Placental Brush-border Membrane Vesicles Research Articles

Mechanism of Nucleoside Uptake in Rat Placenta and Induction of Placental CNT2 in Experimental Diabetes

The purpose of this study was to clarify the transport characteristics of nucleosides in rat placenta and the changes of functional expression of nucleoside transporters in rat placenta with experimental diabetes mellitus. Placental uptake clearances of [(3)H]adenosine and [(3)H]zidovudine from maternal blood was much higher than that of [(14)C]mannitol. Xenopus oocytes injected with rat ENT1 and ENT2 cRNA took up [(3)H]adenosine with K(m) values of 6.1 and 26 µM, respectively. [(3)H]Adenosine transport by rat placental brush-border membrane vesicles (BBMV) was saturable and was inhibited by nitrobenzylthioinosine (NBMPR), a specific ENT inhibitor, in a manner consistent with involvement of both rat ENT1 and ENT2. [(3)H]Didanosine was modestly taken up by placenta, and the inhibitory effect of 100 µM NBMPR on [(3)H]ddI uptake by BBMV suggested a role of ENT2-mediated transport. Expression of ENT1, ENT2, ENT3, CNT2, and CNT3 mRNAs was detected in placenta of control and streptozotocin (STZ)-induced diabetic pregnant rats, and CNT2 (SLC28A2) expression was significantly increased in STZ-induced diabetic rats. Consistently, Na(+)-dependent adenosine uptake by BBMV from STZ-induced diabetic pregnant rats was higher than that from control rats. These results suggest the involvement of placental ENT2 as well as ENT1 in nucleoside uptake from maternal blood, and the induction of CNT2 in experimental diabetes mellitus.

Proton-Coupled Erythromycin Antiport at Rat Blood-Placenta Barrier

The aim of the present study was to characterize the mechanism of erythromycin transport at the blood-placenta barrier, using TR-TBT 18d-1 cells as a model of rat syncytiotrophoblasts. [(14)C]Erythromycin was taken up by TR-TBT 18d-1 cells with a Michaelis constant of 466 microM. Although the uptake was not dependent on extracellular Na(+) or Cl(-), it was increased at weakly alkaline pH. Significant overshoot of [(14)C]erythromycin uptake by placental brush-border membrane vesicles was observed in the presence of an outwardly directed proton gradient. These results indicate that erythromycin is transferred by the H(+)-coupled transport system in syncytiotrophoblasts. To address the physiological transport of erythromycin in rat placenta, fetal-to-maternal transport clearance was estimated by means of the single placental perfusion technique. Clearance of [(14)C]erythromycin was higher than that of [(14)C]inulin, a paracellular pathway marker, and was decreased by the addition of 5 mM erythromycin, indicating that saturable efflux system from fetus to mother is involved. The effect of various transporter inhibitors on [(14)C]erythromycin efflux from TR-TBT 18d-1 cells was evaluated. cyclosporin A, fumitremorgin C, and probenecid had no effect, whereas ethylisopropylamiloride, a specific inhibitor of Na(+)/H(+) exchangers (NHEs), was significantly inhibitory. These results suggest that erythromycin efflux transport at the rat blood-placenta barrier is mediated by an erythromycin/H(+) antiport system, driven by H(+) supplied by NHEs.

Placental Folate Transport during Pregnancy

The aim of this study was to elucidate the mechanism of folate transport in the placenta over the course of pregnancy. We found that folate receptor alpha (FRalpha) and reduced folate carrier (RFC) localized on the apical side of human placental villi. Since folate binding to placental brush-border membrane vesicles (BBMVs) was strongly inhibited by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, it is possible that FRalpha, a glycosyl phosphatidylinositol linked glycoprotein, is a candidate for folate uptake from maternal blood to the placenta. Moreover, additional inhibitory effects of thiamine pyrophosphate (TPP) and hemin on folate uptake after PI-PLC treatment suggested that not only FRalpha but also RFC and heme carrier protein 1 (HCP1) are involved in the folate transport mechanism in the human placenta. It was also found that accumulation of folate after intravenous injection increased with the progress of gestation in the rat placenta and the fetus. Furthermore, increases in the expression levels of mRNA of rFRalpha, rRFC, and rHCP1 in the rat placenta during pregnancy were observed. These findings suggest that FRalpha, RFC, and HCP1 are important carriers of folate in the placenta during pregnancy. The results of this study suggest that increases in the expression levels of FRalpha, RFC, and HCP1 in the placenta play an important role in the response to increased need for folate for the placenta and fetus during development with the progress of gestation.

