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

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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.

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Acknowledgments

The authors thank Mr. Humberto Herrera and Mrs. Lin Ai for their excellent technical assistance. This study was supported in part by the National Heart, lung, and Blood Institute grant HL085133 and the Department of Veterans Affairs Merit Review (JMP).

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Authors and Affiliations

  1. Department of Medicine, University of Florida College of Medicine, Gainesville, FL, 32608-1197, USA

    Tarun E. Hutchinson, Jianliang Zhang, Shen-Ling Xia, Sudeep Kuchibhotla, Edward R. Block & Jawaharlal M. Patel

  2. Research Service (151), Malcom Randall Department of Veterans Affairs Medical Center, 1601 S.W. Archer Road, Gainesville, FL, 32608-1197, USA

    Shen-Ling Xia, Edward R. Block & Jawaharlal M. Patel

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  1. Tarun E. Hutchinson
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  2. Jianliang Zhang
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Correspondence to Jawaharlal M. Patel.

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Hutchinson, T.E., Zhang, J., Xia, SL. et al. Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells. Mol Cell Biochem 360, 309–320 (2012). https://doi.org/10.1007/s11010-011-1070-4

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