Vasostatins (VSs) are vasoactive peptides derived from Chromogranin A, a protein contained in secretory granules of chromaffin and other cells. 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. However, the mechanisms of action of VSs need furher investigations. Our study aims at defining the signaling pathways activated by VS-1 in mammalian ventricular myocardium and in cultured endothelial cells that lead to the modulation of cardiac contractility. We recently shown that VS-1 induces a PI3K-dependent-nitric oxide (NO) release by endothelial cells, contributing to explaining the mechanism of its cardio-suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exherted by this peptide are still unknown, as typical high affinity receptors have not been identified. Here we hypothesized that in endothelial cells VS-1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparane sulfate proteoglycans (HSPG) and activating eNOS phosphorylation (Ser1179) through a PI3K-dependent, endocytosis-coupled mechanism. In bovine aortic endothelial cells (BAE-1 cells) endocytic 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 studied 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 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-β-cyclodextrin (MβCD) abolish the VS-1 dependent eNOS phosphorylation (PSer1179eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPG interaction and caveolae endocytosys, coupled with a PI3K dependent eNOS phosphorylation.
Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan-dependent mechanism
RAMELLA, Roberta;ALLOATTI, Giuseppe;LEVI, Renzo;GALLO, Maria Pia
2010-01-01
Abstract
Vasostatins (VSs) are vasoactive peptides derived from Chromogranin A, a protein contained in secretory granules of chromaffin and other cells. 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. However, the mechanisms of action of VSs need furher investigations. Our study aims at defining the signaling pathways activated by VS-1 in mammalian ventricular myocardium and in cultured endothelial cells that lead to the modulation of cardiac contractility. We recently shown that VS-1 induces a PI3K-dependent-nitric oxide (NO) release by endothelial cells, contributing to explaining the mechanism of its cardio-suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exherted by this peptide are still unknown, as typical high affinity receptors have not been identified. Here we hypothesized that in endothelial cells VS-1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparane sulfate proteoglycans (HSPG) and activating eNOS phosphorylation (Ser1179) through a PI3K-dependent, endocytosis-coupled mechanism. In bovine aortic endothelial cells (BAE-1 cells) endocytic 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 studied 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 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-β-cyclodextrin (MβCD) abolish the VS-1 dependent eNOS phosphorylation (PSer1179eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPG interaction and caveolae endocytosys, coupled with a PI3K dependent eNOS phosphorylation.
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