Study on the relaxing effect of salbutamol combined with Y-27632 on porcine airway smooth muscle_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

FANG Zhanglan , TIAN Yi ,  LUO Ling

Department of Internal Medicine, Chongqing University Cancer Hospital, Chongqing 400030, P. R. China;

Corresponding author：

LUO Ling, Email: ling.luo.cqch@hotmail.com

Keywords：

Salbutamol; Rho associated coiled-coil forming protein kinase inhibitor; airway smooth muscle; synergy

DOI：

10.7507/1671-6205.202208029

Video：

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Objective To explore the effect of salbutamol combined with Rho associated coiled-coil forming protein kinase (ROCK) inhibitor Y-27632 on airway smooth muscle and to find a new way for drug treatment of asthma. Methods Pig tracheal smooth muscle tissue strips were prepared, and after treatment they were divided into an electrical stimulation group (F_max, 50%F_max) and a blank group. The smooth muscle tissue strips were quickly frozen to determine the expression level of Rock-Ⅱ and the phosphorylation level of MLC₂₀. The F_max and 50%F_max electrical stimulation groups were divided into a blank group, a salbutamol group, a Y-27632 group, and a salbutamol combined with Y-27632 group according to different intervention drugs. The relaxation of smooth muscle strips was observed. Results In the blank group, 50%F_max group and Fmax group, the expression level of Rock-Ⅱ and the phosphorylation of MLC₂₀ in smooth muscle tissue showed an increasing trend, with statistically significant differences (P<0.05). In the 4 subgroups of the 50%F_max group intervention with different drugs (blank group, salbutamol group, Y-27632 group, salbutamol plus Y-27632 group), the diastolic ratio smooth muscle tissue strips showed an increasing trend. When the time reaches 10 min, the diastolic ratios were 0.7%, 2.5%, 6.0%, and 15.0%. the diastolic ratios were 1.8%, 4.5%, 7.5%, and 21.0% at the time of 20 min. the diastolic ratios were 1.9%, 7.5%, 7.9% and 22.0% at the time of 40 min. the diastolic ratios were 2.0%, 8.0%, 8.8%, and 22.5% at the time of 60 min. In the four subgroups of the F_max electrical stimulation group, the relaxation ratio of smooth muscle tissue strips also showed an increasing trend. When the time reaches 10 min, the diastolic ratios were 1.0%, 3.0%, 7.0%, and 17.0%. the diastolic ratios were 2.6%, 5.5%, 9.0%, and 24.0% at the time of 20 min. the diastolic ratios were 2.8%, 9.0%, 9.5%, and 27.5% at the time of 40 min. diastolic ratios were 2.9%, 10.5%, 10.5%, and 28.0% at the time of 60 min. The analysis of difference between groups showed that at the same time, the diastolic ratio of smooth muscle in salbutamol combined with Y-27632 group was significantly higher than that in salbutamol alone group and Y-27632 group (P<0.05). In addition, the smooth muscle diastolic ratio of combined intervention was also better than the the mathematical sum effect of both single drug intervention (P<0.05). Conclusions The contractility and intensity of smooth muscle are positively correlated with the expression level of ROCK and the phosphorylation level of MLC20. Salbutamol combined with Y-27632 can enhance the relaxation of porcine airway smooth muscle, which may have a synergistic effect.

Citation： FANG Zhanglan, TIAN Yi, LUO Ling. Study on the relaxing effect of salbutamol combined with Y-27632 on porcine airway smooth muscle. Chinese Journal of Respiratory and Critical Care Medicine, 2022, 21(9): 651-657. doi: 10.7507/1671-6205.202208029 Copy

