Pharmacology 14: 97 103 (1976)
Elec trophy siological Studies of 6-(N,N-Diethylamino)-Hexyl3,4,5,-Trimetlioxybenzoate on Ventricular Muscle and Conduction System Philip Posner and C. Y. Chiou Department of Physiology and Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Fla.
Key Words. Action potential amplitude • Calcium flux • Canine Purkinje fibres • Feline papillary muscles • 6-(N,N-diethylamino)-hexyl-3,4,5-trimethoxybenzoatc • Resting membrane potential • Spontaneous rate • Threshold Abstract. The agent, 6-(N,N-diethylamino)-hexyl-3,4,5-trimethoxybenzoate (TMB-6) which relaxes smooth and skeletal muscle by interfering with intracellular Ca** availability was tested for its electrophysiological effects on canine cardiac Purkinje fibres and feline papillary muscles. In both tissues the drug causes a decrease in resting membrane potential and action potential amplitude as well as spontaneous rate in Purkinje fibres, and an increase in stimulus needed to reach threshold. The minimum effective dose for both tissues was similar at 7.32 X 10" 5 M. The drug effects were reversible in both tissues upon removal of the TMB-6. The Ca**-dcpendent action potentials of canine cardiac Purkinje fibres were also inhibited by TMB-6 at dose range of 7.32 X 10' 5 -24.4 X 10" s M. Because of its electrophysiological effects on threshold, automaticity and ‘slow response’ action potentials and its reversibility, TMB-6 could become a useful antiarrhythmic drug.
Received: July 23, 1975.
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The pharmacology of a series of co-(N,N-diethylamino)-alkyl-3,4,5-trimethoxybenzoates (TMB compounds) has been studied by Dell ‘Omodarme and Brunori (1959), Sharma (1960, 1962), Lindner el al. (1963), and Robinson (1971). It was concluded that the length of the intermediate alkyl chain directly correlates with the potency and the dose range for blockade of a wide variety of contractile agents in smooth and skeletal muscles (Robinson, 1971). 8-(N Jvl-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) has the longest intermediate
Posner/Chiou
98
alkyl chain synthesized and is the most potent skeletal and smooth muscle relaxant of the group (Malagodi and Chiou, 1974a, b). It has been shown by Chiou and Malagodi (1975) to exert its relaxing effect by interfering with Ca++ movement. Since another Ca++ inhibitor, verapamil, has been found to be effica cious in the therapy of cardiac arrhythmias (Schamroth et al., 1972; Gotsman et ai, 1972), the possibility that 6-(N,N-diethylamino)-hexyl-3,4,5-trimethoxybenzoate (TMB-6), structurally closest to TMB-8 but much easier to synthesize, would also possess antiarrhythmic capabilities was tested. This study elucidates further the action mechanism of TMB-6 at the electrophysiological level and supports the previous finding, showing that TMB-6 is capable of correcting car diac arrhythmias (Chiou et al., 1975).
Canine cardiac Purkinje fibres and feline papillary muscles which were less than 1 mm in diameter were excised from the ventricles of anaesthetized dogs and cats (sodium pento barbital, 30 mg/kg i.v. and i.p. respectively). The preparations were placed in a constant temperature bath (37 °C) and superfused with Tyrode’s solution (NaCl, 136.9 mA/; KC1, 5.4 mM; NaHC03, 11.9 mA/; NaH2PO«, 1.78 mA/; MgCl2, 1.05 mA/; CaCl2, 2.7 niA/; glu cose, 5.55 mAf; 98 % 0 2 - 2 % C 0 2; pH = 7.35-7.40) with a sigmamotor pump at a rate of 1 ml/min. TMB-6 was added to the bath where total mixing occurred with a Harvard syringe pump at 0.01 ml/min to give the desired concentration of the drug in the bath. The tissue was driven through stainless steel pins, insulated, except at the tip, connected to a Grass SD5 stimulator. The preparation was driven at a rate of 42/min with a 20 % suprathreshold pulse. The threshold was that the voltage stimulus delivered for 2 msec which produced an action potential 50 % of the time. Transmembrane potentials were monitored continuously with glass microelectrodes filled with 