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Acetylcholine protects endothelial cells from hypoxia/reoxygenation injury
A study in the American Heart Association Journal of Arteriosclerosis, Thrombosis, and Vascular Biology has found acetylcholine (ACh) treatment effective in protecting human umbilical vein endothelial cells (HUVECs) from hypoxia/reoxygenation (H/R) injury by reducing interactions between mitochondria and the endoplasmic reticulum (ER). The goal of this study was to investigate the role of ER-mitochondria calcium cross talk in epithelial cell recovery from H/R damage, a common result of chronic heart failure and other conditions involving a dysfunctional circulatory system. The study also seeks to define the protective effects of ACh, which has been documented to have antioxidative and anti-inflammatory effects in cardiomyocytes, but has yet to have records on how it affects calcium overload (a main factor in H/R damage) in endothelial cells.
To begin the study, the researchers decided to arrange a time course displaying how calcium levels in oxygen-deprived endothelial cells typically responded to reoxygenation, without any presence of acetylcholine. They used the calcium indicator Cal-520 to track calcium flux for up to 24 hours after reoxygenation of their HUVECs, which had been subjected to 8 hours of hypoxia. Through this initial day-long interval of reperfusion, the researchers determined the peak difference in calcium level to be at 2 hours after the start of reoxygenation. This 2 hour time point was noted and used in subsequent calcium monitoring experiments under this study as the point of highest calcium level and potential overload. The researchers would then proceed to compare this information to calcium level data acquired with HUVECs under the treatment of acetylcholine, and continue exploring calcium dynamics in relation to mitochondria-ER interplay.
To acquire their (2 hour) baseline indicating when calcium levels were highest upon reoxygenation, the study had to utilize a calcium dye that would remain emitting a detectable fluorescence even 24 hours after application. With that requirement known, the selected Cal-520 is one of the best calcium dyes for such situations. Unlike other more popular dyes like Fluo-4, which generates a signal that fades away relatively quickly, Cal-520 is enhanced for significantly improved intracellular retention qualities that make it especially suitable for long-duration calcium detection and imaging.
To begin the study, the researchers decided to arrange a time course displaying how calcium levels in oxygen-deprived endothelial cells typically responded to reoxygenation, without any presence of acetylcholine. They used the calcium indicator Cal-520 to track calcium flux for up to 24 hours after reoxygenation of their HUVECs, which had been subjected to 8 hours of hypoxia. Through this initial day-long interval of reperfusion, the researchers determined the peak difference in calcium level to be at 2 hours after the start of reoxygenation. This 2 hour time point was noted and used in subsequent calcium monitoring experiments under this study as the point of highest calcium level and potential overload. The researchers would then proceed to compare this information to calcium level data acquired with HUVECs under the treatment of acetylcholine, and continue exploring calcium dynamics in relation to mitochondria-ER interplay.
To acquire their (2 hour) baseline indicating when calcium levels were highest upon reoxygenation, the study had to utilize a calcium dye that would remain emitting a detectable fluorescence even 24 hours after application. With that requirement known, the selected Cal-520 is one of the best calcium dyes for such situations. Unlike other more popular dyes like Fluo-4, which generates a signal that fades away relatively quickly, Cal-520 is enhanced for significantly improved intracellular retention qualities that make it especially suitable for long-duration calcium detection and imaging.
References
- He, Xi, Xue-yuan Bi, Xing-zhu Lu, Ming Zhao, Xiao-jiang Yu, Lei Sun, Man Xu, W. Gil Wier, and Wei-jin Zang. "Reduction of Mitochondria-Endoplasmic Reticulum Interactions by Acetylcholine Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury." Arteriosclerosis, thrombosis, and vascular biology (2015): ATVBAHA-115.
- Cal-520®, AM. AAT Bioquest, n.d. Web. 1 July 2016
Original created on November 27, 2019, last updated on November 27, 2019
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