Chronic and selective inhibition of basolateral membrane Na-K-ATPase uniquely regulates brush border membrane Na absorption in intestinal epithelial cells

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-[3H]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...

Source: AJP: Cell Physiology - April 15, 2015 Category: Cytology Authors: Manoharan, P., Gayam, S., Arthur, S., Palaniappan, B., Singh, S., Dick, G. M., Sundaram, U. Tags: ARTICLES Source Type: research