Introduction

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Nourishment of the developing fetus and the placenta occurs as a result of uptake and transfer of nutrients from the maternal circulation. Nutrient uptake across the maternal surface of the placenta is mediated by nutrient-specific, active and passive transport mechanisms present in the apical membrane of syncytiotrophoblast cells (Smith et al., 1992).

Choline and ethanolamine phosphoglycerides account for most of the phospholipid composition of mammalian cell membranes, including the human placental brush-border membrane (Kelley et al., 1979; Ogbimi and Johnson, 1980). The synthesis of phosphatidylethanolamine may arise from ethanolamine generated intracellularly from the decarboxylation of phosphatidylserine and subsequent base exchange with serine (Bremer et al., 1960). However, micromolar concentrations of ethanolamine are also present in the circulation of humans and other species and therefore may be considered an additional source available for phospholipid synthesis (Baba et al., 1984; Kruse et al., 1985; Milakofsky et al., 1985). Indeed, the presence of transport mechanisms mediating uptake of ethanolamine and the subsequent use of transported ethanolamine in phosphatidylethanolamine synthesis does suggest a possible role for circulating ethanolamine in cellular phospholipid synthesis (Zelinski and Choy, 1982; Pu and Anderson, 1984; Yorek et al., 1985; Lipton et al., 1990). Ethanolamine is a primary amine possessing a positive charge at physiological pH, and therefore a role for circulating ethanolamine in fetal and placental phospholipid synthesis would require the presence of a mediated transport pathway in the placental brush-border membrane. Previously, we identified and functionally characterized a facilitated diffusion mechanism mediating transport of the quaternary amine choline across the human placental brush-border membrane (Grassl, 1994). Substrate specificity studies of the mechanism mediating choline transport suggested ethanolamine as a possible substrate for transport by the same mechanism. Accordingly, the possible presence of a mediated transport pathway for ethanolamine uptake across the brush-border membrane was assessed in the present investigation using membrane vesicles. The results obtained identify and functionally characterize the presence of a facilitated diffusion mechanism that mediates transport of both choline and ethanolamine across the brush-border membrane of human placenta.

	Experimental Procedures

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Materials. [1,2-³H]Ethanolamine hydrochloride (specific radioactivity, 11 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA) and [methyl-³H]choline chloride (specific activity, 87 Ci/mmol) was purchased from PerkinElmer Life Science Products (Boston, MA). Methylamine, dimethylamine, trimethylamine, ethylamine, (2-hydroxyethyl)-triethylammonium, tetrakis(2-hydroxy-ethyl) ammonium, ethanolamine, N-methylethanolamine, N₁N-dimethylamine, N-ethylethanolamine, N₁N-diethylethanolamine, diethanolamine, 3-amino-1-propanol, 4-amino-1-butanol, butyrylcholine, butyrobetaine, hemicholinium-3, and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) were purchased from Aldrich (Milwaukee, WI). Tetramethylammonium, taurine, phosphoethanolamine, choline, acetylcholine, propranolol, amiloride, procainamide, terbutaline, and valinomycin were purchased from Sigma (St. Louis, MO). Trimethylphenylammonium was purchased from Eastman Kodak (Rochester, NY), and imipramine was purchased from RBI/Sigma (Natick, MA). N₁N-bis-(2-methyl-5,6-diethoxy-9-quinolyl)-diaza[15]-crown-5 (SQI) was purchased from Texas Fluorescence Labs, Inc. (Austin, TX), and homocholine was a gift from Dr. O. M. Brown, Department of Pharmacology, SUNY Health Science Center (Syracuse, NY). All other chemicals were of analytical grade, and all solutions were prepared with distilled-deionized water and filtered through 0.22-µm Millipore filters (Millipore Corporation, Bedford, MA). Valinomycin and FCCP were dissolved in 95% ethanol and added to membrane suspensions in a 1:100 dilution. SQI was dissolved in dimethylsulfoxide and added to membrane suspensions in a 1:200 dilution.

