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Home , Bretylium, Lidocaine, Procainamide

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 2223-2227, April 1986 Medical Sciences blockade: A mechanism for suppressing (ion channels////spontaneous ) MARVIN B. BACANER*, JOHN R. CLAYt, ALVIN SHRIERt, AND RICHARD M. BROCHUt *Department of Physiology, University of Minnesota, Minneapolis, MN 55455; tLaboratory of Biophysics, Intramural Research Program, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health at the Marine Biological Laboratory, Woods Hole, MA 02543; and tDepartment of Physiology, McGill University, Montreal, Quebec H3G 1Y6 Canada Communicated by James Serrin, December 2, 1985

ABSTRACT The suppression of ventricular fibrillation by negative) to -50, -40, -30, ... +30 mV with a rest interval antidysrhythmic drugs is well correlated with their ability to of 5 sec between each step. Drugs tested were bretylium, block potassium channels in nerve and cardiac membranes. bethanidine, , , meobentine, and Blockade of potassium channels reduces electrical inhomoge- . neities in and conduction parameters that lead Chicken Heart Cells. Voltage-clamp measurements were to ventricular fibrillation. These actions tend to effectively performed on embryonic chicken heart cell aggregates before decrease the electrical size of the heart, which suggests a and after addition of bretylium to the external medium. mechanism for antifibrillatory drug action. The sites Chicken hearts were dissected from 7- to 12-day-old embryos for antifibrillatory drug action (IK blockade) appear to be on and dissociated into their component cells with trypsin the outside of the cardiac membrane whereas receptors for (0.05%). An innoculum of cells was placed into a flask antiarrhythmic drug action (INa blockade) appear to be on the containing tissue culture medium, which had been gassed inside of the cardiac membrane. with an atmosphere of 5% C02, 10% 02, and 85% N2. The flask was placed on a gyratory shaker for 48 to 72 hr. The Ventricular fibrillation, the major cause of sudden cardiac temperature was maintained constant at 370C. Aggregation of death, has long been regarded as an extension of less lethal cells into spherical clusters occurs during this process. The ventricular arrhythmias that could be prevented by class 1 clusters were transferred to a plastic culture dish to which antiarrhythmic drugs (1). However, almost every controlled they adhered within 1 hr. Mineral oil was layered over the study has failed to show a reduction in the incidence of medium to prevent evaporation and maintain an unclouded ventricular fibrillation by prophylactic administration of view of the cells. Temperature was maintained at 370C, and lidocaine even when arrhythmias per se were suppressed pH was maintained at 7.3. Membrane currents were mea- (2-11). In contrast, ventricular fibrillation can be suppressed sured with a standard two-microelectrode voltage-clamp by bretylium tosylate and its pharmacologic analog, technique (43). Electrode resistance was typically 50 MQ. bethanidine sulphate (2, 12-31). Both drugs (termed class 3) (1 ,4M) was used to block the fast sodium increase ventricular fibrillation threshold manyfold, facilitate current component (INa). External potassium ion concentra- electrical defibrillation, and induce episodes of pharmaco- tion was 1.3 mM. After control measurements were made, logic (spontaneous) defibrillation under conditions where it bretylium was added to the tissue culture dish at a final does not normally occur (refs. 2, 12, 21-31 and Fig. la). Class concentration of 1 mM. 1 antiarrhythmic drugs such as lidocaine and procainamide do Dog Heart. Electrical ventricular fibrillation threshold not have comparable antifibrillatory effects (12, 30, 31). (VFT) was measured in the hearts of 68 open-chested dogs These drugs also differ from bretylium and bethanidine in before and after intravenous drug infusion in each animal. A their effects on parameters (33-37), single constant current 10 msec in duration was applied which further indicates that antifibrillatory and antiar- during the vulnerable period of the cardiac cycle by means of rhythmic drug actions are not directly related. This report fishhook electrodes embedded within the ventricle. The demonstrates that these differing drug actions are correlated lowest current amplitude that induced sustained fibrillation with differences in the blockade of membrane ionic currents. lasting 30 sec, or longer, was taken as the VFT (12, 23, 29). Preliminary reports of these experiments have been pub- Drugs used were bretylium, bethanidine, lidocaine, pro- lished (38, 39, 44). cainamide, meobentine, guanethidine, , tetramethylammonium, 4-aminopyridine, and cesium. The METHODS doses are given in Fig. 1 and Table 1. Experiments were performed on internally perfused squid RESULTS giant axons, chicken embryonic heart cell aggregates, and anesthesized mongrel dog hearts by using standard tech- In the dog heart bretylium and bethanidine each caused a niques for each preparation (12, 40-43). significant sustained increase in VFT. In contrast, lidocaine Squid Axon. Membrane currents were measured by axial typically induced a small transient increase in VFT and wire voltage-clamp technique in squid axon before and after procainamide had virtually no effect (Fig. la). addition of test drugs to either the internal or the external Squid Axon. The effects of these drugs on voltage-clamped bathing medium. The voltage-clamp protocol consisted of squid axons are shown in Fig. 2. In each case depolarizing serial depolarizations of membrane potential lasting several voltage steps elicited an inward sodium current ('Na) and an milliseconds from a holding potential of -80 mV (inside outward potassium current (IK), similar to the original results of Hodgkin and Huxley (45). Bretylium, bethanidine, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: I, current; VFT, ventricular fibrillation threshold; in accordance with 18 U.S.C. §1734 solely to indicate this fact. APD, action potential duration. 2223 2224 Medical Sciences: Bacaner et al. Proc. Natl. Acad. Sci. USA 83 (1986) a Table 1. Drug effects on membrane currents and on fibrillation threshold Block of IK in squid Increase of axons, %* VFT in infarcted Drug a b c dog hearts, %t E Bretylium 87 88 89 504 (18) Bethanidine 70 75 72 327 (12) U- Meobentine 68 64 70 278 (3)t Lidocaine 51 49 47 53 (12) Procainamide 41 36 33 17 (7) Guanethidine 32 40 46 14 (6)t *Drug concentration in the internal perfusate equal to 5 mM. Data taken from a total of 3 axons (a, b, and c) for each drug. tAverage percentage increase. Parentheses, number of dogs tested. Time, min Drug concentration equal to 20 mg/kg (therapeutic dose) for bretylium, bethanidine, and meobentine. The doses for the other b TMA 4-AP drugs are equivalent to the maximum doses used clinically 100 (lidocaine, 7 mg/kg; procainamide, 9.4 mg/kg; and guanethidine, 15 mg/kg). SNormal hearts without coronary ligation. 50 10 mg/kg

