title: 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 (Ca(2+)) 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 Ca(2+) release in internalization of P1, PKC-α modulation by phorbol ester (PMA), PKC-α knockdown, and Ca(2+) 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 (Ca(2+)) release since the Ca(2+) 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.
authors.affiliation: Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32608-1197, USA.
authors.forename: Tarun E
authors.initials: TE
authors.lastname: Hutchinson
authors.forename: Jianliang
authors.initials: J
authors.lastname: Zhang
authors.forename: Shen-Ling
authors.initials: SL
authors.lastname: Xia
authors.forename: Sudeep
authors.initials: S
authors.lastname: Kuchibhotla
authors.forename: Edward R
authors.initials: ER
authors.lastname: Block
authors.forename: Jawaharlal M
authors.initials: JM
authors.lastname: Patel
mesh_terms: D000818:Animals; D020013:Calcium Signaling; D021941:Caveolae / Q000187:drug effects / Q000201:enzymology*; D051242:Caveolin 1 / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D005372:Filipin / Q000494:pharmacology; D008168:Lung / Q000166:cytology*; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D051242:Caveolin 1; D057846:Cell-Penetrating Peptides; D020536:Enzyme Activators; D010455:Peptides; C000610352:Ser-Ser-Trp-Arg-Arg-Lys-Arg-Lys-Glu-Ser-Ser; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005372:Filipin; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate