Detection of C1(-) flux in the apical microenvironment of cultured foetaldistal lung epithelial cells

摘要

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 Jet 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 Ja was then measured as a function of [Cl-](e) from polarised IDLE cultures grown on permeable supports, At [Cl-](e)<50 mmoll(-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)>100mmol l(-1), isoproterenol (basolaterally applied, 10mol l(-1)) activated a Cl- influx of 561 pmol cm(-2)s(-1), whereas UTP (apically applied, 100 mu mol l(-1)) stimulated a Cl- efflux of 300 pmol cm(-2)s(-1) In all cases, 50-70 % of Jet was abolished by Cl- channel blockade using 10 mu mol 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 beta (2) adrenoreceptor and P2Y receptor agonists.