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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4287-4292
Copyright © 2003 by The Endocrine Society

L-Arginine Transport across the Basal Plasma Membrane of the Syncytiotrophoblast of the Human Placenta from Normal and Preeclamptic Pregnancies

P. F. Speake, J. D. Glazier, P. T.-Y. Ayuk, M. Reade, C. P. Sibley and S. W. D’Souza

Academic Unit of Child Health, University of Manchester, St. Mary’s Hospital (P.F.S., J.D.G., C.P.S., S.W.D.), Manchester, United Kingdom M13 0JH; Nuffield Department of Obstetrics and Gynecology, John Radcliffe Hospital, University of Oxford (P.T.-Y.A.), Oxford, United Kingdom OX3 9DU; and Department of Human Anatomy and Genetics, University of Oxford (M.R.), Oxford, United Kingdom OX1 3QX

Address all correspondence and requests for reprints to: Dr. P. F. Speake, Human Development and Reproductive Health Academic Group, Academic Unit of Child Health, University of Manchester, St. Mary’s Hospital, Hathersage Road, Manchester, United Kingdom M13 0JH. E-mail: paul.speake{at}man.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cord blood levels of nitrate/nitrite, as a measure of nitric oxide (NO), are generally increased in preeclampsia. As L-arginine is the precursor for NO synthesis, we hypothesized that L-arginine transport across the syncytiotrophoblast basal plasma membrane (BM) of placentas from preeclamptic patients is also increased. Glutamine-sensitive and -insensitive [3H]L-arginine uptakes into BM vesicles were measured and expressed as femtomoles per milligram of protein per minute. Total L-arginine uptake was 418 ± 15 (mean ± SEM; n = 9) in BM from control placentas (CBM) and 495 ± 27 (n = 7) in BM from preeclamptic placentas (PE BM; P < 0.05, by two-tailed t test). Glutamine insensitive (system y+) uptake was 45 ± 3 (n = 6) in CBM, with a significantly higher uptake of 97 ± 23 (n = 5) into PE BM (P < 0.05, by two-tailed t test). There was no significant difference in glutamine-sensitive uptake between the two groups. The expression of mRNA for human cationic amino acid transporter (hCAT) 1, 2, and 4 (system y+ genes) and 4F2hc (heavy chain of system y+L) was not different in homogenates of whole placenta from the two groups. Western blotting data showed that hCAT-1 protein expression in PE BM was higher than that in CBM. These data suggest increased activity of the BM system y+ cationic amino acid transporter in preeclampsia. If reflected in vivo, a similar increase in transporter activity could alter the delivery of L-arginine to syncytiotrophoblast eNOS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREECLAMPSIA, CHARACTERIZED BY hypertension, edema, and proteinuria, affects about 10% of all pregnancies (1). It is often associated with intrauterine growth restriction, with impairment in blood flow to the uterine circulation (2). Nitric oxide (NO), is an endogenous modulator of vascular tone (3) and has been shown to modulate fetoplacental and uteroplacental blood flow (4, 5, 6). In preeclampsia there is good evidence that fetoplacental NO synthase (NOS) activity and NO concentrations in the umbilical circulation are altered. Some studies report decreased or unchanged placental NOS activity (7, 8, 9) in preeclampsia. However, both Lyall and colleagues (10) and Norris and colleagues (11) found elevated nitrite/nitrate concentrations in umbilical vein blood from preeclamptic patients compared with that in similar samples from control patients. They suggest that this is indicative of increased NO production in the fetoplacental unit in preeclampsia, perhaps to increase blood flow in this vascular bed and compensate for decreased uteroplacental blood flow. NO in the fetoplacental circulation is most likely derived from endothelial NOS (eNOS) activity, found predominantly in the syncytiotrophoblast, although eNOS also occurs in the fetal endothelium of the placenta (5). Immunocytochemistry revealed increased expression of eNOS in stem villous vessels and in the endothelium of small vessels of the vasculature of placentas from preeclamptic patients compared with control tissue, and a significantly more basal distribution of eNOS within the syncytiotrophoblast of these placentas (12, 13).

