The epithelial Sodium Channel (ENaC) in BeWo cells

DEL MÓNACO SILVANA; ASSEF YANINA; DAMIANO ALICIA E; IBARRA CRISTINA; KOTSIAS BASILIO

Los Cocos, Córdoba- Argentina

Simposio; III Latin-American Symposium on Maternal-Fetal Interaction and Placenta:Basic & Clinical Research; 2007

Placental Association of America

Trophoblast plasma membranes contain various Na+ transport systems that participate in nutrient transfer to the fetus and in maintaining cytosol homeostasis. In our laboratory we confirmed the presence of the Epithelial Sodium Channel (ENaC) in normal syncytiotrophoblast. The study was performed to explore sodium currents in BeWo cells, using patch clamp techniques. These cells comprise a monolayer-forming human trophoblast derived cell line, which displays many of the biochemical and morphological properties similar to those reported for the utero proliferative cytotrophoblast. For whole-cell patch clamp experiments we used 8 Br-cAMP, a membrane permeable cAMP analogue to induce channel exposition into the cell surface in cells treated for 12 hs with 100 nM aldosterone. Under these experimental conditions, BeWo cells showed an amiloride sensitive ion current fraction ¿IC50 of 1.25 uM?. Permeability coefficients were 0.2for PNMDG/PNa+, 0.6 for PK+/PNa+ and 1.3 for PLi+/PNa+ >Na+ indicating permeability rank order of Li+>K+>NMDG. With the inside out configuration of the patch clamp we detected two sodium channel conductance groups in aldosterone treated cells. A first group showed a non rectifying single channel conductance of 5.61 ± 0.68 pS (n=10) over the voltage range studied (-120 to 120 m), with a frequency of appearance of approximately 1 every 3 active channels. The second group showed a non rectifying conductance of 8.96 ± 0.91 pS after patch excision, and with a frequency of appearance of approximately 1 every 6 active channels (n=5). Both groups showed a low P0 (P0 of 0.1± 0.05 and 0.09±0.07 at -80 mV for the first and second channel group, respectively) and this P0 was voltage independent, (p>0.05). Expression of the ENaC gene product was determined by RT-PCR and confirmed by immunocytochemistry and western blot analysis. In BeWo cells cultivated in control conditions, RT-PCR studies showed alpha-ENaC, but not beta and gamma ENaC products whereas in aldosterone treated cells (100 nM, 12 h) alpha, beta and gamma ENaC fragments were detected. ENaC subunit proteins were also determined by western blot analysis and immunocytochemistry with specific ENaC antibodies. In summary our results indicate that this cell line express ENaC subunits and that aldosterone exposure was able to modulate a selective response by generating amiloride sensitive sodium currents compatible with Epithelial Sodium Channels.