Video-microscopy for analysis of molecular dynamics in cells

Ozawa, Koichiro; Tamura, Atsushi; Ikeda, Kayo; Kawai, Eriko; Kondo, Tadashi; Fukano, Yuya; Nomura, Seiji; Ishihara, Yuko; Masujima, Tsutomu

doi:10.1016/s0731-7085(97)00036-8

Cited by 10 publications

(4 citation statements)

References 16 publications

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“…Therefore, the drugs activating beating cilia are of particular importance to prevent or improve respiratory diseases. Ambroxol (ABX), a frequently prescribed secretolytic agent for respiratory diseases, has been shown to increase the CBF (ciliary beat frequency) and CBA (ciliary bend angle, an index of amplitude) in ciliated airway epithelial cells [33,37]. However, the ABX-stimulated mechanism enhancing the CBF and CBA is still controversial.…”

Section: Introductionmentioning

confidence: 99%

Ambroxol-enhanced frequency and amplitude of beating cilia mediated by pHi and [Cl-]i in lung airway epithelial cells of mice

Nakahari,

Suzuki,

Kawaguchi

et al. 2023

Preprint

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Ambroxol (ABX), a frequently prescribed secretolytic agent that stimulates Ca2+ release from acidic stores and Ca2+ entry through CaV1.2, enhanced the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30% in ciliated lung airway epithelial cells (c-LAECs) of mice. An increase in the intracellular Ca2+ concentration ([Ca2+]i) stimulated by ABX triggers two signals in c-LAECs; an increase in pHi (pH pathway) and a decrease in [Cl-]i (Cl- pathway). The pH pathway, which was activated by the HCO3- entry through Na+-HCO3- cotransporter (NBC) and inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), enhanced the CBF (by 30%) and CBA (by 15-20%) and the Cl- pathway, which was activated by Cl- secretion through anoctamine 1 (ANO1) and inhibited by Cl- channel blockers (NPPB and T16Ainh), enhanced the CBA (by 10-15%). The enhancement of the CBF and CBA stimulated by ABX was decreased to 50% by a Ca2+-free solution or nifedipine (an inhibitor of L-type Ca2+ channels) and was abolished by BAPTA-AM in c-LAECs, indicating that an increase in [Ca2+]i stimulated by ABX is essential for activating the pH pathway and the Cl- pathway. The enhancement of CBF and CBA enhanced by ABX was mimicked by applying a CO2/HCO3--free Cl--free solution, which increased the pHi and decreased the [Cl-]i. In conclusion, ABX increased the CBF and CBA by 30% via the pH pathway and the Cl- pathway triggered by the [Ca2+]i increase in c-LAECs of mice.

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Section: Introductionmentioning

confidence: 99%

Ambroxol-enhanced frequency and amplitude of beating cilia mediated by pHi and [Cl-]i in lung airway epithelial cells of mice

Nakahari,

Suzuki,

Kawaguchi

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Therefore, the drugs activating beating cilia are of particular importance to prevent or improve respiratory diseases. Ambroxol (ABX), a frequently prescribed secretolytic agent for respiratory diseases, which increases [Ca 2+ ] i by activating a voltage-gated Ca 2+ channel, Ca V 1.2, in the ciliated lung airway 2 of 19 epithelial cells (c-LAECs), has been shown to increase the CBF (ciliary beat frequency) and CBA (ciliary bend angle, an index of amplitude) [5,6]. However, the Ca 2+ -regulated signals following Ca V 1.2 activation, which may enhance CBF and CBA, remain uncertain.…”

Section: Introductionmentioning

confidence: 99%

Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl−]i Decrease in the Lung Airway Epithelial Cells of Mice

Nakahari,

Suzuki,

Kawaguchi

et al. 2023

IJMS

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Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl−]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3− cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15–20%), and a decrease in [Cl−]i, which was induced by the Cl− release via anoctamine 1 (ANO1), enhanced the CBA (by 10–15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl−]i in the c-LAECs. In conclusion, the pHi increase and the [Cl−]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

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“…In vitro, dynein activity and ciliary motility are stimulated by mucus and medium conditions; for this reason ciliary beat frequency (CBF) informs on the temporary motility status, but remains uninformative on the degree of differentiation [Ingels et al, 1992]. Up to now, evaluation of respiratory epithelium cultured in vitro has mostly been performed by measurement of CBF [Umeda et al, 1995;Ozawa et al, 1997]. Although multiple ciliated cells may be analysed by CBF measurement, only limited regions of interest within the culture may be analysed and no information is provided on ciliary function of dormant cells.…”

Section: Introductionmentioning

confidence: 99%

Adenosine Triphosphatase Expression Correlates with Ciliary Activity of Respiratory Epithelium in vitro

Schütz

Foerster²,

Bücheler³

2002

Cells Tissues Organs

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In vitro culture of respiratory epithelium is of great utility for pharmacological investigations and tissue engineering. Up to now, the degree of differentiation of respiratory cells cultured in vitro has exclusively been estimated by measuring ciliary beat frequency (CBF). Ciliary motility is dependent on the function of the motor protein dynein that is composed of at least two heavy chains, sharing attributes of adenosine triphosphatases (ATPases). CBF is further dependent on medium conditions and does not allow to draw any accurate conclusion on the proportion of fully differentiated ciliated cells in culture. For this reason we introduced the immunohistochemical detection of a 100-kD ATPase subunit as a correlation with dynein activity in human respiratory cell tissue culture. Our results show that the amount of immunohistochemically detectable ATPase-subunit-positive cells strongly correlates with ciliary motility in vitro. Cultures without ciliary activity exhibited no ATPase staining, whereas in cell cultures with excessive ciliary beat, up to 15.1% of the cells were ATPase positive. Immunohistochemical detection of ATPase in respiratory cell cultures seems to be a sensitive and reproducible complement for the characterization of cultured ciliated epithelium.

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Video-microscopy for analysis of molecular dynamics in cells

Cited by 10 publications

References 16 publications

Ambroxol-enhanced frequency and amplitude of beating cilia mediated by pHi and [Cl-]i in lung airway epithelial cells of mice

Ambroxol-enhanced frequency and amplitude of beating cilia mediated by pHi and [Cl-]i in lung airway epithelial cells of mice

Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl−]i Decrease in the Lung Airway Epithelial Cells of Mice

Adenosine Triphosphatase Expression Correlates with Ciliary Activity of Respiratory Epithelium in vitro

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