Amiodarone. Biochemical aspects and haemodynamic effects.

The mechanisms underlying the non-competitive beta-antagonistic properties of amiodarone were investigated, and the haemodynamic responses to exercise following the administration of oral amiodarone or intravenous propranolol were compared in dogs with a healed myocardial infarction submitted to a graded treadmill exercise. In radioligand binding studies, amiodarone, up to 10 mumol/L did not compete with 125I-iodocyanopindolol for binding to rat heart beta-adrenoceptors. Exposure of cardiac membranes to greater concentrations of amiodarone induced a significant decrease in the number of beta-adrenoceptors without affecting their affinity for 125I-iodocyanopindolol. Similar results were observed ex vivo, in rats after single or multiple dose administration. When added in vitro to rat heart membranes, amiodarone non-competitively inhibited the activation of adenylate cyclase by isoprenaline, glucagon and secretin. Stimulation of adenylate cyclase by those agents which act at more internal sites in the sarcolemmal membrane such as GppNHp, sodium fluoride or forskolin, was much less affected by amiodarone. In dogs performing at a submaximal work level, amiodarone significantly reduced heart rate and tended to increase coronary flow and to reduce left ventricular end-diastolic pressure, but did not affect left ventricular dP/dt. During submaximal exercise, propranolol had similar effects on heart rate, but dramatically reduced myocardial contractility.

Cardiac beta-adrenoceptor modulation by amiodarone.

beta-Antiadrenergic properties are part of the pharmacological characteristics of amiodarone. In the present study, the action of amiodarone on rat-heart beta-adrenoceptors was investigated. [125I]Cyanopindolol (CYP) was used to label beta-adrenoceptors in crude rat-heart microsomes. In competition binding experiments, amiodarone up to 10(-6) M did not displace [125I]CYP from cardiac beta-adrenergic receptors. The effects of amiodarone on the number and affinity for [125I]CYP of beta-adrenoceptors were evaluated in saturation experiments. In vitro exposure of cardiac microsomes to 10(-5) M amiodarone did not modify these parameters. At higher concentrations the beta-receptor number decreased while the affinity for [125I]CYP was not affected. In vivo experiments showed a significant decrease in beta-adrenoceptor density after a single oral dose of 50 mg/kg amiodarone. In chronically treated animals, the same decrease in beta-receptor number was observed 24 hr after the last administration of the drug. 5'-Nucleotidase activity, another specific marker of the plasma membrane, was unaffected by the treatment. These results suggest that part of the beta-adrenergic antagonism of amiodarone is due to a decrease in the beta-adrenoceptor density at the surface of the myocardial cell.

Effect of amiodarone on myosin isoenzymic distribution in rat ventricular myocardium.

Rats were given amiodarone (50 mg X kg-1 X day-1, orally) for 4 weeks and the distribution of ventricular isomyosins, a sensitive index of the effects of thyroid hormones on cardiac tissue, was analyzed. Amiodarone treatment induced a marked increase in both T4 and rT3 and tended to decrease T3 serum levels. At the pharmacologically active dosage we used, the drug induced a moderate redistribution of ventricular isomyosins in favour of V, at the expense of V1. Our results do not support the hypothesis that the major mechanism of action of amiodarone is mediated through hypothyroid-like effects.

Amiodarone is a potent calmodulin antagonist.

The possible interaction between amiodarone, a potent antiarrhythmic and antianginal agent, and calmodulin (CaM) was investigated by three avenues of approach: (a) Effect of amiodarone on cardiac and vascular Ca2+/calmodulin-activated cyclic nucleotide phosphodiesterase (CaM-PDE); (b) Effect on the CaM-activated (Ca2+ + Mg2+)-ATPase from human erythrocytes; (c) Direct interaction between amiodarone and calmodulin measured by the effect of the drug on the fluorescence of 9-anthroylcholine (9AC) bound to calmodulin. Results show that amiodarone did not interact with basal activities of CaM-PDE and other isolated CaM-insensitive PDE forms as well as with (Ca2+ + Mg2+)-ATPase. Amiodarone inhibited calmodulin-activation of aortic CaM-PDE (Ki = 650 nM, substrate cGMP) and calmodulin-activation of erythrocyte ghosts (Ca2+ + Mg2+)-ATPase (IC50 = 4.5 microM) in an apparently competitive manner. Amiodarone decreased the fluorescence of the hydrophobic probe 9AC bound to calmodulin (IC50 = 5 microM). It is concluded that amiodarone is a potent calmodulin antagonist.

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.

Uncoupling between beta-adrenoceptors and adenylate cyclase in dog ischemic myocardium.

