Journal of Pharmacological Sciences 137 (2018) 98e100
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Short Communication
Selectivity of Ca2þ channel blockers for dilator actions on the isolated lower esophageal sphincter and aorta from rats Akira Takahara*, Shuhei Nozaki, Akane Ishiguro, Kaori Okamura, Xin Cao, Megumi Aimoto, Yoshinobu Nagasawa Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 November 2017 Received in revised form 4 January 2018 Accepted 22 January 2018 Available online 19 April 2018
We compared dilator actions of representative four Ca2þ channel blockers on the isolated lower esophagus sphincter (LES) and thoracic aorta from rats. Verapamil, diltiazem, nifedipine and cilnidipine suppressed KCl-induced contractions of LES and thoracic aorta in a concentration-dependent manner. The order of selectivity for LES, which was calculated as ratio of IC50 value for thoracic aorta divided by that for LES, was diltiazem > verapamil > nifedipine > cilnidipine. These results suggest that diltiazem more preferentially dilates the LES whereas cilnidipine is expected to have lower potential risk of gastroesophageal dysfunction during the antihypertensive therapy. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Ca2þ channel blockers Lower esophagus sphincter Thoracic aorta
The lower esophagus sphincter (LES) is located at the junction between the esophagus and stomach, and separates esophagus from the gastric environment by remaining tonic contraction. In a previous study using the isolated human LES, Ca2þ influxes through L-type Ca2þ channels (Cav1.2) have been demonstrated to play an important role in the maintenance of the LES tone.1 Indeed, Ca2þ channel blockers including verapamil decreased resting pressure of the LES in normal subjects and patients with achalasia.2 In a double-blind crossover study using patients with achalasia, however, oral nifedipine or verapamil made little impact on the clinical symptomatology, despite the reduction in the LES pressure and contraction amplitude of the distal esophageal body.3 More recently, increased risk of exacerbating gastrointestinal disease has been suggested among elderly patients with cardiovascular diseases following treatment with Ca2þ channel blockers in a retrospective cohort study.4 Since little is known about tissue specificity of Ca2þ channel blockers for the LES, we assessed selectivity of representative Ca2þ channel blockers, verapamil, diltiazem, nifedipine and cilnidipine, for dilator actions on the isolated LES and aorta from rats. All experiments were approved by the Ethics Committee of Toho University and performed in accordance with the Guiding
* Corresponding author. Fax: þ81 47 472 3225. E-mail address:
[email protected] (A. Takahara). Peer review under responsibility of Japanese Pharmacological Society.
Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society. The thoracic aorta and lower esophagus were isolated from male Wistar rats (Sankyo Labo Service, Tokyo) weighing 168e270 g, and placed into the normal Tyrode's solution constantly aerated with 95% O2 and 5% CO2. Composition of the normal Tyrode's solution (in mM) was NaCl 137, KCl 5.4, CaCl2 2, MgCl2 1, NaH2PO4 0.4, NaHCO3 11.9, glucose 5.6 (pH 7.4 at 37 C). The isolated aorta and esophagus including the LES were cleaned of the fat and connective tissue, and cut into 3 and 5 mm wide rings, respectively. The aorta was denuded of endothelium and mounted with one stainless-steel wire in a 20-ml organ bath filled with the normal Tyrode's solution, and the other wire through the vessel lumen was connected to an isometric force transducer (Fort 10) in a Myobath 4 system (World Precision Instruments, Sarasota, Fla, USA). The preparation was gently stretched up to 1 g. The LES ring preparation was placed in a 20-ml organ bath filled with the normal Tyrode's solution. The LES ring was tied to a stainless-steel hook at one end of the organ bath, and the other end was connected to an isometric force transducer (TB-651T, Nihon Kohden, Tokyo). The preparation was gently stretched up to 0.5 g. The chambers were continuously gassed with 95% O2 and 5% CO2, and maintained at 36.5 ± 0.5 C. After the equilibration period, 50 mM KCl-induced contraction was elicited by switching the normal Tyrode's solution to the high-K Tyrode's solution of the following composition (in mM): NaCl 92.4, KCl 50, CaCl2 2, MgCl2 1, NaH2PO4 0.4, NaHCO3 11.9, glucose 5.6 (pH 7.4 at 37 C). After a maximum response (control response) was
https://doi.org/10.1016/j.jphs.2018.01.012 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Takahara et al. / Journal of Pharmacological Sciences 137 (2018) 98e100
Fig. 1. Representative traces of an effect of diltiazem on the isolated lower esophagus sphincter (upper panel) and aorta (lower panel) from rats. Diltiazem at a concentration of 108 M inhibited KCl-induced contraction by 70% and 36% in the lower esophagus sphincter and aortic preparations, respectively. LES; lower esophagus sphincter.
