Effects of Quinidine and Verapamil on Human Cardiovascular α1-Adrenoceptors
Background—The antiarrhythmic drugs quinidine and verapamil are known to block α1-adrenoceptors (α1ARs). α1ARs are a heterogeneous family of three subtypes (α1A, α1B, and α1D), and little is known about the effects of quinidine and verapamil on the different human α1AR subtypes.
Methods and Results—Reverse transcriptase–polymerase chain reaction showed that all α1AR subtypes are expressed in both human heart (atrium and ventricle) and the mesenteric artery. Pharmacological profiles of quinidine and verapamil actions on the α1AR subtypes were characterized with Chinese hamster ovary cells stably expressing cloned human α1AR subtypes. Radioligand binding studies showed that quinidine and verapamil had high affinities for all α1AR subtypes. Also, both drugs synergistically inhibited α1AR-mediated inositol 1,4,5-triphosphate production at the clinical effective concentration range (1 μmol/L quinidine and 0.1 μmol/L verapamil).
Conclusions—The results show that all α1AR subtypes are expressed in the human cardiovascular system and that quinidine and verapamil may have a potent, synergistic inhibitory effect on the α1ARs. Clinically observed hypotension after quinidine plus verapamil can be explained by their synergistic inhibitory effects on human α1ARs.
Quinidine is used for the treatment of ventricular arrhythmia and supraventricular tachycardia.1 Verapamil is a calcium channel blocker that has been used in the treatment of vasospasm and supraventricular arrhythmia, and its vasodilator effect makes it useful for the treatment of other disorders, such as angina pectoris and hypertension.2 Because quinidine and verapamil are widely used for the treatment of a variety of cardiovascular disorders, their interaction is of potential significance. Serious hypotension, which responds to epinephrine,3 has been reported after combined therapy with verapamil and quinidine, and radioligand binding studies using native tissues (rat heart and kidney for α1AR and human platelets for α2AR) have shown the interaction to be due to an additive blockade of αAR.4 5
α1ARs play an important role in human cardiovascular physiology.6 Recently, the heterogeneity of α1ARs has been recognized,7 8 and three distinct cDNAs encoding human α1AR subtypes (α1A, α1B, and α1D) have been cloned.9 10 11 Little is known, however, about the effects of quinidine and verapamil on each α1AR subtype. The present study was therefore designed to assess this effect on human α1AR subtypes by use of human cloned α1ARs.
Reverse Transcriptase–Polymerase Chain Reaction
Autopsy samples of myocardium and mesenteric artery were analyzed. Total RNA from each tissue was reverse transcribed, and first-strand cDNA was used as a template in PCR (94°C for 60 seconds, 55°C for 30 seconds, and 72°C for 60 seconds) as previously described.9 The primers CATCGTGGTCGGCTGCT TCGTCCTCTGCTG (sense) and TCCCACGGGGATGCGCAC CATGTCCTTGTG (antisense) were chosen for amplification of α1AAR, CCTGTGCGCCATCTCCATCGATCGCTAC (sense) and ATGAAGAAGGGTAGCCAGCACAAGATGAA (antisense) for α1BAR, CTCTGCACCATCTCCGTGGACCGGTAC (sense) and AAAGAAGAAAGGGAACCAGCAGAGCACGAA (antisense) for α1DAR, and ATGGGGGCAACCCGGGAAC (sense) and AGATCT GCGGAGTCCATGCC (antisense) for β-actin.
Incorporation of [α-32P]dCTP during the PCR reaction showed that PCR products were exponentially generated from the 23rd to the 35th cycle for α1AAR, the 29th to the 35th cycle for α1BAR, and the 29th to the 35th cycle for α1DAR. Therefore, PCR experiments were performed within these ranges.
Radioligand binding studies with the transfected CHO cells were performed as described previously.12 Briefly, measurement of specific [125I]HEAT binding was performed by incubating 0.1 mL of membrane preparation (≈10 μg of protein) with [125I]HEAT (2200 Ci/mmol) in a final volume of 0.25 mL assay buffer for 60 minutes at 25°C in the presence or absence of competing drugs. The incubation was terminated by addition of ice-cold buffer and immediate filtration through glass-fiber filters. Each filter was collected, and the radioactivity was measured. The protein concentration was measured with the bicinchoninic acid protein assay kit (Pierce).
