Effects of nipradilol (K-351) on the electrophysiological properties of canine cardiac tissues: Comparison with propranolol and sotalol

European Journal of Pharmacology, 104 (1984) 335-344

335

Elsevier

E F F E C T S OF N I P R A D I L O L (K-351) ON T H E E L E C T R O P H Y S I O L O G I C A L P R O P E R T I E S OF CANINE CARDIAC T I S S U E S : C O M P A R I S O N W I T H P R O P R A N O L O L AND S O T A L O L H A R U A K I N A K A Y A *, S H I N I C H I KIMURA, YASUO N A K A O and MORIO K A N N O

Department of Pharmacolog~~, Hokkaido University School of Medicine, Sapporo 060, Japan Received 21 February 1984, revised MS received 29 May 1984, accepted 19 June 1984 H. N A K A Y A , S. K I M U R A , Y. N A K A O a n d M. K A N N O , Effects of nipradilol (K-351) on the electrophysiological properties of canine cardiac tissues: comparison with propranolol and sotalol, E u r o p e a n J. P h a r m a c o l . 104 (1984)

335-344. The electrophysiological effects of K-351 (0.3-300 /tM), a new fl-adrenoceptor blocking drug, on canine Purkinje and ventricular muscle fibers were examined and compared with those of propranolol (0.3-30 txM) and sotalol (3-300 t~M) using standard microelectrode techniques. K-351 and propranolol dose dependently reduced the maximum upstroke velocity of phase 0 and action potential amplitude in both cardiac tissues. Action potential durations (APD) of Purkinje fibers were shortened by both propranolol and K-351 but prolonged by sotalol. However, in ventricular muscle fibers K-351 and sotalol significantly prolonged APD and effective refractory periods which contrasted with the unchanged APD after propranolol. These results suggest that K-351 is a fl-blocking drug possessing a membrane stabilizing action (class I antiarrhythmic properties) and in ventricular muscles it can also exert an APD-prolonging effect (class III antiarrhythmic properties). Nipradilol K-351 Action potential duration

Sotalol

Propranolol

1. Introduction

Membrane stabilizing action

ran) (fig. 1) is a newly developed nonselective fl-adrenoceptor blocking drug possessing a-antagonistic and vasodilating properties (Asada et al., 1982; Uchida et al., 1983). In the present study, the electrophysiological actions of K-351 on transmembrane action potentials were evaluated in normally polarized canine Purkinje fibers and ventricular muscles. The electrophysiological actions were compared with those of propranolol and sotalol, supposed to be representative fl-blocking drugs possessing a membrane stabilizing action (class I action) and APD-prolonging action (class III action), respectively (Davis and Temte, 1968; Strauss et al., 1970; Vaughan Williams, 1970).

Despite the widespread clinical use of fl-adrenoceptor blocking drugs as antiarrhythmic agents, their mode of action has not been determined with certainty (Wit et al., 1975). Although fl-adrenergic antagonism, is undoubtedly related to their antiarrhythmic effect, especially in the presence of excessive catecholamines, the direct membrane stabilizing action of fl-adrenoceptor blocking drugs should also be taken into consideration (Kupersmith et al., 1976; Kupersmith and Shiang, 1978). In addition, recent studies have suggested that the prolongation of the action potential duration elicited by acute or chronic administration of flblocking drugs may also be important in their effectiveness (Echt et al., 1982; Cobbe et al., 1983). Nipradilol (K-351, 3,4-dihydro-8-(2-hydroxy-3isopropylamino)propoxy-3-nitroxy-2H-l-benzopy-

2.1. Tissue preparations and solutions

* To whom all correspondence should be addressed.

Mongrel dogs of either sex, weighing 5-12 kg, were anesthetized with pentobarbital sodium (30

(X)14-2999/84/$03.00 ~ 1984 Elsevier Science Publishers B.V.

