Nitric Oxide Production Stimulated by the Basic Fibroblast Growth Factor Requires the Synthesis of Ceramide

Authors

  • SARA ARENA,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • ALESSANDRA PATTAROZZI,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • STEFANO THELLUNG,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • VALENTINA VILLA,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • ALESSANDRO CORSARO,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • ALESSANDRO MASSA,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • FABRIZIO DIANA,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • GIUSEPPE SPOTO,

    1. Department of Applied Sciences of Oral and Dental Diseases, University of G. D'Annunzio of Chieti, Chieti, Italy
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  • SABRINA FORCELLA,

    1. Department of Applied Sciences of Oral and Dental Diseases, University of G. D'Annunzio of Chieti, Chieti, Italy
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  • GIANLUCA DAMONTE,

    1. Department of Experimental Medicine, University of Genoa, Genoa, Italy
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  • MIRELLA FILOCAMO,

    1. Diagnostic Laboratory of Pre- and Postnatal Metabolic Disorders, Istituto G. Gaslini, Genoa, Italy
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  • UMBERTO BENATTI,

    1. Department of Experimental Medicine, University of Genoa, Genoa, Italy
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  • GENNARO SCHETTINI,

    1. Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy
    2. Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy
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  • TULLIO FLORIOA

    Corresponding author
    1. Section of Pharmacology, Department of Biomedical Sciences, University of G. D'Annunzio of Chieti, Chieti, Italy
      Address for correspondence: Prof. Tullio Florio, Unità Neuroscienze, Centro Biotecnologie Avanzate, Largo Rosanna Benzi 10, 16132 Genoa, Italy. Voice: +39-010-5737255; fax: +39-010-5737257; florio@cba.unige.it.
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Address for correspondence: Prof. Tullio Florio, Unità Neuroscienze, Centro Biotecnologie Avanzate, Largo Rosanna Benzi 10, 16132 Genoa, Italy. Voice: +39-010-5737255; fax: +39-010-5737257; florio@cba.unige.it.

Abstract

Abstract: Nitric oxide (NO) is an intracellular and intercellular mediator involved in the modulation of many physiologic and pathologic processes including the regulation of neoangiogenesis. We analyzed the effects of basic fibroblast growth factor (bFGF) on NO production in CHO-K1 cells and the intracellular mechanisms involved. bFGF induces NO production through activation of the endothelial NO synthase (eNOS), causing a subsequent increase in cGMP levels. In most systems, eNOS activation is a Ca2+-calmodulin-dependent process. In CHO-K1 cells, NO production by bFGF is Ca2+ and MAP kinase independent, because it was not reverted by pretreatment with intracellular Ca2+ chelators or MEK inhibitors. Translocation of the eNOS from the plasma membrane, where it is bound to caveolin 1, to the cytosol is the crucial step in the synthesis of NO. We demonstrate that the cytosolic translocation of eNOS is caused by increased synthesis of ceramide dependent by the bFGF activation of sphingomyelinase. Indeed, in the presence of the sphingomyelinase inhibitors D609 or desipramine, bFGF-dependent NO production is abrogated. To support this evidence we evaluated ceramide concentration using HPLC-electrospray ionization-mass spectrometry in controls and in bFGF-treated cells: after bFGF stimulation, a substantial increase in ceramide levels was observed. These data were further confirmed by the lack of NO production in response to fibroblast growth factor in fibroblasts derived from Niemann Pick patients who genetically lack the enzyme sphingomyelinase. In conclusion, ceramide in CHO-K1 cells is responsible for a novel Ca2+/calmodulin-independent mechanism for eNOS activation after fibroblast growth factor stimulation.

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