The substrate for NO production by eNOS is L-arginine (14). Evidence from other cells suggests that extracellular L-arginine and therefore transport of the amino acid across the plasma membrane are rate limiting for eNOS activity (15), possibly because of compartmentalization of the enzyme (16). Uptake of extracellular L-arginine into the syncytiotrophoblast occurs across two plasma membranes: the microvillous (MVM; maternal facing) and basal (BM; fetal facing) plasma membranes. We have shown previously that there are two cationic amino acid transporters in term syncytiotrophoblast, systems, y+ and y+L (17). System y+ only transports cationic amino acids such as L-arginine and L-lysine, is electrogenic, and has a low affinity (17). System y+ is the product of the cationic amino acid transporter (CAT) family of genes: human (h) CAT-1, -2, and -4 mRNA are expressed by the placenta (18), although hCAT-4 may not be a functioning transporter (19). The system y+ transport system is found predominantly in the MVM and, to a much lesser extent, in BM (17). System y+L exchanges cationic amino acids and neutral (leucine and glutamine) amino acids in the presence of sodium; it is electroneutral and has a higher affinity (17) than system y+. This transporter is found in both the MVM and BM (17). System y+L is one of a family of heterodimeric amino acid transporters made up of a heavy chain 4F2hc and a light chain, y+LAT1 or y+LAT2 (20). mRNA for 4F2hc, y+LAT1, and y+LAT2 is expressed by the placenta (21).

We hypothesized that L-arginine transport across the plasma membranes of the syncytiotrophoblast and fetal capillary endothelium is increased in preeclampsia and may contribute to the increased NO production in the fetoplacental unit in this condition. We have recently reported that the activities of systems y+ and y+L in the MVM (17) are unaffected by preeclampsia (22). Here, we have investigated the uptake of [3H]L-arginine into vesicles isolated from the BM of placentas from normal and preeclamptic pregnancies. The results show that in vitro the system y+ transporter is more active in BM isolated from placentas of preeclamptic patients than in control placentas. Real-time quantitative PCR showed no difference in mRNA expression of hCAT-1, -2, or -4 between the two groups of placenta. However, Western blotting showed that hCAT-1 protein expression was higher in the BM from preeclamptic placentas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

2-Ethoxyethanol, potassium chloride, potassium dihydrogen orthophosphate, sodium chloride, disodium orthophosphate, and sucrose were purchased from Merck & Co. (Poole, UK). L-Arginine, choline (chloride), HEPES (free acid), mannitol, polyoxyethylene sorbitan monolaurate (Tween 20), Tris[hydroxymethyl]amino methane) hydrochloride (Tris-HCl), Tris[hydroxymethyl]amino methane (Tris-base), and valinomycin were purchased from Sigma-Aldrich Corp. (Poole, UK). L-Glutamine was purchased from ICN Biochemicals, Inc. (Baisingstoke, UK); [3H]dihydroalprenolol (final concentration, 50 nM) and [3H]arginine (final concentration, 0.20 µM) were purchased from NEN Life Science Products (Hounslow, UK). Optiphase Hisafe2 liquid scintillation fluid was obtained from Fisher Chemicals (Leicester, UK). Human placental cDNA was obtained from Clontech (BD Biosciences, Oxford, UK).

BM vesicle preparation

Placental tissue was obtained from patients admitted to the Central Delivery Unit at St. Mary’s Hospital (Manchester, UK). Informed consent was obtained according to our local research ethical committee guidelines. Placentas were collected from women who had had an uncomplicated pregnancy and from women whose pregnancy was complicated by preeclampsia. Preeclampsia was defined as gestational proteinuric hypertension, and patients were only selected when all of the following criteria were met: 1) nulliparity, 2) normotensive (blood pressure, 140/90 mm Hg) and nonproteinuric at the time of booking for antenatal care (<20 wk gestation), 3) development of hypertension (blood pressure, >140/90 mm Hg on at least two occasions, at least >4 h apart) at more than 20 wk gestation, and 4) development of proteinuria (300 mg urinary protein excretion over 24 h or >2+ on reagent strip testing) at more than 20 wk gestation [based on definitions described previously (23, 24)].

