(-)[3H]AMLODIPINE BINDING TO RAT CARDIAC MEMBRANES

Amlodipine is a newly developed long-acting dihydropyridine-based calcium antagonist. To characterize the binding properties of this compound, saturation binding studies were undertaken, using (-)[H-3]amlodipine and rat cardiac membrane fragments. (-)[H-3]Amlodipine bound to a single population of high-affinity binding sites with a K(D) of 1.68 +/- 0.12 nM, a B(max) of 0.34 +/- 0.08 pmol/mg protein, and a Hill coefficient approaching unity. Binding required up to 5 h to reach asymptote, and was pH- and temperature-sensitive. The specific binding was totally inhibited by (-) amlodipine and (-) D600 (IC50 values of 9.20 +/- 5.56 and 6.58 +/- 6.57 nM, respectively) and only partially inhibited by (+) PN 200-110, (-) Bay K 8644, (+) D600, and d-cis diltiazem (IC50 values of 60 +/- 10, 160 +/- 20, 250 +/- 40, and 200 +/- 30 nM, respectively). These results indicate that in addition to its ability to bind to the dihydropyridine and benzothiazepine recognition sites in rat cardiac membrane fragments, (-)[H-3]amlodipine also binds strongly to the recognition sites for the phenylalkylamine-based calcium antagonists. The results also show that the inhibition of (-)[H-3]amlodipine binding by D600 is sterospecific with (-) > (+)D600. Dissociation of bound (-)[H-3]amlodipine was slowed under acidotic (pH 6.0) and accelerated under alkalotic (pH 10.0) conditions.