Study on changes in placental L-alanine transport activity during gestation (using microvillous membrane vesicles

Using microvillous membrane vesicles prepared from human normal early and full term placenta, we investigated the transport mechanism of L-alanine and the change in its transport activity during gestation. We estimated the purity of microvillous membrane vesicles prepared from human placenta from the relative specific activities (microvilli versus homogenate) of the membrane's maker enzymes, alkaline phosphatase (ALP), 5'-nucleotidase, and gamma-glutamyltranspeptidase ( gamma-GTP). In early pregnancy (12-15 weeks gestational age), the relative specific activities (microvilli versus homogenate) were calculated to be: ALP: 15.3, 5'-nucleotidase: 14.0, gamma-GTP: 8.3, and in full term pregnancy (37-40 weeks gestational age) the relative specific activities (microvilli versus homogenate) were calculated to be: ALP: 16.0, 5'-nucleotidase: 14.8, gamma-GTP: 7.5. The uptake of L-alanine into microvillous membrane vesicles was Na+ electrochemical gradient (extravesicular greater than intravesicular) dependent and this Na+ dependent uptake was membrane-potentially sensitive both in early pregnancy and in full term pregnancy. The kinetics parameter of the initial L-alanine uptake into microvillous membrane vesicles were calculated to be: Km: 0.78 +/- 0.20 mM, Vmax: 0.62 +/- 0.21 nmol/mg protein/20 sec in early pregnancy, Km: 0.80 +/- 0.24 mM, Vmax: 3.53 +/- 0.70 nmol/mg protein/20 sec in full term pregnancy. In conclusion, the placental transport mechanisms of L-alanine in both early and full term pregnancy were the same, and the L-alanine transport activity of full term pregnancy was much greater than that of early pregnancy.     

Study on human placental beta-alanine and taurine transport mechanism (using microvillous membrane vesicles

Using microvillous membrane vesicles prepared from human full term placenta, we studied the placental beta-amino acid transport mechanism. The transport of amino acids into microvillous membrane vesicles was studied by a filtration technique using a millipore filter. The uptake of beta-alanine into microvillous membrane vesicles was dependent on Na+ electrochemical gradient (extravesicular greater than intravesicular). The initial rate of this Na+ gradient dependent beta-alanine transport exhibited saturation kinetics with respect to the beta-alanine concentration: an apparent Km of 0.24 mM and Vmax of 46 pmol/mg protein/20 sec were calculated. Taurine inhibited beta-alanine uptake into microvillous membrane vesicles, but on the other hand L-alanine didn't inhibit this beta-alanine uptake. The L-alanine uptake into microvillous membrane vesicles was Na+ electrochemical gradient dependent and the initial rate of this Na+ dependent L-alanine uptake into vesicles was faster than the uptake of Na+ itself into vesicles. On the other hand, the initial rate of Na+ dependent beta-alanine and taurine uptake into vesicles was slower than the uptake of Na+ itself into vesicles. These results indicated that there existed a beta-amino acid specific transport system in human placental microvillous membrane, and placental taurine transport was carried out by this system. And it was also indicated that this placental beta-amino acid transport mechanism is quite different from that of L-alanine.     

A study on the mechanism of bile acid transport in the human placenta (the passive transport system of taurocholate across microvillous membrane)

The uptake of taurocholate into microvillous membrane vesicles prepared from human normal full term placenta was studied using a rapid filtration technique. The taurocholate uptake into microvillous membrane vesicles was sensitive to extravesicular osmolarity, and preincubation with the taurocholate increased the uptake of taurocholate into the vesicles. These findings indicate that the uptake of taurocholate by microvillous membrane vesicles represents transport into vesicles. The uptake of taurocholate into vesicles was not dependent on sodium electrochemical gradient (extravesicular greater than intravesicular). But, this uptake was markedly increased when the intravesicular space was rendered electrically more positive by the use of lowly permeant anions or K+ diffusion potentials via valinomycin. These findings indicated that taurocholate was transported into microvillous membrane vesicles as anion. Cholic acid inhibited the uptake of taurocholate into vesicles, but taurine didn't inhibit this uptake. The initial rate of taurocholate transport exhibited saturation kinetics with respect to the taurocholate concentration; an apparent Km of 67 microM and Vmax of 0.30n mol/mg protein/20 sec were calculated. These results indicated that placental taurocholate transport was not active but passive (facilitated diffusion), and taurocholate was transported from fetus to mother via placenta because the blood concentration was higher in fetus than mother.     

