NHE2 is an epithelial Na/H exchanger (NHE). Although immunolocalization studies showed that NHE2 is co-localized with another epithelial exchanger, NHE3, on the brush border (bb) membranes of intestinal and renal epithelial cells, NHE2 is inconsistent with known pharmacological and functional properties of bb isoform exchangers being amiloride sensitive and stimulated by phorbol esters. Therefore, in the present study, we examined the membrane location (bb vs basolateral(bl)) of NHE2 stably expressed in polarized Madin-Darby Canine Kidney (MDCK) cells, by measuring NHE activity and by immunocytochemistry. NHE activity was measured fluorometrically with the pH sensitive dye BCECF and [Na]o=140mM. Untransfected cells contained no bb NHE activity but a potent bl NHE which was completely blocked by 10uM HOE694. The bl NHE is NHE1 since the IC50 of NHE1 and NHE2 for HOE694 are 1uM and 30uM, respectively. Further, by Western blotting, untransfected MDCK cells contain only NHE1 but not NHE2 and NHE3. When NHE2 was stably transfected with lipofectin in MDCK cells, a clonal cell line, MDCK/NHE2/Tl was obtained. An anti-NHE2 antibody, Ab597, identified NHE2 by Western blotting as 85kD and 75kD proteins. To determine the polarity of NHE2 expression by function, differential sensitivity of NHE1 and NHE2 to 10uM HOE694 was used. NHE2 activity was found predominantly in the bb of MDCK/NHE2/Tl cells although small but detectable NHE2 activity was also found in the bl membranes (10% of bb). By immunocytochemistry. NHE2 was expressed in the bb of MDCK/NHE2/Tl cells with minimal bl NHE2. Thus, we conclude that NHE2 in transfected MDCK cells is predominantly expressed on the bb. This MDCK/NHE2/Tl cells will be a useful epithelial cell culture model for studying function and growth factor regulation of polarized NHE2.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology