IRREVERSIBLE BINDING OF N-METHYL-N-[(1S)-1-(4-ISOTHIOCYANATOPHENYL)-2-(1-PYRROLIDINYL)ETHYL-3,4-DI CHLOROPHENYLACETAMIDE TO THE CLONED RAT KAPPA OPIOID RECEPTOR

摘要

N-Methyl-N-[(1S)-1-(4-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl-3,4-di chlorophenylacetamide (MITPD)(1), is an isothiocyanate derivative of the kappa agonist ICI-199,441. In this study, interaction of MITPD with cloned mu, delta, and kappa opioid receptors was characterized. MITPD inhibited [H-3]diprenorphine binding to kappa receptors with high affinity and with similar to 700- and similar to 870-fold selectivity over mu and delta receptors. Pretreatment with MITPD followed by extensive washing reduced kappa receptor binding with an IC50 value of 3.7 nM, but did not affect mu or delta binding at I 0.1 mu M. Preincubation with I mu M MITPD abolished [H-3]diprenorphine binding, while pretreatment with 1 mu M ICI-199,441 increased K-d of [H-3]diprenorphine binding with no change in B-max. Thus, MITPD is a selective kappa irreversible ligand. The region of the kappa receptor that conferred selectivity for MITPD was determined by examining its binding to four mu/kappa chimeras. IC50 values of MITPD for inhibition of [H-3]diprenorphine binding were determined to be 430 nM for Chimera III (kappa 1-141/mu 151-398), 1.8 nM for Chimera IV (mu 1-150/kappa 142-380), 40 nM for Chimera XI (mu 1-268/kappa 263-380) and 14 nM for Chimera XII (kappa 1-262/mu 269-398). Pretreatment with MITPD followed by extensive washing reduced binding to chimera IV with an IC50 value of 75 nM, but did not affect III, XI or XII binding (IC50 >1 mu M). Thus, the region from the third transmembrane helix to the C-terminus of the kappa receptor is important for the binding of MITPD. [References: 38]