Anti-dihydropyridine Antibodies Exhibit [3H]Nitrendipine Binding Properties Similar to the Membrane Receptor for the 1,4-Dihydropyridine Ca2+ Channel Antagonists

Abstract

The [H]nitrendipine binding properties of antidihydropyridine antibodies were characterized and compared with the known [H]nitrendipine binding properties of the membrane receptor for the 1,4-dihydropyridine Ca channel antagonists. Immunization of rabbits with dihydropyridine-protein conjugates resulted in the production of antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca channel antagonists. Anti-dihydropyridine antibodies were found specifically to bind [H]nitrendipine, [H]nimodipine, [H]nisoldipine, and [H]PN200-110 with high affinity, while [H]verapamil, [H]diltiazein, and [H]trifluoperazine were not recognized. [H]Nitrendipine binding to antidihydropyridine antibodies was characterized by a competitive radioimmunoassay. The average dissociation constant of the [H]nitrendipine-antibody complex was 0.06 ± 0.02 nM for an antiserum studied in detail, and ranged from 0.01 to 0.24 nM for all antisera studied. The average dissociation constant determined from rate constants of association (k1 = 2.7 × 10 M s) and dissociation (k = 1.8 × l0s) of [H]nitrendipinefronianti-dihydropyridine antibodies immobilized on Protein-A Sepharose was 0.66 nM Inhibition of [H]nitrendipine binding was specific for the 1,4-dihydropyridine Ca channel modifiers, and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM; structurally unrelated Ca channel antagonists did not modify [H]nitrendipine binding to the anti-dihydropyridine antibodies. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca channel antagonists and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca channel antagonists.