Inhibition of Amino Acid Transport System A by Interleukin-1β in Trophoblasts

The current study sought to investigate the influence of interleukin-1beta (IL-1beta) on the function of the amino acid transport system A in trophoblasts. BeWo choriocarcinoma cells were exposed to recombinant human IL-1beta in serum-free medium. Cells incubated with serum-free medium in the absence of IL-1beta were used as control. System A activity was determined in control and treated cells by measuring the uptake of alpha-(methylamino)isobutyric acid. The results obtained were confirmed by measuring system A activity in placental brush border membrane vesicles isolated from pregnant rats injected with IL-1beta. Treatment of BeWo cells with IL-1beta resulted in a time- and dose- dependent inhibition of system A. Treatment with IL-1beta also inhibited the uptake of arginine, and glutamate but had no significant effect on the uptake of leucine, tryptophan, and ascorbate. The inhibition of system A activity by IL-1beta was abolished in the presence of IL-1beta receptor antagonist. The inhibitory effect was associated with a decrease in the maximal velocity of the transport system with no effect on the substrate affinity. Steady-state levels of both SNAT1 and SNAT2 mRNA were reduced by IL-1beta treatment as evidenced by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In rat placental brush border membrane vesicles isolated from IL-1beta-treated pregnant rats, system A activity was found to be decreased by approximately 40% compared to activity in control membrane vesicles. IL-1beta decreases SNAT1 and SNAT2 mRNA levels in trophoblasts, which is associated with a decrease in system A-mediated transport activity at the functional level. These findings may have important consequences under both physiologic conditions and pathologic conditions during pregnancy that are associated with elevated levels of IL-1beta.

L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2.

Maternofetal transport of l-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that l-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K(m) = 11.09 +/- 1.32 microM; V(max) = 41.75 +/- 0.94 pmol.mg protein(-1).min(-1)), and was unchanged over the pH range from 5.5 to 8.5. l-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of l-carnitine, including d-carnitine, acetyl-d,l-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-l-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na(+)-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for l-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans.

Interaction between anticonvulsants and human placental carnitine transporter.

To examine the inhibitory effect of anticonvulsants (AEDs) on carnitine transport by the human placental carnitine transporter. Uptake of radiolabeled carnitine by human placental brush-border membrane vesicles was measured in the absence and presence of tiagabine (TGB), vigabatrin (VGB), gabapentin (GBP), lamotrigine (LTG), topiramate (TPM), valproic acid (VPA), and phenytoin (PHT). The mechanism of the inhibitory action of TGB was determined. Most of the AEDs inhibited placental carnitine transport. Kinetic analyses showed that TGB had the greatest inhibitory effect [50% inhibitory concentration (IC50, 190 microM)], and the order of inhibitory potency was TGB > PHT > GBP > VPA > VGB, TPM > LTG. Further studies showed that TGB competitively inhibited carnitine uptake by the human placental carnitine transporter, suggesting that it may be a substrate for this carrier. Although the involvement of carnitine deficiency in fetal anticonvulsant syndrome requires further evaluation, potential interference with placental carnitine transport by several AEDs was demonstrated. Despite the higher inhibitory potency of TGB, given the therapeutic unbound concentrations, the results for VPA and PHT are probably more clinically significant.

Kinetic analysis of P-glycoprotein-mediated transport by using normal human placental brush-border membrane vesicles.

