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Sodium Channel-Blocking Properties of Spiradoline, a κ Receptor Agonist, are Responsible for Its Antiarrhythmic Action in the Rat

Pugsley, Michael K.; Saint, David A.*; Hayes, Eric S.; Kramer, David; Walker, M. J. A.

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Journal of Cardiovascular Pharmacology: December 1998 - Volume 32 - Issue 6 - p 863-874
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Abstract

Several kappa (κ) receptor agonists have "nonopioid" actions on both sodium and potassium currents in heart (1,2) and in neuronal tissue (3-5). Other studies confirm that the antiarrhythmic actions are due to block of the cardiac ion channel(s) (1,2,6,7). Such studies used nonopioid enantiomers such as (+)PD 129,289, an arylbenzacetamide similar in chemical structure to U-50,488H and opioid antagonists, such as naloxone and MR 2266 (1,8).

Spiradoline (U-62,066E) is a centrally acting, selective κ opioid receptor agonist (9). In mice and rats, spiradoline is 5-25 times more potent in analgesia tests than U-50,488H and 1.5-7 times as potent as morphine (10). Spiradoline does not produce physical dependence (9) but is analgesic (10) and has cross-tolerance with U-50,488H on prolonged exposure (9). In dogs and cats, spiradoline produces a naloxone-reversible cardiovascular depression (11) and is a potent diuretic (12).

In this study we examined the actions of spiradoline in isolated rat hearts and in anesthetized rats in an attempt to determine its cardiac and cardiovascular actions and its effectiveness against both electrically induced and ischemia-induced arrhythmias. Studies were performed in the absence and presence of naloxone in an attempt to identify nonopioid properties. Changes observed in the ECG suggested blockade of cardiac ionic currents, and so an electrophysiologic profile study was performed in isolated rat myocytes for the actions on sodium and the transient outward (Ito) and sustained delayed-rectifier plateau (IKsus) potassium currents. These electrophysiologic studies helped to explain actions observed in vivo. It was shown that spiradoline produces "mixed" cardiac ion channel blockade suggestive of class Ia antiarrhythmic actions and also produces tonic and use-dependent blockade of myocardial sodium currents not readily observed with related κ-agonists such as U-50,488H.

MATERIALS AND METHODS

Male Sprague-Dawley rats (U.B.C. Animal Care Center) weighing between 200 and 300 g were used for whole animal and isolated heart studies at U.B.C. Male Wistar rats (200-300 g) were used for isolated cell studies in Canberra. All studies conducted at U.B.C. were performed according to the guidelines established by the Animal Care Committee, whereas those performed in Australia were conducted according to guidelines established by the Australian National University.

Isolated perfused rat hearts

Hearts were mounted on a modified Langendorff apparatus (13) and perfused at an aortic root pressure of 100 mm Hg with Krebs-Henseleit solution (pH 7.4), bubbled with 5% CO2 in O2 at a temperature of 35°C. A compliant saline-filled balloon at a diastolic pressure of 10 mm Hg was used to measure developed pressure and the derived maximal rate of intraventricular pressure development (+dP/dtmax) by means of a Grass differentiator (model 7P20C; Quincy, MA, U.S.A.). ECGs were recorded (band width, 0.1-40 Hz) from silver-ball electrodes placed on the epicardial surfaces of right atrium and left ventricle.

Animal preparation

Intact rats were anesthetized with pentobarbital (60 mg/kg, i.p.), and the trachea cannulated for artificial ventilation at a stroke volume of 10 ml/kg and at a rate of 60 strokes/min. A rectal thermometer and heating lamp were used to keep body temperature between 36 and 37°C. The right jugular vein and left carotid artery were cannulated for administration of drugs and recording of blood pressure (BP), respectively. The ECG was recorded according to the method of Penz et al. (14).

Spiradoline dose-response curves in vivo

Dose-response data were obtained for spiradoline, in the absence and presence of naloxone, in pentobarbital anesthetized, artificially ventilated rats (n = 5). Animals randomly received either an i.v. injection of saline (0.2 ml) or naloxone (8.0 μmol/kg) before dosing with spiradoline. Cumulative infusions of spiradoline (1.0, 2.0, 4.0, 8.0, and 16.0 μmol/kg/min, i.v.) were made, each over a 5-min period, and recordings were taken at the end of the infusion period just before the next infusion. The dose of naloxone was sufficient to provide sustained opioid receptor blockade without influencing ECG, BP, or heart rate (1,8).

Heart rate was calculated from the RR interval of ECG traces recorded on a Grass polygraph (model 7D), whereas the PR, QRS, and QaT intervals were measured directly. The QaT interval was not corrected for heart rate because, in the rat, this interval does not change with rate (15). RSh, an index of sodium channel blockade, also was measured (14,16).

Electrical-stimulation studies

By using the intact rats described, electrical stimulation of the left ventricle was accomplished via two Teflon-coated silver wire stimulating electrodes inserted through the chest wall and implanted into the left ventricle, as described by Walker and Beatch (17). This placement technique usually produced an interelectrode distance of <1 mm. Square-wave stimulation was used to determine threshold current (it-μA) and pulse width (tt-ms) for induction of extrasystoles. Ventricular fibrillation threshold (VFt-μA), maximal following frequency (MFF-Hz), and effective refractory period (ERP-ms) were determined according to Howard and Walker (18). Although MFF is approximately the inverse of ERP, we measured both values because there are certain differential sensitivities of each to various drugs (17). Drug was infused according to a random and blind protocol at the doses described. Electrical-stimulation measures were taken 3 min after increasing the dose of the drug by doubling the rate of infusion.

