Basic Research in
Cardiology
Basic Res Cardio186:355-362 (1991 )
The effect of selected antiarrhythmic drugs on neutrophil free oxygen radicals production measured by chemiluminescence T. Siminiak 1, H. Wysocki 1, A. Veit 2, and H. R. Maurer 2 1 Academy of M e d i d n e , Institute of Internal Medicine, Department of Intensive Therapy Poznafi, Poland (Head: Prof. Dr. H. Wysocki), and 2 Free University of Berlin, Institute of Pharmacy, Biochemical Department, Berlin, F R G (Head: Prof. Dr, R. H. Maurer)
Summary: The evidence that free oxygen radicals produced by polymorphonuclear neutrophils (PMN) participate in generation of reperfusion arrhythmias is well documented. The in vitro effect of selected antiarrhythmic drugs on PMN free radicals production was evaluated by luminol- (LuCL) and lucigenin- (LgCL) amplified chemiluminescence stimulated with opsonized zymosan (o.z.) and phorbol myristate acetate (PMA). Fast sodium channel inhibitors varied in the influence on PMN chemiluminescence: from an inhibition in all models studied by procainamide, to lack of an effect by mexiletine. Propafenone, similarly to ajmaline and verapamil, inhibited LuCL stimulated with PMA, as well as LgCL after stimulation with both PMA and o.z. Bretylium tosylate decreased LuCL stimulated with both inducers, with no effect on LgCL. Amiodarone in high concentrations inhibited both LuCL and LgCL. [3-blockers propranolol and practolol impaired LuCL stimulated with o.z., as well as LgCL induced with PMA, whereas a-blocker phentolamine inhibited LuCL and LgCL stimulated with both inducers. The drugs' effect on PMN free oxygen radicals production may constitute an additional mechanism of their activity. Key words: Reperfusion Larrhythmias;_freeoxygen radicals; chemiluminescence; antiarrhythmic drugs Introduction
Increasing evidence obtained in recent years indicates the concept that, in certain clinical situations like ischemia and repeffusion, free oxygen radicals are involved in generation of arrhythmias. Reactive oxygen species such as the superoxide anion (O~-), hydroxyl radical ( O H ' ) , and hydrogen peroxide (H202) affect myocardium calcium ion transfer (11) and membrane lipid metabolism (20, 24) and, subsequently, result in electrophysiological disturbances in myocyte membranes and generation of reperfusion arrhythmias. Various sources of enhanced free radical production have been studied and results prove participation of myocyte mitochondria (35), activated platelets (19), endothelium, xanthine oxidase (18), and catecholamines (30). However, it is well accepted that the most effective source of free oxygen radicals in ischemic myocardium is activated polymorphonuclear neutrophils (PMN). Perfusion of ischemic/reperfused myocardial tissue with neutrophildepleted blood resulted in limitation of myocardial injury and markedly reduced the incidence of reperfusion alThythmias (9). Similar results were obtained after pharmacological interventions that decreased PMN accumulation in ischemic myocardium (10). We have recently evaluated the effect of selected antiarrhythmic drugs on PMN superoxide anion production in vitro as measured by cytochrome C reduction in a whole blood assay (38). However, large amounts of superoxide dismutase contained in erythrocytes might affect the final results. The goal of the present study was to evaluate the in vitro effect 681
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of selected antiarrhythmic drugs on production of reactive oxygen species by isolated neutrophils as m e a s u r e d by luminol- (LuCL) and lucigenin (LgCL) d e p e n d e n t chemiluminescence. Materials and methods To obtain polymorplaonuclear neutrophils, heparinized blood from healthy laboratory personnel was separated by one-step centrifugation procedure using Gradisol-G medium (Polfa). Residual erythrocytes were removed by hypotonic lysis. Then PMN were washed twice in PBS and resuspended in the same buffer. Cells were then incubated 15 min at 37 °C with sMine or investigated drug solution in saline in final concentrations calculaled from minimal (Cmln) and maximal (Cmax) concentrations achieved in blood after therapeutical doses as follows: 0.5 Cmln, (Cmin + Cmax)/2, 2Cmax- Following final drug concentrations were used: ajmaline (Gilurytmal, Giulini) 2, 7, and 20 rag/l; amiodarone (Cordarone, Krka) 0.5, 2, and 6 mg/1; bretylium tosylate (Bretylate, Wellcome) 0.3. 1, and 2.8 mg]l; disopiramide (Rythmodan, Russel) 1.5, 4.5, and 12 rag/l; lidocaine (Lignocainum, Polfa) 0.7, 3.25, and t0 rag/l; mexiletine (Mexitil, Boehringer Ingelheim) 0.5, 1.5, and 4 rag/l; phentolamine (Regitin, Ciba) 0.5, 1.5, and 4 rag/l; practolol (Eraldin, ICI) 0.25, 0.75, and 2 rag/l; procainamide (Procainamidum, Potfa) 2, 7, and 20 mg/1; propafenone (Rytmonorm, Knoll) 2, 7, and 20 rag/l; propranolol (Polfa) 25, 75, and 200 ~g/l; verapamil (Isoptin, Knoll) 20, 55, and 70 mg/l. To opsonize the zymosan particles, zymosan A (Sigma) was boiled for 30 rain, washed twice, and resuspended to 15 mg/ml in saline and then incubated for 20 min at 37 °C with 20 % fresh pooled human serum. Then opsonized zymosan was washed twice in PBS and resuspended 5 mg/ml in HBSS.
Table 1. The effect of selected antiarrhythmic drugs on luminol-dependent chemiluminescence of neutrophils stimulated with opsonized zymosan (percent values of control samples ± SD).