Measurement of Ins(1,4,5)P3
The cells at 50% confluence in 35-mm culture dishes treated with NE (1 μmol/L) for 10 seconds were immediately added by ice-cold 20% perchloric acid. After centrifugation, the supernatant was adjusted to pH 7.0 with HEPES-KOH solution, and the sediment was eliminated by centrifugation. Amounts of Ins(1,4,5)P3 in a sample were measured by a radioreceptor assay with a d-myo-inositol 1,4,5-triphosphate [3H] assay kit, TRK 1000 (Amersham).
Analysis of saturation and competition binding data was performed with LIGAND,13 a nonlinear curve-fitting program. All results are shown as mean±SEM. All experiments were conducted at least three times.
The results of RT-PCR are shown in Fig 1⇓. All α1AR subtype mRNAs were detected in both human heart (atrium and left ventricle) and the mesenteric artery; the mRNA of all α1ARs was more abundant in mesenteric artery than in the atrium or ventricle.
Membrane preparations from CHO cells stably expressing the cloned human α1ARs showed saturable binding of [125I]HEAT; Bmax values were 1.3±0.2, 5.5±0.1, and 1.1±0.1 pmol/mg protein, with Kd values of 110±21, 60±1, and 300±26 pmol/L, for the α1A-, α1B-, and α1DARs (n=3 each), respectively. Competition isotherms showed the expected pharmacology for each receptor subtype.12 The abilities of quinidine and verapamil to compete with specific [125I]HEAT binding at each α1AR subtype are shown in Table 1⇓. Both quinidine and verapamil showed small differences in their binding potencies for the different α1AR subtypes (Fig 2⇓). The Hill coefficient for quinidine obtained for each α1AR subtype was close to unity, indicating that quinidine displaces at one site. Conversely, the Hill coefficient for verapamil was 1.5 to 1.7 (significantly different from 1), suggesting a positive cooperativity. Verapamil could be an allosteric modifier that can displace [125I]HEAT binding.
We also examined the abilities of these drugs to inhibit [125I]HEAT binding to the three cloned human α1AR subtypes at their clinically effective concentration ranges (1 μmol/L quinidine and 0.1 μmol/L verapamil).4 14 As shown in Table 2A⇓, quinidine (1 μmol/L) potently inhibited the binding at all α1AR subtypes, whereas verapamil (0.1 μmol/L) had little displacing effect. When the two drugs were combined, little additive effect on [125I]HEAT binding was observed.
To determine whether quinidine and verapamil exert agonistic or antagonistic effects on α1ARs, we further examined their effects on α1AR-mediated Ins(1,4,5)P3 production. As shown in Table 2B⇑, quinidine (1 μmol/L) inhibited NE-induced Ins(1,4,5)P3 production for each α1AR subtype. Interestingly, verapamil (0.1 μmol/L) had an inhibitory effect on NE-induced Ins(1,4,5)P3 production. Moreover, quinidine (1 μmol/L) plus verapamil (0.1 μmol/L) almost abolished NE-induced Ins(1,4,5)P3 production in all α1AR subtypes. In all cells examined, neither quinidine nor verapamil alone had a stimulatory effect (data not shown), and phentolamine (10 μmol/L) abolished NE-induced Ins(1,4,5)P3 production.
Our RT-PCR study showed that α1ARs are expressed in the human cardiovascular system, and binding and functional studies using cloned human α1ARs showed for the first time that quinidine and verapamil used together clinically may exert a potent, synergistic inhibitory effect on human α1ARs.
Our radioligand studies using cloned human α1ARs provide more direct evidence for an interaction of quinidine and verapamil with human α1ARs. Compared with the previous binding studies using nonhuman α1AR,4 15 our study showed the affinity for quinidine and verapamil to be generally comparable to that for the cloned human α1AR, although data obtained from the natively expressed α1AR cannot simply be compared with those obtained with the cloned α1AR, because mixed populations of α1AR subtypes are contained in native tissues. Our approach using cells engineered by molecular biology may provide a useful model for assessing the pharmacological properties of receptors naturally expressed in human tissue.