Coronary artery occlusion studies

The surgical procedures used were based on those described by Au et al. (19). In brief, rats were anesthetized and ventilated as described earlier. The left carotid artery cannula was used for recording BP and to remove blood samples (0.1 ml) for determination of serum K+ concentrations (Ionetics Potassium Analyzer, Cosa Mesa, CA, U.S.A.). A left thoracotomy between ribs five and six was used to expose the heart so that a polyethylene occluder could be placed around the left main coronary artery. The chest was closed, and the animal was allowed to recover for 20 min after surgical preparation.

Surgically prepared animals were given an initial injection of either saline or naloxone (8 μmol/kg) followed by a random and blind infusion of saline (at a rate of 0.016 ml/min) or spiradoline (2.5 μmol/kg/min). BP and ECG measures were taken 5 min after beginning infusion, whereas a blood sample (0.25 ml) was taken just before occlusion for determination of serum K+ levels.

ECG, arrhythmias, BP, heart rate, and mortality were monitored for 30 min after occlusion. Arrhythmias, consisting of ventricular premature beats (VPBs), ventricular tachycardia (VT), and ventricular fibrillation (VF) were recorded and summarized by the Arrhythmia Score (AS) of Curtis and Walker (20). At the end of the 30-min observation period, a second blood sample was taken from surviving animals. Hearts were then removed and perfused with cardiogreen dye (1.0 mg/ml) to reveal the underperfused occluded zone (zone at risk). Experimental design and analyses were performed according to guidelines established by the Lambeth Conventions (21) with replacement of lost animals to maintain experimental group balance.

Isolated ventricular myocyte studies

Enzymatic isolation of cardiac myocytes was performed according to the method of Farmer et al. (22). Male Wistar rats (300-350 g) were killed by cervical dislocation followed by exsanguination. Hearts were removed, washed in an ice-cold, oxygenated, Ca2+-free Tyrode's solution for 5 min before being perfused, via an aortic cannula, with the same Ca2+-free Tyrode's solution warmed to 37°C. The Tyrode's solution contained (in mM): NaCl, 134; TES (N-tris-(hydroxymethyl)methyl-2-amino ethanesulphonic acid), 10; KCl, 4; MgCl2, 1.2; NaH2PO4, 1.2; glucose, 11, and adjusted to pH 7.4 with 1.0 M NaOH. After a 5-min wash, the heart was enzymatically dissociated in Tyrode's solution supplemented with protease (0.1 mg/ml, Sigma Type XIV), collagenase (1.0 mg/ml, Worthington CLS II), fetal calf serum (1.0 μg/ml), and 25 μM Ca2+. Approximately 35-40 min later, the heart became pale and flaccid. The ventricles were removed in one-third sections, each of which was carefully cut into small pieces in fresh 25 μM Ca2+-Tyrode's solution and gently dissociated into separate myocytes. Cell suspensions were centrifuged, washed in a 200 μM Ca2+-Tyrode's solution, and resuspended in a 1 mM Ca2+-containing Tyrode's solution, 1 h before being plated onto glass coverslips. Prepared cells were stored at room temperature (22-24°C) and used within 12 h of isolation.

Solutions and drugs

All myocyte experiments were performed at room temperature in a bath solution containing (in mM): NaCl, 70; TES, 10; KCl, 5.4; MgCl2, 1.0; CaCl2, 2; CoCl2, 5; CsCl, 5; glucose, 10; choline Cl, 60; and adjusted to pH 7.4 with 1.0 M NaOH. The pipette solution used in conjunction with pulse patterns generated by custom software allowed recording of Na+ and K+ currents. It contained (in mM): KCl, 140; TES, 10; MgCl2, 1; K-EGTA, 10; CaCl2, 2; ATP-disodium, 10; and was pH adjusted to 7.4 with 1.0 M KOH. During the recording of Na+ currents, the pipette KCl solution was replaced with CsCl, and while we recorded K+ currents, 50 μM tetrodotoxin (TTX) was added to the bath solution.

Spiradoline (U-62,066E) (a gift from Dr. P.F. Von Voigtlander, The Upjohn Co., Kalamazoo, MI, U.S.A.), naloxone (Sigma Chemical Co., St. Louis, MO, U.S.A.), and TTX were initially solubilized in distilled water before i.v. injection or dissolved in external bath solution used for isolated hearts and single cardiac myocytes to give the desired concentrations. A low volume (0.5-1.0 ml) plexiglass recording bath allowed rapid exchange (1-2 s) between control and experimental solutions from gravity-flow reservoirs. A suction device ensured that drug solutions superfused cells at a flow rate of 1-2 ml/min and maintained a constant fluid level.

Electrophysiologic recording

Experiments were performed in a small plexiglass tissue bath mounted on an inverted Nikon microscope. Only cells that were quiescent, rod-shaped, and had clear striations were used. Electrodes were prepared from borosilicate glass by using a two-stage puller (Narishige Scientific Instruments, Tokyo, Japan). Only microelectrodes with a resistance between 1 and 5 MΩ were used. Myocyte currents were recorded 10 min after achieving a whole-cell patch-clamp configuration (23). All current measurements were performed by using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA, U.S.A.), which allowed compensation and reduction of both capacitance transients and leak currents from computer-generated voltage commands. Final capacitance and leak compensation was performed at the time of analysis by subtraction of the current produced by a 20-mV hyperpolarizing current pulse, which always preceded the test pulse. Currents were filtered at 5 kHz, sampled at 10 kHz by using a 12-bit A-D converter, and records saved on the hard drive for subsequent analysis.

