arachidonyltrifluoromethane and 7-nitroindazole

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.

Topics: Adenine; Allopurinol; Animals; Antioxidants; Apoptosis; Arachidonic Acids; Benzoquinones; Blotting, Western; Calcium; Carbon; Caspase 3; Caspases; Cell Death; Cell Survival; Chick Embryo; Chromans; Coloring Agents; Cytosol; DNA Fragmentation; Enzyme Inhibitors; Glutathione; In Situ Nick-End Labeling; Indazoles; Metalloporphyrins; Mitochondria; Neurons; Nitric Oxide Synthase; Oligomycins; Protein Isoforms; Reactive Oxygen Species; Retina; Rotenone; Staurosporine; Tetrazolium Salts; Thiazoles; Time Factors; Ubiquinone; Uncoupling Agents; Xanthine Oxidase

Glutamate toxicity is a viable hypothesis to explain the expanding tissue degeneration occurring after traumatic or ischemic spinal cord injury. One important component in this process is the acute, excessive release of glutamate. In the current communication, the glycolytic inhibitor iodoacetate was used to induce metabolic inhibition in spinal cord slices and thereby provide an in vitro model to study the mechanisms of pathological glutamate release in the spinal cord. The evoked glutamate release was not Ca2+-dependent. Exclusion of NaCl reduced the evoked release of endogenous glutamate by 56%, while excluding Na+ increased release. Glutamate release was also reduced by the PLA2 inhibitors indomethacin (40%), arachidonyltrifluoromethyl ketone (45%) and 4-bromophenacyl bromide (36%). Blocking reverse glutamate transport by preincubation with 1 mM dihydrokainic acid reduced evoked release by 41%. However, when the dihydrokainic acid and arachidonyltrifluoromethyl ketone treatments were combined, no additive effect of the two substances was seen. These findings suggest that glutamate is released by three mechanisms from the energy compromised spinal cord: (1) in response to cellular swelling, most likely by the regulatory volume decrease, (2) by PLA2-mediated breakdown of the cell membrane and diffusion of glutamate down its concentration gradient, and (3) through reversal of the glutamate transporter.

Topics: Adenosine; Animals; Arachidonic Acids; Baclofen; Calcium; Energy Metabolism; Enzyme Inhibitors; GABA Agonists; Glutamic Acid; In Vitro Techniques; Indazoles; Iodoacetates; Male; Neurotoxins; Phospholipases; Rats; Rats, Sprague-Dawley; Spinal Cord