Division of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Division of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Microvasc Res. 2021 Jan;133:104075. doi: 10.1016/j.mvr.2020.104075. Epub 2020 Sep 18.
Phosphorylation plays an essential role in the regulation of endothelial nitric oxide synthase (eNOS) activity. However, the phosphorylation of eNOS under hypoglycemia and whether hypoglycemia changes eNOS activity is unknown. This paper aims to clarify the regulation of eNOS phosphorylation and its activity change under hypoglycemia.
Bovine aortic endothelial cells (BAECs) and Sprague-Dawley rats were treated with hypoglycemia, and the phosphorylation of eNOS was subjected to western blot. Blood nitric oxide (NO) concentration was determined by NO kit and endothelial-dependent vasodilation was detected by multi-wire myograph.
In both BAECs and rats' thoracic aorta, hypoglycemia induced eNOS phosphorylation decrease specifically on Threonine (Thr) 497. Inhibition of ubiquitination of protein kinase C α subunit (PKCα) reverses the decrease of eNOS phosphorylation in hypoglycemia. Ubiquitinated PKCα can be reversed by AMPK knockdown. In rats, insulin induced hypoglycemia increased the concentration of NO in arterial blood, and progressively enhanced the endothelium-dependent vasodilation of the thoracic and mesenteric aorta.
In vitro, the activation of AMPK may lead to the expression of PKCα by regulating ubiquitination, resulting in a decrease in the level of P-eNOS Thr497 phosphorylation under hypoglycemia. In vivo, insulin-induced hypoglycemia produces a beneficial cardiovascular effect on rats.
磷酸化在调节内皮型一氧化氮合酶（eNOS）活性中起着至关重要的作用。然而，在低血糖状态下 eNOS 的磷酸化情况以及低血糖是否会改变 eNOS 活性尚不清楚。本文旨在阐明低血糖状态下 eNOS 磷酸化的调节及其活性变化。
采用牛主动脉内皮细胞（BAEC）和 Sprague-Dawley 大鼠进行低血糖处理，通过 Western blot 检测 eNOS 的磷酸化。通过一氧化氮试剂盒测定血液中一氧化氮（NO）浓度，通过多导丝肌描记器检测内皮依赖性血管舒张。
在 BAEC 和大鼠胸主动脉中，低血糖均可特异性诱导 eNOS 磷酸化在苏氨酸（Thr）497 上的减少。蛋白激酶 Cα 亚基（PKCα）的泛素化抑制可逆转低血糖诱导的 eNOS 磷酸化减少。AMPK 敲低可逆转泛素化的 PKCα。在大鼠中，胰岛素诱导的低血糖可增加动脉血中 NO 的浓度，并逐渐增强胸主动脉和肠系膜动脉的内皮依赖性血管舒张。
在体外，AMPK 的激活可能通过调节泛素化导致 PKCα 的表达，从而导致低血糖下 P-eNOS Thr497 磷酸化水平降低。在体内，胰岛素诱导的低血糖对大鼠产生有益的心血管作用。
