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/10(6) cells, as assessed by Scatchard analysis of the equilibrium binding at 25 degrees C. The Kd 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(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl2 and BaCl2 increased the Bmax, but the Kd 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 Kd 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.
Sumimoto
K
K
Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Hirata
M
M
Kuriyama
H
H
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D003331:Coronary Vessels / Q000378:metabolism; D004594:Electrophysiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D009543:Nifedipine / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D013552:Swine; D014316:Tritium
D002121:Calcium Channel Blockers; D002412:Cations; D014316:Tritium; D009543:Nifedipine