10 mg/kg effect (34). Our results with guanethidine and meobentine 1 argue against this view. Guanethidine, which has the same E 0 antiadrenergic effect as bretylium, has little antifibrillatory effect (12, 53). Furthermore, meobentine, the 0-methyl analog of bethanidine, has no antiadrenergic action but it increases VFT significantly (ref. 54 and Table 1) and also induces pharmacologic defibrillation (unpublished observa- tion, M.B.B.). The results with both drugs are well correlated 7,.5 mg/kg with their effects on IK in squid axons (Table 1). These 10 mg/kg experiments further indicate that blockade of IK underlies 100 0 50 100 antifibrillatory drug action. Cardiac Cells. To test our prediction that bretylium would Time, min also block IK in cardiac cells, voltage-clamp studies on FIG. 1. (a) Representative effect of antidysrhythmic drugs on chicken embryonic heart cell aggregates were carried out. VFT in dog heart. Control threshold was determined for each animal When bretylium (1 mM) was placed in the external medium, at zero time. Drugs were administered intravenously shortly there- outward current was significantly reduced as shown by the after. Drug dosages based on the upper limits that are used clinically reduction in IK at the end of the voltage steps (Fig. 4). In were as follows: procainamide, 9.4 mg/kg; lidocaine, 7 mg/kg; seven preparations bretylium consistently blocked both a bretylium, 20 mg/kg; bethanidine, 20 mg/kg. Additional drug dos- time-dependent outward current and a time-independent ages were administered at approximately the 3-hr mark in the (background) component. Blockade of the time-dependent bretylium and bethanidine experiments. (b) Representative effect of outward current is especially evident by the standard potassium channel blockers on fibrillation threshold in dog reduction of the heart by using the same experimental protocol as in a. Note that tail current amplitude (Fig. 4, Inset) upon return to the tetraethylammonium (TEA), tetramethylammonium (TMA), 4- holding potential (-55 mV). Blockade of the background aminopyridine (4-AP), and cesium (Cs) increased fibrillation thresh- component is apparent in the reduction of the initial current old to 100 mA, the maximum output of our current generator. jump immediately following the voltage step (Fig. 4, Inset). The time-dependent component has been demonstrated to be lidocaine, and procainamide partially blocked both the INa a potassium ion-selective current (A.S. and J.R.C., unpub- and IK current components when the drugs were added to the lished observations). The time-independent, or background internal perfusing solution (Fig. 2, Table 1). The effects of component, is also carried at least partly by potassium ions these drugs on IK are further illustrated in Fig. 3 along with (A.S. and J.R.C., unpublished observations). the effects of tetraethylammonium, which is a standard Site of Action. Our data provide some insight into which potassium . Tetrodotoxin was added to the side of the membrane the receptors are located for drugs that external solution in these experiments to block the INa block sodium and potassium channels. Lidocaine and component. These results demonstrate that equimolar con- procainamide partially blocked INa and IK in squid axons centrations (5 mM) of bretylium and bethanidine produce a when the drugs were added to either the internal or external significantly greater blockade of IK than either lidocaine or solution (Figs. 2 and 5). Bretylium and bethanidine (Fig. 5) procainamide. had no effects on either current component when they were Fibrillation Threshold. To further relate our observations in added to the external solution in contrast to their strong squid axon to dog heart, we tested the effects oftetraethylam- effects when they were added to the internal solution (Figs. monium (3 dogs), 4-aminopyridine (3 dogs), cesium (2 dogs), 2 and 3). These results indicate that the receptor sites for drug and tetramethylammonium (2 dogs) on fibrillation threshold. blockade ofIK and INa in squid axons are on the internal side These diverse chemical agents, all of which are known of the axonal membrane. The differing actions of external blockers of potassium current in nerve and cardiac mem- lidocaine and bretylium can be attributed to differing lipid branes (46-52), significantly increased VFT in dog hearts solubilities of these agents. Lidocaine is able to cross the (Fig. lb). squid axon membrane to reach the sites for blockade of 'K Adrenergic Blockade. Bretylium inhibits transmitter re- and INa in contrast to bretylium, a quaternary ammonium lease from sympathetic , which may suggest that compound, with poor lipid solubility. The lack of effect of adrenergic blockade is the mechanism for its antifibrillatory external bretylium on INa in squid axons is consistent with its Medical Sciences: Bacaner et al. Proc. Natl. Acad. Sci. USA 83 (1986) 2225 Antiarrhythmic Antifibril latory

