We have previously demonstrated that Na⁺-H⁺ exchanger 1 (NHE1) protein is expressed and activity differentially regulated throughout gestation in the microvillous membrane (MVM) of the human placenta (Hughes et al. 2000). Furthermore, preliminary data have shown that NHE1 mRNA expression is lower in first trimester than term placenta (Lacey et al. 2001). Oxygen tension in the intervillous space of the placenta rapidly increases between 8 and 11 weeks of pregnancy, concomitant with the onset of blood flow through the spiral arteries (Jauniaux et al. 2001). Here we test the hypothesis that NHE1 protein expression in the placenta is regulated by oxygen tension. Villous explants were dissected from first trimester (n = 2, 11+6 and 10+5 weeks) and term (n = 3) placental tissue (5 mg wet weight) and maintained in explant culture on Netwell porous supports for 4-5 days, by which time term placental villous explants have undergone resyncytialisation (Siman et al. 2001). Explants were maintained in 3 or 21 % oxygen in 5 % carbon dioxide at 37 °C. On day 4-5 of culture, several explants were harvested, washed in PBS and protein homogenate prepared at 4 °C in PBS + 1.0 mM serine protease inhibitor. 20 mg protein homogenate from first trimester and term placental explants cultured at 3 and 21 % oxygen, together with MVM (positive control for NHE1; Hughes et al. 2000), were loaded on a 7 % reducing SDS-PAGE gel and electroblotted. Blotted membranes were probed with 1:100 mouse anti-human NHE1 primary monoclonal antibody (Becton Dickenson), followed by incubation with goat anti-mouse-HRP conjugated secondary antibody (1:1000) and detection by enhanced chemiluminescence. A band of 94 kDa representing NHE1 was observed in all samples. Intensity of NHE1 signal was calculated as optical density X area (Molecular Analyst Software, BioRad). In first trimester explants (n = 3), the median change in NHE1 protein expression, quantified as % change in optical density between 3 and 21 %, was -1.83 % (-63.08/+78.2 %, 95 % confidence interval), compared with term explants, n = 5, 27.85 % (-21.8/+106.1 %, 95 % confidence interval). We conclude that NHE1 protein expression in term human placenta may be affected by oxygen tension.

This work was supported by The Wellcome Trust.

All procedures accord with current local guidelines.