The method used for isolation of BM vesicles from human placental syncytiotrophoblast was based on that described by Kelleyet al. (25) with slight modifications (26) as described previously (17). Vesicle purity was determined by measuring the enrichment of dihydroalprenolol binding, a marker for BM (27), and the nonenrichment of alkaline phosphatase, a marker for MVM (28), as described previously (26, 29). Enrichment was expressed as the ratio of activity in the BM vesicles compared with that in the whole tissue homogenate. The protein concentrations of BM and placental homogenate were measured by the Lowry method (30).

[3H]L-Arginine uptake

BM vesicles were suspended in intravascular buffer (containing 50 mM KCl, 50 mM choline chloride, 100 mM mannitol, and 20 mM HEPES-Tris, pH 7.4) and stored at 4 C until use, which was usually within 24 h of isolation. Before uptakes, vesicles were diluted to 7.5 mg protein/ml with intravascular buffer and incubated with 4 µM valinomycin for 60 min at room temperature (mean ± SEM, 23 ± 0.4 C; n = 20).

Uptake of arginine across the BM may be by three possible routes (17). Firstly, system y+L, previously reported to be the predominant route of [3H]L-arginine uptake across BM (17), exchanges cationic amino acid for neutral amino acid (31). The activity of this transporter can be measured as [3H]L-arginine uptake, which is inhibitable by 10 mM glutamine (17). Secondly, system y+, which only transports cationic amino acids, is therefore measured as the glutamine-insensitive component of uptake (17). The third remaining route is via a noncarrier-mediated component, probably representing diffusion across the plasma membrane, demonstrated as the component of [3H]L-arginine uptake not saturated by excess unlabeled L-arginine. We previously showed that 20 mM arginine saturates mediated [3H]L-arginine uptake (17).

To discriminate among these three possible routes of arginine transport, uptakes of [3H]L-arginine (0.2 µM) were measured in the presence and absence of 10 mM glutamine and in the presence and absence of 20 mM arginine in extravesicular buffers (EVB; containing 50 mM KCl, 50 mM NaCl, 100 mM mannitol, and 20 mM HEPES-Tris, pH 7.4). In a second set of experiments [3H]L-arginine uptake was measured over a range of unlabeled L-arginine concentrations over 60 sec to derive values of Km and Vmax as described previously (22). All kinetic data were analyzed using computer models of Michaelis-Menton equation (PRISM, version 3.0, GraphPad, Inc., San Diego, CA). The uptakes were normalized to vesicle membrane protein and expressed as femtomoles per milligram of protein. This protocol is identical to that recently reported in our study of arginine uptake by MVM from normal and preeclamptic placentas (22).

Real-time quantitative RT-PCR (qRT-PCR).

mRNA expression for hCAT-1, hCAT-2, hCAT-4, and 4F2hc was determined by real-time qRT-PCR. RNA was isolated from homogenates of placentas obtained from normal and preeclamptic pregnancies, and the expression levels were determined using specific primers and probes, exactly as described previously (32). The quantity of mRNA in each sample was expressed as a proportion of expression of that mRNA in human placental cDNA (Clontech, Palo Alto, CA), as described previously (32).