The role of glutathione on placental amino acid transport (using microvillous membrane vesicles)

To elucidate the role of glutathione (GSH) on placental amino acid transport, we investigated L-lysine transport using microvillous membrane vesicles prepared from full term human placenta. 1. The transport of L-lysine into microvillous membrane vesicles was not affected by glutathione. 2. The transport of L-lysine into microvillous membrane vesicles was inhibited by inorganic mercury (Hg2+), and 0.1mM Hg2+ inhibited 34% of this transport and 1mM Hg2+ inhibited 50%. 3. The transport of L-lysine inhibited by Hg2+ was almost completely restored when glutathione was added simultaneously. These results indicated that glutathione defended the inhibitory action of inorganic mercury on L-lysine transport across microvillous membrane.     

The study of placental L-ascorbate (vitamin C) transport mechanism (using microvillous membrane vesicles)

To investigate the placental L-ascorbate (Vitamin C) transport mechanism, the uptake of L-ascorbate into microvillous membrane vesicles prepared from human term placenta was studied using the rapid filtration technique. The uptake of L-ascorbate into microvillous membrane vesicles was osmotically sensitive. This finding indicated that the uptake of L-ascorbate into microvillous membrane vesicles represented transport into the vesicles. The uptake of L-ascorbate into microvillous membrane vesicles was not dependent on sodium electrochemical gradient. The initial rate of uptake was not changed when the intravesicular space was rendered electrically more negative by membrane diffusion potential induced by the use of highly permeant anions. The initial rate of L-ascorbate transport exhibited saturation kinetics with respect to the L-ascorbate concentration; an apparent Km of 1.33 mM and Vmax of 47p mol/mg protein/20 sec was calculated. The uptake of L-ascorbate into microvillous membrane vesicles was competitively inhibited by D-isoascorbate. These results indicated that transport of L-ascorbate across the placental microvillous membrane vesicles was carrier mediated and was passive transport.     

Placental transport of taurine in brush border microvilli

Placental transport of taurine was studied in isolated brush border microvillous plasma membrane vesicles by a rapid filtration technique. Brush border microvillous plasma membrane vesicles were prepared from syncytio trophoblast of human term placenta by a method of differential centrifugation and calcium precipitation. The specific activities of alkaline phosphatase, 5' nucleotidase and gamma-GTP in the membrane preparation were enriched to 13-14 times, 12-13 times, and 5-6 times respectively, as high as those in the homogenate. The membrane vesicles exhibit uptake of 3H-labeled taurine into an osmotically reactive intravesicular space. Taurine uptake by vesicles was stimulated specifically by an inward sodium gradient, and replacement of NaCl in the transport medium by KCl, LiCl, and choline chloride had no effect on the transport activity of the vesicles. Taurine transport is inhibited competitively by the presence of beta alanine and GABA. The initial rate of transport followed saturation kinetics with respect to the taurine concentration: An apparent Km of 0.22mM and Vmax of 67 pmol/mg protein were calculated in 20 seconds. These results indicate that transport of taurine across the placental brush border membrane is sodium dependent and carrier mediated.     

The study on human placental DHA-S transport mechanism (using placental microvillous membrane vesicles)

To investigate the placental DHA-S (dehydroepiandrosterone sulfate) transport mechanism, the uptake of DHA-S into microvillous membrane vesicles prepared from human term placenta was studied using the rapid filtration technique. 1. The uptake of DHA-S into microvillous membrane vesicles was not dependent on both Na+ electrochemical gradient and membrane potential difference. 2. The uptake of DHA-S into microvillous membrane vesicles was dependent on temperature. The initial uptake rate of DHA-S at 37 degrees C was three times as great as at 4 degrees C. 3. The initial rate of DHA-S transport exhibited saturation kinetics with respect to the DHA-S concentration; an apparent Km of 0.067 mM and Vmax of 1.01 nmol/mg protein/20 sec were calculated. 4. The uptake of DHA-S into microvillous membrane vesicles was inhibited by DHA, but not by estriol. These results indicated that placental DHA-S transport was carrier mediated and a passive one.     

