α₁-Adrenoceptor Blockade by Class I Antiarrhythmic Drugs in Guinea Pig Thoracic Aorta as Revealed by Mechanical and Fluorescence Displacement Analysis

Abstract

The effects of thirteen Vaughn Williams class I antiarrhythmic drugs on the α₁-adrenergic receptor-mediated contraction were examined in thoracic aorta tissue preparations isolated from the guinea pig. Cibenzoline, quinidine, aprindine, and ranolazine, as well as prazosin, inhibited the phenylephrine-induced contraction with pA₂ values of 5.64, 5.59, 5.61, 5.08, and 8.50, respectively, but not prostaglandin F_2α-induced. These drugs reduced the staining of the smooth muscle layer by fluorescent prazosin. Propafenone inhibited the phenylephrine-induced contraction with an apparent pA₂ value of 5.31 and reduced the staining by fluorescent prazosin, but also inhibited the prostaglandin F_2α-induced contraction. Other class I antiarrhythmic drugs, disopyramide, pirmenol, procainamide, lidocaine, mexiletine, flecainide, pilsicainide, and GS-458967, affected neither the contraction by phenylephrine nor the fluorescent staining by prazosin. These results indicate that among the class I antiarrhythmic drugs, cibenzoline, aprindine, and propafenone, as well as quinidine and ranolazine, have α₁-adrenoceptor-blocking activity at therapeutically relevant concentrations.

INTRODUCTION

Vaughn Williams class I antiarrhythmic drugs have been used in the pharmacological treatment of various types of arrhythmias. They reduce the maximum rate of rise of the action potential by blocking the Na⁺ channels and inhibit the propagation of ectopic and/or reentrant excitation through the myocardium.^1,2) Although class I antiarrhythmic agents are moderately effective against various types of arrhythmias, the treatment results are not satisfactory, and sometimes interfered by their side effects. To optimize the clinical usage of class I antiarrhythmic drugs, understanding the precise pharmacological profiles of these drugs is essential.

Class I antiarrhythmic drugs are subclassified based on their mode of action on the Na⁺ channel.^1,2) Class Ia drugs (cibenzoline, disopyramide, pirmenol, procainamide, and quinidine) moderately block the Na⁺ channel and also prolong the action potential duration through the additional blocking effect on the K⁺ channel. Class Ib drugs (aprindine, lidocaine, and mexiletine) mildly block the Na⁺ channel and show selectivity towards ventricular myocardium. Class Ic drugs (flecainide, pilsicainide, and propafenone) markedly block the Na⁺ channel and are often used in the treatment of atrial fibrillation. Class Id is a subclassification proposed relatively recently including drugs such as ranolazine and GS-458967, which preferentially blocks the persistent component of the Na⁺ channel current also known as the late Na⁺ current.^2–4)

Besides their blockade of the Na⁺ channel, class I antiarrhythmic drugs also have other pharmacological effects via receptors and transporters.^1,2) Most of the class Ia drugs block the muscarinic acetylcholine receptor and the class Ic drug propafenone blocks the β-adrenergic receptor at clinically relevant concentrations; these effects are considered to affect the overall therapeutic potential and side effects of the drugs. Concerning the α₁-adrenergic receptor, the blockade by the classical drug quinidine and a relatively new drug ranolazine is known,^5,6) but whether other class I antiarrhythmic drugs have such effect has not been systematically examined. In the present study, the effects of thirteen class I antiarrhythmic drugs on α₁-adrenergic receptor-mediated contraction were studied in thoracic aorta tissue preparations isolated from the guinea pig using mechanical and fluorescence ligand displacement analyses. The results showed that cibenzoline, aprindine, and propafenone, as well as quinidine and ranolazine, have α₁-adrenoceptor blocking effects at clinically relevant concentrations.

MATERIALS AND METHODS

All experiments were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society and the Guide for the Care and Use of Laboratory Animals at the Faculty of Pharmaceutical Sciences, Toho University (Approval Number: 22-507, accredited on March 31, 2022).