3 A/ KC1 connected via Zeltex ZA 801 D1 operational amplifiers to the input of a 3A3 amplifier of a Tektronix 564 B oscilloscope. Photographs were taken with a Nikon-Kohden 35 mm oscilloscope camera. In order to determine the effect of TMB-6 on spontaneous rate, the Purkinje fibre was equilibrated for 1 h (when it had achieved a stable rate of automaticity) in 2.7 mA/ KC1 Tyrode solution. At the end of equilibration, TMB-6 was administered and a new stable rate achieved. Then TMB-6 was washed out. The effect was expressed as percent of the mean control and recovery rate. Each dose was tested in this manner. Ca*‘-dependent action potentials were produced in canine cardiac Purkinje fibres, driven at a rate of 42/min, using the method of Aronson and Cranefield (1973). The fibres were equilibrated in a solution containing tetraethylammonium chloride, 128 mA/; CaCl2, 16.2 mA/; KC1, 2.7 mA/; MgCl2, 0.5 mA/; glucose, 5.5 mM; and Tris, 5.0 mA/ at 37 °C, pH 7.4 for 2 h before intracellular recordings were performed. After equilibration, resting mem brane potential, action potential amplitude and action potential duration were measured using the standard microelectrode technique. After control readings were made, the fibre was exposed to TMB-6 in increasing concentrations for a period of 30 min for each dose. Measurements of resting membrane potential, action potential amplitude, and action poten tial duration were made during each exposure and expressed as percent of control. TMB-6 was synthesized in our laboratory with the method published previously by Malagodi and Chiou (1974a).
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Methods
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Etectrophysiological Studies o f TMB-6
Table I. Effects of TMB-6 on canine cardiac Purkinjc fibre (mean t SE) Dose, M X 10"5
RMP'
AP amplitude2
threshold
100 74 ± 8J
100 55 ± 17* 78 t 19 59 t 123
100 152 t 53 250 ± 126 344 * 872
+1
ii 6 3 3
Percent of control of
co CO
0 7.32 8.54 9.76
n
77 t 75
‘ Resting membrane potential. The control absolute value is 72 * 1 mV. 2 Action potential amplitude. The control absolute value is 91 t 3 mV. 3 p < 0.05, compared with control.
Fig. 1. Action potentials recorded from an excised canine cardiac Purkinje fibre. The records have been retouched. A Control driven at 42/min with a stimulus of 6 V, 2 msec. B Same preparation as A , 30 min after starting an infusion of 9.76 X 10*5 M TMB-6. Threshold stimulus is 20 V, 2 msec.
The results of canine Purkinje fibre experiments are summarized in table I and a typical experiment is shown in figure 1. In the dose range of 7.32—9.76 X 10~SM of TMB-6. the cell was depolarized and the action potential amplitude was depressed (fig. 1). The voltage necessary for stimulation increased and spon taneous activity ceased with the drug level tested. These effects could be re versed by washing out the TMB-6. The duration of action potential did not change significantly. The results of experiments on cat papillary muscle are summarized in table 11 and a typical experiment shown in figure 2. As in the Purkinje fibres, the drug above 7.32 X 10"5 M reduced resting membrane potential and action po tential amplitude, and it raised stimulus threshold. These effects were reversible
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Results
100
Posner /Chiou
Fig. 2. Action potentials recorded from an excised cat papillary muscle. A Control driven at 42/min with a stimulus of 6 V, 2 msec. B Same preparation as A. 30 min after starting 9.76 X 10"s M TMB-6. Threshold stimulus is 30 V, 2 msec.
Table II. Effects of TMB-6 on feline papillary muscle (mean t SE) M x 1 0 '5
n
0 7.32 8.54 9.76
19 5 2 6
Percent of control of RMP1
AP amplitude2
threshold
100 85 t 83 74 ± 143 75 ± 123
100 66 ± 183 69 ± 93 30 t 193
100 241 ± 94 292 ± 423 425 t 110
1 Resting membrane potential. The control absolute value is 80 ± 3 mV. 2 Action potential amplitude. The control absolute value is 96 ± 5 mV. 3 p < 0.05, compared with control.
