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Abstract

Data obtained in studies of the nature of the correlation which we have previously observed [10,17] between mitochondrial depolarization and the level of disruption of Ca2+ homeostasis in cultivated brain neuronsare summarized. Experiments were performed on cultured cerebellar granule cells loaded with Fura-2-AM or rhodamine 123 to measure changes in cytoplasmic Ca2+ and mitochondrial potential during pathogenic treatments of the cells. Prolonged exposure to 100 microM glutamate induced a reversible increase in [Ca2+]i, which was accompanied by only a small degree of mitochondrial depolarization. A sharp increase in this mitochondrial depolarization, induced by addition of 3 mM NaCN or 300 microM dinitrophenol (DNP) to the glutamate-containing solution, resulted in further increase in [Ca2+]i, due to blockade of electrophoretic mitochondrial Ca2+ uptake. Prolonged exposure to CN- or DNP in the post-glutamate period maintained [Ca2+]i at a high level until the metabolic inhibitors were removed. In most cells, this plateau was characterized by low sensitivity to removal of external Ca2+, demonstrating that the mechanisms of Ca2+ release from neurons were disrupted. Addition of oligomycin, a blocker of mitochondrial ATP synthase/ATPase, to the solution containing glutamate and CN- or DNP eliminated the post-glutamate plateau. Parallel experiments with direct measurements of intracellular ATP levels ([ATP]) showed that profound mitochondrial depolarization induced by CN- or DNP sharply enhanced the drop in ATP due to glutamate, while oligomycin significantly weakened this effect of the metabolic inhibitors. Analysis of these data led to the conclusion that blockade of mitochondrial Ca2+ uptake and inhibition of ATP synthesis resulted from mitochondrial depolarization and plays a key role in the mechanism disrupting [Ca2+]i homeostasis after toxic exposure to glutamate.

Changes in ATP content in cerebellar granule cells during hyperstimulation of glutamate receptors: Possible role of NO and nitrite ions

“…The procedure of isolation of cell suspension for neuron culturing was described elsewhere [3,5,6]. The cells in culture were washed with a control solution containing (in mM): 130 NaCl, 5.6 KCl, 1.8 CaCl 2 , 1.0 MgCl 2 , 20 HEPES, 5.0 glucose, pH 7.4.…”

“…Address for corre spondence: sorokina@nczd.ru. E. G. SorokinaThe decrease in ATP content in neurons during brain hypoxia and hyperstimulation of glutamate receptors can disturb intracellular and intercellular signaling in cerebral neurons, in particular, ionic homeostasis, activity of glycolytic and oxidative phosphorylation enzymes, mitochondrial Ca 2+ uptake, and protein synthesis [6][7][8]10,11,13]. Previous studies showed that ATP content in cultured cerebellar granule cells (CGC) decreased immediately after application of glutamate (Glu) [1,2,8,3].…”

Proteinase-Activated Type 1 Receptors are Involved in the Mechanism of Protection of Rat Hippocampal Neurons from Glutamate Toxicity

“…Cell suspension for culturing was obtained as described elsewhere [3]. This suspension was put on cover glasses (1 hippocampus, 200 ml per 1 glass) coated with poly-D-lysine (10 mg/ml).…”

“…Selective peptide agonist of proteinase-activated type 1 receptors simulated, but receptor antagonist suppressed the neuroprotective effect of thrombin. Our results suggest that peptide antagonist of type 1 receptors play a role in the mechanisms of neuronal protection from glutamate toxicity.Hyperstimulation of glutamate receptors underlies death of brain neurons during ischemic and hemorrhagic strokes, brain traumas, and some neurodegenerative diseases [3,4,7]. These disorders are associated with increased permeability of the blood-brain barrier, which results in the appearance of serine thrombin proteinase in the nervous tissue.…”

“…Hyperstimulation of glutamate receptors underlies death of brain neurons during ischemic and hemorrhagic strokes, brain traumas, and some neurodegenerative diseases [3,4,7]. These disorders are associated with increased permeability of the blood-brain barrier, which results in the appearance of serine thrombin proteinase in the nervous tissue.…”