Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan-dependent mechanism

• Published in: Journal of cellular biochemistry 2010-05, Vol.110 (1), p.70-79
• Main Authors: Ramella, Roberta, Boero, Ombretta, Alloatti, Giuseppe, Angelone, Tommaso, Levi, Renzo, Gallo, Maria Pia
• Format: Article
• Language: English
• Subjects: Androstadienes - pharmacology; Animals; Cattle; Caveolae - drug effects; Caveolae - metabolism; Caveolin 1 - metabolism; chromogranin A; Chromogranin A - pharmacology; endocytosis; Endocytosis - drug effects; endothelial cell; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Enzyme Activation - drug effects; Heparin Lyase - pharmacology; nitric oxide synthase; Nitric Oxide Synthase Type III - metabolism; Peptide Fragments - pharmacology; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation - drug effects; Protein Transport - drug effects; Proteoglycans - metabolism; Transport Vesicles - drug effects; Transport Vesicles - metabolism; vasostatin 1
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.22510

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‐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: HSPGs interaction and caveolae endocytosis, coupled with a PI3K‐dependent eNOS phosphorylation. J. Cell. Biochem. 110: 70–79, 2010. © 2010 Wiley‐Liss, Inc.