Hartley strain male guinea pigs were exsanguinated under deep isoflurane (more than 3%) anesthetia. The thoracic aorta was isolated, and ring preparations were made using standard procedures.⁷⁾ The intimal surface of the preparations was rubbed with cotton buds to remove the endothelium. The preparations were mounted in an organ bath filled with a physiological salt solution of the following composition: 118.4 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 24.9 mM NaHCO₃, and 11.1 mM glucose (pH = 7.4, 37 °C), gassed with 95% O₂-5% CO₂ and maintained at 36 ± 0.5 °C. A basal tension of 1 g was applied to the preparations, and the contractile force was recorded isometrically previously described.⁷⁾ The functional absence of endothelium was confirmed by the absence of the relaxation by 10 μM acetylcholine in the preparations precontracted with 10 μM phenylephrine.

Fluorescence displacement analysis of binding to α₁-adrenoceptors was performed with procedures similar to those reported.⁸⁾ The ring preparations were cut open and incubated for 2 h in the physiological salt solution containing BODIPY FL-prazosin (50 nM) and Hoechst 33342 (12.5 μg/mL) with or without the addition of non-fluorescent prazosin (30 nM) or various class I antiarrhythmic drugs (30 μM). After washout, the preparations were placed in a glass bottom chamber luminal side down, and the smooth muscle layer was observed with a laser confocal microscope A1R (Nikon, Tokyo, Japan). The fluorescence of BODIPY FL-prazosin in the smooth muscle layer was imaged with excitation and emission wavelengths of 488 nm and 500 to 550 nm, respectively, and that of Hoechst 33342 with 405 nm and 425 to 475 nm, respectively. The objective lens was ×40 Apochromat water immersion (Nikon).

Aprindine, cibenzoline, and lidocaine were purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan), disopyramide and Hoechst 33342 from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), flecainide and mexiletine, phenylephrine, prazosin, and procainamide from Sigma-Aldrich (St. Louis, MO, U.S.A.), GS-458967 from MedKoo Biosciences (Durham, NC, U.S.A.), pilsicainide from Alomone Labs (Jerusalem, Israel), pirmenol from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan), propafenone from LKT Laboratories, Inc. (St. Paul, MN, U.S.A.), quinidine and ranolazine from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and prostaglandin F_2α (PGF_2α) and BODIPY FL-prazosin from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Disopyramide, flecainide, GS-458967, lidocaine, prazosin, propafenone, quinidine, and ranolazine were dissolved and diluted with dimethyl sulfoxide, PGF_2α with ethanol, and other chemicals with distilled water. Small aliquots of drug solutions were added to the solution in the organ bath to obtain the desired final concentrations.

Curve fitting was performed using GraphPad PRISM 9.1.1 software (GraphPad Software Inc., San Diego, CA, U.S.A.). α₁-Adrenoceptor antagonist potencies were expressed as pA₂ values, which were calculated from a Schild plot analysis.⁹⁾ Data were expressed as means ± standard error of the mean (S.E.M). Statistical significance between means was evaluated by the one-way ANOVA with Dunnett՚s multiple comparisons using the GraphPad PRISM software. A p-value less than 0.05 was considered significant.

RESULTS

Prazosin (1 to 30 nM) inhibited the contraction induced by 10 μM phenylephrine in a concentration-dependent manner but had no effect on the contraction induced by 10 μM PGF_2α (Fig. 1A). Among the Vaughn Williams class I antiarrhythmic drugs examined, cibenzoline, quinidine, aprindine, propafenone and ranolazine, inhibited the phenylephrine-induced contraction in a concentration-dependent manner. All of these drugs had no effect on the PGF_2α-induced contraction (Figs. 1B–1F) except for propafenone, which inhibited the PGF_2α-induced contraction only at the concentration of 30 μM. These class I antiarrhythmic drugs, as well as prazosin, caused a rightward shift of the concentration–response curves of phenylephrine (Fig. 2). The pA₂ value for prazosin, cibenzoline, quinidine, aprindine, propafenone, and ranolazine was 8.50, 5.64, 5.59, 5.61, 5.31, and 5.08, respectively.