Study on the mechanism of placental transport of phosphate (using human placental microvillous (brush border) membrane vesicles)

Using the rapid filtration technique, the uptake of phosphate into microvillous (brush border) membrane vesicles isolated from human placental trophoblast was investigated. The microvillous membrane vesicles exhibit the uptake of phosphate into an osmotically reactive intravesicular space and preincubation with the phosphate increased the uptake of phosphate into the vesicles. These findings indicate that the uptake of phosphate by placental trophoblast microvillous membranes represents transport into membrane vesicles. In the presence of sodium electrochemical gradient (extravesicular greater than intravesicular) the uptake of phosphate by vesicles shows an overshoot phenomenon. Sodium-dependent phosphate uptake is about two times higher at pH 6.0 as in the uptake observed at pH 8.0. The initial rate of sodium-dependent phosphate transport exhibited saturation kinetics with respect to phosphate concentration: An apparent Km of 0.28 mM and Vmax of 330 pmol/mg protein in 20 seconds were calculated. These results indicate that the transport of phosphate across the microvillous membranes is carrier mediated. Experiments with different anions (SCN-, Cl-, gluconate-) and ionophore (valinomycin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of electrical potential across the vesicle membranes. It was concluded that placental trophoblast microvillous membranes contain an electroneutral Na+/phosphate co-transport system.     

The character of human placental glucose and amino acid transport activity (using microvillous membrane vesicles)

To elucidate the character of human placental D-glucose and L-alanine transport activity, we investigated the uptake of D-glucose and L-alanine into microvillous membrane vesicles prepared from human early and term placenta using the rapid filtration technique. 1. The uptake of D-glucose into microvillous membrane vesicles did not depend on the Na+ gradient (extravesicular greater than intravesicular). The uptake of D-glucose into vesicles was three times as great as that of L-glucose. And phloretin prominently inhibited the uptake of D-glucose into vesicles. So, it was indicated that the transport mechanism of D-glucose across microvillous membrane was facilitated diffusion. 2. The uptake of L-alanine into microvillous membrane vesicles depended on Na+ gradient (extravesicular greater than intravesicular), so that the transport mechanism of L-alanine across microvillous membrane was a secondary active one. The transport of L-alanine into vesicles prepared from term placenta increased prominently compared to that of early placenta. On the other hand, the transport activity of D-glucose into vesicles prepared from term placenta did not differ from that of early placenta.     

The effect of inorganic mercury on placental amino acid transport using microvillous membrane vesicles

To study the toxic effect of inorganic mercury on the placenta we elucidate the effect of inorganic mercury on placental amino acid transport using microvillous membrane vesicles isolated from human normal full term placenta. The transport of amino acids into microvillous membrane vesicles was studied by a rapid filtration technique using a millipore filter. The transport of L-alanine across placental microvillous membrane was Na+ electrochemical gradient dependent and 0.1 mM inorganic mercury inhibited 77% of this Na+ dependent L-alanine transport and 1 mM inorganic mercury inhibits 90% of this Na+ dependent L-alanine transport. The transport of L-lysine across microvillous membrane vesicles was sodium independent and 0.1 mM inorganic mercury inhibited 34% of this transport and 1 mM inorganic mercury inhibited 50% of this transport. These results indicated that one of the toxic effects of inorganic mercury on placenta-fetus unit was the inhibition of placental nutrient transport.     

The changes in EPH gestosis placental amino acid transport activity (using human placental microvillous membrane vesicles)

To elucidate the change in EPH gestosis placental amino acid transport activity, we investigated the uptake of L-alanine into microvillous membrane vesicles prepared from EPH gestosis placenta and from normal placenta by using a rapid filtration technique. 1. Alkaline phosphatase (ALP) was the marker enzyme of microvillous membrane vesicles (MMV). The ALP activity of mild EPH gestosis placental MMV didn't differ from that of normal placental MMV. On the other hand, the ALP activity of severe EPH gestosis placental MMV decreased compared to that of normal placental MMV. 2. The uptake of L-alanine into human placental MMV was dependent on the Na+ electrochemical gradient, so the transport across human placental MMV was a secondarily active one. The L-alanine transport activity of mild EPH gestosis placental MMV didn't differ from that of normal placental MMV. On the other hand, the L-alanine transport activity of severe EPH gestosis placental MMV decreased prominently compared to that of normal placental MMV.     

Study on Na+ and L-alanine cotransport of the human placenta using microvillous membrane vesicles

Using microvillous membrane vesicles prepared from human normal term placenta, placental cotransport system of Na+ and L-alanine was studied using rapid filtration technique. The uptake of L-alanine into microvillous membrane vesicles was Na+ ion electrochemical gradient (extravesicular greater than intravesicular) dependent and showed typical overshoot phenomenon. Both Na+ dependent L-alanine uptake and Na+ ion uptake into microvillous membrane were membrane potential dependent and were markedly increased when the intravesicular space was rendered electrically more negative by membrane diffusion potentials, induced by the use of highly permeant anions. L-alanine gradient (extravesicular greater than intravesicular) induced a temporary accumulation of Na+ ions. These results indicated that L-alanine and Na+ ions were cotransported across microvillous membrane and this cotransport was dependent on the electrical potential difference in the membrane.     