Drugs
Percent values of chemiluminescence Low Medium concentration concentration
High concentration
Class I a: Ajmaline Disopiramide Procainamide
100.3 + 6.7 88.8 + 5.3 96.2 + 5.5
99.4 +±16.2 89.9 ± 12.0 83.4 ± 2.4
95.9 ± 14.2 85.9 ± 7.4 71.2 _+4.2
Class Ib: Lidocaine Mexyletine
101.3 ± 13.2 97.3 ± 4.7
98.6 _+4.1 98.8 -+ 8.1
98.9 + 13.8 96.4 _+5.1
Class I c: Propafenone
92.6 _+6.4
96.2 ± 8.1
98.1 + 6.1
Class Ih Practolol Propranolol
88.9 -+ 5.5 80.3 _+5.1
86.1 ± 5.7 82.5 ± 8,1
87.2 +_11.0 80.1 ± 7.0
107,4 + 3.2 86.3 ± 5.1
104.3 ± 8.0 81..4 ± 5.9
31.4 +_20.4 73.2 ± 7.1
Class IIh Amiodarone Bretytium tosyl Class IV: Verapamil
98.9 ± 11.4
104.5 ± 10,1
110.9 ± 24.6
a-adrenoceptor blocker Phentolamine
90.9 -!--6.3
93.3 ± 7.8
78.9 ± 11.9
Siminiak et al., Antiarrhythmic drugs and free radicals
357
Chemiluminescence measurements were performed according to a previously described method (2) using a microtiter-plate-based luminescence analyzer Ameflite (Amersham Bucher, FRG) allowing simultaneous estimation of 96 cell samples. 50 ~1 of cell suspension 5 x 106/ml was placed in each separated well of a white microtiter plate (Dynatech) and 100 ~tl of amplifier luminol or lucigenin in concentration of 10-4M in HBSS was added. Then the background was measured for 15 min. To activate the system 100 ~xl of opsonized zymosan (o.z.) 5 mg/ml or phorbol myristate acetate (PMA) 750 nM was added to each well. Then the light emission was measured continuously for 60 rain. Experiments were performed in triplicate. The peak values of light index of each well were taken into further consideration. Results are given as percent of light emission of control sample ± SD of transformed data.
Results D e t a i l e d m e a n p e a k values of c h e m i l u m i n e s c e n c e as well as S D of t h r e e s e p a r a t e d e x p e r i m e n t s are s h o w n in t h e tables. Class I A of a n t i a r r h y t h m i c drugs e x e r t e d various effects o n P M N c h e m i l u m i n e s c e n c e . P r o c a i n a m i d e i n h i b i t e d b o t h L u C L a n d LgCI, s t i m u l a t e d with o.z. in a d o s e - d e p e n d e n t m a n n e r , b u t only t h e highest c o n c e n t r a t i o n t e s t e d was effective w h e n P M A was used as C L stimulus. D i s o p i r a m i d e slightly d i m i n i s h e d luminol amplified light e m i s s i o n a f t e r P M N i n d u c t i o n with o.z. a n d did n o t affect o t h e r e x p e r i m e n t a l m o d e l tested. A j m a l i n e e x e r t e d a d o s e - r e l a t e d i n h i b i t o r y effect o n fi'ee oxygen radical p r o d u c t i o n , e v a l u a t e d by L g C L as well as P M A s t i m u l a t e d LgCL.
Table 2. The effect of selected antiarrhythmic drugs on luminol-dependent chemiluminescence of neutrophils stimulated with phorbol myristate acetate (percent values of control samples + SD).
Drugs
Class I a: Ajmaline Disopiramide Procainamide Class Ib: Lidocaine Mexyletine
Percent values of chemiluminescence in Low Medium concentration concentration
90.0 + 10,2 97.3 + 8.8 95.4 + 4.6 105.2 _+8.2 106.4 ± 3.4
High concentration
85,1 ± 8.5 91,9 ± 12.3 93.2 _.+_,7.5
77.5 _+ 11.6 94.7 + 6.4 86.9 _+6.8
97.4 ± 12.0 96.7 _+ 14.1
95.7 ± 1.1 92.4 _+6.3
Class I c: Propafenone
88.7 + 14.8
89.9 ± 6.4
84.9 ± 5.9
Class It: Practolol Propranolol
91.0 ± 10.3 96.5 ± 15.2
92.6 _+ 12.2 96.2 ± 14,6
92.3 + 8.4 91,5 _+ 13,0
Class III: Amiodarone Bretylium tosyI
95.5 ± 9.0 83.3 __ 12.5
110.3 +_5.0 82.0 _+ 12.1
19.8 + 5.0 69,3 ± 14.8
Class IV: Verapamil
96.6 _+0.7
80.1 ± 10.6
81.5 ± 2.3
ct-adrenocepmr blocker Phentolamine
96.4 ± 4.1
98.6 ± 11.5
77.1 ± 14.6
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Among class IB drugs only mexiletine was completely inactive in affecting neutrophil chemiluminescence, whereas lidocaine slightly impaired both o,z. and P M A stimulated LgCL. Class IC drugs, propafenone impaired LgCL stimulated by both o.z. and P M A in a dosedependent manner and exerted a slight effect on P M A induced LuCL. Lucigenin amplified light emission was limited by increasing amiodarone concentrations up to near complete blockade at 2Cm~x; this concentration also extremely decreased LuCL. Bretytium tosylate exerted a dose-dependent inhibitory effect of PMN light emission amplified by luminol, but with no effect of LgCL. The calcium channel blocker verapamil did not affect LuCL stimulated with o.z., and decreased CL in a dose-dependent manner in other models studied. Both t3-receptor blocking agents propranolol and practolol slightly decreased o.z.-stimulated LuCL, as well as - in highest concentration studied - PMA-induced LgCL. The a-blocker phentolamine inhibited neutrophil CL in all models studied. However, LuCL inhibition by phentolamine was significant only in C2max. Discussion Chemiluminescence (CL) is an energy product of PMN, oxygenation activity being defined as the removal of one or more electrons or reducing equivalents from a substrata atom or molecule. Chemically amplified CL by luminol (LuCL) and lucigenin (LgCL) is used for detection of reactive oxygen species production following PMN stimulation (2). Table 3. The effect of selected antiarrhythmic drugs on lucigenin-dependent chemiluminescence of neutrophils stimulated with opsonized zymosan (percent values of control samples _+ SD).