Fig. 1. Effect of Class I Antiarrhythmic Drugs on the Phenylephrine (PE)-Induced and Prostaglandin (PG) F_2α-Induced Contractions

Typical traces for the inhibition of PE-induced (a) and PGF_2α-induced contractions (b) and the summarized concentration–response relationships (c) for prazosin (A), cibenzoline (B), quinidine (C), aprindine (D), propafenone (E) and ranolazine (F). Closed and open circles in c indicate values obtained in PE-induced and PGF_2α-induced contractions, respectively. The developed tensions in the presence of drugs were expressed as a percentage of that in their absence. The gray bars indicate the therapeutic concentration range of class I antiarrhythmic drugs (Supplementary Table 1). Symbols and vertical bars indicate the mean ± S.E.M. (n = 5).

Fig. 2. Blocking Effect of Class I Antiarrhythmic Drugs on the Phenylephrine-Induced Contractions

Concentration–response curves (a) and the Shild plot (b) for the effect of prazosin (A), cibenzoline (B), quinidine (C), aprindine (D), propafenone (E) and ranolazine (F). Symbols and vertical bars indicate the mean ± S.E.M. (n = 5).

Other class I antiarrhythmic drugs, disopyramide, pirmenol, procainamide, lidocaine, mexiletine, pilsicainide, and GS-458967, did not decrease the contraction by phenylephrine; the contraction after the application of 30 μM of the drugs was 99.6 ± 0.4, 99.4 ± 3.0, 97.4 ± 0.7, 98.8 ± 1.3, 96.9 ± 3.2, 98.7 ± 0.8, and 97.7 ± 1.3% of that before application, respectively (Fig. 3). Flecainide also did not affect the phenylephrine-induced contraction at less than 10 μM. However, the inhibitory effect of contraction by phenylephrine was observed more than 30 μM, which was higher concentration than the therapeutic concentration; the contraction after the application of 30 μM of flecainide was 83.4 ± 6.5% of that before application (Fig. 3F).

Fig. 3. Effect of Class I Antiarrhythmic Drugs on the Phenylephrine (PE)-Induced Contractions

Typical traces for the inhibition of PE-induced contractions (a) and the summarized concentration–response relationships (b) for disopyramide (A), pirmenol (B), procainamide (C), lidocaine (D), mexiletine (E), flecainide (F), pilsicainide (G) and GS-458967 (H). The gray bars indicate the therapeutic concentration range of class I antiarrhythmic drugs (Supplementary Table 1). Symbols and vertical bars indicate the mean ± S.E.M. (n = 5).

To confirm the binding of the class I antiarrhythmic drugs to the α₁-adrenergic receptors, displacement analysis was performed with fluorescent prazosin as previously described.⁸⁾ The BODIPY FL-prazosin appeared to bind the smooth muscle layer (Fig. 4A, Control), and this fluorescence signal was reduced in the presence of non-fluorescent prazosin (Fig. 4A, Prazosin). The fluorescence intensity of the smooth muscle layer by BODIPY FL-prazosin was also decreased when the incubation was performed in the presence of cibenzoline, quinidine, aprindine, propafenone, and ranolazine at 30 μM; these drugs reduced the staining of the smooth muscle layer by BODIPY FL-prazosin to about 60% (Fig. 4). Other class I antiarrhythmic drugs, disopyramide, pirmenol, procainamide, lidocaine, mexiletine, flecainide, pilsicainide and GS-458967, at 30 μM, did not affect the staining by BODIPY FL-prazosin.

Fig. 4. Effect of Class I Antiarrhythmic Drugs on the BODIPY FL-Prazosin Fluorescence

(A) Typical images of the smooth muscle layer stained with BODIPY FL-prazosin in the absence (Control) and presence of drugs. The same amount of dimethyl sulfoxide was added to the control group. The fluorescence of BODIPY FL-prazosin is shown in green and Hoechst 33342 in blue. The image field size is 50 × 50 μm. (B) Summarized results for the BODIPY FL-prazosin fluorescence. The fluorescence intensity of BODIPY FL-prazosin in the presence of drugs was expressed as percentages of that in their absence (Control). Columns and vertical bars indicate the mean ± S.E.M. (n = 10), * p < 0.05 vs. Control.