P-Glycoprotein (Pgp) plays an important role in drug disposition and excretion in various tissues such as the brain, intestine, and kidney. Moreover, we have demonstrated that Pgp is expressed on the brush-border membranes of trophoblast cells in the placenta and restricts drug transfer from the maternal circulation to the fetus. However, the transport kinetics of physiologically expressed Pgp has scarcely been investigated. In this study, we assessed the functional kinetics of transport mediated by Pgp that is physiologically expressed in normal tissue by using human placental brush-border membrane vesicles (BBMVs). Digoxin and vinblastine were used as typical substrates of Pgp. The uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly increased in the presence of an ATP-regenerating system. The ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs exhibited saturable kinetics. The Michaelis constants (Kt values) were 2.65 +/- 1.80 microM and 21.9 +/- 3.37 microM, respectively. In the presence of a Pgp inhibitor such as verapamil, cyclosporine A, or progesterone, the ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly reduced. Anti-Pgp monoclonal antibody C219 completely inhibited the uptake of [3H]digoxin. The transport kinetics of [3H]digoxin and [3H]vinblastine by physiologically expressed Pgp were successfully evaluated by using BBMVs prepared from normal human placenta. The present method enabled us to evaluate the function of physiologically expressed Pgp and is superior to the use of cultured transfectants in terms of the yield of vesicles. The present method may also be applicable to investigating the influence of various factors such as the genotype of the MDR1 gene or various pathophysiologic states of neonates on the function of Pgp.

Proton gradient-dependent transport of valproic acid in human placental brush-border membrane vesicles.

To investigate the transport mechanism of valproic acid across the human placenta, we used human placental brush-border membrane vesicles and compared them with that of lactic acid. Transport of [3H]valproic acid and [14C]lactic acid was measured by using human placental brush-border membrane vesicles. The uptakes of [3H]valproic acid and [14C]lactic acid into brush-border membrane vesicles were greatly stimulated at acidic extravesicular pH. The uptakes of [3H]valproic acid and [14C]lactic acid were inhibited by various fatty acids, p-chloromercuribenzene sulfonate, alpha-cyano-4-hydroxycinnamate, and FCCP. A kinetic analysis showed that it was saturable, with Michaelis constants (Kt) of 1.04 +/- 0.41 mM and 1.71 +/- 0.33 mM for [3H]valproic acid and [14C]lactic acid, respectively. Furthermore, lactic acid competitively inhibited [3H]valproic acid uptake and vice versa. These results suggest that the transport of valproic acid across the microvillous membrane of human placenta is mediated by a proton-linked transport system that also transports lactic acid. However, some inhibitors differentially inhibited the uptakes of [3H]valproic acid and [14C]lactic acid, suggesting that other transport systems may also contribute to the elevated fetal blood concentration of valproic acid in gravida.

Human placental transport of vinblastine, vincristine, digoxin and progesterone: contribution of P-glycoprotein

To elucidate the role of P-glycoprotein in human placenta, we examined its expression in placenta, and the transcellular transport and uptake of P-glycoprotein substrates in cultured human placental choriocarcinoma epithelial cells (BeWo cells). The uptake of [ 3H]vinblastine and [ 3H]vincristine into BeWo cells was increased in the presence of a metabolic inhibitor, sodium azide. The basolateral-to-apical transcellular transport of [ 3H]vinblastine, [ 3H]vincristine and [ 3H]digoxin was greater than the apical-to-basolateral transcellular transport. In the presence of cyclosporin A, the basolateral-to-apical transcellular transport of [ 3H]vinblastine, [ 3H]vincristine and [ 3H]digoxin was significantly increased, and the apical-to-basolateral transcellular transport was decreased. The uptake of [ 3H]vinblastine, [ 3H]vincristine and [ 3H]digoxin into BeWo cells was significantly enhanced in the presence of several inhibitors, such as verapamil or mouse monoclonal antibody anti- P-glycoprotein MX-MDR (MRK16) as well as cyclosporin A. Although progesterone significantly enhanced the uptake of [ 3H]vinblastine, [ 3H]vincristine and [ 3H]digoxin into BeWo cells, the uptake of [ 3H]progesterone was not affected by these inhibitors. Immunoblot analysis revealed that P-glycoprotein with a molecular weight of 172 kDa was expressed in BeWo cells and isolated trophoblast cells. Furthermore, P-glycoprotein was detected in human placental brush-border membrane vesicles, but not in human placental basolateral membrane vesicles. In conclusion, these data suggest that P-glycoprotein is expressed on the brush-border membrane (maternal side) of human placental trophoblast cells. P-Glycoprotein is considered to regulate the transfer of several substances including vinblastine, vincristine and digoxin from mother to fetus, and to protect the fetus from toxic substances.

