Br. J. Pharmacol. (1990), 101, 406-410
-0 Macmillan Press Ltd, 1990
Influence of N-ethylmaleimide on action potential and force of contraction of guinea-pig papillary muscles Corona von Poehl, H. Iven & 'H. Brasch Department of Pharmacology, Medical University of Lubeck, Ratzeburger Allee 160, D-2400 Lubeck, F.R.G. 1 Standard microelectrode techniques were used to investigate the influence of N-ethylmaleimide on the action potential, slow response action potential and force of contraction of guinea-pig papillary muscles. 2 N-ethylmaleimide, 3 x 10' to 10-'moll concentration-dependently increased the force of contraction. The positive inotropic effect developed quickly and, with the largest drug concentration, was followed by a progressive decline of the contractile force. The action potential duration was progressively shortened by N-ethylmaleimide. 3 The effects of N-ethylmaleimide were not prevented in the presence of tetrodotoxin 3 x 10-8 mollI and propranolol 4 x 10- 6mol 11 or by a reduction of the Na+-concentration to 70 mmol 1-. 4 Verapamil, 10-6 mol I1, reduced the positive inotropic, but not the action potential shortening effect of N-ethylmaleimide. 5 In K+-depolarized muscles in the presence of propranolol and tetrodotoxin, N-ethylmaleimide i0' mol I- increased the maximum depolarization velocity and the duration of the slow response action potential. The latter effect was transient and was followed by a progressive reduction of the action potential duration. 6 The most likely explanation for the action potential shortening effect of N-ethylmaleimide seems to be an increase of an outward potassium current while the transient inotropic effect of the drug may be caused, at least in part, by an increase of the slow inward calcium current. P,
Introduction N-ethylmaleimide (NEM) increases the force of contraction of cat papillary muscles (Bennett et al., 1958), guinea-pig papillary muscles (Fricke, 1978) and guinea-pig atria (From, 1970; Ueno, 1971; Yamamoto et al., 1973; Temma et al., 1978). A release of endogenous noradrenaline (Yamamoto et al., 1973) and an inhibition of Na'-K+-ATPase (Skou & Hilberg, 1965; Fujita et al., 1968; Tobin & Akera, 1975) have been implicated in the positive inotropic drug effect, but other explanations cannot be ruled out. During prolonged exposure to NEM, an increase of the resting tension, a decline of the contractile force and the appearance of arrhythmias are often observed (From, 1970; Yamamoto et al., 1973). The latter effect suggests that NEM might alter the membrane currents and the time course of the action potential. Indeed, Toda & Konishi (1969) observed a slowing of diastolic depolarization and a progressive shortening of the action potential duration of sinoatrial pacemaker cells by NEM. Braun & Sperelakis (1988), on the other hand, found the action potential of guinea-pig atrium unchanged after a 25 min exposure to NEM, whereas no information is available as to the effect of the drug on ventricular action potentials. We therefore decided to study the effect of NEM on action potential and force of contraction of guineapig papillary muscles with standard microelectrode techniques. The results revealed multiple sites of drug action, and the influence of NEM on the calcium current in K+depolarized muscles suggests a new interpretation of the transient inotropic effect of this drug.
Methods
Experimental procedure Male guinea-pigs (250-500 g) were killed by a blow on the head. The heart was rapidly excised and papillary muscles, from the right ventricle, were prepared in cooled, oxygenated Krebs-Henseleit solution. Muscles with a diameter of less than 1 mm were then mounted horizontally in a 20 ml organ bath filled with Krebs-Henseleit solution of the following composi' Author for correspondence.
tion (mmol -1): NaCl 117.6, KCl 5.8, CaCl2 2.5, NaHCO3 25, NaH2PO4 1.2, MgSO4 1.2 and glucose 5.5. The solution was kept at 32 + 0.20C and was gassed with a mixture of 95% 02 and 5% CO2. The muscles were stimulated via pointed platinum electrodes at a rate of 1 Hz with square pulses of 1 ms duration and 1.5 times threshold voltage. The force of contraction was registered isometrically by a K 30 force-displacement transducer (Hugo Sachs, Hugstetten, F.R.G.). Action potentials were recorded with standard glass microelectrodes which were filled with 3 molI- ' KCl and had a tip resistance of 510 MCI. The maximum depolarization velocity V,. was obtained by passive signal differentiation. The RC-circuit with a time constant of 2 x 10-4 s had been tested and found to be linear over the desired range in a previous study (Iven & Brasch, 1977). The action potential, ,.,. and force of contraction were displayed on a Tektronix 502 A dual-beam oscilloscope (Tektronix Inc., Beaverton, OR, U.S.A.) and photographed with a Grass C4 Camera (Grass Medical Instruments, Quincy, MA, U.S.A.) for subsequent analysis. Drugs were added to the organ bath after an equilibration period of at least 90min. The effect of N-ethylmaleimide (NEM) was observed for 60 min. To test the reversibility of the drug effect, the organ bath was then perfused with drug-free Krebs-Henseleit solution for 20min (4mlmin-') and the remaining drug effect was evaluated after a further 30min recovery period. An experiment was included in the study only if the microelectrode remained in the same cell during the whole time course of the experiment. In experiments with low sodium Krebs-Henseleit solution, 73.8 mol P1 NaCl was replaced by 147.6 mmol P` saccharose. To induce Ca2 +-mediated slow-response action potentials, the papillary muscles were depolarized by increasing the KCI concentration to 26 mmolI- 1. The muscles were then stimulated at a rate of 0.1 Hz with square pulses of 3 ms duration and 1.5 times threshold voltage. In these experiments, the Krebs Henseleit solution contained no MgSO4 but 0.2mmoll-' BaCl2 to reduce the outward potassium current (Ehara & Inazawa, 1980). To avoid indirect drug effects caused by a release of endogenous catecholamines the effects of NEM and Bay K 8644 were tested in the presence of propranolol and tetrodotoxin. Propranolol 4 x 10-66mo11-' and tetrodotoxin 3 x 10-6 mol PI were added to the organ bath 1 h before the
INOTROPIC EFFECT OF N-ETHYLMALEIMIDE
407
application of NEM or Bay K 8644 to allow sufficient time for establishing steady-state concentrations in the tissues.
