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Department of Physiology and Pharmacology, Institute of Medical Biology, University of Southern Denmark-Odense University, DK-5000 Odense C, Denmark
Na+/H+-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na+-dependent processes to acid extrusion, 2) sensitivity to Na+/H+ exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pHi) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na+ concentration ([Na+]o) during pHi recovery decreased H+ efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na+. The Na+/H+ exchange inhibitors ethylisopropylamiloride and amiloride inhibited H+ efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed 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 propose that acid extrusion in duodenal epithelial cells involves Na+/H+ exchange by isoforms NHE1, NHE2, and NHE3.
intracellular pH; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; ethylisopropylamiloride; amiloride; fluorescence spectroscopy; reverse transcriptase-polymerase chain reaction; Western blot; duodenum; sodium/hydrogen exchanger isoforms
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