Cytosolic and mitochondrial ROS in staurosporine-induced retinal cell apoptosis

• Published in: Free radical biology & medicine 2003-12, Vol.35 (11), p.1500-1514
• Main Authors: Gil, Joana, Almeida, Sandra, Oliveira, Catarina R., Rego, A.Cristina
• Format: Article
• Language: English
• Subjects: Adenine - chemistry; Allopurinol - pharmacology; Animals; Antioxidants; Antioxidants - pharmacology; Apoptosis; Arachidonic Acids - chemistry; Benzoquinones - pharmacology; Blotting, Western; Calcium; Calcium - chemistry; Carbon - chemistry; Caspase 3; Caspases - metabolism; Cell Death; Cell Survival; Chick Embryo; Chromans - pharmacology; Coloring Agents - pharmacology; Cytosol - metabolism; DNA Fragmentation; Enzyme Inhibitors - pharmacology; Free radicals; Glutathione - pharmacology; In Situ Nick-End Labeling; Indazoles - pharmacology; Metalloporphyrins - pharmacology; Mitochondria; Mitochondria - metabolism; Neurons - metabolism; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase - metabolism; Oligomycins - chemistry; Protein Isoforms; Reactive Oxygen Species; Retina - cytology; Retina - pathology; Retinal cells; Rotenone - pharmacology; Staurosporine; Staurosporine - pharmacology; Tetrazolium Salts - pharmacology; Thiazoles - pharmacology; Time Factors; Ubiquinone - analogs & derivatives; Uncoupling Agents - pharmacology; Xanthine Oxidase - antagonists & inhibitors
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2003.08.022

In this study, we investigated the involvement of reactive oxygen species (ROS) and calcium in staurosporine (STS)-induced apoptosis in cultured retinal neurons, under conditions of maintained membrane integrity. The antioxidants idebenone (IDB), glutathione-ethylester (GSH/EE), trolox, and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly reduced STS-induced caspase-3-like activity and intracellular ROS generation. Endogenous sources of ROS production were investigated by testing the effect of the following inhibitors: 7-nitroindazole (7-NI), a specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS); arachidonyl trifluoromethyl ketone (AACOCF 3), a phospholipase A 2 (PLA 2) inhibitor; allopurinol, a xanthine oxidase inhibitor; and the mitochondrial inhibitors rotenone and oligomycin. All these compounds decreased caspase-3-like activity and ROS generation, showing that both mitochondrial and cytosolic sources of ROS are implicated in this mechanism. STS induced a significant increase in intracellular calcium concentration ([Ca 2+] i), which was partially prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation. These two antioxidants and the inhibitors allopurinol and 7-NI also reduced the number of TdT-mediated dUTP nick-end labeling-positive cells. Thus, endogenous ROS generation and the rise in intracellular calcium are important inter-players in STS-triggered apoptosis. Furthermore, the antioxidants may help to prolong retinal cell survival upon apoptotic cell death.