Drugs
z
The following drugs were used in the study: N-ethylmaleimide (Sigma, Deizenhofen, F.R.G.); tetrodotoxin (Sankyo Co., Tokyo, Japan); propranolol hydrochloride (Rhein-Pharma, Heidelberg, F.R.G.); verapamil hydrochloride (Knoll, Ludwigshafen, F.R.G.) and ouabain (Sigma, Deizenhofen, F.R.G.). Racemic Bay K 8644 (1,4-dihydro-2,6-dimethyl-3-nitro-442trifluorophenyl) pyridine-5-carboxylic acid) was a gift from the Bayer AG (Leverkusen, F.R.G.). Bay K 8644 was dissolved in DMSO and 20u1 of the solution was added to the 20 ml organ bath. The experiments with Bay K 8644 were carried out under light from a sodium vapour lamp. All other drugs were dissolved in twice distilled water, and stock solutions were prepared daily.
Statistics Arithmetic means and their standard errors (s.e.) were calculated for the pre-drug values of all parameters. Drug effects were expressed as changes from the pre-drug values for each individual experiment. Mean changes were then calculated and assessed for statistical significance by Student's t test for paired data. P < 0.05 for the two-tailed test was fixed as criterion for the acceptance of statistical significance throughout.
Results
Effects ofN-ethylmaleimide Action potential parameters remained stable in control experiments, but a slow decline of the contractile force was seen during the 60 min observation period (Table 1, Figures 1 and 2). NEM concentration-dependently increased the force of contraction. The positive inotropic effect developed quickly
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4045 50 55 60 W Time (min) Figure 1 Effect of N-ethylmaleimide (NEM) on the contractile force of guinea-pig papillary muscles. Abscissa scale: time after drug addition (min). W = effect remaining after washout of drug. Ordinate scale: change of the force of contraction (mN). (0) Control experiments, no drug; (V) NEM 3 x 10-5moll-1; (0) NEM 5.6 x 10-5mollP1; (A) NEM 10-'mollP1. The symbols indicate mean changes and vertical lines show s.e.mean (n = 6). Asterisks indicate changes that are statistically significant (t test for paired data, P < 0.05 two tailed). Pre-drug values of the contractile force were 0.46 ± 0.13 mN in control experiments and 0.05 ± 0.15, 0.34 ± 0.08 and 0.41 + 0.12 mN in the experiments with the three concentrations of NEM. 15 20 25 30 35
and reached a maximum 10min after drug addition (Figure 1). With 3 x i0ob and 5.6 x 10-5moll-' NEM, the contractile force remained elevated until the end of the 1 h period of drug exposure. But with 10-4moll- NEM the force of contraction declined quickly after the maximum was reached (Figures 1 and 3) and a negative inotropic effect of the drug was obvious towards the end of the observation period, which could not be reversed by washout (Figure 1). During the 50min after the end of drug exposure (20min of perfusion
Table 1 Effect of N-ethylmaleimide (NEM) on action potential parameters and force of contraction of guinea-pig papillary muscles MRP (mV)
APA (mV)
P.. (Vs-,)
APD20 (Ms)
APD80 (Ms)
Fc (mN)
C A 10 min Alh
-79.8 + 1.1 -0.3 + 0.8 -0.5 + 1.5
116.7 + 2.1 +0.7 + 0.5 +0.5 ± 1.4
92.3 ± 10.1 -0.5 + 2.2 +1.8 ± 2.5
112.7 ± 8.5 -0.3 + 1.0 +4.7 ± 2.2
187.8 + 7.8
+0.8 ± 2.7
0.46 + 0.13 -0.03 ± 0.02 -0.06 + 0.02*
NEM 30jumol I -
C A 10 min Alh
-78.5 + 1.0 +0.7 + 0.7 +0.7 + 0.9
115.2 ± 4.4 +0.8 ± 0.9 -1.5 ± 2.6
92.3 ± 5.9 + 1.7 ± 1.7 +6.5 + 3.5
124.2 ± 11.1 +4.7 ± 1.3* -9.8 + 8.9
210.5 + 13.2 +1.7 ± 3.9 -16.8 + 12.7
0.50 ± 0.15 +0.12 ± 0.04* +0.12 ± 0.08
56gmol 1-
C A 10min Alh
-79.3 + 1.3 -2.2 + 0.8* -2.3 ± 1.1
114.3 + 2.8 +2.8 ± 2.2 -5.2 ± 1.5*
85.0 + 7.9 + 1.7 ± 3.2 -4.7 ± 4.6
