CardiovascularResearch 1992;26:1199-1205
1199
Sodium channel states control binding and unbinding behaviour of antiarrhythmic drugs in cardiac myocytes from the guinea pig Shin-ichi Koumi, Ryoichi Sato, Ryo Katori, Ichiro Hisatome, Kouichi Nagasawa, and Hirokazu Hayakawa Objective: The aim was to investigate whether cardiac sodium channel states (rested, activated, inactivated) regulate the binding and unbinding behaviour of antiarrhythmic drugs on the receptor sites. Methods: Single ventricular myocytes of adult guinea pig heart were obtained by an enzymatic dissociation method in the Langendorff manner. The channel state dependent blocking effects on cardiac sodium current (IN,) of quinidine and disopyramide were studied under the whole cell variation of the patch clamp technique. Results: 10 p,M quinidine and 20 pM disopyramide produced similar levels of tonic block and use dependent block. The steady state inactivation curve (h- curve) was shifted parallel in the negative potential direction by quinidine (10 kM) and disopyramide (20 pM) to the same extent (-10 mV). Removal of the fast inactivation process of IN^ by chloramine-T did not reduce tonic and use dependent block by these drugs. Onset block study using a double pulse protocol revealed that block developments by both drugs were fitted to the sum of double exponential functions. However, time constant of fast phase of block by disopyramide was faster than that by quinidine, while slow phase was not significantly different. Definition of time courses of unbinding (recovery) at -140 mV indicated that quinidine dissociated relatively slowly as compared to disopyramide. Conclusions: Quinidine produces more potent tonic and use dependent block of IN, by binding to sodium channels at both rested and inactivated states, while disopyramide has a higher affinity for activated state. Therefore, sodium channel states regulate the binding and unbinding behaviour of antiarrhythmic drugs. Furthermore, the fast inactivation process is not essential in producing tonic and use dependent block by antiarrhythmic drugs. Cardiovascular Research 1992;26: 1199-1205
0
uinidine and disopyramide, both of which are classified as class Ia antiarrhythmic drugs, produce similar electrophysiological effects in cardiac -tissues. Both drugs depress the maximum rate of depolarisation, increase the conduction time, and prolong the terminal phase of repolarisation. By using the single cell preparation, the effect of quinidine and disopyramide on the maximum rate of depolarisation was found to be brought about by sodium channel blockade.’” In order to explain drug-sodium channel interaction, two different hypotheses have been proposed: the modulated receptor hypothesis4 and the guarded receptor hypothesis.6 According to the former hypothesis, drugs bind to a sodium channel receptor site, and the affinity of the receptor of the drug is modulated by states of channel. The drug associated channels differ from the drug free channels in that they do not conduct, and their ability to be activated is shifted toward more negative potential; the affinity of the binding site for drugs is higher when the channel is open or inactivated than when the channel is in the rested state. In contrast, the guarded receptor hypothesis assumes use dependent block as the result of transient access to a binding site of constant
affinity, controlled by the channel gate conformation. According to these hypotheses, Gruber and Carmeliet’ performed a study in which they attempted to determine whether drug dissociation from the binding site or the channel gating mechanism controls the unbinding of the drugs from use dependent block. They pointed out some opposite behaviours between quinidine and disopyramide. For example, quinidine does not show voltage dependency of recovery like that obtained with disopyramide. Weld et a18 showed that the rate constant for dissociation of quinidine and quinidine blocked channels becomes larger as the transmembrane voltage becomes negative, differing from the behaviour of disopyramide. In terms of the size of the molecule, quinidine resembles to disopyramide. Thus they suggested that some characteristics such as the stereochemical properties of a molecule might be important for different properties. The aim of the present study is to clarify the mechanism whereby difference in sodium channel state dependent blocking effect of antiarrhythmic drugs on IN^ occurs in isolated heart cells. We compared the potency for producing tonic and use dependent block effects on IN, by quinidine and
First Department of Internal Medicine, Nippon Medical School, Tokyo 113, Japan: S Koumi, K Nagasawa, H Hayakawa; First Department of Internal Medicine, Kinki University School of Medicine, Osaka 589, Japan: R Sato, R Katori; First Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan: I Hisatome. Correspondence to Dr Koumi at: Reingold ECG Center (Department of Medicine), Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 6061 1-3008, USA.
