ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia |
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Authors: | Blaug, Sasha Hybiske, Kevin Cohn, Jonathan Firestone, Gary L. Machen, Terry E. Miller, Sheldon S. |
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Abstract: | Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na+ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(RT) are 5.9 mV and 829 · cm2, respectively. The apical membranepotential (VA) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (RA/RB) is 2.8. Apicalamiloride hyperpolarized VA by 19.7 mV andtripled RA/RB. AcAMP-elevating cocktail depolarized VA by 17.6 mV, decreased RA/RB by60%, increased short-circuit current by 6 µA/cm2,decreased RT by 155 · cm2, and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na+ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm2 · h1). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa+ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na+] and [Cl]. |
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