Epidermal growth factor receptors on isolated human placental syncytiotrophoblast plasma membrane

Receptor sites for mEGF that are specific, saturable and reversible have been demonstrated on isolated syncytiotrophoblast microvillous plasma membrane from term human placentae. Analysis of the binding data indicated the presence of two receptor populations. The high affinity population had 1.7 X 10(13) sites per mg membrane protein with an affinity constant (Ka) of 5.4 X 10(9)/M. The receptors of lower affinity had a Ka of 4.1 X 10(8)/M and 3.1 X 10(13) sites per mg membrane protein.     

Diverse calcium channel types are present in the human placental syncytiotrophoblast basal membrane

The functional expression of calcium channels has been scarcely studied in human placental syncytiotrophoblast. We have presently sought to characterize Ca(2+) 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(2+) (relative PCa/PK up to 99.5) using K(+) solutions in symmetric conditions. Recordings performed in Ba(2+) gradients showed Ba(2+)-conducting channels in 100% of experiments. Ba(2+) total patch currents were consistently blocked by addition of NiCl(2), Nifedipine (L-type voltage-gated calcium channel blocker) or Ruthenium Red (TRPV5-TRPV6 channel blocker); Nifedipine and Ruthenium Red exerted a synergic blocking effect on Ba(2+) total patch currents. Immunohistochemistry of placental villi sections evidenced presence of alpha(1) subunit of voltage-gated calcium channels and TRPV5-TRPV6 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(2+) transport, but could participate in other relevant trophoblast processes, such as exocytosis and Ca(2+) transport regulation.     

A description of human placental syncytiotrophoblast membrane glycosphingolipids

The glycosphingolipid (GSL) structure of isolated human term placental syncytiotrophoblast microvillous plasma membrane (StMPM) has been investigated by Folch solvent extraction and thin-layer chromatography. StMPM preparations were shown to contain unique hydrophilic GSL species, including two highly glycosylated (greater than 15 saccharide residues/molecule) components. This fraction consistently included four major gangliosides (sialylated GSL), as well as two highly-sialylated (greater than or equal to 4 sialic acid residues/molecule) minor gangliosides.     

A placental sub-proteome: the apical plasma membrane of the syncytiotrophoblast

As a highly vascularized tissue, the placenta mediates gas and solute exchange between maternal and fetal circulations. In the human placenta, the interface with maternal blood is a unique epithelial structure known as the syncytiotrophoblast. Previously we developed a colloidal-silica based method to generate highly enriched preparations of the apical plasma membrane of the syncytiotrophoblast. Using similar preparations, a proteomics assessment of this important sub-proteome has identified 340 proteins as part of this apical membrane fraction. The expression of 38 of these proteins was previously unknown in the human placental syncytiotrophoblast. Together with previous studies, the current proteomic database expands our knowledge of the proteome of the syncytiotrophoblast apical plasma membrane from normal placentas to include more than 500 proteins. This database is a valuable resource for future comparisons to diseased placentas. Additionally, this data set provides a basis for further experimental studies of placenta and trophoblast function.     

Isolation of a transferrin receptor structure from sodium deoxycholate-solubilized human placental syncytiotrophoblast plasma membrane

A human placental transferrin receptor structure has been isolated as a complex with doubly-radiolabelled (125I, 59Fe) human transferrin following gel filtration and affinity chromatography of sodium deoxycholate-solubilized protein from isolated syncytiotrophoblast microvillous plasma membrane vesicle preparations. The molecular weight of this complex has been determined to be 150 000 by Sepharose 6B gel filtration. The molecular weights of the constituent components of the complex have been determined by sodium dodecyl sulphate/polyacrylamide disc gel electrophoresis to be 80 000 (representing transferrin) and 65 000 (representing the deoxycholate-solubilized receptor structure). The binding activity of this placental transferrin receptor structure is maintained in sodium deoxycholate and is not requisite on being an integral component of the cell membrane.     

