Interaction of the antiarrhythmic agents SR 33589 and amiodarone with the beta-adrenoceptor and adenylate cyclase in rat heart.

1. The effects of SR 33589 and amiodarone on the cardiac beta-adrenoceptor were studied in vitro and after chronic treatment by means of [125I]-(-)-iodocyanopindolol ([125I]-(-)-CYP) binding and measurement of adenylate cyclase activity. 2. Binding of [125I]-(-)-CYP was inhibited in a dose-dependent manner by SR 33589 (IC50=1.8 +/- 0.4 microM, nH=0.93 +/- 0.06) and amiodarone (IC50=8.7 +/- 2.0 microM, nH=9.2 +/- 0.03). Saturation binding experiments indicated a non-competitive interaction such that SR 33589 (1 and 3 microM) and amiodarone (5 and 10 microM) reduced the Bmax of [125I]-(-)-CYP binding without any effect on the KD. Kinetic studies showed that the rate of association of [125I]-(-)-CYP was unchanged while the rate of dissociation was increased both in the presence of SR 33589 (10 microM) and amiodarone (30 microM).3. Under the same conditions, the receptor stimulated adenylate cyclase activity was inhibited in a dose-dependent, but non-competitive manner, by SR 33589 (isoprenaline-, glucagon- and secretin-stimulated enzyme inhibited 50% at 6.8 +/- 0.6 microM, 31 +/- 10 microM and 12 +/- 3 microM, respectively) while the basal, GTP- and GPP(NH)p-stimulated enzyme was inhibited by 5-10% and the NaF and forskolin-stimulated enzyme by 50% at 500 microM. Amiodarone exhibited a similar pattern of inhibition. 4. After chronic oral treatment (50, 100, 150 mg kg(-1) per day, 14 days), both SR 33589 and amiodarone produced a dose-dependent decrease in Bmax without any effect on KD as determined from [125I]-(-)-CYP saturation experiments and a decrease of the isoprenaline- and glucagon-stimulated adenylate cyclase activity without any effect on basal enzyme activity or activity when stimulated by agents acting directly on regulatory catalytic units. 5. Unlike amiodarone, SR 33589 does not contain iodine substituents. Plasma levels of T3, T4, and rT3 were changed after SR 33589 treatment except a decrease in T4 level at the highest dose whilst the T4 T3 ratio and the level of rT3 were dose-dependently increased by amiodarone treatment. 6. In vitro, SR 33589 and amiodarone were characterized as non-competitive beta-adrenoceptor antagonists. Chronic treatment led to a down-regulation of the beta-adrenoceptor; the down-regulation cannot be attributed to an indirect effect mediated by the thyroid hormones. To reconcile these opposing observations, we propose that SR 33589 and amiodarone interact with the beta-adrenoceptor at a site close to the intracellular loops which are involved in the coupling with Gs and contain the phosphorylable sites.

Characterization of the binding of [3H]SR 33805 to the slow Ca2+ channel in rat heart sarcolemmal membrane.

SR 33805 is a representative of a new class of compounds (indole sulfone) that inhibits L-type Ca2+ channels. [3H]SR 33805 has been shown to bind with a high affinity (Kd approximately 20 pM calculated from saturation isotherms and association/dissociation kinetics) to a single site in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various cations (Na+, Ca2+, Cd2+, and La3+) were shown to inhibit specific [3H]SR 33805 binding, with La3+ being the most potent. Using a range of receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace [3H]SR 33805. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenyl-butylpiperidines were found to inhibit [3H]SR 33805 in a non-competitive manner as demonstrated by displacement experiments in addition to dissociation kinetics. In contrast, the interaction of SR 33805 with fantofarone has been found to be competitive. Binding of [3H]SR 33805 (and [3H]fantofarone) is entropy driven as opposed to that of the [3H]nitrendipine which is enthalpy driven. From these results we suggest that SR 33805 binds with a high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes. This site is equivalent to that of fantofarone and in allosteric interaction with that of the dihydropyridines, phenylalkylamines and benzothiazepines.

SR 33557, a novel calcium-antagonist: interaction with [3H]-(+/-)-nitrendipine and [3H]-(-)-desmethoxy-verapamil binding sites in cerebral membranes.

We investigated the pharmacological properties of SR 33557, a novel compound with calcium-antagonist properties, in both functional tests in vitro and radioligand binding studies. SR 33557 potently antagonized calcium-induced contraction of potassium-depolarized rat aorta in vitro with an IC50 value of 5.6 +/- 0.9 nM, but was a much weaker inhibitor of noradrenaline-induced contraction of the same tissue (IC50 = 96 +/- 22 nM). SR 33557 totally inhibited [3H]-(+/-)-nitrendipine binding to rat brain membranes with a Ki value of 0.19 +/- 0.03 nM. Diltiazem, which used alone increases [3H]-(+/-)-nitrendipine binding, reversed this inhibition indicating that SR 33557 allosterically regulates [3H]-(+/-)-nitrendipine binding. SR 33557 also fully inhibited [3H]-(-)-desmethoxyverapamil binding to cerebral membranes, but inhibition curves were biphasic. IC50 value calculated for that part of the curve which reflects the high affinity binding site of SR 33557 (IC50 = 0.20 +/- 0.02 nM) was very similar to the Ki value determined for inhibition of [3H]-(+/-)-nitrendipine binding. Kinetic evidences indicate that SR 33557 binds to a site which is distinct from the 1,4-dihydropyridine or the phenylalkylamine binding sites associated with the calcium channel. To test the pharmacological specificity of these interactions, the ability of SR 33557 to interact with eight other receptors in cerebral or heart membranes was assessed by binding assays. No high-affinity interaction was observed between SR 33557 and any of the receptors investigated. We conclude that SR 33557 binds specifically and with a high affinity to a site closely associated with the voltage-operated calcium channel in cerebral membranes.

