Na+/H+ exchangers are integral plasma membrane proteins that exchange extracellular Na+ for intracellular H+ with a stoichiometry of one for one. They are inhibitable by the diuretic amiloride and have multiple cellular functions, including intracellular pH homeostasis, cell volume control, and electroneutral NaCl absorption in epithelia. The presence of multiple forms of the exchangers was demonstrated by the recent cloning of four mammalian Na+/H+ exchangers, NHE1, NHE2, NHE3, and NHE4. All of these cloned Na+/H+ exchangers have 10-12 putative transmembrane helixes and a long cytoplasmic carboxyl domain. Despite the structural similarity, these Na+/H+ exchanger isoforms differ in their tissue distribution, kinetic characteristics, and response to external stimuli. The present review deals with the recent developments in the molecular identification of the Na+/H+ exchanger gene family, the functional characteristics, and the short-term regulation of Na+/H+ exchange at molecular and cellular levels.

NHE3, a cloned intestinal and renal brush border Na+/H+ exchanger, has previously been shown to be both stimulated and inhibited by different protein kinases/growth factors. For instance, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by protein kinase C. In the present study, we used a series of NHE3 C terminus truncation mutants to identify separate regions of the C-terminal cytoplasmic tail responsible for stimulation and inhibition by protein kinases/growth factors. Five NHE3 C terminus truncation mutant stable cell lines were generated by stably transfecting NHE3 deletion cDNAs into PS120 fibroblasts, which lack any endogenous Na+/H+ exchanger. Using fluorometric techniques, the effects of the calcium/calmodulin (CaM) inhibitor W13, calcium/CaM kinase inhibitor KN-62, phorbol myristate acetate, okadaic acid, FGF, and fetal bovine serum on Na+/H+ exchange were studied in these transfected cells. Inhibition of basal activity of full-length NHE3 is mediated by CaM at a site C-terminal to amino acid 756; this CaM effect occurs through both kinase dependent and independent mechanisms. There is another independent inhibitory domain for protein kinase C between amino acids 585 and 689. In addition, there are at least three stimulatory regions in the C-terminal domain of NHE3, corresponding to amino acids 509-543 for okadaic acid, 475-509 for FGF, and a region N-terminal to amino acid 475 for fetal bovine serum. We conclude that separate regions of the C terminus of NHE3 are involved with stimulation or inhibition of Na+/H+ exchange activity, with both stimulatory and inhibitory domains having several discrete subdomains. A conservative model to explain the way these multiple domains in the C terminus of NHE3 regulate Na+/H+ exchange is via an effect on associated regulatory proteins.

In terminally differentiated ileal villus Na+-absorptive cells, epidermal growth factor (EGF) stimulates NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors, and as we confirm here, a brush border tyrosine kinase. In the present study we show that brush border phosphatidylinositol 3-kinase (PI 3-kinase) is involved in EGF stimulation of NaCl absorption and brush border Na+/H+ exchange. In rabbit ileum studied with the Ussing chamber-voltage clamp technique, EGF stimulation of active NaCl absorption is inhibited by the selective PI 3-kinase inhibitor wortmannin. PI 3-kinase, a largely cytosolic enzyme, translocates specifically to the brush border of ileal absorptive cells following EGF treatment. This translocation occurs as early as 1 min after EGF treatment and remains increased at the brush border for at least 15 min. EGF also causes a rapid (1 min) and large (4-5-fold) increase in brush border PI 3-kinase activity. Involvement of PI 3-kinase activity in intestinal Na+ absorption is established further by studies done in the human colon cancer cell line, Caco-2, stably transfected with the intestinal brush border isoform of the Na+/H+ exchanger, NHE3 (Caco-2/NHE3 cells). Brush border Na+/H+ exchange activity was measured using the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)5-(6)-carboxyfluorescein. EGF added to the basolateral surface but not apical surface of Caco-2/NHE3 cells increased brush border Na+/H+ exchange activity. The EGF-induced increase in brush border Na+/H+ exchange activity was completely abolished in cells pretreated with wortmannin. EGF treatment caused increased tyrosine phosphorylation of PI 3-kinase in both ileal brush border membranes and Caco-2/NHE3 cells, suggesting that a tyrosine kinase upstream of the PI 3-kinase is involved in the EGF effects on Na+ absorption. In conclusion, the present study provides evidence in two separate intestinal models, the ileum and a human colon cancer cell line, that PI 3-kinase is an intermediate in EGF stimulation of intestinal Na+ absorption.