Proteins of the apical and basal plasma membranes of the human placental syncytiotrophoblast: Immunochemical and electrophoretic studies

Proteins of the basal and microvillous plasma membranes of the human placental syncytiotrophoblast were compared to elucidate the basis for structural and functional differences in the two membranes. Among the proteins common to both membranes were actin, alkaline phosphatase, albumin, transferrin, immunoglobulin G, proteins of Mr 35,000, 55,000 and 180,000, and five immunochemically detected proteins. Each membrane also contained unique proteins. Major microvillous cytoskeleton proteins of Mr 68,000 80,000 and 105,000 (alpha-actinin) were lacking or absent from basal membrane cytoskeletons which instead contained unique proteins of Mr 14,000, 16,000, 220,000 and 240,000. In addition, immunochemical analyses revealed four glycoprotein antigens unique to microvillous membrane and five unique to basal membrane. Fibronectin was also found to be exclusive to basal membrane. The difference in membrane-associated cytoskeletal proteins correlated with the different organization of actin microfilaments at the two membranes. The protein antigens unique to each of the two membranes provided further evidence for the polarization of membrane proteins and functions in the syncytium.     

Proteins associated with activity of Fc receptors on isolated human placental syncytiotrophoblast microvillous plasma membranes

To identify Fc receptors from human placental microvilli, proteins that were liberated by detergents from human placental synctiotrophoblast microvillous membranes (StMPM) were characterized by their abilities to bind human IgG in immune complexes with sheep or goat anti-human IgG and to monomeric rabbit anti-dinitrophenol (DNP) IgG bound to DNP-lysine Sepharose. Three placental IgG-binding proteins coprecipitated with immune complexes (Mr = 68,000, 52,000-56,000, 40,000) and were designated pIBP68, pIBP56 and pIBP40, respectively. Of the three proteins only pIBP56 bound to immobilized monomeric rabbit IgG. It was isolated from detergent lysates of StMPM and LDS/phenol glycoprotein extracts of placental plasma membranes suggesting that pIBP56 was a glycoprotein FcR previously reported (Mikulska et al, 1982). The binding specificities of pIBP56 and pIBP40 appeared to be detergent dependent. Photoaffinity crosslinking of StMPM surface proteins in situ to monomeric rabbit derivatized with N-succinimidyl(4-azidophenyl)-I, 3-dithiopropionate identified IgG-binding proteins identical in size to pIBP56 and pIBP40. Crosslinking further suggested that monomeric IgG covalently bound to a complex of StMPM proteins with a total size of 110,000-120,000 Mr. The findings suggest that pIBP68, pIBP56 and pIBP68 are responsible for IgG binding activity of placental StMPM.     

Implication of ATP and sodium in arachidonic acid incorporation by placental syncytiotrophoblast brush border and basal plasma membranes in the human

The human placental syncytiotrophoblast is the main site of exchange of nutrients and minerals between the mother and her fetus. In order to characterize the placental transport of some fatty acids, we studied the incorporation of arachidonic acid, a fetal primordial fatty acid, in purified bipolar syncytiotrophoblast brush border (BBM) and basal plasma membranes (BPM) from human placenta. The basal arachidonic acid incorporation in BBM and BPM was time dependent and reached maximal values of 0.75+/-0.10 and 0.48+/-0.18 pmol/mg protein, respectively, after 2.5 min. The presence of adenosine triphosphate (ATP) (3 m m) increases significantly the maximal incorporation of arachidonic acid by sixfold (4.75+/-0.35 pmol/mg) and ninefold (4.40+/-0.84 pmol/mg) in BBM and BPM, respectively. Moreover, an increase in the arachidonic acid incorporation was also obtained in the presence of sodium where the values achieved 7.68+/-0.98 (10x) and 6.53 pmol/mg (13.6x) for BBM and BPM, respectively. We also showed that the combination of both Na(+)and ATP increases significantly the maximal incorporation of arachidonic acid in BPM to 7.89+/-0.15 pmol/mg protein, while in BBM it did not modify its incorporation (8.18+/-0.25 pmol/mg protein), as compared to the presence of sodium alone. Our results demonstrate that arachidonic acid is incorporated by both placental syncytiotrophoblast membranes, and is ATP and sodium-linked. However, different mechanisms seem to be involved in this fatty acid incorporation through BBM and BPM, since the presence of Na(+)or ATP increased it, while the association of these two elements increased it only in BPM. We also demonstrated by osmolarity experiments that both membranes bind arachidonic acid, potentially involving one or more fatty acids binding proteins.     