Human Placental Brush-Border Membrane Preparation. Brush-border membrane vesicles were isolated from the maternal surface of syncytiotrophoblasts by divalent cation aggregation and differential centrifugation (Grassl, 1994). Briefly, the villous tissue of the placenta, obtained within 15 min of an elective caesarean section, was quickly dissected and minced into small (~1 cm) fragments at 4°C. The tissue fragments were rinsed three times in 300 mM mannitol and 10 mM HEPES/tetramethylammonium, pH 7, and gently stirred for approximately 30 min using a motor-driven spatula. The tissue suspension was filtered through sterile cotton gauze, and phenylmethylsulfonyl fluoride was added to a final concentration of 0.2 mM. The filtrate was centrifuged at 8,100 rpm for 15 min using an SS-34 rotor (Sorvall, Newton, CT). The low-speed pellet was discarded, and the supernatant was centrifuged at 19,000 rpm for 40 min. The high-speed pellet was gently resuspended, and magnesium chloride was added to a final concentration of 12 mM. After incubating for 10 min, the membrane suspension was centrifuged at 5,000 rpm for 15 min to pellet the magnesium-induced membrane aggregates. The low-speed supernatant was centrifuged at 19,000 rpm for 40 min, and the resulting pellet (brush-border membrane vesicles) was resuspended and washed twice in buffers designated for each experiment. Membrane vesicles were stored frozen (70°C) and used within 3 weeks of preparation. The brush-border membrane vesicle preparation was typically enriched, 25.4 ± 1.3-fold (n = 7), in alkaline phosphatase activity compared with homogenates of villous tissue.

Isotopic Flux Measurements. The timed uptake of [³H]ethanolamine and [³H]choline was assayed at 37°C in the presence of intra- and extravesicular solutions described for each experiment in the figure and table legends. Intravesicular substrate content was determined by rapid filtration (Grassl, 1994). An aliquot of media (40-97.5 µl) containing radiolabeled substrate was placed at the bottom of a glass test tube, and an aliquot of membrane suspension (2.5-10 µl) was positioned on the test tube wall immediately above the radiolabeled substrate-containing media. Vesicle uptake of radiolabeled substrate was initiated by rapidly mixing the 2 aliquots using a vortex, and after a predetermined time interval, the uptake was terminated by rapid dilution with isosmotic potassium chloride, tetramethylammonium chloride, and 20 mM MES/tetramethylammonium, pH 6, kept at 4°C. The diluted membrane suspension was passed through a 0.65-µm-Millipore filter (DAWP), and the filter was immediately washed with additional buffer. To minimize the nonspecific association of radiolabeled ethanolamine with the filter and vesicles, an excess (5 mM) of ethanolamine was included in the wash buffer. The filters were dissolved in Ready Safe (Beckman Coulter, Inc., Fullerton, CA) and counted by scintillation spectroscopy.

Data Analysis. Substrate uptake at each time point was assayed at least in triplicate, and all experiments were performed at least in triplicate using a different membrane preparation in each instance. The timed uptake values obtained were corrected by the nonspecific retention of isotope by the filters. Although absolute substrate uptake values, expressed per milligram of protein, varied from membrane preparation to preparation and were possibly due to unknown placental pathobiologies affecting transporter expression levels and/or regulation of transporter activity, the relative changes in substrate uptake resulting from the conditions set forth in each experiment were highly reproducible. Where appropriate, statistical significance has been determined using an unpaired t test for two means, where p < 0.05 is taken as the limit denoting significant differences.