111.2 + 9.6 +4.7 ± 2.7 -53.5 ± 9.3*
201.0 + 8.7 -3.0 ± 2.6 -70.7 ± 5.1*
0.34 ± 0.08 +0.19 ± 0.04* +0.07 ± 0.02*
NEM 100nmoll-
C A 10 min Alh
-79.7 + 1.4 +0.8 + 1.2 -1.8 + 1.4
117.5 + 2.4 -1.8 + 0.8 -17.7 ± 6.7*
88.0 + 7.9 +0.3 ± 2.3 -16.2 + 5.1*
112.5 ± 3.3 +2.8 ± 4.5 -70.0 + 7.7*
200.8 ± 4.6 -7.7 + 3.5 - 106.0 + 11.4*
0.41 + 0.12 +0.41 + 0.10* -0.19 ± 0.09
C
205.7 + 4.7 -6.5 ± 3.3 - 119.5 + 8.2*
0.38 + 0.10 +0.54 ± 0.11* -0.20 + 0.09
Drug None
NEM
+1.7±1.8
NEM 100pmol I + TrX 30 nmol I-' + Prop 4pmolI'
A 10min Alh
-79.8 ± 0.9 -2.2 + 0.5* -2.8 + 1.1*
111.5 ± 1.4 +1.2 ± 0.6 -20.7 ± 2.9*
84.3 + 3.1 -0.3 ± 3.0 -16.8 ± 6.4*
119.2 + 5.7 -0.8 ± 3.1 -82.0 + 4.9*
NEM 100jmol I + Ver 1 ymol I -
C A 10 min Alh
-78.5 ± 0.9 -2.3 ± 0.6* -2.5 + 1.4
107.8 ± 1.3 +0.7 ± 0.7 -20.8 ± 10.7
135.2 + 8.0 +2.3 + 1.7 -35.7 ± 20.2
109.7 ± 4.4 +8.3 ± 3.0* -64.8 + 9.8*
200.2 ± 6.8 +6.0 ± 3.8 -98.0 ± 14.6*
0.05 ± 0.01 +0.11 ± 0.03* +0.07 ± 0.05
NEM 100lumolI'
C A 10 min Alh
-79.3 + 0.7 +0.3 + 0.9 +1.0+ 1.1
107.7 + 0.9 +2.7 ± 2.2 -1 1.0 ± 1.0*
68.3 ± 11.3 +1.7 ± 4.4 +0.0 + 5.8
81.7 + 8.3 + 10.0 ± 0.5* -48.3 + 8.3*
155.0 + 12.6 + 11.7 + 1.7* -85.0 ± 15.3*
0.35 + 0.10 +0.37 + 0.04* -0.27 + 0.08
Na' 70mmolI-1
Pre-drug values (C) and their mean changes 10min (A 10min) and 1 h (A 1 h) after addition of NEM are given (Ax ±s.e.mean, n= 6). Asterisks indicate changes that are statistically significant (Student's t test for paired data, P 0.05, two-tailed). Abbreviations: MRP=membrane resting potential; APA=action potential amplitude; ,'V =maximum depolarization velocity; APD20, APD80= action potential duration at 20% and 80% of full repolarization; Fc=force of contraction; TTX=tetrodotoxin 3 x lO moll-1; Prop = propranolol 4 x 10-6molP1-; Ver= verapamil 106 mol 1: a= 3 experiments only.
408
C. VON POEHL et al.
-a 20
~.-~~i-
-40
C-70
-so~~~*,
-50
''*ti
-80
0135
in'
1015202530354045505560
W
Time (min)
Figure 2 Effect of N-ethylmaleimide (NEM) on the action potential duration at 80% of full repolarization (APD80). Abscissa scale: time alter drug addition (min). W = effect remaining after washout of drug. Ordinate scale: change in action potential duration (ins). (0) Control experiments, no drug; (v) NEM 3 x lO-5moll-t; (0) NEM 5.6 x 10-5moll-1; (-) NEM 10-4moll-1. The symbols indicate mean changes and vertical lines show s.e.mean (n = 6). Asterisks mark changes that are statistically significant (t test for paired data, P < 0.05 two-tailed). Pre-drug values of the AP duration were 187.8 + 7.8ms in control experiments and 210 + 13.2, 201.0 + 8.7 and 200.8 + 4.6ms in the experiments with the three concentrations of NEM.
with drug-free solution and 30 further mmn of recovery without perfusion; see Methods) the force of contraction decreased even further. It is not clear whether this was due to a persistent effect of NEM or merely a consequence of the long duration of these experiments. In many muscles a transient prolongation of the duration of the action potential in the plateau range was observed after the addition of NEM (APD20; Table 1). The overall effect, however, was slight and statistically significant only with the lowest concentration. With 3 x 10 5mol P' NEM, the only further change was a minor reduction of the total action potential duration towards the end of the observation period. However, with 5.6 x i0-5 and 10-4 mol P-1 NEM a progressive shortening of the action potential duration developed (Table 1, Figure 2). The action potential amplitude and the maximum depolarization velocity were likewise reduced. All these effects were only incompletely reversible after washout.