The effect of antibiotics (gentamicin) on placental amino acid transport activity (using human placental microvillous membrane vesicles)

In order to elucidate the effect of antibiotics (gentamicin) on placental amino acids transport, we investigated L-alanine transport using microvillous membrane vesicles prepared from full-term human placental by a rapid filtration technique. 1. The active transport of L-alanine into microvillous membrane vesicles was dependent on Na+ electrochemical gradient (extravesicular greater than intravesicular). And the double reciprocal plot of this Na+ dependent initial uptake rate versus L-alanine concentration exhibited an apparent Km of 0.79 + 0.23mM and a Vmax of 3.56 + 0.70n mol/mg protein/20 sec. 2. Gentamicin did not affect the Km value of this Na+ dependent L-alanine transport kinetics (0.77 + 0.19 mM [lmM gentamicin], 0.79 + 0.21mM [10mM gentamicin]). On the other hand, gentamicin apparently decreased, the Vmax value of this transport kinetics (1.99 + 0.48n mol/mg protein/20 sec [1mM gentamicin], 1.12 + 0.32n mol/mg protein/20 sec [10mM gentamicin]).     

Transport of sugars across human placental membranes measured by light scattering

The goal of this research was to investigate movement of sugars across placental plasma membranes. Changes in vesicle volume produced by solute uptake were measured by light scattering. Analysis, performed by fitting of the light scattering data to exponentials, revealed that for certain sugars such as glucose, a rapid component and a second, slower transport process were present. Measurements in the presence of the glucose transport inhibitor phloretin, comparison with the transport of mannitol and analysis of the concentration dependence of the two transport components were used to demonstrate that these two processes are consistent with protein-mediated and lipid-diffusional transport of glucose. Calculation of glucose flux rates using the time constants which define these processes provided values similar to those determined by radioisotopic methods. Glucose, 2-deoxyglucose and galactose were transported both by carrier-mediated and diffusional processes, while mannitol, fructose, ribose and 2-deoxyribose were transported solely by the latter process and not by a protein carrier. The rate of glucose transport across the syncytiotrophoblast basal membrane was slightly greater than that across the microvillous membrane, in contrast to that predicted previously by immunoblotting. In addition, measurements of hexose transmembrane diffusion showed that microvillous and basal transport rates were similar and lower than previously determined. We conclude that this new technique represents a simple and rapid method for investigating sugar transport across placental membranes.     

Study on placental L-glutamine transport mechanism using microvilli vesicles

Using microvillous (brush border) membrane vesicles prepared from human term placenta, the uptake of L-glutamine was studied using a rapid filtration technique. The uptake of L-glutamine into the vesicles was osmotically sensitive. A Na+ electrochemical gradient (extravesicular greater than intravesicular) stimulated the initial rate of L-glutamine uptake and the Na+ dependent uptake of L-glutamine into vesicles showed a typical overshoot phenomenon. This overshoot and the initial rate of uptake were markedly increased when the intravesicular space was rendered electrically more negative by membrane diffusion potentials, induced by the use of highly permeant anions. A similar stimulation of L-glutamine uptake was observed when membrane potential (inside negative) was imposed by K+ diffusion potentials via valinomycin. These results indicated that a sodium dependent uptake of L-glutamine into the microvillous membrane vesicles was dependent on the electrical potential difference of membrane. The initial rate of L-glutamine transport exhibited saturation kinetics with respect to L-glutamine concentration; the apparent Km of 0.42 mM and Vmax of 1.54 nmol/mg protein/20 sec were calculated. The uptake of L-glutamine into the vesicles was competitively inhibited by L-alanine.     

Placental glucose transport in gestational diabetes mellitus

OBJECTIVE: We have previously reported that type 1 diabetes mellitus with hyperglycemia during the first trimester is associated with an up-regulation of placental glucose transport at term. We speculated that glucose concentrations regulate placental glucose transporters only during early pregnancy. To test this hypothesis we studied placental glucose transport in gestational diabetes mellitus, which is associated with hyperglycemia mainly during the second half of pregnancy. STUDY DESIGN: Syncytiotrophoblast microvillous membrane vesicles and basal membrane vesicles were isolated from uneventful pregnancies (control group, n = 32) and pregnancies complicated by gestational diabetes mellitus (n = 18). Glucose uptake and glucose transporter 1 expression were studied by means of radiolabeled tracers and Western blotting, respectively. RESULTS: Gestational diabetes mellitus was not associated with alterations in placental glucose transport. Separate analysis of 6 patients in the gestational diabetes mellitus group with large-for-gestational-age babies did not affect these results. CONCLUSION: These findings are consistent with the hypothesis that the sensitivity of placental glucose transporters to regulation by nutrient availability is limited to early pregnancy.     

