Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells |
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Authors: | Bachetti Tiziana Comini Laura Curello Salvatore Bastianon Daniela Palmieri Michela Bresciani Giuliana Callea Francesco Ferrari Roberto |
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Affiliation: | Cardiovascular Pathophysiology Research Centre, Fondazione Salvatore Maugeri IRCCS, Via Pinidolo, 23, 25064 Gussago (Brescia), Italy. tbachetti@fsm.it |
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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. |
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Keywords: | Nitric oxide Endothelial factors Cell culture/isolation Cytokines Infection/inflammation |
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