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obtained, the organ bath was refilled with the normal Tyrode's solution. An initial concentration of a Ca2þ channel blocker was applied to the organ bath 30 min before induction of a contraction by 50 mM KCl. To observe a concentration-dependent action, a 10 times higher concentration of the drug was similarly applied in turn. Verapamil hydrochloride, diltiazem hydrochloride and nifedipine were obtained from Wako Pure Chemical Industries (Osaka), whereas cilnidipine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Verapamil hydrochloride and diltiazem hydrochloride were dissolved in distilled water. Nifedipine and cilnidipine were dissolved in dimethyl sulfoxide (DMSO). A small aliquot of each drug was added to the organ bath to obtain the desired final concentration (final DMSO concentration: 0.1%). All other chemicals were commercial products of the highest available quality. Data are presented as the mean ± S.E.M. The amplitude of the response was measured and normalized with reference to the control response. Half-maximal inhibitory concentration (IC50) values were obtained by least squares method. Contractions of the LES and aorta appeared immediately after the application of 50 mM KCl, as shown in Fig. 1 (left panel), and the maximum changes in their tension were 1.41 ± 0.17 g (n ¼ 24) and 1.27 ± 0.12 g (n ¼ 24), respectively. Representative traces of an effect of diltiazem on the LES and aorta are shown in Fig. 1, where diltiazem at a concentration of 108 M inhibited KCl-induced contraction by 70% and 36% in the LES and aortic preparations, respectively. Fig. 2 summarizes effects of diltiazem, verapamil, nifedipine and cilnidipine on the KCl-induced contractions of LES and aorta, and Table 1 represents their pIC50 values. In the LES preparations, diltiazem, verapamil and nifedipine inhibited the KCl-induced contractions in a concentration-dependent manner. Based on their IC50 values, the order of the potency was diltiazem > verapamil > nifedipine >> cilnidipine. In the aortic preparations, all the drugs inhibited the KCl-induced contractions in a concentration-
Fig. 2. Effects of diltiazem, verapamil, nifedipine and cilnidipine on the KCl-induced contractions of the isolated lower esophagus sphincter and aorta from rats. Data are presented as the mean ± S.E.M of 6 experiments. LES; lower esophagus sphincter.
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A. Takahara et al. / Journal of Pharmacological Sciences 137 (2018) 98e100
Table 1 Experimental pIC50 values for inhibition of 50 mM KCl-induced contraction in the isolated LES and aorta from rats. LES Diltiazem Verapamil Nifedipine Cilnidipine
9.1 8.1 7.7 5.1
Aorta ± ± ± ±
0.3 0.3 0.3 0.2
7.2 7.3 8.0 9.0
± ± ± ±
Selectivity for LES 0.1 0.1 0.2 0.5
71.6 6.0 0.46 0.00013
Data are presented as mean ± S.E.M of 6 experiments. Selectivity for lower esophagus sphincter (LES) was calculated as ratio of IC50 value for aorta divided by that for LES.
