Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells

Tiziana Bachetti, Laura Comini, Salvatore Curello, Daniela Bastianon, Michela Palmieri, Giuliana Bresciani, Francesco Callea, Roberto Ferrari

Research output: Contribution to journalArticle

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 languageEnglish
Pages (from-to)939-945
Number of pages7
JournalJournal of Molecular and Cellular Cardiology
Volume37
Issue number5
DOIs
Publication statusPublished - 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

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