Store-operated Ca2+ entry in first trimester and term human placenta.

We have examined whether store-operated Ca2+ entry, a common pathway for Ca2+ entry in non-excitable tissue, is apparent in the syncytiotrophoblast of both first trimester and term human placenta. Expression of transient receptor potential (TRPC) homologues, a family of channels thought to be involved in store-operated Ca2+ entry, was also studied at the mRNA and protein levels. [Ca2+]i in syncytiotrophoblast of first trimester and term placental villous fragments was measured by microfluorimetry using the Ca2+-sensitive dye fura-2. Store-operated Ca2+ entry was stimulated using 1 microM thapsigargin in Ca(2+)-free Tyrode buffer (no added Ca2+ + 1 mM EGTA) followed by superfusion with control (Ca2+-containing) buffer. In term fragments, this protocol resulted in a rapid increase in [Ca2+]i, which was inhibited in the presence of 150 microM GdCl3, 200 microM NiCl2, 200 microM CoCl2 or 30 microM SKF96365 but was unaffected by addition of 10 microM nifedipine. It was not possible to stimulate such a rise in [Ca2+]i in first trimester fragments. Messenger RNA encoding TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 was identified in both first trimester and term placentas. From Western blotting, TRPC3 and TRPC6 proteins were detected in term, but not in first trimester, placentas, while TRPC1 protein was not detected. By immunocytochemistry, TRPC3 and TRPC4 were localised to cytotrophoblast cells in first trimester placentas and to the syncytiotrophoblast in term placentas. TRPC6 staining was present in the syncytiotrophoblast of both first trimester and term placenta, but the intensity was much greater in the latter. We propose that store-operated Ca2+ entry may be an important route for Ca2+ entry into the syncytiotrophoblast of term, but not first trimester placentas, and that in human placenta TRPC channels may underlie this entry mechanism.

ATP-stimulated Ca(2+)-activated K(+) efflux pathway and differentiation of human placental cytotrophoblast cells.

The aim of this study was to determine whether extracellular ATP ([ATP](o)) stimulated a Ca(2+)-activated K(+) efflux in trophoblast cells that was dependent on extracellular Ca(2+) ([Ca(2+)](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 (86)Rb as a trace marker. Intracellular Ca(2+) ([Ca(2+)](i)) was examined by microfluorometry using fura 2. [ATP](o) significantly increased (86)Rb 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 [Ca(2+)](o) significantly reduced (86)Rb efflux in both groups as did application of 150 microM GdCl(3). [ATP](o) significantly increased [Ca(2+)](i) in both groups of cells. The response was reduced by removing [Ca(2+)](o) and applying 150 microM GdCl(3). For both (86)Rb efflux and microfluorometry experiments, the response to [ATP](o) was more dependent on [Ca(2+)](o) in 66-h cells compared with 18-h cells (approximately 70% greater). Cytotrophoblast cells exhibit an [ATP](o)-stimulated Ca(2+)-activated K(+) efflux. The dependency of this pathway on [Ca(2+)](o) is greater in the 66-h multinucleate syncytiotrophoblast-like cells, suggesting that the mechanism for Ca(2+) entry may be altered during differentiation of trophoblast cells.

Inwardly rectifying K(+) current and differentiation of human placental cytotrophoblast cells in culture.

Ion transport is important for driving nutrient transport across the syncytiotrophoblast and yet is poorly understood. We have examined K(+)currents under basal conditions in cultured cytotrophoblast cells, at various stages of differentiation, using the whole cell patch clamp technique. Cytotrophoblast cells were isolated from human term placenta and maintained in culture for up to 3 days. Cells were studied at four stages of progressive morphological differentiation: (i) mononuclear cells, (ii) mononuclear cells in aggregates, (iii) small multinucleate cells and (iv) large multinucleate syncytiotrophoblast-like cells. In the conditions of whole cell recording the only K(+) selective current identified in all cell types was a strong inwardly rectifying current which was sensitive to Ba(2+) and Cs(+). This current was unaffected by intracellular ATP whereas intracellular GTPgammas caused either run down of the current or activated a linear current. The characteristics of the current described are consistent with those of the inwardly rectifying K(+) channel Kir2.1. The inwardly rectifying K(+) current was observed in three out of 19 (16 per cent ) mononuclear cells, seven out of 21 (33 per cent ) mononuclear aggregates, eight out of 21 (38 per cent ) small multinucleate cells and 16 out of 19 (84 per cent ) large multinucleate cells. This inwardly rectifying K(+) current is likely to have an important role in determining net K(+) diffusion across the syncytiotrophoblast cell membrane, perhaps increasing in importance as the cells terminally differentiate.

Expression of the Kir2.1 (inwardly rectifying potassium channel) gene in the human placenta and in cultured cytotrophoblast cells at different stages of differentiation.