Drugs
Percent values of chemiluminescence in Low Medium concentration concentration
High concentration
Class I a: Ajmaline Disopiramide Procainamide
98.9 _+5.5 93.6 -+ 6.3 79,2 -+ 4.2
76,4 ± 3,1 93.9 ± 5,9 70,2 ± 8.4
58.1 _+8.2 93.8 _+3.6 60.7 +_8.4
Class I b: Lidocaine Mexyletine
90.4 ± 9.9 96.4 + 5,5
90.9 ± 4.5 99.1 -+ 5.1
88.1 _+4,0 90.5 ± 3.9
Class I c: Propafenone
89.4 + 8,2
78,7 _+4.5
64.4 _+6,2
Class II: Practolol Propranolol
91.9 - 7.8 101.6 ± 2.9
91,1 ± 5.1 93.7 _+2.7
91.5 ± 8.4 93.7 _+6.6
Class IIh Amiodarone Bretylium tosyl
98.5 ± 5.3 100.0 ± 2.8
69.6 -+ 5.7 91.5 ± 12.5
12.6 ± 8.1 93.9 + 15.7
Class IV: Verapamil
78.8 + 7.3
69.1 ± 13.1
64.1 _+12.4
c~-adrenoeeptor blocker Phentolamine
87.2 + 4.1
83.1 ± 0.8
63.3 ± 4.6
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Siminiak et al., Antiarrhythmic drugs and free radicals
Table 4. The effect of selected antiarrhythmic drugs on lucigenin-dependent chemiluminescence of neutrophils stimulated with phorbol myristate acetate (percent values of control samples ± SD). Drugs Class I a: Ajmaline Disopiramide Procainamide
Percent values of chemiluminescence in bw Medium concentration concentration 96.2 ± 0A 84.9 ± 12.1 85,5 ± 9,4
80.2 + 8.0 91.5 ± 8.5 82.8 + 14.2
High concentration 55.1 ± 3.5 88.7 _+10.2 78.4 _+6.1
Class Ib: Lidocaine Mexyletine
82.4 _+8.1 100,3 ± 2.6
84.0 + 7.5 101.6 + 1.8
84.4 + 9.1 91.9 ± 9.4
Class I c: Propafenone
90.6 _+1.1
85.8 ± 7.3
50.5 + 12.5
Class II: Practolol Propranolol
97.9 ± 1.2 88.5 ± 8.6
92.5 + 1.8 91.9 + 1.5
86.6 +±2.6
Class IIh Amiodarone Bretylium tosyl
73.1 ± 19.0 98.1 ± 4.5
70.4 + 10.9 t04.5 ± 12.1
3.9 + 2.4 98.0 ± 17.7
Class IV: Verapamil
81.2 ± 8.3
41.8 ± 9.9
28.9 ± 7.6
a-adrenoceptor blocker Regityne
88.5 ± 6.2
91.9 ± 3.4
70.0 ± 12.7
79.9 ± 1.8
Activation of PMN oxidative metabolism by o.z. occurs via FclgG and/or complement receptor stimulation, whereas PMA exerts its effect directly on protein kinase C, resulting in a subsequent PMN "respiratory burst". Both CL amplifiers detect different oxygenation activity (2). Luminol amplified light is generated mainly by H202 and related intermediate singlet oxygen (102) , whereas LgCL is an effect of superoxide anion production by PMN. Participation of free oxygen radicals produced by PMN in generation of reperfusion arrhythmias is well documented. Reactive oxygen species produce enhanced lipid peroxidation and calcium movements and both of these factors have been implicated in arrhythmogenesis. Conflicting results obtained in clinical and experimental studies on drug effectiveness in the treatment of reperfusion arrhythmias as well as the evidence that PMN delivered free radicals participate in generation of this kind of arrhythmias indicate the expediency of evaluation of the drugs influences on PMN oxidative metabolism. Such an effect may constitute an additional mechanism of their antiarrhythmic activity. Fast sodium channel inhibitors are widely used as first-line drugs in the treatment of reperfusion arrhythmias, however, the experimental studies on the effectiveness of these drugs in this kind of arrhythmia give conflicting results. Procainamide was found to be effective in the treatment of reperfusion arrhythmias in the rat and in the dog, whereas in the pig it was shown to be ineffective (3). In our study, procainamide decreased free oxygen radicals production (as estimated by CL) in a dose-dependent manner, and this confirms previously reported inhibition of O~ and H202 production by PMN under in vitro influence of this drug (32). Furthermore, it was found that PMN were involved in the metabolism of
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procainamide to the toxic procainamide-hydroxylamine and H202 produced by PMN "respiratory burst" was shown to be responsible (27). Lidocaine is commonly used in clinical practice. However, this drug was found to be ineffective in treatment of reperfusion arrhythmias in several experimental studies in the dog (3, 15) and pig (13). lit was suggested (14) that reperfnsion-induced arrhythmias in the dog occur due to both re-entry and enhanced automaticity, and lidocaine was suggested to suppress those arrhythmias attributed to increased automaticity, but not to re-entry. It may be speculated that due to membrane lipid peroxidation and calcium influx, free radicals may increase automaticity. Thus, the previously reported (32) and non confirmed (by LuCL) slight effect of tidocaine on PMN oxygen metabolism could be involved in limiting this kind of arrhythmia. Ajmaline inhibited both o.z. and PMA stimulated LgCL, suggesting decreased O~ detection. This effect is probably related to the previously reported increase of the amount of O~ reacting with superoxide dismutase by ajmaline (29) and subsequent scavenge of superoxide anion. The influence of ajmatine on PMN derived superoxide anion scavenge might be responsible for the effectiveness of this drug in the treatment of reperfusioninduced arrhythmias (21). Amiodarone, especially in the higher concentrations studied inhibited free radical production by PMN as measured by CL. Treatment with amiodarone was reported to result in a dose-related development of multilamellar inclusion bodies in PMN (1). It may be therefore speculated that this effect is rather more related to modification of PMN functions, than it is a scavenger effect. However, the mechanim of possible modification of PMN oxygen metabolism is at the present unknown. Bretylium tosylate was shown to reduce the incidence of reperfusion-induced ventricular fibrillation (5) in an experimental model. Due to the inhibition of H202 production by PMN as estimated by LuCL the antiarrhythmic activity of bretylium tosylate