Non-gastric H+/K+ ATPase is present in the microvillous membrane of the human placental syncytiotrophoblast

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.     

Loss and regeneration of the microvilli of human placental syncytiotrophoblast

In order to explore the functional role of microvilli of the syncytiotrophoblast of the human full-term placenta, 16 placentas were examined by scanning electron microscopy (SEM). Our results showed that the microvilli projecting from the apical portion of the syncytiotrophoblast appeared to be highly pleomorphic and showed regional variations in their distribution. This has been correlated to the difference in the stage of growth of microvilli following certain obvious examples of loss. Such a process of distortion and renewal or regeneration may suggest a dynamic functional activity of the microvilli on the villous surface.     

Barium, TEA and sodium sensitive potassium channels are present in the human placental syncytiotrophoblast apical membrane

The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without paracellular routes, forming the main barrier for materno-fetal exchange. There is ample evidence suggesting the presence of potassium (K(+)) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K(+) currents of isolated apical membranes from human term placenta using electrophysiological methods: reconstitution of ion channels from apical membranes into giant liposomes (single channel recordings, patch clamp method) or their functional transplantation into Xenopus laevis oocytes (total currents recording, voltage clamp method). Single channel recording experiments show the presence of K(+) channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba(+2)). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba(+2) respectively. The more frequent conductance was approximately 73pS, however several levels of current were detected suggesting the presence of more than one type of K(+) channel. In addition, sodium (Na(+)) sensitivity was detected in the patch current thus, over 10 mmol/L Na(+) reduced the seal current to 38%. These results were corroborated by the total current experiments where the K(+) current elicited in injected oocytes with apical purified membrane was blocked by Ba(+2) and TEA. The total current was also affected by Na(+), becoming larger when a Na(+)-free solution was used. Our results show the existence of at least two types of Ba(+2)-sensitive K(+) channels including a TEA sensitive sub-population, and some of them Na(+) sensitive K(+) channels. These channels could be the conductive pathways proposed previously for this cation in placental hSTB. Our novel contribution has been to successfully obtain K(+) channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.     

Identification of two leucine-sensitive lysine transport activities in human placental basal membrane

The recent demonstration of multiple high-affinity leucine-sensitive cationic transport systems prompted this investigation of their role in lysine uptake in basal cell membrane. Transport of lysine by basal membrane was saturable at both 22 and 37°C and linear in time to I min and 30 sec, respectively. At 22°C, at least two systems were active. The portion of uptake inhibited by the sulphydryl binding reagent N-ethylmaleimide (NEM) but not by leucine in the absence of sodium had a high K_m and high V_max and was attributed to system y⁺. NEM-insensitive uptake was fitted by a one-system model with K_m ( ± s.e.) of 4 ± 1 μm and a V_max of 0.9 ± 0.1 pmol/mg protein/min. This component was completely inhibited by leucine in the absence of sodium but not by glutamine in the presence of sodium. Therefore, it was attributed to system b^o,+. At 37°C, at least three systems were active. For essentially the same reasons as above the NEM inhibitable uptake was attributed to system y⁺. NEM-insensitive uptake was fitted by a one-system model with K_m of 26 ± 7 μm and V_max of 11.1 ± 2.8 pmol/mg protein/30 sec. Inhibition studies, however, indicated its heterogeneity. NEM-insensitive saturable uptake was only partially inhibited by either leucine in the absence of sodium (system b^o,+) or by glutamine in the presence of sodium (system y⁺L). It is concluded that the NEM-insensitive portion of lysine uptake at 37°C represents activity of both system b^o,+ and the temperature-sensitive system y⁺L. As a previous investigation indicates, only one of these (system y⁺L) is present in the more specialized microvillous membrane. The demonstration of functional differences in the high affinity leucine transporters of basal and microvillous membrane in this and our previous investigations suggest that the two membranes possess different transport or modifier proteins.     