	Results

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Conductive Ethanolamine Uptake. The possible presence of a mechanism mediating facilitated diffusion, and therefore conductive uptake, of the organic cation ethanolamine across the human placental brush-border membrane was first investigated by determining the ability of an inside-negative voltage difference to serve as a driving force for intravesicular ethanolamine accumulation. As shown in Fig. 1, compared with ethanolamine uptake measured in the absence of potassium, the imposition of an outwardly directed potassium gradient resulted in a marked stimulation of ethanolamine uptake in membrane vesicles pretreated with, but not without, the potassium ionophore valinomycin. Notably, potassium gradient-dependent ethanolamine uptake exceeded equilibrium only in valinomycin pretreated membranes, which suggests that an inside-negative voltage difference may serve as a driving force for concentrative accumulation of intravesicular ethanolamine via a conductive uptake pathway. To the extent that a conductive uptake pathway is present in placental brush-border membrane, maneuvers designed to limit or offset the formation of an inside-negative voltage difference should also result in a decreased level of intravesicular ethanolamine accumulation. As shown in Fig. 2, valinomycin and potassium gradient-dependent uptake of ethanolamine was decreased when measured in the presence of the protonophore FCCP. This observation suggests that charge-compensating movements of protons across FCCP-treated membranes partially dissipated the valinomycin and potassium gradient-induced inside-negative diffusion potential, which in turn resulted in a decreased level of conductive ethanolamine uptake. Furthermore, given the increased influx of protons expected in the presence of FCCP and the possible formation of an inside-acid pH gradient, the decreased level of ethanolamine uptake measured in FCCP-treated membranes would also suggest an absence of a mechanism mediating ethanolamine/proton exchange. Indeed, in other experiments not shown, ethanolamine uptake was indistinguishable when measured in the presence (pH_o 6/pH_i 5) and absence (pH_o 6/pH_i 6) (pH_o 5/pH_i 5) of a pH gradient.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of membrane potential on ethanolamine influx. Brush-border membrane vesicles were pre-equilibrated with 20 mM MES/N-methyl-D-glucamine, pH 6, and either 150 mM N-methyl-D-glucamine chloride or 100 mM N-methyl-D-glucamine chloride, and 50 mM potassium chloride. Uptake of ethanolamine (25 µM) occurred from extravesicular solutions containing 20 mM MES/N-methyl-D-glucamine, pH 6, and either 150 mM N-methyl-D-glucamine chloride () or 147.5 mM N-methyl-D-glucamine chloride, and 2.5 mM potassium chloride (×, ). Where indicated, membrane vesicles were incubated with valinomycin (VAL, 0.25 mg/ml) or an equivalent volume of ethanol for a minimum of 30 min. A representative experiment of three independent observations, each performed with a different membrane preparation, is shown.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of FCCP on ethanolamine influx. Brush-border membrane vesicles were pre-equilibrated with 100 mM N-methyl-D-glucamine chloride, 50 mM potassium chloride, and 20 mM MES/N-methyl-D-glucamine, pH 6. Uptake of ethanolamine (25 µM) occurred from extravesicular solutions containing 20 mM MES/N-methyl-D-glucamine, pH 6, and either 100 mM N-methyl-D-glucamine chloride and 50 mM potassium chloride () or 147.5 mM N-methyl-D-glucamine chloride and 2.5 mM potassium chloride (×, ). Membrane vesicles were incubated for a minimum of 30 min with valinomycin (VAL, 0.25 mg/ml) and where indicated with FCCP (1.25 mM) or an equivalent volume of ethanol. A representative experiment of three independent observations, each performed with a different membrane preparation, is shown.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of SQI-induced sodium diffusion potential on ethanolamine influx. Brush-border membrane vesicles were pre-equilibrated with 100 mM sodium gluconate, 50 mM potassium gluconate, and 20 mM MES/N-methyl-D-glucamine, pH 6. Uptake of ethanolamine (25 µM) occurred from extravesicular solutions containing 50 mM potassium gluconate, 20 mM MES/N-methyl-D-glucamine, pH 6, and either 100 mM sodium gluconate () or 5 mM sodium gluconate and 95 mM N-methyl-D-glucamine gluconate (×, ). Membrane vesicles were preincubated for a minimum of 30 min with SQI (50 µM) and either valinomycin (VAL, 0.25 mg/mol) or an equivalent volume of ethanol. A representative experiment of three independent observations, each performed with a different membrane preparation, is shown.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of thiocyanate gradient on ethanolamine influx. Brush-border membrane vesicles were pre-equilibrated with 75 mM N-methyl-D-glucamine gluconate, 20 mM MES/N-methyl-D-glucamine, pH 6, and either 75 mM potassium thiocyanate or 75 mM potassium gluconate. Uptake of ethanolamine (25 µM) occurred from extravesicular solutions containing 75 mM N-methyl-D-glucamine gluconate, 20 mM MES/N-methyl-D-glucamine, pH 6, and either 75 mM potassium thiocyanate () or 71 mM potassium thiocyanate, and 4 mM potassium gluconate (×, ). Membrane vesicles were preincubated for a minimum of 30 min with valinomycin (VAL, 0.25 mg/ml) or an equivalent volume of ethanol. A representative experiment of three independent observations, each performed with a different membrane preparation, is shown.

