Overexpressed p32 localized in the endoplasmic reticulum and mitochondria negatively regulates calcium‑dependent endothelial nitric oxide synthase activit

Choi,Kwanhoon; Koo,Bon‑Hyeock; Yoon,Byeong   Jun; Jung,Minkyo; Yun,Hye  Young; Jeon,Byung   Hwa; Won,Moo‑Ho; Kim,Young‑Myeong; Mun,Ji   Young; Lim,Hyun   Kyo; Ryoo,Sungwoo

doi:10.3892/mmr.2020.11307

Overexpressed p32 localized in the endoplasmic reticulum and mitochondria negatively regulates calcium‑dependent endothelial nitric oxide synthase activit

Authors:
- Kwanhoon Choi
- Bon‑Hyeock Koo
- Byeong Jun Yoon
- Minkyo Jung
- Hye Young Yun
- Byung Hwa Jeon
- Moo‑Ho Won
- Young‑Myeong Kim
- Ji Young Mun
- Hyun Kyo Lim
- Sungwoo Ryoo
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Affiliations: Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine, Wonju, Gangwon 26426, Republic of Korea, Department of Biological Sciences, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea, Department of Neural Circuits Research, Korea Brain Research Institute, Dong, Daegu 41068, Republic of Korea, Department of Physiology, School of Medicine, Chungnam National University, Daejeon 34134, Republic of Korea, Department of Neurobiology, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea, Department of Molecular and Cellular Biochemistry, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea
Published online on: July 6, 2020 https://doi.org/10.3892/mmr.2020.11307
Pages: 2395-2403
Copyright: © Choi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The p32 protein plays a crucial role in the regulation of cytosolic Ca2+ concentrations ([Ca2+]c) that contributes to the Ca2+‑dependent signaling cascade. Using an adenovirus and plasmid p32‑overexpression system, the aim of the study was to evaluate the role of p32 in the regulation of [Ca2+] and its potential associated with Ca2+‑dependent endothelial nitric oxide synthase (eNOS) activation in endothelial cells. Using electron and confocal microscopic analysis, p32 overexpression was observed to be localized to mitochondria and the endoplasmic reticulum and played an important role in Ca2+ translocation, resulting in increased [Ca2+] in these organelles and reducing cytosolic [Ca2+] ([Ca2+]c). This decreased [Ca2+]c following p32 overexpression attenuated the Ca2+‑dependent signaling cascade of calcium/calmodulin dependent protein kinase II (CaMKII)/AKT/eNOS phosphorylation. Moreover, in aortic endothelia of wild‑type mice intravenously administered adenovirus encoding the p32 gene, increased p32 levels reduced NO production and accelerated reactive oxygen species (ROS) generation. In a vascular tension assay, p32 overexpression decreased acetylcholine (Ach)‑induced vasorelaxation and augmented phenylephrine (PE)‑dependent vasoconstriction. Notably, decreased levels of arginase II (ArgII) protein using siArgII were associated with downregulation of overexpressed p32 protein, which contributed to CaMKII‑dependent eNOS phosphorylation at Ser1177. These results indicated that increased protein levels of p32 caused endothelial dysfunction through attenuation of the Ca2+‑dependent signaling cascade and that ArgII protein participated in the stability of p32. Therefore, p32 may be a novel target for the treatment of vascular diseases associated with endothelial disorders.

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