in Table 1, were similar to the control values in the untreated groups of muscles. In the presence of propranolol and tetrodotoxin NEM 10- mol 1' still exerted a transient positive inotropic effect which was followed by a rapid decline of the contractile force (Table 1, Figure 3). The time course was similar to that in the experiments with NEM alone. Propranolol and tetrodotoxin did not prevent the decrease of the action potential duration by NEM (Table 1, Figure 3). Verapamil alone 10-6 molI 1- , given 1 h before the addition of NEM, greatly reduced the force of contraction of the muscles, but had little influence on the action potential characteristics (see control values in Table 1). In the presence of verapamil, 10i4mollP NEM still exerted a positive inotropic effect (Figure 3). However, the force increased more slowly than with NEM alone, reached a maximum only 30min after drug addition and remained elevated until the end of the experiments. The maximum increase was much smaller than with NEM alone (Table 1). In contrast, the shortening of the action potential duration by NEM was not attenuated by verapamil (Table 1, Figure 3). In low sodium Krebs-Henseleit solution, the positive inotropic effect of 10i4mollP NEM was not reduced (Table 1). A rapid increase was again followed by a negative inotropic effect of NEM. The progressive shortening of the action potential duration was also still observed.
Effects of ouabain Ouabain, 10 6moll , had a prominent positive inotropic effect (Figure 4). The effect developed more slowly than with NEM. The force of contraction increased continuously during 1 h of drug exposure. The magnitude of the ouabain effect was b
a
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z
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I
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- - I
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Modif'ication of N-ethylmaleimide effects by other drugs The addition of propranolol (4 x 10-6mollP1, 40 mm before NEM) and tetrodotoxin (3 x 10- 8mol P'- , 20 mmn before NEM) had no effect on the action potential or the, force of contraction. After pretreatment with both drugs the control values immediately before addition of NEM, which are shown
Figure 4 Typical examples of the effect of ouabain 106 molI ' on action potential (upper traces) and force of contraction (lower traces) of papillary muscles. (a) Effect of ouabain at normal (143.8 mmol -1) sodium concentration. (b) Effect of ouabain at reduced (70mmolI-1) sodium concentration. Solid line: control, before drug addition; broken line, 1 h after addition of ouabain. c
I I
.'. ...
1 mN/
1 mN
0.4 mN
- ...... ...........-
,-,
Figure 3 Typical examples for the effect of I0' mol PI N-ethylmaleimide (NEM) on action potential (upper traces) and force of contraction (lower traces) of papillary muscles. (a) Effect of NEM alone. (b) Effect of NEM in the presence of propranolol 4 x 106 moll-' and tetrodotoxin 3 x 10-8molPl-. (c) Effect of NEM in the presence of verapamil 10-6molPl1. ( ) Control, before addition of NEM; (---) 10 min after addition of NEM; (.....) 30 min after addition of NEM.
INOTROPIC EFFECT OF N-ETHYLMALEIMIDE
response action potentials from 14.3 + 2.0Vs-s to 21.7 + 4.0 Vs- (P < 0.05) and increased the action potential duration by 41.7 + 6.0ms (from 273.3 + 34.7ms to 315.0 + 30.4 ms; P < 0.05). The effect of Bay K 8644 remained constant for 1h and subsequent addition of 10- moll-P NEM in these muscles caused no further increase of V,.,x and only a marginal (+15 + 2.9 ms) additional increase of the action potential duration. However, a progressive shortening of the action potential duration was again evident when the exposure to NEM lasted longer than 20min. Ouabain, in concentrations up to 10 5moll-', had no detectable effect on slow response action potentials (preliminary results, data not shown).
greatly influenced by the sodium concentration (Figure 4). While a 145+8% increase of the force was seen in normal Krebs-Henseleit solution, a mere 37+10% increase was obtained in sodium-depleted solution (3 experiments each). The decreases of the action potential duration at 80% of full repolarization and of the action potential amplitude (Figure 4), which were prominent in normal solution (-25 + 5% and -13 + 3%, respectively) became marginal when the sodium concentration was reduced (-6 + 3% and -1 + 0.5%, respectively).
