title: ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia.
abstract: 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(+) 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(T)) are -5.9 mV and 829 Omega x cm(2), respectively. The apical membrane potential (V(A)) 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 microA/cm(2), decreased R(T) by 155 Omega x cm(2), and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na(+) currents [linear current-voltage (I-V) relation] and forskolin-stimulated Cl(-) currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2--4 microl x cm(-2) x h(-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(+) transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl(-) 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(+)] and [Cl(-)].
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
authors.forename: S
authors.initials: S
authors.lastname: Blaug
authors.forename: K
authors.initials: K
authors.lastname: Hybiske
authors.forename: J
authors.initials: J
authors.lastname: Cohn
authors.forename: G L
authors.initials: GL
authors.lastname: Firestone
authors.forename: T E
authors.initials: TE
authors.lastname: Machen
authors.forename: S S
authors.initials: SS
authors.lastname: Miller
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D017097:Electric Impedance; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology; D053503:Epithelial Sodium Channels; D007150:Immunohistochemistry; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D012016:Reference Values; D015222:Sodium Channels / Q000502:physiology*
chemical_list: D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000584:Amiloride; D000255:Adenosine Triphosphate; D000242:Cyclic AMP