2. Materials and methods

336 mg.kg i.v.). Their hearts were quickly excised and placed in cool, oxygenated Tyrode solution containing (mmol/1) NaC1 125, KCI 4, N a H C O 3 25, N a H 2 P O 4 1.8, MgC12 0.5, CaC12 2.7, and dextrose 5.5. Free-running false tendons and ventricular trabecular muscles were dissected from the hearts and mounted in a 3 ml bath perfused with Tyrode solution which was gassed with a mixture of 95% O 2 and 5% CO 2 and maintained at 37.0 + 1.0°C. The pH of the solution was 7.4 + 0.05.

OH I

OCH2CHCH2NHCH(CH3)2

ONO 2

OH 0 CH2CHCH2NHCH(CH3)2 Desnitro 1<-551

2.2. Electrical arrangements All preparations were stimulated electrically with a stimulator (Nihon Kohden, SEN-6100, Tokyo, Japan) at 1.0 Hz using rectangular pulses that were twice the diastolic threshold and 2 ms duration. The stimuli were delivered through a thin bipolar electrode interfaced with a stimulus isolation unit (Nihon Kohden SS-101J, Tokyo, Japan). Transmembrane potentials were recorded using glass microelectrodes filled with 3 M KCI. Electrodes with resistance of 10 to 30 MI2 were coupled to the input stage of a high-impedance capacitance neutralizing amplifier (Nihon Kohden 8201, Tokyo, Japan). The first derivative of the transmembrane potential was obtained by a calibrated electronic differentiator that was linear up to 800 V/s. The amplified signals were displayed on an oscilloscope (Nihon Kohden VC-9, Tokyo, Japan) and photographed on 35 mm film. The effective refractory period (ERP), defined as the longest interval at which the premature stimulus failed to generate an active response, was determined by delivering a single test stimulus at progressively earlier diastolic intervals, applied after every 10th basic beat.

2.3. Drugs and experimental protocol The drugs and their concentrations (/~M) were as follows: K-351 (Kowa Pharmaceutical Company, Japan), 0.3, 3, 30 and 300; desnitro K-351, 3, 30 and 300; propranolol hydrochloride (Sigma Chemical Co., U.S.A.), 0.3, 3 and 30; sotalol hydrochloride (Bristol-Myers, Japan), 3, 30 and 300. Desnitro K-351 (fig. 1) is a major metabolite of K-351 and a less potent fl-blocker than its parent compound (Uchida et al., 1983). K-351 was

Nipradilol (K-~51)

OH Fig. 1. Chemical structures of nipradilot (K-351) and desnitro K-351. dissolved in equimolar HC1 and other drugs in distilled water. These stock solutions were added to the Tyrode solution to obtain the desired concentrations. It was confirmed that the vehicle did not cause any appreciable pH changes of the Tyrode solution. After an equilibration period of 60 rnin, control recordings were taken and ERP was determined. The preparations were then exposed to a solution containing the lowest concentration of a fl-blocking drug and the concentration increased stepwise at a 60 min interval. The recording of transmembrane potential was repeated every 10 min and ERP was determined at the end of the 60 min drug superfusion period. When conduction block appeared at 1 Hz stimulation during the exposure to the highest concentration of K-351, superfusion of the solution containing the drug was stopped. All preparations were washed out for 60 min with a drug-free solution to verify the reversibility of drug effects. A relatively long exposure time (60 min) was selected because it reportedly takes over 60 rain for propranolol to exhibit steady state accumulation and drug effects in isolated cardiac tissues (Pruett et al., 1980). It was sometimes difficult to keep single impalement throughout the study and reimpalement was carried out when dislodging occurred.

2.4. Statistics All data are presented as mean values + standard errors. Statistical analyses of drug-in-

337

duced changes in action potential characteristics were performed using the paired t-test. Significance was established when the probability value was less than 0.05.

