RADIOLIGAND AND FUNCTIONAL ESTIMATES OF THE INTERACTION OF THE 1,4-DIHYDROPYRIDINES, ISRADIPINE AND LACIDIPINE, WITH CALCIUM CHANNELS IN SMOOTH-MUSCLE

1 The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2 We studied the effect of lacidipine on contraction evoked by 100 mm KCl solution in rat aorta, treated by N(omega)-nitro-L-arginine (0.1 mm), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) m-1 min-1) and the dissociation constant (K(D)) = k-1/k1 = 115 pm) which was close to the IC50 value in steady-state conditions (160 pM). 3 The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mm KCl-solution. Washout with drug-free 40 mm K+-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4 Radioligand binding studies were performed with [H-3]-lacidipine and [H-3]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for [H-3]-lacidipine: K(D) = 23 +/- 2.6 pM B(max) = 380 +/- 21 fmol mg-1 protein in membranes from aorta; K(D) = 23 +/- 3.1 pM, B(max) = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [H-3]-isradipine: K(D) = 140 +/- 46 pM, B(max) 350 +/- 64 fmol mg-1 protein in membrane from aorta; K(D) = 68 +/- 14 pM, B(max) = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [H-3]-lacidipine and [3 H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (k-1) were 0.0257 min-1 and 0.0595 min-1, for [H-3]-lacidipine and [H-3]-isradipine, respectively. 5 The non specific binding of [H-3]-lacidipine and [H-3]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine (1 mum). After incubation with [H-3]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt after 35 h washout in drug-free solution. After incubation with [H-3]-isradipine 47.2 +/- 0.4 pm for 2 h it was 2.26 0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution. 6 It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a non specific binding in a tissue compartment different from the plasma membrane.