Abstract
Carbocisteine (CCis), a mucoactive agent, is widely used to improve respiratory diseases. This study demonstrated that CCis increases ciliary bend angle (CBA) by 30% and ciliary beat frequency (CBF) by 10% in mouse airway ciliary cells. These increases were induced by an elevation in intracellular pH (pHi; the pHi pathway) and a decrease in the intracellular Cl− concentration ([Cl−]i; the Cl− pathway) stimulated by CCis. The Cl− pathway, which is independent of CO2/HCO3−, increased CBA by 20%. This pathway activated Cl− release via activation of Cl− channels, leading to a decrease in [Cl−]i, and was inhibited by Cl− channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid and CFTR(inh)-172). Under the CO2/HCO3−-free condition, the CBA increase stimulated by CCis was mimicked by the Cl−-free NO3− solution. The pHi pathway, which depends on CO2/HCO3−, increased CBF and CBA by 10%. This pathway activated HCO3− entry via Na+/HCO3− cotransport (NBC), leading to a pHi elevation, and was inhibited by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid. The effects of CCis were not affected by a protein kinase A inhibitor (1 μM PKI-A) or Ca2+-free solution. Thus, CCis decreased [Cl−]i via activation of Cl− channels including CFTR, increasing CBA by 20%, and elevated pHi via NBC activation, increasing CBF and CBA by 10%.
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References
Afzelius BA (2004) Cilia-related diseases. J Pathol 204:470–477
Arora K, Huang Y, Mun K, Yariagadda S, Sundram N, Kessler MM, Hannig G, Kurtz CB, Silos-Santiago I, Helmrath M, Palermo JJ, Clancy JP, Steinbrecher KA, Naren AP (2017) Guanylate cyclase 2C agonism corrects CFTR mutants. JCI Insight 2(19):e93686. https://doi.org/10.1172/jci.insight.93686
Barbar E, Kleinman B, Imhoff D, Li M, Hays TS, Hare M (2001) Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein. Biochemistry 40:1596–1605
Benedetto R, Ousingswat J, Wanitchakool P, Zhang Y, Holtzman MJ, Amaral M, Rock JR, Schreiber R, Kunzelmann K (2017) Epithelial chloride transport by CFTR requires TMEM16A. Sci Rep 7:12397. https://doi.org/10.1038/s41598-017-10910-0
Bridges RJ (2012) Mechanisms of bicarbonate secretion: lessons from the airways. Cold Spring Harb Prespect Med 2:a015016
Brokaw CJ, Kamiya R (1987) Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil Cytoskeleton 8:68–75
Brokaw CJ (1994) Control of flagellar bending: a new agenda based on dynein diversity. Cell Motil Cytoskeleton 28:199–204
Brown CDA, Dudley AJ (1996) Chloride channel blockers decrease intracellular pH in cultured renal epithelial LLC-PK1 cells. Br J Pharmacol 118:443–444
Chen WY, Xu WM, Chen ZH, Ni Y, Yuan YY, Zhou SC, Zhou WW, Tsang LL, Chung YW, Höglund P, Chan HC, Shi QX (2009) Cl− is required for HCO3 − entry necessary for sperm capacitation in guinea pig: involvement of a Cl−/HCO3 − exchanger (SLC26A3) and CFTR. Biol Reprod 80:115–123
Colombo B, Turconi P, Daffonchio L, Fedele G, Omini C, Cremaschi D (1994) Stimulation of Cl− secretion by the mucoactive drug S-carboxymethylcysteine-lysine salt in the isolated rabbit trachea. Eur Respir J 7:1622–1628
Delmotte P, Sanderson MJ (2006) Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices. Am J Respir Cell Mol Biol 35:110–117
Guizzardi F, Rodighiero S, Binelli A, Saino S, Bononi E, Dossena S, Garavaglia ML, Bazzini C, Bottà G, Conese M, Daffonchio L, Novellini R, Paulmichl M, Meyer G (2006) S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium. J Mol Med 84:97–107
Hahn A, Faulhaber J, Srisawang L, Stortz A, Salomon JJ, Mall MA, Frings S, Möhrlen F (2017) Cellular distribution and function of ion channels involved in transport processes in rat tracheal epithelium. Physiol Rep 5(12):e13290. doi: https://doi.org/10.14814/phy2.13290
Hahn A, Salomon JJ, Leitz D, Feigenbutz D, Korsch L, Lisewski I, Schrimpf K, Millar-Büchner P, Mall MA, Frings S, Möhrlen F (2018) Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research. Pflügers Arch 470:1335–1348. https://doi.org/10.1007/s00424-018-2160-x
