Hypoxia reduces expression and function of system A amino acid transporters in cultured term human trophoblasts.

We tested the hypothesis that hypoxia diminishes the expression and transport of neutral amino acids by system A in full-term human trophoblasts. Cytotrophoblasts from normal human placentas were cultured in standard conditions of 20% O(2) or in 1% and 3% O(2) for 24 h before assay. Neutral amino acid transport for systems A, ASC, and L was assayed at 24 and 72 h by the cluster-tray technique.

Lysine uptake by cloned hCAT-2B: comparison with hCAT-1 and with trophoblast surface membranes.

To study the cationic amino-acid transporter hCAT-2B of human placenta, total RNA was harvested from primary cultured trophoblast and from the BeWo choriocarcinoma cell line (b30 clone) and used for reverse transcription (RT) and polymerase chain reaction (PCR). Primers based on published sequences identified expression of mRNA for hCAT-2B. RT-PCR yielded a 2.

Stable polarized expression of hCAT-1 in an epithelial cell line.

Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP).

Polarized distribution of interleukin-1 receptors and their role in regulation of serotonin transporter in placenta.

We investigated the expression of interleukin-1 (IL-1) receptors and their involvement in the regulation of the serotonin transporter gene expression in human placenta. IL-1beta is an activator of the serotonin transporter gene expression in JAR human placental choriocarcinoma cells as demonstrated by an increase in the steady-state levels of the transporter mRNA and in serotonin transport activity. This activation is blocked by IL-1 receptor antagonist.

IGF binding protein-1 (IGFBP-1) is preferentially associated with the fetal-facing basal surface of the syncytiotrophoblast in the human placenta.

IGF receptors are expressed in a spatially polarized manner on the syncytiotrophoblast cell membrane. We therefore examined the hypothesis that IGFBPs expressed at the maternal-fetal interface interact with distinct surfaces of the syncytiotrophoblast membrane to modulate IGF function. Membrane vesicles were prepared specifically from the maternal-facing, microvillous membrane (MVM) and the fetal-facing, basal membrane (BM) surfaces of the syncytiotrophoblast.

Identification of three cationic amino acid transporters in placental trophoblast: cloning, expression, and characterization of hCAT-1.

The concentrative transfer of amino acids from maternal to fetal blood is essential to fetal growth and metabolism. Cationic amino acids are transported across the placental microvillous and basal membranes by multiple pathways which act to mediate maternal/fetal transport. To identify the cationic amino acid transporters of human placenta, total RNA was harvested from cultured trophoblast and from the BeWo choriocarcinoma cell line, b30 clone, and used for reverse transcription (RT) and polymerase chain reaction (PCR).

Sodium-independent lysine uptake by the BeWo choriocarcinoma cell line.

Transport of L-lysine by a cultured placental trophoblast cell line was investigated by characterization of L-[3H]lysine uptake. In the mononuclear form of the BeWo clone b30 choriocarcinoma cell, at least two sodium-independent systems are present. Concentration dependence data were fitted by a two system model with Km values (+/- s.

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 degrees C and linear in time to 1 min and 30 sec, respectively. At 22 degrees C, at least two systems were active.

Spatial polarization of insulin-like growth factor receptors on the human syncytiotrophoblast.

The expression of IGF receptors on the maternal-facing, microvillous membrane (MVM) surface and the fetal-facing, basal membrane (BM) surface of the syncytiotrophoblast was studied using standard ligand binding assays, covalent cross-linking techniques, and immunoblot analysis. Scatchard analysis of [125I]IGF-I and -II binding revealed the presence of both high and low affinity binding sites associated with each membrane preparation that did not clearly distinguish between the two membrane preparations. Cross-linking analysis, however, demonstrated type I and type II IGF receptors associated primarily with MVM, suggesting that nonreceptor binding sites may contribute to total membrane binding.

Lysine uptake by human placental microvillous membrane: comparison of system y+ with basal membrane.

Transport of lysine by microvillous membranes was investigated by characterization of L-[3H]lysine uptake in membrane vesicles isolated from human placentas. At least one Na(+)-independent system was observed at 22 degrees C and two systems at 37 degrees C. Lysine concentration dependence data were fit by a one- or two-system model with a Michaelis-Menten constant (Km) of 124 +/- 28 microM and a maximum velocity (Vmax) of 33.

Functional characterization of L-alanine transport in a placental choriocarcinoma cell line (BeWo).

The substrate selectivity of the neutral amino acid transport systems of the b30 clone of the choriocarcinoma cell line (BeWo) were characterized. Three transport systems were identified in undifferentiated (without forskolin) and two transport systems in differentiated syncytial cells (with forskolin). In the undifferentiated b30 cells were two sodium-dependent systems with one having substrate selectivity patterns resembling system A (e.

ASC system activity is altered by development of cell polarity in trophoblast from human placenta.

Pathways of neutral amino acid uptake were investigated in vitro during differentiation of primary cultures of trophoblast isolated from full-term human placentas and a clone (b30) of the BeWo cell line. Inhibition of initial alanine (0.1 microM) uptake by 2-(methylamino)isobutyric acid and unlabeled alanine revealed two Na(+)-dependent systems and one Na(+)-independent transporter.

Human placental syncytiotrophoblast expresses two pharmacologically distinguishable types of Na(+)-H+ exchangers, NHE-1 in the maternal-facing (brush border) membrane and NHE-2 in the fetal-facing (basal) membrane.

We investigated whether highly purified preparations of basal (fetal-facing) membrane isolated from normal term human placentas possess Na(+)-H+ exchanger activity. Uptake of Na+ into basal membrane vesicles was stimulated many-fold by an outwardly directed H+ gradient. This H(+)-gradient-dependent uptake was inhibitable by amiloride and its analogues.

Two cationic amino acid transport systems in human placental basal plasma membranes.

Transport of cationic amino acids in basal (fetal facing) plasma membranes was investigated by characterization of L-[3H]lysine and L-[3H]arginine uptake in membrane vesicles isolated from term human placentas. At least two Na(+)-independent systems were present. Lysine concentration dependence data were fit by a two-system model with Km values of 1.