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-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.
| Original language | English |
|---|---|
| Pages (from-to) | 939-945 |
| Number of pages | 7 |
| Journal | Journal of Molecular and Cellular Cardiology |
| Volume | 37 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Nov 2004 |
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Keywords
- Cell culture/isolation
- Cytokines
- Endothelial factors
- Infection/inflammation
- Nitric oxide
ASJC Scopus subject areas
- Molecular Biology
- Cardiology and Cardiovascular Medicine
Cite this
Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells. / Bachetti, Tiziana; Comini, Laura; Curello, Salvatore; Bastianon, Daniela; Palmieri, Michela; Bresciani, Giuliana; Callea, Francesco; Ferrari, Roberto.
In: Journal of Molecular and Cellular Cardiology, Vol. 37, No. 5, 11.2004, p. 939-945.Research output: Contribution to journal › Article
}
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
UR - http://www.scopus.com/inward/record.url?scp=7444220194&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=7444220194&partnerID=8YFLogxK
U2 - 10.1016/j.yjmcc.2004.07.006
DO - 10.1016/j.yjmcc.2004.07.006
M3 - Article
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 -