Human Placental Brush-border Membrane Research Articles

Opiates inhibit paclitaxel uptake by P-glycoprotein in preparations of human placental inside-out vesicles

The use of either methadone or buprenorphine for treatment of the pregnant opiate-dependent patient improves maternal and neonatal outcome. However, patient outcomes are often complicated by neonatal abstinence syndrome (NAS). The incidence and severity of NAS should depend on opiate concentration in the fetal circulation. Efflux transporters expressed in human placental brush border membranes decrease fetal exposure to medications by their extrusion to the maternal circulation. Accordingly, the concentration of either methadone or buprenorphine in the fetal circulation is, in part, dependent on the activity of the efflux transporters. The objective of this study was to characterize the activity of P-gp and its interaction with opiates in the placental apical membrane. Therefore, brush border membrane vesicles were prepared from human placenta. The vesicles were oriented approximately 75% inside-out, exhibited saturable ATP-dependent uptake of P-gp substrate [ 3H]-paclitaxel with an apparent K t of 66 ± 38 nM and V max of 20 ± 3 pmol mg protein −1 min −1. Methadone, buprenorphine, and morphine inhibited paclitaxel transport with apparent K i of 18, 44, and 90 μM, respectively. Our data indicate that a method has been established to determine the activity of the efflux transporter P-gp, expressed in placental brush border membranes, and the kinetics for the transfer of its prototypic substrate paclitaxel. Furthermore, the method was used to determine the effects of methadone, buprenorphine, and morphine on paclitaxel transfer by placental P-gp and revealed that they have higher affinity to the transporter than its classical inhibitor verapamil ( K i, 300 μM).

287: Transport activity of human placental P-glycoprotein

Efflux transporters expressed in human placental brush border membranes have an important role in protecting the fetus from xenobiotics and drugs. Accordingly, the extent of fetal exposure to a therapeutic administered to the pregnant woman could, in part, be dependent on the activity of efflux transporters. Therefore, we developed a method for determining the activity of P-glycoprotein, expressed in term human placental brush border membranes, for efflux of its prototypic substrate Taxol.

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.

Sulphydryl modification inhibits taurine transport in human placental brush border membranes

Taurine, 2-aminoethanesulphonic acid, is a -amino acid required for mammalian development. Although the human fetus accumulates taurine in many tissues, it has limited capacity for synthesis. The majority of fetal taurine is derived from the mother via placental transfer. The objective of this study was to analyse the functional groups involved in the taurine transport system of human placental brush-border membranes. Sulphydryl modifying reagents N-ethylmaleimide (NEM) and pyridyldithioethylamine (PDA) caused a dose-dependent inhibition of taurine uptake by brush-border membrane vesicles. Inhibition by PDA was reversible upon reduction by dithiothreitol but not by glutathione indicating that sulphydryl group(s) are located within the bilayer. Preincubation of brush-border membranes with taurine but not with taurocholate, before exposure to NEM, protected taurine transport function. Labelling studies using NEM and chemical cross-linking indicated that a 37.5 kDa protein was protected. These results demonstrate that sulphydryls located within the membrane bilayer are important for taurine transport in human placental brush-border membranes and suggest that a 37.5 kDa protein may be associated with Na +-dependent regulation of the taurine transporter.

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.

Na+ transport by human placental brush border membranes: are there several mechanisms?

Na+ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na+ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na+ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na+ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na+ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na+ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na+ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na+ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na+. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na+ uptake, whereas benzamil, the Na+ channel blocker, had no effect, it was concluded that the amiloride sensitive Na+ uptake was predominantly or exclusively due to a Na+-H+ exchanger activity. K+ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na+ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 microM or 1 mM dibutyryl cAMP, 0.1 or 1 microM phorbol myristate acetate, 10(-7) M insulin, 10(-10) M angiotensin II, or 10(-8) M human parathyroid hormone (PTH) did not influence Na+ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na+-H+ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of cosubstrates such as phosphate and aminoacids, the Na+-H+ exchange is probably the unique mechanism of Na+ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane.

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).

A chloride channel from human placenta reconstituted into giant liposomes

OBJECTIVE: Ion channels play important roles in epithelial transport, but they are difficult to access for conventional electrophysiologic studies in intact placenta. The purpose of this work was to explore the suitability of purified trophoblast plasma membrane as a source of ion channels for reconstitution in artificial lipid membranes. STUDY DESIGN: Human placental brush border membranes were purified by differential and gradient centrifugation and fused with small liposomes. Giant liposomes were then generated by a cycle of dehydration and rehydration. These giant liposomes are suitable for electrophysiologic studies and were probed for the presence of active ion channels by the patch-clamp method. RESULTS: The results reported here indicate the presence of a high conductance chloride channel showing some similarities with “maxi” chloride channels described in secreting and absorbing epithelia. The channel had a slight outward rectification with conductances of 232 and 300 pS at negative and positive potentials, respectively. CONCLUSIONS: For the first time successful reconstitution of a human placental ion channel is achieved in a system suited for electrophysiologic studies. The chloride channel described might play a role in transplacental transport.

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.

Solubilization and purification of the retinol-binding protein receptor from human placental membranes

The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.

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.

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.

Multiple carbohydrate moieties on the Na+/H+ exchanger.

Affinity-purified antibodies against the C-terminal region of the Na+/H+ exchanger (NHE-1) were used to analyse the carbohydrate moiety of the protein. The Na+/H+ exchanger in human placental brush-border membranes has an apparent molecular mass of 105 kDa. Incubation of intact or detergent-solubilized membranes with glycopeptidase F removed the carbohydrate moiety and increased the apparent mobility of the exchanger. Digestion with endoglycosidase-F caused a similar change in mobility, but endoglycosidase-H had no effect, suggesting that the placental Na+/H+ exchanger is a glycoprotein of the biantennary complex type. Removal of the carbohydrate moiety with glycopeptidase F had no effect on the ability of the protein to promote the exchange of Na+ for H+, and had no detectable effect on the sensitivity of the exchanger to trypsin. Limited digestion with glycopeptidase F and neuraminidase indicated the presence of two intermediate forms between the fully glycosylated and the deglycosylated protein. This suggests the presence of at least two, and possibly three, N-linked carbohydrate moieties.