dependent manner, and the order of the potency was cilnidipine > nifedipine > verapamil ¼ diltiazem. The important observation of this study is the rank of the potency of the Ca2þ channel blockers in the LES; namely, diltiazem > verapamil > nifedipine >> cilnidipine based on their IC50 values (Table 1), which is in inverse relation to their effects on the aorta, showing that their selectivity for dilator action on the LES and aorta is quite different. It may be noteworthy to mention a binding of each drug to the L-type Ca2þ channels in the LES, since resting membrane potential of the LES smooth muscle has been reported to be more positive (40 to 50 mV) than that of the esophageal body (50 to 60 mV).1 In addition, phenotype diversity of the pore-forming a1C-subunit of the Ca2þ channels may provide possible mechanisms. A smooth muscle Cav1.2 splice variant (Cav1.2SM) has been found as the smooth muscle form Cav1.2b channel lacking exon 33, which underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine compared with Cav1.2b channel,5 indicating a possibility that Cav1.2SM is more densely localized in the smooth muscles sensitive to dihydropyridine type of Ca2þ channel blockers, like vascular smooth muscle cells. Since the variant in the LES muscle is identified as Cav1.2b,1 we speculate that a difference of inhibitory effects of each Ca2þ channel blocker on Cav1.2b and Cav1.2SM may partly determine selectivity for dilator action on the LES and aorta. In this study, diltiazem more preferentially suppressed the contraction of the LES among the Ca2þ channel blockers examined, which may become an assumed cause of gastroesophageal dysfunctions during antihypertensive therapy. Although clinical data from Interview Form published by pharmaceutical companies in Japan reveal that heartburn appeared as a gastroenterological adverse effect in 0.11, 0.10 and 0.02% patients receiving diltiazem, nifedipine and cilnidipine, respectively,6e8 information is extremely limited regarding directly examined relationship between Ca2þ channel blockers and their adverse reaction in the esophagus like gastroesophageal reflux disease.9,10 In epidemiological studies conducted by community pharmacies in Japan and Australia, where initial prescription of antisecretory drugs or recall of reflux symptom by patients is regarded as an outcome, risk of exacerbating gastrointestinal disease has been suggested among
elderly patients with cardiovascular diseases following treatment with Ca2þ channel blockers.4,11 However, it has never been investigated in those studies which Ca2þ channel blocker more often induced such symptoms. Based on the current results, cilnidipine is expected to become an antihypertensive drug with lower potential risk to gastroesophageal function among Ca2þ channel blockers, and this information will be useful for patients and prescribers. In conclusion, we found that Ca2þ channel blockers have different selectivity for the lower esophagus and blood vessel. Diltiazem more preferentially dilates the LES, whereas cilnidipine is expected to have lower potential risk of gastroesophageal dysfunction during the antihypertensive therapy with Ca2þ channel blockers. Conflict of interest The authors indicated no potential conflicts of interest. Acknowledgments This study was supported by EA Pharma Co., Ltd. References 1. Kovac JR, Preiksaitis HG, Sims SM. Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle. Am J Physiol Gastrointest Liver Physiol. 2005;289:G998eG1006. 2. Becker BS, Burakoff R. The effect of verapamil on the lower esophageal sphincter pressure in normal subjects and in achalasia. Am J Gastroenterol. 1983;78:773e775. 3. Triadafilopoulos G, Aaronson M, Sackel S, Burakoff R. Medical treatment of esophageal achalasia. Double-blind crossover study with oral nifedipine, verapamil, and placebo. Dig Dis Sci. 1991;36:260e267. 4. Hamada A, Ishii J, Doi K, et al. Increased risk of exacerbating gastrointestinal disease among elderly patients following treatment with calcium channel blockers. J Clin Pharm Ther. 2008;33:619e624. 5. Liao P, Yu D, Li G, et al. A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine. J Biol Chem. 2007;282:35133e35142. 6. Mitsubishi Tanabe Pharma Corporation. Interview form: HERBESSER® Tablets. 11th ed. [Japanese]. [Internet]. Osaka (Japan): Mitsubishi Tanabe Pharma Corporation; [updated 2016 Feb; cited 2018 Jan 4]. Available from: http://www. info.pmda.go.jp/go/interview/1/400315_2171006F1224_1_110_1F. 7. Bayer Yakuhin, Ltd. Interview form: Adalat®-CR. 5th ed. [Japanese]. [Internet]. Tokyo (Japan): Bayer Yakuhin, Ltd.; [updated 2016 Oct; cited 2018 Jan 4]. Available from: http://www.info.pmda.go.jp/go/interview/1/630004_2171014G3022_1_002_1F. 8. EA Pharma Co., Ltd. Interview form: ATELEC®Tab. 7th ed. [Japanese]. [Internet]. Tokyo (Japan): EA Pharma Co., Ltd., [updated 2016 Feb; cited 2018 Jan 4]. Available from: http://www.info.pmda.go.jp/go/interview/2/111890_2149037F1032_ 2_1F. 9. Ishikawa H, Iwakiri K, Sugiura T, Kobayashi M. Effect of nifedipine administration (10 mg) on esophageal acid exposure time. J Gastroenterol. 2000;35: 43e46. 10. Hoshino E, Umeda N, Matsueda K, et al. Double cardia. An unusual sequela of reflux esophagitis with ulcer. Dig Dis Sci. 1990;35:638e640. 11. Hughes J, Lockhart J, Joyce A. Do calcium antagonists contribute to gastrooesophageal reflux disease and concomitant noncardiac chest pain? Br J Clin Pharmacol. 2007;64:83e89.
