title: Cholera toxin enhances Na(+) absorption across MCF10A human mammary epithelia.
abstract: Cellular mechanisms to account for the low Na(+) concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short-circuit current (Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na(+) channel ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride-sensitive Isc at all time points tested (2 h to 7 days), an effect that is not reduced with Ctx washout for 12 h. Amiloride-sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002), and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and Western blot analyses indicate no significant increase in either the mRNA or protein expression for α-, β-, or, γ-ENaC subunits. Ctx increases the abundance of both β- and γ-ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na(+) concentration in milk. Importantly, the results suggest that Ctx increases the expression but reduces the activity of the E3 ubiquitin ligase Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na(+) transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body.
authors.affiliation: Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Schultz
mesh_terms: D000042:Absorption; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D062686:Epithelial Sodium Channel Blockers / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D017136:Ion Transport; D042361:Mammary Glands, Human / Q000187:drug effects* / Q000378:metabolism; D008564:Membrane Potentials; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D012333:RNA, Messenger; C507045:SCNN1A protein, human; C507048:SCNN1B protein, human; C507052:SCNN1G protein, human; D002772:Cholera Toxin; D012964:Sodium; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D006854:Hydrocortisone
keywords: ENaC; Isc; amiloride; cholera toxin; epithelial Na+ channel; mammary gland; short-circuit current