoceptors and [3H]quinuclidinyl benzylate at muscarinic acetylcholine receptors of synaptosomal membranes from rat cerebral cortex demonstrated that the receptors bound two ligand molecules each and consisted of two discrete pools of high- and low-affinity receptors. Similar results were obtained for the displacement of [3H]propranolol, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate by propranolol, dihydroalprenolol and quinuclidinyl benzylate.