1.	Lang DM. Severe asthma: epidemiology, burden of illness, and heterogeneity. Allergy Asthma Proc, 2015, 36(6): 418-424.
2.	Sheehan WJ, Phipatanakul W. Difficult-to-control asthma: epidemiology and its link with environmental factors. Curr Opin Allergy Clin Immunol, 2015, 15(5): 397-401.
3.	李锋, 张旻, 周新. 氧化应激与慢性气道疾病. 中国呼吸与危重监护杂志, 2012, 11(1): 97-100.
4.	Kawayama T, Kinoshita T, Matsunaga K, et al. Role of regulatory T cells in airway inflammation in asthma. Kurume Med J, 2018, 64(3): 45-55.
5.	Kume H. Role of airway smooth muscle in inflammation related to asthma and COPD. Adv Exp Med Biol, 2021, 1303(1): 139-172.
6.	廖增华, 李超乾. 白细胞介素-1家族新成员IL-36和IL-38在哮喘作用机制中的研究进展. 中国呼吸与危重监护杂志, 2022, 21(6): 452-456.
7.	Camoretti-Mercado B, Lockey RF. Airway smooth muscle pathophysiology in asthma. J Allergy Clin Immunol, 2021, 147(6): 1983-1995.
8.	Seow CY. Passive stiffness of airway smooth muscle: the next target for improving airway distensibility and treatment for asthma. Pulm Pharmacol Ther, 2013, 26(1): 37-41.
9.	Luo L, Wang L, Paré PD, et al. The huxley crossbridge model as the basic mechanism for airway smooth muscle contraction. Am J Physiol Lung Cell Mol Physiol, 2019, 317(2): L235-L246.
10.	Ding F, Yin Z, Wang HR. Ubiquitination in Rho signaling. Curr Top Med Chem, 2011, 11(23): 2879-2887.
11.	Ke X, Do DC, Li CJ, et al. Ras homolog family member A/Rho-associated protein kinase 1 signaling modulates lineage commitment of mesenchymal stem cells in asthmatic patients through lymphoid enhancer-binding factor 1. J Allergy Clin Immunol, 2019, 143(4): 1560-1574. e6.
12.	Loirand G. Rho kinases in health and disease: from basic science to translational research. Pharmacol Rev, 2015, 67(4): 1074-1095.
13.	Álvarez-Santos MD, Álvarez-González M, Estrada-Soto S, et al. Regulation of myosin light-chain phosphatase activity to generate airway smooth muscle hypercontractility. Front Physiol, 2020, 11: 701.
14.	Gao N, Tsai MH, Chang AN, et al. Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle. J Physiol, 2017, 595(19): 6231-6247.
15.	Gosens R, Schaafsma D, Grootte Bromhaar MM, et al. Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent. Eur J Pharmacol, 2004, 494(1): 73-76.
16.	Schaafsma D, Bos IS, Zuidhof AB, et al. The inhaled Rho kinase inhibitor Y-27632 protects against allergen-induced acute bronchoconstriction, airway hyperresponsiveness, and inflammation. Am J Physiol Lung Cell Mol Physiol, 2008, 295(1): L214-L219.
17.	Wei B, Shang YX, Li M, et al. Cytoskeleton changes of airway smooth muscle cells in juvenile rats with airway remodeling in asthma and the RhoA/ROCK signaling pathway mechanism. Genet Mol Res, 2014, 13(1): 559-569.
18.	Schaafsma D, Bos IS, Zuidhof AB, et al. Inhalation of the Rho-kinase inhibitor Y-27632 reverses allergen-induced airway hyperresponsiveness after the early and late asthmatic reaction. Respir Res, 2006, 7(1): 121.
19.	Morgan SJ, Deshpande DA, Tiegs BC, et al. β-Agonist-mediated relaxation of airway smooth muscle is protein kinase A-dependent. J Biol Chem, 2014, 289(33): 23065-23074.