Trans-Stimulation of Ethanolamine Uptake by Ethanolamine. Although the observed coupling of ethanolamine uptake to an inside-negative voltage difference suggests the presence of a mediated transport process, conductive ethanolamine uptake may also occur via some form of leak pathway, possibly introduced during membrane vesicle preparation and/or storage. An attempt was made to distinguish between these two possibilities by testing the effect of trans-ethanolamine on the rate and magnitude of radiolabeled ethanolamine uptake measured in the presumed absence of membrane potential. Membrane vesicles were preloaded with or without 20 mM ethanolamine and diluted 40-fold into an extravesicular solution containing, in both instances, 500 µM radiolabeled ethanolamine. As shown in Fig. 5, compared with those vesicles initially devoid of intravesicular ethanolamine, both the rate and magnitude of radiolabeled ethanolamine uptake was increased in vesicles where a large outward ethanolamine gradient was initially imposed. The observed trans-stimulation of radiolabeled ethanolamine uptake by intravesicular ethanolamine demonstrates the property of counterflow, a membrane transport phenomena arising from the presence of a carrier-mediated transport process (Rosenberg and Wilbrandt, 1957; Heinz, 1978). Significantly, this observation furthermore suggests conductive ethanolamine uptake occurs by a mediated transport process and is not an artifact resulting from the preparation, handling, or storage of membrane vesicles.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of intravesicular ethanolamine on ethanolamine influx. Brush-border membrane vesicles were pre-equilibrated with 130 mM potassium chloride, 20 mM MES/N-methyl-D-glucamine, pH 6, and 20 mM N-methyl-D-glucamine chloride () or 20 mM ethanolamine chloride (). Uptake of ethanolamine (500 µM) occurred from an extravesicular solution containing 130 mM potassium chloride, 19.5 mM N-methyl-D-glucamine chloride, and 20 mM MES/N-methyl-D-glucamine, pH 6. Membrane vesicles were preincubated for a minimum of 30 min with valinomycin (0.25 mg/ml). A representative experiment of three independent observations, each performed with a different membrane preparation, is shown.

Cis-Inhibition of Ethanolamine Uptake by Choline. The functional properties of the brush-border membrane transport mechanism mediating conductive uptake of ethanolamine were next assessed with regard to its possible interaction with the quaternary amine choline. Previous membrane vesicle studies (Grassl, 1994) identified and functionally characterized a transport mechanism mediating facilitated diffusion of choline across the human placental brush-border membrane. The requirement for recognition of transportable substrates by the transport protein mediating conductive choline uptake was assessed by testing the ability of various chemical analogs of choline to decrease conductive choline uptake. Among the compounds examined, the primary amine ethanolamine was observed to significantly reduce the level of conductive choline uptake suggesting both choline and ethanolamine may be transportable substrates of the same mechanism. To the extent that choline and ethanolamine share the same transport mechanism for uptake across the placental brush-border membrane, a concentration-dependent inhibition of conductive ethanolamine uptake by choline would be anticipated. Accordingly, the conductive uptake of ethanolamine was measured in the presence of increasingly larger choline concentrations, and as shown in Table 1, a choline concentration-dependent decrease in conductive ethanolamine uptake was observed (IC₅₀  250 µM). The inhibition of conductive choline uptake by ethanolamine and the inhibition of conductive ethanolamine uptake by choline further suggests, but does not prove, transport of choline and ethanolamine by a common facilitated diffusion mechanism. In both instances, the decreased conductive uptake observed may have resulted from only a competition between substrates for access to or interaction with sites of substrate recognition within the transport protein, but not necessarily transport of the competing substrate by a common mechanism. Furthermore, the decreased conductive uptake observed may have also resulted from a more rapid dissipation of the inside-negative voltage difference, the driving force for conductive uptake, due to the possible presence of two separate transport mechanisms mediating conductive uptake of choline and ethanolamine.