The aim of this study was to investigate whether the Kir2.1 gene is expressed by the human placenta throughout pregnancy and in cytotrophoblast cells at different stages of differentiation in culture. RNA was extracted from cytotrophoblast cells isolated from term placentas and maintained in culture for 18, 66 and 114 h and from first, second and third trimester placentas. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers, a cDNA product of 1.2 kb, as expected for Kir2.1 gene expression, was detected in all the RNA samples from cytotrophoblast cells and from placentas. The RT-PCR products were verified by sequencing and by detection of the expected transcript size for the Kir2.1 mRNA at 5.6-5.7 kb on Northern blots, using the 1.2 kb cDNA generated by RT-PCR. Northern blot quantification, using a control 28S rRNA probe, showed no significant difference in Kir2.1 mRNA expression between any of the three stages of cytotrophoblast cell differentiation studied (ANOVA; n = 3 RNA samples from each stage). These data demonstrate that the Kir2.1 gene is expressed by the human placenta and, specifically, by cytotrophoblast cells, at all stages of development and differentiation.

Expression of the facilitated glucose transporters (GLUT1 and GLUT3) by a choriocarcinoma cell line (JAr) and cytotrophoblast cells in culture.

The expression of GLUT1 and GLUT3 mRNA and protein in human placental trophoblast-derived cells was investigated. A dividing choriocarcinoma derived cell line (JAr) was compared to differentiating cytotrophoblast cells, isolated from human term placenta, following 18 (mononucleate) and 66 h (multinucleate) in culture. JAr cells treated with 8-bromoadenosine, which inhibits growth and induces differentiation, were also studied. GLUT1 mRNA and protein expression were similar in the four groups of cells. However, GLUT3 mRNA expression was significantly higher (six- to sevenfold) in both control and 8-bromoadenosine-treated JAr cells compared to cytotrophoblast cells and was also significantly higher in untreated versus treated JAr cells. Western blotting showed that GLUT3 protein was undetectable in either cytotrophoblast cell groups, but was abundant in both groups of JAr cells. GLUT3 protein in JAr cells treated with 8-bromoadenosine was also significantly lower than in untreated JAr cells, in agreement with the mRNA data. We conclude that GLUT1 expression is unaffected by either growth or differentiation of trophoblast cells whereas GLUT3 expression is associated with dividing cells. We propose that in the placenta, GLUT3 may be involved in maintaining metabolic requirements of dividing trophoblast cells, rather than having a direct role in transport of glucose to the fetus.

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

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.

A Ca2+-activated whole-cell Cl- conductance in human placental cytotrophoblast cells activated via a G protein.

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24-48 hr after their isolation from term placentas. Cl--selective currents were examined using K+-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mM GTPgammaS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPgammaS was found to be Ca2+-dependent since GTPgammaS failed to activate currents when included in a Ca2+-free electrode solution. In addition, similar currents could be activated by increasing the [Ca2+] of the pipette solution to 500 nM. The Ca2+-activated conductance was judged to be Cl--selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl-. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 microM. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca2+-activated Cl- conductance in human placental trophoblast are discussed.

Membrane potential difference and intracellular cation concentrations in human placental trophoblast cells in culture.

1. The electrochemical gradients for Na+ and K+ were assessed in a cell culture model of trophoblast differentiation. 2. Membrane potential difference (Em), intracellular water and Na+ and K+ contents were measured in choriocarcinoma cells (JAr cell line; 96% of which are undifferentiated trophoblast cells) and in mononucleate and multinucleate (differentiated) cytotrophoblast cells isolated from the human placenta at term. 3. There was a significant fall in Em from -57 mV in JAr cells, to -48 and -40 mV in mono-and multinucleate cytotrophoblast cells, respectively. Treatment with ouabain (1 mM for 15 min) depolarized the JAr cell membrane by 15 mV but did not affect cytotrophoblast cell membrane potential. 4. Intracellular K+ concentration was similar in JAr, mono- and multinucleate cytotrophoblast cells but Na+ concentration was higher in mononucleate cytotrophoblast cells compared with JAr cells. 5. Ouabain treatment (3 mM for 15 min) caused a small increase (4.5%) in cell water in mononucleate cytotrophoblast cells but lowered K+ (approximately 30%) and increased Na+ concentration (approximately 125%) in all the trophoblast cells studied. 6. The K+ equilibrium potential (EK) was more negative than Em in all cells and the difference between EK and Em was smaller in JAr cells (-25 mV) than in mono- and multinucleate cytotrophoblast cells (-33 and -43 mV, respectively). 7. The Na+ equilibrium potential (ENa) was positive in the trophoblast cells and the difference between ENa and Em was 122, 100 and 100 mV in JAr, mono- and multinucleate cytotrophoblast cells, respectively. 8. These results suggest that the electrochemical gradient for K+ is affected by the stage of trophoblast cell differentiation. In contrast, the electrochemical gradient for Na+ is similar in mono- and multinucleate cytotrophoblast cells.