DISCUSSION

In the present study, we examined the α₁-adrenergic blocking effects of thirteen class I antiarrhythmic drugs in isolated guinea pig thoracic aorta with mechanical and fluorescence ligand displacement analyses. It was previously reported that phenylephrine-induced contraction in guinea pig thoracic aorta was inhibited by prazosin but not α₂ adrenoceptor antagonists, yohimbine and idazoxan, which indicated that phenylephrine-induced contraction was triggered by the α₁-adrenergic receptor.⁷⁾ Our result that the pA₂ value of prazosin was 8.50 was consistent with the previous report,⁷⁾ which indicated that the contraction in the present study was mediated by the α₁-adrenergic receptor.

The results showed that cibenzoline, quinidine, aprindine, and ranolazine have blocking effects on the α₁-adrenergic receptor with similar potency; their pA₂ values falling in the range of 5.08 to 5.64. α₁-Adrenoceptor blockade has been well documented for quinidine and ranolazine,^5,6) but this is the first report for cibenzoline and aprindine. Concerning propafenone, the rightward shift of the concentration–response curve at lower concentrations and the displacement of BODIPY FL-prazosin suggested that it blocks the α₁-adrenoceptor. However, in the present study, propafenone also inhibited the PGF_2α-induced contraction. This could be partly explained by the reported blockade of the voltage-dependent Ca²⁺ channel²⁾ and suppression of the Ca²⁺ release from intracellular stores¹⁰⁾ by propafenone. Thus, the pA₂ value of 5.31 for propafenone towards the α₁-adrenoceptor obtained in the present study, may not be accurate.

