Abstract： Aim: To explore the effects of benactyzine (BEN) on the action potential and contractile force in guinea pig papillary muscles.Methods: Conventional microelectrode technique was used to record the fast action potentials (FAP) and slow action potentials (SAP) of guinea pig papillary muscles.Results: Benactyzine 5,10,50 μmol^. L^-1 suppressed the maximal upstroke velocity (v_max ) of FAP and contractile force (Fc) concentration-dependently while prolonged the action potential duration at 50%,90% repolarization (APD₅₀ ,APD₉₀ ) and effective refractory period (ERP) of FAP.The suppression on the v_max was frequency-dependent.Benactyzine 5,10,50μmol^. L^-1 lengthened the APD50,APD90 of SAP induced by isoprenaline or histamine when perfused with KCl 22 mmol^. L^-1 Tyrode's solution.The v_max of the SAP was not decreased by benactyzine 5,10 μmol^. L^-1 but by 50 μmol^. L^-1 .The effects on the SAP were antagonized by elevation of the extracellular calcium from 2.0 to 5.6 mmol^. L^-1 .The effects of benactyzine on SAP elicited by tetrodotoxin resembled that by isoprenaline or histamine except the more pronounced suppression on v_max and action potential amplitude (APA).The persistent rapid spontaneous activity and triggered tachyarrhythmia induced by ouabain were also abolished immediately by benactyzine 5 μmol^. L^-1 .Conclusion: Benactyzine can inhibit Na⁺ ,K⁺ ,Ca²⁺ transmembrane movement and intracellular Ca²⁺ mobilization in the myocardium,and this may be the electrophysiological basis of its effects against experimental arrhythmias.

INTRODUCTION
Benactyzine is a synthetic blocker of M-receptor possessing smooth muscle relaxant properties and quinidine-like action on heart^[1] .Benactyzine is effective in restoring circulatory function disturbed by organophosphate poisoning^[2,3] .Previous studies suggested that benactyzine had antiarrhythmic effects in animals^[4～6] and calcium antagonistic action on smooth muscle^[7,8] .The electrophysiological effects of benactyzine on myocardium have not been examined.The purpose of this study is to explore its electrophysiological effects and calcium antagonistic action on myocardium.

MATERIALS AND METHODS
Drugs and chemicals
benactyzine (Sigma);tetrodotoxin (Dalian Institute of Marine Materia Medica,Liaoning Province,China);ouabain (Merck);isoprenaline (Shanghai Tianfeng Pharmaceutical Factory);histamine (Shanghai Institute of Biochemistry,Shanghai,China).
Animals
Fifty-six guinea pigs of either sex,weighting 250～350 g were supplied by Experimental Animal Center of Henan Medical University (Certificate No 981002).
Guinea pigs were stunned and papillary muscles were taken from the right ventricle and pinned in a 1.5 ml chamber.The preparations of papillary muscle were perfused with Tyrode's solution (8 ml/min) aerated with 95% O₂ + 5% CO₂ at (32±0.5)℃ and pH 7.2 ～7.4 and electrically driven with pulses of 1 ms duration and 1.5 times threshold voltage at 1Hz frequency from a stimulator.For fast action potential (FAP),the Tyrode solution contained(mmol^. L^-1 ):NaCl 134,KCl 4.0,MgCl₂ 0.5,CaCl₂ 2.0,NaHCO₃ 12,NaH₂ PO₄ 0.42,Glucose 5.5.
Induction of fast action potential and contraction
Contractile force (Fc) of papillary muscle was measured with an isometric force transducer connected to the tendinous end of the muscle.Initial length was adjusted to yield maximal contraction amplitude upon stimulation.Transmembrane action potentials were recorded with a conventional glass microelectrode filled with KCl 3.0 mol^. L^-1 having a resistance of 15 ～ 30 MΩ.The maximal upstroke velocity (v_max ) was obtained by an electronic differentiator.The electronic and mechanical signals were displayed on the screen of oscilloscope and photographed with camera.
Induction of slow action potential
After the impalement of the microelectrode was stabilized for 30 min,the preparations were changed to be driven with pulses of 3 ms and 0.5 Hz and fast Na⁺ channels were inactivated to induce slow action potential (SAP) either by perfusing preparations with high K⁺ (22 mmol^. L^-1 ) Tyrode solution or by tetrodotoxin 20 μmol^. L^-1 .When perfused with high K⁺ Tyrode solution,isoprenaline 1 μmol^. L^-1 or histamine 10 μmol^. L^-1 was added to induce SAP.
Induction of triggered tachyarrhythmia
Oubaine 1.5 μmol^. L^-1 was added to Tyrode solution to induce oscillatory potential or triggered activity.When tachyarrhythmia was induced and persisted for 10 min,benactyzine was given to observe its effect.After induced action potentials were stabilized for 40 min,benactyzine 5,10,50 μmol^. L^-1 was administered in accumulative manner to observe its effects on FAP,Fc and SAP,only the results obtained from a single cell throughout the whole experimental period were subjected to statistical analysis.