1200
Koumi, Sato, Katori, Hisatome, Nagasawa, Hayakawa
disopyramide, as well as the kinetics of sodium channel block according to the sodium channel states. Preliminary results have been reported in abstract form.' Methods Cell preparation Single ventricular myocytes of adult guinea pig heart were obtained by an enzymatic dissociation method similar to that described Briefly, guinea pigs of either sex weighing 300-400 g, were anaesthetised with intraperitoneal diethyl ether (1 m1.kg-I). The chest was opened under artificial ventilation and the aorta was cannulated in situ. The heart was excised and mounted on a Langendorff type apparatus. Blood was washed out of the coronary arteries by retrograde perfusion with 30 ml of Tyrode's solution under a hydrostatic pressure of about 70 cm HzO. The heart was then perfused with 50 ml of nominally Ca" free Tyrode's solution followed by perfusion with Ca" free Tyrode's solution containing 0.04% collagenase (Sigma, type I, St Louis, MO, USA) for 10-20 min. The collagenase was washed out by perfusion with 60 ml of a high potassium, low chloride Krebs buffer solution. All perfumes were bubbled with 100% 0 2 and warmed to 37°C. After washing out the collagenase, the heart was gently agitated in the Krebs buffer solution, and then stored in this solution at 4°C for at least h before starting the experiments. Only those cells which were Ca-' tolerant, clearly striated. and rod shaped without any blebs on the surface were used in these experiments. Animal maintenance and the procedures performed in the present study conformed with the Guide for the care and use of laboratory animals published by the US National Institutes of Health. Soluticm and drugs The control Tyrode's solution contained (in mmol4tre-I): NaCl 136.5, KCI 5.4, CaCh 1.8, MgClz 0.53, glucose 10. and N-2-hydroxyethylpiperazine-N'-2-ethanesulphonicacid (HEPES)-NaOH buffer 5.0 (pH=7.4). Ca" free Tyrode's solution was made by omitting CaCh from the normal Tyrode's solution. The composition of the external solution (mmoLlitre-') was: NaCl 25, CsCl 5, CaClz 1.8, MgCh 0.5, CoCh 1.0. tetraethylammonium chloride 90, HEPES 20. and glucose 10; the pH was adjusted to 7.4 with tetraethylammonium hydroxide. A low external Na' concentration was used to reduce the magnitude of IN^, so the membrane potential could be adequately controlled. The standard pipette solution (internal solution) contained (in mmol4itre-I) NaF 10, CsF 125, and HEPES 5 , and the pH was adjusted to 7.2 by adding CsOH. Using these solutions resulted in INa being the only measurable current generated in response to the applied test potentials. Replacing K' with Cs' and adding tepaethylammonium to the external solutions eliminated K' currents. Ca-' currents were blocked by internal F and external Co*'. The Co" in the external solution did not alter the time course or voltage dependence of INa. The modified Krebs buffer solution had the following composition (in mmol.litre-I): taurine 20, oxalic acid 10, glutamic acid 70, KCI 25, KH~POJ10, MgSOl 5.0, HEPES 10, glucose I I , and ethyleneglycolbis (p aminoethy1ether)N N'-tetra-acetic acid (EGTA) 0.5; the pH was adjusted to 7.2 with KOH." l 3 The desired concentrations of quinidine and disopyramide were made by diluting a 1 mM stock solution. No recordings were made for 5-10 min after adding quinidine or disopyramide to the external solution to allow INI to reach steady state. Quinidine (quinidine hydrochloride, MW 324.4).
disopyramide (disopyramide phosphate, MW 339.5), and chloramine-T (N-chloro-p-toluensulphonamide sodium salt) were obtained from Sigma.