Characterization of the slow calcium channel binding sites for [3H]SR 33557 in rat heart sarcolemmal membranes.

SR 33557 represents a new class of compounds (indolizine sulfone) that inhibit L-type Ca2+ channels. [3H]SR 33557 has been shown to bind with high affinity (Kd congruent to 0.36 nM, calculated from saturation isotherms and association/dissociation kinetics) to a single class of sites in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various divalent cations (Mg2+, Mn2+, Ca2+, Ba2+, and Cd2+) were shown to inhibit specific [3H]SR 33557 binding, with Cd2+ being the most potent. Among several receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace [3H]SR 33557. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylbutylpiperidines were found to inhibit [3H]SR 33557 in a noncompetitive manner as demonstrated by displacement and saturation experiments in addition to dissociation kinetics. From these results, we suggest that SR 33557 binds with high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes.

SR 33557, a novel calcium entry blocker. II. Interactions with 1,4-dihydropyridine, phenylalkylamine and benzothiazepine binding sites in rat heart sarcolemmal membranes.

We have assessed the binding characteristics of a structurally novel calcium entry blocker, SR 33557, to purified rat heart sarcolemma. SR 33557 prevented completely the binding of (+)-[3H]PN200-110, (-)-[3H]D888 and cis-(+)-[3H]diltiazem to their specific binding sites in an apparently competitive manner (nH congruent to 1.0) and with a high affinity (Ki = 0.5-2.0 nM). Equilibrium and kinetic studies suggest that SR 33557 does not act as a simple competitive antagonist at the 1,4-dihydropyridine, the phenylalkylamine or the benzothiazepine-selective sites associated with the L-type calcium channel: 1) inhibition of (-)-[3H]D888 and cis-(+)-[3H]diltiazem binding by SR 33557 resulted in a decrease in maximum binding, 2) cis-(+)-diltiazem and (+)-PN200-110 allosterically increased the inhibition of (+)-[3H]PN200-110 binding and of (-)-[3H]D888 and cis-(+)-[3H]diltiazem binding by SR 33557, respectively and 3) dissociation kinetics of the three radioligands were accelerated by SR 33557. Calcium (in millimolar concentrations) decreased the apparent affinity of SR 33557 for its high-affinity binding sites. This observation was similar to that seen with the phenylalkylamines and cis-(+)-diltiazem, but contrasted from that seen with the 1,4-dihydropyridines. These results indicate that SR 33557 interacts with a high affinity to a novel binding site associated with the L-type calcium channel and has a strong negative allosteric interaction with the well-characterized binding sites for 1,4-dihydropyridines, phenylalkylamines and benzothiazepines.

Allosteric modulation of [3H]nitrendipine binding to cardiac and cerebral cortex membranes by amiodarone.

The possible interaction between the antianginal and antiarrhythmic drug amiodarone and the slow calcium channel was investigated by competition binding experiments in guinea-pig cerebral cortex and rat heart membranes using [3H]nitrendipine as radioligand. Amiodarone displaced specifically bound [3H]nitrendipine from cerebral cortex and cardiac membranes in an apparently competitive manner. In saturation binding experiments, apparent affinity for [3H]nitrendipine progressively decreased with increasing concentrations of amiodarone, whereas maximal binding capacity (Bmax remained unchanged. Both diltiazem and verapamil reversed the inhibitory effect of amiodarone on [3H]nitrendipine binding to cerebral cortex membranes. Together these results suggest that amiodarone exerts a pseudocompetitive inhibition on [3H]nitrendipine binding by acting at a site in allosteric interaction with the 1,4 dihydropyridine binding site associated with the calcium channel. The data are compatible with the existence of a common binding site for diltiazem, verapamil, and amiodarone. These observations are discussed in connection with the pharmacological properties of the drug.

Prevention of calcium overload and down-regulation of calcium channels in rat heart by SR 33557, a novel calcium entry blocker.

The effect of SR 33557, a novel calcium entry blocker, on calcium overload, and regulation of calcium channels and beta-adrenergic receptors was investigated in the rat heart. Calcium overload and infarct-like lesions were produced by a large dose of isoproterenol (40 mg/kg, subcutaneously) to rats. Calcium overload was maximal 8 hours after administration of isoproterenol (control: 5.7 mmol Ca2+/kg dry weight, isoproterenol: 34.9 mmol Ca2+/kg dry weight). At that time, a decrease in the total number of beta-adrenergic receptors (-27%) and calcium channels (-20% and -23%) was observed. Intravenous injection of SR 33557 (0.5-10 mg/kg), 30 minutes before administration of isoproterenol, attenuated the increase in calcium content in a dose-related manner, such that 5 mg/kg SR 33557 reduced calcium overload by 50%. At this dose, SR 33557 had no effect on the number of beta-adrenergic receptors but prevented the decrease in the number of calcium channels. The total number of binding sites and the dissociation constants of each radioligand were estimated from saturation isotherms. The dissociation constants were unchanged when animals given isoproterenol or SR 33557 and isoproterenol were compared to the control group. The results indicate that SR 33557 is able to protect against the calcium overload induced by sympathetic over-stimulation. This over-stimulation of the sympathetic system causes a down-regulation of the number of active beta-adrenergic receptors and calcium channels. The down-regulation of calcium channels is selectively reduced by earlier administration of SR 33557.