An improved method for the preparation of human placental syncytiotrophoblast microvilli

A simple procedure is described for the further purification of placental microvillus preparations. Based on previously published methods for the isolation of microvilli from other tissues, it depends on the preferential aggregation of containing structures by Mg2+. In the purified microvillus preparation, the two placental microvillar marker enzymes, alkaline phosphatase and 5'-nucleotidase, were enriched 24-fold and were obtained in 5 per cent yield. Five other microvilla enzymes were also further enriched by the Mg2+-treatment. Marker enzymes for other subcellular components showed that this treatment completely removed contamination by mitochondria and endoplasmic reticulum and contamination by lysosomes was decreased three-fold. (Na+ + K+)-activated ATPase was depleted by the Mg2+-treatment as was beta2-microglobulin.     

Isoforms of amino acid transporters in placental syncytiotrophoblast: plasma membrane localization and potential role in maternal/fetal transport

Many cell proteins exist as isoforms arising either from gene duplication or alternate RNA splicing. There is growing evidence that isoforms with different, but closely related, functional characteristics are often directed to discrete cellular locations. Thus, specialized functions may be carried out by proteins of similar evolutionary origin in different membrane compartments. In polarized epithelial cells, this mechanism allows the cell to control amino acid transport independently at each of its specialized apical and basolateral plasma membrane domains. Investigations of isoform localization in these membranes have generally been performed in epithelia other than the placental trophoblast.This review of placental amino acid transporter isoforms first provides an overview of their properties and preliminary plasma membrane localization. We then discuss studies suggesting various roles of isoform localization in trophoblast function. To provide insights into the molecular basis of this localization in trophoblast, we present a review of current knowledge of plasma membrane protein localization as derived from investigations with a widely used epithelial model cell line. Finally, we discuss a potential approach using cultured trophoblast-derived cells for studies of transporter isoform localization and function. We hope that this review will stimulate investigation of the properties of trophoblast transporter isoforms, their membrane localization and their contribution to the cellular mechanism of maternal-fetal nutrient transport.     

Transferrin and somatomedin C receptors in the human ovarian follicles

Transferrin and somatomedin C receptors (TFr and SMCr) were studied in human ovaries by immunostaining using monoclonal antisera. The oocyte of evoluting follicles was intensely positive for both types of receptors, but this positivity decreases in large follicles. An evident positivity for both TFr and SMCr was shown in granulosa cells of evoluting follicles. However, not all of these cells were equally immunoreactive. An intense positivity was present in the developing thecal cells of early cavitary follicles as well as in thecal cells of follicles of medium and large size (greater than 6 mm) and in some cells of involuting follicles in initial atresia. These results seem to demonstrate that TFr and SMCr are present in different cellular components of the human developing and early involuting follicles.     

A high molecular weight species of placental alkaline phosphatase in human syncytiotrophoblast microvilli

A high molecular weight form of human placental alkaline phosphatase has been detected in extracts of placenta at term by electrophoresis in starch gels containing 0.5 per cent Triton X-100. The enzyme has a mobility intermediate between the previously described A and B forms of the enzyme and has been called the 'M' form of placental alkaline phosphatase. The M form is the major form of the enzyme found in microvilli extracted from syncytiotrophoblast, though trace amounts of membrane-associated M form can be found in extracts of placentae which had previously been experimentally depleted of microvilli. The M form is present in both of the two recently described subfractions of placental microvilli (see Davies, Parry and Sutcliffe, 1981; Truman, Wakefield and Ford, 1981). A variety of experiments show that the M form is not an artefact of extraction. The characteristic mobility of the M form in starch/Triton gels is the same, whether the microvilli are extracted in butanol, chloroform/methanol, Nonidet P40, Triton X-100 or Na deoxycholate. Serological, heat-stability and genetic studies showed that the A and M forms contain the same enzymatic polypeptide. Gel filtration of butanol/H2O and butanol/saline extracts of microvilli provided an estimated molecular weight of the A form of 127000 and of the M form of 725000; these values were unaffected by the presence of Triton in the medium.