Nitric oxide production stimulated by the basic fibroblast growth factor requires the synthesis of ceramide.
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 Ca(2+)-calmodulin-dependent process. In CHO-K1 cells, NO production by bFGF is Ca(2+) and MAP kinase independent, because it was not reverted by pretreatment with intracellular Ca(2+) 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 Ca(2+)/calmodulin-independent mechanism for eNOS activation after fibroblast growth factor stimulation.
Arena
Sara
S
Pharmacology and Neuroscience, National Institute for Cancer Research (IST), c/o Advanced Biotechnology Center (CBA), 16132 Genoa, Italy.
Pattarozzi
Alessandra
A
Thellung
Stefano
S
Villa
Valentina
V
Corsaro
Alessandro
A
Massa
Alessandro
A
Diana
Fabrizio
F
Spoto
Giuseppe
G
Forcella
Sabrina
S
Damonte
Gianluca
G
Filocamo
Mirella
M
Benatti
Umberto
U
Schettini
Gennaro
G
Florioa
Tullio
T
D000818:Animals; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D016466:CHO Cells; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism*; D006224:Cricetinae; D006152:Cyclic GMP / Q000378:metabolism; D003891:Desipramine / Q000494:pharmacology; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D009542:Niemann-Pick Diseases; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009636:Norbornanes; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D012016:Reference Values; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology
D001952:Bridged-Ring Compounds; D002518:Ceramides; D009636:Norbornanes; D010726:Phosphodiesterase Inhibitors; D013859:Thiocarbamates; D013871:Thiones; D016222:Fibroblast Growth Factor 2; D009569:Nitric Oxide; C046498:tricyclodecane-9-yl-xanthogenate; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; D003891:Desipramine