20.	Pera T, Penn RB. Bronchoprotection and bronchorelaxation in asthma: new targets, and new ways to target the old ones. Pharmacol Ther, 2016, 164: 82-96.
21.	Fonseca FV, Raffay TM, Xiao KH, et al. S-nitrosylation is required for β2AR desensitization and experimental asthma. Mol Cell, 2022, 82(16): 3089-3102. e7.
22.	Schaafsma D, Gosens R, Zaagsma J, et al. Rho kinase inhibitors: a novel therapeutical intervention in asthma. Eur J Pharmacol, 2008, 585(2-3): 398-406.
23.	Zhang WW, Bhetwal BP, Gunst SJ. Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle. J Physiol, 2018, 596(16): 3617-3635.
24.	Puetz S, Lubomirov LT, Pfitzer G. Regulation of smooth muscle contraction by small GTPases. Physiology (Bethesda), 2009, 24(6): 342-356.
25.	Chiba Y, Ueno A, Shinozaki K, et al. Involvement of RhoA-mediated Ca²⁺ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice. Respir Res, 2005, 6(1): 4.
26.	Do DC, Zhang Y, Tu W, et al. Type II alveolar epithelial cell-specific loss of RhoA exacerbates allergic airway inflammation through SLC26A4. JCI Insight, 2021, 6(14): e148147.
27.	Yang JQ, Kalim KW, Li Y, et al. Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation. J Leukoc Biol, 2019, 106(5): 1139-1151.
28.	Chiba Y. Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma. Nihon Yakurigaku Zasshi, 2020, 155(6): 364-368.
29.	Ito S, Kume H, Honjo H, et al. Possible involvement of Rho kinase in Ca²⁺ sensitization and mobilization by MCh in tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol, 2001, 280(6): L1218-L1224.
30.	Bhattacharya M, Sundaram A, Kudo M, et al. IQGAP1-dependent scaffold suppresses RhoA and inhibits airway smooth muscle contraction. J Clin Invest, 2014, 124(11): 4895-4898.
31.	吴丰芹, 朱述阳, 何彩霞, 等. 法舒地尔对支气管哮喘小鼠肺组织Rho激酶-1表达及气道炎症的影响. 中华结核和呼吸杂志, 2009, 32(11): 847-851.
32.	Pazhoohan S, Raoufy MR, Javan M, et al. Effect of Rho-kinase inhibition on complexity of breathing pattern in a guinea pig model of asthma. PLoS One, 2017, 12(10): e0187249.
33.	Yamagata S, Ichinose M, Sugiura H, et al. Effect of a calcium sensitization modulator, Y-27632, on isolated human bronchus and pulmonary artery. Pulm Pharmacol Ther, 2000, 13(1): 25-29.
34.	Zhang Y, Saradna A, Ratan R, et al. RhoA/Rho-kinases in asthma: from pathogenesis to therapeutic targets. Clin Transl Immunology, 2020, 9(5): e01134.
35.	Raqeeb A, Jiao Y, Syyong HT, et al. Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment. J Appl Physiol (1985), 2012, 112(3): 337-346.
36.	Gosens R, Schaafsma D, Meurs H, et al. Role of Rho-kinase in maintaining airway smooth muscle contractile phenotype. Eur J Pharmacol, 2004, 483(1): 71-78.
37.	Kume H, Ito S, Ito Y, et al. Role of lysophosphatidylcholine in the desensitization of beta-adrenergic receptors by Ca²⁺ sensitization in tracheal smooth muscle. Am J Respir Cell Mol Biol, 2001, 25(3): 291-298.
38.	Kume H. RhoA/Rho-kinase as a therapeutic target in asthma. Curr Med Chem, 2008, 15(27): 2876-2885.
39.	Schellenberg LM, Regenthal R, Abraham G. The Rho kinase (ROCK) inhibitor Y-27632 reduces the β₂-adrenoceptor density but enhance cAMP formation in primary equine bronchial epithelial cells. Eur J Pharmacol, 2021, 907: 174323.