Sodium-dependent high-affinity binding of carnitine to human placental brush border membranes

The interaction of carnitine with human placental brush-border membrane vesicles was investigated. Carnitine was found to associate with the membrane vesicles in a Na +-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na + gradient-driven transport process. The absolute requirement for Na + was noticeable whether the association of carnitine with the vesicles was measured with a short time incubation or under equilibrium conditions, indicating Na +-dependent binding of carnitine to the human placental brush-border membranes. The binding was saturable and was of a high-affinity type with a dissociation constant of 1.37 ± 0.03 μM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analyses revealed that Na + increased the affinity of the binding process for carnitine and the Na +/carnitine coupling ration for the binding process was 1. The dissociation constant for the interaction of Na + with the binding of carnitine was 24 ± 4 mM. This constitutes the first report on the identification of Na +-dependent high-affinity carnitine binding in the plasma membrane of a mammalian cell. Studies with purified rat renal brush-border membrane vesicles demonstrated the presence of Na + gradient-driven carnitine transport but no Na +-dependent carnitine binding in these membrane vesicles. In contrast, purified intestinal brush-border membrane vesicles possess neither Na + gradient-driven carnitine transport nor Na +-dependent carnitine binding.

Sulfate transport in human placental brush-border membrane vesicles

Membrane transport pathways for transplacental transfer of sulfate were investigated by assessing the possible presence of a bicarbonate-coupled anion exchange mechanism for sulfate in the maternal facing membrane of human placental epithelial cells. The presence of a SO 4 2−/HCO 3 − exchange mechanism was determined from 35S0 4 2− tracer flux measurements in preparations of purified brush-border membrane vesicles. Under 10% CO 2/90% N 2 the imposition of an outwardly directed bicarbonate gradient (pH o 6/pH i 7.5) stimulated sulfate uptake to levels approximately 4-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, [ K +] o = [ K +] i ) significantly reduced bicarbonate gradient-induced sulfate uptake but concentrative accumulation of sulfate persisted. Early time point determinations performed in the presumed absence of membrane potential suggest the reduced level of bicarbonate gradient-induced sulfate uptake resulted from a more rapid dissipation of the imposed bicarbonate gradient. Concentrative accumulation of sulfate was not observed in the presence of a pH gradient alone under 100% N 2, suggesting a preference of bicarbonate over hydroxyl ions as substrates for exchange. Static head determinations of opposing sulfate and bicarbonate gradients resulting in zero net flux of sulfate suggests the anion exchange mechanism mediates the electroneutral exchange of 2 bicarbonate or 1 carbonate for each sulfate. Sulfate uptake was increased with increasing intravesicular concentrations of carbonate at constant bicarbonate but was constant with increasing intravesicular concentrations of bicarbonate at constant carbonate suggesting carbonate as a substrate for anion exchange. The mechanism mediating bicarbonate gradient-induced sulfate uptake was sensitive to inhibition by stilbene derivatives, furosemide, bumetanide and probenecid. Substrate specificity studies suggest possible interactions of the anion exchange mechanism with salicylate, butyrate, thiosulfate, sulfite, selenate, chromate and oxalate. The results of this study provide evidence for the presence of a bicarbonate-coupled anion exchange mechanism as an electroneutral pathway for sulfate transport across the maternal-facing membrane of human placental epithelial cells.

Dipeptide transport in brush-border membrane vesicles (BBMV) prepared from human full-term placentae

