Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells.
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.
Bachetti
Tiziana
T
Cardiovascular Pathophysiology Research Centre, Fondazione Salvatore Maugeri IRCCS, Via Pinidolo, 23, 25064 Gussago (Brescia), Italy. tbachetti@fsm.it
Comini
Laura
L
Curello
Salvatore
S
Bastianon
Daniela
D
Palmieri
Michela
M
Bresciani
Giuliana
G
Callea
Francesco
F
Ferrari
Roberto
R
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
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