Detection of C1(-) 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(C l)) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-](e)) and in respo nse to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillati on of a Cl--selective microelectrode between known points, micrometres apar t, orthogonal to the plasma membrane, Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced s ignal) 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 s tatistically 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 pe rmeable supports, At [Cl-](e)<50 mmoll(-1), an apical-to-basolateral (inwar d) flux, maximal at 400 pmol cm(-2)s(-1) was observed; this reverted to a c ontinuous 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 (basolater ally applied, 10<mu>mol 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- efflu x 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 (DP C) 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 re gion of lung epithelia that varies in both direction and magnitude as a fun ction of external [Cl-](e) and in response to Cl(-)channel blockade and to beta (2) adrenoreceptor and P2Y receptor agonists.