NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na+/H+-exchangers (NHE) mediate acid extrusion from duodenal epithelial cel ls, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na+-dependent processes to acid extrusi on, 2) sensitivity to Na+/H+ exchange inhibitors, and 3) molecular expressi on of NHE isoforms. By fluorescence spectroscopy the recovery of intracellu lar pH (pH(i)) was measured on suspensions of isolated acidified murine duo denal epithelial cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfl uorescein. Expression of NHE isoforms was studied by RT-PCR and Western blo t analysis. Reduction of extracellular Na+ concentration ([Na+](o)) during pH(i) recovery decreased H+ efflux to minimally 12.5% of control with a rel atively high apparent Michaelis constant for extracellular Na+. The Na+/Hexchange inhibitors ethylisopropylamiloride and amiloride inhibited H+ effl ux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were express ed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na+](o) and inhibitors we p ropose that acid extrusion in duodenal epithelial cells involves Na+/H+ exc hange by isoforms NHE1, NHE2, and NHE3.