may be related to its antioxidant effect. Certain experimental data (4, 25) indicate a potentially beneficial effect of verapamil in the treatment of reperfusion arrhythmias. Since calcium ions are required for PMN activation the inhibitory effect of the calcium channel blocker verapamil on PMN function cited in many studies (37), has been confirmed by CL. Furthermore, verapamil has direct antioxidant properties (17) and therefore decreases lipid peroxidation (22). Alpha-adrenoceptor blocking compounds are usually not used in the treatment of arrhythmias. However, these substances were found to be extremely effective in reducing the incidence of reperfusion arrhythmias in experimental models (8, 28, 33, 34). Due to their multiple pharmacological properties it is not fully established if these compounds act via blockade of the alpha receptor or some other mechanisms. The number of alpha receptors was shown to increase during ischemia and early reperfusion (7), and alpha blockade was shown to reduce the incidence of reperfusion-induced arrhythmias (6, 7, 34). Furthermore, phentolamine exerts a direct membrane stabilizing effect (26), inhibits platelet aggregation (23), and stimulates insulin secretion (16), and these mechanisms may be involved in the effectiveness of this drug in the treatment of reperfusion arrhythmias. Among all substances tested only the alpha-blocker phentolamine inhibited neutrophil free radical production in all models studied. Our findings indicate that also inhibition of free oxygen radicals production by neutrophils may be considered an additional mechanism of phentolamine antiarrhythmic activity. Blockade of [3-adrenoceptors with propranolol was shown to reduce neutrophil-related myocardial injury after permanent coronary artery occlusion, but failed to protect myocardium during ischemia followed by reperfusion (31). This divergence may be explained in part by an increase in PMN aggregation under influence of propranolol (36) and subsequent
Siminiak et al., Antiarrhythmic drugs and free radicals
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modification of "no-reflow" phenomenon being a result of increased PMN aggregation during reperfusion (9). Our results indicate decreased o.z.-stimulated LuCL, as well as (in higher concentrations) PMN-induced LgCL. Propranolol was shdwn to act as a xantine oxidase inhibitor (12) and, therefore, to protect against free radical mediated lipid peroxidation (17). Reperfusion arrhythmias generated during relief of coronary artery spasm, cardiac surgery, fibrynolysis, and angioplasty are of high clinical importance. Due to partipication of neutrophil-delivered free radicals in the development of reperfusion arrhythmias, further research on the effect of antiarrhythmic drugs on neutrophil oxygen metabolism may constitute a new approach to control of this kind of arrhythmia. References
1. Adams PC, Sloan P, Morley AR, Holt DW (1986) Peripheral neutrophil inclusions in amiodarone treated patients. Br J Clin Pharmacol 22 (6):736--8 2. Allen RC (1986) Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymot 133:449-493 3. Bergey JL, Nocella K, McCallum JD (1982) Acute coronary artery occlusion-reperfusiominduced arrhythmias in rats, dogs and pigs: anti-arrhythmic evaluation of quinidine and lidocaine. Eur J Pharmacol 81:205-216 4. Brooks WW, Verrier RL, Lown B (1980) Protective effect of verapamil on vulnerability to ventricular fibrillation during myocardial ischemia and reperfusion. Cardiovasc Res 14:295-302 5. Coker SJ (1989) Anesthetized rabbit as a model for ischemia- and reperfusion~induced arrhythmias: effects of quinidine and bretylium. J Pharmacol Methods 21:263-279 6. Corr PB, Penkoske PB, Sobel BE (1978) Adrenergic influences on arrhythmias due to coronary occlusion and reperfusion. Br Heart J 40 8uppl, 62-70 7. Corr PB, Shayman JA, Kramer JB, Kipnis RJ (1981) Increased alpha-adrenergic receptors in ischemic cat myocardium: a potential mediator of electrophysiological derangements. J Clin Invest 67:1232-1326 8. Corr PB, Witowski FX (1983) Potential electrophysiolic mechanisms responsible for dysrhythmias associated with reperfusion of ischaemic myocardium. Circulation 68 suppl I:16-24 9. Engler RL, Dahlgren MD, Morris DD, Peterson MA, Schmid-Schoenbein GW (1986) Role of leukocytes in the response to acute myocardial ischemia and reflow in dogs. Am J Physiol 251 :H314-H323 10. Gruber HE, Hoffer ME, McAllister DR, Laikind PK, Lane TA, Schmid-Schoenbein GW, Engler RL (1989) Increased adenosine concentration in blood from ischemie myocardium by AICA riboside. Effects on flow, granulocytes and injury. Circulation 80:1400-141I 11. Hess ML, Okabe E, Kontos HA (1981) Proton and free oxygen radical interactions with the calcium transport system of cardiac sarcomplasmic reticulum. J Mol Cell Cardiol 13:767-772 12. Janero DR, Lopez R, Pittman J, Burghardt B (1989) Propranolol as xanthine oxidase inhibitor: implications for antioxidant activity. Life Sci 44:1579-1588 13. Janse MJ (1982) Electrophysiological changes in the acute phase of myocardial ischemia and mechanisms of ventricular arrhythmias. In: Parratt JR (ed) Early arrhythmias resulting from myocardial ischemia. MacMillan Press, London, pp 57-80 14. Kabell G, Scherlag BJ, Hope RR, Lazzara R (1980) Reperfusion arrhythmias: differential effect of lidocaine on reentry and enhanced automaticity (abstr). Am J Cardiol suppl 474 15. De Lorgeril M, Rousseau G, Basmadjian A, Latour JG (1988) Lignocaine in experimental myocardial infarction: failure to prevent neutrophil accumulation and ventricular fibrillation and to reduce infarct size. Cardiovasc Res 22:439-446 16. Majid PA, Saxton C, Dykes JR, Galvin MC, Taylor SH (1970) Automatic control of insulin secretion and the treatment of heart failure. Br Med J 4:320323 17. Mak IT, Weglicki WB (1988): Protection by beta-blocking agents against free radical-mediated sarcolemmal lipid peroxidation. Circ Res 63:262-266 18. Manning AS, Coltart DJ, Hearse DJ (1984): Ischemia and reperfusion-induced arrhythmias in the rat: Effect of xantine oxidase inhibition with allopurinol. Circ Res 55:545-548