Electrical measurement The membrane currents were studied using the whole cell patch clamp technique described by Hamill et The electrodes were pulled in two stages from microhaematocrit tubes (Drummond Scientific Co, USA), using a vertical microelectrode puller (Type PE-2, Narishige, Tokyo, Japan). The electrodes had diameters of 2.5 to 3.5 FM and resistances of 0.5 to 0.8 MR after being fire polished and tilled with the internal solution. Inverted voltage clamp pulses were applied to the bath through an Ag-AgCI pellet-KCI agar bridge. The pipette potential was maintained at ground level. The head stage of the voltage clamp circuit had an ultra low bias current operational amplifier. The patch electrode was connected to the negative input with a feedback register of 100 M a The voltage error due to the series resistance, attributed to the pipette tip and the cell interior, was compensated by summing a fraction of the converted current signal to the command potential, and feeding it to the positive input of the operational amplifier. The capacitative transient remaining after series resistance compensation was constant throughout the experiment. The pipette potential was adjusted to give zero current when both the pipette and the bath contained normal Tyrode's solution. After positioning the pipette tip against the cell surface, a gigaohm seal was formed by applying 20-30 cm HzO of suction to the pipette. After waiting several minutes to ensure complete exchange of the intrapipette solution, the cell membrane was ruptured by briefly applying additional suction to the pipette. Membrane rupture was indicated by an increase in the capacitative transient. Series resistance compensation was done to minimise the capacitative surge. To obtain rapid and uniform control of the membrane potential and minimise voltage errors related to the flow of IN^ across the series resistance, membrane current was recorded using low resistance electrodes (0.5-0.8 MR) and an external solution with 25 mM Na' at a temperature of 17- 19°C. Moreover, all other ionic currents that could have interfered with the measurement of I N s were eliminated as described previously. Under these conditions, leak current at the test potential (-30 mV) was less than 0.1 nA. Cell capacitance (Cm) was estimated from the current transient produced by a small (10 mV) voltage clamp step, and determined by integrating the current transient; Cm=74 (SD 6) pF (n=8). Series resistance (Rs) was determined by fitting exponentials to the current transient, which was well described by a single exponential. Rs was estimated from the time constant ( 7 ) of the capacitative transient on the assumption of ?.=RsCm. Mean time constant was 98(8) ks (n=8). and Rs was 1.3(0.4) M a Data analysis The voltage and current signals were displayed on a storage oscilloscope (Type 51 13, Tektronix) and were stored on digital audio tape (R-60DM. Maxell) using a PCM data recording system (RD-IOOT. Teac). Analysis of the data was performed on a computer (PC-9801. NEC) using custom software. All curve fitting was done with a nonlinear least squares algorithm using a Marquardt routine." The results are expressed as mean(SD). Statistical analysis was done using paired Student's t test, and the results were considered to be significant when the p value was less than 0.05.
A
Figure 1 Blockade of IN(, by 10 pM
quinidine or 20 pM disopyramide. Panel A (part a): pulse protocol. Holding potential was kept at -140 mV and depolarised by test pulses ranging from -70 mV to +SO mV with 30 ins duration in every 30 s. Families of IN^ before and after application of quinidine are shown in parts (b) and ( c ) respectively. The peak amplitude was reduced 22.5% by quinidine. Panel B shows I-V relationships for peak IN"shown in panel A. Empty circles indicate the control IN,,,while jilled circles represent IN"following exposure to quinidine. Peak potential and reversal potential remained constant, at -30 mV and +42 m y respectively. Panel C: I-V curve before and afer application of 20 pM disopyramide. Empty circles denote the control IN"and jilled circles indicate disopyramide treated IN'$.The peak amplitude was reduced 18.7% by disopyramide. The shape ($1-V curve was not changed by discipyrainide.
(a)
(b) Control
(c) Quinidine 10 (LM
L
Nu' channel states control blocking behaviour of drugs
1201
100
C
0 .-c .-
r
.-C m
z
0 41
lo-'
10
Dose (FM)
50,
10-3
t
1
Figure 2 Dose dependent inhibitory effect of IN^ by quinidine (circles) and disopyramide (squares). Percent inhibition of peak IN^ on I-V curve corresponding to that of the control value is plotted against drug concentration. Holding potential was -140 m C: and test pulses of 30 ms duration were applied to -30 mCI The dissociation constant (KD)was 17(SD 4.1) p M in quinidine and 36(5.4) pV in disopyramide. Values are means, bars=SD, for four preparations. The continuous curves are least squares fitting to equation (I) in the text.
Results Tonic and use dependent block Blocking effects of quinidine (10 FM) and disopyramide (20 FM) on INn are shown in fig 1. Families of IN^ recordings before and after treatment with 10 FM quinidine are illustrated in fig 1A. Quinidine produced a decrease in INa, but this inhibitory effect was reversible after washing out the drug. The peak current-voltage (I-V) relationships presented in fig 1B were made using the IN, traces shown in fig 1A. Quinidine (10 FM) had no effect on the threshold, peak, or reversal potentials of IN,. However, peak amplitude of IN^ was suppressed by 22(SD 3.8)% (n=5). Disopyramide (20 p,M) also depressed IN^ by 18(3.1)% (n=5) (fig 1C). This result showed that both quinidine and disopyramide produce tonic block of IN,. Figure 2 shows a plot of the dose-effect relationship of the both drugs on IN,. The half blocking concentrations (KD) of quinidine and disopyramide were 17(4.1) FM and 36(5.4) FM (n=5), respectively. The data were well fitted by the equation: Percent inhibition of IN,= 1/(1 +KD/[M])
(1)
where [MI is the drug concentration and KD is the apparent dissociation constant. Hill plots of the data of quinidine and disopyramide had slopes of 0.92 and 0.95, respectively. These findings may indicate the presence of one to one stoichiometry of the drug molecule-sodium channel reaction proposed for the action of both drugs. In addition to producing tonic block of IN,, both quinidine and disopyramide suppressed IN, in a use dependent manner. Whereas IN, amplitude remained constant during repetitive stimuli under the control conditions, drastic decreases were observed in the presence of quinidine of disopyramide. The time course of use dependent block is clearly seen in fig 3 in which the current amplitude associated with each pulse is plotted as a function of pulse number. As shown in fig 3, quinidine had a higher potency for producing use dependent block compared with disopyramide. A similar level of use
10
I
30
20
Pulse number
Figure 3 Use dependent block of IN" by quinidine and disopyramide. The membrane potentid was held at -140 m y arid depalarised to -30 mVfiir 16 ms at u frequency of 2 Hz as shown in inset. Peak IN,,, expressed as a percentage of peak IN'(for the first depolarising puOe, is plotted as ti function of pulse number for each of the two different concentrations o j quinidine and disopyrumide: empty circles indicate 10 p M yuinidine; filled circles 20 phi quinidine; empty squares 10 pA4 disopyramide; and filled squares 20 pM disopyramide. Find blocked ratio for each concentration was 24.7% for 10 p M quinidine, 30.6% for 20 pkl quinidine, 18.1% for 10 pV disopyramide, and 26.5% for 20 pM disopyramide.
dependent blocking effect was obtained at the concentrations of 10 FM with quinidine and 20 p,M with disopyramide.
Effects of ubolishing IN
1199
Sodium channel states control binding and unbinding behaviour of antiarrhythmic drugs in cardiac myocytes from the guinea pig Shin-ichi Koumi, Ryoichi Sato, Ryo Katori, Ichiro Hisatome, Kouichi Nagasawa, and Hirokazu Hayakawa Objective: The aim was to investigate whether cardiac sodium channel states (rested, activated, inactivated) regulate the binding and unbinding behaviour of antiarrhythmic drugs on the receptor sites. Methods: Single ventricular myocytes of adult guinea pig heart were obtained by an enzymatic dissociation method in the Langendorff manner. The channel state dependent blocking effects on cardiac sodium current (IN,) of quinidine and disopyramide were studied under the whole cell variation of the patch clamp technique. Results: 10 p,M quinidine and 20 pM disopyramide produced similar levels of tonic block and use dependent block. The steady state inactivation curve (h- curve) was shifted parallel in the negative potential direction by quinidine (10 kM) and disopyramide (20 pM) to the same extent (-10 mV). Removal of the fast inactivation process of IN^ by chloramine-T did not reduce tonic and use dependent block by these drugs. Onset block study using a double pulse protocol revealed that block developments by both drugs were fitted to the sum of double exponential functions. However, time constant of fast phase of block by disopyramide was faster than that by quinidine, while slow phase was not significantly different. Definition of time courses of unbinding (recovery) at -140 mV indicated that quinidine dissociated relatively slowly as compared to disopyramide. Conclusions: Quinidine produces more potent tonic and use dependent block of IN, by binding to sodium channels at both rested and inactivated states, while disopyramide has a higher affinity for activated state. Therefore, sodium channel states regulate the binding and unbinding behaviour of antiarrhythmic drugs. Furthermore, the fast inactivation process is not essential in producing tonic and use dependent block by antiarrhythmic drugs. Cardiovascular Research 1992;26: 1199-1205
0
uinidine and disopyramide, both of which are classified as class Ia antiarrhythmic drugs, produce similar electrophysiological effects in cardiac -tissues. Both drugs depress the maximum rate of depolarisation, increase the conduction time, and prolong the terminal phase of repolarisation. By using the single cell preparation, the effect of quinidine and disopyramide on the maximum rate of depolarisation was found to be brought about by sodium channel blockade.’” In order to explain drug-sodium channel interaction, two different hypotheses have been proposed: the modulated receptor hypothesis4 and the guarded receptor hypothesis.6 According to the former hypothesis, drugs bind to a sodium channel receptor site, and the affinity of the receptor of the drug is modulated by states of channel. The drug associated channels differ from the drug free channels in that they do not conduct, and their ability to be activated is shifted toward more negative potential; the affinity of the binding site for drugs is higher when the channel is open or inactivated than when the channel is in the rested state. In contrast, the guarded receptor hypothesis assumes use dependent block as the result of transient access to a binding site of constant