Western blotting

BM were prepared as described above, and protein (30 µg) was subjected to SDS-PAGE under reducing conditions and probed for 4F2hc, the heavy chain subunit of system y+L, CD98 goat antihuman polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as described previously (17). For detection of hCAT-1, the samples were prepared in a similar manner with 60 µg protein/lane. Membranes were blocked with 5% milk protein in Tris-buffered saline/0.05% Tween for 1 h, followed by incubation with hCAT-1 rabbit polyclonal antibody (1:1000; donated by S. I. Zharikov and E. R. Block, University of Florida, Gainesville, FL) for 1 h at room temperature (33). Negative controls were prepared by omission of primary antibody. Immunoreactive species for 4F2hc were detected with horseradish peroxidase-conjugated rabbit antigoat (1:2000; DAKO, Ely, UK) and for hCAT-1 goat antirabbit (1:2000; DAKO) antibodies by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL). The density of immunoreactive species was assessed using a GS 700 imaging densitometer (Bio-Rad Laboratories, Hercules, CA) with Molecular Analyst software (version 1.5), and lanes were compared by unpaired t test.

Statistics

Uptakes are expressed as the mean ± SEM; n is the number of placentas. Statistical differences were tested using t test and were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
There was no difference in the gestational ages between the two placental groups: 39 ± 1 yr (n = 9; range, 37–41 yr) and 39 ± 1 yr (n = 7; range 35–39 yr) in control and preeclamptic groups, respectively. The babies from the preeclamptic pregnancies were significantly smaller (3.05 ± 0.2 kg; n = 7) than those from normal pregnancies (3.63 ± 0.23 kg; n = 9; P < 0.05, by two-tailed t test).

The BM vesicle preparations exhibited no enrichment in alkaline phosphatase [control, 0.94 ± 0.14 (n = 9); preeclampsia, 1.27 ± 0.29 (n = 7)], with no significant difference between the groups. The BM vesicles were enriched for dihydroalprenolol binding (control, 23.3 ± 2.1; preeclampsia, 30.2 ± 7.4), with no significant difference between the groups. These enrichments fall within the range reported previously for BM vesicle preparations by us and others (25, 26, 34).

The total uptake of [3H]L-arginine was linear between 10 and 60 sec and was significantly inhibited by 10 mM glutamine in both groups (P < 0.0001, by two-tailed t test; Fig. 1Go, only control uptakes shown). Extrapolation of the data to time zero gave a significant intersect on the y-axis. However there was no difference between the control (154 ± 16) and the preeclamptic (134 ± 17; P = NS, by two-tailed t test) group. Total uptake of [3H]L-arginine, determined as the slope of uptakes over 10–60 sec was 418 ± 15 fmol/mg protein·min (n = 9) in BM vesicles from control placenta. In BM vesicles isolated from placentas of preeclamptic women, this was significantly elevated to 495 ± 27 fmol/mg protein·min (n = 7; P < 0.05, by two-tailed t test).



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  		FIG. 1. Initial rate of uptake of [3H]L-arginine into BM vesicles isolated from placenta from normal pregnancies (n = 9). Uptakes were performed in EVB containing 50 mM KCl, 50 mM NaCl, 100 mM mannitol, and 20 mM HEPES-Tris, pH 7.4, in the absence ({blacksquare}) or presence (•) of 10 mM glutamine. The slope of the uptakes in control vesicles was 5.11 ± 0.43 fmol/mg protein·sec in the absence of glutamine (r2 = 0.97) and 0.88 ± 0.11 fmol/mg protein·sec in the presence of glutamine (r2 = 0.94).

 
[3H]L-Arginine uptakes over a range of unlabeled L-arginine concentrations was measured to enable calculation of Michaelis-Menten constants, Km and Vmax (Fig. 2Go). Data were fitted to a one-site model as analysis showed that this was a better fit than a two site model. The Km was 36 ± 3 and 43 ± 10 µM in control and preeclamptic placental groups, respectively, and the Vmax was 77 ± 4 pmol/mg protein·min in control (n = 6) compared with 88 ± 24 pmol/mg protein·min (n = 6) in the preeclamptic placental group. These values were not significantly different.



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  		FIG. 2. Kinetic determination of the uptake of [3H]L-arginine into BM vesicles. Uptakes were performed in EVB containing 50 mM KCl, 50 mM NaCl, 100 mM mannitol, and 20 mM HEPES-Tris in the presence of 0.2 µM to 20 mM arginine in control (n = 6; {blacksquare}) or preeclamptic (n = 6; •) BM vesicles.

