Glutamate is shown to induce increases in intracellular Ca2+ concentrations ([Ca2+]i), increases in 45Ca2+ influx, decreases in the activity of Na+,K+-ATPase activity, and activation of the Na+/Ca2+ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with alpha-tocopherol, superoxide dismutase, and ganglioside GM1 normalized [Ca2+]i, 45Ca2+ influx, and Na+,K+-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM1 activated the Na+/K+ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca2+ influx into cells, decreases in Na+,K+-ATPase activity, and significant increases in [Ca2+]i, though this could be prevented by antioxidants and gangliosides.
The significant increase of free calcium concentration ([Ca2+]i) was found in rat cerebral cortex synaptosomes and hippocampal crude synaptosomal fraction after their exposure to glutamate. But no change of [Ca2+]i was revealed in cerebellar synaptosomes, the slight increase of [Ca2+]i in striatal synaptosomes was not significant. The presence of Ng-nitro-L-arginine methyl ester (L-NAME) in the incubation medium practically prevented the increase of [Ca2+]i initiated by glutamate in cerebral cortex synaptosomes, but not in hippocampal ones. The significant diminution of [Ca2+]i in the presence of this inhibitor was shown in striatal synaptosomes exposed to glutamate. Na+,K+-ATPase activity is significantly lower in cerebral cortex, striatal and hippocampal synaptosomes exposed to glutamate. L-NAME prevented the inactivation of this enzyme by glutamate. In cerebellar synaptosomes the tendency to the decrease of enzymatic activity in the presence of L-NAME was on the contrary noticed. Thus, the data obtained provide evidence of the protective effect of NO synthase inhibitor in brain cortex and striatal synaptosomes, but not in cerebellar synaptosomes. Synaptosomes appear to be an adequate model to study the regional differences in the mechanism of toxic effect of excitatory amino acids.
Glutamate significantly increased intracellular calcium concentration, enhanced 45Ca2+ entry, and activated Na+/Ca 2 § exchanger in synaptosomes from rat cerebral cortex. Preincubation with GMI ganglioside reduced the glutamate-induced increase in intracellular calcium and 45Ca2+ entry. Gangliosides and glutamate stimulated Na+/Ca 2 § exchanger showing additive effects.Key Words: GM 1 ganglioside," glutamate; calcium; cerebral cortex synaptosomes Neuronal damage in ischemia, brain trauma, and some neurological diseases is largely determined by toxic effects of high concentrations of glutamate released into the intercellular space [2,3,12]. Excitotoxin-induced disturbance in intracellular calcium ([Ca2+]i) homeostasis is considered to be the major cause of neuronal death under these conditions [4].Activation of N-methyl-D-aspartate receptors triggering this process is a necessary but not sufficient condition for sustained elevation of [Ca:+]i. An important role in the development of pathological processes initiated by excitotoxins is played by activation of free radical reactions [1,13] and other processes stimulating Ca 2+ entry and inactivating the system stabilizing calcium homeostasis.Gangliosides, the most complex animal glicosphingolipids, improve viability of cultured nervous cells exposed to glutamate [3,6] and significantly decrease [Ca 2+] ~, which seems to underlie their neuroprotective activity [12]. However, the mechanism of this effect remains unclear. We found no reports on the effects of gangliosides on glutamate-induced activation of Ca 2+ entry or activity of Na+/Ca 2+ exchanger.Our aim was to study the mechanism of stabilizing effect of gangliosides on Ca 2+ homeostasis dis- MATERIALS AND METHODSSynaptosomes isolated from the brain of male Wistar rats (150-180 g) as described elsewhere [9] were resuspended in buffer A containing (in mM): 135 NaC1, 1.2 MgSO4, 2.0 KC1. 10 glucose, and 20 tris-HC1 (pH=7.4), centrifuged at 15,000g for 15 min, and resuspended again in buffer A (4-5 mg/ml protein concentration).[Ca2+]i was measured with Fura-2M (Sigma), a fluorescent calcium probe. Synaptosomes (1 ml) were mixed with 1 ml buffer A containing 0.5% BSA (flee from fatty acids) and 8 gM Fura-2M dissolved in 0.4% DMSO and incubated for 40 min at 30~ [16]. Preincubation with 50 nM GM~ ganglioside was performed simultaneously with Fura-2M loading, which was stopped by adding 20-fold volume of buffer A followed by 15-min centrifugation at 12,000g. The sediment was resuspended in MgSQ-free buffer B containing (in mM): 135 NaC1. 2.0 KC1, 1.0 CaC 89 10 glucose. and 20 tris-HC1 (pH=7.4) and incubated with glutamate and other test compounds for 15 min at 30~ Fluorescence was measured on a MPF-2A fluorimeter (Hitachi) at 340 and 380 nm excitation and 510 nm 9 0007-4888/00/0001-36525.00
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