Statistical analysis

Values are shown as the mean ± SEM for group size of n. Statistical analyses were performed by using an NCSS statistical package (24) at an α-level of 0.05. A general linear model analysis of variance (ANOVA) followed by Duncan's test for critical differences between means were used throughout the experiments, except in arrhythmia studies, in which VPB (ventricular premature beat) number was log10 transformed to facilitate quantification and statistical application. Mainland's contingency tables were used to determine significance between the incidence of events in different groups (25). Isolated heart data were expressed as percentage change from predrug values in the absence, or presence, of naloxone.

RESULTS

Isolated heart studies: contractility and ECG effects of spiradoline

Spiradoline produced concentration-dependent changes in heart rate and the PR interval and QRS duration of the isolated rat heart ECG. Figure 1A shows the concentration-related reduction in heart rate by spiradoline from 266 ± 12 beats/min in control to 74 ± 10 beats/min with 100 μM spiradoline (n = 5). PR-interval prolongation is shown in Fig. 1B. The PR interval changed from 69 ± 4 ms in control hearts to 189 ± 15 ms in 100 μM spiradoline; QRS width increased from 29 ± 2 ms in control hearts to 76 ± 14 ms (data not shown). Cardiac contractility assessed from left ventricle peak systolic pressure (+dP/dtmax) was reduced in a concentration-dependent manner by spiradoline. Peak systolic pressure was reduced by 54% with 100 μM spiradoline from a control of 93 ± 5 to 43 ± 11 mm Hg. The rates of intraventricular pressure development (+dP/dtmax) and relaxation (−dP/dtmax) also were reduced by spiradoline. The maximal control rates of contractility and relaxation were 2,080 ± 139 and 1,770 ± 167 mm Hg/s, and after 100 μM spiradoline exposure, the values were 850 ± 295 and 300 ± 91 mm Hg/s, respectively. Naloxone was devoid of any such actions at the concentrations examined and did not block any of the responses to spiradoline. Thus these experiments in isolated hearts suggest that spiradoline produces a direct depression of cardiac contractility, which may be related to ion channel blockade.

FIG. 1
FIG. 1:
Concentration-dependent effects in perfused rat hearts of spiradoline on heart rate (A), and PR interval of the ECG (B). A: Effects on heart rate in the absence (▵) and presence (▴) of 1 μM naloxone. B: The PR interval in the absence (▵) and presence (▴) of 1 μM naloxone. Values expressed as mean ± SEM for five animals/group. The effects of naloxone alone (□) as well as control (•) values are shown.

Effects in intact rats

Blood pressure, heart rate, and ECG actions of spiradoline. In saline control (n = 5) and 8.0 μmol/kg naloxone-treated (n = 5) animals, BP and heart rate were stable over the duration of the drug-infusion period (data not shown). Spiradoline, in the absence and presence of naloxone, produced a dose-dependent reduction in both BP and heart rate. At a dose of 32 μmol/kg/min, spiradoline reduced BP from 126 ± 7 to 51 ± 4 mm Hg (or −59%) and heart rate from 352 ± 14 to 124 ± 7 beats/min (or −65%). In the presence of naloxone, BP was equally reduced (from 118 ± 6 to 57 ± 7 mm Hg or −52%); however, heart rate was reduced by 44% (or from 360 ± 19 to 201 ± 17 beats/min).

ECG measures also were influenced in a dose-related manner by spiradoline. The highest dose, 32 μmol/kg/min, produced a 59% increase in the PR interval (from 61 ± 1 to 97 ± 2 ms). In animals administered naloxone before spiradoline, a 53% increase in PR-interval prolongation resulted at the same dose (from 62 ± 2 to 95 ± 3 ms; Fig. 2A). In a similar manner, the QRS width was prolonged by 50% (or from 30 ± 1 to 45 ± 2 ms) in the naloxone-pretreated spiradoline group, whereas spiradoline alone prolonged the QRS width 57%, from 29 ± 0.5 to 47 ± 3 ms (Fig. 2B). The QaT interval of the ECG, as well as the ERP, was prolonged by spiradoline infusion. In Fig. 2C, it can be seen that spiradoline, at doses >8 μmol/kg/min, produced an increase in the QaT interval. QaT was increased 44% by 32 μmol/kg/min spiradoline alone and 53% in the presence of naloxone. RSh, an index of sodium channel blockade in the rat, also was increased in a dose-dependent manner by spiradoline (Fig. 2D). The RSh interval was increased from 0.51 ± 0.05 to 1.03 ± 0.05 mV by 32 μmol/kg/min. In the presence of naloxone, the same dose increased RSh from 0.49 ± 0.03 to 1.15 ± 0.06 mV. The vehicle control did not affect any of the ECG measures over the duration of the experiment.

FIG. 2
FIG. 2:
Dose-related effects of spiradoline in pentobarbital-anesthetized rats on PR interval (A), QRS width (B), QaT interval (C), and RSh interval (D) in the absence and presence of 8.0 μmol/kg naloxone. Values expressed as mean ± SEM for n = 5. *Significant difference from pretreatment at p < 0.05 for spiradoline (▵), spiradoline + naloxone (▴), 8.0 μmol/kg naloxone alone (□), and saline vehicle (•), respectively.

Electrical-stimulation studies. The pattern of action of spiradoline in isolated hearts and intact rats indicated that spiradoline may block both sodium and potassium currents. To determine whether such properties protected against arrhythmias, we examined the effectiveness of spiradoline against electrically induced arrhythmias in the rat. Figures 3A and B show that spiradoline dose-dependently increased threshold current for capture or induction of extrasystoles (it) and ventricular fibrillation threshold (VFt)). The spiradoline threshold for capture, either in the absence or presence of naloxone, was increased 173 and 202% (from 86 ± 6 μA and 84 ± 9 μA to 235 ± 9 μA and 254 ± 11 μA, respectively), as shown in Fig. 3A. The dose-dependent changes in VFt produced by spiradoline were similar to those by it. VFt increased from 135 ± 9 to 534 ± 19 μA with spiradoline alone and from 124 ± 11 to 580 ± 26 μA with naloxone at the highest dose of spiradoline examined.

