Suppression of the increasing level of acetylcholine‑stimulated intracellular Ca2+ in guinea pig airway smooth muscle cells by mabuterol

Song,Xirui; Zhao,Chao; Dai,Cailing; Ren,Yanxin; An,Nan; Wen,Huimin; Pan,Li; Cheng,Maosheng; Zhang,Yuyang

doi:10.3892/br.2015.502

Suppression of the increasing level of acetylcholine‑stimulated intracellular Ca2+ in guinea pig airway smooth muscle cells by mabuterol

Authors:
- Xirui Song
- Chao Zhao
- Cailing Dai
- Yanxin Ren
- Nan An
- Huimin Wen
- Li Pan
- Maosheng Cheng
- Yuyang Zhang
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Affiliations: Department of Pharmacology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P.R. China, Department of Medicine Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P.R. China
Published online on: August 4, 2015 https://doi.org/10.3892/br.2015.502
Pages: 778-786
Copyright: © Song et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The present study aimed to establish an effective method for the in vitro culture of guinea pig airway smooth muscle (ASM) cells, and also investigate the suppressive effect of mabuterol hydrochloride (Mab) on the increased level of intracellular Ca2+ in ASM cells induced with acetylcholine (Ach). Two different methods, i.e. with or without collagenase to pretreat tracheal tissues, were applied to the manufacture of ASM cells. Cell viability was determined with the 3‑(4,5‑dimethylthinazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. Immunocytochemistry and immunofluorescence were used for the identification of ASM cells. Different concentration levels (10‑3, 10‑4, 10‑5, 10‑6 and 10‑7 mmol/l) of Mab were administered 5 min before Ach (10‑4 M) treatment, respectively. The Ca2+ fluorescent probe, Fura‑2/AM or Fluo‑3/AM were applied to the inspection of Ca2+ fluorescent intensity with Varioskan Flash, immunocytometry systems and an inverted system microscope, respectively. The results showed that the fresh method, in which isolated tracheal tissues were previously treated with collagenase for 20 min, was more advantageous for the preparation of guinea pig ASM cells compared to when the enzyme was not used. The time for the ASM cells to initially migrate out of the ‘tissue blocks’ and the culture having to be generated due to the thick cell density was significantly less. On identification with immunocytochemistry or immunofluorescent staining, >95% of the cells were ASM cells. Mab (10‑3‑10‑7 mmol/l) significantly suppressed the elevation of intracellular Ca2+ induced by Ach in a concentration‑dependent manner. The inhibitory rates of intracellular Ca2+ by different concentrations of Mab, from low to high, were 14.93, 24.73, 40.06, 48.54 and 57.13%, respectively, when Varioskan Flash was used for determination. In conclusion, this novel method has a shorter harvesting period for ASM cells. Mab can suppress the increasing level of intracellular Ca2+ induced by Ach in guinea pig ASM cells. Further investigation into the precise mechanisms of action is required.