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19. Maza SR, Frishman WH (1987): Therapeutic options to minimize flee radical damage and thrombogenicity in ischemic/reperfused myocardium. Am Heart J 114/5:1206-1215 20. Meerson FZ, Kagan VE, Kozlow YuP, Belkina LM, Arkipienko YnV (1982) The role of lipid peroxidation in pathogenesis of ischemic damage and the antioxidant protection of the heart. Bas Res Cardiol 77:465-485 21. Okumura K, Hashimoto Y, Yasuhara M, Hori R (1988) Regional myocardial ajmaline concentration and antiarrhythmic activity for ischaemia- and reperfusion-induced arrhythmias in rats. Br J Phatanacol 93/4:827-832 22. Ondrias K, Misik V, Gergel D, Stasko A (1989) Lipid peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine. Biochim Biophys Acta 1003 (3):238-245 23. Pfister B, Imhof PR (1977) Inhibition of adrenaline-induced platelet aggregation by the orally administrated alpha-adrenergic blocker phentolamine (Regitine). Eur J Clin Pharmacol 11:7-14 24. Rao PS, Cohen MV, Mueller HS (1985) Production of free radicals and lipid peroxides in early experimental myocardial ischemia. J Moll Cell Cardiol 17:485-493 25. Ribiero LGT, Brandon TA, Debauch TL, Maroko PR, Miller RR (1981) Anti-arrhythmic and hemodynamic effects of calcium channel blocking agents during coronary arterial reperfusion. Am J Cardiol 48:69-74 26. Rosen MR, Gelband H, Hoffman B (1971) Effects of phentolamine on electrophysiologic properties of isolated canine Purkinje fibers. J Pharmacol Exp Ther 179:586-593 27. Rubin RL, Curnutte JT (1989) Metabolism of procainamide to the cytotoxic hydroxylamine by neutrophils activated in vitro. J Clin Invest 83/4:1336-1343 28. Sheridan DJ, Penkoske PA, Sobel BE, Corr PB (1980) Alpha adrenergic contributions to dysrhythmia during myocardial ischemia and reperfnsion cats. J Clin Invest 65:161-171 29. Shridi F, Robak J (1988) The influence of calcium channel blockers on superoxide anions. Pharmacol Res Comm 20:13-21 30. Singal PK, Kaput N, Beamish RE, Das PK, Dhalla NS (1984) Antioxidant protection against epinephrine-induced arrhythmias. In: Beamish RE, Singal PK, Dhalla NS (eds): Stress and heart disease. Martinus Nijhoff, pp 190-201 31. Smith EF 3d, Egan JW, Griswold DE (1989) Effect of propranolol on ischemic myocardial damage and left ventricular hypertrophy following permanent coronary artery occlusion or occlusion followed by reperfusion. Pharmacolo~ ~38 (5):298-309 32. Stelzner TJ, Welsh CH, Berger E, McCutlongh RG, Morris K, Repine JE, Well JV (1987) Antiarrhythmic agents diminish thiourea-induced pulmonary vascular protein leak in rats. J Appl Physiol 63/5:1877-1883 33. Steward JR, Bnrmeister WE, Burmeister J, Lucchesi BR (1980) Electrophysiologic and antiaxrhythmic effects of phentolamine in experimental coronary artery occlusion and reperfusion in the dog. J Cardiovasc Pharmacol 2:77-91 34. Thandroyen FT, Worthington MG, Higginson L, Opie LH (1983) The effect of alpha-adrenoceptor antagonist agents on reperfusion ventricutar fibrillation and metabolic status in the isolated perfused rat heart. J Am Coll Cardiol 1:1056-1066 35. Turners JF, Bovanis A (1980) Generation of the superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J 156:435-444 36. Weisdorf D J, Jacob HS (1987) Beta-adrenergic blockade: augmentation of neutrophil-mediated inflammation. J Lab Clin Med 109/2:120-126 37. Weisdorf DJ, Thayer MS (1989) Occult intracellular calcium pools: relevance to neutrophil oxidant production. J Lab Clin Med 114:260-265 38. Wysoeka E, Wysocki H, Siminiak T, Szczepanik A (1989) Effect of selected antiarrhythmic drugs on the superoxide anion production by polymorphonuclear neutrophils in vitro. Cardiology 76/4:264270 Received April 18, 1991 Authors' address: Dr. med. Tomasz Siminiak, Academy of Medicine, Institute of Internal Medicine, Dept. of Intensive Therapy, ul. Przybyszewskiego 49, PL-60-355 Poznafi, Poland
Cardiology
Basic Res Cardio186:355-362 (1991 )
The effect of selected antiarrhythmic drugs on neutrophil free oxygen radicals production measured by chemiluminescence T. Siminiak 1, H. Wysocki 1, A. Veit 2, and H. R. Maurer 2 1 Academy of M e d i d n e , Institute of Internal Medicine, Department of Intensive Therapy Poznafi, Poland (Head: Prof. Dr. H. Wysocki), and 2 Free University of Berlin, Institute of Pharmacy, Biochemical Department, Berlin, F R G (Head: Prof. Dr, R. H. Maurer)
Summary: The evidence that free oxygen radicals produced by polymorphonuclear neutrophils (PMN) participate in generation of reperfusion arrhythmias is well documented. The in vitro effect of selected antiarrhythmic drugs on PMN free radicals production was evaluated by luminol- (LuCL) and lucigenin- (LgCL) amplified chemiluminescence stimulated with opsonized zymosan (o.z.) and phorbol myristate acetate (PMA). Fast sodium channel inhibitors varied in the influence on PMN chemiluminescence: from an inhibition in all models studied by procainamide, to lack of an effect by mexiletine. Propafenone, similarly to ajmaline and verapamil, inhibited LuCL stimulated with PMA, as well as LgCL after stimulation with both PMA and o.z. Bretylium tosylate decreased LuCL stimulated with both inducers, with no effect on LgCL. Amiodarone in high concentrations inhibited both LuCL and LgCL. [3-blockers propranolol and practolol impaired LuCL stimulated with o.z., as well as LgCL induced with PMA, whereas a-blocker phentolamine inhibited LuCL and LgCL stimulated with both inducers. The drugs' effect on PMN free oxygen radicals production may constitute an additional mechanism of their activity. Key words: Reperfusion Larrhythmias;_freeoxygen radicals; chemiluminescence; antiarrhythmic drugs Introduction
