Amiodarone: biochemical evidence for binding to a receptor for class I drugs associated with the rat cardiac sodium channel.

Amiodarone has multiple pharmacological effects in heart. Electrophysiological data suggest that among its other effects, amiodarone is a sodium channel blocker. Using a radioligand assay, we determined whether amiodarone interacted with a previously described receptor for type I agents associated with the cardiac sodium channel. The radioligand was [3H]batrachotoxinin A 20 alpha-benzoate ([ 3H]BTXB), a toxin that binds to the activated state of the sodium channel. We have previously shown that class I antiarrhythmic drugs inhibit [3H]BTXB binding. The purpose of this study was to assess whether amiodarone and other class III agents interact with this receptor. Amiodarone inhibited [3H]BTXB binding in a dose-dependent fashion, with an estimated IC50 value of 3.6 microM. This IC50 value is similar to reported clinically effective serum concentrations of amiodarone. In contrast to amiodarone, the IC50 values for other class III drugs (bretylium, sotalol, bethanidine, N-acetylprocainamide) were much higher than their therapeutic concentrations and bore no relation to them. Scatchard analysis of [3H]BTXB binding showed that amiodarone reduced the maximal binding for [3H]BTXB; this finding indicates irreversible inhibition or (more likely) allosteric inhibition by amiodarone. The latter agrees with electrophysiological data suggesting that amiodarone binds to inactivated sodium channels. Sodium channel blockade by amiodarone may contribute to its overall electrophysiological effect.

Mexiletine-quinidine in isolated hearts: an interaction involving the sodium channel.

Combination therapy with mexiletine and quinidine has been shown to be more effective than either monotherapy in treating patients with ventricular tachycardia. This enhanced efficacy was associated with prolongation of ventricular refractoriness and conduction time in the infarct zone. As sodium channel activity is a determinant of both conduction time and refractoriness we formed the hypothesis that the mexiletine-quinidine interaction was due at least in part to interactions involving the sodium channel. To assess the role of sodium channel blockade in the enhanced anti-arrhythmic activity of mexiletine-quinidine combination we determined whether the electrophysiological and anti-arrhythmic effects of tetrodotoxin combined with mexiletine or quinidine mimicked the effect seen with mexiletine combined with quinidine. Eighty isolated perfused rabbit hearts were treated with mexiletine, quinidine and tetrodotoxin either alone or in combination before and after circumflex occlusion-reperfusion. Ventricular fibrillation occurred in response to single extrastimuli in all 24 hearts treated with a saline control infusion. Combinations of mexiletine and quinidine at concentrations which alone had little electrophysiological activity produced anti-arrhythmic activity greater than that seen with high concentrations of mexiletine or quinidine alone. The combination of similarly low concentrations of tetrodotoxin and quinidine also produced enhanced anti-arrhythmic efficacy and enhanced prolongation of ventricular refractoriness and conduction which mimicked the effect of mexiletine and quinidine in combination. In contrast, the combination of mexiletine and tetrodotoxin did not produce enhanced anti-arrhythmic and electrophysiological activity. Since tetrodotoxin is a highly specific sodium channel blocker, these data suggest that the enhanced antiarrhythmic activity of mexiletine-quinidine combination therapy involves, at least in part, blockade of the cardiac sodium channel.

Tetrodotoxin: sodium channel specific anti-arrhythmic activity.

Many Class I anti-arrhythmic drugs not only block the cardiac sodium channel but also block the calcium and/or potassium channels. The hypothesis tested in this study was that sodium channel blockade without blockade of calcium or potassium channels produced anti-arrhythmic activity in the treatment of malignant ventricular arrhythmias. The arrhythmia model consists of ventricular fibrillation induced by critically timed single extrastimuli at twice diastolic pacing threshold following 15 minutes of ischaemic injury in a rabbit heart perfused in vitro. Preparations were randomly assigned to either tetrodotoxin (a selective sodium channel blocking toxin) or vehicle. Ventricular fibrillation occurred in all vehicle treated preparations in response to single extrastimuli following ischaemic injury. Treatment with tetrodotoxin at concentrations of 0.1 to 1.0 micromolar protected some hearts from fibrillation, while at concentrations above 3 micromolar ventricular fibrillation was not inducible. Tetrodotoxin produced concentration dependent increases in ventricular effective refractory period and conduction time in the infarct zone which were associated with anti-arrhythmic activity. No concentration dependent change in action potential duration was seen with tetrodotoxin. Thus the electrophysiological and anti-arrhythmic activities of tetrodotoxin in this model demonstrate that the property of selective sodium channel blockade is sufficient to produce anti-arrhythmic activity.

Stereospecific interaction of tocainide with the cardiac sodium channel.

