Basic Fibroblast Growth Factor Stimulates Surface Expression and Activity of Na+/H+ Exchanger NHE3 via Mechanism Involving Phosphatidylinositol 3-Kinase

Abstract

Na؉ /H ؉ exchanger NHE3 is a plasma membrane (PM) protein, which contributes to Na ؉ absorption in the intestine. Growth factors stimulate NHE3 via phosphatidylinositol 3-kinase (PI3-K), but mechanism of this process is not clear. To examine the hypothesis that growth factors stimulate NHE3 by modulating NHE3 recycling, and that PI3-K participates in this mechanism, we used PS120 fibroblasts expressing a fusion protein of NHE3 and green fluorescent protein. At steady state, ϳ25% of cellular NHE3 content was expressed at PM. Inhibition of PI3-K decreased PM expression of NHE3, which correlated with retention of the exchanger in recycling endosomal compartment. In contrast, basic fibroblast growth factor (bFGF) increased PM expression of NHE3, which was associated with a 2-fold increase in rate constant for exit of the exchanger from the recycling compartment. Qualitatively similar effects of bFGF were observed in cells pretreated with PI3-K inhibitors, but their magnitude was only ϳ50% of that in intact cells. These data suggest that: (i) bFGF stimulates NHE3 by increasing PM expression of the exchanger; (ii) PI3-K mediates PM expression of NHE3 in both basal and bFGF-stimulated conditions, and (iii) not all of the effects of bFGF on NHE3 expression are mediated by PI3-K, suggesting additional regulatory mechanisms.In the mammalian intestine, sodium and water are reabsorbed by multiple mechanisms which include the activity of Na ϩ /H ϩ exchanger NHE3. 1 This transmembrane protein is expressed in the epithelium of renal tubules, intestine, gall bladder, and salivary gland, where it was localized to the apical microvillar domain and, at least in the kidney and in the intestine, to an yet undefined cytoplasmic compartment (1-4).In the small intestine, NHE3 participates in neutral NaCl absorption, and in the increase in Na ϩ absorption that occurs via neurohormonal stimulation after meals (5). The activity of NHE3 is acutely regulated by multiple mechanisms involving growth factors and protein kinases (6). We and others have shown that stimulation of NHE3 activity by growth factors, okadaic acid, and serum occurs via an increase in the maximal velocity (V max ) of the exchange, whereas phorbol ester and carbachol inhibits NHE3 via a decrease in V max (6). These effects were observed in non-polarized mesenchymal cells as well as in epithelial cells, and they suggested that at least part of the acute regulation might be accomplished by rapid changes in the number of active exchanger molecules at the plasma membrane.Over the last few years, a growing body of evidence has indicated that NHE3 might, indeed, be regulated by redistribution of the exchanger molecules between the cytoplasm and the plasma membrane. Thus, recycling of NHE3 has been suggested in kidney epithelial cells based on the results of subcellular fractionation experiments (7,8), and on the presence of an intracellular compartment accumulating NHE3 (1). Moreover, the protein kinase C-mediated inhibition of endogenous NHE3 in the human colonic adenocarcino...

Gs Protein-coupled Receptor Agonists Induce Transactivation of the Epidermal Growth Factor Receptor in T84 Cells

“…Another possibility is that PI3K may regulate trafficking of transport proteins to and from the plasma membrane. In support of this hypothesis it has been previously shown that PI3K is intimately involved in cytoskeletal reorganization and membrane trafficking in a number of systems, including intestinal epithelial cells (51)(52)(53)(54)(55). Furthermore, a growing body of evidence suggests that epithelial secretory responses to cAMP-dependent agonists are not only dependent on phosphorylation and activation of CFTR channels resident in the apical membrane but are also dependent on rapid trafficking of the channel between endosomes and the plasma membrane (56 -59).…”

Ezrin regulates NHE3 translocation and activation after Na + -glucose cotransport

“…The best-characterized mechanism of NHE3 up-regulation is recruitment to the plasma membrane (22,23). The data show that this is also the mechanism by which Na ϩ -glucose cotransport activates NHE3.…”

“…We considered NHE3 phosphorylation as a potential regulatory mechanism (21), but, although documented, the role of phosphorylation in NHE3 regulation remains to be established. We also considered trafficking of NHE3 between intracellular vesicles and the apical membrane, which has been described as a mechanism of acute NHE3 up-regulation (22,23). To explore this possibility, we measured surface NHE3 before and after initiation of Na ϩ -glucose cotransport.…”

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

“…Basal NHE3 activity was inhibited by exposure to the PI3K inhibitors, viz. LY594002 (31) and wortmannin (32,33) at low doses, and acutely stimulated NHE3 was reduced by the same inhibitors between 50 and 100% based on the cell type expressing NHE3 and the ligand studied (FGF in PS120 cells by 50% (32); lysophosphatidic acid in opossum kidney cells by 100%) (31). These previous studies of PI3K dependence of basal and acutely stimulated NHE3 activity plus the rapid stimulation of NHE3 activity when PI(3,4,5) 3 was added to the Table 1.…”