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

Abstract

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-α 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-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were incubated in culture medium containing Rhodamine red-labeled P1 (100 μM) for 0–120 min. Confocal examinations indicate that P1 internalization is time-dependent and reaches a plateau at 60 min. Caveolae disruption by methyl-β-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-α and increases intracellular calcium (Ca2+) release in intact cells suggesting that P1 internalization is regulated by PKC-α in ECs. To confirm the roles of increased phosphorylation of PKC-α and Ca2+ release in internalization of P1, PKC-α modulation by phorbol ester (PMA), PKC-α knockdown, and Ca2+ scavenger BAPTA-AM model systems were used. PMA-stimulated phosphorylation of caveolar PKC-α is associated with significant reduction in P1 internalization. In contrast, PKC-α deficiency and reduced phosphorylation of PKC-α enhanced P1 internalization. P1-mediated increased phosphorylation of PKC-α 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-α play crucial roles in the regulation of P1 internalization in lung ECs.

Peptide-stimulated angiogenesis: Role of lung endothelial caveolar signaling and nitric oxide

“…The caveolae enriched membrane fractions were isolated using detergent-free method as we previously described [10, 24]. The isolated membrane fractions were analyzed by western blots using 50 μg of protein to determine the level of the caveolae marker protein Cav-1.…”

“…The western blots developed as previously described [3 and 10]. In brief, cell lysate, immunoprecipitates, or caveolar enriched fraction obtained as described in Methods.…”

“…Caveolae are cholesterol/sphingolipid-enriched microdomain invaginations of the plasma membrane in EC [8]. The putative functions of caveolae include transmembrane transport by endocytosis, potocytosis, and signal transduction [8-10]. The loss and/or modulation of caveolae integrity can result in impaired NO production, calcium signaling, and associated pathophysiologic responses including angiogenesis [10-13].…”

“…A number of studies have reported that EC caveolae localizes multiple receptors and signaling modules including PI3K, PLC-γ, and PKC-α [8-10]. An agonist-stimulated Ca 2+ wave in EC has been shown to originate in the caveole-rich region and to propagate throughout the cell [14].…”

“…For example, P1-stimulation of EC and/or PA resulted in : i) activation of eNOS independent of increased expression or phosphorylation of eNOS leading to NO-dependent vasorelaxation of contracted PA [23] and ii) enhanced compartmentalization and activation of eNOS in lung EC [24]. More recently we reported that P1-stimulation modulates caveolar PKC-α signaling that enhances eNOS activation, NO production, intracellular Ca 2+ release, and phosphorylation of PKC-α and regulates internalization of P1 in lung EC [10]. P1-mediated activation of eNOS is associated with increased phosphorylation of Cav-1 and Cav-1/eNOS dissociation in caveolae-enriched fractions of EC and in an intact EC [24].…”