title: Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase.
abstract: In certain cases of endothelial dysfunction l-arginine becomes rate-limiting for NO synthesis in spite of sufficiently high plasma concentrations of the amino acid. To better understand this phenomenon, we investigated routes of substrate supply to endothelial nitric oxide synthase (eNOS). Our previous data with human umbilical vein (HUVEC) and EA.hy.926 endothelial cells demonstrated that eNOS can obtain its substrate from the conversion of l-citrulline to l-arginine and from protein breakdown. In the present study, we determined the quantitative contribution of proteasomal and lysosomal protein degradation and investigated to what extent extracellular peptides and l-citrulline can provide substrate to eNOS. The RFL-6 reporter cell assay was used to measure eNOS activity in human EA.hy926 endothelial cells. Individual proteasome and lysosome inhibition reduced eNOS activity in EA.hy926 cells only slightly. However, the combined inhibition had a pronounced reducing effect. eNOS activity was fully restored by supplementing either l-citrulline or l-arginine-containing dipeptides. Histidine prevented the restoration of eNOS activity by the dipeptide, suggesting that a transporter accepting both, peptides and histidine, mediates the uptake of the extracellular peptide. In fact, the peptide and histidine transporter PHT1 was expressed in EA.hy926 cells and HUVECs (qRT/PCR). Our study thus demonstrates that l-citrulline and l-arginine-containing peptides derived from either intracellular protein breakdown or from the extracellular space seem to be good substrate sources for eNOS.
authors.affiliation: Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Karbach
authors.forename: Alexandra
authors.initials: A
authors.lastname: Simon
authors.forename: Anita
authors.initials: A
authors.lastname: Slenzka
authors.forename: Isabel
authors.initials: I
authors.lastname: Jaenecke
authors.forename: Alice
authors.initials: A
authors.lastname: Habermeier
authors.forename: Ursula
authors.initials: U
authors.lastname: Martiné
authors.forename: Ulrich
authors.initials: U
authors.lastname: Förstermann
authors.forename: Ellen I
authors.initials: EI
authors.lastname: Closs
mesh_terms: D001120:Arginine / Q000378:metabolism / Q000494:pharmacology; D050197:Atherosclerosis / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000378:metabolism / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017930:Genes, Reporter; D006639:Histidine / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects
chemical_list: D004151:Dipeptides; D007976:Leupeptins; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D002956:Citrulline; D009569:Nitric Oxide; D006639:Histidine; D002738:Chloroquine; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin