Unique Regulation of Na-K-ATPase during Growth and Maturation of Intestinal Epithelial Cells

Abstract

Na-K-ATPase on the basolateral membrane provides the favorable transcellular Na gradient for the proper functioning of Na-dependent nutrient co-transporters on the brush border membrane (BBM) of enterocytes. As cells mature from crypts to villus, Na-K-ATPase activity doubles, to accommodate for the increased BBM Na-dependent nutrient absorption. However, the mechanism of increased Na-K-ATPase activity during the maturation of enterocytes is not known. Therefore, this study aimed to determine the mechanisms involved in the functional transition of Na-K-ATPase during the maturation of crypts to villus cells. Na-K-ATPase activity gradually increased as IEC-18 cells matured in vitro from day 0 (crypts) through day 4 (villus) of post-confluence. mRNA abundance and Western blot studies showed no change in the levels of Na-K-ATPase subunits α1 and β1 from 0 to 4 days post-confluent cells. However, Na-K-ATPase α1 phosphorylation levels on serine and tyrosine, but not threonine, residues gradually increased. These data indicate that as enterocytes mature from crypt-like to villus-like in culture, the functional activity of Na-K-ATPase increases secondary to altered affinity of the α1 subunit to extracellular K⁺, in order to accommodate the functional preference of the intestinal cell type. This altered affinity is likely due to increased phosphorylation of the α1 subunit, specifically at serine and tyrosine residues.

Keywords: Na-dependent nutrient co-transport; Na/K-ATPase; cell maturation; crypt cells; intestinal absorption; villus cells.

Figure 1

Alkaline phosphatase (ALP) activity to validate IEC-18 cell maturation. Values are represented as means ± standard error of the mean (SEM), n = 4. Values not sharing common superscripted letters are significantly different at p < 0.001.

Figure 2

Increase in Na-dependent glucose uptake as IEC-18 cells matured. Uptake was performed in the presence and absence of phlorizin (1 mM) in reaction medium containing [³H]-OMG tracer. Values are represented as means ± SEM, n = 6 independent experiments. Values not sharing common superscripted letters are significantly different at p < 0.001.

Figure 3

Na-K-ATPase activity as measured by P_i release in IEC-18 cells. Na-K-ATPase activity was measured in the presence or absence of ouabain (1 mM). The absolute Na-K-ATPase activity presented was calculated by subtracting P_i release in the presence of ouabain from that in the absence of ouabain. (A) Cellular homogenates. (B) Plasma membrane preparations. Values are represented as means ± SEM, n = 5. Values not sharing common superscripted letters are significantly different at p < 0.01.

Figure 4

Na-K-ATPase activity as measured by ⁸⁶Rb⁺ uptake in IEC-18 cells. Values are represented as means ± SEM, n = 5. Values not sharing common superscripted letters are significantly different at p < 0.01.

Figure 5

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of IEC-18 cells on different days of post confluence. Values are relative to 0-day and normalized to β-actin. (A). Na-K-ATPase α1. (B). Na-K-ATPase β1. Values are represented as mean ± SEM, n = 4.

Figure 6

Western blot analysis of IEC-18 cells in cellular homogenates. (A). Representative blot of Na-K-ATPase α1, Na-K-ATPase β1 and internal control β-actin. Densitometric quantitation of Western blots. (B). Na-K-ATPase α1 and (C). Na-K-ATPase β1. Values are relative to 0-day and normalized to β-actin. Values are represented as mean ± SEM, n = 4.

Figure 7

Western blot analysis of IEC-18 cells in plasma membrane fractions. (A). Representative blots of Na-K-ATPase α1 and Na-K-ATPase β1. Densitometric quantitation of Western blots. (B). Na-K-ATPase α1 and (C). Na-K-ATPase β1. Equal amount of proteins were loaded. Values are relative to 0-day and represented as mean ± SEM, n = 4.

Figure 8

Western blot analysis of phosphorylation levels of Na-K-ATPase α1 in plasma membrane during different days post confluence. (A). Representative blot of p-tyrosine, p-serine, p-threonine and Na-K-ATPase α1 after immunoprecipitation with Na-K-ATPase α1 antibody. Densitometric quantitation of Western blots. (B). p-tyrosine. (C). p-Serine. (D). p-Threonine. Values are relative to 0-day and normalized to Na-K-ATPase α1. Values are represented as mean ± SEM, n = 3. Values not sharing common superscripted letters are significantly different at p < 0.05.

Figure 9

Western blot analysis of plasma membrane Na-K-ATPase α1-Ser23 at different days of post confluence. (A). Representative blot of experiment done in triplicate. (B). Densitometric analysis of Western blots. Values are relative to 0-day and normalized to Na-K-ATPase α1. Values are represented as mean ± SEM, n = 3. Values not sharing common superscripted letters are significantly different at p < 0.01.