ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid tra nsport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENa C) message, and immunomicroscopy showed apical membrane staining for both p roteins. CFTR was also localized to the apical membrane of native human mam mary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance (R-T) are -5.9 mV and 829 Omega .cm(2), respectively. The apical membrane potential (VA) is -40.7 mV, and the mean ratio of apical to basolateral membrane resistance (R-A/R-B) is 2.8. Apical amiloride hyperpolarized V-A by 19.7 mV and tripled R-A/R-B. A cAMP-elevating cocktail depolarized V-A by 17.6 mV, decreased R-A/R-B by 60%, increased short-circuit current by 6 muA/cm(2), decreased R-T by 155 Omega .cm(2), and largely eliminated responses to amiloride. Whole cell pat ch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage (I-V) relation] and forskolin-stimulated Cl- currents (lin ear I-V relation). A capacitance probe method showed that in the control st ate, MEC monolayers either absorbed or secreted fluid (2-4 mul.cm(-2).h(-1) ). Fluid secretion was stimulated either by activating CFTR (cAMP) or block ing ENaC (amiloride). These data plus equivalent circuit analysis showed th at 1) fluid absorption across MEC is mediated by Na+ transport via apical m embrane ENaC, and fluid secretion is mediated, in part, by Cl- transport vi a apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, E NaC, and tight junctions allow MEC to either absorb or secrete fluid and, i n situ, may help control luminal [Na+] and [Cl-].