The uptakes of the tritiated, hydrolysis-resistant cationic (d-Phe-L-Lys), neutral (D-Phe-L-Ala) and anionic (D-Phe-L-Glu) peptides into human full-term placental brush-border membrane vesicles (BBMV) were time-dependent and into an osmotically-active space. Uptakes of D-Phe-L-Lys and D-Phe-L-Glu were temperature-dependent. Uptake of D-Phe-L-Lys was electroneutral (either cation exchange or anion co-transport), whereas D-Phe-L-Ala and D-Phe-L-Glu were both stimulated by an increasingly inside-positive membrane potential (explained by either cation exchange or anion co-transport, or translocation alone, respectively). Uptake of D-Phe-L-Ala was stimulated (approximately 50 per cent) by an inwardly-directed proton gradient (pHin = 7.4, pHout = 5.5), whereas D-Phe-L-Glu was unaffected, and D-Phe-L-Lys uptake was inhibited (approximately 50 per cent) but was unaffected by the organic cation-exchange inhibitors 1,1-diethyl-2,2-cyanine (decynium22) and 5-(N-methyl-N-isobutyl)amiloride (MIBA). Over the concentration range studies, the peptides did not self-inhibit, and the only cross-inhibition was by D-Phe-L-Glu on D-Phe-L-Lys uptake (estimated K(I) 24.2 +/- 1.36 mM), suggesting very low affinity transporter(s). Under conditions favouring its transport by PepT1, D-Phe-L-Glu uptake was unaffected by diethylpyrocarbonate (DEPC); neither D-Phe-L-Ala nor D-Phe-L-Lys was inhibited by DEPC under maximally proton-stimulated conditions of uptake. We conclude that Pep-T-like transporters are not responsible for peptide uptake into human placental BBMV; while the molecular identity of the transporter(s) involved remains unclear, we hypothesize that they could be similar to the as yet unidentified epithelial basolateral peptide transporter(s).

Human placental monoamine transporters as targets for amphetamines

OBJECTIVES: The use of amphetamine and its derivatives during pregnancy is known to have adverse effects on the outcome of pregnancy. These effects are at least partly a result of impairment of placental function caused by these abusable drugs. We hypothesized that the two monoamine transporters, namely, the serotonin transporter and the norepinephrine transporter, that are expressed in the human placenta are direct targets for these drugs. STUDY DESIGN: The interaction of amphetamine and methamphetamine with human placental serotonin and norepinephrine transporters was examined. Activity of the serotonin tarnsporter was assessed by serotonin uptake in both maternal-facing brush border membrane vesicles isolated from normal term human placentas and in JAR choriocarcinoma cells. Activity of the norepineprhine transporter was assessed by dopamine uptake and nisoxetine binding in placental brush border membrane vesicles. RESULTS: Amphetamine and methamphetamine are potent inhibitors of the serotonin and norepinephrine transporters expressed in the human placenta. The inhibitory potency of amphetamine is greater than that of methamphetamine. In each case, the S(+)diastereoisomer is more potent than the corresponding R(−)diastereoisomer. The sensitivity of the norepinephrine transporter to inhibition by these drugs is at least two orders of magnitude greater than that of the serotonin transporter. At concentrations known to occur in the plasma of users, these drugs cause a marked inhibition of the norepinephrine transporter. CONCLUSIONS: The results show that the norepinephrine transporter and, to a lesser extent, the serotonin transporter are cellular targets in the human placenta for the abusable drugs amphetamine and methamphetamine.

Human placenta as a target organ for cocaine action: Interaction of cocaine with the placental serotonin transporter

The interaction of cocaine and its analog 2 beta-carbomethoxy-3 beta-(4-iodophenyl) tropane (RTI-55) with the human placental serotonin transporter was investigated. The function of the placental serotonin transporter was assayed using three different approaches: serotonin uptake in purified human placental brush border membrane vesicles, paroxetine binding in placental brush border membrane vesicles, and serotonin uptake in cultured human placental choriocarcinoma cells. The interaction of cocaine and RTI-55 with the transporter was studied by determining the effects of these compounds on the transporter function. Cocaine and RTI-55 were found to be potent inhibitors of the transporter in all three approaches. The inhibition was competitive in nature in all cases. The inhibition constant (Ki) for cocaine was not influenced significantly by the duration of time allowed for the compound to interact with the transporter. In contrast, the inhibition constant for RTI-55 was influenced markedly by this parameter. The longer the time allowed for interaction of RTI-55 with the transporter, the smaller was the Ki value. These results suggest that the association kinetics for the interaction of cocaine and RTI-55 with the placental serotonin transporter are significantly different. When equilibrium interaction was allowed, cocaine inhibited the function of the transporter with a Ki of 0.09 microM. It is concluded that cocaine and its analog RTI-55 are potent inhibitors of the function of the serotonin transporter that is expressed in the normal human placenta and in cultured human placental choriocarcinoma cells. Since the reported values for cocaine concentration in the blood of cocaine users are several-fold higher than the inhibition constant for cocaine, the present study strongly suggests that the function of the placental serotonin transporter may be severely impaired by maternal use of cocaine during pregnancy. These findings may be relevant to fetal and placental complications of cocaine abuse during pregnancy.

