King Nicola, McGivan John D, Griffiths Elinor J, Halestrap Andrew P, Suleiman M-Saadeh
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK.
J Mol Cell Cardiol. 2003 Aug;35(8):975-84. doi: 10.1016/s0022-2828(03)00182-2.
Glutamate loading has been shown to protect single isolated perfused cardiomyocytes against metabolic inhibition and wash-off. The mechanism underpinning this protection is unknown. This study aimed to investigate whether reactive oxygen species (ROS) are generated by single isolated perfused cardiomyocytes and whether the protective effect of glutamate loading on cell metabolism is linked to ROS. Single rat cardiomyocytes were isolated with or without glutamate to stimulate glutamate loading. ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate in various stressful conditions including metabolic inhibition and wash-off with/without antimycin A or myxothiazol; simulated ischaemia (without cyanide) and glucose reintroduction; and H(2)O(2) perfusion. Reduced glutathione (GSH) levels were measured in control and glutamate-loaded cells with/without exposure to H(2)O(2). Finally, the effect of glutamate on glutathione reductase and glutathione peroxidase activity was measured. In every stressful condition studied, ROS production was significantly lower in glutamate-loaded cells compared to controls. This occurred regardless of whether ROS were produced intracellularly (e.g. from the respiratory chain inhibited with antimycin A) or via the extracellular precursor H(2)O(2). Glutamate-loaded cells also maintained their morphological integrity at higher H(2)O(2) concentrations than control cells. Furthermore, during H(2)O(2) exposure GSH levels decreased in glutamate-loaded cells but stayed constant in control cells. Glutamate stimulated the activity of glutathione peroxidase in a concentration-dependent fashion. These results provide new evidence to show that the cardioprotective effect of glutamate loading may be mediated through an enhanced ability to destroy ROS in the cell.
已表明谷氨酸加载可保护单个分离的灌注心肌细胞免受代谢抑制和洗脱。这种保护作用的潜在机制尚不清楚。本研究旨在调查单个分离的灌注心肌细胞是否产生活性氧（ROS），以及谷氨酸加载对细胞代谢的保护作用是否与ROS相关。用或不用谷氨酸分离大鼠单个心肌细胞以刺激谷氨酸加载。在各种应激条件下，包括代谢抑制和用/不用抗霉素A或粘噻唑洗脱、模拟缺血（无氰化物）和重新引入葡萄糖以及灌注H₂O₂，使用5-（和-6）-氯甲基-2'，7'-二氯二氢荧光素二乙酸酯测量ROS产生。在有/无H₂O₂暴露的对照细胞和谷氨酸加载细胞中测量还原型谷胱甘肽（GSH）水平。最后，测量谷氨酸对谷胱甘肽还原酶和谷胱甘肽过氧化物酶活性的影响。在研究的每种应激条件下，与对照相比，谷氨酸加载细胞中的ROS产生显著降低。无论ROS是在细胞内产生（例如由抗霉素A抑制呼吸链产生）还是通过细胞外前体H₂O₂产生，均会出现这种情况。与对照细胞相比，谷氨酸加载细胞在更高的H₂O₂浓度下也能保持其形态完整性。此外，在H₂O₂暴露期间，谷氨酸加载细胞中的GSH水平降低，但对照细胞中的GSH水平保持恒定。谷氨酸以浓度依赖性方式刺激谷胱甘肽过氧化物酶的活性。这些结果提供了新的证据，表明谷氨酸加载的心脏保护作用可能是通过增强细胞内破坏ROS的能力来介导的。
