title: Chronic and selective inhibition of basolateral membrane Na-K-ATPase uniquely regulates brush border membrane Na absorption in intestinal epithelial cells.
abstract: Na-K-ATPase, an integral membrane protein in mammalian cells, is responsible for maintaining the favorable intracellular Na gradient necessary to promote Na-coupled solute cotransport processes [e.g., Na-glucose cotransport (SGLT1)]. Inhibition of brush border membrane (BBM) SGLT1 is, at least in part, due to the diminished Na-K-ATPase in villus cells from chronically inflamed rabbit intestine. The aim of the present study was to determine the effect of Na-K-ATPase inhibition on the two major BBM Na absorptive pathways, specifically Na-glucose cotransport and Na/H exchange (NHE), in intestinal epithelial (IEC-18) cells. Na-K-ATPase was inhibited using 1 mM ouabain or siRNA for Na-K-ATPase-α1 in IEC-18 cells. SGLT1 activity was determined as 3-O-methyl-D-[(3)H]glucose uptake. Na-K-ATPase activity was measured as the amount of inorganic phosphate released. Treatment with ouabain resulted in SGLT1 inhibition at 1 h but stimulation at 24 h. To further characterize this unexpected stimulation of SGLT1, siRNA silencing was utilized to inhibit Na-K-ATPase-α1. SGLT1 activity was significantly upregulated by Na-K-ATPase silencing, while NHE3 activity remained unaltered. Kinetics showed that the mechanism of stimulation of SGLT1 activity was secondary to an increase in affinity of the cotransporter for glucose without a change in the number of cotransporters. Molecular studies demonstrated that the mechanism of stimulation was not secondary to altered BBM SGLT1 protein levels. Chronic and direct silencing of basolateral Na-K-ATPase uniquely regulates BBM Na absorptive pathways in intestinal epithelial cells. Specifically, while BBM NHE3 is unaffected, SGLT1 is stimulated secondary to enhanced affinity of the cotransporter.
authors: Department of Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio;
authors: Palanikumar
authors: P
authors: Manoharan
authors: Section of Digestive Diseases, West Virginia University, Morgantown, West Virginia;
authors: Swapna
authors: S
authors: Gayam
authors: Department of Clinical and Translational Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia; and.
authors: Subha
authors: S
authors: Arthur
authors: Department of Clinical and Translational Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia; and.
authors: Balasubramanian
authors: B
authors: Palaniappan
authors: Department of Clinical and Translational Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia; and.
authors: Soudamani
authors: S
authors: Singh
authors: Center for Cardiovascular and Respiratory Sciences, West Virginia University, Morgantown, West Virginia.
authors: Gregory M
authors: GM
authors: Dick
authors: Department of Clinical and Translational Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia; and sundaramu@marshall.edu.
authors: Uma
authors: U
authors: Sundaram
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D003048:Coccidiosis / Q000469:parasitology / Q000473:pathology; D004539:Eimeria / Q000276:immunology / Q000472:pathogenicity; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007408:Intestinal Absorption / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000201:enzymology; D007421:Intestine, Small / Q000166:cytology; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering; D011817:Rabbits; D051381:Rats; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; C495676:Slc5a1 protein, rat; C000613387:Slc9a3 protein, rat; D051269:Sodium-Glucose Transporter 1; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Na-K-ATPase; Na-glucose cotransporter; SGLT1; intestinal inflammation; ouabain
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