Increasing evidence obtained in recent years indicates the concept that, in certain clinical situations like ischemia and repeffusion, free oxygen radicals are involved in generation of arrhythmias. Reactive oxygen species such as the superoxide anion (O~-), hydroxyl radical ( O H ' ) , and hydrogen peroxide (H202) affect myocardium calcium ion transfer (11) and membrane lipid metabolism (20, 24) and, subsequently, result in electrophysiological disturbances in myocyte membranes and generation of reperfusion arrhythmias. Various sources of enhanced free radical production have been studied and results prove participation of myocyte mitochondria (35), activated platelets (19), endothelium, xanthine oxidase (18), and catecholamines (30). However, it is well accepted that the most effective source of free oxygen radicals in ischemic myocardium is activated polymorphonuclear neutrophils (PMN). Perfusion of ischemic/reperfused myocardial tissue with neutrophildepleted blood resulted in limitation of myocardial injury and markedly reduced the incidence of reperfusion alThythmias (9). Similar results were obtained after pharmacological interventions that decreased PMN accumulation in ischemic myocardium (10). We have recently evaluated the effect of selected antiarrhythmic drugs on PMN superoxide anion production in vitro as measured by cytochrome C reduction in a whole blood assay (38). However, large amounts of superoxide dismutase contained in erythrocytes might affect the final results. The goal of the present study was to evaluate the in vitro effect 681
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of selected antiarrhythmic drugs on production of reactive oxygen species by isolated neutrophils as m e a s u r e d by luminol- (LuCL) and lucigenin (LgCL) d e p e n d e n t chemiluminescence. Materials and methods To obtain polymorplaonuclear neutrophils, heparinized blood from healthy laboratory personnel was separated by one-step centrifugation procedure using Gradisol-G medium (Polfa). Residual erythrocytes were removed by hypotonic lysis. Then PMN were washed twice in PBS and resuspended in the same buffer. Cells were then incubated 15 min at 37 °C with sMine or investigated drug solution in saline in final concentrations calculaled from minimal (Cmln) and maximal (Cmax) concentrations achieved in blood after therapeutical doses as follows: 0.5 Cmln, (Cmin + Cmax)/2, 2Cmax- Following final drug concentrations were used: ajmaline (Gilurytmal, Giulini) 2, 7, and 20 rag/l; amiodarone (Cordarone, Krka) 0.5, 2, and 6 mg/1; bretylium tosylate (Bretylate, Wellcome) 0.3. 1, and 2.8 mg]l; disopiramide (Rythmodan, Russel) 1.5, 4.5, and 12 rag/l; lidocaine (Lignocainum, Polfa) 0.7, 3.25, and t0 rag/l; mexiletine (Mexitil, Boehringer Ingelheim) 0.5, 1.5, and 4 rag/l; phentolamine (Regitin, Ciba) 0.5, 1.5, and 4 rag/l; practolol (Eraldin, ICI) 0.25, 0.75, and 2 rag/l; procainamide (Procainamidum, Potfa) 2, 7, and 20 mg/1; propafenone (Rytmonorm, Knoll) 2, 7, and 20 rag/l; propranolol (Polfa) 25, 75, and 200 ~g/l; verapamil (Isoptin, Knoll) 20, 55, and 70 mg/l. To opsonize the zymosan particles, zymosan A (Sigma) was boiled for 30 rain, washed twice, and resuspended to 15 mg/ml in saline and then incubated for 20 min at 37 °C with 20 % fresh pooled human serum. Then opsonized zymosan was washed twice in PBS and resuspended 5 mg/ml in HBSS.
Table 1. The effect of selected antiarrhythmic drugs on luminol-dependent chemiluminescence of neutrophils stimulated with opsonized zymosan (percent values of control samples ± SD).
Drugs
Percent values of chemiluminescence Low Medium concentration concentration
High concentration
Class I a: Ajmaline Disopiramide Procainamide
100.3 + 6.7 88.8 + 5.3 96.2 + 5.5
99.4 +±16.2 89.9 ± 12.0 83.4 ± 2.4
95.9 ± 14.2 85.9 ± 7.4 71.2 _+4.2
Class Ib: Lidocaine Mexyletine
101.3 ± 13.2 97.3 ± 4.7
98.6 _+4.1 98.8 -+ 8.1
98.9 + 13.8 96.4 _+5.1
Class I c: Propafenone
92.6 _+6.4
96.2 ± 8.1
98.1 + 6.1
Class Ih Practolol Propranolol
88.9 -+ 5.5 80.3 _+5.1
86.1 ± 5.7 82.5 ± 8,1
87.2 +_11.0 80.1 ± 7.0
107,4 + 3.2 86.3 ± 5.1
104.3 ± 8.0 81..4 ± 5.9
31.4 +_20.4 73.2 ± 7.1
Class IIh Amiodarone Bretytium tosyl Class IV: Verapamil
98.9 ± 11.4
104.5 ± 10,1
110.9 ± 24.6
a-adrenoceptor blocker Phentolamine
90.9 -!--6.3
93.3 ± 7.8
78.9 ± 11.9
Siminiak et al., Antiarrhythmic drugs and free radicals
357
Chemiluminescence measurements were performed according to a previously described method (2) using a microtiter-plate-based luminescence analyzer Ameflite (Amersham Bucher, FRG) allowing simultaneous estimation of 96 cell samples. 50 ~1 of cell suspension 5 x 106/ml was placed in each separated well of a white microtiter plate (Dynatech) and 100 ~tl of amplifier luminol or lucigenin in concentration of 10-4M in HBSS was added. Then the background was measured for 15 min. To activate the system 100 ~xl of opsonized zymosan (o.z.) 5 mg/ml or phorbol myristate acetate (PMA) 750 nM was added to each well. Then the light emission was measured continuously for 60 rain. Experiments were performed in triplicate. The peak values of light index of each well were taken into further consideration. Results are given as percent of light emission of control sample ± SD of transformed data.
