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

• Main Author: Daskoulidou, Nikoleta
• Other Authors: Xu, Shang-Zhong
• Published: University of Hull 2013
• Subjects: Medicine
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.769082

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.