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 transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial [Na.sup.+] channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ([R.sub.T]) are -5.9 mV and 829 [Omega] [multiplied by] [cm.sup.2], respectively. The apical membrane potential ([V.sub.A]) is -40.7 mV, and the mean ratio of apical to basolateral membrane resistance ([R.sub.A]/[R.sub.B]) is 2.8. Apical amiloride hyperpolarized [V.sub.A] by 19.7 mV and tripled [R.sub.A]/[R.sub.B]. A cAMP-elevating cocktail depolarized VA by 17.6 mV, decreased [R.sub.A]/[R.sub.B] by 60%, increased short-circuit current by 6 [micro]A/[cm.sup.2], decreased [R.sub.T] by 155 [Omega] [multiplied by] [cm.sup.2], and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited [Na.sup.+] currents [linear current-voltage (I-V) relation] and forskolin-stimulated [Cl.sup.-] currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2-4 [micro]l [multiplied by] [cm.sup.-2] [multiplied by] [h.sup.-1]). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by [Na.sup.+] transport via apical membrane ENaC, and fluid secretion is mediated, in part, by [Cl.sup.-] transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [[Na.sup.+]] and [[Cl.sup.-]]. amiloride; diphenylamine-2-carboxylate; milk secretion; patch clamp; microelectrodes; electrophysiology; cystic fibrosis; tight junctions; leaky and tight epithelia; epithelial sodium channel; cystic fibrosis transmembrane conductance regulator