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Ouabain Regulates Caveolin-1 Vesicle Trafficking by a Src-Dependent Mechanism

Harris, Tanoya L.
http://rave.ohiolink.edu/etdc/view?acc_num=mco1333732028

Abstract Details

2012, Doctor of Philosophy (PhD), University of Toledo, College of Medicine.
Na/K-ATPase interacts with Src to form a functional receptor for endogenous cardiotonic steroids such as ouabain. Binding of ouabain to this receptor stimulates Src. We have previously shown that knockdown of Na/K-ATPase activates Src and increases scission of Cav-1 vesicles. We hypothesize that ouabain may have a hormone effect and regulate Cav-1 vesicle trafficking through Src activation. Using live cell time-lapse microscopy we observed that ouabain increased scission of Cav-1 vesicles from the plasma membrane in LLC-PK1 cells in a dose-dependent manner indicating that ouabain stimulates the endocytosis of caveolae. For confirmation, we incubated cells with cholera toxin B, a protein complex that enters the cell via caveolae endocytosis. We observed an increase in cholera toxin B internalization by ouabain. Next, we treated cells with Src inhibitor, PP2 and a specific antagonist of ouabain, pNaKtide, and both inhibitors blocked ouabain-induced scission of Cav-1 vesicles. In addition, we mutated Cav-1 Y14 to Y14F to prevent Src-mediated tyrosine phosphorylation of Cav-1. We saw a decrease in scission of Cav-1 vesicles in response to ouabain stimulation. Dynamin is involved in the scission of caveolae. Since dynamin is a known effector of Src, we tested the role of Dyn2. These studies revealed that ouabain activated Dyn2 in LLC-PK1 cells. Moreover, inhibition of Dyn2 completely obliterated ouabain-induced scission of Cav-1 vesicles. Cholesterol is very important for the fluidity of mammalian cells and also regulates cellular distribution of Cav-1. We depleted plasma membrane cholesterol with Mβ-CD and compound U18666A. Both treatments showed an increase in Cav-1 vesicle scission. To confirm our findings we incubated cells with cholera toxin B plus Mβ-CD and observed an increase in internalization. These findings provide evidence that ouabain can induce scission of Cav-1 vesicle trafficking through Na/K-ATPase by a Src-dependent mechanism.
Zi-Jian Xie, PhD (Committee Chair)
Amir A. Askari, PhD (Committee Member)
David Giovannucci, PhD (Committee Member)
Lijun Liu, PhD (Committee Member)
Sandrine V. Pierre, PhD (Committee Member)
100 p.
Molecular Biology; Pharmacology; Physiology
ouabain; caveolae; caveolin-1; hormone; Src; trafficking

Recommended Citations

Citations

  • Harris, T. L. (2012). Ouabain Regulates Caveolin-1 Vesicle Trafficking by a Src-Dependent Mechanism [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1333732028

    APA Style (7th edition)

  • Harris, Tanoya. Ouabain Regulates Caveolin-1 Vesicle Trafficking by a Src-Dependent Mechanism. 2012. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1333732028.

    MLA Style (8th edition)

  • Harris, Tanoya. "Ouabain Regulates Caveolin-1 Vesicle Trafficking by a Src-Dependent Mechanism." Doctoral dissertation, University of Toledo, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=mco1333732028

    Chicago Manual of Style (17th edition)

mco1333732028
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© 2012, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.
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