Table III. Effect of TMB-6 on Ca4+-dependent action potentials of canine cardiac Purkinje fibres (mean t SE) TMB-6 MX 10-5
0 7.32 14.6 24.4
n
7 4 7 4
Overshoot
Percent of control of RMP'
AP amp2
duration3
100 97 t 4 87 ± 5 92 ± 16
100 80 ± 94 72 t 64 48 ± 194
100 100 ± 0 79 ± 11 68 t 18
10 -5 -2 -2 2
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1 Resting membrane potential. The control absolute value is 50 ± 6 mV. 2 Action potential amplitude. The control absolute value is 60 t 7 mV. 3 Action potential duration. The control absolute value is 260 ± 47 msec. 4 p < 0.05 compared with control.
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Fig. 3. Ca**-dependent action potentials recorded from an excised canine cardiac Purkinje fibre. A Control driven at 42/min with a stimulus of 15 V and 12 msec. B, C Same preparation as A, 30 min after an infusion of TMB-6 at concentrations of 7.32 X 10"s M and 1.46 X 1 0 '4 M, respectively.
upon removal of the drug. No significant change of action potential duration was observed in these experiments. It can be seen that the slopes of all dose-response curves were quite steep. Table III shows the inhibition of Ca++-dependent action potential amplitude by TMB-6 (7.32-24.4 X I0~5 M). The resting membrane potential was affected only slightly, while the slight depression of action potential duration was not statistically significant. Figure 3 shows tracings of a typical experiment. These results indicate that tire slow inward Ca++ current is also depressed by TMB-6. All data were tested using Student’s t test.
The data show that in the effective dose range TMB-6 depolarizes Purkinje fibre and papillary muscle cells, decreases the amplitude of the action potential and raises the stimulus threshold. The effect on amplitude and threshold is probably the result of the depolarization ( Weidmann, 1955; Trautwein, 1963). The depolarization could be caused by (a) a decrease in K+ conductance, (b) an inhibition of the Na*-K+ pump, or (c) an increase in Ca++ or Na+ conductance. If
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Discussion
Posner /Chiou
102
TMB-6 were depressing K+ conductance, one would expect to see a lengthening of the action potential; since none was observed and in fact a shortening oc curred in some instances, one would conclude that this is not the primary mechanism of depolarization caused by TMB-6. The second possible cause, in hibition of the Na+-K+ pump, has not been tested directly. However, preliminary experiments show that TMB-8, a more potent analog of the TMB series, inhibits isolated cardiac phosphodiesterase, which would raise the level of cyclic-AMP to stimulate rather than to inhibit the Na+-K+ pump (Vassalle and Barnabei, 1971). Finally, since in smooth and skeletal muscle, TMB-8 was shown to affect Ca++ movements in the tissue (Malagodi and Chiou, 1974a, b; Chiou and Malagodi, 1975), it is reasonable to postulate such an effect occurred in cardiac tissue also. Preliminary data from our laboratory show TMB-6 to first enhance and then depress contractions in atrial and papillary muscle preparations. Thus, if TMB-6 were dislodging Ca+* from myocardial membrane sites and hence increasing intracellular Ca++ concentration, the initial effect would be an enhanced contrac tion and a depolarization of the cell. Thus, the depolarization recorded in the presence of TMB-6 is probably the result of an increased Ca++ influx, although it is also possible that pNa/pK is increased. Since TMB-6 blocks the ‘slow’ Ca++ channel, it would tend to reduce further influx of Ca++ and to exert a late negative inotropic effect. This effect could also be induced via blockade of Ca-release from sarcoplasmic reticulum by the drug (Chiou and Malagodi, 1975). Three current mechanisms thought to cause cardiac arrhythmias would all be suppressed by TMB-6. (1) In the event of a rapidly firing ectopic focus, depolarization of that focus and elevation of threshold would result in a de creased automaticity and spontaneous rate (Hoffman and Bigger, 1971). (2) Reentrant arrhythmias would be suppressed by the decreased conduction of low amplitude action potentials through areas of elevated threshold; hence, the reentrant pathway would conduct decrementally and would ultimately die out (Hoffman and Bigger, 1971). (3) In the case of arrhythmias caused by a ‘slow’ response (Cranefield et al, 1971) where they are the result of an inward Ca++ current (Cranefield et al., 1974), the blockade of slow inward Ca++ current by TMB-6 would convert the rhythm disturbance by depressing or abolishing these foci. Thus, TMB-6 shows promise as a useful drug in combating arrhythmias. These results provide further evidence to elucidate the action mechanism of TMB-6 as an antiarrhythmic drug reported previously (Chiou et al, 1975).
