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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 3, 2003 as doi:10.1096/fj.02-0916fje. |
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,2
* Leukocyte Biology, Division of Biomedical Sciences, Imperial College School of Medicine, London SW7 2AZ; and
Academic Unit of Child Health, University of Manchester, St. Mary’s Hospital, Manchester, M13 0JH, UK
2Correspondence: Academic Unit of Child Health, University of Manchester, St. Mary’s Hospital, Hathersage Road, Manchester M13 0JH, UK. E-mail:jglazier{at}fs3.scg.man.ac.uk
SPECIFIC AIMS
1) To investigate the expression of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC) using RT-PCR, Western blot, and immunohistochemistry. 2) To characterize active arginine transporters in HPMEC by measuring radiolabeled arginine uptake with and without ion substitutions in the presence and absence of neutral amino acids. 3) To measure nitric oxide (NO) production by HPMEC as an index of eNOS activity.
PRINCIPAL FINDINGS
1. HPMEC express mRNA for a broad range of cationic amino acid transporters
RT-PCR revealed expression of genes encoding proteins that could mediate cationic amino acid transport in HPMEC. Amplicons of the predicted size, which comigrated with signal from positive control tissues, were observed for CAT1, CAT 2A, CAT2B, CAT4, 4F2hc (CD98), y+LAT1, y+LAT2, rBAT, and b0+AT1. No PCR product was detectable for system B0+ in HPMEC.
2. HPMEC express 4F2hc (CD98) and CAT1 at the protein level
Western blot under nonreducing conditions for 4F2hc (CD98) detected a 135 kDa protein as the major species in HPMEC lysates. Under reducing conditions, this resolved into two immunoreactive species of 85 kDa and 135 kDa representing 4F2hc monomer and 4F2hc light chain heterodimers, respectively. Cytochemical staining for 4F2hc revealed the protein was uniformly spread over the cell surface, with no evidence of membrane polarization being present throughout the cytoplasm.
A doublet of 79 kDa and 90 kDa was detected in HPMEC using a CAT1-specific antibody and Western blot. This contrasted with the single protein species of 67 kDa detected in HUVEC (included for comparison as an example of a macrovascular endothelial cell), which concurs with the predicted size of nonglycosylated CAT1 protein. This difference in CAT1 molecular mass between these two endothelial cell types suggests that CAT1 in HPMEC is glycosylated, with both potential glycosylation sites complexed. The 79 kDa band had a greater intensity than the 90 kDa band, suggesting that the lesser glycosylated CAT1 species predominates in HPMEC. Cytochemical staining for CAT1 demonstrated a punctate distribution with evidence of membrane association. Cytoplasmic staining was also apparent, showing partial polarization to the apical side.
3. Arginine transport in HPMEC is mediated by systems y+ and y+L
Figure 1
shows that arginine uptake by HPMEC was independent of extracellular Na+ or Cl– but was inhibitable by the neutral amino acid glutamine and leucine, although a noninhibitable component remained. Both this neutral amino acid-sensitive component (taken as system y+L) and the neutral amino acid-insensitive component of uptake (taken as system y+) were inhibitable by arginine and lysine. Cystine, however, had no effect in the absence of Na+, eliminating system b0+ as a contributor to transport. Concentration-dependent glutamine inhibition of arginine uptake exhibited Na+ dependency, demonstrating a reduced affinity in the absence of Na+, a key feature of system y+L. Further support for two transport systems mediating arginine uptake in HPMEC was obtained by kinetic modeling, which resolved two transport components with high and low affinities. Taking these to represent systems y+L and y+, respectively, the data are consistent with our demonstration of 4F2hc (CD98, the heavy chain subunit of system y+L) and CAT1 (mediator of system y+ activity) proteins in HPMEC.
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4. Expression of eNOS by HPMEC is relatively low and eNOS activity is undetectable
Using Western blot we were unable to detect any eNOS signal in HPMEC, although this was readily detectable in macrovascular endothelial cells such as HUVEC and an aortic endothelial cell line, as well as in placenta (Fig. 2
). However, weak eNOS reactivity was observed in HPMEC by immunocytochemistry, with partial polarization to the apical surface. The cellular content of eNOS in HPMEC was confirmed by ELISA to be markedly lower (by two orders of magnitude) than that in HUVEC or placenta.
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Basal production of NO by HPMEC was undetectable by the Griess reaction or when using NO-sensing electrodes. Attempts to stimulate NO production by addition of extracellular arginine, VEGF, or A23187 were unsuccessful and did not result in detectable intracellular accumulation of cGMP.
CONCLUSIONS AND SIGNIFICANCE
The fetal capillary endothelium and the syncytiotrophoblast of human placenta constitute the two cellular layers across which maternofetal exchange occurs. The fetal capillary endothelium forms an extensive microvascular bed capable of restricting permeability to large hydrophilic solutes. Cells (HPMEC) isolated from this part of the placental vascular tree exhibit an endothelial phenotype that shows barrier properties consistent with the placental capillary bed and that of other tissues. HPMEC therefore can provide useful information relating not only to the endothelium of placenta, but also to other endothelia in human organs. Although the endothelial phenotype of these cells has been well characterized, there has been no functional characterization of transporter activity in these cells. As such, this study represents the first report examining a transport function in this cell type.
We elected to study arginine transport in HPMEC as this amino acid is the precursor of NO synthesis and NO-induced vasodilation is important in the maintenance of low vascular resistance within the fetoplacental circulation. Our data indicate that two transport systems, system y+L and system y+, mediate arginine uptake in HPMEC. These transport systems have been implicated in arginine transport by other types of endothelial cells, such as HUVEC. The syncytiotrophoblast of human placenta employs these same two transport systems to mediate cationic amino acid transport, highlighting a common transport mechanism in these different placental cell types.
This common transport feature, however, appears to bear no relationship to the relative expression of eNOS in these cell types. Nevertheless, our eNOS data fit with the broad observation that there appears to be a relationship between the regional distribution of eNOS immunoreactivity and placental vessel size in situ: eNOS expression diminishing with decreasing vessel size. The low/undetectable eNOS phenotype in HPMEC therefore mirrors the low level of eNOS immunoreactivity in the fetal capillary endothelium in situ, strengthening our assertion that HPMEC are derived from this part of the placental vascular tree.
This differential localization of eNOS along the placental vasculature implies there are regional differences in vascular NO production. Using a variety of techniques, we found that production of NO by HPMEC was below the threshold of detection. If this reflects the situation in vivo, it seems unlikely that NO derived from the fetal microvasculature would make a significant contribution to the maintenance of low vascular tone in the fetoplacental circulation. HPMEC therefore represents an unusual endothelial phenotype with respect to eNOS and NO synthesis. Perhaps the close apposition of HPMEC to an epithelial layer with abundant eNOS, the syncytiotrophoblast, results in down-regulation of eNOS expression and activity in the former cell type.
The ability of HPMEC to transport cationic amino acids despite the absence of eNOS activity indicates these transporters are important for other metabolic requirements of these cells. However, it is also possible that this may contribute to the overall maternofetal transport of cationic amino acids such as lysine and arginine by affecting the concentration of amino acids in the interstitial space, influencing the transplacental concentration gradient. Such a role of the endothelium in addition to that of the syncytiotrophoblast has not been considered, and would be dependent on a polarized distribution of transporters.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj. org/cgi/doi/10.1096/fj.02-0916fje 
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