We evaluated the effects of ischemic injury on the myocardial adenylate cyclase system, 5 h after ligation of the left anterior descending coronary in 5 anesthetized dogs. Crude cardiac membrane preparations were isolated from control and ischemic areas of ventricular myocardium and tested for: 1. L-(125I)iodocyanopindolol binding, in the absence and presence of +/- -isoprenaline and GTP, and 2. adenylate cyclase activity. The density of beta-adrenoceptors increased by 35% in membranes from ischemic areas while the proportion of receptors in a high affinity state for +/- -isoprenaline decreased from 43% to 20%. Adenylate cyclase activities in the basal state and under stimulation with NaF, forskolin, Gpp(NH)p, +/- -isoprenaline and VIP were all markedly and similarly reduced, being only about 30% of comparable activities in membranes from control areas. The +/- -isoprenaline subsensitivity of cardiac adenylate cyclase can, thus, be attributed to a defective enzymatic system and not to a reduction in the number of beta-adrenoceptors implying that the internal components of the system were more sensitive to acute ischemia than the outward oriented hormone receptors. It is tempting to ascribe this uncoupling to a functional depletion in the guanine nucleotide-binding regulatory protein Ns that might reflect a loss of high energy phosphate stores including GTP.

Haemoglobin messenger RNA in spleen of anaemic rabbits.

Haemoglobin meseenger RNA was isolated from spleen erythroïd cells of anaemic rabbits. The mRNA fraction looks quite homogeneous when analyzed by sucrose gradient centrifugation; it sediments in the 9S region. When added to an ascites cell free system, spleen 9S RNA stimulates the incorporation of radioactive leucine into protein. The synthesized product has been characterized and identified as α and ß chains of rabbit globin.

Protective effects of amiodarone pretreatment on mitochondrial function and high energy phosphates in ischaemic rat heart.

The effects of the antianginal and antiarrhythmic drug amiodarone on mitochondrial function and high-energy phosphate content were assessed during normothermic ischaemic cardiac arrest and reperfusion in Langendorff-perfused rat heart. Total ischaemia for 30 min at 37 degrees C produced highly significant changes in mitochondrial oxidative phosphorylation and high-energy phosphate content. Pretreatment of the rats with one single dose of amiodarone (20 mg/kg i.v., 30 min before killing) markedly attenuated the deleterious effect of ischaemia on mitochondrial function and slightly reduced ATP depletion. In normally perfused hearts, amiodarone pretreatment did not modify any parameter of mitochondrial respiratory function nor did it influence high-energy phosphate or glycogen content. After reperfusion for 15 min, amiodarone-treated hearts showed improved recovery of mitochondrial oxidative phosphorylation and tissue high-energy phosphate content as compared to control hearts. Pretreatment of hearts with amiodarone did not reduce ischaemia-induced leakage of total adenylic nucleotides but highly significantly reduced lactate dehydrogenase release during reperfusion. These results indicate that amiodarone could exert substantial protection on the infarcting myocardium.

Globin messenger RNA from anaemic rabbit spleen. Size of its polyadenylate segment.

The size of the polyadenylate segment of globin messenger RNA isolated from spleens of anaemic rabbits was estimated by comparison of its electrophoretic migration in polyacrylamide gels to that of synthetic poly(A) segments of known lengths. Conditions of enzymic degradation of mRNA with pancreatic ribonuclease and T1 ribonuclease were carefully established in order to ensure complete degradation of the heteropolymeric part of mRNA without affecting the polyadenylate sequence. The poly (A) segments of spleen globin mRNA were found to be 25-90 nucleotides long whilst those of peripheral blood reticulocytes from the same animals were only 10-30 residues long. Since spleen contains young erythroid cells and since anucleated blood reticulocytes constitute a statistically older population of the same cell line, these results support the idea that the poly(A) segment of mRNA shortens when the message ages.

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.

Molecular modifications associated with Aging of globin messenger RNA in vitro.

Using polyacrylamide gel elution-electrophoresis in aqueous medium, highly purified rabbit globin mRNA can be fractionated into several populations of molecules differing by their mean poly(A) content. Both alpha and beta globin mRNA are heterogenous with respect to their electrophoretic mobilities. With the conditions used no separation of alpha and beta globin mRNA occurs during electrophoresis. From the specific radioactivity distribution in the different mRNA fractions one can conclude that the polyadenylate sequence at the 3' end of globin mRNA molecules becomes shorter with aging. This shortening occurs on alpha as well as beta, globin mRNAs and the extent of heterogeneity in poly(A) content is similar for both globin mRNAs. Furthermore, using two different methods of mRNA fractionation (polyacrylamide gel elution-electrophoresis and elution of poly (U)-Sepharose-bound mRNA at increasing temperatures) it is shown that old mRNA molecules differ from relatively young messages in their ability to direct cell-free globin synthesis. Modifications reducing template activity in vitro thus seem to take place during globin mRNA aging.

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.