This study was partially supported by grants from the Canaveral Area Heart Associa tion and Pharmaceutical Manufacturers Association (Research Starter Grant). The authors wish to thank Mr. Chartes R. Lambert for his excellent technical assistance.
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References
Dr. C.Y. Chiou, Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610 (USA)
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Aronson, R.S. and Cranefield, P.F.: The electrical activity of canine cardiac Purkinje fibres in sodium-free, calcium-rich solutions. J. gen. Physiol. 61: 786-808 (1973). Chiou, C. Y. and Malagodi, M.H.: Studies on the mechanism of action of a new Ca** antago nist, 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), in smooth and skeletal muscles. Br. J. Pharmacol. 53: 279-285 (1975). Chiou, C.Y.: Malagodi, M.H.: Sastry, B.V.R., and Posner. P.: Effects of Ca-antagonist, 6-(N,N-dicthylamino) hexyl 3,4,5-trimethoxybenzoate. on cardiac contractions and digitalis-induced arrhythmias. J. Pharmac. exp. Thcr. (in press). Cranefield. P.F.; Aronson, R.S., and Wit, A.L.: Effect of verapamil on the normal action potential and on a calcium dependent slow response of canine cardiac Purkinje fibers. Circulation Res.34: 204-213 (1974). Cranefield, P.F.: Klein, H.O., and Hoffman, B.F.: Conduction of the cardiac impulse. 1. Delay, block, and one-way block in depressed Purkinje fibers. Circulation Res. 28: 199-219 (1971). Dell ‘Omodarme, G. and Brunori, F.: Hypotensive and neurodepressive action of some trimethoxybenzoic acid derivatives. Rass. Med. sper. 6: 279- 295 (1959). Gotsman, M.S.: Lewis, B.S.: Bakst, A., andMitlia, A.: Verapamil in life-threatening tachyar rhythmias. S. Afr. med. J .46: 2017-2019 (1972). Hoffman, B.F. and Bigger, J.T., jr.: Antiarrhythmic drugs; in Dipalma Drill’s pharmacology in medicine, pp. 824-852 (McGraw-Hill, New York 1971). Lindner, A.; Claasen, V.: Hendriksen, R.W.J., and Kralt, R.: Reserpine analogs: phencthylamine derivatives. J. med. Chem. 6: 97-101 (1963). Malagodi, M.H. and Chiou, C.Y.: Pharmacological evaluation of a new Ca** antagonist, 8-(N,N-diethylamino)octyl 3,4,5,-trimethoxybenzoatc hydrochloride (TMB-8): Studies in smooth muscles. Eur. J. Pharmacol. 27; 25-33 (1974a). Malagodi, M.H. and Chiou, C.Y.: Pharmacological evaluation of a new Ca** antagonist, 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). Studies in skeletal muscles. Pharmacology 12: 20-31 (1974b). Robinson, C.: Pharmacomctrics of cc-(N,N-diethylamino) n-alkyl 3,4,5-trimethoxybenzoates patterned after the structure of reserpine on the nervous system; diss., Vanderbilt University (1971). Schamroth, L.: Krikler, D.M., and Garrett, C.: Immediate effects of intravenous verapamil in cardiac arrhythmias. Br. med. J. i; 660-662 (1972). Sharma, V.N.: Bronchodilator activity of twelve recently synthesized local anesthetics. Archs int. Pharmacodyn. Thcr. 125: 304-310 (1960). Sharma, V.N.: Antiacetylcholine, antiaccelerator and local anesthetic activity of some quinidine-like drugs. Archs int. Pharmacodyn. Ther. 137: 410-427 (1962). Trautwein, W.: Generation and conduction of impulses in the heart as affected by drugs. Pharmac. Rev. 15: 277-332 (1963). Vassalle, M. and Barnabei, O.: Norepinephrine and potassium fluxes in cardiac Purkinje fibers. Pflügers Arch. ges. Physiol. 322: 287-303 (1971). Weidmann, S.: The effect of the cardiac membrane potential on the rapid availability o f the sodium-carrying system. J. Physiol., Lond. 127: 213 224 (1955).
Elec trophy siological Studies of 6-(N,N-Diethylamino)-Hexyl3,4,5,-Trimetlioxybenzoate on Ventricular Muscle and Conduction System Philip Posner and C. Y. Chiou Department of Physiology and Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Fla.