Effect of N-ethylmaleimide on slow response action potentials NEM, 10-'moll-j, increased the maximum depolarization velocity and the duration of slow response action potentials. lvaxwas nearly doubled (from 11.3 + 1.1 to 21.3 + 1.9Vs-1; P < 0.05) after 25min of NEM exposure and remained elevated for the rest of the observation period (Figure 5). The increase of the action potential duration (pre-drug value 224.3 + 12.54ms), on the other hand, was transient. The maximum prolongation after 15min of NEM exposure was followed by a progressive shortening (Figure 5). After 40min the mean reduction was 21.7 + 12.54ms and during the next 20min three of the six preparations became inexcitable. The amplitude of the action potential (pre-drug value 77.8 + 1.3 mV) was increased and decreased with a similar time course, but the changes were small (about 2 mV) and not statistically significant. The membrane resting potential (-42.8 + 0.6 mV) was not affected by NEM. In three preliminary experiments 5 x 10 7moll-P of the Ca2+-channel activator Bay K 8644 increased V,. of slow
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-0 Macmillan Press Ltd, 1990
Influence of N-ethylmaleimide on action potential and force of contraction of guinea-pig papillary muscles Corona von Poehl, H. Iven & 'H. Brasch Department of Pharmacology, Medical University of Lubeck, Ratzeburger Allee 160, D-2400 Lubeck, F.R.G. 1 Standard microelectrode techniques were used to investigate the influence of N-ethylmaleimide on the action potential, slow response action potential and force of contraction of guinea-pig papillary muscles. 2 N-ethylmaleimide, 3 x 10' to 10-'moll concentration-dependently increased the force of contraction. The positive inotropic effect developed quickly and, with the largest drug concentration, was followed by a progressive decline of the contractile force. The action potential duration was progressively shortened by N-ethylmaleimide. 3 The effects of N-ethylmaleimide were not prevented in the presence of tetrodotoxin 3 x 10-8 mollI and propranolol 4 x 10- 6mol 11 or by a reduction of the Na+-concentration to 70 mmol 1-. 4 Verapamil, 10-6 mol I1, reduced the positive inotropic, but not the action potential shortening effect of N-ethylmaleimide. 5 In K+-depolarized muscles in the presence of propranolol and tetrodotoxin, N-ethylmaleimide i0' mol I- increased the maximum depolarization velocity and the duration of the slow response action potential. The latter effect was transient and was followed by a progressive reduction of the action potential duration. 6 The most likely explanation for the action potential shortening effect of N-ethylmaleimide seems to be an increase of an outward potassium current while the transient inotropic effect of the drug may be caused, at least in part, by an increase of the slow inward calcium current. P,
Introduction N-ethylmaleimide (NEM) increases the force of contraction of cat papillary muscles (Bennett et al., 1958), guinea-pig papillary muscles (Fricke, 1978) and guinea-pig atria (From, 1970; Ueno, 1971; Yamamoto et al., 1973; Temma et al., 1978). A release of endogenous noradrenaline (Yamamoto et al., 1973) and an inhibition of Na'-K+-ATPase (Skou & Hilberg, 1965; Fujita et al., 1968; Tobin & Akera, 1975) have been implicated in the positive inotropic drug effect, but other explanations cannot be ruled out. During prolonged exposure to NEM, an increase of the resting tension, a decline of the contractile force and the appearance of arrhythmias are often observed (From, 1970; Yamamoto et al., 1973). The latter effect suggests that NEM might alter the membrane currents and the time course of the action potential. Indeed, Toda & Konishi (1969) observed a slowing of diastolic depolarization and a progressive shortening of the action potential duration of sinoatrial pacemaker cells by NEM. Braun & Sperelakis (1988), on the other hand, found the action potential of guinea-pig atrium unchanged after a 25 min exposure to NEM, whereas no information is available as to the effect of the drug on ventricular action potentials. We therefore decided to study the effect of NEM on action potential and force of contraction of guineapig papillary muscles with standard microelectrode techniques. The results revealed multiple sites of drug action, and the influence of NEM on the calcium current in K+depolarized muscles suggests a new interpretation of the transient inotropic effect of this drug.
Methods
Experimental procedure Male guinea-pigs (250-500 g) were killed by a blow on the head. The heart was rapidly excised and papillary muscles, from the right ventricle, were prepared in cooled, oxygenated Krebs-Henseleit solution. Muscles with a diameter of less than 1 mm were then mounted horizontally in a 20 ml organ bath filled with Krebs-Henseleit solution of the following composi' Author for correspondence.
tion (mmol -1): NaCl 117.6, KCl 5.8, CaCl2 2.5, NaHCO3 25, NaH2PO4 1.2, MgSO4 1.2 and glucose 5.5. The solution was kept at 32 + 0.20C and was gassed with a mixture of 95% 02 and 5% CO2. The muscles were stimulated via pointed platinum electrodes at a rate of 1 Hz with square pulses of 1 ms duration and 1.5 times threshold voltage. The force of contraction was registered isometrically by a K 30 force-displacement transducer (Hugo Sachs, Hugstetten, F.R.G.). Action potentials were recorded with standard glass microelectrodes which were filled with 3 molI- ' KCl and had a tip resistance of 510 MCI. The maximum depolarization velocity V,. was obtained by passive signal differentiation. The RC-circuit with a time constant of 2 x 10-4 s had been tested and found to be linear over the desired range in a previous study (Iven & Brasch, 1977). The action potential, ,.,. and force of contraction were displayed on a Tektronix 502 A dual-beam oscilloscope (Tektronix Inc., Beaverton, OR, U.S.A.) and photographed with a Grass C4 Camera (Grass Medical Instruments, Quincy, MA, U.S.A.) for subsequent analysis. Drugs were added to the organ bath after an equilibration period of at least 90min. The effect of N-ethylmaleimide (NEM) was observed for 60 min. To test the reversibility of the drug effect, the organ bath was then perfused with drug-free Krebs-Henseleit solution for 20min (4mlmin-') and the remaining drug effect was evaluated after a further 30min recovery period. An experiment was included in the study only if the microelectrode remained in the same cell during the whole time course of the experiment. In experiments with low sodium Krebs-Henseleit solution, 73.8 mol P1 NaCl was replaced by 147.6 mmol P` saccharose. To induce Ca2 +-mediated slow-response action potentials, the papillary muscles were depolarized by increasing the KCI concentration to 26 mmolI- 1. The muscles were then stimulated at a rate of 0.1 Hz with square pulses of 3 ms duration and 1.5 times threshold voltage. In these experiments, the Krebs Henseleit solution contained no MgSO4 but 0.2mmoll-' BaCl2 to reduce the outward potassium current (Ehara & Inazawa, 1980). To avoid indirect drug effects caused by a release of endogenous catecholamines the effects of NEM and Bay K 8644 were tested in the presence of propranolol and tetrodotoxin. Propranolol 4 x 10-66mo11-' and tetrodotoxin 3 x 10-6 mol PI were added to the organ bath 1 h before the
INOTROPIC EFFECT OF N-ETHYLMALEIMIDE
407
application of NEM or Bay K 8644 to allow sufficient time for establishing steady-state concentrations in the tissues.
