Cell penetrating peptides can be used as therapeutic agents via modulation of selective cell
functions. Nitric oxide (NO) generated by vascular endothelial NO synthase (eNOS) plays a critical
role in the NO/ cyclic guanosine 5’-monophosphate (cGMP)-mediated pulmonary vascular function.
Here we examined whether internalization of a fifteen amino acid (KRFNSISCSSWRRKR) synthetic
peptide (P3) enhances the catalytic activity of eNOS via caveolae/eNOS dissociation leading to NO release
and increased cGMP production in pulmonary artery endothelial cells (EC). ECs were treated
with varying concentrations of P3 and used to monitor internalization, isolation of caveolae-enriched
fraction, the catalytic activity of eNOS, NO/cGMP production, and intracellular Ca2+ release. Confocal images show timedependent
internalization of P3 in EC. Treatment of EC with P3, but not scrambled P3, increased the catalytic activity of
eNOS in a dose-dependent manner without change in eNOS expression or phosphorylation. Treatment of EC with P3
stimulated intracellular Ca2+ release, increased the catalytic activity of phospatidylinsositide 3 kinase (PI3K) and resulted
in eNOS/caveolae-1 (Cav-1) dissociation leading to translocation of eNOS to intracellular compartment in EC. P3-
mediated activation of eNOS was abolished by intracellular Ca2+ chelator 1,2-bis(2-aminophenooxy)ethane-N,N,N’,N’-
tertraacetic acid-AM (BAPTA-AM), PI3K inhibition, or by siRNA-mediated Cav-1 suppression. These results demonstrate
that exogenous peptide consisting of cationic amino acids can internalize and enhance the catalytic activity of eNOS
via modulation of caveolar signaling and intracellular Ca2+ release in EC.