关键词：自参比电极(self-referencing electrode,SrE);lung(肺);囊性纤维化(cystic Fibrosis);β2肾上腺素受体(β2 adrenoreceptor);氯离子流(Cl^- flux)

参考文献：LAND SC, et al. The Journal of Experimental Biology, 2001, 204, 785-795

全文下载：http://jeb.biologists.org/cgi/reprint/204/4/785

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 (pmolcm^-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<50mmoll^-1, an apical-tobasolateral (inward) flux, maximal at 400 pmolcm^-2 s^-1, was observed; this reverted to a continuous basolateralto-apical (outward) flux of 203 pmolcm^-2 s^-1 at [Cl]_e> 100 mmoll^-1. At [Cl^-]_e>100mmoll^-1, isoproterenol (basolaterally applied, 10 mmol l^-1) activated a Cl^- influx of 561 pmolcm^-2 s^-1, whereas UTP (apically applied, 100 mmol l^-1) stimulated a Cl^- efflux of 300 pmolcm^-2 s^-1. In all cases, 50–70 % of J_^Cl was abolished by Cl^- channel blockade using 10 mmol 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.
Key words: self-referencing electrode, fluid transport, lung, cystic fibrosis, β2 adrenoreceptor, P2Y receptor, Cl^- flux.