A.J. Moe

Functional expression of the plasma membrane serotonin transporter but not the vesicular monoamine transporter in human placental trophoblasts and choriocarcinoma cells

We investigated the functional expression of the plasma membrane serotonin transporter and the vesicular monoamine transporter in choriocarcinoma cells and normal trophoblasts. The RBL 2H3 cells, a rat basophilic leukaemia cell line, which express both transporters were used for comparison. The choriocarcinoma cells JAr and BeWo were found to possess the plasma membrane serotonin transporter as assessed by the presence of serotonin transport activity in intact cells that was Na(+)-dependent and was sensitive to inhibition by tricyclic and non-tricyclic antidepressants. The activity of the vesicular monoamine transporter in these cells was determined by measuring serotonin transport in digitonin-permeabilized cells. The transport in permeabilized cells was very slow, was not stimulated by ATP and was insensitive to inhibition by reserpine. Under similar conditions, the vesicular monoamine transporter activity was demonstrable in RBL cells, which was stimulated by ATP and was inhibitable by reserpine, bafilomycin A1 (an inhibitor of the V-type H(+)-pump) and carbonyl cyanide p-trifluoromethoxy phenylhydrazone (a protonophore which dissipates transmembrane H+ gradients). In corroboration with these findings, mRNA transcripts hybridizable to the vesicular monoamine transporter cDNA probe were detectable in RBL cells but not in JAr choriocarcinoma cells. Similarly, there was no evidence for the expression of the vesicular monoamine transporter as assessed by Northern blot analysis in normal trophoblasts which were maintained in culture to differentiate to form multinucleated syncytial cells. It is concluded that the trophoblasts and choriocarcinoma cells express the plasma membrane serotonin transporter but not the vesicular monoamine transporter.

Selective expression of the high-affinity isoform of the folate receptor (FR-α) in the human placental syncytiotrophoblast and choriocarcinoma cells

The folate receptor (FR), an essential component in the process of folate uptake in various cells, is known to exist in three isoforms, FR-alpha, FR-beta and FR-gamma, with differential tissue expression. Transfer of folate across the human placenta from mother to fetus involves participation of a folate receptor expressed in the syncytiotrophoblast, but the isoform identity of this receptor has not been established. Based on the tissue/cell type from which these isoforms have been cloned, it is currently believed that FR-alpha is the isoform expressed in adult tissues whereas FR-beta is the isoform expressed in fetal tissues including placenta. The present study, undertaken primarily to establish the isoform identity of the FR expressed in the placental syncytiotrophoblast, does not support this currently prevailing nomenclature. Reverse transcription coupled with polymerase chain reaction (RT-PCR) of total/poly(A)+ RNA from placenta, cultured trophoblast cells and JAR choriocarcinoma cells with primer pairs specific for either FR-alpha or FR-beta reveals that while both isoforms are detectable in the whole placental tissue, only FR-alpha is present in the normal trophoblast cells and in the choriocarcinoma cells. Northern analysis with probes designed to distinguish between the mRNA transcripts coding for these two isoforms corroborate the RT-PCR findings. Furthermore, the nucleotide sequences of the PCR products obtained from the trophoblast cells and JAR cells are identical to the nucleotide sequence of the FR-alpha cDNA. These studies establish that it is the FR-alpha isoform, and not the FR-beta isoform, which is selectively expressed in the placental trophoblast cells. FR-beta, which is known to be present in the placenta, most likely arises from the maternal decidua normally associated with this tissue.

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.g. sensitive to MeAIB and a broad range of neutral amino acids) and the other resembling system ASC (e.g. MeAIB insensitive and inhibited by alanine, serine and cysteine). In addition a sodium-independent system was identified with characteristics resembling system l. The differentiated syncytial cells possessed the system A and system l-like activities but not the system ASC-like activity of the system A-like or system l-like activities. The b30 clone is apparently an appropriate model for placental neutral amino acid transport systems.

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.e.) of 2 +/- 0.7 and 94 +/- 31 microM and Vmax values (+/- s.e.) of 0.7 +/- 0.3 and 25 +/- 6.0 nM/mg DNA/min. A portion of sodium-independent uptake was inhibited by the sulphydryl modifying reagent N-ethylmaleimide (NEM). Following NEM treatment, the data were fitted by a single system with Km = 10 +/- 2 microM AND Vmax = 5.1 +/- 0.8 nM/mg DNA/min. In the absence of sodium, NEM-resistant uptake was sensitively inhibited by leucine whereas NEM-sensitive uptake was not inhibited by leucine. It is concluded that like placental basal membrane, the mononuclear BeWo cell possesses two sodium-independent L-lysine transport systems. The high-capacity, NEM-sensitive, leucine-insensitive system resembles the widespread system y+. The high-affinity, NEM-resistant, leucine-sensitive system resembles system b(0,+).

Isolation and characterization of brush border membrane vesicles from pig small intestine

1. Brush border membrane vesicles (BBMV) were isolated from swine mid-intestine by a MgCl2 precipitation and sucrose density gradient centrifugation. 2. Transport of D-glucose and L-alanine were Na+-stimulated and into an osmotically sensitive space. 3. Estimates of kinetic parameters for Na+-dependent D-glucose transport were: apparent Kt = 1.8 mM and Jmax = 16.8 nmol/mg protein/min. 4. Results of experiments with the delta pH sensitive fluorescent probe 9-aminoacridine indicated independent mechanisms for Na+-dependent glucose transport and Na+/H+ exchange. 5. This study demonstrates that pig BBMV provide a useful model for investigating intestinal membrane transport.

Proton efflux from rat intestinal basal-lateral membrane vesicles is stimlated by ATP and NA+

1. ATP-stimulated 22Na uptake and 14C-methylamine efflux were studied in inside-out rat intestinal basal-lateral membrane vesicles (BLMV). 2. Uptake of 22Na by basal-lateral membrane vesicles was stimulated by addition of ATP and by an acidic vesicle interior. 3. Efflux of 14C-methylamine was stimulated by ATP and Na+. 4. 14C-methylamine efflux was not influenced by vanadate or amiloride by themselves but was inhibited by the presence of both agents. 5. These data are consistent with a basal-lateral proton translocation mechanism which may be responsible for alkalinization of the lateral intercellular space and implicates the Na+-pump in this mechanism.