Date of Award

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

John Lemasters

Second Advisor

Yuri Peterson

Third Advisor

Kathryn Appleton

Fourth Advisor

Rick G. Schnellmann

Fifth Advisor

John Woodward

Sixth Advisor

Robin C. Muise-Helmericks

Abstract

3-isobutyl 1-methylxanthine (IBMX) is a commonly used inhibitor of cyclic nucleotide phosphodiesterases (PDE) and G protein coupled adenosine receptors. Administration of IBMX to retina photoreceptor-derived 661w cell line results in decreased mitochondrial respiration followed by cell death, mimicking phenomena observed in inherited retina neurodegeneration in humans and animals and thus suggesting their use as a cell-based model of retina degeneration. In this dissertation, the temporal relationship between pathways altered by IBMX and how these pathways affect mitochondrial physiology are evaluated. These studies utilize a novel high content microscopic method for simultaneously measuring mitochondrial morphology and membrane potential in living cultured cells to identify an increase in networked morphology (pro-fusion) and mitochondrial membrane potential within 1-4 h of IBMX administration that is sustained and accompanied by decreased uncoupled respiration at 24 h. cGMP PDE inhibition was observed to recapitulate both the immediate and sustained physiological alterations as selective inhibitors of PDE3 and PDE5 increased mitochondrial membrane potential and interconnected morphology at 1 h which was followed by a decrease in mitochondrial respiration at 24 h similar to that elicited by IBMX. Administration of selective inverse agonist of adenosine A2B receptor recapitulated the latent (24 h), but not early (1 h) IBMX-induced alterations in mitochondrial physiology. Administration of a selective agonist and antagonist prior to IBMX abrogated latent but not early IBMX-induced alterations. A similar pattern of reductions in latent but not early alterations were achieved by activating adenylate cyclase or protein kinase C. Inhibition of protein kinase A resulted in increased mitochondrial membrane potential at 1 h and 24 h. The concomitant increase of mitochondrial membrane potential with decreased respiration suggested decreased permeability of the mitochondrial inner membrane to inward proton flux, perhaps due to lack of ADP availability or decreased mitochondrial Complex V activity. Inhibiting mitochondrial permeability transition pore (mPTP) regulator GSK-3β prior to IBMX administration prevented increases in both membrane potential and networked morphology, suggesting GSK-3β activation follows IBMX. Taken together, this work demonstrates that IBMX first increases cGMP via inhibition of PDEs at 1 h to modulate a rise in mitochondrial membrane potential and networked morphology, followed by inverse agonism at the adenosine A2B receptor at 24 h. Downstream pathways invoked at 24 h include decreased adenylate cyclase and PKA signaling, as well as PKC. GSK-3β activation following loss in PKA and PKC activity may integrate these PDE- and adenosine A2B receptor-dependent signals.

Rights

All rights reserved. Copyright is held by the author.

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