dizocilpine-maleate and leupeptin

Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5 mM CuSO4 for 24 h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Regulatory Proteins; Calpain; Caspases; Cell Line, Tumor; Chelating Agents; Chromatin; Copper Sulfate; Dizocilpine Maleate; Gene Expression Regulation; Glutathione; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Leupeptins; Mice; Neoplasm Proteins; Neurons; Nifedipine; Oxidation-Reduction; Oxidative Stress; Protease Inhibitors; Protein Kinase Inhibitors; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Teratocarcinoma

The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl(-) concentration ([Cl(-)](i)) that forms the basis for hyperpolarizing GABA(A) receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg(2+)-free solution led to a fast reduction in KCC2-mediated Cl(-) transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.

Topics: Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Calcium; Calcium Ionophores; Calpain; Cycloheximide; Cysteine Proteinase Inhibitors; Dipeptides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Emetine; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Ionomycin; K Cl- Cotransporters; Leupeptins; Magnesium; Male; Membrane Potentials; N-Methylaspartate; Patch-Clamp Techniques; Protein Synthesis Inhibitors; Pyramidal Cells; Rats; Rats, Wistar; Statistics, Nonparametric; Symporters; Valine

Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture.

Topics: Animals; Cells, Cultured; Cerebellum; Dizocilpine Maleate; Egtazic Acid; Excitatory Amino Acid Antagonists; Fluorometry; Glutamic Acid; Leupeptins; Neurons; Protease Inhibitors; Pterins; Rats; Rats, Wistar; Temperature; Xanthine Oxidase; Xanthopterin