The Journal of Physiological Sciences

, Volume 68, Issue 2, pp 191–199 | Cite as

Measurement of [Cl]i unaffected by the cell volume change using MQAE-based two-photon microscopy in airway ciliary cells of mice

  • Yukiko Ikeuchi
  • Haruka Kogiso
  • Shigekuni Hosogi
  • Saori Tanaka
  • Chikao Shimamoto
  • Toshio Inui
  • Takashi NakahariEmail author
  • Yoshinori Marunaka
Technical Note


MQAE is a ‘non-ratiometric’ chloride ion (Cl)-quenched fluorescent indicator that is used to determine intracellular Cl concentration ([Cl]i). MQAE-based two-photon microscopy is reported to be a useful method to measure [Cl]i, but it is still controversial because a change in cell volume may alter the MQAE concentration, leading to a change in the fluorescence intensity without any change in [Cl]i. In an attempt to elucidate the effect or lack of effect of cell volume on MQAE concentration, we studied the effects of changes in cell volume, achieved by applying different levels of osmotic stress, on the intensity of MQAE fluorescence in airway ciliary cells. To study solely the effect of changes in cell volume on MQAE fluorescence intensity, i.e., excluding the effect of any change in [Cl]i, we first conducted the experiments in a Cl-free nitrate (NO3) solution to substitute NO3 (non-quenching anion for MQAE fluorescence) for Cl in the intracellular fluid. Hypo- (− 30 mM NaNO3) or hyper-osmotic stress (+ 30 mM NaNO3) effected changes in cell volume, but the stress did not result in any significant change in MQAE fluorescence intensity. The experiments were also carried out in Cl-containing solution. Hypo-osmotic stress (− 30 mM NaCl) increased both MQAE fluorescence intensity and cell volume, while hyper-osmotic stress (+ 30 mM NaCl) decreased both of these properties. These results suggest that the osmotic stress-induced change in MQAE fluorescence intensity was caused by the change in [Cl]i and not by the MQAE concentration. Moreover, the intracellular distribution of MQAEs was heterogeneous and not affected by the changes in osmotic stress-induced cell volume, suggesting that MQAEs are bound to un-identified subcellular structures. These bound MQAEs appear to have enabled the measurement of [Cl]i in airway ciliary cells, even under conditions of cell volume change.


MQAE Intracellular Cl concentration Two-photon microscopy Cell volume NO3 



This work is partly supported by Grants-in Aid for Scientific Research from the Japan Society of the Promotion of Science to SH and TN (No. 17K08545).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The procedures and protocols for the experiments were approved by the Animal Research Committee of Kyoto Prefectural University of Medicine (No. 26-263), and the experiments were carried out in accordance with the guidelines of this committee, which are identical to those of the Physiological Society of Japan.


