Synthesis and characterization of an affinity label for brain receptors to psychotomimetic benzomorphans: differentiation of σ-type and phencyclidine receptors

Abstract

Brain σ-type receptors and phencyclidine receptors are though to mediate the psychotomimetic effects of benzomorphans and phencyclidine in humans. Recently, we reported the characterization of a selective σ receptor ligand, 1,3-di-o-tolyl-guanidine (DTG), that shows negligible crossreactivity with phencyclidine receptors. Here we describe the synthesis and characterization of an isothiocynate derivative of DTG, di-o-tolyl-guanidine-isothiocyanate (DIGIT). Guinea pig brain membranes treated with nanomolar doses of DIGIT followed by extensive washing exhibit a dose dependent reduction of [ 3H]DTG and (+)[ 3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)[ 3H]3-PPP) binding to σ receptors. Binding of radiolabelled ligands for phencyclidine, μ-opioid, benzodiazepine and dopamine-D 2 receptors is not affected by membrane treatment with DIGIT, indicating specificity of the affinity label for σ-type receptors. Treatment of DIGIT-derivatized membranes with 2 M NaCl does not result in recovery of σ binding activity, suggesting that DIGIT's interaction with σ receptors is not of an ionic nature. Equilibrium saturation binding experiments show that the inhibition of [ 3H]DTG binding to σ receptors by DIGIT pretreatment of membranes is attributable to an irreversible reduction in the affinity (increase in K d) of σ receptors for DTG. The finding that σ receptors are irreversibly modified by DIGIT whereas phencyclidine receptors are not affected suggests that σ receptors are physically separate from phencyclidine receptors. The availability of a selective affinity label for the σ receptor should facilitate the purification of the receptors and the characterization of σ-type pharmacological effects in vivo and in vitro.