Key Words. Action potential amplitude • Calcium flux • Canine Purkinje fibres • Feline papillary muscles • 6-(N,N-diethylamino)-hexyl-3,4,5-trimethoxybenzoatc • Resting membrane potential • Spontaneous rate • Threshold Abstract. The agent, 6-(N,N-diethylamino)-hexyl-3,4,5-trimethoxybenzoate (TMB-6) which relaxes smooth and skeletal muscle by interfering with intracellular Ca** availability was tested for its electrophysiological effects on canine cardiac Purkinje fibres and feline papillary muscles. In both tissues the drug causes a decrease in resting membrane potential and action potential amplitude as well as spontaneous rate in Purkinje fibres, and an increase in stimulus needed to reach threshold. The minimum effective dose for both tissues was similar at 7.32 X 10" 5 M. The drug effects were reversible in both tissues upon removal of the TMB-6. The Ca**-dcpendent action potentials of canine cardiac Purkinje fibres were also inhibited by TMB-6 at dose range of 7.32 X 10' 5 -24.4 X 10" s M. Because of its electrophysiological effects on threshold, automaticity and ‘slow response’ action potentials and its reversibility, TMB-6 could become a useful antiarrhythmic drug.
Received: July 23, 1975.
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The pharmacology of a series of co-(N,N-diethylamino)-alkyl-3,4,5-trimethoxybenzoates (TMB compounds) has been studied by Dell ‘Omodarme and Brunori (1959), Sharma (1960, 1962), Lindner el al. (1963), and Robinson (1971). It was concluded that the length of the intermediate alkyl chain directly correlates with the potency and the dose range for blockade of a wide variety of contractile agents in smooth and skeletal muscles (Robinson, 1971). 8-(N Jvl-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) has the longest intermediate
Posner/Chiou
98
alkyl chain synthesized and is the most potent skeletal and smooth muscle relaxant of the group (Malagodi and Chiou, 1974a, b). It has been shown by Chiou and Malagodi (1975) to exert its relaxing effect by interfering with Ca++ movement. Since another Ca++ inhibitor, verapamil, has been found to be effica cious in the therapy of cardiac arrhythmias (Schamroth et al., 1972; Gotsman et ai, 1972), the possibility that 6-(N,N-diethylamino)-hexyl-3,4,5-trimethoxybenzoate (TMB-6), structurally closest to TMB-8 but much easier to synthesize, would also possess antiarrhythmic capabilities was tested. This study elucidates further the action mechanism of TMB-6 at the electrophysiological level and supports the previous finding, showing that TMB-6 is capable of correcting car diac arrhythmias (Chiou et al., 1975).
Canine cardiac Purkinje fibres and feline papillary muscles which were less than 1 mm in diameter were excised from the ventricles of anaesthetized dogs and cats (sodium pento barbital, 30 mg/kg i.v. and i.p. respectively). The preparations were placed in a constant temperature bath (37 °C) and superfused with Tyrode’s solution (NaCl, 136.9 mA/; KC1, 5.4 mM; NaHC03, 11.9 mA/; NaH2PO«, 1.78 mA/; MgCl2, 1.05 mA/; CaCl2, 2.7 niA/; glu cose, 5.55 mAf; 98 % 0 2 - 2 % C 0 2; pH = 7.35-7.40) with a sigmamotor pump at a rate of 1 ml/min. TMB-6 was added to the bath where total mixing occurred with a Harvard syringe pump at 0.01 ml/min to give the desired concentration of the drug in the bath. The tissue was driven through stainless steel pins, insulated, except at the tip, connected to a Grass SD5 stimulator. The preparation was driven at a rate of 42/min with a 20 % suprathreshold pulse. The threshold was that the voltage stimulus delivered for 2 msec which produced an action potential 50 % of the time. Transmembrane potentials were monitored continuously with glass microelectrodes filled with 3 A/ KC1 connected via Zeltex ZA 801 D1 operational amplifiers to the input of a 3A3 amplifier of a Tektronix 564 B oscilloscope. Photographs were taken with a Nikon-Kohden 35 mm oscilloscope camera. In order to determine the effect of TMB-6 on spontaneous rate, the Purkinje fibre was equilibrated for 1 h (when it had achieved a stable rate of automaticity) in 2.7 mA/ KC1 Tyrode solution. At the end of equilibration, TMB-6 was administered and a new stable rate achieved. Then TMB-6 was washed out. The effect was expressed as percent of the mean control and recovery rate. Each dose was tested in this manner. Ca*‘-dependent action potentials were produced in canine cardiac Purkinje fibres, driven at a rate of 42/min, using the method of Aronson and Cranefield (1973). The fibres were equilibrated in a solution containing tetraethylammonium chloride, 128 mA/; CaCl2, 16.2 mA/; KC1, 2.7 mA/; MgCl2, 0.5 mA/; glucose, 5.5 mM; and Tris, 5.0 mA/ at 37 °C, pH 7.4 for 2 h before intracellular recordings were performed. After equilibration, resting mem brane potential, action potential amplitude and action potential duration were measured using the standard microelectrode technique. After control readings were made, the fibre was exposed to TMB-6 in increasing concentrations for a period of 30 min for each dose. Measurements of resting membrane potential, action potential amplitude, and action poten tial duration were made during each exposure and expressed as percent of control. TMB-6 was synthesized in our laboratory with the method published previously by Malagodi and Chiou (1974a).
