The Na⁺/H⁺ exchanger (NHE) isoforms NHE1, NHE2 and NHE3 are expressed on both maternal facing, microvillous plasma membrane (MVM) and fetal facing, basal plasma membrane (BM) of the human placental syncytiotrophoblast (Pepe et al. 2001). We used fragments of placental tissue to investigate the functional activity of NHE isoforms in the control of intracellular pH in the syncytiotrophoblast.

Fragments of term placenta were incubated in Tyrode buffer (containing (mM): NaCl 135, KCl 5, CaCl₂ 1.8, MgCl₂ 1, Mops 10 and glucose 5) with 1 µM BCECF (Molecular Probes) for 30 min at 37 °C, washed in buffer without BCECF and then incubated for 2 X 15 min at 37 °C with fresh Tyrode solution. Fragments were plated onto coverslips and BCECF excited with 440 and 490 nm wavelengths using a monochromator (Cairn Research Limited). Emitted light was detected using a CoolSnap camera (Roper Scientific) and the data acquired using MetaFluor software (Universal Imaging Corporation). NHE activity was assessed as the rate of Na⁺-induced recovery of cytosolic pH (pH_i) following an acid load, imposed by prepulsing with 20 mM NH₄Cl, expressed as pH units min^-1, mean ± S.E.M., n = number of placentas.

Control rate of recovery from an acid load was 0.18 ± 0.03 pH units min^-1 (n = 18) and in Na⁺-free solutions (Na⁺ substituted with choline 135 mM) was -0.04 ± 0.03 (n = 6). In tissue incubated in Na⁺-containing Tyrode solution, with amiloride (500 µM), a non-specific inhibitor of NHE transport (Mahnensmith et al. 1985) or HOE 694 (100 µM inhibits NHE1; Counillon et al. 1993), the rate of recovery from an acid load was significantly reduced to 0.01 ± 0.01 (n = 5) and -0.04 ± 0.03 pH units min^-1 (n = 6), respectively (P < 0.05, ANOVA followed by Bonfferoni multiple comparisons test). S3226 at 1 µM (inhibits NHE3; Schwark et al. 1998) had no effect on the rate of recovery, 0.12 ± 0.04 pH units min^-1 (n = 9).

In conclusion, isolated placental fragments exhibited a Na⁺-dependent recovery from an acid load. Both amiloride and HOE694 blunted such recovery, but S3226 had no effect. These data support evidence from membrane vesicles (Speake et al. 2002) showing that NHE3 is not functionally active in the term syncytiotrophoblast under basal conditions.

This work was supported by The Wellcome Trust. HOE 694 and S3226 were kindly donated by Dr Jurgen Punter, Aventis Pharma.