FIG. 3
FIG. 3:
Effects of spiradoline in pentobarbital-anesthetized rats on the threshold current for capture (it) (A) and ventricular fibrillation threshold (VFt) (B) in the absence (▵) and presence (▴) of 8.0 μmol/kg naloxone. Neither saline vehicle (•) or 8.0 μmol/kg naloxone (□) produced any effect on either measure. Values (mean ± SEM for five animals/group) were measured after three infusions.

Maximal following frequency (MFF) was significantly decreased by spiradoline. Spiradoline (32 μmol/kg/min) reduced MFF from 14.6 ± 0.8 Hz in control animals to 7.5 ± 1.1 Hz. In the presence of naloxone, the change was 13.5 ± 0.5 to 6.6 ± 0.5 Hz (Fig. 4A). The effective refractory period, ERP, also was dose-dependently prolonged by spiradoline in the absence and presence of naloxone pretreatment (Fig. 4B). At 32 μmol/kg/min, spiradoline prolonged ERP from 56 ± 3 to 129 ± 5 ms in the absence and 58 ± 4 to 152 ± 7 ms in the presence of naloxone. Saline control values for thresholds (it and VFt) and refractoriness (MFF and ERP) were constant over the treatment period.

FIG. 4
FIG. 4:
Electrical-stimulation measures of refractoriness in cardiac muscle, effective refractory period (ERP) (A) and maximal following frequency (MFF) (B), are dose-dependently increased and decreased, respectively, with spiradoline in the absence (▵) and presence (▴) of 8.0 μmol/kg naloxone. Neither saline vehicle (•) or 8.0 μmol/kg naloxone (□) produced an effect. Values expressed as mean ± SEM for five animals/group.

Ischemic arrhythmia study. In the coronary occlusion-arrhythmia study, a dose of spiradoline (2.0 μmol/kg/min) was chosen that produced minimal changes in the ECG and that only slightly reduced BP (Table 1). None of the changes produced by spiradoline (2.5 μmol/kg/min) were prevented by naloxone pretreatment. Spiradoline (2.5 μmol/kg/min) statistically significantly reduced arrhythmias induced by coronary occlusion (Table 2). The incidence of both ventricular tachycardia and fibrillation were reduced almost equally by spiradoline. Naloxone itself appeared to reduce arrhythmia incidence and, when given in combination with spiradoline, abolished VF.

TABLE 1
TABLE 1:
The cardiovascular and ECG effects of 2.5 μmol/kg/min spiradoline, in the absence and presence of 8.0 μmol/kg naloxone-antiarrhythmic study
TABLE 2
TABLE 2:
Antiarrhythmic effect of spiradoline in the absence and presence of 8.0 μmol/kg naloxone against coronary artery occlusion-induced arrhythmias in pentobarbital-anesthetized rats

The reduction in arrhythmia incidence could not be ascribed to changes in either occlusion-zone size (zone at risk) or serum potassium concentration. Table 3 shows that there were no significant differences between the occluded-zone size between groups, so the arrhythmic insult to the myocardium was assumed to be the same. Similarly, serum potassium concentrations were not influenced by drug treatment. Postocclusion serum potassium levels were only slightly increased but not significantly different between groups. The time to ST- and R-wave maxima, indices of ischemia, were prolonged after spiradoline treatment, regardless of naloxone pretreatment. Naloxone itself slowed the time to development of an R-wave maximum.

TABLE 3
TABLE 3:
The actions of 2.5 μmol/kg/min spiradoline, in the absence and presence of naloxone, on ECG changes, mortality, and serum potassium levels produced by coronary artery occlusion in pentobarbital-anesthetized rats

Whole-cell voltage-clamp studies in cardiac myocytes

Concentration-response curves. Spiradoline produced a concentration-dependent reduction in the magnitude of the sodium current in isolated rat myocytes at concentrations of spiradoline that produced marked effects on isolated hearts. Figure 5A shows data obtained from an experiment in which sodium currents were evoked by a voltage step to 0 mV from a prepulse potential of −150 mV. These voltage steps were given at 6-s intervals, and currents generated were plotted as a function of time. Spiradoline (15-500 μM) produced a reversible concentration-dependent block of sodium current. Concentration-response experiments were repeated in four cells, and a similar dose-response relation was obtained for each cell. Figure 5B describes the concentration-response curve (n = 5 cells) for the degree of sodium current block by spiradoline from individual cells. An estimate of the half-maximal sodium current-blocking concentration (EC50) was made from these data by using the best fits to the equation fractional block of INa = 1/1 + (KA/[A]). The EC50 was 66 μM. In the presence of 1 μM naloxone, there was no change in the concentration-response curve (data not shown).

FIG. 5
FIG. 5:
Effect of spiradoline on sodium currents in rat cardiac myocytes. A: Sodium currents evoked by a voltage-step from a prepulse potential of −150 mV to a potential of 0 mV. The voltage step was delivered at 6-s intervals, and spiradoline was added to the bath solution at the concentrations indicated. The recontrol current was indistinguishable from predrug controls. Spiradoline was added to the bath solution for 2 min before evoking currents. B: Concentration-response curve for the degree of blockade of sodium current by spiradoline. The degree of block, 1-[I(control)-I(block)/I(control)], is shown as a function of log10 concentration of spiradoline. Data from five individual cells are plotted with the line of best fit for the equation fractional block of INa = 1/(1 + (Ka/[A])) shown.