The antiarrhythmic action of type I antiarrhythmic drugs may be mediated via binding of the drugs to a receptor associated with the cardiac sodium channel. This suggested that the effects of type I drugs might be stereospecific. We measured the effect of the tocainide stereoisomers (which have stereospecific antiarrhythmic effects) on conduction time and on radioligand binding to the cardiac sodium channel. The concentration-dependent effects of the individual enantiomers of tocainide on interventricular conduction time measured during constant rate ventricular pacing at 350 msec were assessed in 47 isolated perfused rabbit heart preparations. Significant increases (p less than 0.05) in conduction time occurred for both R-(-)-tocainide (75 microM, 10 +/- 5 msec) and S-(+)-tocainide (150 microM, 4 +/- 1 msec). R-(-)-Tocainide was more potent than the S-(+)-tocainide in prolonging conduction time (p less than 0.05). This stereospecific prolongation of conduction time suggested a stereospecific interaction with the sodium channel. The affinities of the enantiomers for the channel were measured with a radioligand binding assay using [3H]batrachotoxinin benzoate and freshly isolated cardiac myocytes. Both enantiomers inhibited [3H]batrachotoxin benzoate binding, but the IC50 (+/- SD) values were different: R-(-)-tocainide 184 +/- 8 microM; S-(+)-tocainide, 546 +/- 37 microM (p less than 0.003). Tocainide isomers are stereospecific in terms of prolonging conduction time and in binding to the sodium channel. The stereospecific electrophysiologic effects of tocainide may result from binding to a receptor associated with the cardiac sodium channel.

A receptor for type I antiarrhythmic drugs associated with rat cardiac sodium channels.

We assessed the effects of type I antiarrhythmic drugs on the binding of ligands to receptors on voltage-sensitive sodium channels of rat cardiac myocytes. The radioligand was [3H]batrachotoxinin A 20 alpha-benzoate ([3H]BTXB), a toxin that binds to the sodium channel. The 8 drugs tested inhibited [3H]BTXB binding in a dose-dependent fashion with IC50 values from 1.34 microM for O-demethylencainide to 811 microM for procainamide. A log-log plot of IC50 versus mean therapeutic serum concentration yielded a regression line with slope of 1.17 and r of 0.95. Scatchard analysis of [3H]BTXB binding showed that lidocaine reduced the maximal binding without altering the KD for [3H]BTXB binding, indicating allosteric inhibition. The inhibition by lidocaine of [3H]BTXB binding was reversible within 30 minutes when the samples were diluted from 390 to 39 microM lidocaine. In other studies, the stereoisomers of tocainide were shown to have a threefold to fourfold difference in IC50 for inhibition of [3H]BTXB binding. The binding of antiarrhythmic drugs to this site is saturable, reversible, and stereospecific and occurs at pharmacologically relevant concentrations with similar rank order of potency in vivo and in vitro. This suggests that binding at this site relates to pharmacologic activity.

Binding of [3H]batrachotoxinin A benzoate to specific sites on rat cardiac sodium channels.

Radiolabeled neurotoxins have been used to study the structure and function of sodium channels. We studied the binding of [3H] batrachotoxinin A 20 alpha-benzoate [( 3H]BTX-B) to specific sites on sodium channels on rat cardiac myocytes. Calcium-tolerant myocytes were prepared by collagenase dispersion of adult rat hearts and were 75-83% viable. As with the nerve channel, specific binding of [3H]BTX-B to its receptor site was seen only in the presence of sea anemone toxin (ATX). The affinity of ATX for its binding sites may be estimated from its concentration-dependent stimulatory effect on [3H]BTX-B binding. These results suggest that, in the presence of 5.4 mM KCl, the myocytes have two affinities for ATX with estimated dissociation constants of 0.52 microM and 12.9 microM. Depolarization of the myocytes with either 65 mM KCl or 0.1 mM BaCl2 results in the loss of the 0.52 microM component, suggesting that it is voltage sensitive. The 0.52 microM and 12.9 microM components have maximal binding capacities corresponding to 4 and 11 sites/micron 2 of myocyte surface area, respectively. Scatchard analysis of [3H]BTX-B binding in the presence of ATX demonstrates a single class of sites with a KD of 25-35 nM. These results demonstrate that [3H]BTX-B can be used as a radioligand probe of the adult rat sodium channel and will facilitate a biochemical approach to the study of the interaction between antiarrhythmic drugs and the sodium channel.

The effects of commercial coaching or the NBME Part I Examination.

A total of 55 second-year medical students from the University of Alabama in Birmingham utilized the services of a commercial test-coaching company to assist them in preparations for the June 1977, 1978, and 1979 National Board of Medical Examiners Part I examination. These students scored significantly higher on the examination than students with comparable basic science grade-point averages. Students were surveyed to determine the nature of the course and to identify the salient points which they believed contributed to higher Part I scores. The study raises questions concerning the impact of commercial coaching on the licensure process for both domestic students and students of foreign medical schools entering the U.S. medical profession.

Recent experience with tetanus in Alabama.

In recent years, all cases of tetanus reported in the United States have occurred either in unimmunized persons, partially immunized persons, or persons whose immunization history was uncertain. We present our experience with three unimmunized individuals who acquired this disease. One patient became ill after a "clean, minor wound." Present guidelines do not encourage immediate protection with human tetanus immune globulin (TIG) for unimmunized patients with this type of injury. We believe serious consideration should be given to modification of current recommendations to include the more liberal use of TIG, especially in nonimmune persons.