Comparison of High Affinity Binding of 3H‐Proadifen and 3H‐(‐)‐Cocaine to Rat Liver Membranes

Abstract

The characteristics of the binding of 3H-proadifen to rat liver membranes were studied and compared to those of 3H-cocaine. It was found that 3H-proadifen was bound reversibly with high affinity (KD = 1.8 +/- 0.5 nM) and large capacity (Bmax = 2010 +/- 340 pmol/g wet tissue) to liver membranes. The corresponding values for the 3H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of 3H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 microM CdCl2 in the incubation buffer it was possible to differentiate between two 3H-cocaine binding sites with Kd values of 1.6 and 7.7 nM and Bmax values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of 3H-proadifen and 3H-cocaine with high affinity (IC50 = 2.2 nM and 0.4 nM, respectively). The R-enantiomer was 100 to 300 times less potent. Cocaine inhibited the binding of 3H-proadifen (IC50 = 10 nM) and proadifen that of 3H-cocaine (IC50 = 1 nM). There was a high correlation coefficient (rr = 0.972; P < 0.01; n = 12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Besides some potent alaproclate analogues a couple of compounds had moderately high affinity (IC50 = 100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl2, ZnCl2 and CuCl2 inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)