Current-voltage effects of spiradoline-activation/inactivation kinetics. The characteristics of sodium current block by spiradoline were examined for the voltage dependence of activation and inactivation (h∞). The effect of 150 μM spiradoline was examined on the voltage dependence of activation of the sodium current by using voltage steps to various test potentials between −70 and +50 mV from a fixed prepulse potential of −150 mV. The peak current is shown in Fig. 6A plotted against the prepulse potential. To examine effects on activation kinetics, we examined peak sodium conductance (GNa) versus membrane potential (EM). Conductance (GNa) was calculated by using the Hodgkin-Huxley model [INa = gNa × (V − Erev)] and approximated by a Boltzmann equation (Equation (1)) where Gmax is the maximal channel conductance for sodium, V′ is the voltage at which GNa is half-maximal, k the slope factor, and Erev the reversal potential for sodium. In Fig. 6A, the control peak Gmax was 1.49 nS, with a V′ of −42 mV, a slope factor (k) of 3.0 mV per e-fold change in INa and a reversal potential (Erev) of 47 mV. After exposure to 150 μM spiradoline, values were 0.22 nS, −42 mV, 3.0 mV per e-fold change in INa, and Erev of 47 mV. Thus spiradoline reduced INa by decreasing Gmax and not by producing a change in the voltage dependence of the current.

FIG. 6
FIG. 6:
Effect of spiradoline on the current-voltage relation (A) and inactivation kinetics (h) (B) of sodium currents. A: Currents were evoked by a voltage pulse to potentials of −70 to +50 mV from a potential of −150 mV. Peak current amplitude is plotted against potential for control data (•) and in the presence of 150 μM spiradoline (✦). Conductance (GNa) was calculated from the Boltzmann equation INa = Gmax/[1 + exp(V − V′/k)] × (V − Erev). B: sodium currents were evoked by a voltage step to −20 mV from a prepulse potential that varied between −140 and −30 mV. The peak current amplitude is shown plotted against the prepulse potential for control and recontrol data (•) and in the presence of 150 μM spiradoline (✦). The data are shown by the best-fit line(s) with the Boltzmann equation y = Gmax/1 + exp[(V − V′)/k]. The best fits for the control curve were obtained for values of Gmax = 0.92 nS, V′ = −58.6 mV and k = 7.0 mV per e-fold change in INa. The dotted-line curve shows the data in the presence of spiradoline scaled to the same maximum as control/recontrol data. This scaled curve resulted in values of Gmax = 0.93 nS (from a Gmax of 0.17 nS), V′ = −78.2 mV, and k = 8.5 mV per e-fold shift in INa.

The effects of spiradoline on inactivation were studied by using voltage steps to −20 mV from prepulse potentials from −140 to −30 mV. These studies showed that spiradoline changed the steady-state inactivation, as shown in Fig. 6B. A Boltzmann equation (Equation (2)) was used to derive a curve for the data obtained under control conditions and during the application of 150 μM spiradoline. The presence of 50 and 150 μM spiradoline concentration was accompanied by a shift in the voltage dependence of INa by 21 ± 2.9 and 24 ± 2.2 mV, respectively, to hyperpolarized potentials (n = 6 cells each, data for 50 μM concentrations not shown in Fig. 6B). This shift is revealed only when the curve in the presence of spiradoline is scaled to the control maximum. As well, 150 μM spiradoline increased the slope factor, k, from 7 mV in control to 8.5 mV per e-fold change in INa after drug exposure. However, the maximal current amplitude was greatly reduced by 150 μM spiradoline, even at very negative values of the prepulse potential, which indicates that the shift in the voltage dependence of inactivation may not be the principal means by which current block is produced.

Tonic and use-dependent components of spiradoline block. In an attempt to determine whether spiradoline produced frequency-dependent blockade, we evoked sodium currents at various frequencies. As can be seen with pulses at 1-s intervals (Fig. 7), 50 μM spiradoline produced a slowly developing block. However, peak current was reduced from 10.2 to 6.9 nA at 6.0 Hz, 10.0 to 6.0 nA at 13 Hz, and 10.1 to 3.6 nA at 30 Hz. The average peak amplitude in (d), control current at 25 Hz, was 10.35 ± 0.22 nA.

FIG. 7
FIG. 7:
Frequency-dependent block of the sodium current by spiradoline on a 1-s time scale. Sodium currents were evoked by a voltage step to 0 mV for 10 ms from a prepulse potential of −150 mV at various frequencies. The traces are representative of a 1-s train of such stimuli at 6 Hz (a), 13 Hz (b), and 30 Hz (c), all in the presence of 50 μM spiradoline. Cells were exposed to the drug for 2 min before delivering frequency pulse trains. For comparison, the 1-s train of stimuli shown in trace (d) is the evoked sodium current at 25 Hz in control. Results were obtained in five additional cells.

Figure 8A shows the frequency-dependent block produced by 50 μM spiradoline. The voltage steps were delivered in frequency trains of 0.7, 3.3, 6.6, and 16 Hz (n = 5 cells) with 3 min of recovery time between trains in the presence of 50 μM spiradoline. Cells were exposed to the drug for 2 min before pulsing. Peak currents (normalized to the first current evoked) were plotted against the times at which they were evoked for each frequency. In control solution, no reduction in peak current was seen at frequencies ≤40 Hz (data not shown, see Fig. 7D for 25-Hz representation). The time to block development decreased markedly as the rate of stimulation increased.

