title: Functional expression of the human thiazide-sensitive NaCl cotransporter in Madin-Darby canine kidney cells.
abstract: The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which is expressed on the apical membrane of epithelial cells lining the distal convoluted tubule, is responsible for the reabsorption of 5% to 10% of filtered Na(+) and Cl(-). To date, functional studies on the structural and regulatory requirements for localized trafficking and ion-transporting activity of NCC have been hampered by lack of a suitable cell system expressing this cotransporter. Reported here is the functional expression of human NCC (hNCC) in a polarized mammalian cell of renal origin-that is, the high-resistance Madin-Darby canine kidney (MDCK) cell. Western blot testing revealed that the cells predominantly expressed the complex glycosylated (approximately 140 kD) form of hNCC. hNCC was present primarily in the apical part of the cell. The functionality of hNCC was demonstrated by the gain of thiazide-sensitive Na(+) uptake and transepithelial transport activity. Na(+) uptake was significantly increased after short-term (15 min) treatment with forskolin, whereas cyclic guanosine monophosphate, wortmannin, phorbol 12-myriatate 13-acetate, and staurosporine were without effect. This indicates that hNCC activity is regulated through cyclic adenosine monophosphate, rather than via cyclic guanosine monophosphate, phospho-inositide 3-kinases or protein kinase C. Aldosterone did not alter Na(+) uptake in the short term (15 min) but significantly increased the transport activity in the long term (16 h). The latter effect of aldosterone was due to an effect on the cytomegalovirus promoter/enhancer driving the expression of hNCC. hNCC-MDCK cells are a good model for the study of the regulation of apical trafficking and ion-transporting activity of hNCC.
mesh_terms: D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D001581:Benzothiadiazines; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D004232:Diuretics; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D012964:Sodium / Q000493:pharmacokinetics; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics* / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D001581:Benzothiadiazines; D002273:Carcinogens; D004232:Diuretics; C578877:SLC12A2 protein, human; D049993:Sodium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D005576:Colforsin; D000450:Aldosterone; D012964:Sodium; D013755:Tetradecanoylphorbol Acetate