Tyrode solution had the following characteristics: maximum diastolic potential (MDP), - 9 3 . 1 + 0.7 mV; maximum upstroke velocity of phase 0 depolarization (Vmax), 566.4+ 19.9 V / s ; action potential amplitude (APA), 128.3 + 0.7 mV; action potential duration at 50% repolarization (APDs0) and 90% repolarization (APDg0), 242.7 + 8.1 ms and 319.0 + 8.4 ms, respectively (19 preparations). There were no significant differences in any of these parameters between the subgroups of cells exposed to the various/3-blocking drugs. Representative examples of action potential changes observed 60 min after addition of the

3. Results

3.1. Effects of ~-adrenoceptor blocking drugs on the Purkinje fiber action potentials Action potentials recorded from canine Purkinje fibers at the basic driving rate of 1 Hz in drug-free

Control

30 JJM

K351

Propranolol

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.

.

.

i

_

I.

-

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50

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Sotalol

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Fig. 2. Effects of ,8-blocking drugs on transmembrane action potentials of Purkinje fibers. Action potentials recorded before drug superfusion (Control, left panels) and 60 min after superfusion with 30 ~tM of K-351, propranolol and sotalol (right panels) are shown. The upper, middle and lower tracing in each panel indicates 0 potential, the transmembrane potential and its differential, respectively. Single impalement was kept throughout each experiment shown in this figure.

3 3,oo¢Mio.

338 03

3

30

300)J~ W.O. i

-20

3o

i

l

-2O

-40

-40

O E "> -60

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-80

-801

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-I00

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0 K351 I Z~ Pro~'anolol J D Sotolo

-IOC

20 40y

40

20

, ~ A

,'

&

)o ,,~ -2O
G_ -2o

-4o

-40

-60

-60

-8c

-80

Fig. 3. Effects of K-351 (O), propranolol (A) and sotalol (D) on electrophysiological properties of Purkinjc fibers. Percent changes of maximum upstroke velocity of phase 0 (Vmax), action potential amplitude (APA). action potential duration at 50% repolarization (APDs0) and 90% repolarization (APDg0) from control values are indicated on the ordinates and concentrations of B-blocking drugs are on the abscissae. W.O. means the data obtained after a 60 rain washout period. * P < 0.05 compared to each control value. Results are expressed as mean + S.E. for 5 experiments in each group.

same concentration (30/~M) of 3/~-blocking drugs are shown in fig. 2. These are tracings from the experiments in which the same impalement was maintained throughout. K-351 and propranolol reduced ~rmax, APA, APDso and APD9o. On the contrary, sotalol had little effect on "~ma*and APA, and prolonged APDs0 and APDg0. The drug-induced changes in action potential parameters of 5 Purkinje fibers in each group are summarized in fig. 3. Sotalol produced concentration-dependent increases in APDso and APD90. "V'ma~ was reduced only insignificantly by sotalol. Propranolol dose dependently decreased Vma*, APA, APDs0 and APDgo. M D P was not signifi-

cantly affected at any concentration of sotalol o~ propranolol. K-351 also concentration dependently reduced Vmax, APA, APD50 and APDg0. At concentrations of 30 p~M or greater, the decreases in these parameters were statistically significant. However, these changes of action potential characteristics after K-351 were less than those after propranolol when compared at the same concentration of each drug. Conduction block (2:1 block) appeared in all 5 fibers 15-50 min after the exposure to the highest concentration (300 t~M) ot K-351, indicating that the refractory periods exceeded 1000 ms. The action potential parameters after 300 # M of K-351 presented in fig. 3 were

339

obtained from the recording just prior to the appearance of conduction block. The highest concentration of K-351 depolarized M D P by 4.2 + 1.4 mV, which was statistically significant. All the changes induced by the 3 fl-blocking drugs reverted toward control levels after wash-out with drug-free superfusion solution although recovery was incomplete. The effects of desnitro K-351 on transmembrane action potentials were evaluated in 4 Purkinje fibers. In concentrations of 3 and 30/~M the metabolite produced slight and non-significant decreases in Vmax, APA, APDs0 and APD90. After 60 min superfusion with a solution containing 300 Control

/~M of desnitro K-351, the Vmax, APA, APD50 and APDg0 were significantly reduced by 38.9 + 2.8%, 7.1 _+ 1.4%, 30.9 + 2.5% and 11.1 + 3.4%, respectively. These results indicate that the membrane stabilizing action of the metabolite is far less than that of its parent compound.

