sq-23377 and diacetyldichlorofluorescein

The overexcitation of glutamate receptors is believed to be the cause of several neurodegenerative disorders. The determination of calcium fluxes, mitochondrial membrane potential (MMP) variations or the production of reactive oxygen species (ROS) in mammalian cells are usually measured during the development of potentially useful drugs that might interfere in the events induced by glutamate receptor activation. By using flow cytometry with dissociated cerebellar granule cells, we have developed a rapid and economical method to measure changes in biochemical parameters that are involved in neuronal cell death. The formation of intracellular ROS is measured using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The mitochondrial membrane potential is assessed by the retention of rhodamine 123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. Finally, intracellular calcium increases are detected by using the calcium-selective indicator Indo-1. Cell viability is also assessed by using propidium iodide (PI) which stains DNA strands of permeabilized cells. This method might be useful for the screening of new drugs with potential neuroprotective activity, with improved cost/effectiveness ratio compared to other techniques.

Topics: Animals; Antioxidants; Calcium; Chromans; Cytoplasm; Flow Cytometry; Fluoresceins; Free Radicals; Glutamic Acid; Ionomycin; Ionophores; Mammals; Membrane Potentials; Mitochondria; Neurons; Piperazines; Pregnatrienes; Rats; Rats, Sprague-Dawley; Staining and Labeling

The implication of reactive oxygen species for the Ca2+ ionophore ionomycin-induced apoptosis was investigated in cultured cortical neurons from embryonic rats. Ionomycin increased the production of intracellular peroxides as measured by flow cytometric analysis with 6-carboxy-2'7'-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester). Low doses of ionomycin increased the level of manganese-superoxide dismutase (Mn-SOD). In addition, N-acetyl-L-cysteine prevented apoptotic neuronal death induced by ionomycin in a dose-dependent manner. Buthionine sulfoximine suppressed the effect of N-acetyl-L-cysteine. These results suggest that peroxides and redox-regulation play an important role in the apoptosis of neurons induced by elevation of intracellular Ca2+ concentration.

Topics: Acetylcysteine; Animals; Apoptosis; Buthionine Sulfoximine; Calcium; Cell Nucleus; Cells, Cultured; Cerebral Cortex; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Indoles; Ionomycin; Ionophores; Kinetics; Neurons; Peroxides; Rats; Rats, Wistar; Superoxide Dismutase

Effect of N,N-diethyldithiocarbamate (DDC), an inhibitor for cytosolic superoxide dismutase, on an ionomycin-induced increase in oxidative metabolism was examined in cerebellar neurons dissociated from the rats, using a flow cytometer and 2',7'-dichlorofluorescin diacetate and fluo-3-AM, fluorescent dyes for intracellular hydrogen peroxide and Ca2+, respectively. DDC reduced the ionomycin-induced augmentation of 2',7'-dichlorofluorescin fluorescence in a dose-dependent manner. DDC did not affect cellular content of 2',7'-dichlorofluorescin and ionomycin-induced increase in intracellular Ca2+ concentration. Results indicate that ionomycin increases the formation of superoxide anion in brain neuron.

Topics: Aniline Compounds; Animals; Cerebellum; Ditiocarb; Fluoresceins; Fluorescent Dyes; Ionomycin; Neurons; Oxidation-Reduction; Rats; Rats, Wistar; Xanthenes