affinity, controlled by the channel gate conformation. According to these hypotheses, Gruber and Carmeliet’ performed a study in which they attempted to determine whether drug dissociation from the binding site or the channel gating mechanism controls the unbinding of the drugs from use dependent block. They pointed out some opposite behaviours between quinidine and disopyramide. For example, quinidine does not show voltage dependency of recovery like that obtained with disopyramide. Weld et a18 showed that the rate constant for dissociation of quinidine and quinidine blocked channels becomes larger as the transmembrane voltage becomes negative, differing from the behaviour of disopyramide. In terms of the size of the molecule, quinidine resembles to disopyramide. Thus they suggested that some characteristics such as the stereochemical properties of a molecule might be important for different properties. The aim of the present study is to clarify the mechanism whereby difference in sodium channel state dependent blocking effect of antiarrhythmic drugs on IN^ occurs in isolated heart cells. We compared the potency for producing tonic and use dependent block effects on IN, by quinidine and
First Department of Internal Medicine, Nippon Medical School, Tokyo 113, Japan: S Koumi, K Nagasawa, H Hayakawa; First Department of Internal Medicine, Kinki University School of Medicine, Osaka 589, Japan: R Sato, R Katori; First Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan: I Hisatome. Correspondence to Dr Koumi at: Reingold ECG Center (Department of Medicine), Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 6061 1-3008, USA.
1200
Koumi, Sato, Katori, Hisatome, Nagasawa, Hayakawa
disopyramide, as well as the kinetics of sodium channel block according to the sodium channel states. Preliminary results have been reported in abstract form.' Methods Cell preparation Single ventricular myocytes of adult guinea pig heart were obtained by an enzymatic dissociation method similar to that described Briefly, guinea pigs of either sex weighing 300-400 g, were anaesthetised with intraperitoneal diethyl ether (1 m1.kg-I). The chest was opened under artificial ventilation and the aorta was cannulated in situ. The heart was excised and mounted on a Langendorff type apparatus. Blood was washed out of the coronary arteries by retrograde perfusion with 30 ml of Tyrode's solution under a hydrostatic pressure of about 70 cm HzO. The heart was then perfused with 50 ml of nominally Ca" free Tyrode's solution followed by perfusion with Ca" free Tyrode's solution containing 0.04% collagenase (Sigma, type I, St Louis, MO, USA) for 10-20 min. The collagenase was washed out by perfusion with 60 ml of a high potassium, low chloride Krebs buffer solution. All perfumes were bubbled with 100% 0 2 and warmed to 37°C. After washing out the collagenase, the heart was gently agitated in the Krebs buffer solution, and then stored in this solution at 4°C for at least h before starting the experiments. Only those cells which were Ca-' tolerant, clearly striated. and rod shaped without any blebs on the surface were used in these experiments. Animal maintenance and the procedures performed in the present study conformed with the Guide for the care and use of laboratory animals published by the US National Institutes of Health. Soluticm and drugs The control Tyrode's solution contained (in mmol4tre-I): NaCl 136.5, KCI 5.4, CaCh 1.8, MgClz 0.53, glucose 10. and N-2-hydroxyethylpiperazine-N'-2-ethanesulphonicacid (HEPES)-NaOH buffer 5.0 (pH=7.4). Ca" free Tyrode's solution was made by omitting CaCh from the normal Tyrode's solution. The composition of the external solution (mmoLlitre-') was: NaCl 25, CsCl 5, CaClz 1.8, MgCh 0.5, CoCh 1.0. tetraethylammonium chloride 90, HEPES 20. and glucose 10; the pH was adjusted to 7.4 with tetraethylammonium hydroxide. A low external Na' concentration was used to reduce the magnitude of IN^, so the membrane potential could be adequately controlled. The standard pipette solution (internal solution) contained (in mmol4itre-I) NaF 10, CsF 125, and HEPES 5 , and the pH was adjusted to 7.2 by adding CsOH. Using these solutions resulted in INa being the only measurable current generated in response to the applied test potentials. Replacing K' with Cs' and adding tepaethylammonium to the external solutions eliminated K' currents. Ca-' currents were blocked by internal F and external Co*'. The Co" in the external solution did not alter the time course or voltage dependence of INa. The modified Krebs buffer solution had the following composition (in mmol.litre-I): taurine 20, oxalic acid 10, glutamic acid 70, KCI 25, KH~POJ10, MgSOl 5.0, HEPES 10, glucose I I , and ethyleneglycolbis (p aminoethy1ether)N N'-tetra-acetic acid (EGTA) 0.5; the pH was adjusted to 7.2 with KOH." l 3 The desired concentrations of quinidine and disopyramide were made by diluting a 1 mM stock solution. No recordings were made for 5-10 min after adding quinidine or disopyramide to the external solution to allow INI to reach steady state. Quinidine (quinidine hydrochloride, MW 324.4).