Results D e t a i l e d m e a n p e a k values of c h e m i l u m i n e s c e n c e as well as S D of t h r e e s e p a r a t e d e x p e r i m e n t s are s h o w n in t h e tables. Class I A of a n t i a r r h y t h m i c drugs e x e r t e d various effects o n P M N c h e m i l u m i n e s c e n c e . P r o c a i n a m i d e i n h i b i t e d b o t h L u C L a n d LgCI, s t i m u l a t e d with o.z. in a d o s e - d e p e n d e n t m a n n e r , b u t only t h e highest c o n c e n t r a t i o n t e s t e d was effective w h e n P M A was used as C L stimulus. D i s o p i r a m i d e slightly d i m i n i s h e d luminol amplified light e m i s s i o n a f t e r P M N i n d u c t i o n with o.z. a n d did n o t affect o t h e r e x p e r i m e n t a l m o d e l tested. A j m a l i n e e x e r t e d a d o s e - r e l a t e d i n h i b i t o r y effect o n fi'ee oxygen radical p r o d u c t i o n , e v a l u a t e d by L g C L as well as P M A s t i m u l a t e d LgCL.
Table 2. The effect of selected antiarrhythmic drugs on luminol-dependent chemiluminescence of neutrophils stimulated with phorbol myristate acetate (percent values of control samples + SD).
Drugs
Class I a: Ajmaline Disopiramide Procainamide Class Ib: Lidocaine Mexyletine
Percent values of chemiluminescence in Low Medium concentration concentration
90.0 + 10,2 97.3 + 8.8 95.4 + 4.6 105.2 _+8.2 106.4 ± 3.4
High concentration
85,1 ± 8.5 91,9 ± 12.3 93.2 _.+_,7.5
77.5 _+ 11.6 94.7 + 6.4 86.9 _+6.8
97.4 ± 12.0 96.7 _+ 14.1
95.7 ± 1.1 92.4 _+6.3
Class I c: Propafenone
88.7 + 14.8
89.9 ± 6.4
84.9 ± 5.9
Class It: Practolol Propranolol
91.0 ± 10.3 96.5 ± 15.2
92.6 _+ 12.2 96.2 ± 14,6
92.3 + 8.4 91,5 _+ 13,0
Class III: Amiodarone Bretylium tosyI
95.5 ± 9.0 83.3 __ 12.5
110.3 +_5.0 82.0 _+ 12.1
19.8 + 5.0 69,3 ± 14.8
Class IV: Verapamil
96.6 _+0.7
80.1 ± 10.6
81.5 ± 2.3
ct-adrenocepmr blocker Phentolamine
96.4 ± 4.1
98.6 ± 11.5
77.1 ± 14.6
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Among class IB drugs only mexiletine was completely inactive in affecting neutrophil chemiluminescence, whereas lidocaine slightly impaired both o,z. and P M A stimulated LgCL. Class IC drugs, propafenone impaired LgCL stimulated by both o.z. and P M A in a dosedependent manner and exerted a slight effect on P M A induced LuCL. Lucigenin amplified light emission was limited by increasing amiodarone concentrations up to near complete blockade at 2Cm~x; this concentration also extremely decreased LuCL. Bretytium tosylate exerted a dose-dependent inhibitory effect of PMN light emission amplified by luminol, but with no effect of LgCL. The calcium channel blocker verapamil did not affect LuCL stimulated with o.z., and decreased CL in a dose-dependent manner in other models studied. Both t3-receptor blocking agents propranolol and practolol slightly decreased o.z.-stimulated LuCL, as well as - in highest concentration studied - PMA-induced LgCL. The a-blocker phentolamine inhibited neutrophil CL in all models studied. However, LuCL inhibition by phentolamine was significant only in C2max. Discussion Chemiluminescence (CL) is an energy product of PMN, oxygenation activity being defined as the removal of one or more electrons or reducing equivalents from a substrata atom or molecule. Chemically amplified CL by luminol (LuCL) and lucigenin (LgCL) is used for detection of reactive oxygen species production following PMN stimulation (2). Table 3. The effect of selected antiarrhythmic drugs on lucigenin-dependent chemiluminescence of neutrophils stimulated with opsonized zymosan (percent values of control samples _+ SD).
Drugs
Percent values of chemiluminescence in Low Medium concentration concentration
High concentration
Class I a: Ajmaline Disopiramide Procainamide
98.9 _+5.5 93.6 -+ 6.3 79,2 -+ 4.2
76,4 ± 3,1 93.9 ± 5,9 70,2 ± 8.4
58.1 _+8.2 93.8 _+3.6 60.7 +_8.4
Class I b: Lidocaine Mexyletine
90.4 ± 9.9 96.4 + 5,5
90.9 ± 4.5 99.1 -+ 5.1
88.1 _+4,0 90.5 ± 3.9
Class I c: Propafenone
89.4 + 8,2
78,7 _+4.5
64.4 _+6,2
Class II: Practolol Propranolol
91.9 - 7.8 101.6 ± 2.9
91,1 ± 5.1 93.7 _+2.7
91.5 ± 8.4 93.7 _+6.6
Class IIh Amiodarone Bretylium tosyl
98.5 ± 5.3 100.0 ± 2.8
69.6 -+ 5.7 91.5 ± 12.5
12.6 ± 8.1 93.9 + 15.7
Class IV: Verapamil
78.8 + 7.3
69.1 ± 13.1
64.1 _+12.4
c~-adrenoeeptor blocker Phentolamine
87.2 + 4.1
83.1 ± 0.8
63.3 ± 4.6
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Table 4. The effect of selected antiarrhythmic drugs on lucigenin-dependent chemiluminescence of neutrophils stimulated with phorbol myristate acetate (percent values of control samples ± SD). Drugs Class I a: Ajmaline Disopiramide Procainamide
Percent values of chemiluminescence in bw Medium concentration concentration 96.2 ± 0A 84.9 ± 12.1 85,5 ± 9,4
80.2 + 8.0 91.5 ± 8.5 82.8 + 14.2
High concentration 55.1 ± 3.5 88.7 _+10.2 78.4 _+6.1
Class Ib: Lidocaine Mexyletine
82.4 _+8.1 100,3 ± 2.6
84.0 + 7.5 101.6 + 1.8
84.4 + 9.1 91.9 ± 9.4
Class I c: Propafenone
90.6 _+1.1
85.8 ± 7.3
50.5 + 12.5
Class II: Practolol Propranolol
97.9 ± 1.2 88.5 ± 8.6
92.5 + 1.8 91.9 + 1.5
86.6 +±2.6
Class IIh Amiodarone Bretylium tosyl
73.1 ± 19.0 98.1 ± 4.5
70.4 + 10.9 t04.5 ± 12.1
3.9 + 2.4 98.0 ± 17.7
Class IV: Verapamil
81.2 ± 8.3
41.8 ± 9.9
28.9 ± 7.6
a-adrenoceptor blocker Regityne
88.5 ± 6.2
91.9 ± 3.4
70.0 ± 12.7
79.9 ± 1.8
Activation of PMN oxidative metabolism by o.z. occurs via FclgG and/or complement receptor stimulation, whereas PMA exerts its effect directly on protein kinase C, resulting in a subsequent PMN "respiratory burst". Both CL amplifiers detect different oxygenation activity (2). Luminol amplified light is generated mainly by H202 and related intermediate singlet oxygen (102) , whereas LgCL is an effect of superoxide anion production by PMN. Participation of free oxygen radicals produced by PMN in generation of reperfusion arrhythmias is well documented. Reactive oxygen species produce enhanced lipid peroxidation and calcium movements and both of these factors have been implicated in arrhythmogenesis. Conflicting results obtained in clinical and experimental studies on drug effectiveness in the treatment of reperfusion arrhythmias as well as the evidence that PMN delivered free radicals participate in generation of this kind of arrhythmias indicate the expediency of evaluation of the drugs influences on PMN oxidative metabolism. Such an effect may constitute an additional mechanism of their antiarrhythmic activity. Fast sodium channel inhibitors are widely used as first-line drugs in the treatment of reperfusion arrhythmias, however, the experimental studies on the effectiveness of these drugs in this kind of arrhythmia give conflicting results. Procainamide was found to be effective in the treatment of reperfusion arrhythmias in the rat and in the dog, whereas in the pig it was shown to be ineffective (3). In our study, procainamide decreased free oxygen radicals production (as estimated by CL) in a dose-dependent manner, and this confirms previously reported inhibition of O~ and H202 production by PMN under in vitro influence of this drug (32). Furthermore, it was found that PMN were involved in the metabolism of
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procainamide to the toxic procainamide-hydroxylamine and H202 produced by PMN "respiratory burst" was shown to be responsible (27). Lidocaine is commonly used in clinical practice. However, this drug was found to be ineffective in treatment of reperfusion arrhythmias in several experimental studies in the dog (3, 15) and pig (13). lit was suggested (14) that reperfnsion-induced arrhythmias in the dog occur due to both re-entry and enhanced automaticity, and lidocaine was suggested to suppress those arrhythmias attributed to increased automaticity, but not to re-entry. It may be speculated that due to membrane lipid peroxidation and calcium influx, free radicals may increase automaticity. Thus, the previously reported (32) and non confirmed (by LuCL) slight effect of tidocaine on PMN oxygen metabolism could be involved in limiting this kind of arrhythmia. Ajmaline inhibited both o.z. and PMA stimulated LgCL, suggesting decreased O~ detection. This effect is probably related to the previously reported increase of the amount of O~ reacting with superoxide dismutase by ajmaline (29) and subsequent scavenge of superoxide anion. The influence of ajmatine on PMN derived superoxide anion scavenge might be responsible for the effectiveness of this drug in the treatment of reperfusioninduced arrhythmias (21). Amiodarone, especially in the higher concentrations studied inhibited free radical production by PMN as measured by CL. Treatment with amiodarone was reported to result in a dose-related development of multilamellar inclusion bodies in PMN (1). It may be therefore speculated that this effect is rather more related to modification of PMN functions, than it is a scavenger effect. However, the mechanim of possible modification of PMN oxygen metabolism is at the present unknown. Bretylium tosylate was shown to reduce the incidence of reperfusion-induced ventricular fibrillation (5) in an experimental model. Due to the inhibition of H202 production by PMN as estimated by LuCL the antiarrhythmic activity of bretylium tosylate may be related to its antioxidant effect. Certain experimental data (4, 25) indicate a potentially beneficial effect of verapamil in the treatment of reperfusion arrhythmias. Since calcium ions are required for PMN activation the inhibitory effect of the calcium channel blocker verapamil on PMN function cited in many studies (37), has been confirmed by CL. Furthermore, verapamil has direct antioxidant properties (17) and therefore decreases lipid peroxidation (22). Alpha-adrenoceptor blocking compounds are usually not used in the treatment of arrhythmias. However, these substances were found to be extremely effective in reducing the incidence of reperfusion arrhythmias in experimental models (8, 28, 33, 34). Due to their multiple pharmacological properties it is not fully established if these compounds act via blockade of the alpha receptor or some other mechanisms. The number of alpha receptors was shown to increase during ischemia and early reperfusion (7), and alpha blockade was shown to reduce the incidence of reperfusion-induced arrhythmias (6, 7, 34). Furthermore, phentolamine exerts a direct membrane stabilizing effect (26), inhibits platelet aggregation (23), and stimulates insulin secretion (16), and these mechanisms may be involved in the effectiveness of this drug in the treatment of reperfusion arrhythmias. Among all substances tested only the alpha-blocker phentolamine inhibited neutrophil free radical production in all models studied. Our findings indicate that also inhibition of free oxygen radicals production by neutrophils may be considered an additional mechanism of phentolamine antiarrhythmic activity. Blockade of [3-adrenoceptors with propranolol was shown to reduce neutrophil-related myocardial injury after permanent coronary artery occlusion, but failed to protect myocardium during ischemia followed by reperfusion (31). This divergence may be explained in part by an increase in PMN aggregation under influence of propranolol (36) and subsequent
Siminiak et al., Antiarrhythmic drugs and free radicals
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modification of "no-reflow" phenomenon being a result of increased PMN aggregation during reperfusion (9). Our results indicate decreased o.z.-stimulated LuCL, as well as (in higher concentrations) PMN-induced LgCL. Propranolol was shdwn to act as a xantine oxidase inhibitor (12) and, therefore, to protect against free radical mediated lipid peroxidation (17). Reperfusion arrhythmias generated during relief of coronary artery spasm, cardiac surgery, fibrynolysis, and angioplasty are of high clinical importance. Due to partipication of neutrophil-delivered free radicals in the development of reperfusion arrhythmias, further research on the effect of antiarrhythmic drugs on neutrophil oxygen metabolism may constitute a new approach to control of this kind of arrhythmia. References
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