 
To investigate the relative contributions of system y+ and y+L, experiments were performed where vesicles were incubated with either glutamine (10 mM) or arginine (20 mM). The uptakes in the presence and absence of glutamine were then used to calculate a glutamine-sensitive component of uptake (total minus that in 10 mM glutamine) and glutamine-insensitive component (that in the presence of glutamine). There was a difference in the glutamine-insensitive uptake [Fig. 3Go; 89 ± 4 fmol/mg protein·min (n = 6) in BM from control placentas compared with 151 ± 23 in BM from preeclamptic placentas (n = 5); P < 0.05, by two-tailed t test].



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  		FIG. 3. Uptake of [3H]L-arginine into BM vesicles isolated from placenta from normal pregnancies ({square}) or from placenta from patients with preeclampsia ({blacksquare}) in femtomoles per milligrams of protein per minute (in parentheses is the number of placentas). To assess the contribution of the transporters present in the BM of the placental syncytiotrophoblast, uptakes were performed in the absence or presence of glutamine and arginine as described previously (12 ). *, P < 0.05, by t test.

 
This glutamine-insensitive component of [3H]L-arginine uptake is comprised of two possible components: an arginine-saturable component (system y+), and a component not saturable with arginine (nontransporter-mediated uptake via diffusion). To evaluate the contributions of these two modes of uptake, [3H]L-arginine uptake was measured in the presence and absence of 20 mM L-arginine. Uptake in the presence of 20 mM arginine was not different between control and preeclamptic vesicles [Fig. 3Go; 44 ± 4 fmol/mg protein·min (n = 6) and 54 ± 8 fmol/mg protein·min (n = 5), respectively]. These values were subtracted from the [3H]arginine uptakes in the presence of 10 mM glutamine to give a glutamine-insensitive, arginine-saturable component, assumed to be uptake via system y+. This was significantly different between the two groups [45 ± 3 fmol/mg protein·min (n = 6) in controls and 97 ± 23 fmol/mg protein·min (n = 5) in preeclamptic vesicles; P < 0.05, by two-tailed t test].

We proceeded from these uptake data to determine whether there were any changes in mRNA expression of transporters between the two groups. Data are expressed as a ratio of that measured in placental reference tissue (mean ± SEM; n = 6 in both groups). qRT-PCR analysis of 4F2hc (1.30 ± 0.27 in control compared with 0.81 ± 0.11 in preeclampsia) and hCAT-1 (0.47 ± 0.09 in control compared with 0.34 ± 0.05 in preeclampsia), hCAT-2 (1.26 ± 0.30 in control compared with 0.91 ± 0.19 in preeclampsia), and hCAT-4 (0.62 ± 0.19 in control compared with 0.69 ± 0.25 in preeclampsia) mRNA in whole placenta showed no significant difference between the two groups. Confidence in this negative result stems from the following: 1) we isolated a sufficient quantity of high quality RNA from these placentas in which to detect a difference if there was one; 2) the specificity of the PCR was confirmed by visualization of the products by agarose gel electrophoresis; and 3) we have used this technique previously, with the same primers, probes, and reaction conditions, to demonstrate significant effects of similar clinical conditions on hCAT mRNA expression in other cell types (32).

Western blotting with an antibody to 4F2hc under reducing conditions detects both the heavy chain and heavy-light chain dimers of system y+L (17). We were previously unable to detect 4F2hc in BM from placenta using 10 µg protein/lane (17), and this was clarified here using a higher protein loading (30 µg protein/lane). Immunoreactive bands were detected at 85 and 135 kDa, respectively, in BM from both control and preeclamptic placentas (Fig. 4Go). Signal was abolished by preincubation of antibody with antigen. Densitometry, measured as OD x area, showed no significant differences in the signal intensity between the groups for each band (Fig. 5AGo).