FIG. 8
FIG. 8:
A: Frequency dependence of block by spiradoline. Sodium currents were evoked by a voltage step to 0 mV from a prepulse potential of −150 mV at 0.7 Hz, 3.3 Hz, 6.6 Hz, and 16 Hz. Cells were exposed to 50 μM spiradoline for 2 min before testing. The peak current (normalized to the first record) is shown plotted against the time at which it was evoked, at each of the frequencies tested. In the absence of spiradoline, no reduction in peak current was seen at frequencies ≤40 Hz (data not shown, see Fig. 7d for 25 Hz). B: Relation between peak sodium current amplitude and the number of pulses applied. A train of depolarizing pulses of 10-ms duration was used. Sodium currents were evoked by depolarizing to 0 mV from a prepulse potential of −150 mV at a stimulation frequency of 10 Hz. In control solution, no noticeable reduction of current with successive pulses was seen (data not shown, see Fig. 7d for 25 Hz). Tests were performed after 2-min exposure to spiradoline (15-50 μM).

Because the rate of block development by antiarrhythmic drugs is concentration dependent, we determined whether this occurred with spiradoline, as shown by a plot of peak sodium current amplitude and the number of pulses applied (Fig. 8B). A train of depolarizing pulses of 10-ms duration to 0 mV from a prepulse potential of −150 mV were given at a stimulation frequency of 10 Hz. In control solutions, no noticeable reduction of current with successive pulses was seen (data not shown). Spiradoline block was not complete at either low concentrations (15 and 50 μM) or for ≤12 pulses delivered at 10 Hz. However, block occurred with 150 μM spiradoline after only seven pulses at 10 Hz. The slow development of the use-dependent block component is reminiscent of drugs that have slow kinetics such as quinidine (26).

Effects on itoand iksuspotassium currents. In addition to examining the effects of spiradoline on sodium currents, we examined its effects on two potassium currents in isolated myocytes (Fig. 9). We examined the effects of 50 and 150 μM spiradoline on the transient outward (ito) and sustained outward plateau (iksus) potassium currents evoked by depolarization to +50 mV from a prepulse potential of −150 mV for a duration of 300 ms. The potassium current amplitude was measured at the peak (∼2 ms after evoking the outward current), and after 300 ms, at which time the iksus trace remained after complete inactivation of ito. Spiradoline produced a concentration-dependent block of iksus, reduced from a control of 5.20 nA to 1.52 and 0.55 nA by 50 and 150 μM spiradoline, respectively. The peak component of ito in this cell was reduced from a control of 10.3 nA to 7.2 and 5.0 nA by 50 and 150 μM spiradoline, respectively. Spiradoline also increased the rate of decay of the transient component of ito. A tangent to the decay curve of ito gave an approximation for effects on the rate of decay of ito assuming monoexponential decay (27). In Fig. 9, the control rate of decay of ito is ∼0.12 nA/ms, while at a concentration of 50 μM, the rate of decay increased threefold to 0.35 nA/ms and then only marginally increased to 0.38 nA/ms with 150 μM spiradoline. The speeding of inactivation by spiradoline was such that ito inactivation was almost complete within 50 ms as compared with a control of 100 ms (Fig. 9). Similar results were obtained in four additional cells.

FIG. 9
FIG. 9:
Effects of spiradoline (50 and 150 μM) on the depolarization-induced transient outward (ito) and sustained outward plateau (iksus) potassium currents. Currents were elicited by depolarization to +50 mV from a prepulse potential of −150 mV for 300 ms. Spiradoline was applied in the presence of 50 μM tetrodotoxin (TTX), and currents evoked 2 min later. The sustained outward current remained after complete inactivation of ito. The potassium current amplitude was measured at the peak and after 300 ms, at which time the sustained outward current trace was stable. Note that this sustained component remained essentially unchanged at the end of the pulse, even with prolonged depolarization.

DISCUSSION

Nonopioid actions of the benzacetamide κ-agonist, U-50,488H1 (8), and the benzofuranacetamides PD129,289 and PD129,2902 have been well characterized in vivo and in vitro. In this study, we examined a more potent κ-receptor agonist, the oxaspirobenzacetamide, spiradoline (U-62066E) and determined that in addition to its antiarrhythmic properties, it possessed a frequency-dependent sodium channel-blocking action.

In a manner similar to that for other arylacetamides, spiradoline reduced contractility and prolonged the PR and QRS intervals of the ECG in isolated rat hearts, and in anesthetized rats, increased RSh and QaT. Such changes were independent of κ receptor actions because they occurred in the presence of naloxone. Thus spiradoline induced sodium and potassium channel blockade in the heart, as seen with class I and III antiarrhythmic agents.

Electrical-stimulation studies confirm the possibility of such ion channel blockade because spiradoline produced dose-dependent increases in thresholds for capture or induction of extrasystoles (it) and ventricular fibrillation (VFt) (28). Prolongation of ERP and MFF suggested potassium channel blockade. In the whole animal, it is difficult to differentiate drug-channel preference for either sodium or potassium channels. Thus spiradoline blocks both sodium and potassium currents, but sodium current blockade occurs at lower doses than potassium.

Presumably the mechanisms underlying the antiarrhythmic actions of spiradoline may relate to such ion channel-blocking actions. In previous studies, we suggest that sodium channel block with arylbenzacetamides accounted for the antiarrhythmic properties of these drugs (2,29). In this study, we showed that spiradoline produces tonic and use-dependent block of cardiac sodium currents and also blocks repolarizing potassium currents.