A comparison of binding and Ins(1,4,5)P3 studies revealed an interesting property of the quinidine and verapamil interaction on α1ARs. Quinidine had an α1AR-blocking effect, whereas verapamil had little effect on the α1AR site; however, verapamil showed an inhibitory effect on α1AR-mediated Ins(1,4,5)P3 production, suggesting that it inhibits α1AR signal transduction by a mechanism other than α1AR blockade.14 It is particularly noteworthy that the two drugs, when given together at their clinically effective concentrations, inhibited the α1AR-mediated response much more potently than would be expected from their additive effect. Hence, the results suggest that the synergistic inhibition of α1AR function via the quinidine and verapamil interaction may occur not only through antagonism at α1AR sites but also through a postreceptor mechanism.
In summary, our radioligand and functional approaches using cloned human α1ARs provide more direct evidence for an interaction between quinidine and verapamil on human α1ARs. Our results show that quinidine and verapamil may have a potent, synergistic inhibitory effect on α1ARs. Clinically observed hypotension after quinidine plus verapamil can be explained by their synergistic inhibitory effects on human α1ARs.
Selected Abbreviations and Acronyms
|CHO||=||Chinese hamster ovary|
|PCR||=||polymerase chain reaction|
This work was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science, and Culture of Japan; a grant-in-aid from the Japan Health Science Foundation; and a grant from the Ministry of Human Health and Welfare, Tokyo, Japan.
- Received January 8, 1998.
- Revision received February 12, 1998.
- Accepted February 12, 1998.
- Copyright © 1998 by American Heart Association
Marcus FI, Opie LH. Antiarrhythmic agents. In: Opie LH, ed. Drugs for the Heart. Philadelphia, Pa: W.B. Saunders; 1995:207–246.
Opie LH, Fishman WH, Thadani U. Calcium channel antagonists (calcium entry blockers). In: Opie LH, ed. Drugs for the Heart. Philadelphia, Pa: W.B. Saunders; 1995:50–82.
Motulsky HJ, Maisel AS, Snavely MD, Insel PA. Quinidine is a competitive antagonist at α1- and α2-adrenergic receptors. Circ Res. 1984;55:376–381.
Nichols JA, Ruffolo JRR. Functions mediated by α-adrenoceptors. In: Ruffolo JRR, ed. α-Adrenoceptors: Molecular Biology, Biochemistry, and Pharmacology. Basel, Switzerland: Karger; 1991:115–179.
Han C, Abel PW, Minneman KP. Heterogeneity of α1-adrenergic receptors revealed by chlorethylclonidine. Mol Pharmacol. 1987;32:505–510.
Tsujimoto G, Tsujimoto A, Suzuki E, Hashimoto K. Glycogen phosphorylase activation by two different α1-adrenergic receptor subtypes: methoxamine selectively stimulates a putative α1-adrenergic receptor subtype (α1a) that couples with Ca2+ influx. Mol Pharmacol. 1989;36:166–176.
Esbenshade TA, Hirasawa A, Tsujimoto G, Tanaka T, Yano J, Minneman KP, Murphy TJ. Cloning of the human α1A/D-adrenergic receptor and inducible expression of three human subtypes in SKNMC cells. Mol Pharmacol. 1995;47:977–985.
Shibata K, Foglar R, Horie K, Obika K, Sakamoto A, Ogawa S, Tsujimoto G. KMD-3213, a novel, potent, α1a-adrenoceptor-selective antagonist: characterization using recombinant human α1-adrenoceptors and native tissues. Mol Pharmacol. 1995;48:250–258.
Nishimura J, Kanaide H, Nakamura M. Binding of [3H]prazosin to porcine aortic membranes: interaction of calcium antagonists with vascular alpha-1 adrenoceptors. J Pharmacol Exp Ther. 1985;236:789–793.