Trans-Stimulation of Choline Uptake by Ethanolamine. The possible transport of choline and ethanolamine by the same facilitated diffusion mechanism was further investigated by assessing the mechanistic flux coupling of radiolabeled choline influx to efflux of intravesicular ethanolamine. Membrane vesicles were preloaded with or without 20 mM ethanolamine and diluted 40-fold into an extravesicular solution containing, in both instances, 1 mM radiolabeled choline. As shown in Fig. 6, both the rate and magnitude of radiolabeled choline uptake was increased in vesicles where a large outward ethanolamine gradient was imposed, compared with choline uptake measured in vesicles in the absence of an ethanolamine gradient. Given that the uptake of choline was measured in the presumed absence of membrane potential, the observed ethanolamine gradient-induced stimulation of choline uptake suggests a mechanistic coupling of choline influx to ethanolamine efflux. Consistent with the transport of choline and ethanolamine by a common facilitated diffusion mechanism, the observed stimulation of choline uptake is suggested to arise from the accelerated appearance of extravesicular substrate binding sites due to mediated efflux of intravesicular ethanolamine.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of intravesicular ethanolamine on choline influx. Brush-border membrane vesicles were pre-equilibrated as described in the legend to Fig. 5. Uptake of choline (1 mM) occurred from extravesicular solutions described in the legend to Fig. 5. Membrane vesicles were preincubated for a minimum of 30 min with valinomycin (0.25 mg/ml). A representative experiment of three independent observations, each performed with a different membrane preparation, is shown.

Effect of Ethanolamine on the Kinetics of Choline Uptake. The presence of a placental brush-border membrane transport mechanism mediating facilitated diffusion of both choline and ethanolamine was further investigated by observing the effect of ethanolamine on the kinetics of the mechanism mediating conductive choline uptake. Initial rates of choline uptake were determined from the uptake of choline measured after a 3-s incubation and at the choline concentrations shown in Fig. 7 in the presence and absence of 5 mM ethanolamine. As shown in Fig. 7, the presence of ethanolamine shifted the kinetic plot of [choline]/V versus [choline] to the left, resulting in an apparent 2.7-fold increase in the K_m value for choline without affecting the maximal rate of choline transport. The observed effect of ethanolamine to decrease the apparent affinity of the transport mechanism for choline without effecting its maximal capacity to transport choline suggests ethanolamine and choline compete for access to, and/or occupancy of, a substrate recognition site within a common transport protein.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of ethanolamine on the kinetics of conductive choline influx. Brush-border membrane vesicles were pre-equilibrated with 105 mM N-methyl-D-glucamine chloride, 50 mM potassium chloride, 20 mM MES/N-methyl-D-glucamine, pH 6. A 3-s choline uptake was measured at the choline concentrations shown in the absence and presence of a potassium gradient from extravesicular solutions containing 20 mM MES/N-methyl-D-glucamine, pH 6, and either 100 mM N-methyl-D-glucamine chloride and 50 mM potassium chloride or 147.5 mM N-methyl-D-glucamine chloride and 2.5 mM potassium chloride. Where indicated, choline uptake was determined in the presence of 5 mM ethanolamine chloride () or 5 mM N-methyl-D-glucamine chloride (). Membrane vesicles were preincubated for a minimum of 30 min with valinomycin (0.25 mg/ml). The mean ± S.E.M. of nine experiments, each performed with a different membrane preparation, is shown.