3.2. Effects of fl-adrenoceptor blocking drugs on oentricular muscle fiber action potentials The action potential parameters of ventricular muscle fibers under control conditions had values as follows: MDP, - 8 8 . 0 + 1.2 mV; Vmax, 168.6 __+ 9.6 V/S; APA, 109.9 + 1.8 mV; APD20 (action

30 ~uM

K 351

\

Propranolol

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I

I

.

i

V "~

Sotalol

, I

II

i

•

, 15°mv

. . . . .

"ljlOOV/s

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I

Fig. 4. Effects of fl-blocking drugs on transmembrane action potentials of ventricular muscle fibers. Action potentials recorded before drug superfusion (left panels) and 60 rnin after exposure to 30/zM of K-351, propranolol and sotalol (right panels) are shown. Upper, middle and lower tracings in each panel are the same as in fig. 2. Single impalement was also maintained throughout each experiment shown in this figure.

340

potential duration at 20% repolarization), 78.7 + 4.7 ms; APDs0, 141.0 + 5.4 ms and APDg0, 183.8 + 4.7 ms. Again, there were no significant differences in the values of any of these parameters between the three subgroups of 5 ventricular muscle fibers. Typical changes of transmembrane action potentials of ventricular trabeculae after 30 ~M of 3 /3-blocking drugs are shown in fig. 4. V,,~ax and APA were reduced by propranolol and K-351. A P D was increased by sotalol and K-351, especially at the 90% repolarization level. The results of 5 experiments in each group are summarized in fig. 5. Sotalol produced concentration-dependent increases in APDso and APDg0 without any significant effect on Vmax and APA. The prolongation of A P D was not reversible on superfusion with drug-free Tyrode solution for 60 min. Moreover, APDs continued to increase during washout in all 5 fibers. Propranolol reduced Vm~ and APA concentration dependently as had

been observed in Purkinje fibers. APDs were not significantly affected by propranolol with the exception that, at 3 /~M, it slightly but significantly prolonged APDg0. K-351 also reduced V..... and APA, but slightly less than did propranolol when compared on a concentration basis. K-351 concentration dependently prolonged APDg0 and the prolongation after 30/~M and 300 #M was statistically significant. Conduction block appeared in two trabecular muscles 18 and 30 min after the application of 300 ~M of K-351. These electrophysiological changes after K-351 were partially reversible after 60 min in drug-free Tyrode solution.

3.3. Effects of fl-adrenoceptor blocking drugs on effective refractory periods The changes of the ERP and ERP/APDg0 ratio in Purkinje fibers and ventricular muscle fibers exposed to various/3-blocking drugs are shown in fig. 6. Under control conditions ERP and the ratio

20

30 0

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g

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-4C 4C

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0

A ~ 50 I < -20 ,q

0 K351 I /k Propronolol [] Sololol -4C

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Q3

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300~M ~0.

-20"-

Fig. 5. Effects of K-351 (O), propranolol (,x) and sotalol (El) on electrophysiological properties of ventricular muscle fibers. Percent changes of maximum upstroke velocity of phase 0 ('Qma~), action potential amplitude (APA), action potential duration at 20% (APD20), 50% (APDs0) and 90% repolarization (APDgo) from control values are indicated on the ordinates and concentration o[ B-blocking drugs are on the abscissae. W.O. means the data obtained after a 60 min washout period. * P < 0.05 compared to each control value. Results are expressed as m e a n + S.E. for 5 experiments in each group.

