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

Abstract

The aim of this study was to determine whether extracellular ATP ([ATP]o) stimulated a Ca2+-activated K+ efflux in trophoblast cells that was dependent on extracellular Ca2+([Ca2+]o). Cytotrophoblast cells, isolated from human placenta, were examined following 18 h (relatively undifferentiated) and 66 h (multinucleate cells) of culture. Potassium efflux was measured using 86Rb as a trace marker. Intracellular Ca2+ ([Ca2+]i) was examined by microfluorometry using fura 2. [ATP]osignificantly increased 86Rb efflux to a peak that declined to control (18-h cells) or an elevated plateau (66-h cells) and was inhibited by 100 nM charybdotoxin. Removing [Ca2+]o significantly reduced86Rb efflux in both groups as did application of 150 μM GdCl3. [ATP]o significantly increased [Ca2+]i in both groups of cells. The response was reduced by removing [Ca2+]o and applying 150 μM GdCl3. For both 86Rb efflux and microfluorometry experiments, the response to [ATP]o was more dependent on [Ca2+]o in 66-h cells compared with 18-h cells (∼70% greater). Cytotrophoblast cells exhibit an [ATP]o-stimulated Ca2+-activated K+ efflux. The dependency of this pathway on [Ca2+]o is greater in the 66-h multinucleate syncytiotrophoblast-like cells, suggesting that the mechanism for Ca2+ entry may be altered during differentiation of trophoblast cells.

Purinergic receptors in human placenta: evidence for functionally active P2X4, P2X7, P2Y2, and P2Y6

“…86 Rb efflux. 86 Rb efflux was assessed from cytotrophoblast cells under control conditions and in response to a panel of P2Y and P2X receptor agonists (2-MeS-ADP, 2-MeS-ATP, 5-BrUTP, ␣␤-meATP, ADP, ATP, BzATP, UDP, and UTP) using the methods previously described (12). Briefly, cytotrophoblast cells were loaded for 2 h with a 3 Ci/ml stock solution of 86 Rb in control Tyrode's solution (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5.6 mM glucose, and 10 mM HEPES, adjusted to pH 7.4 with 10 M NaOH) at room temperature.…”

“…In an initial screening study, each agonist was applied at a single concentration (100 M) to cells isolated from the same placenta, to eliminate variability between placentas [our group previously found that 100 M ATP produced maximal 86 Rb efflux from cytotrophoblast cells (12)]. From these data, the rate constant of the fall in cellular isotope with time in controls was compared with that in the presence of each agonist on cells from the same isolate.…”

“…To compare the responses to agonists in the presence and absence of extracellular Ca 2ϩ , we expressed the data as the percentage change in ratio above control. This avoids the complications associated with calibrating fura-2 measurements to estimate [Ca 2ϩ ]i concentration, and percentage changes have been used previously to express [Ca 2ϩ ]i in cytotrophoblast cells after ATP stimulation (12).…”

“…Accordingly, these cells have been widely used as an in vitro model of the syncytiotrophoblast and a system in which to examine trophoblast cell differentiation from the mono-to multinucleate phenotype (11,22). Our group (12) previously demonstrated that extracellular ATP elevates [Ca 2ϩ ] i and promotes K ϩ efflux from multinucleate cytotrophoblast cells, although the receptors involved were not identified. In the present study, we tested the hypothesis that multiple purinergic receptor subtypes are expressed by mono-and multinucleate cytotrophoblast cells and that their activation by extracellular nucleotides modulates ion (K ϩ ) efflux and [Ca 2ϩ ] i .…”

Intermediate Conductance Ca2+-Activated K+ Channels Modulate Human Placental Trophoblast Syncytialization

“…86 Rb efflux was measured in cytotrophoblasts at 66 h of culture using a technique previously described [53]. Briefly, cells plated onto 35 mm dishes were removed from the incubator and washed in control Tyrode’s buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, 5.6 mM glucose, pH 7.4; osmolality ∼283 mOsm/kgH 2 O, isotonic compared to maternal plasma at term [56]; osmolality measured by freezing point depression).…”