Substrate Specificity of Ethanolamine and Choline Uptake. The possible transport of both choline and ethanolamine by the same facilitated diffusion mechanism in placental brush-border membrane may be suggested by a comparison of the substrate specificity of conductive choline and ethanolamine uptake. To the extent that choline and ethanolamine are substrates transported by the same facilitated diffusion mechanism, a quantitatively similar profile of inhibition by the same panel of chemical analogs would be expected. The effect of structurally similar chemical analogs of choline and ethanolamine on conductive choline and ethanolamine uptake is shown in Table 2. The pattern and rank order inhibition of choline and ethanolamine uptake by the compounds shown in Table 2 is remarkably similar and sufficient to further suggest a common facilitated diffusion mechanism mediating uptake of choline and ethanolamine across the brush-border membrane of human placenta. The substrate specificity studies also suggest at least two chemical determinants, which appear necessary for substrate recognition by the facilitated diffusion mechanism mediating conductive choline and ethanolamine uptake. In general, these would be the presence of a terminal hydroxyl group or other moiety capable of hydrogen bonding to a site within the transport protein and the presence of a positively charged nitrogen, the affinity for which is determined by its degree of alkylation.

	Discussion

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Previously, we identified and functionally characterized a facilitated diffusion mechanism for choline transport across the human placental brush-border membrane (Grassl, 1994). Substrate specificity studies of the mechanism mediating choline transport suggested that ethanolamine may also be transported by the same mechanism. Here we show, for the first time, a common facilitated diffusion mechanism mediating transport of both choline and ethanolamine across the human placental brush-border membrane. A common facilitated diffusion mechanism is suggested by observing trans-stimulation of choline uptake by ethanolamine and a mutual inhibition of conductive choline and ethanolamine uptake by ethanolamine and choline, respectively. The competitive interaction of choline and ethanolamine for a common substrate binding site(s), indicated by the effect of ethanolamine on the kinetics of conductive choline uptake, is further consistent with the presence of a transport mechanism mediating facilitated diffusion of both choline and ethanolamine. Moreover, the pattern and rank order inhibition of conductive choline and ethanolamine uptake by the same panel of chemical analogs is sufficiently similar to suggest that both choline and ethanolamine transport occurs by the same facilitated diffusion mechanism.

Ethanolamine transport has been studied in Y79 human retinoblastoma cell cultures (Yorek et al., 1985), rabbit retina (Pu and Anderson, 1984), hamster heart (Zelinski and Choy, 1982), and bovine aortic endothelial cells (Lipton et al., 1988). The results obtained suggest that ethanolamine uptake occurs by both a high-affinity and sodium-dependent mechanism as well as a low-affinity and sodium-independent mechanism. The latter is similar to the ethanolamine transport mechanism observed in human placental brush-border membrane vesicles. Evidence from kinetic, inhibitor, and substrate specificity studies of ethanolamine uptake also suggest that transport of both choline and ethanolamine may occur by a common mechanism (Yorek et al., 1985; Lipton et al., 1988). Similarly, kinetic, inhibitor, and substrate specificity studies of radiolabeled choline uptake in hamster heart (Zelinski and Choy, 1984), human retinoblastoma cell cultures (Yorek et al., 1986), hamster kidney cell cultures (Zha et al., 1992), rabbit renal brush-border membrane vesicles (Wright et al., 1992), and rat proximal tubules (Ullrich and Rumrich, 1996) also suggest that both choline and ethanolamine may be transported by the same mechanism. The presence of a facilitated diffusion mechanism mediating conductive uptake of choline in renal brush-border membrane vesicles and proximal tubules is suggested by the ability of the membrane potential to serve as a driving force and induce net choline transport (Wright et al., 1992; Ullrich and Rumrich, 1996). Similarly, membrane potential is also observed to serve as a driving force for choline (Grassl, 1994) as well as for ethanolamine accumulation in placental brush-border membrane vesicles. Thus, facilitated diffusion of choline and ethanolamine across the apical membrane of renal and placental epithelia may be mediated by isoforms of the same transport protein.

In conclusion, a transport mechanism mediating facilitated diffusion of ethanolamine across the brush-border membrane of human placenta has been identified and functionally characterized. The evidence described indicates that both choline and ethanolamine are transportable substrates of the same facilitated diffusion mechanism in placental brush-border membrane. Future studies will determine whether other physiologic and pharmacologic amines are also transported by the same mechanism in human placenta.