Lidocaine Procainamide Bretyl ium Tosylate Bethonidine

cm3 BR CM3C CH2NHC =NCH3 Q NHCOCH2N IC2H512 H2N ec~- CONRCH2CH2N ICH2CH3J12 D CH2N-C245H3C -SO3 NHCH3 Cl3 CM3

% Control I

I

2 mA.cm2

Test s (5 mM) 5c-- 5 msec

FIG. 2. Effects of antidysrhythmic drugs on membrane currents in internally perfused squid giant axons. The control results immediately below the chemical structure of each drug are superimposed membrane current responses to voltage clamp depolarizations 7 msec in duration to -50, -40, ... +30 mV with a 5-sec rest interval between each step. The downward deflections show INa (inward); the upward deflections show 'K (outward). These results were corrected for leakage and capacitance currents. Holding potential was -80 mV (inside negative). Temperature was 7°C. Membrane-current responses for each axon with the same voltage-clamp protocol following addition ofthe corresponding drug to the internal perfusate at a concentration of 5 mM are shown below the control result. failure to decrease upstroke velocity (Vmax) of the normal antiarrhythmic drug action (INa blockade) is on the internal cardiac action potential (33-35), which suggests that side of the cardiac membrane, which bretylium does not bretylium is also unable to cross the cardiac membrane. The reach. In contrast, the primary site for antifibrillatory drug lack of effect of external bretylium and bethanidine on IK in action (IK blockade) appears to be on the external side of the squid axons is attributable to the lack of an external receptor cardiac membrane, although effects on IK by drugs that cross for most potassium channel blockers in this preparation, in the cardiac membrane to internal receptors are not excluded. contrast to other nerve and cardiac preparations where it is present (46, 47). Our results on chicken heart cell aggregates DISCUSSION (Fig. 4) indicate that bretylium is, in fact, an effective blocker Ischemic injury results in asynchronous excitability states of IK components in cardiac membrane when the drug is between normal and injured cardiac cells causing decreased added to the external solution. Failure ofbretylium to reduce conduction velocity, local conduction blocks, conduction Vmax, in cardiac cells suggests that the site for primary over aberrant pathways, and decreased space constant (2,

Control 8r mA-cm-2 Procainamide -6 0

Lidocaine -4

Bethanidine -2 Bretylium

Ia, I I ITEA I -40, , 20 40 60 80 100 -20, mV

Aa.