The study on the human placental L-lactate transport mechanism (using placental microvillous membrane vesicles)

To elucidate the human placental L-lactate transport mechanism, we investigated L-lactate uptake by microvillous membrane vesicles (MMV) prepared from full termed human placenta using the rapid filtration technique. 1. The transport of L-lactate into the MMV was not dependent on the Na+ electrochemical gradient (extravesicular greater than intravesicular). 2. The transport of L-lactate into the MMV was dependent on the H+ gradient (extravesicular greater than intravesicular) and this H+ dependent L-lactate uptake showed a tendency to overshoot. This overshoot disappeared with the addition of H+ ionophore (FCCP). 3. The initial rate of this H+ dependent L-lactate transport into the MMV exhibited saturation kinetics with respect to the L-lactate concentration: An apparent Km of 4.35mM and Vmax of 2.78n mol/mg protein/20sec were calculated. These results indicated that there existed a H+ and L-lactate transport system in human placental MMV.     

Studies on the amino acid transport systems in the human placenta--L-alanine and L-leucine uptake by microvillous brush border membrane vesicles

To characterize the placental amino acid transport systems, L-alanine and L-leucine uptakes were studied using microvillous brush border membrane vesicles prepared from human placenta. The specific activities of alkaline phosphatase and 5'-nucleotidase in the membrane preparation were enriched 9-11 times as high as those in the homogenate. Intravesicular water (IVW) volume determined with 3-O-methyl-D-glucose was 0.59 microliters/mg protein. The saturation kinetics of L-leucine uptake by the vesicles equilibrated with Na+ gave a single set of Km (4.2mM) and Vmax (1.16 mumol/ml IVW/30s). These parameters were clearly different from those for L-alanine uptake reported previously (Asai et al.: Biochem. Int., 4:377, 1982). In the presence of an inward Na+-gradient L-leucine uptake was stimulated about 2 times, but transient accumulation was not observed differing from L-alanine uptake. Discrimination of the neutral amino acid transport systems in the presence of an inward 100mM Na+-gradient revealed that the relative contributions of A, ASC and L systems, and simple diffusion were 55, 20, 15 and 10% for L-alanine, and 45, 0, 15 and 40% for L-leucine, respectively. The results indicate that the neutral amino acid transport systems in the human placental microvillous membranes are clearly different between L-alanine and L-leucine.     

Neutral Amino Acid Uptake by Microvillous and Basal Plasma Membrane Vesicles from Appropriate- and Small-for-Gestational Age Human Pregnancies

Fetal growth depends upon nutrient availabilty in the maternal circulation as well as appropriate transfer across the placenta via known transport mechanisms in the maternal- and fetal-facing plasma membranes of the syncytiotrophoblast. In this study, microvillous (maternal-facing) and basal (fetal-facing) plasma membrane vesicles were isolated from the placentas of appropriate-for-gestational age (AGA) and small-for-gestational age (SGA) human pregnancies and the uptakes of tritiated leucine, alanine, and glycine were determined. There was no significant difference in the uptakes of either alanine or leucine by the microvillous and basal vesicles from AGA and SGA pregnancies. In contrast, glycine uptake by both microvillous and basal membrane vesicles from SGA pregnancies was significantly less than vesicles from AGA pregnancies. Glycine is delivered to the umbilical circulation in amounts only minimally in excess of its accretion into protein resulting in a narrow margin of safety for fetal growth. The relative decrease in glycine uptake by SGA vesicles may reflect compromised in vivo availability with resultant depletion of marginal reserves and impaired fetal growth.     

Dipeptide transport in brush-border membrane vesicles isolated from normal term human placenta

We studied the transport of glycylsarcosine by the brush-border membrane vesicles isolated from normal term human placentas. This dipeptide resisted hydrolysis by placental membrane vesicles and was transported intact into an osmotically responsive intravesicular space. The transport process was Na+-independent and probably occurred down a concentration gradient. Many peptides inhibited the transport of glycylsarcosine whereas amino acids had no effect. These results demonstrate, for the first time, the presence of a dipeptide transport system in the human placenta.