Detection of Cl- flux in the apical microenvironment of cultured foetal distal lung epithelial cells.
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.
Land
S C
SC
Centre for Research into Human Development, Tayside Institute of Child Health, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, UK. s.c.land@dundee.ac.uk
Collett
A
A
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016764:Cell Polarity; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D005260:Female; D017136:Ion Transport / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D008839:Microelectrodes; D009579:Nitrobenzoates / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D020545:Respiratory Mucosa / Q000166:cytology* / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D000318:Adrenergic beta-Agonists; D002121:Calcium Channel Blockers; D018118:Chloride Channels; D002712:Chlorides; D016895:Culture Media, Serum-Free; D009579:Nitrobenzoates; D062367:ortho-Aminobenzoates; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C001209:fenamic acid; D007545:Isoproterenol