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Detection of Cl- flux in the apical microenvironment of cultured foetal distal lung epithelial cells.
- Source :
-
The Journal of experimental biology [J Exp Biol] 2001 Feb; Vol. 204 (Pt 4), pp. 785-95. - Publication Year :
- 2001
-
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 x cm(-2) x 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 x l(-1), an apical-to-basolateral (inward) flux, maximal at 400 pmol x cm(-2 ) x s(-1), was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol x cm(-2 ) x s(-1) at [Cl]e>100 mmol x l(-1). At [Cl-]e>100 mmol x l(-1), isoproterenol (basolaterally applied, 10 micromol x l(-1)) activated a Cl- influx of 561 pmol x cm(-2 ) x s(-1), whereas UTP (apically applied, 100 micromol x l(-1)) stimulated a Cl- efflux of 300 pmol x cm(-2) x s(-1). In all cases, 50-70 % of J(Cl) was abolished by Cl- channel blockade using 10 micromol x 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 beta2 adrenoreceptor and P2Y receptor agonists.
- Subjects :
- Adrenergic beta-Agonists pharmacology
Animals
Calcium Channel Blockers pharmacology
Cell Polarity
Cells, Cultured
Chloride Channels antagonists & inhibitors
Culture Media, Serum-Free
Female
Isoproterenol pharmacology
Lung metabolism
Microelectrodes
Nitrobenzoates pharmacology
Pregnancy
Rats
Rats, Sprague-Dawley
ortho-Aminobenzoates pharmacology
Chloride Channels metabolism
Chlorides metabolism
Ion Transport physiology
Lung cytology
Respiratory Mucosa cytology
Respiratory Mucosa metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0022-0949
- Volume :
- 204
- Issue :
- Pt 4
- Database :
- MEDLINE
- Journal :
- The Journal of experimental biology
- Publication Type :
- Academic Journal
- Accession number :
- 11171361
- Full Text :
- https://doi.org/10.1242/jeb.204.4.785