Regulation of intestinal epithelial brush border Na+/H+ exchanger isoforms, NHE2 and NHE3, in C2bbe cells

Until recently, studies to characterize the intestinal epithelial Na+/H+ ex changers had to be done in nonepithelial, mutated fibroblasts. In these cel ls, detection of any Na+/H+ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to mea sure NHE activity. Because changes in pH, only approximate Na+/H+ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used (22)[Na] unidirection al apical to cell uptake to measure activities specific to NHE2 or NHE3. Fu rthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental preconditi on. Both brush border NHEs, when expressed in the well-differentiated, inte stinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical do main and are regulated by second messengers in the same way they are regula ted in vivo. Increases in intracellular calcium and cAMP inhibit both isofo rms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca+ involves changes to both K-Na and V-max. In contrast, the same two second messengers inhibit NHE3 by a decrease in V-max exclusively. Phorbol ester activation of protein kinase C alters both V-max and K-Na of NHE3, suggesti ng a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in ep ithelial cells, are basally active and are differentially regulated by sign al transduction pathways.