FIG. 3. Drug effects on potassium channel current-voltage relations in internally perfused squid axon. Tetrodotoxin (1 AM) was added to the external medium to block INa and the external potassium ion concentration was elevated to 300 mM (equal to inside). The membrane potential was clamped to 0 mV to activate potassium channels followed by a second step to -60, -40, ... + 100 mV (Inset). Holding potential was -80 mV. The membrane current was measured 100 ,sec following the second step. Drug concentration in the internal perfusate was 5 mM, except for tetraethylammonium for which 10 mM was used. 2226 Medical Sciences: Bacaner et al. Proc. Natl. Acad Sci. USA 83 (1986) action potential inhomogeneities. Bretylium has been shown a b to decrease such inhomogeneities in conduction times be- * tween normal and ischemic myocardium (58). Thus, the large heart or the ischemically injured h~art may take on some characteristics of the small heart following administration of . bretylium, bethanidine, or meobentine, as indicated by epi- . sodes of pharmacologic defibrillation and an increase in VFT . to supernormal levels. The blockade of IK explains the most consistent elec- trophysiologic action of bretylium, which is to increase the action potential duration (APD) (33-35). Bethanidine and meobentine have also been reported to have similar effects on -50 i APD in rat heart (37). Moreover, clofilium, another class 3 -60 0 - -40 -30 -20 -10 antifibrillatory drug, has been reported to block IK in guinea mV pig heart cells (60). Blockade of IK could exert an important antifibrillatory action by blocking an increase in potassium FIG. 4. Representative voltage-clamp study of chicken heart cell conductance (61) with shortening of APD that often imme- aggregates. The control current-voltage relation inwardly rectified at diately precedes the onset offibrillation (61, 62). In ischemic V > -30 mV. Bretylium in the external medium significantly reduced or infarcted myocardium, bretylium prolongs APD in both outward current at potentials positive to -40 mV with a region of the uninvolved normal cells and in injured cells with abnor- negative slope conductance positive to V = -30 mV. (Inset) Membrane currents in response to 5-sec duration voltage-clamp mally shortened APD so that duration is prolonged about steps in control (a) and following addition ofbretylium to the external equally in both groups (63). Since bretylium has little effect medium (b). Holding potential was -55 mV. Step potential was -25 on APD in ischemic injured cells where it is already abnor- mV in (a) and -22 mV in (b). Time-dependent and time-independent mally prolonged, the net effect is a reduction in the temporal (background) currents were elicited by the voltage step. The time- and spatial disparity of APDs between normal and injured dependent component is most accurately represented by the ampli- cells throughout the heart (33). Increased homogeneity of tude of tail current following return to the holding potential. The both APD and conduction parameters (58) would result in time-independent component is most accurately represented by the more synchronous and uniform coupling of excitation and instantaneous current jump immediately following the voltage step. contraction. These actions tend to effectively reduce the Both currents were reduced by bretylium. Similar results were observed in seven preparations. Control, solid squares; 1 mM electrical size of the heart, which could explain the increased bretylium, open diamonds. resistance to initiating and sustaining fibrillation induced by antifibrillatory drugs. Our results suggest that one important mechanism underlying this effect is the blockade of one or 55-59). These effects can cause ventricular fibrillation when more potassium ion currents. the disruption ofnormal ion transport causes action potential and conduction inhomogeneities that critically desynchron- This work was supported in part by a grant from the Medical ize normal excitation and contraction coupling over a suffi- Research Council of Canada to A.S. Bretylium was donated by cient mass of myocardium (2). American Critical Care (Waukegan, IL). It been known that sustained fibrillation is related has long 1. Lown, B., Fakhro, A. M. & Hood, W. B. (1967) J. Am. Med. to the absolute size ofthe heart (32). For example, ventricular Assoc. 199, 188-198. fibrillation usually terminates spontaneously in the hearts of 2. Bacaner, M. B. (1983) Int. J. Cardiol. 4, 133-152. small animals (cat, rat, and rabbit). In large hearts (dog, beef, 3. Editorial (1975) Br. Med. J. 1, 473-474. and man) ventricular fibrillation persists until death unless 4. Pentecost, B. L., De Giovanni, J. V., Lamb, P., Cadigan, terminated by electrical countershock, because the large P. J., Evemy, K. L. & Flint, E. J. (1981) Br. Heart J. 45, and conduction make spontaneous 42-47. number ofcells long paths 5. Editorial (1979) Lancet i, 193-194. electrical and contractile resynchronization improbable. 6. O'Brian, K. P., Taylor, P. M. & Croyson, R. S. (1973) Med. J. Similarly, the ischemic or infarcted myocardium can become Aust. Special Suppl. 2, 36-37. effectively "enlarged" electrophysiologically because of ab- 7. Chopra, M. P., Thudani, U., Protal, R. W. & Aber, C. P. normal and aberrant conduction characteristics caused by (1971) Br. Med. J. 3, 668-670. 8. Bennet, M. A., Wilner, J. M. & Pentecost, B. L. (1970) Lan- cet i, 909-911. External External 9. Lie, K. I., Loen, L. L., Louridtz, W. J., Janse, M. J., Wil- Bethanidine Lidocoine lebrands, A. F. & Durrer, D. (1978) Am. J. Cardiol. 42, / 486-488. 10. Darby, S., Bennett, M. A. & Cruickshank, J. C. (1972) Lancet Control ;, 817-819. 11. Julian, D. G. (1980) R. Coll. Physicians (London) 14, 17-21. 12. Bacaner, M. B. (1968) Am. J. Cardiol. 21, 504-512.

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