title: Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells.
abstract: Despite originally identified in neurones, the neuronal type of nitric oxide synthase (nNOS) is present also in cardiac and skeletal myocytes. Whether nNOS is functionally expressed in human endothelial cells--as the endothelial enzyme (eNOS)--is unknown. Human umbilical vein endothelial cells (HUVEC) were studied under control culture conditions and after 48 h treatment with cytomix (human tumour necrosis factor-alpha, interferon-gamma and E. coli endotoxin). We tested: (i) localisation and expression of nNOS and eNOS proteins by immunostaining and immunoblotting; (ii) activity of nNOS and eNOS by measuring L-arginine to L-citrulline conversion with 1-(2-trifluoromethylphenyl)imidazole (TRIM), a specific nNOS antagonist, in sub-cellular fractions; (iii) intracellular cGMP levels, as a marker for nitric oxide production, after TRIM pre-treatment, by radioimmunoassay. nNOS protein was expressed in the cytosolic fraction and immunolocalised in cultured HUVEC, and co-localised with the eNOS protein in frozen sections of the human umbilical cord. nNOS protein contributed to total L-citrulline production as TRIM selectively and dose-dependently reduced L-citrulline synthesis in the cytosolic but not particulate fraction of HUVEC. Similarly, TRIM reduced intracellular cGMP content both at baseline and after stimulation with a calcium ionophore. Cytomix down-regulated the expression and function of both nNOS and eNOS while no inducible NOS (iNOS) was detected. In conclusion, a functional neuronal type of NOS is co-expressed with the endothelial NOS type in HUVEC, suggesting a possible role for nNOS in regulation of blood flow.
authors.affiliation: Cardiovascular Pathophysiology Research Centre, Fondazione Salvatore Maugeri IRCCS, Via Pinidolo, 23, 25064 Gussago (Brescia), Italy. tbachetti@fsm.it
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bachetti
authors.forename: Laura
authors.initials: L
authors.lastname: Comini
authors.forename: Salvatore
authors.initials: S
authors.lastname: Curello
authors.forename: Daniela
authors.initials: D
authors.lastname: Bastianon
authors.forename: Michela
authors.initials: M
authors.lastname: Palmieri
authors.forename: Giuliana
authors.initials: G
authors.lastname: Bresciani
authors.forename: Francesco
authors.initials: F
authors.lastname: Callea
authors.forename: Roberto
authors.initials: R
authors.lastname: Ferrari
mesh_terms: D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002956:Citrulline / Q000096:biosynthesis; D006152:Cyclic GMP / Q000032:analysis / Q000378:metabolism; D003600:Cytosol / Q000276:immunology / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014470:Umbilical Cord / Q000166:cytology
chemical_list: D007093:Imidazoles; D008070:Lipopolysaccharides; D009419:Nerve Tissue Proteins; D014409:Tumor Necrosis Factor-alpha; C098186:1-(2-trifluoromethylphenyl)imidazole; D002956:Citrulline; D009569:Nitric Oxide; D007371:Interferon-gamma; C496320:NOS1 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; D002118:Calcium