RESULTS
The effects of benactyzine on FAP and Fc of guinea pig papillary muscles
Benactyzine 5,10,50 μmol^. L^-1 obviously suppressed v_max and Fc concentration-dependently while prolonged APD₅₀ ,APD₉₀ and effective refractory period (ERP) without affecting resting potential (RP),action potential amplitude (APA) and action potential duration at 20% (APD₂₀ ) (Tab 1).

Tab 1 Effects of benactyzine on FAP and Fc of guinea pig papillary muscles( n= 7，)

^a P > 0.05,^b P < 0.05,^c P < 0.01 vs control using paired t-test

The suppression on v_max was frequency-dependent (Tab 2,Fig 1).

Tab 2 Use-dependent inhibition of benactyzine 10 μmol^. L^-1 on steady state v_max of guinea pig papillary muscles (n=5,)

^a P > 0.05,^b P < 0.05,cP < 0.01 vs control using paired t-test,^d P < 0.01 between means using ANOVA.

Fig 1 Use-dependent inhibition of benactyzine on v_max of guinea pig papillary muscles.

The effects of benactyzine on SAP of guinea pig papillary muscles
After Na⁺ channels were inactivated by KCl 22 mmol^. L^-1 SAP were induced by isoprenaline 1 μmol^. L^-1 or histamine 10 μmol^. L^-1 .Benactyzine 5,10,50 μmol^. L^-1 lengthened the APD₅₀ and APD₉₀ .The v_max was not decreased by benactyzine 5,10 μmol^. L^-1 but by 50 μmol^. L^-1 .All the effects of benactyzine were antagonized by elevation of the extracellular calcium from 2.0 to 5.6 mmol^. L^-1 (Tab 3).

Tab 3 Effects of benactyzine (BEN) on slow action potential induced by isoprenaline (Iso,1 μmol^. L^-1 ) or histamine (His,10 μmol^. L^-1 ) in guinea pig papillary muscles perfused with KCl 22 mmol^. L^-1 Tyrode solution and antagonistic effect of increase of Ca²⁺ from 2.0 to 5.6 mmol^. L^-1 ( n = 7,)

^a P > 0.05,^b P < 0.05,^c P < 0.01 vs BEN 0 μmol^. L^-1 using paired t-test

Tetrodotoxin 20 μmol^. L^-1 suppressed the v_max of FAP to 62%.The effects of benactyzine on SAP elicited by tetrodotoxin resembled that by isoprenaline or histamine except the more pronounced suppression on v_max and APA (Tab 4).

Tab 4 Effects of benactyzine on slow action potential induced by tetrodotoxin 20 μmmol^. L^-1 in guinea pig papillary muscles perfused with KCl 4.0 mmol^. L^-1 Tyrode solution (n = 6,)

^a P > 0.05,^b P < 0.05,^c P < 0.01,vs control using paired t-test

The effects of benactyzine on abnormal rhythms of guinea pig papillary muscles
During the process of stabilizing after impalement of microelectrode persistent rapid spontaneous activities were usually observed.Both the persistent rapid spontaneous activity and triggered tachyarrhythmia induced by oubaine 1.5 μmol^. L^-1 were abolished immediately by benactyzine 5 μmol^. L^-1 .

