Regulation of store-operated channel molecules ORAI and STIM by oxidative stress in blood vessels

Daskoulidou, Nikoleta

Medicine
March 2013

Thesis or dissertation


Rights
© 2013 Nikoleta Daskoulidou. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Abstract

ORAI and STIM genes are recently identified store-operated calcium channel molecules that play important roles in human physiology. In this thesis, the effects of oxidative stress conditions including high glucose, homocysteine and H₂O₂ on the expression of ORAI and STIM, Ca²⁺ influx, ORAI channel activity and potential underlying mechanisms were investigated using cell models and in vivo tissue samples from diabetic patients and mice.

ORAI1-3 and STIM1-2 were detected in vascular endothelial cells and smooth muscle cells using RT-PCR, western blotting and immunostaining. Their expression was upregulated by chronic treatment with high glucose in cell models. The upregulation was also observed in human aorta from Type 2 diabetic patients and kidney tissues from streptozotocin-induced and Akita Type 1 diabetic mouse models. The high glucose-induced gene upregulation was prevented by the calcineurin inhibitor cyclosporin A and store-operated channel blocker diethylstilbestrol. H₂O₂ also upregulated ORAI1-3 and STIM1-2, however, homocysteine increased STIM1-2 expression, but downregulated ORAI1-3.

Ca²⁺ influx and ORAI channel activity were investigated using Ca²⁺ imaging and whole-cell patch clamp. Chronic treatment with high glucose enhanced storeoperated Ca²⁺ influx in endothelial cells, but there was no effect if treated acutely. In HEK-293 cells overexpressing STIM1/ORAI1-3, high glucose had no acute effect on ORAI1-3 currents, but homocysteine decreased the currents. The cytosolic STIM1 movement was monitored by live-cell fluorescence imaging. Oxidative stress did not change STIM1-EYFP translocation and clustering after Ca²⁺ store-depletion. The effect of hyperosmolarity on STIM and ORAI expression and channel activity was also investigated. Hyperosmolarity inhibited ORAI1-3 currents and downregulated ORAI1-3 and STIM1-2 gene expression, but did not alter cytosolic STIM1-EYFP translocation.

It is concluded that store-operated channel molecules, STIMs and ORAIs, are new proteins regulated by oxidative stress, especially in diabetes, which may provide a novel concept for the abnormality of Ca²⁺ homeostasis in blood vessels from patients with diabetes.

Publisher
Hull York Medical School, The University of Hull and The University of York
Supervisor
Xu, Shang-Zhong
Sponsor (Organisation)
University of Hull
Qualification level
Doctoral
Qualification name
PhD
Language
English
Extent
4 MB
Identifier
hull:17230
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