Drugs
z
The following drugs were used in the study: N-ethylmaleimide (Sigma, Deizenhofen, F.R.G.); tetrodotoxin (Sankyo Co., Tokyo, Japan); propranolol hydrochloride (Rhein-Pharma, Heidelberg, F.R.G.); verapamil hydrochloride (Knoll, Ludwigshafen, F.R.G.) and ouabain (Sigma, Deizenhofen, F.R.G.). Racemic Bay K 8644 (1,4-dihydro-2,6-dimethyl-3-nitro-442trifluorophenyl) pyridine-5-carboxylic acid) was a gift from the Bayer AG (Leverkusen, F.R.G.). Bay K 8644 was dissolved in DMSO and 20u1 of the solution was added to the 20 ml organ bath. The experiments with Bay K 8644 were carried out under light from a sodium vapour lamp. All other drugs were dissolved in twice distilled water, and stock solutions were prepared daily.
Statistics Arithmetic means and their standard errors (s.e.) were calculated for the pre-drug values of all parameters. Drug effects were expressed as changes from the pre-drug values for each individual experiment. Mean changes were then calculated and assessed for statistical significance by Student's t test for paired data. P < 0.05 for the two-tailed test was fixed as criterion for the acceptance of statistical significance throughout.
Results
Effects ofN-ethylmaleimide Action potential parameters remained stable in control experiments, but a slow decline of the contractile force was seen during the 60 min observation period (Table 1, Figures 1 and 2). NEM concentration-dependently increased the force of contraction. The positive inotropic effect developed quickly
E 0
-0.2-
01 3 5
hf~ 10
I*
*
__2|
4045 50 55 60 W Time (min) Figure 1 Effect of N-ethylmaleimide (NEM) on the contractile force of guinea-pig papillary muscles. Abscissa scale: time after drug addition (min). W = effect remaining after washout of drug. Ordinate scale: change of the force of contraction (mN). (0) Control experiments, no drug; (V) NEM 3 x 10-5moll-1; (0) NEM 5.6 x 10-5mollP1; (A) NEM 10-'mollP1. The symbols indicate mean changes and vertical lines show s.e.mean (n = 6). Asterisks indicate changes that are statistically significant (t test for paired data, P < 0.05 two tailed). Pre-drug values of the contractile force were 0.46 ± 0.13 mN in control experiments and 0.05 ± 0.15, 0.34 ± 0.08 and 0.41 + 0.12 mN in the experiments with the three concentrations of NEM. 15 20 25 30 35
and reached a maximum 10min after drug addition (Figure 1). With 3 x i0ob and 5.6 x 10-5moll-' NEM, the contractile force remained elevated until the end of the 1 h period of drug exposure. But with 10-4moll- NEM the force of contraction declined quickly after the maximum was reached (Figures 1 and 3) and a negative inotropic effect of the drug was obvious towards the end of the observation period, which could not be reversed by washout (Figure 1). During the 50min after the end of drug exposure (20min of perfusion
Table 1 Effect of N-ethylmaleimide (NEM) on action potential parameters and force of contraction of guinea-pig papillary muscles MRP (mV)
APA (mV)
P.. (Vs-,)
APD20 (Ms)
APD80 (Ms)
Fc (mN)
C A 10 min Alh
-79.8 + 1.1 -0.3 + 0.8 -0.5 + 1.5
116.7 + 2.1 +0.7 + 0.5 +0.5 ± 1.4
92.3 ± 10.1 -0.5 + 2.2 +1.8 ± 2.5
112.7 ± 8.5 -0.3 + 1.0 +4.7 ± 2.2
187.8 + 7.8
+0.8 ± 2.7
0.46 + 0.13 -0.03 ± 0.02 -0.06 + 0.02*
NEM 30jumol I -
C A 10 min Alh
-78.5 + 1.0 +0.7 + 0.7 +0.7 + 0.9
115.2 ± 4.4 +0.8 ± 0.9 -1.5 ± 2.6
92.3 ± 5.9 + 1.7 ± 1.7 +6.5 + 3.5
124.2 ± 11.1 +4.7 ± 1.3* -9.8 + 8.9
210.5 + 13.2 +1.7 ± 3.9 -16.8 + 12.7
0.50 ± 0.15 +0.12 ± 0.04* +0.12 ± 0.08
56gmol 1-
C A 10min Alh
-79.3 + 1.3 -2.2 + 0.8* -2.3 ± 1.1
114.3 + 2.8 +2.8 ± 2.2 -5.2 ± 1.5*