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  		FIG. 4. Western blotting for 4F2hc (30 µg protein/lane; A) and hCAT-1 (60 µg protein/lane; B) in BM from different term placentas. C1–C3, Control; P1–P5, preeclampsia. A, An approximately 135-kDa and an approximately 85-kDa specific band were detected in BM from both control and preeclampsia placentas (exposure was 3 min, and the signal was abolished by preabsorption of antibody with blocking peptide). B, For hCAT-1 bands were detected at approximately 147 and 124 kDa in BM from both control and preeclampsia placentas (exposure was 20 min, and the signal was abolished in the absence of primary antibody).

 


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  		FIG. 5. Densitometry analysis (mean ± SEM) of expression of 4F2hc (A) and hCAT-1 (B) in BM isolated from placentas from normal (n = 3) or preeclamptic (n = 5) pregnancies as shown in Fig. 4Go. There was no significant difference between the expression of 4F2hc in the two groups. The expression of the 124-kDa band of hCAT-1 in BM was significantly higher in BM isolated from placentas from preeclamptic (n = 5) compared with normal (n = 3) pregnancies. *, P < 0.05, by t test.

 
Probing an identical Western blot, using the same BM samples as for 4F2hc, with an antibody specific to hCAT1 revealed two bands, at 147 and 124 kDa. Signal was abolished in the absence of primary antibody. Densitometry showed that although there was no difference in the intensity of the upper band between the two groups, that of the lower band was significantly greater in the preeclamptic group compared with the control group (Fig. 5BGo; P < 0.05, by unpaired t test).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we investigated whether there are any differences in the transport of L-arginine, the precursor of NO, between BM of placentas from normal pregnancies compared with BM of placentas from pregnancies complicated by preeclampsia. We found that total L-arginine transporter activity is higher in the BM from preeclamptic placentas compared with those from control placentas. This is predominantly due to increased activity of a glutamine-insensitive, arginine-sensitive activity, which we ascribe to system y+ (17, 22). This system y+ activity in the BM is very low, as measured by [3H]L-arginine uptake, in placentas from normal pregnancies (this study and Ref. 17) and is most likely due predominantly to hCAT-1 expression (35).

The preeclamptic placentas we obtained in this study were obtained at random from the delivery suite. By chance, they were delivered relatively close to term, and there was no significant difference in gestation compared with our control group. The babies born to the preeclamptic mothers were significantly lighter than those born to the women with normal pregnancies. Therefore, it is important to note that cationic amino acid transporter activity in BM of placentas from women delivering intrauterine growth restriction babies in the absence of preeclampsia is significantly lower than normal (36). Therefore, the increased activity observed here is most likely related directly to preeclampsia.

The uptake data measured over 10–60 sec did not extrapolate to a zero intercept on the y-axis at time zero. This could be due to binding of tracer to the plasma membrane or a very rapid component of uptake. Further work is required to distinguish between these two possibilities. The presence of a rapid component of uptake would result in error in our absolute kinetic measurements. However, there was no significant difference in the y-intercept between the control and preeclamptic group, and therefore we believe our observation of a difference in uptake activity over 10–60 sec is secure.

Despite the differences in uptake rate, we were unable to determine a difference in Km or Vmax in L-arginine transport between the two sets of BM (Fig. 4Go). This is most likely due to the inability of our protocol to discriminate differences related to the small component of [3H]L-arginine uptake that is mediated by system y+ in this in vitro system.

Our failure to detect differences in hCAT mRNA between the two groups could suggest that the effect of preeclampsia we observed on system y+ activity reflects a difference in posttranscriptional processing. This is consistent with the observation of a difference in BM activity, but not MVM activity (22). It is also consistent with our Western blotting data, showing a significant increase in the expression of the smaller of the hCAT-1 bands in the BM from the preeclamptic group. This trend was specific to this hCAT-1 species, as the higher molecular weight hCAT-1 band showed no difference between groups. Furthermore, probing the same samples for 4F2hc revealed no difference in heavy or light chain expression between the two groups, consistent with a lack of difference in the system y+L transporter activity data. The molecular weights of the two bands of hCAT-1 observed here are both larger than would be predicted by the amino acid sequence of the core protein (67 kDa) (37). However, the sequence suggests glycosylation sites (38), and larger glycosylated species have been reported (37). Therefore, it is possible that the two bands we observed in the BM are differentially glycosylated forms of the hCAT-1 protein. This requires further investigation, as does the protein expression of other hCAT isoforms.