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Methods
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Etectrophysiological Studies o f TMB-6
Table I. Effects of TMB-6 on canine cardiac Purkinjc fibre (mean t SE) Dose, M X 10"5
RMP'
AP amplitude2
threshold
100 74 ± 8J
100 55 ± 17* 78 t 19 59 t 123
100 152 t 53 250 ± 126 344 * 872
+1
ii 6 3 3
Percent of control of
co CO
0 7.32 8.54 9.76
n
77 t 75
‘ Resting membrane potential. The control absolute value is 72 * 1 mV. 2 Action potential amplitude. The control absolute value is 91 t 3 mV. 3 p < 0.05, compared with control.
Fig. 1. Action potentials recorded from an excised canine cardiac Purkinje fibre. The records have been retouched. A Control driven at 42/min with a stimulus of 6 V, 2 msec. B Same preparation as A , 30 min after starting an infusion of 9.76 X 10*5 M TMB-6. Threshold stimulus is 20 V, 2 msec.
The results of canine Purkinje fibre experiments are summarized in table I and a typical experiment is shown in figure 1. In the dose range of 7.32—9.76 X 10~SM of TMB-6. the cell was depolarized and the action potential amplitude was depressed (fig. 1). The voltage necessary for stimulation increased and spon taneous activity ceased with the drug level tested. These effects could be re versed by washing out the TMB-6. The duration of action potential did not change significantly. The results of experiments on cat papillary muscle are summarized in table 11 and a typical experiment shown in figure 2. As in the Purkinje fibres, the drug above 7.32 X 10"5 M reduced resting membrane potential and action po tential amplitude, and it raised stimulus threshold. These effects were reversible
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Results
100
Posner /Chiou
Fig. 2. Action potentials recorded from an excised cat papillary muscle. A Control driven at 42/min with a stimulus of 6 V, 2 msec. B Same preparation as A. 30 min after starting 9.76 X 10"s M TMB-6. Threshold stimulus is 30 V, 2 msec.
Table II. Effects of TMB-6 on feline papillary muscle (mean t SE) M x 1 0 '5
n
0 7.32 8.54 9.76
19 5 2 6
Percent of control of RMP1
AP amplitude2
threshold
100 85 t 83 74 ± 143 75 ± 123
100 66 ± 183 69 ± 93 30 t 193
100 241 ± 94 292 ± 423 425 t 110
1 Resting membrane potential. The control absolute value is 80 ± 3 mV. 2 Action potential amplitude. The control absolute value is 96 ± 5 mV. 3 p < 0.05, compared with control.
Table III. Effect of TMB-6 on Ca4+-dependent action potentials of canine cardiac Purkinje fibres (mean t SE) TMB-6 MX 10-5
0 7.32 14.6 24.4
n
7 4 7 4
Overshoot
Percent of control of RMP'
AP amp2
duration3
100 97 t 4 87 ± 5 92 ± 16
100 80 ± 94 72 t 64 48 ± 194
100 100 ± 0 79 ± 11 68 t 18
10 -5 -2 -2 2
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1 Resting membrane potential. The control absolute value is 50 ± 6 mV. 2 Action potential amplitude. The control absolute value is 60 t 7 mV. 3 Action potential duration. The control absolute value is 260 ± 47 msec. 4 p < 0.05 compared with control.