Choline transport in human placental brush-border membrane vesicles

Pathways for transport of choline by human placental epithelia were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of choline transport mechanisms mediating Na +-choline cotransport, choline/H + exchange and facilitated diffusion were assessed from [ 3H]choline tracer flux measurements. The rate and magnitude of intravesicular choline accumulation was unaffected by the imposition of an inwardly directed Na + gradient suggesting an absence of a mechanism mediating brush border membrane Na +-choline cotransport. The imposition of inside-acid or inside-alkaline pH gradients was observed to have no significant effect on choline uptake suggesting choline is not a substrate for placental epithelial organic cation/H + exchange. Conditions favoring the development of an inside-negative K + diffusion potential was observed to induce a concentrative accumulation of choline to levels exceeding equilibrium suggesting the presence of a conductive uptake pathway for choline in placental brush border membrane. Evidence to suggest conductive choline uptake resulted from a mediated transport process includes a demonstration of the counterflow phenomena, the concentration-dependent inhibition by hemicholinium-3 ( I C50 ≅ 100 μ M ) and the saturable rate of conductive choline uptake ( K m ≅ 300 μ M , V max ≅ 30 nmol/mg per min ). Substrate specificity studies of the mechanism mediating conductive choline uptake suggest the interaction of choline with the transport protein occurs at a minimum of two sites: a site of negativity with the positively charged nitrogen group and a site of hydrogen bonding to the primary alcohol. Several commonly prescribed pharmaceuticals known to cross the placental barrier including imipramine, verapamil, propranolol, quinine, flurazepam, amiloride and ritodrin were observed to inhibit conductive choline uptake suggesting an interaction with the mechanism mediating conductive choline transport. Conductive choline uptake was unaffected by the presence of the basic amino acids lysine, arginine and histidine; the neurotransmitters serotonin, dopamine and histamine and the vitamins thiamine and carnitine which suggests the mechanism mediating conductive choline transport is not a pathway for placental uptake of these compounds.

Human placenta as a target organ for cocaine action: Interaction of cocaine with the placental serotonin transporter

The interaction of cocaine and its analog 2β-carbomethoxy-3β-(4-iodophenyl) tropane (RTI-55) with the human placental serotonin transporter was investigated. The function of the placental serotonin transporter was assayed using three different approaches: serotonin uptake in purified human placental brush border membrane vesicles, paroxetine binding in placental brush border membrane vesicles, and serotonin uptake in cultured human placental choriocarcinoma cells. The interaction of cocaine and RTI-55 with the transporter was studied by determining the effects of these compounds on the transporter function. Cocaine and RTI-55 were found to be potent inhibitors of the transporter in all three approaches. The inhibition was competitive in nature in all cases. The inhibition constant (K i) for cocaine was not influenced significantly by the duration of time allowed for the compound to interact with the transporter. In contrast, the inhibition constant for RTI-55 was influenced markedly by this parameter. The longer the time allowed for interaction of RTI-55 with the transporter, the smaller was the K i value. These results suggest that the association kinetics for the interaction of cocaine and RTI-55 with the placental serotonin transporter are significantly different. When equilibrium interaction was allowed, cocaine inhibited the function of the transporter with a K i of 0.09 μ m. It is concluded that cocaine and its analog RTI-55 are potent inhibitors of the function of the serotonin transporter that is expressed in the normal human placenta and in cultured human placental choriocarcinoma cells. Since the reported values for cocaine concentration in the blood of cocaine users are several-fold higher than the inhibition constant for cocaine, the present study strongly suggests that the function of the placental serotonin transporter may be severely impaired by maternal use of cocaine during pregnancy. These findings may be relevant to fetal and placental complications of cocaine abuse during pregnancy.

