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. Hypoxia during the initial 24 h of culture reduced system A transport by 82% in 1% O(2) and by 37% in 3% O(2) (P < 0.01) compared with standard conditions. Hypoxia during the latter 24 h of the 72 h in culture reduced system A transport by 55% in 1% O(2) and by 20% in 3% O(2) (P < 0.05) compared with standard conditions at 72 h. Hypoxia (1% O(2)) also reduced total amino acid transport by 40% in the more differentiated syncytiotrophoblasts present at 72 h. Northern analysis of trophoblasts in standard conditions showed that subtypes of human amino acid transporter A (hATA1 and hATA2) were each expressed in cytotrophoblasts and syncytiotrophoblasts. Hypoxia decreased expression of hATA1 and hATA2 in both trophoblast phenotypes. We conclude that hypoxia downregulates system A transporter expression and activity in cultured human trophoblasts.

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 1.0 +/- 0.8 and 223 +/- 57 microM and Vmax values of 0.06 +/- 0.03 and 24.0 +/- 5.8 pmol.mg protein-1.min-1. In the presence of either 10 mM L-leucine or Na+ plus 10 mM L-homoserine, the data were fit by single system models with kinetic parameters similar to the higher and lower Km systems seen in the absence of inhibitors. Uptake of 10 or 20 microM L-lysine in the absence of Na+ showed the higher Km system was inhibited completely by L-arginine, L-homoarginine, and L-histidine. In the presence of Na+, the higher Km system was inhibited completely by L-alanine, L-homoserine, L-leucine, L-phenylalanine, and L-norleucine. The lower Km system was inhibited completely by L-arginine, L-homoarginine, L-histidine, L-leucine, and L-methionine. Time course studies of uptake demonstrated that uptake by either system alone filled the total vesicular space. The basal membrane of human placental syncytiotrophoblast possesses two transport systems for lysine and arginine, resembling the ubiquitous y+ system and the bo,+ system previously described in mouse blastocysts. The higher Vmax of the y+ system suggests that in utero it may mediate transfer of cationic amino acids from the syncytiotrophoblast to the fetus. The role of the high-affinity low-capacity bo,+ system remains to be determined.

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. Na+ uptake in these vesicles did not occur via a Na+ channel, as it was not influenced by changes in membrane potential and, in addition, was inhibited by benzamil only at high micromolar concentrations. The results indicate that the human placental basal membrane possesses Na(+)-H+ exchanger activity. We then studied whether this exchanger is similar to or distinct from the Na(+)-H+ exchanger described in brush border (maternal-facing) membrane preparations. For this purpose, we compared the pharmacological characteristics of the basal membrane Na(+)-H+ exchanger with those of the brush border membrane Na(+)-H+ exchanger. The basal membrane exchanger was about 20-fold less sensitive to inhibition by amiloride and about 70-fold less sensitive to inhibition by dimethylamiloride than was the brush border membrane exchanger. The exchanger activity in both membrane preparations was inhibitable by clonidine and cimetidine, but the inhibition patterns with these compounds were markedly different between basal and brush border membrane preparations. These data demonstrate that the basal membrane Na(+)-H+ exchanger is distinct from the brush border membrane Na(+)-H+ exchanger. The pharmacological profiles of these exchangers indicate that the human placental brush border membrane possesses the housekeeping or non-epithelial type Na(+)-H+ exchanger (NHE-1), whereas the basal membrane possesses the epithelial or apical type Na(+)-H+ exchanger (NHE-2).

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. Characterization of these transporters, by selective inhibition, suggested system A, ASC, and L-like transporters. Concomitant with formation of microvillous membrane and syncytium, system ASC activity decreased from 16.1 +/- 2.8 pmol.mg DNA-1.min-1 at 24 h to 2.4 +/- 1.1 pmol.mg DNA-1.min-1 at 72 h. Na(+)-independent alanine uptake increased from 6.0 +/- 2.0 to 12.9 +/- 0.9 pmol.mg DNA-1.min-1 at 24 and 72 h, respectively. Similarly, alpha-(methylamino)isobutyric acid-insensitive, Na(+)-dependent activity in b30 cells (100 microM alanine) decreased from 6.5 +/- 1.6 to 1.2 +/- 1.2 nmol.mg DNA-1.min-1 for control and forskolin-treated cells, respectively. We conclude that membrane specialization accompanying fusion and differentiation of the cytotrophoblast to form syncytiotrophoblast results in a polarization of neutral amino acid transport systems.