Proteins of the apical and basal plasma membranes of the human placental syncytiotrophoblast: Immunochemical and electrophoretic studies (original) (raw)
Placenta, 1999
In order to establish a gestational profile for placental transcellular permeabilities to water, urea and mannitol, syncytiotrophoblast microvillous (MVM) and basal membrane (BM) vesicles were isolated from human placentae obtained from 16 weeks of gestation to term. Using stop-flow/light-scattering techniques the rate of change in vesicle volume in response to an osmotic challenge was measured and osmotic water permeabilities (P f ) and solute permeabilities (P s ) calculated. Membrane fluidity was assessed by steady-state DPH anisotropy. Permeability of MVM to water and solutes increased by 20-30 per cent in mid-pregnancy and declined again after the 36th week of gestation. In BM, this pattern was apparent only for water permeability; solute permeabilities were not significantly altered. MVM cholesterol content was approx two-fold higher and membrane fluidity lower compared to BM. Cholesterol content in BM, but not in MVM, increased during the late third trimester. Membrane fluidity did not change consistently during gestational development. We conclude that syncytiotrophoblast plasma membranes exhibit small but significant changes in passive permeability to water and non-electrolytes from 16 weeks of gestation to term. It is suggested that an increased water permeability of the syncytiotrophoblast plasma membranes might contribute substantially to the gestational increase in water exchange across the human placenta observed in vivo.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1999
The objective of this study was to determine placental membrane permeabilities to water, urea and mannitol in intrauterine growth restriction (IUGR) and compare them to normal gestational age matched controls. Further, we wished to investigate whether potential changes in permeability were related to changes in membrane fluidity, cholesterol or phospholipid fatty acid content of the membranes. Syncytiotrophoblast microvillous (MVM) and basal membranes (BM) were isolated from normal and IUGR placentas at term. Passive permeability to water, urea, and mannitol showed no significant alterations in IUGR compared to controls. Cholesterol content in BM, but not in MVM, was lower in placentas from pregnancies complicated by IUGR. However, membrane fluidity did not change in these pregnancies. The phospholipid fatty acid composition of the plasma membranes isolated from all placentas showed a predominance of unsaturated fatty acid species in the BM and saturated species in the MVM. In the MVM from IUGR, mead acid (20:3), behenic acid (22:0) and nervonic acid (24:1) constituted higher percentages of the total when compared to normally grown controls. In the BM from IUGR, mead acid (20:3) was increased relative to the total phospholipid fatty acid content. In conclusion, the syncytiotrophoblast membranes exhibit only minor changes in passive permeability and composition when the pregnancy is complicated by IUGR. ß 0005-2736 / 99 / $^see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 -2 7 3 6 ( 9 9 ) 0 0 0 9 6 -6 * Corresponding
Placenta, 2004
In humans, the non-gastric H + /K + ATPase (ATP1AL1) has previously been shown to be expressed in the epithelia of skin, kidney and colon. In this study we tested the hypothesis that the non-gastric H + /K + ATPase is localized to the syncytiotrophoblast, the transporting epithelium of the human placenta. Microvillous (MVM) and basal plasma membranes (BM) of the syncytiotrophoblast were isolated from term placenta and membrane proteins were separated using SDS-PAGE. The ATP1AL1 protein was identified as a 114 kD band in both MVM and BM by Western blot, however, the protein was more abundant in the MVM. Using immunocytochemistry H + /K + ATPase protein was localized in MVM but not BM. We constructed primers specific for ATP1AL1 and performed RT-PCR on RNA isolated from human placenta and human kidney. A product of the expected size could be detected in both tissues after 30 cycles of amplification. The sequence identity of this 517 nucleotide product was confirmed by sequencing and found to be identical to the human non-gastric H + /K + ATPase. The activity of this proton pump appears to be low in normal healthy placental at term, however, it is speculated that MVM non-gastric H + /K + ATPase may be important in pathological states. In conclusion, non-gastric H + /K + ATPase is present in the microvillous plasma membrane of the transporting epithelia of the human placenta.
Distinct Lipid Rafts in Subdomains from Human Placental Apical Syncytiotrophoblast Membranes
Journal of Membrane Biology, 2008
We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal–fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and β-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.
