Characterization of [³H]nifedipine binding to intact vascular smooth muscle cells

Specific binding of the dihydropyridine Ca²⁺ antagonist [³H]nifedipine to dispersed smooth muscle cells of the porcine coronary artery was investigated and the findings were compared with the binding to microsomes of smooth muscles. Specific binding to intact cells was saturable and reversible. The dissociation constant was 1.93 ± 0.42 nM and the maximal binding capacity was 59.6 ± 12.4 fmol/10⁶ cells, as assessed by Scatchard analysis of the equilibrium binding at 25°C. The K(d) value with intact cells was slightly higher than that observed with microsomes. Specific binding of [³H]nifedipine to intact cells was completely displaced by unlabeled dihydropyridine derivatives. Among other Ca²⁺ antagonists, verapamil and d-cis-diltiazem partially and flunarizine completely inhibited the binding. In the case of microsomes, d-cis-diltiazem stimulated the binding of [³H]nifedipine. These results suggest that there may be multiple binding sites for different subclasses of Ca²⁺ antagonists. Polyvalent cations had no effect on the binding to intact cells. In the case of ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl₂ and BaCl₂ increased the B(max), but the K(d) value remained unchanged. MnCl₂ and CdCl₂ had stimulatory or inhibitory effects, depending on the concentrations, whereas LaCl₃ had no effect. The effect of membrane depolarization on the binding was also examined. When the intact cells were incubated in high [K⁺](o) solution for 60 min, the K(d) was lowered to 1.4 nM from the control value of 2.0 nM, thereby indicating that [³H]nifedipine binds to Ca²⁺ channels, with a higher affinity, at depolarized states.