1. Lang DM. Severe asthma: epidemiology, burden of illness, and heterogeneity. Allergy Asthma Proc, 2015, 36(6): 418-424.
2. Sheehan WJ, Phipatanakul W. Difficult-to-control asthma: epidemiology and its link with environmental factors. Curr Opin Allergy Clin Immunol, 2015, 15(5): 397-401.
3. 李锋, 张旻, 周新. 氧化应激与慢性气道疾病. 中国呼吸与危重监护杂志, 2012, 11(1): 97-100.
4. Kawayama T, Kinoshita T, Matsunaga K, et al. Role of regulatory T cells in airway inflammation in asthma. Kurume Med J, 2018, 64(3): 45-55.
5. Kume H. Role of airway smooth muscle in inflammation related to asthma and COPD. Adv Exp Med Biol, 2021, 1303(1): 139-172.
6. 廖增华, 李超乾. 白细胞介素-1家族新成员IL-36和IL-38在哮喘作用机制中的研究进展. 中国呼吸与危重监护杂志, 2022, 21(6): 452-456.
7. Camoretti-Mercado B, Lockey RF. Airway smooth muscle pathophysiology in asthma. J Allergy Clin Immunol, 2021, 147(6): 1983-1995.
8. Seow CY. Passive stiffness of airway smooth muscle: the next target for improving airway distensibility and treatment for asthma. Pulm Pharmacol Ther, 2013, 26(1): 37-41.
9. Luo L, Wang L, Paré PD, et al. The huxley crossbridge model as the basic mechanism for airway smooth muscle contraction. Am J Physiol Lung Cell Mol Physiol, 2019, 317(2): L235-L246.
10. Ding F, Yin Z, Wang HR. Ubiquitination in Rho signaling. Curr Top Med Chem, 2011, 11(23): 2879-2887.
11. Ke X, Do DC, Li CJ, et al. Ras homolog family member A/Rho-associated protein kinase 1 signaling modulates lineage commitment of mesenchymal stem cells in asthmatic patients through lymphoid enhancer-binding factor 1. J Allergy Clin Immunol, 2019, 143(4): 1560-1574. e6.
12. Loirand G. Rho kinases in health and disease: from basic science to translational research. Pharmacol Rev, 2015, 67(4): 1074-1095.
13. Álvarez-Santos MD, Álvarez-González M, Estrada-Soto S, et al. Regulation of myosin light-chain phosphatase activity to generate airway smooth muscle hypercontractility. Front Physiol, 2020, 11: 701.
14. Gao N, Tsai MH, Chang AN, et al. Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle. J Physiol, 2017, 595(19): 6231-6247.
15. Gosens R, Schaafsma D, Grootte Bromhaar MM, et al. Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent. Eur J Pharmacol, 2004, 494(1): 73-76.
16. Schaafsma D, Bos IS, Zuidhof AB, et al. The inhaled Rho kinase inhibitor Y-27632 protects against allergen-induced acute bronchoconstriction, airway hyperresponsiveness, and inflammation. Am J Physiol Lung Cell Mol Physiol, 2008, 295(1): L214-L219.
17. Wei B, Shang YX, Li M, et al. Cytoskeleton changes of airway smooth muscle cells in juvenile rats with airway remodeling in asthma and the RhoA/ROCK signaling pathway mechanism. Genet Mol Res, 2014, 13(1): 559-569.
18. Schaafsma D, Bos IS, Zuidhof AB, et al. Inhalation of the Rho-kinase inhibitor Y-27632 reverses allergen-induced airway hyperresponsiveness after the early and late asthmatic reaction. Respir Res, 2006, 7(1): 121.
19. Morgan SJ, Deshpande DA, Tiegs BC, et al. β-Agonist-mediated relaxation of airway smooth muscle is protein kinase A-dependent. J Biol Chem, 2014, 289(33): 23065-23074.
20. Pera T, Penn RB. Bronchoprotection and bronchorelaxation in asthma: new targets, and new ways to target the old ones. Pharmacol Ther, 2016, 164: 82-96.
