Detection of Cl-flux in the apical microenvironment of cultured foetal distal lung epithelial cells

S. C. Land; A. Collett

Detection of Cl-flux in the apical microenvironment of cultured foetal distal lung epithelial cells

S. C. Land, A. Collett

Research output: Contribution to journal › Article › peer-review

Abstract

A self-referencing Cl^--selective microelectrode (Cl^- SrE) was developed and used to detect changes in the direction and magnitude of the Cl^- flux (J_Cl) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl^- concentration ([Cl^-]_e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl^--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl^- flux (pmol cm^-2 s^-1). A Cl^- effusion gradient was used to confirm that empirical measurements of J_Cl using the Cl^- SrE were statistically similar to predicted flux values calculated from the fall in [Cl^-] with distance from the tip of the effusion source. Apical J_Cl was then measured as a function of [Cl^-]_e from polarised FDLE cultures grown on permeable supports. At [Cl^-]_e<50 mmol l^-1, an apical-to-basolateral (inward) flux, maximal at 400 pmol cm^-2 s^-1, was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol cm^-2 s^-1 at [Cl]_e>100 mmol l^-1. At [Cl^-]_e>100 mmol l^-1, isoproterenol (basolaterally applied, 10 μmol l^-1) activated a Cl^- influx of 561 pmol cm^-2 s^-1, whereas UTP (apically applied, 100 μmol l^-1) stimulated a Cl^- efflux of 300 pmol cm^-2 s^-1. In all cases, 50-70 % of J_Cl was abolished by Cl^- channel blockade using 10 μmol l^-1 diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl^- SrE resolves a Cl^- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl^-]_e and in response to Cl^- channel blockade and to β₂ adrenoreceptor and P2Y receptor agonists.

Original language	English
Pages (from-to)	785-795
Number of pages	11
Journal	Journal of Experimental Biology
Volume	204
Issue number	4
Early online date	1 Feb 2001
Publication status	Published - Feb 2001
Externally published	Yes

Access to Document

https://jeb.biologists.org/content/204/4/785Licence: Unspecified

Cite this

@article{07dc0b30df784811a8bf0d3560310458,

title = "Detection of Cl-flux in the apical microenvironment of cultured foetal distal lung epithelial cells",

abstract = "A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (JCl) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmol cm-2 s-1). A Cl- effusion gradient was used to confirm that empirical measurements of JCl using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical JCl was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50 mmol l-1, an apical-to-basolateral (inward) flux, maximal at 400 pmol cm-2 s-1, was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol cm-2 s-1 at [Cl]e>100 mmol l-1. At [Cl-]e>100 mmol l-1, isoproterenol (basolaterally applied, 10 μmol l-1) activated a Cl- influx of 561 pmol cm-2 s-1, whereas UTP (apically applied, 100 μmol l-1) stimulated a Cl- efflux of 300 pmol cm-2 s-1. In all cases, 50-70 % of JCl was abolished by Cl- channel blockade using 10 μmol l-1 diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl-]e and in response to Cl- channel blockade and to β2 adrenoreceptor and P2Y receptor agonists.",

keywords = "β adrenoreceptor, Cl-flux, Cystic fibrosis, Fluid transport, Lung, P2Y receptor, Self-referencing electrode",

author = "Land, {S. C.} and A. Collett",

year = "2001",

month = feb,

language = "English",

volume = "204",

pages = "785--795",

journal = "Journal of Experimental Biology",

issn = "0022-0949",

publisher = "Company of Biologists Ltd",

number = "4",

}

TY - JOUR

T1 - Detection of Cl-flux in the apical microenvironment of cultured foetal distal lung epithelial cells

AU - Land, S. C.

AU - Collett, A.

PY - 2001/2

Y1 - 2001/2

N2 - A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (JCl) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmol cm-2 s-1). A Cl- effusion gradient was used to confirm that empirical measurements of JCl using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical JCl was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50 mmol l-1, an apical-to-basolateral (inward) flux, maximal at 400 pmol cm-2 s-1, was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol cm-2 s-1 at [Cl]e>100 mmol l-1. At [Cl-]e>100 mmol l-1, isoproterenol (basolaterally applied, 10 μmol l-1) activated a Cl- influx of 561 pmol cm-2 s-1, whereas UTP (apically applied, 100 μmol l-1) stimulated a Cl- efflux of 300 pmol cm-2 s-1. In all cases, 50-70 % of JCl was abolished by Cl- channel blockade using 10 μmol l-1 diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl-]e and in response to Cl- channel blockade and to β2 adrenoreceptor and P2Y receptor agonists.

AB - A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (JCl) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmol cm-2 s-1). A Cl- effusion gradient was used to confirm that empirical measurements of JCl using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical JCl was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50 mmol l-1, an apical-to-basolateral (inward) flux, maximal at 400 pmol cm-2 s-1, was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol cm-2 s-1 at [Cl]e>100 mmol l-1. At [Cl-]e>100 mmol l-1, isoproterenol (basolaterally applied, 10 μmol l-1) activated a Cl- influx of 561 pmol cm-2 s-1, whereas UTP (apically applied, 100 μmol l-1) stimulated a Cl- efflux of 300 pmol cm-2 s-1. In all cases, 50-70 % of JCl was abolished by Cl- channel blockade using 10 μmol l-1 diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl-]e and in response to Cl- channel blockade and to β2 adrenoreceptor and P2Y receptor agonists.

KW - β adrenoreceptor

KW - Cl-flux

KW - Cystic fibrosis

KW - Fluid transport

KW - Lung

KW - P2Y receptor

KW - Self-referencing electrode

UR - http://www.scopus.com/inward/record.url?scp=0035746139&partnerID=8YFLogxK

M3 - Article

C2 - 11171361

AN - SCOPUS:0035746139

VL - 204

SP - 785

EP - 795

JO - Journal of Experimental Biology

JF - Journal of Experimental Biology

SN - 0022-0949

IS - 4

ER -

Detection of Cl-flux in the apical microenvironment of cultured foetal distal lung epithelial cells

Abstract

Access to Document

Other files and links

Fingerprint

Cite this