Identification and molecular characterization of the isoquinoline rat intestinal binding site using 6,7-dimethoxy-4-(4'-amino-3'-(/sup 125/I)iodobenzyl) isoquinoline
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'-(/sup 125/I)iodobenzyl) isoquinoline (/sup 125/I-DMABI) binds reversibly to rat intestinal membranes. Binding is rapid, saturable, and temperature dependent. The binding of /sup 125/I-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 /sup 125/I-DMABI-labeled protein with molecular weight of 36,000 and two minor proteins with molecular weights of 52,000 and 26,000.
- Research Organization:
- Equipe de Recherches de Neuroendocrinologie du Systeme Digestif, Villejuif, France
- OSTI ID:
- 6992673
- Journal Information:
- Mol. Pharmacol.; (United States), Journal Name: Mol. Pharmacol.; (United States) Vol. 4; ISSN MOPMA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METALS
ANIMALS
AROMATICS
AZAARENES
AZINES
BARIUM
BETA DECAY RADIOISOTOPES
BODY
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
ELEMENTS
GASTROINTESTINAL TRACT
HETEROCYCLIC COMPOUNDS
INTERMEDIATE MASS NUCLEI
INTESTINES
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
METALS
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PROTEINS
PYRIDINES
QUINOLINES
RADIOISOTOPES
RADIORECEPTOR ASSAY
RATS
RECEPTORS
RODENTS
SPECIFICITY
TRACER TECHNIQUES
VERTEBRATES