Publication Date
2018
Document Type
Thesis
Committee Members
Peter Lauf, M.D. (Advisor); Norma Adragna, Ph.D. (Committee Member); Ulas Sunar, Ph.D. (Committee Member)
Degree Name
Master of Science (MS)
Abstract
The primary function of Na⁺/K⁺ATPase (NKA) discovered by Jens Christian Skou in 1958, through electrogenic exchange of 3 Na⁺ for 2 K⁺ ions (Robert L Post, 1958), is the homeostatic preservation of their electrochemical gradient across the plasma membrane (PM) of almost all species and their derived cell lines. Cardiotonic steroids (CTS), such as cardenolides or bufadienolides, are well established inhibitors of NKA. Fox glove plant extracts containing CTS, including digitalis, were first described by William Withering (1744-91) for their positive inotropic effect in heart failure patients. Later research identified the mechanism of their action through direct inhibition of NKA with simultaneous cytosolic Na⁺ accumulation and due to reversal of the Na⁺/Ca²⁺ exchanger located in series with NKA in the plasma membrane. Subsequent increases in cytoplasmic Ca²⁺ levels lead to higher contractility of the heart, the inotropic effect of CTS. The Strophantus gratus-derived CTS ouabain is experimentally widely used. During the past two decades, ouabain binding to NKA, resulting in the here so coined NKA-ouabain receptor complex (NORC), has been shown to elicit at least two signaling pathways involving protein kinases with sequential phosphorylation/ transphosphorylation reactions leading to transcriptional upregulation of proteins iv involved in cell proliferation, survival, and death. More recent studies suggest NORC internalization through the plasmalemmal membrane and its cytosolic appearance. Mechanisms of NORC internalization and ensuing putative actions of NKA or ouabain or both have yet to be identified. In the present work, it was hypothesized that during its membrane to cytosol descent, NORC utilizes a variety of cytosolic and cytoskeletal proteins through protein/protein interactions as an escalator to reach endosomes and lysosomes for subsequent breakup into its NKA receptor and ligand ouabain. NORC internalization would then serve as an alternate model to explain novel transcriptional gene upregulation pathways which are different from early protein/protein interactionbased kinase upregulation. In the present work, human lens epithelial (B-3) cells were used as an experimental model, where the presence of only NKA a1 isoform was established by RTPCR, and the membrane transport properties have been well defined (Lauf et al., 2006). Proteins potentially interacting with NKA, such as the oncogenic protein BclXL (B-cell lymphoma derived), α-tubulin, caveolin-1, EEA1 (endosomeassociated antigen), and LAMP1 (lysosomal-associated membrane protein) in the presence and absence of ouabain, were analyzed by SDS-electrophoresis and Western Blot. Colocalization and cytosolic internalization of NORC by Bodipy-fluorescent ouabain (BFO) and anti-α1 specific Cy3-red labeled antibodies (AB) is presented by immunocytochemical evidence. B-3 cells incubated with unlabeled ouabain at > nanomolar concentrations showed increases in cell size and redistribution of Cy3-labeled anti-α1 AB, signifying ouabain-induced cell swelling presumably due to Na⁺ and water entry. Anti-α1 AB Cy3-red labeled NORC colocalization with Alexa-Fluor green marked AB against, α-tubulin increased at higher ouabain concentrations, while decreasing with v acetylation resulting from exposure to the histone deacetylase inhibitor Tricostatin (TSA). As expected, NKA also colocalized with caveolin-1 and with EEA1 and LAMP1. These findings suggest that NORC-colocalizing proteins provide a rough outline of its cytosolic fate and form the basis for a preliminary model delineating its cytosolic fate. This information may contribute to our understanding of clinically observed attenuating effects of CTS in cancer biology and chronic kidney disease (CKD).
Page Count
58
Department or Program
Department of Pharmacology and Toxicology
Year Degree Awarded
2018
Copyright
Copyright 2018, all rights reserved. My ETD will be available under the "Fair Use" terms of copyright law.
