title: Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan-dependent mechanism.
abstract: Accumulating evidences point to a significant role for the chromogranin A (CgA)-derived peptide vasostatin 1 (VS-1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS-1 induces a PI3K-dependent-nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio-suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high-affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS-1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K-dependent, endocytosis-coupled mechanism. In bovine aortic endothelial cells (BAE-1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water-soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS-1-dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS-1 induces a marked increase in the caveolae-dependent endocytosis, (115 +/- 23% endocytotic spots/cell/field in VS-1-treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 +/- 15% above control) and Wortmannin (Wm, 7 +/- 22% above control). Heparinase, Wortmannin, and methyl-beta-cyclodextrin (MbetaCD) abolish the VS-1-dependent eNOS phosphorylation (P(Ser1179)eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K-dependent eNOS phosphorylation.
authors.affiliation: Dipartimento di Biologia Animale e dell'Uomo, Università di Torino, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Ramella
authors.forename: Ombretta
authors.initials: O
authors.lastname: Boero
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Alloatti
authors.forename: Tommaso
authors.initials: T
authors.lastname: Angelone
authors.forename: Renzo
authors.initials: R
authors.lastname: Levi
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Gallo
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D053379:Chromogranin A / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D019763:Heparin Lyase / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011509:Proteoglycans / Q000378:metabolism*; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D051242:Caveolin 1; D053379:Chromogranin A; D010446:Peptide Fragments; D011509:Proteoglycans; C064110:vasostatin I; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D019763:Heparin Lyase; D000077191:Wortmannin