Platelet aggregation inhibiting activity of human placental chorioepithelial brush border membrane vesicles—the role of alkaline phosphatase

Human placental chorioepithelial brush border membrane, which is in direct contact with maternal blood flow, has platelet aggregation inhibiting activity. In the present study, the mechanism of this action has been examined in relation to ADP (adenosine diphosphate) degrading activity and alkaline phsophatase activity of brush border membrane vesicles (BBMV). BBMV prepared from human early and term placental villi, inhibited platelet aggregation induced by ADP. BBMV had potent ADP degrading (ADPase) activity. ADP was quickly degraded by BBMV. ADP degrading activity of BBMV was not so different between early and term placenta. Alkaline phosphatase activity of late placental BBMV was about three times greater than that of early placental BBMV. On the other hand, ADP degrading activity of late placental BBMV was almost the same as that of early placental BBMV. Inhibiting activity of platelet aggregation induced by ADP and ADP degrading activity of BBMV, were not inhibited by levamisole (alkaline phosphatase inhibitor).

Amiloride-sensitive sodium uptake into human placental brush border membrane vesicles

Sodium transport into human placental brush border membrane vesicles was examined in the presence of an outwardly directed sodium gradient leading to the formation of an intravesicular negative charge. 22Na entered the vesicles in a time dependent fashion. The activation energy of the uptake process was calculated and was found to be 11.2 kcal/mol, similar to the value of ionic diffusion in free solution. Amiloride inhibited Na uptake in a concentration dependent fashion with an IC 50 value of 3.08 μM. Neither ouabain nor bumetanide had an effect on Na Uptake at6concentrations up to 100 or 1000 μM, respectively. The system presented here indicates Na transport via channels without involvement of the NaK-ATPase or the NaKCl cotransporter. The system may be useful in investigating Na transport defects in cystic fibrosis.

Platelet-aggregation inhibiting activity of human placental chorioepithelial brush border membrane vesicles and basal plasma membrane vesicles

This laboratory investigated the platelet-aggregation inhibiting activity of human placental brush border membrane vesicles (BBMV) and basal plasma membrane vesicles (BpMV), and obtained the following results. o 1) A strong platelet-aggregation inhibiting activity exists in placental BBMW. In a 20 μg/ml protein concentration, the BBMV completely inhibited the secondary platelet aggregation induced by ADP. In a 40 μg/ml protein concentration, the BBMV completely inhibited the platelet aggregation induced by arachidonic acid and collagen, and in a 200 μg/ml protein concentration, the BBMV completely inhibited the platelet aggregation induced by ristocetin. However, placental BpMV did not show prominent platelet-aggregation inhibiting activity on platelet aggregation induced by ADP, arachidonic acid, collagen, or ristocetin. 2) A strong ADP degrading activity (ADPase activity) in the placental BBMV was found. ADP-degrading activity of the BBMV was 10.5±0.5 μmol/mg protein/minute and ADP-degrading activity of BpMV was 0.6±0.1 μmol/mg protein/minute. 3) The placental BBMV inhibited platelet thromboxane A 2 (TXA 2 ) production. In a 40 μg/ml protein concentration of placental BBMV, a platelet TXA 2 (TXB 2 ) production was almost completely inhibited. BpMV did not show prominent inhibitory activity on platelet TXA 2 (TXB 2 ) production. These results indicate that brush border membrane and basal plasma membrane have a different function in the respect of platelet-aggregation inhibiting activity, and that the brush border membrane may play an important role inhibiting platelet aggregation.

Studies on placental inhibition of platelet aggregation: A comparison of human syncytiotrophoblast brush border and basal plasma membranes

We compared the platelet aggregation inhibiting activity of human placental syncytiotrophoblast brush border membrane vesicles (BBMV) and basal plasma membrane vesicles (BpMV), and obtained the following results. Strong platelet aggregation inhibiting activity is found in placental BBMV. BBMV inhibited platelet aggregation induced by ADP (adenosine diphosphate) and arachidonic acid in a way which depended on the protein concentration of BBMV added. In contrast, BpMV showed no detectable platelet aggregation inhibiting activity. Quite high ADP degrading activity (ADPase activity) was present in the placental BBMV. ADP was quickly degraded by BBMV. In contrast, BpMV did not degrade ADP so quickly. Platelet TXB2 production was almost completely abolished at the protein concentration of 40 micrograms/ml of BBMV. In contrast, BpMV did not significantly inhibit platelet TXA2 (TXB2) production. These results show that syncytiotrophoblast brush border and basal plasma membranes of the human placenta have markedly different properties with respect to platelet aggregation inhibiting activity.