The Role of Protein S-glutathionylation on Ca2+ Signaling in Cultured Aortic Endothelial Cells

In non-excitable cells, oxidative stress impacts both basal and agonist-mediated changes in cytosolic free Ca2+ concentration ([Ca2+]i). An increase in protein S-glutathionylation is a common response to oxidative stress; however, the role of glutathionylation in Ca2+ homeostasis and Ca2+ signaling processes is not well understood. Diamide, a membrane-permeable oxidizing reagent, rapidly and reversibly converts glutathione (GSH) to its disulfide (GSSG), and promotes glutathionylation. In the present Dissertation, the acute effect of diamide on the [Ca2+]i of cultured aortic endothelial cells (ECs) was examined at the single-cell level using the fluorescent Ca2+ indicator, fura-2 and time-lapse video microscopy. Low concentrations (50, 100 ¿¿M) of diamide reversibly increased spontaneous Ca2+ oscillations, whereas high concentrations (250, 500 ¿¿M) of diamide caused an immediate, synchronized Ca2+ oscillation in essentially all cells examined followed by a time-dependent rise in basal [Ca2+]i. Most strikingly, diamide evoked a dose-dependent increase in single-cell Ca2+ oscillations in both the presence, and importantly the absence of extracellular Ca2+. Oscillations in [Ca2+]i were due to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) activation, since they were attenuated by pharmacological inhibition of either phospholipase C or IP3Rs themselves. However, diamide, even at high concentrations, did not increase PIP2 hydrolysis, suggesting diamide increases the sensitivity of IP3Rs to activation. In agreement with this, diamide enhanced both IP3-induced Ca2+-release (IICR) and Ca2+-induced Ca2+-release (CICR) via IP3Rs. Since IP3 is not changing during challenge with diamide, our results indicate diamide primarily increases the sensitivity of IP3Rs to cytosolic Ca2+, i.e. enhanced CICR. Consistent with GSH-dependent thiol-modification, this oscillatory response was dependent upon both the cellular GSH concentration and the ability to recycle GSSG back to GSH. Moreover, exogenous exposure of cells to hydrogen peroxide (H2O2) produced similar changes in IP3R activity and in biochemical assays we found that the IP3R could be reversibly glutathionylated in response to either diamide or H2O2. When taken together, our data demonstrate oxidative stress increases CICR via the IP3R, and reveals an important role for glutathionylation in the coordination of cellular Ca2+ dynamics during both physiological redox signaling processes and during the pathological response to oxidative stress.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Physiology and Biophysics

Grantor dc:publisher

Case Western Reserve University School of Graduate Studies

Year dc:date

2013

Author dc:creator

• Lock, Jeffrey T.

Contributors dc:contributor

• Schilliing, William
• Smith, Corey

• Physiology
• Calcium signaling
• Oxidative Stress
• Glutathionylation
• Diamide
• IP3 receptor
• Calcium oscillations

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=case1354470367

OAI identifier oai:identifier

oai:etd.ohiolink.edu:case1354470367