Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, PC32
Nitrative stress increases K+ (86Rb) efflux from human placental syncytiotrophoblast
P. Diaz1, C. P. Sibley1, S. L. Greenwood1
1. Maternal and Fetal Health Research Centre, University of Manchester, Manchester, United Kingdom.
Successful pregnancy depends on maintenance of syncytiotrophoblast (STB), the transporting epithelium of the human placenta, by cell proliferation, differentiation and apoptosis. Pre-eclampsia, a serious disease of pregnancy, is associated with abnormal STB renewal and increased nitrative stress. In many tissues cell proliferation and apoptosis is regulated by K+ channels, including intermediate conductance Ca2+-activated K+ channels (IKCa), and K+ channel activity can be modulated by reactive nitrogen species (RNS). Here we test the hypothesis that STB expresses functional IKCa channels and that K+ channels can be modulated by acute and chronic exposure to RNS. STB K+ channel activity was assessed by measuring 86Rb (tracer for K+) efflux from cytotrophoblast cells or villous tissue of normal term placentas (n=3-6). Cells/fragments were incubated in 86Rb for 2h and then 86Rb efflux measured every 1 or 2min for 10-16min. Cytotrophoblast cells were maintained in culture for 66hr until syncytia had formed. 86Rb efflux was measured over 15min under control conditions and in response to acute application (over 10-15min) of peroxynitrite (10-4M; ONOO-), the IKCa channel opener EBIO (100μM) or blocker TRAM34 (10μM). To assess chronic exposure to RNS, placental tissue explants were cultured at 6%O2 (normoxia for placenta at term) for 6 days. On days 3-5, explants were treated daily with EBIO (100μM) or the free radical generator SIN-1 (2.5mM) and 86Rb efflux was determined at day 6. %86Rb efflux was expressed as (86Rb effluxt/86Rb in cells or tissueo)x100 and data were analyzed by two-way ANOVA or Wilcoxon signed rank test. EBIO promoted an 8.4 and 1.6-fold increase in 86Rb efflux from cytotrophoblast cells and freshly isolated villous fragments respectively (p<0.04) that was almost completely blocked (>90%) by TRAM34. ONOO- induced a sustained increase in 86Rb efflux from cytotrophoblast cells (2.8x control; p<0.0001) and this efflux was inhibited by 5mM Ba2+ (20%; p<0.01) and TRAM34 (40%; n=2). 86Rb efflux over 10min was increased by 74% and 27% (p<0.01) in placental explants treated with EBIO and SIN-1 respectively. EBIO and SIN-1 did not damage cell integrity as the release of lactate dehydrogenase into the culture medium was similar to control. EBIO stimulation of 86Rb efflux from STB is inhibited by TRAM34 indicating the presence of IKCa channels. Short term nitrative stress (ONOO-) elevates 86Rb efflux in part through activation of IKCa channels. Longer term exposure of placental villous tissue to RNS (SIN-1) promotes 86Rb efflux in common with EBIO. Further experiments are needed to confirm whether IKCa channels in STB are a direct target of RNS and whether activation of these channels by nitrative stress in pre-eclampsia could contribute to altered trophoblast turnover.
Where applicable, experiments conform with Society ethical requirements