Subcellular Redistribution Is Involved in Acute Regulation of the Brush Border Na+/H+ Exchanger Isoform 3 in Human Colon Adenocarcinoma Cell Line Caco-2

Abstract

Na؉ /H ؉ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na ؉ /H ؉ exchanger gene family, plays an important role in reabsorption of Na ؉ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in V max , but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from ϳ4.3 to ϳ2.4. This translocation resulted in 10 -15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes ϳ50% to the overall protein kinase C-induced inhibition of the exchanger.

Bimodal Acute Effects of A1 Adenosine Receptor Activation on Na+/H+ Exchanger 3 in Opossum Kidney Cells

“…Additional work will be required to show whether CPA-dependent control of NHE3 activity is accomplished by altering the distribution of NHE3 between endomembranes and the surface membrane. In support of adenosine's having an effect on cycling of NHE3, one recent study in the Caco-2 cell line showed that TPA, an agonist of PKC, regulates NHE3 acutely by changing the intrinsic NHE3 activity as well as the number of NHE3 surface protein (40).…”

The Apical Na+/H+ Exchanger Isoform NHE3 Is Regulated by the Actin Cytoskeleton

“…In both native (epithelial) and heterologous transfection systems, a sizable fraction of NHE3 is located in endosomes, from which it recycles to the membrane (5,29), and it was recently shown that inhibition of NHE3 activity is observed when an imbalance between the rates of exo-and endocytosis is imposed (7). Unpublished observations from our laboratory indicate that endocytosis is mediated, at least in part, by clathrin-coated vesicles.…”

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

“…Furthermore, stimulation of PKC by phorbol ester has been shown to inhibit NHE3 in PS120 cells (24) and in Caco-2 cells (25). Given the parallel action of NHE3 and DRA, it appeared possible that the inhibition of DRA by calcium involved the activation of PKC.…”