Higashijima T, Ferguson KM, Sternweis PC (1987) Regulation of hormone-sensitive GTP-dependent regulatory proteins by chloride. J Biol Chem 262:3597–3602
Hooper C, Calvert J (2008) The role for S-carboxymethylcysteine (carbocisteine) in the management of chronic obstructive pulmonary disease. Int J COPD 3:659–669
Hosogi S, Kusuzaki K, Inui T, Wang X, Marunaka Y (2014) Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell. J Cell Mol Med 18:1124–1133
Houtmeyers E, Gosselink R, Gayan-Ramirez G, Decramer M (1999) Effects of drugs on mucus clearance. Eur Respir J 14:452–467
Ikeuchi Y, Kogiso H, Hosogi S, Tanaka S, Shimamoto C, Inui T, Nakahari T, Marunaka Y (2018) Measurement of [Cl−]i unaffected by the cell volume change using MQAE-based two-photon microscopy in airway ciliary cells of mice. J Physiol Sci 68:191–199
Keskes L, Giroux-Widemann V, Serres C, Pignot-Paintrand I, Jouannet P, Feneux D (1998) The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH. Mol Reprod Dev 49:416–425
Kim D, Kim J, Burghardt B, Best L, Steward MC (2014) Role of anion exchangers in Cl− and HCO3 − secretion by the human airway epithelial cell line Calu-3. Am J Physiol Cell Physiol 307:C208–C219
Komatani-Tamiya N, Daikoku E, Takemura Y, Shimamoto C, Nakano T, Iwasaki Y, Kohda Y, Matsumura H, Marunaka Y, Nakahari T (2012) Procaterol-stimulated increases in ciliary bend amplitude and ciliary beat frequency in mouse bronchioles. Cell Physiol Biochem 29:511–522
Kogiso H, Hosogi S, Ikeuchi Y, Tanaka S, Shimamoto C, Matsumura H, Nakano T, Sano K, Inui T, Marunaka Y, Nakahari T (2017) A low [Ca2+]i-induced enhancement of cAMP-activated ciliary beating by PDE1A inhibition in mouse airway cilia. Pflügers Arch Eur J Physiol 469:1215–1227
Kogiso H, Hosogi S, Ikeuchi Y, Tanaka S, Inui T, Marunaka Y, Nakahari T (2018) [Ca2+]i modulation of cAMP-stimulated ciliary beat frequency via PDE1 in airway ciliary cells of mice. Exp Physiol 103:381–390
Köttgen M, Busch AE, Hug MJ, Greger R, Kunzelman K (1996) N-acetyl-L-cysteine and its derivatives activate a Cl− conductance in epithelial cells. Pflügers Arch Eur J Physiol 431:549–555
Kurita T, Yamamura H, Suzuki Y, Giles WR, Imaizumi Y (2015) The CLC-7 chloride channel is downregulated by hypoosmotic stress in human chondrocytes. Mol Pharmacol 88:113–120
Liu Y, Zhang H, Qi J, Xu J, Gao H, Du X, Gamper N, Zhang H (2015) Characterization of effects of Cl− channel modulators on TMEM16A and bestrophin-1 Ca2+ activated Cl− channels. Pflügers Arch 467:1417–1430
Marunaka Y (1997) Hormonal and osmotic regulation of NaCl transport in renal distal nephron epithelium. Jpn J Physiol 47:499–511
Marunaka Y (2017) Actions of quercetin, a flavonoid, on ion transporters: its physiological roles. Ann N Y Acad Sci 1398:142–151
Meyer G, Doppierio S, Daffonchio L, Cremaschi D (1997) S-carbocysteine-lysine salt monohydrate and cAMP cause non-additive activation of the cystic fibrosis transmembrane regulator channel in human respiratory epithelium. FEBS Lett 404:11–14
Nakahari T, Marunaka Y (1996) Regulation of cell volume by β2-adrenergic stimulation in rat fetal distal lung epithelial cells. J Membr Biol 151:91–100
Nakajima A, Morita M, Takemura A, Kamimura S, Okuno M (2005) Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm. J Exp Biol 208:4411–4418
Ozawa K, Tamura A, Ikeda K, Kawai E, Kondo T, Fukano Y, Nomura S, Ishihara Y, Masujima T (1997) Video-microscopy for analysis of molecular dynamics in cells. J Pharm Biomed Anal 15:1483–1488
Peitzmann ER, Zaidman NA, Maniak PJ, O’Grady SM (2016) Carvedilol binding to β2-adrenergic receptors inhibits CFTR-dependent anion secretion in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 310:L50–L58
Pier GB, Grout M, Zaidi TS (1997) Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc Natl Acad Sci U S A 94:12088–12093
Rogers DF (2007) Mucoactive agents for airway mucus hypersecretory diseases. Respirr Care 52:1176–1193