Journal of Biological Chemistry, 2004
The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 ؎ 1.9% in the range of ؎80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 ؎ 7.4 pS (control) to 105 ؎ 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (P o ) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at ؎40 mV and at ؎80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxichloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta.
Journal of Proteome Research, 2005
Brush borders (microvilli) are cell membrane specialized structures that function mainly as highthroughput absortive/secretory areas. It has been wellestablished that brush borders are particularly rich in membrane lipids characteristic to lipid rafts. Here, we report 57 proteins identified from microvillous membranes (MVM) isolated from human syncytiotrophoblast cells using an experimental method that avoids the use of nonionic detergents. About 60% of the proteins reported here have been described previously as lipid-raft specific. Well-known lipid raft-markers such as Annexin A2 and alkaline phosphatase were identified. Cytoskeleton structural constituents and proteins related with the control and modulation of the cytoskeletal architecture as well as the regulation of the interaction of cytoskeletal constituents with the cell membrane and particularly with lipid raft domains were found (Ezrin, IQGAP1 and 2, EBP50). Other proteins identified include signal transduction molecules, such as Ras-related protein Rab-1B and Rab-7, and ADP-ribosylation factor 1. Several proteins harbor putative post-translational modifications that favor its localization in the lipid-raft environment, such as GPI (alkaline phosphatase and 5'-nucleotidase) and myristoylation (BASP1 and MARCKS). On the whole, this extensive description demonstrates from the protein composition point of view that brush border membranes are indeed highly enriched in lipid raft microdomains.
2008
It is known that human syncytiotrophoblast (hSCT) actively transports more than 80% of the Ca 2þ that goes from maternal to fetal circulation. Transepithelial transport of Ca 2þ is carried out through channels, transporters and exchangers located in both microvillous (MVM) and basal (BM) plasma membranes. The plasma membrane Ca-ATPase (PMCA) is the most important mechanism of Ca 2þ homeostasis control in the human placenta. In this work, we reexamined the distribution of PMCA in isolated hSCT of term placenta. The PMCA activity was determined in isolated hSCT plasma membranes. A partial characterization of the PMCA activity was performed, including an evaluation of the sensitivity of this enzyme to an in vitro induced lipid peroxidation. Expression of the PMCA in hSCT plasma membranes and tissue sections was investigated using Western blots and immunohistochemistry, respectively. Our study demonstrates, for the first time, a correlation between the activity and structural distribution of PMCA in both MVM and BM of hSCT. It also demonstrates a higher PMCA activity and expression in MVM as compared to BM. Finally, PMCA4 seems to be preferentially distributed in both hSCT plasma membranes, while PMCA1 is shown to be present in the hSCT homogenate. However, the membrane fractions did not show any PMCA1 labeling. Our results must be taken into account in order to propose a new model for the transport of calcium across the hSCT.
Diverse Calcium Channel Types are Present in the Human Placental Syncytiotrophoblast Basal Membrane
Placenta, 2006
The functional expression of calcium channels has been scarcely studied in human placental syncytiotrophoblast. We have presently sought to characterize Ca 2þ currents of the healthy syncytiotrophoblast basal membrane using purified basal membranes reconstituted in giant liposomes subjected to patch-clamp recordings. We detected presence of channels with high permeability to Ca 2þ (relative PCa/PK up to 99.5) using K þ solutions in symmetric conditions. Recordings performed in Ba 2þ gradients showed Ba 2þ-conducting channels in 100% of experiments. Ba 2þ total patch currents were consistently blocked by addition of NiCl 2 , Nifedipine (L-type voltage-gated calcium channel blocker) or Ruthenium Red (TRPV5eTRPV6 channel blocker); Nifedipine and Ruthenium Red exerted a synergic blocking effect on Ba 2þ total patch currents. Immunohistochemistry of placental villi sections evidenced presence of a 1 subunit of voltage-gated calcium channels and TRPV5eTRPV6 channels in basal and apical syncytiotrophoblast plasma membranes; these three calcium channels were also detected in purified basal and apical fractions using Western blot. These results show the presence of three types of calcium channels in the syncytiotrophoblast basal membrane by both functional and molecular means. These basal membrane calcium channels would not be directly involved in mother-to-fetus Ca 2þ transport, but could participate in other relevant trophoblast processes, such as exocytosis and Ca 2þ transport regulation.