DISCUSSION
The contraction of myocardium involves the Ca²⁺ influx in plateau phase and the intracellular Ca²⁺ mobilization,while the APA and v_max of SAP is mainly mediated by slow inward calcium current (I_si )^[9] .That benactyzine inhibited Fc at three concentrations and v_max of SAP only at higher concentrations indicates benactyzine has the calcium antagonistic action which concerns extracellular Ca²⁺ influx and intracellular Ca²⁺ mobilization,that is to say,benactyzine results in excitation-contraction uncoupling^[10] .The depressant effect of benactyzine on calcium influx was further verified by the fact that the elevation of Ca²⁺ .Antagonized the inhibition of benactyzine on v_max of SAP.The mild suppression of benactyzine on SAP induced by isoprenaline or histamine may be attributed to that the external Ca²⁺ accumulation elicited by isoprenaline or histamine attenuated the effect of benactyzine^[11] .The prolongation of APD₅₀ and APD₉₀ may be the result of blockade of K⁺ channels by benactyzine.That benactyzine decreased the v_max of FAP at three concentrations and the v_max of SAP only at higher concentrations infers that benactyzine possesses the Na⁺ channel blocking effect at lower concentration and Ca²⁺ channel blocking effect at higher concentration.The inhibitory effect of Na⁺ ,Ca²⁺ currents was consistent with its effect of abolishing the persistent rapid spontaneous activity and triggered tachyarrhythmia induced by oubaine,for both of the two kinds of arrhythmia are thought to be related to the increase of Na⁺ or Ca²⁺ current.
It is reported that the activation of M-receptor can open the cardiac muscarinic K⁺ channel (K_Ach ) leading the shorting of the APD^[12] .In the study,we observed that benactyzine,the blocker of M-receptor,prolong the APD,therefore we can speculate that the prolongation of APD caused by benactyzine is the result of K⁺ channel blockade due to M-receptor blockade.
Our result indicates that benactyzine can inhibit Na⁺ ,K⁺ ,Ca²⁺ transmembrane movement and intracellular Ca²⁺ mobilization in the myocardium,exhibiting calcium antagonistic action on heart,and this may be the electrophysiological basis of its effects against experimental arrhythmias like quinidine^[1] .

作者简介：DING Zhongren,National Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences,Beijing 100005,China.

REFERENCES：

[1]Reynolds JEF.Martindale:the Extra Pharmacopoeia.the 28th ed.The Pharmaceutical Press,1982.1 505
[2]Kassa J,Fusek J.The positive influence of a cholinergic-anticholinergic pretreatment and antidotal treatment on rats poisoned with supralethal doses of soman.Toxicology,1998,128(1):1
[3]Kassa J,Fusek J.Effect of Panpal pretreatment and antidotal treatment (HI-6 plus benactyzine) on respiratory and circulatory function in soman-poisoned rats.Hum Exp Toxicol,1997,16(10):563
[4]Arora RB,Somani P,Lal A.Effect of chloroquine and chloroquine-tranquillizing drug combination on ectopic ventricular tachycardia following acute myocardial infarction in dogs.Indian J Med Res,1962,50:720
[5]Сенова З Л.К механиэму противоаритм ического действия фармакологических веществ.Хармакология и Токсикоиогия,1966,29(2):177
[6]Агафова ГН,Саляев ВН.Оценка аритмог енной активности адренергических и холинергических средств при хлоркальц иевой аритмии.Хармакология и Токсико иогия,1978,41(5):560
[7]向井呈一,高木敬次郎.モルモツト回肠纵走筋におけるBa 收缩とぞの收缩に及ぼすD-600,BenactyzineおょびPapaverineの抑制作用.日平滑筋志,1983,19:97
[8]向井呈一,高木敬次郎.モルモツト盲肠纽におけるHistamine收缩とぞの收缩に及ぼすD-600,PapaverineおょびBenactyzineの抑制作用.日平滑筋志,1983,19:107
[9]Daleau P,Khalifa M,Turgeon J.Effects of cadmium and nisoldipine on the delayed rectifier potassium current in guinea pig ventricular myocytes.J Pharmacol Exp Ther,1997,281(2):826
[10]Dekker LR,Fiolet JW,Van Bavel E,et al.Intracellular Ca²⁺ ,intercellular electrical coupling,and mechanical activity in ischemic rabbit papillary muscle.Effects of preconditioning and metabolic blockade.Circ Res ,1996,79(2):237
[11]Fleckenstain A.Calcium antagonism in heart and smooth muscle.N.Y.:John Wiley & Sons Inc,1983.81
[12]Yamaguchi H,Sakamoto N,Watanabe Y,et al.Dual effects of endothelins on the muscarinic K⁺ current in guinea pig atrial cells.Am J Physiol,1997,273(4 Pt 2):H1745

(收稿日期：1999-11-17) (DING Zhongren ZHANG Yan ZHANG Guiqing KE Jun HU Xiangjie)