Cytosolic and mitochondrial ROS in staurosporine-induced retinal cell apoptosis.
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.
Gil
Joana
J
Institute of Biochemistry, Faculty of Medicine, University of Coimbra and Center for Neuroscience and Cell Biology of Coimbra, Coimbra, Portugal.
Almeida
Sandra
S
Oliveira
Catarina R
CR
Rego
A Cristina
AC
D000225:Adenine / Q000737:chemistry; D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D001095:Arachidonic Acids / Q000737:chemistry; D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002470:Cell Survival; D002642:Chick Embryo; D002839:Chromans / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007191:Indazoles / Q000494:pharmacology; D008665:Metalloporphyrins / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism; D009840:Oligomycins / Q000737:chemistry; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D012160:Retina / Q000166:cytology / Q000473:pathology*; D012402:Rotenone / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014451:Ubiquinone / Q000031:analogs & derivatives; D014475:Uncoupling Agents / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors
D000975:Antioxidants; D001095:Arachidonic Acids; D016227:Benzoquinones; D002839:Chromans; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D007191:Indazoles; D008665:Metalloporphyrins; D009840:Oligomycins; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D014475:Uncoupling Agents; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; C081565:arachidonyltrifluoromethane; D012402:Rotenone; D014451:Ubiquinone; D000493:Allopurinol; D002244:Carbon; D019001:Nitric Oxide Synthase; D014969:Xanthine Oxidase; D053148:Caspase 3; D020169:Caspases; C022616:thiazolyl blue; D005978:Glutathione; D019311:Staurosporine; C036619:idebenone; D000225:Adenine; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D002118:Calcium; C080122:7-nitroindazole