THE EPITHELIAL SODIUM CHANNEL (ENaC) IN BEWO CELLS

SILVANA DEL MÓNACO, YANINA ASSEF, ALICIA DAMIANO, CRISTINA IBARRA, BASILIO A. KOTSIAS

Los Cocos, Córdoba

Congreso; III Latin American Symposium on maternal-fetal interaction; 2007

International Federation of Placental Association

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 human syncytiotrophoblast. The present 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 in utero proliferative cytotrophoblast. For whole-cell patch clamp experiments we used 8Br-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 µM). Permeability coefficients were 0.2 for PNMDG/PNa, 0.6 for PK/PNa and 1.3 for PLi/PNa indicating a permeability rank order of Li+ > Na+ > 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 mV), 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 Po (Po of 0.1 ± 0.05 and 0.09 ± 0.07 at -80 mV for the first and second channel group, respectively) and this Po 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 a-ENaC, but not b and g-ENaC products whereas in aldosterone treated cells (100 nM, 12 h) a, b and g-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 the Epithelial Sodium Channel.