The α₁-adrenoceptor blocking effect of the above-mentioned class I antiarrhythmic drugs was present at the therapeutic blood concentrations,¹⁾ which implies that it may possibly contribute to their medicinal efficacy and side effects. Quinidine was reported to cause vasodilation and hypotension in human subjects through direct action on the vascular smooth muscle.¹¹⁾ Aprindine and cibenzoline were also reported to have a hypotensive effect when they were given intravenously.^12–14) Ranolazine, which is also used as an antianginal drug, is considered to cause vasodilation through its blocking effect on the α-adrenoceptor as well as on the persistent component of the Na⁺ channel current.⁶⁾ There are some reports suggesting that stimulation of myocardial α₁-adrenoceptors may be involved in arrhythmia. In the atrio-ventricular blocked rabbit, the QT prolongation and induction of torsades de pointes by nifekalant was enhanced by α-adrenoceptor-stimulation.¹⁵⁾ Atrial fibrillation, the most common arrhythmia in clinical practice, is triggered by the repetitive focal activity of the pulmonary vein myocardium.¹⁶⁾ In the isolated pulmonary vein myocardium of the guinea pig, stimulation of α₁-adrenoceptors caused a depolarization of the resting membrane potential and induced automatic firing of action potentials.¹⁷⁾ To what extent the α₁-adrenoceptor blocking effect of class I antiarrhythmic drugs affects their therapeutic potential awaits further investigation in animal models and human patients.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Numbers: JP20K07299, JP20K16013, and JP20K07091.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1)  Ono K, Iwasaki Y, Akao M, et al. JCS/JHRS 2020 guideline on pharmacotherapy of cardiac arrhythmias. Circ. J., 86, 1790–1924 (2022).
• 2)  Lei M, Wu L, Terrar DA, Huang CL. Modernized classification of cardiac antiarrhythmic drugs. Circulation, 138, 1879–1896 (2018).
• 3)  Belardinelli L, Liu G, Smith-Maxwell C, Wang WQ, El-Bizri N, Hirakawa R, Karpinski S, Li CH, Hu L, Li XJ, Crumb W, Wu L, Koltun D, Zablocki J, Yao L, Dhalla AK, Rajamani S, Shryock JC. A novel, potent, and selective inhibitor of cardiac late sodium current suppresses experimental arrhythmias. J. Pharmacol. Exp. Ther., 344, 23–32 (2013).
• 4)  Irie M, Hiiro H, Hamaguchi S, Namekata I, Tanaka H. Involvement of the persistent Na⁺ current in the diastolic depolarization and automaticity of the guinea pig pulmonary vein myocardium. J. Pharmacol. Sci., 141, 9–16 (2019).
• 5)  del Pozo BF, Perez-Vizcaino F, Villamor E, Zaragoza F, Tamargo J. Stereoselective effects of the enantiomers, quinidine and quinine, on depolarization- and agonist-mediated responses in rat isolated aorta. Br. J. Pharmacol., 117, 105–110 (1996).
• 6)  Virsolvy A, Farah C, Pertuit N, Kong L, Lacampagne A, Reboul C, Aimond F, Richard S. Antagonism of Nav channels and α₁-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine. Sci. Rep., 5, 17969 (2015).
• 7)  Obara K, Yoshioka K, De Dios Regadera M, Matsuyama Y, Yashiro A, Miyokawa M, Iura R, Tanaka Y. Phenylephrine-induced contraction in guinea pig thoracic aorta is triggered by stimulation of α_1L-adrenoceptors functionally coupled with store-operated Ca²⁺ channels and voltage-dependent Ca²⁺ channels. Biol. Pharm. Bull., 46, 309–319 (2023).
• 8)  Daly CJ, Ross RA, Whyte J, Henstridge CM, Irving AJ, McGrath JC. Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and ’cannabinoid-like’ receptors in small arteries. Br. J. Pharmacol., 159, 787–796 (2010).
• 9)  Arunlakshana O, Schild HO. Some quantitative uses of drug antagonists. Br. J. Pharmacol. Chemother., 14, 48–58 (1959).
• 10)  Carrón R, Perez-Vizcaino F, Delpon E, Tamargo J. Effects of propafenone on ⁴⁵Ca movements and contractile responses in vascular smooth muscle. Br. J. Pharmacol., 103, 1453–1457 (1991).
• 11)  Mariano DJ, Schomer SJ, Rea RF. Effects of quinidine on vascular resistance and sympathetic nerve activity in humans. J. Am. Coll. Cardiol., 20, 1411–1416 (1992).
• 12)  Hashimoto K, Komori S, Ishii M, Kamiya J. Effective plasma concentrations of aprindine in canine ventricular arrhythmias. J. Cardiovasc. Pharmacol., 6, 12–19 (1984).
• 13)  Yamazaki K, Sasaki Y, Furuta S. Effectiveness of intravenous administration of aprindine for conversion of paroxysmal atrial fibrillation. Therapeutic Research, 14, 229–235 (1993).
• 14)  Rigaud M, Jouret G, Canal M, Bardet J, Flouvat B, Bourdarias JP. Hemodynamic effects of a new antiarrhythmic agent: cibenzoline. J. Pharmacol., 16, 247–257 (1985).
• 15)  Kawakami S, Kambayashi R, Takada K, Aimoto M, Nagasawa Y, Takahara A. Role of cardiac α₁-adrenoreceptors for the torsadogenic action of IKr blocker nifekalant in the anesthetized atrioventricular block rabbit. J. Pharmacol. Sci., 150, 67–73 (2022).
• 16)  Haïssaguerre M, Jaïs P, Shah DC, Takahashi A, Hocini M, Quiniou G, Garrigue S, Le Mouroux A, Le Métayer P, Clémenty J. Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins. N. Engl. J. Med., 339, 659–666 (1998).
• 17)  Irie M, Tsuneoka Y, Shimobayashi M, Hasegawa N, Tanaka Y, Mochizuki S, Ichige S, Hamaguchi S, Namekata I, Tanaka H. Involvement of alpha- and beta-adrenoceptors in the automaticity of the isolated guinea pig pulmonary vein myocardium. J. Pharmacol. Sci., 133, 247–253 (2017).