The patch-clamp studies with spiradoline in isolated cardiac myocytes confirmed a direct ion channel blockade. At concentrations that produced ECG effects in isolated hearts, spiradoline blocked both sodium and potassium currents in myocytes. Antiarrhythmic drugs can interact with either the resting, open, or inactive state of the sodium channel in a voltage- and time-dependent manner (30). Our results indicate that spiradoline may preferentially bind to the inactive state of the sodium channel because a hyperpolarizing shift in the voltage dependence of inactivation resulted with spiradoline block. Thus spiradoline could then reduce the availability of resting-state sodium channels for subsequent activation and reduce cardiac excitability. Under the pathologic conditions associated with ischemia, spiradoline may be especially effective at suppressing arrhythmias because partial membrane depolarization (sodium channel inactivation) occurs in ischemic tissue.

The use-dependent block shows as an additional reduction in INa, which develops in a frequency-dependent manner with trains of pulses. We calculated the predicted recovery-time constant (τ1/2) for channel block by using molecular weight, calculated pKa, and lipophilicity of spiradoline according to an equation developed by Courtney (31). The predicted τ1/2 for spiradoline was 2.3 s, based on an ethanol/water partition coefficient of 1.3 at pH 7.4, pKa 7.8, and molecular weight of 411 Daltons. This value is similar to values calculated by Courtney for antiarrhythmic agents such as procainamide, quinidine, and imipramine (31). Thus spiradoline shows intermediate off-rate kinetics similar to quinidine. Further experimental evidence of intermediate recovery kinetics is suggested by frequency-dependent studies, which show that spiradoline block of the sodium channel takes between seven and 30 pulses to reach a steady-state block. Quinidine, a class Ia antiarrhythmic agent, was shown to interact with the resting, open, and inactive states of the sodium channel (32,33). In keeping with such considerations, patch-clamp studies show that spiradoline effectively reduced arrhythmias at high heart rates such as VF and VT.

Spiradoline also produced a small tonic block component in sodium current (1-7% between 15 and 50 μM). When we examined the effects of spiradoline on cells at several concentrations spanning the range of antiarrhythmic doses, the increase in tonic block with increasing drug concentration was small compared with use-dependent block. Antiarrhythmic doses effective against VF in occlusion studies could be equivalent to 25 μM in the patch-clamp studies. This concentration corresponds to an approximate tonic reduction in sodium current by 20%. Thus although our studies show that spiradoline binds to the inactive state of the cardiac channel and has intermediate off-rate kinetics, further direct experimental evidence is required to elucidate interactions with other states (open and resting) of the sodium channel. Thus the kinetics of spiradoline remain largely undetermined.

Spiradoline blocked at least two repolarizing potassium currents in the rat heart. The dominant transient outward (ito) current responsible for the early phase of repolarization of the rat action potential (34) and the sustained outward plateau current (35), which we ascribe to a delayed rectifier potassium current (iKsus), are differentially affected. Spiradoline increased the rate of decay of ito and markedly reduced the sustained plateau component in a concentration-dependent manner. Thus depression of these potassium currents prolongs refractoriness and, in conjunction with the predicted slow rate of drug-bound inactivated sodium channel recovery, should provide antiarrhythmic activity. Such a mixed action of cardiac ion channel blockade is not surprising for antiarrhythmic agents. Snyders and Hondeghem (32) showed that quinidine blocks sodium and potassium currents in myocardial tissue in such a manner.

Thus spiradoline is the first arylbenzacetamide we have examined that possesses tonic and use-dependent sodium channel-blocking properties and at higher concentrations blocks at least two repolarizing potassium currents in rat cardiac tissue. These channel-blocking properties make it an effective antiarrhythmic agent against both electrical and ischemic arrhythmias. It was suggested and shown that the combination of a class Ib and class III antiarrhythmic agent may be of potential therapeutic benefit (33). Spiradoline and chemically related arylacetamides may provide useful structural and kinetic information for the actions of these novel antiarrhythmic agents at the membrane level and provide a novel area for the development of more effective and less toxic frequency-dependent antiarrhythmic agents for use against ischemic heart injury.

Acknowledgment: This study was supported by the Heart & Stroke Foundation of B.C. & Yukon, the Science Council of B.C., R.S.D., Ltd., The B.C. Health Care Research Foundation, and the National Health and Medical Research Council of Australia.

