&p.1:Abstract We examined the expression and activity of the Na + /H + exchanger in the human choriocarcinoma BeWo cell line. When treated with methotrexate, these cells differentiated from cytotrophoblast-like cells to en- larged multinucleate syncytiotrophoblast-like cells. There was no change in the apparent K m for Na + between undifferentiated and differentiated cells. However, differ- entiated cells could transport more than five times the proton flux of undifferentiated cells. There was no differ- ence in the Hill coefficient between undifferentiated and differentiated cells. However, the maximal flux (J max ) for undifferentiated cells was higher than that for differenti- ated cells. Inhibition of Na + /H + exchange activity by an amiloride analog and Hoe694 revealed a sensitive and a resistant component in both differentiated and undiffer- entiated cells. Northern blot analysis and immunocyto- chemistry suggested that the sensitive component was due to the NHE1 isoform of the protein while the resis- tant component was due to the NHE3 isoform. The NHE1 isoform was localized to the brush border mem- brane of BeWo cells and Western blot analysis showed that the NHE1 protein was more abundant in brush bor- der membranes from differentiated BeWo cells compared to undifferentiated cells. The results show that BeWo cells contain the NHE1 and NHE3 isoforms of the Na + /H + exchanger and that the NHE1 isoform is primari- ly localized to the brush border membrane. &kwd:Key words: Na + /H + exchanger · Placenta · BeWo · NHE1 · NHE3 · Differentiation&bdy: Introduction The Na + /H + exchanger is a mammalian plasma mem- brane protein that exchanges one intracellular proton for an extracellular sodium ion. It is involved in pH regula- tion, control of cell volume and is stimulated by growth factors [6, 18]. Several isoforms of the protein have been identified (NHE1 to NHE5) and their relative abundance in some tissues has been examined [16, 22, 25, 32, 40, 41]. The NHE1 isoform is the most widely distributed type and is present in most, if not all, mammalian cells [35]. NHE2 and NHE3 are found in gastrointestinal tis- sues and renal epithelial tissues with NHE2 having a more diverse distribution than NHE3. Small amounts of rat NHE2 are also found in skeletal muscle, brain, kid- ney, testis, uterus, heart, and lung [44]. NHE3 is more re- stricted to the kidney and gastrointestinal tract. However, small amounts of human NHE3 are found in some other tissues, such as testis, ovary, prostate, thymus, leuko- cytes, brain, spleen, and placenta [7]. Newly found NHE5 is expressed mainly in non-epithelial tissues in brain, testis, spleen, and skeletal muscle [25]. An important feature of the Na + /H + exchanger iso- forms is their inhibition by the diuretic amiloride and the more potent related derivatives. The sensitivity of the various isoforms to these compounds varies. NHE1 is somewhat more sensitive than NHE2, depending on the species and the derivative used. NHE3 is much more re- sistant than NHE2 and NHE1. The apparent inhibitory constants vary between reports; however, for amiloride they range between 1 and 3 μM for NHE1 and NHE2 and between 39 and 100 μM for NHE3 [12, 31, 43, 47, 48]. NHE4 and NHE5 have not been well characterized in this regard. Hoe694 is a relatively new and potent an- tagonist of Na + /H + exchange and inhibits the isoforms with a similar order of sensitivity [12]. The subcellular localization of the NHE1, NHE2, and NHE3 isoforms varies. In non-polarized cells, NHE1 re- sides in the plasma membrane. In most polarized epithe- lial cells it is found in the basolateral membrane [4, 5]. NHE2 may be in varied locations in the basolateral N.L.C.L. Silva · H. Wang · C.V. Harris · D. Singh · L. Fliegel ( ✉ ) Departments of Pediatrics and Biochemistry, Faculty of Medicine, University of Alberta, 417 Heritage Medical Research Center, Edmonton, Alberta, Canada, T6G 2S2 N.L.C.L. Silva Dept. de Imunologia, Centro de Pesquisa Aggeu Magalhaes Fundacao Oswald Cruz, Recife-PE, 50670-420, Brazil&/fn-block: Pflügers Arch – Eur J Physiol (1997) 433:792–802 © Springer-Verlag 1997 ORIGINAL ARTICLE &roles:Norma Lucena C.L. Silva · Huayan Wang Carmen V. Harris · Dyal Singh · Larry Fliegel Characterization of the Na + /H + exchanger in human choriocarcinoma (BeWo) cells &misc:Received: 25 July 1996 / Received after revision: 25 Novembber 1996 / Accepted: 3 December 1996