Salathe M (2007) Regulation of mammalian ciliary beating. Annu Rev Physiol 69:401–422
Shiima-Kinoshita C, Min KY, Hanafusa T, Mori H, Nakahari T (2004) β2-adrenergic regulation of ciliary beat frequency in rat bronchiolar epithelium: potentiation by isosmotic cell shrinkage. J Physiol 554:403–416
Shimamoto C, Umegaki E, Katsu K, Kato M, Fujiwara S, Kubota T, Nakahari T (2007) [Cl−]i modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig. Am J Physiol Gastrointest Liver Physiol 293:G824–G837
Shiozaki A, Miyazaki H, Niisato N, Nakahari T, Iwasaki Y, Itoi H, Ueda Y, Yamagishi H, Marunaka Y (2006) Furosemide, a blocker of Na+/K+/2Cl− cotransporter, diminishes proliferation of poorly differentiated human gastric cancer cells by affecting G0/G1 state. J Physiol Sci 56:401–406
Shpetner HS, Paschal BM, Vallee RB (1988) Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C). J Cell Biol 107:1001–1009
Sutto Z, Conner GE, Salathe M (2004) Regulation of human airway ciliary beat frequency by intracellular pH. J Physiol 560:519–532
Tohda H, Foskett JK, O’Brodovich H, Marunaka Y (1994) Cl− regulation of a Ca2+-activated nonselective cation channel in β-agonist-treated fetal distal lung epithelium. Am J Physiol Cell Physiol 266:C104–C109
Tokuda S, Shimamoto C, Yoshida H, Murao H, Kishima G, Ito S, Kubota T, Hanafusa T, Sugimoto T, Niisato N, Marunaka Y, Nakahari T (2007) HCO3 −-dependent pHi recovery and overacidification induced by NH4 + pulse in rat lung alveolar type II cells: HCO3 −-dependent NH3 excretion from lungs? Pflügers Arch Eur J Physiol 455:223–239
Treharne KJ, Marshall LJ, Mehta A (1994) A novel chloride-dependent GTP-utilizing protein kinase in plasma membranes from human respiratory epithelium. Am J Physiol (Lung Cell Mol Physiol 11) 267:L592–L601
Wang L, Shen M, Guo X, Wang B, Xia Y, Wang N, Zhang Q, Jia L, Wang X (2017) Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation. Sci Rep 7:44265. https://doi.org/10.1038/srep44262
Wang YY, Lin YH, Wu YN, Chen YL, Lin YC, Cheng CY, Chiang HS (2017) Loss of SLC9A3 decreases CFTR protein and causes obstructed azoospermia in mice. PLoS Genet 13:e1006715. https://doi.org/10.1371/journalpgen.1006715
Wanner A, Salathe M, O’riordan TG (1996) Mucociliary clearance in the airways. Am J Respir Crit Care Med 154:1968–1902
Xu WM, Shi QX, Chen WY, Zhou CX, Ni Y, Rowlands DK, Liu GY, Zhu H, Ma ZG, Wang XF, Chen ZH, Zhou SC, Dong HS, Zhang XH, Chung YW, Yuan YY, Yang WX, Chan HC (2007) Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility. Proc Natl Acad Sci U S A 104:9816–9821
Yaghi A, Dolovich MB (2016) Airway epithelial cell cilia and obstructive lung disease. Cells 5(4):E40
Acknowledgements
We thank Osaka Medical College for giving us an opportunity to perform the experiments using the video microscope equipped with a high-speed camera.
Funding
This work was partly supported by Grants-in-Aid for Scientific Research from the Japan Society of the Promotion of Science to YM (No. JP18H03182) and to SH (No. 17K08545).
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The procedures and protocols for the experiments were approved by the Committee for Animal Research of Kyoto Prefectural University of Medicine (No. 26-263) and Ritsumeikan University (No. BKC 2017-050). The animals were cared for, and the experiments were carried out according to the guidelines of this committee. Female mice (C57BL/6J, 6 weeks of age) were purchased from Shimizu Experimental Animals (Kyoto, Japan) and fed standard pellet food and water ad libitum.
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Ikeuchi, Y., Kogiso, H., Hosogi, S. et al. Carbocisteine stimulated an increase in ciliary bend angle via a decrease in [Cl−]i in mouse airway cilia. Pflugers Arch - Eur J Physiol 471, 365–380 (2019). https://doi.org/10.1007/s00424-018-2212-2
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DOI: https://doi.org/10.1007/s00424-018-2212-2