Vasostatin-1 is a new physiological penetrating peptide acting by a heparan sulphate-endocytosis-PI3K-eNOS pathway.

Objectives: Vasostatins (VSs) are vasoactive peptides derived from Chromogranin A, a protein contained in secretory granules of chromaffin and other cell types. The negative inotropic effect and the reduction of isoproterenol-dependent inotropism induced by VSs in the heart, suggest that they play an anti-adrenergic function. Previous studies in our laboratory showed that the Chromogranin A - derived peptide Vasostatin-1 (VS-1) induces a PI3K-dependent-NO release by endothelial cells. However, in the absence of a specific VS-1 membrane receptor, the molecular mechanisms and the cellular processes upstream the eNOS activation by this peptide are still unknown. In this report we investigated the hypothesis that VS-1 acts as a cationic cell penetrating peptide, binding to heparane sulfate receptors and activating eNOS phosphorylation (Ser1179) through a PI3K dependent-endocytosis-coupled mechanism. Materials and methods: Bovine Aortic Endothelial (BAE-1) cells (European Collection of Cell Cultures, Salisbury, Wiltshire, UK) were maintained in Dulbecco's Modified Eagle's medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were maintained in 1% FCS 24h before the experiments. Endocytotic vescicles traffiking was quantified by confocal microscopy with the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to label plasmalemma-derived vesicles. VS-1 dependent eNOS phosphorylation was studied by immunofluorescence and western blot experiments. Confocal fluorimetric measurements were performed using an Olympus Fluoview 200 laser scanning confocal system mounted on an inverted IX70 Olympus microscope, equipped with a 60X oil-immersion objective (NA 1.25). Results: In endocytosis experiments, BAE-1 cells stimulated with VS-1 showed a marked increase in the endocytotic processes, which was blocked by both heparinase III and Wortmannin. To understand the molecular mechanism responsible for the eNOS activity, we investigated on the increase of P(Ser1179) eNOS, by immunofluorescence and western blot experiments. Our results showed that VS-1 (100 nM) induces a significant increase in the level of phosphorylation of eNOS (Ser1179), blocked by Wortmannin. Conclusions: Our results suggest that VS-1 binds to heparane sulphate receptors and stimulates endocytotic vesicles formation associated with the PI3K dependent eNOS phosphorylation.