Identification of dihydropyridine (DHP) binding sites on cultured monkey renal cells (GMRC) with a photoaffinity probe (-)-[3H]-azidopine.

The low affinity binding sites identified in crude membranes from different excitable tissues with the dihydropyridine (DHP) calcium (Ca2+) channel ligands have confused researches in the field of Ca2+ channels as they can represent low affinity state(s) of the DHP receptor, or they can be labelled with DHP-type Ca2+ channel ligands. The aim of this communication was to provide more evidence for the existence of separate DHP binding sites on the surface of cultured green monkey renal cells (GMRC). The saturation ligand binding experiments with [3H]-nitrendipine (NTP) and photoaffinity labelling studies with (-)-[3H]-azidopine (AZI) were performed in order to identify and further characterize the DHP receptor on cultured GMRC. Specific high affinity sites identified on GMRC with [3H]-NTP (Bmax = 0.78 +/- 0.03 pmol/mg protein and KD = 0.06 +/- 0.1 nmol/l in native cells) and photolabelled with AZI represent DHP receptor on L-type Ca2+ channels. The low affinity binding sites photolabelled with AZI on GMRC (9.84 +/- 2.4 pmol/mg protein and KD = 3.21 +/- 1.25 nmol/l in native cells) were significantly increased after preincubation of GMRC with low concentrations of DHPs nitrendipine and nisoldipine. Preincubation of GMRC with Ca2+ channel agonist (-)BAYK 8644 significantly reduced specific photolabelling with AZI on GMRC and increased low affinity labelling. Preincubation of (+)BAYK 8644 was without any effect. Niguldipine (DHP with the voluminous substituent on the port side of the DHP ring) partially inhibited specific photolabelling with AZI on GMRC and also partially reduced the maximal number of low affinity binding sites labelled with AZI. Our results support the hypothesis of separate subsites in the region of DHP receptor of GMRC and the existence of the "marginal" photolabelling of specific DHP binding sites identified on Ca2+ channels.