160
membranes, while in the presence of 5 nM CST green fluorescence appeared
diffused in the cytosol, as a consequence of Cav1 internalization. Of note, CST-
induced Cav1 internalization was significantly reduced by pretreatment with
heparinase.
As CST stimulates endocytosis, induces Cav1 internalization and enhances NO
production in endothelial cells (Bassino et al. 2011 ), we hypothesized that eNOS
activation is mediated by the displacement of the protein from Cav1 binding.
Previous reports have indeed proposed a mechanism of eNOS activation coupled
with caveolae internalization (Maniatis et al. 2006 ; Sanchez et al. 2009 ) and disso-
ciation of eNOS from Cav1 has been shown as a marker of eNOS activation
(Fleming 2010 ; Minshall et al. 2003 ). To verify this hypothesis, we studied cellular
colocalization of Cav1 and eNOS by immunofluorescence experiments. We
observed that, in comparison with control conditions, CST strongly reduced eNOS/
Cav1 colocalization at plasma membrane. The fact that Wm was able to restore this
colocalization confirmed the role of PI3K in mediating CST intracellular signaling.
Our observation that PI3K activity was required in both endocytosis and eNOS/
Cav1 trafficking suggest that PI3K represents the essential key for the CST-activated
intracellular signalling. Our results further confirm the notion that PI3K/Akt medi-
ated Ser^1179 phosphorylation of eNOS represents a common pathway among the
multiple regulatory mechanisms affecting the activity of this enzyme (Fleming
2010 ).
Moreover, PI3K is widely reported to have an important role in membrane bud-
ding and fission in endothelial cells (Mellor et al. 2012 ). Finally, with the last exper-
iments we confirmed our proposed pathway showing that caveolae disruption and
HSPGs removal both abolished the CST-induced eNOS phosphorylation.
Taken together, these results highlight the obligatory role for proteoglycans and
caveolae internalization in the VS-1/CTS-dependent eNOS activation in endothelial
cells. Our results could clarify the mechanism responsible for the physiological
properties of VS-1 and CST on endothelial cells, in particular with respect to their
ability to activate the intracellular PI3K–eNOS pathways in the absence of a typical
high-affinity membrane receptor.
4 Other Mediators Involved in the Action of CgA-Derived
Peptides
Several data suggest that, at least in certain tissues and organs, the effects of CgA-
derived peptides are due to the release or the interaction with other cell-to-cell mes-
sengers, particularly inflammatory mediators. Among these we consider histamine,
ANP, BNP, TNF and bFGF.
G. Alloatti and M.P. Gallo