disopyramide (disopyramide phosphate, MW 339.5), and chloramine-T (N-chloro-p-toluensulphonamide sodium salt) were obtained from Sigma.
Electrical measurement The membrane currents were studied using the whole cell patch clamp technique described by Hamill et The electrodes were pulled in two stages from microhaematocrit tubes (Drummond Scientific Co, USA), using a vertical microelectrode puller (Type PE-2, Narishige, Tokyo, Japan). The electrodes had diameters of 2.5 to 3.5 FM and resistances of 0.5 to 0.8 MR after being fire polished and tilled with the internal solution. Inverted voltage clamp pulses were applied to the bath through an Ag-AgCI pellet-KCI agar bridge. The pipette potential was maintained at ground level. The head stage of the voltage clamp circuit had an ultra low bias current operational amplifier. The patch electrode was connected to the negative input with a feedback register of 100 M a The voltage error due to the series resistance, attributed to the pipette tip and the cell interior, was compensated by summing a fraction of the converted current signal to the command potential, and feeding it to the positive input of the operational amplifier. The capacitative transient remaining after series resistance compensation was constant throughout the experiment. The pipette potential was adjusted to give zero current when both the pipette and the bath contained normal Tyrode's solution. After positioning the pipette tip against the cell surface, a gigaohm seal was formed by applying 20-30 cm HzO of suction to the pipette. After waiting several minutes to ensure complete exchange of the intrapipette solution, the cell membrane was ruptured by briefly applying additional suction to the pipette. Membrane rupture was indicated by an increase in the capacitative transient. Series resistance compensation was done to minimise the capacitative surge. To obtain rapid and uniform control of the membrane potential and minimise voltage errors related to the flow of IN^ across the series resistance, membrane current was recorded using low resistance electrodes (0.5-0.8 MR) and an external solution with 25 mM Na' at a temperature of 17- 19°C. Moreover, all other ionic currents that could have interfered with the measurement of I N s were eliminated as described previously. Under these conditions, leak current at the test potential (-30 mV) was less than 0.1 nA. Cell capacitance (Cm) was estimated from the current transient produced by a small (10 mV) voltage clamp step, and determined by integrating the current transient; Cm=74 (SD 6) pF (n=8). Series resistance (Rs) was determined by fitting exponentials to the current transient, which was well described by a single exponential. Rs was estimated from the time constant ( 7 ) of the capacitative transient on the assumption of ?.=RsCm. Mean time constant was 98(8) ks (n=8). and Rs was 1.3(0.4) M a Data analysis The voltage and current signals were displayed on a storage oscilloscope (Type 51 13, Tektronix) and were stored on digital audio tape (R-60DM. Maxell) using a PCM data recording system (RD-IOOT. Teac). Analysis of the data was performed on a computer (PC-9801. NEC) using custom software. All curve fitting was done with a nonlinear least squares algorithm using a Marquardt routine." The results are expressed as mean(SD). Statistical analysis was done using paired Student's t test, and the results were considered to be significant when the p value was less than 0.05.
