TY - JOUR
T1 - Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells
AU - Bachetti, Tiziana
AU - Comini, Laura
AU - Curello, Salvatore
AU - Bastianon, Daniela
AU - Palmieri, Michela
AU - Bresciani, Giuliana
AU - Callea, Francesco
AU - Ferrari, Roberto
PY - 2004/11
Y1 - 2004/11
N2 - 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-α, interferon-γ 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.
AB - 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-α, interferon-γ 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.
KW - Cell culture/isolation
KW - Cytokines
KW - Endothelial factors
KW - Infection/inflammation
KW - Nitric oxide
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U2 - 10.1016/j.yjmcc.2004.07.006
DO - 10.1016/j.yjmcc.2004.07.006
M3 - Article
C2 - 15522271
AN - SCOPUS:7444220194
VL - 37
SP - 939
EP - 945
JO - Journal of Molecular and Cellular Cardiology
JF - Journal of Molecular and Cellular Cardiology
SN - 0022-2828
IS - 5
ER -