21. Fonseca FV, Raffay TM, Xiao KH, et al. S-nitrosylation is required for β2AR desensitization and experimental asthma. Mol Cell, 2022, 82(16): 3089-3102. e7.
22. Schaafsma D, Gosens R, Zaagsma J, et al. Rho kinase inhibitors: a novel therapeutical intervention in asthma. Eur J Pharmacol, 2008, 585(2-3): 398-406.
23. Zhang WW, Bhetwal BP, Gunst SJ. Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle. J Physiol, 2018, 596(16): 3617-3635.
24. Puetz S, Lubomirov LT, Pfitzer G. Regulation of smooth muscle contraction by small GTPases. Physiology (Bethesda), 2009, 24(6): 342-356.
25. Chiba Y, Ueno A, Shinozaki K, et al. Involvement of RhoA-mediated Ca²⁺ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice. Respir Res, 2005, 6(1): 4.
26. Do DC, Zhang Y, Tu W, et al. Type II alveolar epithelial cell-specific loss of RhoA exacerbates allergic airway inflammation through SLC26A4. JCI Insight, 2021, 6(14): e148147.
27. Yang JQ, Kalim KW, Li Y, et al. Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation. J Leukoc Biol, 2019, 106(5): 1139-1151.
28. Chiba Y. Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma. Nihon Yakurigaku Zasshi, 2020, 155(6): 364-368.
29. Ito S, Kume H, Honjo H, et al. Possible involvement of Rho kinase in Ca²⁺ sensitization and mobilization by MCh in tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol, 2001, 280(6): L1218-L1224.
30. Bhattacharya M, Sundaram A, Kudo M, et al. IQGAP1-dependent scaffold suppresses RhoA and inhibits airway smooth muscle contraction. J Clin Invest, 2014, 124(11): 4895-4898.
31. 吴丰芹, 朱述阳, 何彩霞, 等. 法舒地尔对支气管哮喘小鼠肺组织Rho激酶-1表达及气道炎症的影响. 中华结核和呼吸杂志, 2009, 32(11): 847-851.
32. Pazhoohan S, Raoufy MR, Javan M, et al. Effect of Rho-kinase inhibition on complexity of breathing pattern in a guinea pig model of asthma. PLoS One, 2017, 12(10): e0187249.
33. Yamagata S, Ichinose M, Sugiura H, et al. Effect of a calcium sensitization modulator, Y-27632, on isolated human bronchus and pulmonary artery. Pulm Pharmacol Ther, 2000, 13(1): 25-29.
34. Zhang Y, Saradna A, Ratan R, et al. RhoA/Rho-kinases in asthma: from pathogenesis to therapeutic targets. Clin Transl Immunology, 2020, 9(5): e01134.
35. Raqeeb A, Jiao Y, Syyong HT, et al. Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment. J Appl Physiol (1985), 2012, 112(3): 337-346.
36. Gosens R, Schaafsma D, Meurs H, et al. Role of Rho-kinase in maintaining airway smooth muscle contractile phenotype. Eur J Pharmacol, 2004, 483(1): 71-78.
37. Kume H, Ito S, Ito Y, et al. Role of lysophosphatidylcholine in the desensitization of beta-adrenergic receptors by Ca²⁺ sensitization in tracheal smooth muscle. Am J Respir Cell Mol Biol, 2001, 25(3): 291-298.
38. Kume H. RhoA/Rho-kinase as a therapeutic target in asthma. Curr Med Chem, 2008, 15(27): 2876-2885.
39. Schellenberg LM, Regenthal R, Abraham G. The Rho kinase (ROCK) inhibitor Y-27632 reduces the β₂-adrenoceptor density but enhance cAMP formation in primary equine bronchial epithelial cells. Eur J Pharmacol, 2021, 907: 174323.

Chinese Journal of Respiratory and Critical Care Medicine

Study on the relaxing effect of salbutamol combined with Y-27632 on porcine airway smooth muscle

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