Glutamate loading protects freshly isolated and perfused adult cardiomyocytes against intracellular ROS generation.
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.
King
Nicola
N
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK. n.king@bris.ac.uk
McGivan
John D
JD
Griffiths
Elinor J
EJ
Halestrap
Andrew P
AP
Suleiman
M-Saadeh
MS
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D005978:Glutathione / Q000032:analysis; D005979:Glutathione Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008689:Methacrylates; D008928:Mitochondria / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010477:Perfusion; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D008689:Methacrylates; D016877:Oxidants; D011500:Protein Synthesis Inhibitors; D017382:Reactive Oxygen Species; D013844:Thiazoles; D018698:Glutamic Acid; D000968:Antimycin A; C030517:myxothiazol; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D005978:Glutathione