85.0 + 7.9 + 1.7 ± 3.2 -4.7 ± 4.6
111.2 + 9.6 +4.7 ± 2.7 -53.5 ± 9.3*
201.0 + 8.7 -3.0 ± 2.6 -70.7 ± 5.1*
0.34 ± 0.08 +0.19 ± 0.04* +0.07 ± 0.02*
NEM 100nmoll-
C A 10 min Alh
-79.7 + 1.4 +0.8 + 1.2 -1.8 + 1.4
117.5 + 2.4 -1.8 + 0.8 -17.7 ± 6.7*
88.0 + 7.9 +0.3 ± 2.3 -16.2 + 5.1*
112.5 ± 3.3 +2.8 ± 4.5 -70.0 + 7.7*
200.8 ± 4.6 -7.7 + 3.5 - 106.0 + 11.4*
0.41 + 0.12 +0.41 + 0.10* -0.19 ± 0.09
C
205.7 + 4.7 -6.5 ± 3.3 - 119.5 + 8.2*
0.38 + 0.10 +0.54 ± 0.11* -0.20 + 0.09
Drug None
NEM
+1.7±1.8
NEM 100pmol I + TrX 30 nmol I-' + Prop 4pmolI'
A 10min Alh
-79.8 ± 0.9 -2.2 + 0.5* -2.8 + 1.1*
111.5 ± 1.4 +1.2 ± 0.6 -20.7 ± 2.9*
84.3 + 3.1 -0.3 ± 3.0 -16.8 ± 6.4*
119.2 + 5.7 -0.8 ± 3.1 -82.0 + 4.9*
NEM 100jmol I + Ver 1 ymol I -
C A 10 min Alh
-78.5 ± 0.9 -2.3 ± 0.6* -2.5 + 1.4
107.8 ± 1.3 +0.7 ± 0.7 -20.8 ± 10.7
135.2 + 8.0 +2.3 + 1.7 -35.7 ± 20.2
109.7 ± 4.4 +8.3 ± 3.0* -64.8 + 9.8*
200.2 ± 6.8 +6.0 ± 3.8 -98.0 ± 14.6*
0.05 ± 0.01 +0.11 ± 0.03* +0.07 ± 0.05
NEM 100lumolI'
C A 10 min Alh
-79.3 + 0.7 +0.3 + 0.9 +1.0+ 1.1
107.7 + 0.9 +2.7 ± 2.2 -1 1.0 ± 1.0*
68.3 ± 11.3 +1.7 ± 4.4 +0.0 + 5.8
81.7 + 8.3 + 10.0 ± 0.5* -48.3 + 8.3*
155.0 + 12.6 + 11.7 + 1.7* -85.0 ± 15.3*
0.35 + 0.10 +0.37 + 0.04* -0.27 + 0.08
Na' 70mmolI-1
Pre-drug values (C) and their mean changes 10min (A 10min) and 1 h (A 1 h) after addition of NEM are given (Ax ±s.e.mean, n= 6). Asterisks indicate changes that are statistically significant (Student's t test for paired data, P 0.05, two-tailed). Abbreviations: MRP=membrane resting potential; APA=action potential amplitude; ,'V =maximum depolarization velocity; APD20, APD80= action potential duration at 20% and 80% of full repolarization; Fc=force of contraction; TTX=tetrodotoxin 3 x lO moll-1; Prop = propranolol 4 x 10-6molP1-; Ver= verapamil 106 mol 1: a= 3 experiments only.
408
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Figure 2 Effect of N-ethylmaleimide (NEM) on the action potential duration at 80% of full repolarization (APD80). Abscissa scale: time alter drug addition (min). W = effect remaining after washout of drug. Ordinate scale: change in action potential duration (ins). (0) Control experiments, no drug; (v) NEM 3 x lO-5moll-t; (0) NEM 5.6 x 10-5moll-1; (-) NEM 10-4moll-1. The symbols indicate mean changes and vertical lines show s.e.mean (n = 6). Asterisks mark changes that are statistically significant (t test for paired data, P < 0.05 two-tailed). Pre-drug values of the AP duration were 187.8 + 7.8ms in control experiments and 210 + 13.2, 201.0 + 8.7 and 200.8 + 4.6ms in the experiments with the three concentrations of NEM.
with drug-free solution and 30 further mmn of recovery without perfusion; see Methods) the force of contraction decreased even further. It is not clear whether this was due to a persistent effect of NEM or merely a consequence of the long duration of these experiments. In many muscles a transient prolongation of the duration of the action potential in the plateau range was observed after the addition of NEM (APD20; Table 1). The overall effect, however, was slight and statistically significant only with the lowest concentration. With 3 x 10 5mol P' NEM, the only further change was a minor reduction of the total action potential duration towards the end of the observation period. However, with 5.6 x i0-5 and 10-4 mol P-1 NEM a progressive shortening of the action potential duration developed (Table 1, Figure 2). The action potential amplitude and the maximum depolarization velocity were likewise reduced. All these effects were only incompletely reversible after washout.
