Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells

We previously reported that the vasoactive peptide 1 (P1, SSWRRKRKESS) modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-alpha is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-alpha phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were incubated in culture medium containing Rhodamine red-labeled P1 (100 mu M) for 0-120 min. Confocal examinations indicate that P1 internalization is time-dependent and reaches a plateau at 60 min. Caveolae disruption by methyl-beta-cyclodextrin (CD) and filipin (FIL) inhibited the internalization of P1 in ECs suggesting that P1 internalizes via caveolae. P1-stimulation also enhances phosphorylation of caveolar PKC-alpha and increases intracellular calcium (Ca2+) release in intact cells suggesting that P1 internalization is regulated by PKC-alpha in ECs. To confirm the roles of increased phosphorylation of PKC-alpha and Ca2+ release in internalization of P1, PKC-alpha modulation by phorbol ester (PMA), PKC-alpha knockdown, and Ca2+ scavenger BAPTA-AM model systems were used. PMA-stimulated phosphorylation of caveolar PKC-alpha is associated with significant reduction in P1 internalization. In contrast, PKC-alpha deficiency and reduced phosphorylation of PKC-alpha enhanced P1 internalization. P1-mediated increased phosphorylation of PKC-alpha appears to be associated with increased intracellular calcium (Ca2+) release since the Ca2+ scavenger BAPTA-AM enhanced P1 internalization. These data indicate that caveolar integrity and P1-mediated increased phosphorylation of caveolar PKC-alpha play crucial roles in the regulation of P1 internalization in lung ECs.