Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells.
We investigated the expression and activity of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC). Using RT-PCR amplification products for eNOS, CAT1, CAT2A, CAT2B, CAT4, 4F2hc (CD98), rBAT and the light chains y+LAT1, y+LAT2, and b0+T1 were detected in HPMEC, but not B0+. Immunohistochemistry and Western blotting confirmed the presence of 4F2hc and CAT1 protein in HPMEC. 4F2hc-light chain dimers were indicated by a shift in molecular mass detected under nonreducing conditions. L-Arginine transport into HPMEC was independent of Na+ or Cl- and was inhibited by the neutral amino acid glutamine, but not by cystine. The Ki for glutamine inhibition was greater in the absence of Na+. Kinetic analysis supported a two-transporter model attributed to system y+L and system y+. Expression of eNOS in HPMEC was detectable by immunohistochemistry and ELISA but not by Western blotting. Activity of eNOS in HPMEC, measured over 48 h, either as the basal production of nitric oxide (NO) or as the accumulation of intracellular cGMP was not detectable. We conclude that HPMEC transport cationic amino acids by systems y+ and y+L and that basal eNOS expression and activity in these cells is low.
Leukocyte Biology, Division of Biomedical Sciences, Imperial College School of Medicine, London SW7 2AZ.
J F
JF
Dye
S
S
Vause
T
T
Johnston
P
P
Clark
J A
JA
Firth
S W
SW
D'Souza
C P
CP
Sibley
J D
JD
Glazier
D026923:Amino Acid Transport Systems, Basic / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport; D002196:Capillaries / Q000166:cytology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism*; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000378:metabolism; D006801:Humans; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010920:Placenta / Q000098:blood supply*; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic
D026923:Amino Acid Transport Systems, Basic; D027282:Fusion Regulatory Protein 1, Heavy Chain; D012333:RNA, Messenger; D001120:Arginine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III