Activity and expression of Na(+)‐K(+)‐ATPase in human placental cytotrophoblast cells in culture.

Abstract

1. To determine whether there is a change during differentiation, the activity and expression of Na(+)‐K(+)‐ATPase were studied in mononucleate cytotrophoblast cells (18 h culture) and syncytiotrophoblast‐like cells (66 h culture). A choriocarcinoma‐derived cell line (JAr) which, unlike the cytotrophoblast cells, divides in culture, was also studied for comparison. 2. Na(+)‐K(+)‐ATPase activity was assessed by measurement of ouabain‐sensitive 86Rb+ uptake. Na(+)‐K(+)‐ATPase expression was determined by (i) measurement of [3H]ouabain binding and (ii) Northern hybridization to measure expression of alpha‐1 and beta 1‐subunit mRNA. 3. There was no significant difference in either activity or expression of Na(+)‐K(+)‐ATPase during differentiation of cytotrophoblast cells. However, expression of alpha 1‐ and beta 1‐subunit mRNA was significantly lower in 66 vs. 18 h cultured cytotrophoblast cells. 4. Both Na(+)‐K(+)‐ATPase activity and [3H]ouabain binding was significantly greater in JAr cells than either cytotrophoblast cell groups, although expression of alpha 1‐ and beta 1‐subunit mRNA was the same as cytotrophoblast cells cultured for 18 h. 5. It is concluded that N(+)‐K(+)‐ATPase activity and protein expression does not change during differentiation of cytotrophoblast cells but that there are changes in expression at the transcriptional or post‐transcriptional level.

ATP-stimulated Ca2+-activated K+ efflux pathway and differentiation of human placental cytotrophoblast cells

“…Alternatively, this anomaly might reflect efflux from a mixed population of cells. In the 66-h cytotrophoblast cell group, although the majority of cells are multinucleate and syncytiotrophoblast-like, some mononuclear cytotrophoblast cells are also present (7,8,18). In contrast, only multinucleate cells were selected for microfluorometry studies in the 66-h cytotrophoblast cell group.…”

“…All cells were maintained at 37°C in a humidified incubator gassed with 5% CO 2-95% air for up to 3 days of culture. We have previously shown that during this time cytotrophoblast cells isolated from human term placenta undergo both morphological and biochemical differentiation to form multinuclear syncytiotrophoblast-like cells (7,18). Cells were studied at two time points in culture: after 18 h, when the majority of cells are mononuclear, and after 66 h, when the majority of cells are multinucleate syncytiotrophoblast-like cells.…”

“…Cytotrophoblast cells were isolated using an adaptation of the method of Kliman et al (30) as previously described in detail (7,18). Briefly, human placentas were collected within 30 min of delivery, and the villous tissue was removed, roughly minced, and washed thoroughly with saline.…”

“…This process, which is ongoing throughout gestation, can be reproduced in vitro using cytotrophoblast cells isolated from human term placenta (7,18). Ion exchange across the syncytiotrophoblast is essential for normal fetal homeostasis; however, little is known about the expression and function of ion conductances in the syncytiotrophoblast or during its formation by differentiation, or how these conductances may be regulated by agonists.…”

Anion efflux from cytotrophoblast cells derived from normal term human placenta is stimulated by hyposmotic challenge and extracellular A23187 but not by membrane‐soluble cAMP

“…Following isolation, cells were plated at a density of 5 million per 35 mm culture dish (Nunc) in 2 ml medium (Dulbecco's modified Eagle's medium (DMEM): F12 (diluted l:l), 10 % (v/v) fetal calf serum, 25 mM Hepes, pH 7.4, 0.12 YO (w/v) penicillin, 0.2 % (w/v) streptomycin and 0.6 % (w/v) glutamine) and kept in an incubator in 5 % CO and 95 % 0, at 37 "C for 66 h. The culture medium had a measured osmolality of 330 mosmol (kg H,O)-. All the experiments reported here were on cytotrophoblast cells kept in primary culture for 66 h. We have previously reported that after 66 h of primary culture cytotrophoblast cells aggregate and differentiate to form multinucleated cells which maximally secrete hCG Clarson et al 1996).…”