In considering the physiological significance of our data, it should be remembered that system y+L is a high affinity, low capacity system, whereas system y+ is low affinity, high capacity. Therefore, at the concentrations of L-arginine present in utero, the greater flux of this amino acid will be via system y+. This flux of L-arginine on system y+ will be driven by the electrochemical gradient. L-Arginine concentrations are likely to be higher in the syncytiotrophoblast cytosol (39) than in the maternal or fetal plasma (40), tending to drive the amino acid out of the syncytium across the MVM and BM. However, for a cationic amino acid such as L-arginine, the membrane potential across both membranes will also be an important driving force; this has been measured as -22 mV across the MVM (41) and, by extrapolation from knowledge of the transtrophoblast potential difference (41), is likely to be similar across the BM. We therefore consider it likely that L-arginine normally moves into the syncytiotrophoblast across MVM and BM on system y+. Consequently, the higher system y+ activity in the BM of preeclamptic placentas would drive greater L-arginine flux in vivo into the syncytiotrophoblast. This would be consistent with the observation of a greater basal distribution of eNOS in preeclampsia (13). However, there are clearly a number of untested assumptions in this speculation. Furthermore, any compartmentalization of the transporter and eNOS, as found in other cell types (16), would mean that completely different driving forces would pertain; this needs to be investigated. We have proposed previously that the maternal-fetal flux of cationic amino acids across the BM is driven by Na+-dependent uptake of neutral amino acids in exchange for cationic amino acids by system y+L (17). Net L-arginine flux across the BM will depend on the balance of activity of the two cationic amino acid transporters in vivo and again may be affected by their cellular compartmentalization.

There are at least two possible mechanisms that could explain the up-regulation of L-arginine transport activity in the BM. Firstly, there are data from endothelial cells to suggest that limited availability of L-arginine can up-regulate system y+ (42) and amino acid starvation has been reported to increase CAT-1 mRNA 3-fold in FAO cells (43). Therefore, increased syncytiotrophoblast eNOS activity or expression and L-arginine utilization in preeclampsia (5) could directly up-regulate system y+ amino acid transporter activity. Secondly, preeclampsia has been likened to an inflammatory response (44), and it is interesting to note that increased system y+ activity, but not system y+L activity, has been found in lymphocytes stimulated with mitogens to model infection (45). Both of these possible explanations deserve further study, as does the mechanism by which the system y+ activity on the BM is up-regulated, whereas that on the MVM is not. Finally, it is also of interest to note preliminary data showing that placental microvascular endothelial cells have reduced system y+ activity in preeclampsia (46). This suggests cell-specific transporter responses to preeclampsia in the placenta.


   Acknowledgments
 
We thank Drs. Sergey Zharikov and Edward Block for the kind donation of the hCAT-1 antibody. We thank Mr. Kurt Mynett and Ms. Milly Cretney for their technical assistance, Drs. Tracey Johnston and Sara Vause for their help in obtaining placentas from preeclamptic patients, and the midwives of the Central Delivery Unit of St. Mary’s Hospital, Manchester, for their help in obtaining normal placentas.


   Footnotes
 
This work was supported by the British Heart Foundation and the Wellcome Trust.

Abbreviations: BM, Basal plasma membrane; CAT, cationic amino acid transporter; CBM, BM from control placenta; eNOS, endothelial NOS; EVB, extravesicular buffer; h, human; MVM, microvillous membrane; NO, nitric oxide; NOS, NO synthase; PE, preeclamptic placenta; qRT-PCR, quantitative RT-PCR; Vmax, maximum velocity.

Received January 13, 2003.

Accepted May 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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