Proteins of the apical and basal plasma membranes of the human placental syncytiotrophoblast: Immunochemical and electrophoretic studies (original) (raw)
Related papers
Review: Placental syncytiotrophoblast membranes – domains, subdomains and microdomains
Placenta, 2011
Human placental syncytiotrophoblast (STB) is an epithelium responsible for materno-fetal exchange. Ions play multiple roles in STB, as in other transport epithelia. We have been interested in the character and functional expression of ion channels in STB membrane fractions. Characterization of ion channels and their relationship with different domains, subdomains and microdomains of STB membranes is important to explain the intracellular mechanisms operating in the placental barrier. The aim of this paper is to summarize our work on this subject. We isolated and purified basal membrane (BM) and two fractions from the apical membrane, a classical fraction (MVM) and a light fraction (LMVM). They were used either for reconstitution into giant liposomes or for transplantation into Xenopus oocyte membranes followed by electrophysiological recordings to characterize chloride and cationic channels in STB from term human placenta. In addition, Western blot analysis, using ion channel antibodies, was performed on purified apical and basal membrane fractions. We also reported the presence of two functional microdomains (lipid rafts) in LMVM and MVM, using detergent resistant membranes (DRMs) and cholesterol-sensitive depletion. Moreover we found evidence of cytoskeletal participation in lipid rafts of different composition. Our results contribute to knowledge of the ion channels present in STB membranes and their participation in the physiology of this epithelium in normal and pathological pregnancies.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
Using immunochemlcal techniques, we identified forms ef erythrocyte membrane proteins in apical and basal plasnla membranes of human pia~n~l ~'6plmMast. A wheat germ agglutinin-hlnding inti~lsic pretein pcesent in the mierovillom (maternal [acing) but not the basal (fetal facing) membrane of the syncyliotrophot~ast epithelium, Conversely, erythrocyle-related proteins of the basal membrane inelmled two intrinsic membcane pmCt4m, a 95000 Mr band 3 iseIorm and a form of SlX~ctrin. These four l~roleins were all absent from the microv~$0us me.mbrmle. The basal membrane spectrin isoform was also present in basal membcane skeletons, A 70eO0 M r polypeptidie which reacted with aalibodies to band 3 was present in both m|erovillous and basal l~qtsma membranes. Therefore, cerlain isoforms af red cell membrane pro4eins are polarized between the two sut4'aces of the hummv placental syncytiotrophoblast. We pt'o|mse that the loealizallon of speetrin to the basal membrane is related to the less bundled organization of microfilamenls at this membrane compared with that of the microvillous membrane. The band 3 isoforms are candidates fo¢ participation in maternofetal anion transport. " This investigation was completed in collaboration with Dr, Martin Mordson shortly before his recent death. Abbreviations: DMMA, dimttbyl maleic anhydride; ConA. concan~valin A; WGA, wheat germ asgluti~in; SDS, sodium tlodecyl sulfate; PBS, IS0 mM NaCI. l0 nlM sodium phosphate (pH 7.4); ME, microvilIcos membra.ne e~y~htocyt~re-]atcd protein; BE, b~a] membrane erythrocyte-reiated prote~n.
Human syncytiotrophoblast membrane proteins defined using a heterologous antiserum
Detergent extracts of human trophoblast plasma membanes (TrPM) and of other fetal tissues from the same conceptus were prepared and the proteins separated by use of isoelectric focusing. A major band of isoelectric point (pI) 41 was present in TrPM together with minor bands of (pI) 4 9, 5 9, 7 3, 7 8 and 7 9. These bands were absent from non-trophoblastic fetal tissues. The pI 4-1 protein reacted with an '251-labelled IgG fraction of a heterologous antiserum raised against TrPM and was found to have an apparent molecular weight of 100,000. This protein did not appear to be placental alkaline phosphatase, SP,, transferrin or human chorionic gonadotrophin. Many neoplastic cell lines tested also revealed a major protein band ofpI 4-1 but it has not been established that this was the same protein as found in the TrPM preparations.
Journal of Cellular Physiology, 1991
We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/ K+-adenosine triphosphatase (ATPase), 37-fold in [3H]dihydroalprenoloI binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg'+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a K , Ca2+ of 0.18 k 0.05 pM and V , , , of 0.93 2 0.1 1 nmol/mgimin. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentrationdependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+iMg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose K, Ca2+ was 0.09 t-0.02 pM and V , , , 3.4 +. 0.2 nmolesimgi3 min. This Ca2+ transport requires Mg2+, the optimal concentration of MgL+ being approximately 1 mM. Preincubation of the membrane with lop6 M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.
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.