341

VM

PF

n

DE

6(3

6C

IO 351 ol /k K Propronol 4C CISotalol

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0 -20

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0.80f 0,60

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0.2(3 0 -0.20

Fig. 6. Effects of K-351 (O), propranolol (A) and sotalol (D) on effective refractory periods (ERP) and the ratio of ERP/APDgo. Percent changes of these parameters from control values are indicated on the ordinates and concentrations of B-blocking drugs are on the abscissae. Data obtained from Purkinje fibers (PF) and ventricular muscles (VM) are presented in the left and fight side of this figure, respectively. Results are expressed as mean_+ S.E. and * P < 0.05 from control values.

of ERP/APDgo determined for 19 Purkinje fibers, each stimulated at 1 Hz, were 271 _+ 9 ms and 0.85 _+ 0.02, respectively. Sotalol increased ERP in a dose-dependent manner with little influence on ERP/APDg0, suggesting that the prolongation of ERP was due to an increase in APD. Propranolol at a concentration of 3/~M significantly decreased ERP concomitantly with the shortening of APD9o. Further increasing the concentrations of propranolol to 30 /~M resulted in an increase in ERP/APD90 ratio and the return of ERP to the control level. Although K-351 at concentrations between 0.3 and 30 /~M hardly affected ERP of Purkinje fibers, it significantly increased the ratio of ERP/APDg0 at a concentration of 30 /~M. Desnitro K-351 at a concentration of 300 #M significantly decreased ERP by 7.9 + 1.8%, which was accompanied by the shortened APD90 of 11.1 + 3.4%.

The control ERP and ERP/APDg0 ratio in 15 ventricular trabeculae stimulated at 1 Hz were 174 _+ 6 ms and 0.94 _+ 0.02, respectively. All these fl-blocking drugs increased ERP in a dose-dependent manner (fig. 6). Increases in ERP after sotalol and K-351 were not accompanied by changes of ERP/APDg0 ratio. However, the increased ERP after relatively a low concentration of propranolol was associated with an insignificant increase of the ratio, suggesting that there might be a time-dependent effect of propranolol on sodium channels (Ban, 1977).

4. Discussion

Uchida et al. (1983) have reported recently that K-351 is a unique fl-adrenoceptor blocking drug which shows a long-lasting antihypertensive action

342 in spontaneously hypertensive rats. They also demonstrated that B-blocking action of the compound was nonselective and not accompanied by an intrinsic sympathomimetic activity. In addition, this compound has been shown to block both intra- and extrajunctional a-adrenoceptors in various vascular tissues (Asada et al., 1982; Kou et al., 1982; Kou and Suzuki, 1983). However, the electrophysiological actions of K-351 on normal cardiac tissues have not been evaluated. The present study revealed that K-351 has a membrane stabilizing action concomitant with the APD-prolonging effect on ventricular muscle cells. According to Vaughan Williams' classification, /3-adrenoceptor blocking drugs are categorized as class II antiarrhythmic agents. However, many fl-blocking drugs, such as propranolol (Davis and Temte, 1968), alprenolol (Sada, 1978), pindolol (Kumakura and Somani, 1974), acebutolol and oxprenolol (Sada and Ban, 1980), are known to have a direct membrane effect also (class I antiarrhythmic properties). It is conceivable that concentrations of these fl-adrenoceptor blocking drugs which exert a membrane stabilizing action might be above the therapeutic range for their/~-receptor blocking action. However, the direct membrane effect may play a role in diseased, depolarized cardiac tissues such as ischemic cardiac muscles. Consistent with a previous report (Davis and Temte, 1968), propranolol produced dose-dependent decreases in "Vmaxand APA concurrently with abbreviated A P D in Purkinje fibers but little change of A P D in ventricular muscle fibers. The present study showed that the class I effect of K-351 was slightly less than that of propranolol. As K-351 is twice as potent as propranolol as a /~-blocker (Uchida et al., 1983), its potency for the direct membrane stabilizing action would be several times less than that of propranolol in clinical situations. In this context, desnitro K-351, a major metabolite of K-351 possessing a weak flblocking action but no vasodilating action (Kou and Suzuki, 1983; Uchida et al., 1983), had very little membrane effect in Purkinje fibers, indicating that the nitroxy portion of K-351 also appears to be important in the class I effect. A membrane stabilizing action is shared not only with B-blocking drugs but also with some a-adrenoceptor

