title: The effect of photogenerated site-directed free radicals on surface dihydropyridine binding sites identified with photoaffinity probe (-)-[3H]-azidopine on cultured monkey renal cells.
abstract: With the use of the photoactivable dihydropyridine (DHP)-type calcium (Ca2+ channel antagonist (-)-[3H]-azidopine, a specific photoaffinity probe for Ca2+ channels, we tested the hypothesis of the existence of a separate subsite in the DHP receptor region on native polarized, stimulated depolarized and UV irradiated green monkey renal (GMR) cells preincubated with selected DHPs. Our results demonstrate that specific binding of (-)-[3H]-azidopine on GMR cells is of high affinity, stereoselective and dependent mainly on the inactivation of the membrane bound Ca2+ channel. Preincubation of the GMR cells with the DHP Ca2+ channel agonist BAY-K-8644 significantly reduced specific photolabeling. The site-directed free radicals generated after UV irradiation in DHP-preincubated renal cells inactivated Ca2+ channels and did not significantly affect the specific photoincorporation of (-)-[3H]-azidopine. (+)-Niguldipine, a DHP with the voluminous substituent on the DHP ring, significantly reduced the photolabeling. Low affinity labeling was partially prevented in (+)-nimodipine and (+)-niguldipine preincubated photoirradiated cells. The results strongly support the existence of central and peripheral subsites of the DHP region on GMR cells, with the former incorporating on photoactivation the intrinsically photoactive DHPs and with the latter labeled with a side chain bearing nitrene-generating photoreactive group, the photoaffinity probe, (-)-[3H]-azidopine.
authors.affiliation: Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava.
authors.forename: J
authors.initials: J
authors.lastname: Drímal
authors.forename: L
authors.initials: L
authors.lastname: Bohácik
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type; D002460:Cell Line; D002522:Chlorocebus aethiops; D004095:Dihydropyridines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005609:Free Radicals; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D007700:Kinetics; D008024:Ligands; D008564:Membrane Potentials; D009124:Muscle Proteins / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D009553:Nimodipine / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D000345:Affinity Labels; D001386:Azides; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D005609:Free Radicals; D008024:Ligands; D009124:Muscle Proteins; D009553:Nimodipine; C041130:azidopine; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C054074:niguldipine