Identification and molecular characterization of the isoquinoline rat intestinal binding site using...

The mechanism of the relaxant action of isoquinolines on smooth muscle is conjectural. In order to gain further insight into the intestinal action of isoquinolines, we have synthesized an isoquinoline derivative which can be radioiodinated, resulting in the obtention of a ligand with a high specific activity. 6,7-Dimethoxy-4-(4'-aminobenzyl) isoquinoline (DMABI) is an arylamine analogue of the most relaxating isoquinoline derivative, i.e., 6,7-dimethoxy-4-(4'-chlorobenzyl) isoquinoline. Its iodinated derivative, 6,7-dimethoxy-4-(4'-amino-3'-[125I]iodobenzyl) isoquinoline (125I-DMABI) binds reversibly to rat intestinal membranes. Binding is rapid, saturable, and temperature dependent. The binding of 125I-DMABI to intestinal membranes is competitively inhibited by identical concentrations of unlabeled DMABI or iodo-DMABI in the range between 10(-8) and 10(-5)M. Scatchard analysis indicates the existence of two classes of binding sites: a class with a low capacity (14 +/- 2 pmol/mg of protein) and a Kd = 0.10 +/- 0.02 microM, and a class with a high capacity (240 +/- 31 pmol/mg of protein) and a Kd = 8.0 +/- 1.1 microM. Specific binding of the radioiodinated ligand is inhibited by a variety of 4-benzyl isoquinolines and 1-benzyl isoquinolines. Structure-activity relationship demonstrates the primordial role of C-6 and C-7 methoxy groups and the important role of 4-benzyl on configuration related to the isoquinoline nucleus. A high significant correlation between competitive binding (Ki) and relaxant effect in rat intestine (IC50) is observed and strongly suggests that the isoquinoline-binding site mediates the pharmacologic response. Upon photolysis, this ligand incorporates irreversibly into rat intestinal membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal a major 125I-DMABI-labeled protein with molecular weight of 36,000 and two minor proteins with molecular weights of 52,000 and 26,000. The labeling of these proteins is specific since it is completely abolished by 100 microM DMABI. Scanning of autoradiographs and integration of peaks show that the probe binds with the same apparent affinity to the three proteins. These findings indicate the utility of this novel high affinity radioiodinated probe as a tool for elucidating the mechanism of action of isoquinoline.