Pharmacologic relevance of dihydropyridine binding sites in membranes from rat aorta: kinetic and equilibrium studies.

Abstract

The kinetic features of the interaction of dihydropyridines with rat aortic smooth muscle were investigated in parallel mechanical and binding studies. The inhibitory action of (+ )-PN200-110 and nisoldipine on contractions evoked by potassium chloride depolarization was characterized by a pronounced time dependency, in which the inhibition increased slowly after depolarization to attain a steady-state value, while with (-)-PN200-110 and ( + )-Bay K8644 the inhibition was almost instantaneous. To explain these observations, specific binding sites for dihydropyridines were studied in membranes isolated from rat aorta, using [ 3 H]( + )-PN200-l 10 as a radioligand. We found that the time course of the development of inhibition of potassium chloride-evoked contractions by various concentrations of (+ )-PN200-110 paralleled the time course of [3H]( + )-PN200-l10 binding to isolated membranes and that the level of inhibition was predictable from the level of occupation of these binding sites. These results indicate that depolarization increases the affinity of calcium channels for dihydropyridines in vascular smooth muscle and that the time course of the inhibitory effect on contraction is determined by the time course of association with the high-affinity state of the channel.

Characterization in rat aorta of the binding sites responsible for blockade of noradrenaline‐evoked calcium entry by nisoldipine

“…The contractile response to 100 mM KCI was concentration-dependently inhibited by nisoldipine (10 pM-10 nM); 10 nm nisoldipine completely abolished the response (Figure lb). Contractions measured in the presence of nisoldipine were not sustained; the degree of inhibition markedly increased with the duration of depolarization, as already reported by Wibo et al (1988). IC50 values measured 2 and 35 min after initiation of contraction were 374 and 49 pm, respectively ( Table 1).…”

“…The high affinity dissociation constant measured in intact depolarized tissue is close to the KD value found in membrane preparation (Wibo et al, 1988) indicating that prolonged depolarization and tissue homogenization favoured a similar conformation of calcium channels, which is most likely, according to the modulated receptor model, the inactivated form of the calcium channel (see Bean, 1984;Sanguinetti & Kass, 1984). At lower KCl concentrations, the Kapp value was very sensitive to small changes of KCl concentration and the absolute value of the KD for the low affinity binding site could be much higher than that measured in (Wibo et al, 1988). The IC50 value of nisoldipine on KCI-induced contraction measured after a short depolarization was close to the value of Ki obtained in aorta incubated in physiological solution and reflected the binding to a low proportion of high affinity sites in resting tissue.…”

Voltage‐ and time‐dependent inhibitory effects on rat aortic and porcine coronary artery contraction induced by propafenone and quinidine

“…In the present experiments, after prolonged exposure (40 min) to quinidine at normally polarized membrane potentials the degree of inhibition increased as the time of depolarization was prolonged (i.e., time-dependent inhibition), reaching a steady-state within 20 min of depolarization. Similar behaviour has been described previously with some CCBs of the dihydropyridine family (Morel & Godfraind, 1987;Wibo et al, 1988;Godfraind & Salomone, 1991 (Imaizumi et al, 1989). Propafenone also inhibited in a concentration-dependent manner the contractions induced by high KCL.…”

Interaction of pinaverium (a quaternary ammonium compound) with 1,4‐dihydropyridine binding sites in rat ileum smooth muscle

“…The specific binding of [3H]-(+)-isradipine was measured, as described previously (Wibo et al, 1988), by incubating frac-(D Macmillan Press Ltd, 1992 tions with the radioligand for 60 min at 370C in 2.5 ml of a buffered salt solution of the following composition (mM): NaCl 145, KCl 5, MgCl2 1.25, CaCl2 2.5, Tris 20; pH 7.4 at 370C. Non-specific binding was estimated in the presence of 1 AM nifedipine.…”