REFERENCES

1. Pugsley MK, Penz WP, Walker MJA, Wong TM. Cardiovascular actions of the κ-agonist, U-50,488H in the absence and presence of opioid receptor blockade. Br J Pharmacol 1992a;105:521-6.
2. Pugsley MK, Saint DA, Penz WP, Walker MJA. Electrophysiological and antiarrhythmic actions of the κ-agonist PD129290, and its R,R(+) enantiomer PD129289. Br J Pharmacol 1993;110:1579-85.
3. Alzheimer C, Ten Bruggencate G. Nonopioid actions of the κ-opioid receptor agonists, U-50,488H and U-69593, on electrophysiologic properties of hippocampal CA3 neurons in vitro. J Pharmacol Exp Ther 1990;255:900-5.
4. Zhu Y, Im WB. Block of sodium channel current by anticonvulsant U-54494A in mouse neuroblastoma cells. J Pharmacol Exp Ther 1992;260:110-6.
5. Zhu Y, Im WB, Lewis RA, Von Voigtlander PF. Comparison of pyrrolidinyl and piperidinyl benzamides for their anticonvulsant activity and inhibitory action on sodium channel. Br J Pharmacol 1992;106:49-54.
6. Parratt JR, Sitsapesan R. Stereospecific antiarrhythmic effect of opioid receptor agonists in myocardial ischaemia. Br J Pharmacol 1986;87:621-2.
7. Sarne J, Flitstein A, Oppenheimer E. Antiarrhythmic activities of opioid agonists and antagonists and their stereoisomers. Br J Pharmacol 1991;102:696-8.
8. Pugsley MK, Penz WP, Walker MJA, Wong TM. Antiarrhythmic effects of U-50,488H in rats subject to coronary artery occlusion. Eur J Pharmacol 1992;212:15-9.
9. Von Voigtlander PF, Lewis RA. Analgesic and mechanistic evaluation of spiradoline: a potent kappa opioid. J Pharmacol Exp Ther 1988;246:259-62.
10. Kunihara M, Ohyama M, Nakano M. Effects of spiradoline mesylate, a selective κ-opioid-receptor agonist, on the central dopamine system with relation to mouse locomotor activity and analgesia. Jpn J Pharmacol 1993;62:223-30.
11. Hall ED, Wolf DL, McCall RB. Cardiovascular depressant effects of the kappa opioid receptor agonists U-50,488H and spiradoline mesylate. Circ Shock 1988;26:409-17.
12. Peters GR, Ward NJ, Antal EG, Lai PY, DeMaar EW. Diuretic actions in man of a selective kappa opioid agonist: U-62,066E. J Pharmacol Exp Ther 1987;240:128-31.
13. Curtis MJ, MacLeod BA, Tabrizchi R, Walker MJA. An improved perfusion apparatus for small animal hearts. J Pharmacol Toxicol Methods 1986;15:87-94.
14. Penz WP, Pugsley MK, Hsieh M-Z, Walker MJA. A new ECG measure (RSh) for detecting possible sodium channel blockade in vivo in rats. J Pharmacol Toxicol Methods 1992;27:51-8.
15. Hayes E, Pugsley MK, Penz WP, Adaikan G, Walker MJA. Relationship between Q-aT and RR intervals in rats, guinea pigs, rabbits, and primates. J Pharmacol Toxicol Methods 1994;32:201-7.
16. Woltz M, Schmetterer L, Gouya G, Zanaschka G, Eichler H-G. Is the ECG measure Rsh a promising technique for detecting class I antiarrhythmic drug action in men? J Cardiovasc Pharmacol 1995;26:660-5.
17. Walker MJA, Beatch GN. A new method for electrical stimulation of the heart in rat. Proc West Pharmacol Soc 1988;30:167-70.
18. Howard PG, Walker MJA. Electrical stimulation studies with quinacainol, a putative Ic agent, in the anaesthetised rat. Proc West Pharmacol Soc 1990;33:123-7.
19. Au TLS, Collins GA, Harvie CJ, Walker MJA. The actions of prostaglandins I2 and E2 on arrhythmias produced by occlusion in rat and dog. Prostaglandins 1979;18:207-20.
20. Curtis MJ, Walker MJA. Quantification of arrhythmias using scoring systems: an examination of seven scores in an in vivo model of regional myocardial ischaemia. Cardiovasc Res 1988;22:656-65.
21. Walker MJA, Curtis MJ, Hearse DJ, et al. The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia, infarction, and reperfusion. Cardiovasc Res 1988;22:447-55.
22. Farmer BB, Mancina M, Williams ES, Watanabe AM. Isolation of calcium tolerant myocytes from adult rat hearts: review of the literature and description of a method. Life Sci 1983;33:1-18.
23. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981;391:85-100.
24. Hintze JL. Number cruncher statistical system: version 5.01. Kaysville, UT: NCSS Statistical Software, 1987.
25. Mainland D, Herrera L, Sutcliffe MI. Statistical tables for use with binomial samples-contingency tests, confidence limits and sample size estimates. New York: Department of Medical Statistical Publishers, New York University College of Medicine, 1956.
26. Hondeghem LM, Matsubara T. Quinidine blocks cardiac sodium channels during opening and slow inactivation in guinea pig papillary muscle. Br J Pharmacol 1988;93:311-8.
27. Tseng G, Hoffman BF. Two components of transient outward current in canine ventricular myocytes. Circ Res 1989;64:633-47.
28. Winslow E. Methods in the detection and assessment of antiarrhythmic activity. Pharmacol Ther 1984;24:401-33.
29. Pugsley MK, Hayes ES, Saint DA, Walker MJA. Do related kappa agonists produce similar effects on cardiac ion channels? Proc West Pharmacol Soc 1995;38:25-7.
30. Hondeghem LM, Katzung BG. Time- and voltage-dependent interactions of antiarrhythmic drugs with cardiac sodium channels. Biochim Biophys Acta 1977;472:373-98.
31. Courtney KR. Size-dependent kinetics associated with drug block of sodium current. Biophys J 1984;45:42-4.
32. Snyders DJ, Hondeghem LM. Effects of quinidine on the sodium current of guinea pig ventricular myocytes: evidence for a drug-associated rested state with altered kinetics. Circ Res 1990;66:565-79.
33. Hondeghem LM, Katzung BG. Test of a model of antiarrhythmic drug action: effects of quinidine and lidocaine on myocardial conduction. Circulation 1980;61:1217-24.
34. Josephson IR, Sanchez-Chapula J, Brown AM. Early outward current in rat single ventricular cells. Circ Res 1984;54:157-62.
35. Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes: evidence for a novel delayed rectifier current similar to Kv1.5 cloned channel currents. Circ Res 1993;73:1061-72.
Keywords:

κ-Opioid receptor agonist; Antiarrhythmic; U-62,066E (spiradoline); Cardiac electrophysiology; Sodium current

© 1998 Lippincott Williams & Wilkins, Inc.