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Fig. 3. Ca**-dependent action potentials recorded from an excised canine cardiac Purkinje fibre. A Control driven at 42/min with a stimulus of 15 V and 12 msec. B, C Same preparation as A, 30 min after an infusion of TMB-6 at concentrations of 7.32 X 10"s M and 1.46 X 1 0 '4 M, respectively.
upon removal of the drug. No significant change of action potential duration was observed in these experiments. It can be seen that the slopes of all dose-response curves were quite steep. Table III shows the inhibition of Ca++-dependent action potential amplitude by TMB-6 (7.32-24.4 X I0~5 M). The resting membrane potential was affected only slightly, while the slight depression of action potential duration was not statistically significant. Figure 3 shows tracings of a typical experiment. These results indicate that tire slow inward Ca++ current is also depressed by TMB-6. All data were tested using Student’s t test.
The data show that in the effective dose range TMB-6 depolarizes Purkinje fibre and papillary muscle cells, decreases the amplitude of the action potential and raises the stimulus threshold. The effect on amplitude and threshold is probably the result of the depolarization ( Weidmann, 1955; Trautwein, 1963). The depolarization could be caused by (a) a decrease in K+ conductance, (b) an inhibition of the Na*-K+ pump, or (c) an increase in Ca++ or Na+ conductance. If
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Discussion
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TMB-6 were depressing K+ conductance, one would expect to see a lengthening of the action potential; since none was observed and in fact a shortening oc curred in some instances, one would conclude that this is not the primary mechanism of depolarization caused by TMB-6. The second possible cause, in hibition of the Na+-K+ pump, has not been tested directly. However, preliminary experiments show that TMB-8, a more potent analog of the TMB series, inhibits isolated cardiac phosphodiesterase, which would raise the level of cyclic-AMP to stimulate rather than to inhibit the Na+-K+ pump (Vassalle and Barnabei, 1971). Finally, since in smooth and skeletal muscle, TMB-8 was shown to affect Ca++ movements in the tissue (Malagodi and Chiou, 1974a, b; Chiou and Malagodi, 1975), it is reasonable to postulate such an effect occurred in cardiac tissue also. Preliminary data from our laboratory show TMB-6 to first enhance and then depress contractions in atrial and papillary muscle preparations. Thus, if TMB-6 were dislodging Ca+* from myocardial membrane sites and hence increasing intracellular Ca++ concentration, the initial effect would be an enhanced contrac tion and a depolarization of the cell. Thus, the depolarization recorded in the presence of TMB-6 is probably the result of an increased Ca++ influx, although it is also possible that pNa/pK is increased. Since TMB-6 blocks the ‘slow’ Ca++ channel, it would tend to reduce further influx of Ca++ and to exert a late negative inotropic effect. This effect could also be induced via blockade of Ca-release from sarcoplasmic reticulum by the drug (Chiou and Malagodi, 1975). Three current mechanisms thought to cause cardiac arrhythmias would all be suppressed by TMB-6. (1) In the event of a rapidly firing ectopic focus, depolarization of that focus and elevation of threshold would result in a de creased automaticity and spontaneous rate (Hoffman and Bigger, 1971). (2) Reentrant arrhythmias would be suppressed by the decreased conduction of low amplitude action potentials through areas of elevated threshold; hence, the reentrant pathway would conduct decrementally and would ultimately die out (Hoffman and Bigger, 1971). (3) In the case of arrhythmias caused by a ‘slow’ response (Cranefield et al, 1971) where they are the result of an inward Ca++ current (Cranefield et al., 1974), the blockade of slow inward Ca++ current by TMB-6 would convert the rhythm disturbance by depressing or abolishing these foci. Thus, TMB-6 shows promise as a useful drug in combating arrhythmias. These results provide further evidence to elucidate the action mechanism of TMB-6 as an antiarrhythmic drug reported previously (Chiou et al, 1975).
This study was partially supported by grants from the Canaveral Area Heart Associa tion and Pharmaceutical Manufacturers Association (Research Starter Grant). The authors wish to thank Mr. Chartes R. Lambert for his excellent technical assistance.
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A cknowledgements
Elcctrophysiological Studies of TMB-6
103
References
Dr. C.Y. Chiou, Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610 (USA)
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