  1. 1.
    Marunaka Y (2017) Actions of quercetin, a flavonoid, on ion transporters: its physiological roles. Ann N Y Acad Sci 1398:142–151CrossRefGoogle Scholar
  2. 2.
    Miyazaki H, Shiozaki A, Niisato N, Marunaka Y (2007) Physiological significance of hypotonicity-induced regulatory volume decrease: reduction in intracellular Cl concentration acting as an intracellular signaling. Am J Physiol Renal Physiol 292:F1411–F1417CrossRefGoogle Scholar
  3. 3.
    Nakahari T, Marunaka Y (1996) Regulation of cell volume by beta 2-adrenergic stimulation in rat fetal distal lung epithelial cells. J Membr Biol 151:91–100CrossRefGoogle Scholar
  4. 4.
    Nakajima K, Marunaka Y (2016) Intracellular chloride ion concentration in differentiating neural cell and its role in growing neurite. Biochem Biophys Res Commun 479:338–342CrossRefGoogle Scholar
  5. 5.
    Nakajima K, Niisato N, Marunaka Y (2012) Enhancement of tubulin polymerization by Cl -induced blockade of intrinsic GTPase. Biochem Biophys Res Commun 425:225–229CrossRefGoogle Scholar
  6. 6.
    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 554:403–416CrossRefGoogle Scholar
  7. 7.
    Tohda H, Foskett JK, O’Brodovich H, Marunaka Y (1994) Cl regulation of a Ca2+-activated nonselective cation channel in beta-agonist-treated fetal distal lung epithelium. Am J Physiol Cell Physiol 266:C104–C109CrossRefGoogle Scholar
  8. 8.
    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 267 267:L592–601PubMedGoogle Scholar
  9. 9.
    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 18:1124–1133CrossRefGoogle Scholar
  10. 10.
    Kim D, Kim J, Burghardt B, Best L, Steward MC (2014) Role of anion exchangers in Cl and HCO3 secretion by human airway epithelial cell line Calu-3. Am J Physiol Cell Physiol 307:C208–C219CrossRefGoogle Scholar
  11. 11.
    Yasuda M, Niisato N, Miyazaki H, Hamma T, Dejima K, Hisa Y, Marunaka Y (2006) Epithelial ion transport of human nasal polyp and paranasal sinus mucosa. Am J Respir Cell Mol Biol 36:466–472CrossRefGoogle Scholar
  12. 12.
    Arosio D, Ratto GM (2014) Twenty years of fluorescence imaging of intracellular chloride. Front Cell Neurosci 8:258CrossRefGoogle Scholar
  13. 13.
    Inglefield JR, Schwartz-Bloom RD (1997) Confocal imaging of intracellular chloride in living brain slices: measurement of GABAA receptor activity. J Neurosci Methods 75:127–135CrossRefGoogle Scholar
  14. 14.
    Kaneko H, Nakamura T, Lindemann B (2001) Noninvasive measurement of chloride concentration in rat olfactory receptor cells with use of a fluorecent dye. Am J Physiol 280:C1387–C1393CrossRefGoogle Scholar
  15. 15.
    Koncz C, Daugirdas JT (1994) Use of MQAE for measurement of intracellular [Cl] in cultured aortic smooth muscle cells. Am J Physiol 267:H2114–H2123PubMedGoogle Scholar
  16. 16.
    Marandi N, Konnerth A, Garaschuk O (2002) Two-photon chloride imaging in neurons of brain slices. Pflügers Arch Eur J Physiol 445:357–365CrossRefGoogle Scholar
  17. 17.
    Verkman A, Sellers MC, Chao AC, Leung T, Ketcham R (1989) Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications. Anal Biochem 178:355–361CrossRefGoogle Scholar
  18. 18.
    Marunaka Y (1997) Hormonal and osmotic regulation of NaCl transport in renal distal nephron epithelium. Jpn J Physiol 47:499–511CrossRefGoogle Scholar
  19. 19.
    Lahn M, Dosche C, Hille C (2011) Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl changes in cockroach salivary acinar cells. Am J Physiol Cell Physiol 300:C1323–C1336CrossRefGoogle Scholar
  20. 20.
    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 29:511–522CrossRefGoogle Scholar
  21. 21.
    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 469:1215–1227CrossRefGoogle Scholar
  22. 22.
    Ikeuchi Y, Kogiso H, Tanaka S, Hosogi S, Nakahari T, Marunaka Y (2016) Activation of ciliary beating by carbocistein via modulation of [Cl]i and pHi in bronchiolar ciliary cells in mice. J Physiol Sci 66[Suppl]:S86Google Scholar
  23. 23.
    Ikeuchi Y, Kogiso H, Tanaka S, Hosogi S, Nakahari T, Marunaka Y (2017) Carbocistein-activated bronchiolar ciliary beating via Cl and pH-mediated pathways in mice. J Physiol Sci 67[Suppl]:S137Google Scholar
  24. 24.
    Kogiso H, Hosogi S, Ikeuchi Y, Tanaka S, Shimamoto C, Nakahari T, Marunaka Y (2016) Ciliary beat frequency modulated by PDE1A activity in procaterol stimulated mouse bronchiole. J Physiol Sci 66[Suppl]:S86Google Scholar
  25. 25.
    Kogiso H, Ikeuchi Y, Hosogi S, Tanaka S, Shimamoto C, Nakahari T, Marunaka Y (2017) Ca2+-regulation of cAMP-activated ciliary beating mediated via PDE1 in mouse bronchiolar cilia. J Physiol Sci 67[Suppl]:S176Google Scholar
  26. 26.
    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. CrossRefPubMedGoogle Scholar

Copyright information

© The Physiological Society of Japan and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Yukiko Ikeuchi
    • 1
  • Haruka Kogiso
    • 1
  • Shigekuni Hosogi
    • 1
  • Saori Tanaka
    • 4
  • Chikao Shimamoto
    • 4
  • Toshio Inui
    • 5
  • Takashi Nakahari
    • 1
    • 2
    • 3
    Email author
  • Yoshinori Marunaka
    • 1
    • 2
    • 3
  1. 1.Department of Molecular Cell Physiology, Graduate School of Medical ScienceKyoto Prefectural University of MedicineKyotoJapan
  2. 2.Department of Bio-Ionomics, Graduate School of Medical ScienceKyoto Prefectural University of MedicineKyotoJapan
  3. 3.Japan Institute for Food Education and HealthSt Agnes’ UniversityKyotoJapan
  4. 4.Laboratory of PharmacotherapyOsaka University of Pharmaceutical SciencesTakatsukiJapan
  5. 5.Saisei Mirai ClinicsMoriguchiJapan

Personalised recommendations