A
Figure 1 Blockade of IN(, by 10 pM
quinidine or 20 pM disopyramide. Panel A (part a): pulse protocol. Holding potential was kept at -140 mV and depolarised by test pulses ranging from -70 mV to +SO mV with 30 ins duration in every 30 s. Families of IN^ before and after application of quinidine are shown in parts (b) and ( c ) respectively. The peak amplitude was reduced 22.5% by quinidine. Panel B shows I-V relationships for peak IN"shown in panel A. Empty circles indicate the control IN,,,while jilled circles represent IN"following exposure to quinidine. Peak potential and reversal potential remained constant, at -30 mV and +42 m y respectively. Panel C: I-V curve before and afer application of 20 pM disopyramide. Empty circles denote the control IN"and jilled circles indicate disopyramide treated IN'$.The peak amplitude was reduced 18.7% by disopyramide. The shape ($1-V curve was not changed by discipyrainide.
(a)
(b) Control
(c) Quinidine 10 (LM
L
Nu' channel states control blocking behaviour of drugs
1201
100
C
0 .-c .-
r
.-C m
z
0 41
lo-'
10
Dose (FM)
50,
10-3
t
1
Figure 2 Dose dependent inhibitory effect of IN^ by quinidine (circles) and disopyramide (squares). Percent inhibition of peak IN^ on I-V curve corresponding to that of the control value is plotted against drug concentration. Holding potential was -140 m C: and test pulses of 30 ms duration were applied to -30 mCI The dissociation constant (KD)was 17(SD 4.1) p M in quinidine and 36(5.4) pV in disopyramide. Values are means, bars=SD, for four preparations. The continuous curves are least squares fitting to equation (I) in the text.
Results Tonic and use dependent block Blocking effects of quinidine (10 FM) and disopyramide (20 FM) on INn are shown in fig 1. Families of IN^ recordings before and after treatment with 10 FM quinidine are illustrated in fig 1A. Quinidine produced a decrease in INa, but this inhibitory effect was reversible after washing out the drug. The peak current-voltage (I-V) relationships presented in fig 1B were made using the IN, traces shown in fig 1A. Quinidine (10 FM) had no effect on the threshold, peak, or reversal potentials of IN,. However, peak amplitude of IN^ was suppressed by 22(SD 3.8)% (n=5). Disopyramide (20 p,M) also depressed IN^ by 18(3.1)% (n=5) (fig 1C). This result showed that both quinidine and disopyramide produce tonic block of IN,. Figure 2 shows a plot of the dose-effect relationship of the both drugs on IN,. The half blocking concentrations (KD) of quinidine and disopyramide were 17(4.1) FM and 36(5.4) FM (n=5), respectively. The data were well fitted by the equation: Percent inhibition of IN,= 1/(1 +KD/[M])
(1)
where [MI is the drug concentration and KD is the apparent dissociation constant. Hill plots of the data of quinidine and disopyramide had slopes of 0.92 and 0.95, respectively. These findings may indicate the presence of one to one stoichiometry of the drug molecule-sodium channel reaction proposed for the action of both drugs. In addition to producing tonic block of IN,, both quinidine and disopyramide suppressed IN, in a use dependent manner. Whereas IN, amplitude remained constant during repetitive stimuli under the control conditions, drastic decreases were observed in the presence of quinidine of disopyramide. The time course of use dependent block is clearly seen in fig 3 in which the current amplitude associated with each pulse is plotted as a function of pulse number. As shown in fig 3, quinidine had a higher potency for producing use dependent block compared with disopyramide. A similar level of use
10
I
30
20
Pulse number
Figure 3 Use dependent block of IN" by quinidine and disopyramide. The membrane potentid was held at -140 m y arid depalarised to -30 mVfiir 16 ms at u frequency of 2 Hz as shown in inset. Peak IN,,, expressed as a percentage of peak IN'(for the first depolarising puOe, is plotted as ti function of pulse number for each of the two different concentrations o j quinidine and disopyrumide: empty circles indicate 10 p M yuinidine; filled circles 20 phi quinidine; empty squares 10 pA4 disopyramide; and filled squares 20 pM disopyramide. Find blocked ratio for each concentration was 24.7% for 10 p M quinidine, 30.6% for 20 pkl quinidine, 18.1% for 10 pV disopyramide, and 26.5% for 20 pM disopyramide.
dependent blocking effect was obtained at the concentrations of 10 FM with quinidine and 20 p,M with disopyramide.
Effects of ubolishing IN
