FULL PAPER Pharmacology Benzodiazepines Inhibit the Acetylcholine Receptor-Operated Potassium Current (IK.ACh) by Different Mechanisms in Guinea-pig Atrial Myocytes Muneyoshi OKADA1), Wataru MIZUNO1), Ryu NAKARAI1), Takashi MATADA1), Hideyuki YAMAWAKI1) * and Yukio HARA1) 1)Laboratory of Veterinary Pharmacology, Kitasato University, Towada, Aomori 034–8628, Japan (Received 30 November 2011/Accepted 31 January 2012/Published online in J-STAGE 14 February 2012) ABSTRACT. The anticholinergic effects of 7 benzodiazepines, bromazepam, camazepam, chlordiazepoxide, diazepam, lorazepam, medazepam and triazolam, were compared by examining their inhibitory effects on the acetylcholine receptor-operated potassium current (IK.ACh) in guinea-pig atrial myocytes. All of these benzodiazepines (0.3–300 µM) inhibited carbachol (1 µM)-induced IK.ACh in a concentration-dependent manner. The ascending order of IC50 values for carbachol-induced IK.ACh was as follows; medazepam, diazepam, camazepam, triazolam, bromazepam, lorazepam and chlordiazepoxide (>300 µM). The compounds, except for bromaze- pam, also inhibited IK.ACh activated by an intracellular loading of 100 µM guanosine 5’-[γ-thio]triphosphate (GTPγS) in a concen- tration-dependent manner. The ascending order of IC50 values for GTPγS-activated IK.ACh was as follows; medazepam, diazepam, camazepam, lorazepam, triazolam chlordiazepoxide (>300 µM) and bromazepam (>300 µM). To clarify the molecular mechanism of the inhibition, IC50 ratio, the ratio of IC50 for GTPγS-activated IK.ACh to carbachol-induced IK.ACh, was calculated. The IC50 ratio for camazepam, diazepam, lorazepam, medazepam and triazolam was close to unity, while it for chlordiazepoxide could not be cal- culated. These compounds would act on the GTP binding protein and/or potassium channel to achieve the anticholinergic effects in atrial myocytes. In contrast, since the IC50 ratio for bromazepam is presumably much higher than unity judging from the IC50 values (104.0 ± 30.0 µM for carbachol-induced IK.ACh and >300 µM for GTPγS-activated IK.ACh), it would act on the muscarinic receptor. In summary, benzodiazepines had the anticholinergic effects on atrial myocytes through inhibiting IK.ACh by different molecular mecha- nisms. KEY WORDS: acetylcholine receptor-operated potassium current, atrial myocyte, benzodiazepines, bromazepam, patch clamp method. doi: 10.1292/jvms.11-0538; J. Vet. Med. Sci. 74 (7): 879–884, 2012 Benzodiazepine derivatives are established therapeutic are presumed to affect the ionic currents in the heart. How- tools with relatively low incidence of adverse effects in hu- ever, there is no data available determining the effects of man and veterinary medicine and are used as preanesthetics, benzodiazepines on cardiac ligand-gated currents. tranquilizers, muscle relaxants, and anticonvulsant agents The acetylcholine receptor-operated potassium current [6]. In veterinary clinical fields, benzodiazepine deriva- (IK.ACh), a ligand-gated potassium current, has been known tives are also utilized for animal behavior disorders [17]. to play an important role in the repolarization of the action Virtually, no cardiac effect is observed after ingestion of potential as well as maintenance of the resting potential in therapeutic doses of diazepam in otherwise healthy patients atrial cells [20]. In atrial cells with chronic atrial fibrillation, [2]. While previous in vitro studies have suggested that di- IK.ACh was constitutively active without muscarinic receptor azepam could affect cardiac contractility, the data and their stimulation [7]. The inhibition of IK.ACh appears to be one of interpretation are rather contradictory. For example, both the mechanisms for the termination and prevention of atrial positive and negative inotropic actions, biphasic inotropic flutter and fibrillation [4]. Thus, to explore the influence action, and no effect of diazepam have been reported in dif- of benzodiazepine derivatives on IK.ACh is of great clinical ferent species of animals using various ranges of concentra- significance. In the present study, influences of 7 benzo- tions [1, 5, 8, 23]. Our previous studies have provided one diazepine derivatives including diazepam on IK.ACh were explanation for this contradiction, i.e., diazepam produced examined by a whole-cell patch clamp method in guinea the negative inotropic effect in isolated guinea pig heart pig atrial myocytes. And mechanisms of the anticholinergic through inhibition of the calcium current [15], while at the action of benzodiazepines were explored. same time, it increased calcium sensitivity of the cardiac muscle fiber in the same concentration ranges [13]. From MATERIALS AND METHODS these reports about diazepam, benzodiazepine derivatives This study was performed in compliance with the “Guid- ing Principles for the Care and Use of Laboratory Animals” *CORRESPONDENCE TO: YAMAWAKI, H., Laboratory of Veterinary Pharmacology, Kitasato University, Higashi 23 bancho, 