blocking drugs, such as phenotalmine, prazosin and yohimbine (Rosen et al., 1971; Northover, 1983). It is noteworthy that K-351 is 5 times less potent than phentolamine as a-adrenoceptor blocker (Uchida et al., 1983). Recently much interest has been expressed in the role of the APD-prolonging action (class III action) in the antiarrhythmic effectiveness of flblocking drugs (Echt et al., 1982; Cobbe et al. 1983). In line with the report of Strauss et al. (1970), sotaiol prolonged APD and refractory periods of both Purkinje fibers and ventricular muscles in a parallel fashion. This effect could presumably be ascribed to a reduction of plateau and background outward potassium current after acute application of sotalol (Carmeliet, 1983). K-351 also produced concentration-dependent increases in APDg0 and refractory periods in ventricular muscle fibers although the extent of the prolongation was smaller than that observed after sotalol. It is thus of interest to ask why K-351 produced opposite effects on APD in ventricular muscles and Purkinje fibers. Although we did not study the ionic mechanism for the electrophysiological action, some speculation may be permissible. As suggested by Coraboeuf et al. (1979), K-351 as well as propranolol might inhibit not only the rapid sodium current but also the background sodium current (window current), resulting in the shortening of A P D in Purkinje fibers. However, in ventricular muscle fibers the tetrodotoxin-sensitive background current does not participate in the development of the plateau (Coraboeuf et al., 1979). Therefore, the inhibitory action of K-351 on the outward repolarization current, presumably the background potassium outward current, might become overt leading to the prolonged APD in ventricular trabeculae. In addition to an acute class Ill effect induced by some B-blocking drugs, there is a chronic effect of /3-blocking drugs devoid of acute class III action, such as propranolol, metoprolol and practolol. Given over a long term these drugs prolong APD, refractory periods and QT intervals in the E C G (Vaughan Williams et al., 1975; Raine and Vaughan Williams, 1981). These authors concluded that the chronic class III effect of these B-blocking drugs stemmed from adaptation of the

343 m y o c a r d i a l cells to p r o l o n g e d B - b l o c k a d e . T h u s , c l a s s III a c t i o n o n v e n t r i c u l a r m u s c l e f i b e r s m a y b e a p r o p e r t y c o m m o n to all B - b l o c k i n g d r u g s during long-term treatment. It is well k n o w n t h a t a - a d r e n o c e p t o r s t i m u l a t i o n c a n l e a d to t h e p r o l o n g a t i o n o f A P D ( G i o t t i et al., 1973). O n t h e o t h e r h a n d , a r e c e n t s t u d y suggests

that

some

a-blocking

drugs

such

as

p r a z o s i n m a y a l s o p r o l o n g A P D ( N o r t h o v e r , 1983). The results of the present study do not exclude the possibility that an a-adrenergic blocking property o f K-351 m i g h t c o n t r i b u t e to t h e a c u t e p r o l o n g a tion of APD. A s c o n c e r n s t h e c a r d i a c e l e c t r o p h y s i o l o g i c a l act i o n o f K - 3 5 1 , it c a n b e c o n c l u d e d t h a t t h i s d r u g is a B - b l o c k i n g d r u g w i t h c o m b i n e d c l a s s I a n d III a n t i a r r h y t h m i c p r o p e r t i e s . H o w e v e r , it s h o u l d b e stressed that these electrophysiological actions of K - 3 5 1 are e x e r t e d less p o t e n t l y t h a n t h o s e o f p r o p r a n o l o l a n d s o t a l o l w h i l e K - 3 5 1 is a m o r e p o t e n t B-blocker. The present data therefore cannot be directly e x t r a p o l a t e d to the clinical setting.

Acknowledgements We wish to acknowledge Kowa Pharmaceutical Company for the generous donation of K-351 and desnitro K-351. Sotalol hydrochloride was kindly provided by Bristol-Myers Company.

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