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1	James A, Mauad T, Abramson M and Green F: Airway smooth muscle hypertrophy and hyperplasia in asthma. Am J Respir Crit Care Med. 186:568–569. 2012. View Article : Google Scholar : PubMed/NCBI
2	Siddiqui S, Redhu NS, Ojo OO, Liu B, Irechukwu N, Billington C, Janssen L and Moir LM: Emerging airway smooth muscle targets to treat asthma. Pulm Pharmacol Ther. 26:132–144. 2013. View Article : Google Scholar : PubMed/NCBI
3	Noble PB, Pascoe CD, Lan B, Ito S, Kistemaker LE, Tatler AL, Pera T, Brook BS, Gosens R and West AR: Airway smooth muscle in asthma: Linking contraction and mechanotransduction to disease pathogenesis and remodelling. Pulm Pharmacol Ther. 29:96–107. 2014. View Article : Google Scholar : PubMed/NCBI
4	Koopmans T, Anaparti V, CastroPiedras I, Yarova P, Irechukwu N, Nelson C, PerezZoghbi J, Tan X, Ward JP and Wright DB: Ca² handling and sensitivity in airway smooth muscle: Emerging concepts for mechanistic understanding and therapeutic targeting. Pulm Pharmacol Ther. 29:108–120. 2014. View Article : Google Scholar : PubMed/NCBI
5	Boese M, Busse R, Mülsch A and Schini-Kerth V: Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: Role of cyclic AMP. Br J Pharmacol. 119:707–715. 1996. View Article : Google Scholar : PubMed/NCBI
6	Dimitropoulou C, White RE, Ownby DR and Catravas JD: Estrogen reduces carbachol-induced constriction of asthmatic airways by stimulating large-conductance voltage and calcium-dependent potassium channels. Am J Respir Cell Mol Biol. 32:239–247. 2005. View Article : Google Scholar : PubMed/NCBI
7	Wang IY, Bai Y, Sanderson MJ and Sneyd J: A mathematical analysis of agonist- and KCl-induced Ca(2+) oscillations in mouse airway smooth muscle cells. Biophys J. 98:1170–1181. 2010. View Article : Google Scholar : PubMed/NCBI
8	Yamamoto H, Nagata M, Tabe K, Suzuki S, Maruo H, Sakamoto Y, Yamamoto K and Dohi Y: The inhibitory effect of long-acting beta-adrenergic agonists, mabuterol, clenbuterol and fenoterol on ‘morning dipping’ in patients with asthma. Arerugi. 39:21–27. 1990.(In Japanese). PubMed/NCBI
9	Osada E, Murai T, Ishizaka Y and Sanai K: Pharmacological studies of mabuterol, a new selective beta 2-stimulant. II: Effects on the cardiovascular system and smooth muscle organs. Arzneimittelforschung. 34(11A): 1641–1651. 1984.PubMed/NCBI
10	Akahane K, Furukawa Y, Ogiwara Y, Haniuda M and Chiba S: Beta-adrenoceptor blocking effects of a selective beta 2-agonist, mabuterol, on the isolated, blood-perfused right atrium of the dog. Br J Pharmacol. 97:709–716. 1989. View Article : Google Scholar : PubMed/NCBI
11	Liu J, Zhang Y, Li Q, Zhuang Q, Zhu X, Pan L and Cheng M: An improved method for guinea pig airway smooth muscle cell culture and the effect of SPFF on intracellular calcium. Mol Med Rep. 10:1309–1314. 2014.PubMed/NCBI
12	Mosmann T: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 65:55–63. 1983. View Article : Google Scholar : PubMed/NCBI
13	Orlandi A, Calzetta L, Doldo E, Tarquini C, Matera MG and Passeri D: Brain natriuretic peptide modulates calcium homeostasis and epidermal growth factor receptor gene signalling in asthmatic airways smooth muscle cells. Pulm Pharmacol Ther. 31:51–54. 2015. View Article : Google Scholar : PubMed/NCBI
14	Liu B, Yang J, Wen Q and Li Y: Isoliquiritigenin, a flavonoid from licorice, relaxes guinea-pig tracheal smooth muscle in vitro and in vivo: Role of cGMP/PKG pathway. Eur J Pharmacol. 587:257–266. 2008. View Article : Google Scholar : PubMed/NCBI
15	Pelaia G, Renda T, Gallelli L, Vatrella A, Busceti MT, Agati S, Caputi M, Cazzola M, Maselli R and Marsico SA: Molecular mechanisms underlying airway smooth muscle contraction and proliferation: Implications for asthma. Respir Med. 102:1173–1181. 2008. View Article : Google Scholar : PubMed/NCBI
16	Yamakage M, Hirshman CA and Croxton TL: Volatile anesthetics inhibit voltage-dependent Ca² channels in porcine tracheal smooth muscle cells. Am J Physiol. 268:L187–L191. 1995.PubMed/NCBI
17	Wu BN, Lin RJ, Lo YC, Shen KP, Wang CC, Lin YT and Chen IJ: KMUP-1, a xanthine derivative, induces relaxation of guinea-pig isolated trachea: The role of the epithelium, cyclic nucleotides and K channels. Br J Pharmacol. 142:1105–1114. 2004. View Article : Google Scholar : PubMed/NCBI
18	Bergner A and Sanderson MJ: Acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices. J Gen Physiol. 119:187–198. 2002. View Article : Google Scholar : PubMed/NCBI
19	Perez JF and Sanderson MJ: The frequency of calcium oscillations induced by 5-HT, ACH and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles. J Gen Physiol. 125:535–553. 2005. View Article : Google Scholar : PubMed/NCBI
20	Kawakami Y: First clinical studies on mabuterol. A summarizing report. Arzneimittelforschung. 34(11A): 1699–1700. 1984.PubMed/NCBI
21	Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans A and Meurs H: Acetylcholine: A novel regulator of airway smooth muscle remodelling? Eur J Pharmacol. 500:193–201. 2004. View Article : Google Scholar : PubMed/NCBI
22	Jude JA, Wylam ME, Walseth TF and Kannan MS: Calcium signaling in airway smooth muscle. Proc Am Thorac Soc. 5:15–22. 2008. View Article : Google Scholar : PubMed/NCBI
23	Mahn K, Ojo OO, Chadwick G, Aaronson PI, Ward JP and Lee TH: Ca(2+) homeostasis and structural and functional remodelling of airway smooth muscle in asthma. Thorax. 65:547–552. 2010. View Article : Google Scholar : PubMed/NCBI
24	Gan LL, Wang MW, Cheng MS and Pan L: Trachea relaxing effects and beta2-selectivity of SPFF, a newly developed bronchodilating agent, in guinea pigs and rabbits. Biol Pharm Bull. 26:323–328. 2003. View Article : Google Scholar : PubMed/NCBI
25	Hao Z, Zhang Y, Pan L, Su X, Cheng M, Wang M, Zhao H and Wu Y: Comparison of enantiomers of SPFF, a novel beta2-Adrenoceptor agonist, in bronchodilating effect in guinea pigs. Biol Pharm Bull. 31:866–872. 2008. View Article : Google Scholar : PubMed/NCBI