35–1, approved by the Japanese Pharmacological Society and Towada, Aomori 034–8628, Japan. the Kitasato University. The methods for cell prepara- e-mail: [email protected] tions and current recordings were the same as the previous ones [12, 16]. Briefly, guinea-pig (male, 250–750 g body © The Japanese Society of Veterinary Science 880 M. OKADA ET AL. weight) hearts were isolated under sodium pentobarbital carbachol (1 µM) in the GTP (100 µM)-loaded atrial myo- (50 mg/kg i.p. injection) anesthesia and set on a modified cytes using the whole-cell mode of patch clamp method at Langendorff apparatus for isolation of single atrial myocytes a holding potential of −40 mV. After induction of IK.ACh, by an enzymatic digestion with collagenase. Whole-cell benzodiazepines were added to the bath solution in the patch clamp method was used for recording of IK.ACh as an presence of carbachol. Concentration of benzodiazepines outward current at a holding potential of −40 mV. IK.ACh was increased in a stepwise fashion every 3 min. All of was induced by a superfusion of 1 µM carbachol or by an the benzodiazepines used in the present study inhibited the intracellular application of 100 µM guanosine 5’-[γ-thio] carbachol-induced IK.ACh effectively in a concentration- triphosphate (GTPγS), a nonhydrolysable guanosine 5’-tri- dependent manner (Figs. 1 and 3). The outward current reap- phosphate (GTP) analogue. The normal N-[2-hydroxyethyl] peared after a wash-out of each drug. Inhibitory effect of the piperazine-N’-[2-ethanesulfonic acid (HEPES)-Tyrode solu- maximum concentration of chlordiazepoxide (300 µM) on tion (pH 7.4) and the standard pipette solution were used as the current was weak and did not attain 50% inhibition (45.2 superfusate and inner solution, respectively. The composi- ± 4.8% inhibition, n=8). The IC50 values for the carbachol- tion of HEPES-Tyrode solution was (mM): NaCl 143, KCl induced IK.ACh are shown in Table 1. The ascending order of 5.4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.33, glucose 5.5 and IC50 values for the carbachol-induced IK.ACh was as follows; HEPES 5.0. The composition of the standard pipette solution medazepam (12.9 ± 2.4 µM), diazepam (54.8 ± 10.7 µM), was (mM): K-aspartate 110, KCl 20, MgCl2 1.0, GTP 0.1, camazepam (85.6 ± 7.5 µM), triazolam (93.1 ± 21.8 µM), adenosine-5’-triphosphate (ATP)-K 5.0, ethylene glycol-bis bromazepam (104.0 ± 30.0 µM), lorazepam (134.3 ± 4.6 (2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) 10 µM) and chlordiazepoxide (>300 µM; 45.2 ± 4.8% inhibi- and HEPES 5.0 (pH 7.4, free Ca2+ concentration, pCa 8). tion at 300 µM). All benzodiazepines, bromazepam, camazepam, chlordi- Effects of 7 benzodiazepines on the GTPγS-activated azepoxide, diazepam, lorazepam, medazepam and triazolam, IK.ACh in a single guinea pig atrial myocyte: Effects of 7 are obtained from the Yamanouchi Pharmaceutical Co., Ltd. benzodiazepines (0.3–300 µM) on the the GTPγS-activated (Tokyo, the present company name is Astellas Pharm Inc.) IK.ACh were examined. In these experiments, the pipet solu- and are dissolved in dimethyl sulfoxide (DMSO) as a stock tion containing 100 µM GTPγS instead of GTP was used. solution. The final concentration of DMSO is less than 1% Intracellular loading of GTPγS in atrial myocytes gradually and this concentration of DMSO did not affect IK.ACh record- activated the outward current, i.e., IK.ACh, at a holding poten- ing. tial of −40 mV. Camazepam, diazepam, lorazepam, medaz- Data analysis: In the recordings of the IK.ACh current, the epam and triazolam inhibited the GTPγS-activated IK.ACh in activated current is followed by a continuous decline by a concentration-dependent manner (Figs. 2 and 3). Bromaze- the desensitization [22]. Continuous current decline before pam produced only a slight inhibition of the current even at benzodiazepine derivative treatment was assumed as quasi- the highest concentration (12.6 ± 2.5% inhibition, n=6, at steady state (QSS). We used QSS as a maximum current. 300 µM, Figs. 2 and 3). Chlordiazepoxide 300 µM inhibited All values are presented as mean ± standard error of mean the current by 33.1 ± 7.4% (n=6). The IC50 values for the (S.E.M.). The concentrations required to produce 50% of GTPγS-activated IK.ACh are shown in Table 1. The ascending the maximal inhibitory effect (IC50) were calculated from order of IC50 values for the GTPγS-activated IK.ACh was as concentration-response curves using Math Curve Fitter (Sig- follows; medazepam (20.7 ± 3.6 µM), diazepam (75.9 ± 9.1 maPlot, Systat Software, Inc., San Jose, CA, U.S.A.) to solve µM), camazepam (81.6 ± 5.9 µM), lorazepam (98.8 ± 3.4 nonlinear equations. For comparison of IC50 value, statistical µM), triazolam (125.3 ± 25.5 µM), chlordiazepoxide (>300 analyses were performed using un-paired Student’s t-test. A µM; 33.1 ± 7.4% inhibition at 300 µM) and bromazepam value of P<0.05 was considered to be statistically signifi- (>300 µM; 12.6 ± 2.5% inhibition at 300 µM).