THE EFFECT OF PHOTOGENERATED SITE-DIRECTED FREE-RADICALS ON SURFACE DIHYDROPYRIDINE BINDING-SITES IDENTIFIED WITH PHOTOAFFINITY PROBE (-)-[H-3]-AZIDOPINE ON CULTURED MONKEY RENAL-CELLS

With the use of the photoactivable dihydropyridine (DHP)-type calcium (Ca2+ channel antagonist (-)-[H-3]-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, sti mulated depolarized and UV irradiated green monkey renal (GMR) cells p reincubated with selected DHPs. Our results demonstrate that specific binding of(-)-[H-3]-azidopine on GMR cells is of high affinity, stereo selective and dependent mainly on the inactivation of the membrane bou nd Ca2+ channel. Preincubation of the GMR cells with the DHP Ca2+ chan nel agonist BAY-K-8644 significantly reduced specific photolabeling. T he site-directed free radicals generated after UV irradiation in DHP-p reincubated renal cells inactivated Ca2+ channels and did nor signific antly affect the specific photoincorporation of (-)-[H-3]-azidopine. ( +)-Niguldipine, a DHP with the voluminous substituent on the DHP ring, significantly reduced the photolabeling. Low affinity labeling was pa rtially prevented in (+)-nimodipine and (+)-niguldipine preincubated p hotoirradiated cells. The results strongly support the existence of ce ntral 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-ge nerating photoreactive group, the photoaffinity probe, (-)-[H-3]-azido pine.