in Table 1, were similar to the control values in the untreated groups of muscles. In the presence of propranolol and tetrodotoxin NEM 10- mol 1' still exerted a transient positive inotropic effect which was followed by a rapid decline of the contractile force (Table 1, Figure 3). The time course was similar to that in the experiments with NEM alone. Propranolol and tetrodotoxin did not prevent the decrease of the action potential duration by NEM (Table 1, Figure 3). Verapamil alone 10-6 molI 1- , given 1 h before the addition of NEM, greatly reduced the force of contraction of the muscles, but had little influence on the action potential characteristics (see control values in Table 1). In the presence of verapamil, 10i4mollP NEM still exerted a positive inotropic effect (Figure 3). However, the force increased more slowly than with NEM alone, reached a maximum only 30min after drug addition and remained elevated until the end of the experiments. The maximum increase was much smaller than with NEM alone (Table 1). In contrast, the shortening of the action potential duration by NEM was not attenuated by verapamil (Table 1, Figure 3). In low sodium Krebs-Henseleit solution, the positive inotropic effect of 10i4mollP NEM was not reduced (Table 1). A rapid increase was again followed by a negative inotropic effect of NEM. The progressive shortening of the action potential duration was also still observed.
Effects of ouabain Ouabain, 10 6moll , had a prominent positive inotropic effect (Figure 4). The effect developed more slowly than with NEM. The force of contraction increased continuously during 1 h of drug exposure. The magnitude of the ouabain effect was b
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Modif'ication of N-ethylmaleimide effects by other drugs The addition of propranolol (4 x 10-6mollP1, 40 mm before NEM) and tetrodotoxin (3 x 10- 8mol P'- , 20 mmn before NEM) had no effect on the action potential or the, force of contraction. After pretreatment with both drugs the control values immediately before addition of NEM, which are shown
Figure 4 Typical examples of the effect of ouabain 106 molI ' on action potential (upper traces) and force of contraction (lower traces) of papillary muscles. (a) Effect of ouabain at normal (143.8 mmol -1) sodium concentration. (b) Effect of ouabain at reduced (70mmolI-1) sodium concentration. Solid line: control, before drug addition; broken line, 1 h after addition of ouabain. c
I I
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1 mN/
1 mN
0.4 mN
- ...... ...........-
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Figure 3 Typical examples for the effect of I0' mol PI N-ethylmaleimide (NEM) on action potential (upper traces) and force of contraction (lower traces) of papillary muscles. (a) Effect of NEM alone. (b) Effect of NEM in the presence of propranolol 4 x 106 moll-' and tetrodotoxin 3 x 10-8molPl-. (c) Effect of NEM in the presence of verapamil 10-6molPl1. ( ) Control, before addition of NEM; (---) 10 min after addition of NEM; (.....) 30 min after addition of NEM.
INOTROPIC EFFECT OF N-ETHYLMALEIMIDE
response action potentials from 14.3 + 2.0Vs-s to 21.7 + 4.0 Vs- (P < 0.05) and increased the action potential duration by 41.7 + 6.0ms (from 273.3 + 34.7ms to 315.0 + 30.4 ms; P < 0.05). The effect of Bay K 8644 remained constant for 1h and subsequent addition of 10- moll-P NEM in these muscles caused no further increase of V,.,x and only a marginal (+15 + 2.9 ms) additional increase of the action potential duration. However, a progressive shortening of the action potential duration was again evident when the exposure to NEM lasted longer than 20min. Ouabain, in concentrations up to 10 5moll-', had no detectable effect on slow response action potentials (preliminary results, data not shown).
greatly influenced by the sodium concentration (Figure 4). While a 145+8% increase of the force was seen in normal Krebs-Henseleit solution, a mere 37+10% increase was obtained in sodium-depleted solution (3 experiments each). The decreases of the action potential duration at 80% of full repolarization and of the action potential amplitude (Figure 4), which were prominent in normal solution (-25 + 5% and -13 + 3%, respectively) became marginal when the sodium concentration was reduced (-6 + 3% and -1 + 0.5%, respectively).
Effect of N-ethylmaleimide on slow response action potentials NEM, 10-'moll-j, increased the maximum depolarization velocity and the duration of slow response action potentials. lvaxwas nearly doubled (from 11.3 + 1.1 to 21.3 + 1.9Vs-1; P < 0.05) after 25min of NEM exposure and remained elevated for the rest of the observation period (Figure 5). The increase of the action potential duration (pre-drug value 224.3 + 12.54ms), on the other hand, was transient. The maximum prolongation after 15min of NEM exposure was followed by a progressive shortening (Figure 5). After 40min the mean reduction was 21.7 + 12.54ms and during the next 20min three of the six preparations became inexcitable. The amplitude of the action potential (pre-drug value 77.8 + 1.3 mV) was increased and decreased with a similar time course, but the changes were small (about 2 mV) and not statistically significant. The membrane resting potential (-42.8 + 0.6 mV) was not affected by NEM. In three preliminary experiments 5 x 10 7moll-P of the Ca2+-channel activator Bay K 8644 increased V,. of slow
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