Nervous system cells are highly dependent on adequate tissue oxygenation and are very susceptible to hypoxia, which causes mitochondrial dysfunctions involved in apoptosis and necrosis. In this paper, we examine the effect of a 12-h incubation of differentiated IMR-32 neuroblastoma cells in a hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell viability, modifications of NO, intracellular Ca(2+) concentration [Ca(2+)](i) and membrane potential, the production of phosphorylated ERK, desferoxamine-chelatable free iron and esterified F2-isoprostane levels. The same parameters were evaluated after a subsequent 24-h re-oxygenation period. The NO concentration increased significantly immediately after hypoxia and returned to values similar to those of controls after the reoxygenation period. At the same time, we observed a significant increase of [Ca(2+)](i) immediately after hypoxia. Phosphorylated ERK proteins increased significantly during the first 2 h of hypoxia, then decreased, and remained practically unmodified after 12 h hypoxia and the following reoxygenation period. Moreover, IMR-32 cell mitochondria were significantly depolarized after hypoxia, while membrane potential returned to normal after the reoxygenation period. Finally, desferoxamine-chelatable free iron and F2-isoprostane levels also increased significantly after hypoxia. Our results indicate that 2% O(2) hypoxia induces variations of NO and [Ca(2+)](i) with subsequent mitochondrial depolarization, and it is responsible for oxidative stress, represented by increased free iron and F2-isoprostane, protein carbonyls and 4 hydroxynonenal protein adducts levels.

The NO donor 3-Morpholinosydnonimine (SIN-1) releases NO in the presence of molecular oxygen. In this study, we evaluated the effect of SIN-1 on mitochondria of rat cortical synaptosomes. We demonstrated in vitro that the amount of ONOO(-) generated and H(2)O(2) formation directly correlated with SIN-1 concentration. The mean oxygen consumption by synaptosomal mitochondria was approximately 3.8 nmol of O(2) min(-1) mg(-1) protein, which decreased significantly in the presence of SIN-1 1 mM to 2.5 nmol O(2) min(-1) mg(-1). This decrease was not modified by catalase or Trolox, demonstrating that ONOO(-) was responsible for the effect. The same concentration of SIN-1 caused a significant decrease of ATP production by synaptosomal mitochondria and depolarized the mitochondrial membrane. Moreover, ROS production increased progressively and was completely inhibited by pre-incubation of synaptosomes with Trolox. Finally, phosphatidylserine was externalized and, at the same time, intrasynaptosomal lactate dehydrogenase decreased confirming both, the external membrane breakdown after the addition of SIN-1 and the damage to the synaptosomes.

Nerve cells are very susceptible to hypoxia responsive for mitochondrial dysfunctions involved in the subsequent oxidative stress, apoptosis and necrosis. In this paper, we examined the effect of 12 h incubation of U-373 MG astrocytes in hypoxic environment (73% N 2 : 2% O 2 : 5% CO 2 , v:v) by evaluating cell proliferation, modifications of NO and ATP production, intracellular Ca 2? concentration [Ca 2? ] i , membrane potential, desferoxaminechelatable free iron, esterified F2-isoprostanes levels and the production of phosphorylated ERK. The same parameters were evaluated also after a following re-oxygenation period of 24 h. Immediately after hypoxia the NO concentration increased significantly and returned to values similar to those of controls after the re-oxygenation period. At the same time, ATP levels remained similar to controls and the cell proliferation significantly decreased. This involved a significant increase of [Ca 2? ] i immediately after hypoxia and the value remained significantly elevated after the following re-oxygenation period. Moreover, after hypoxia, astrocytes were slightly although not significantly depolarized. Indeed iron and F2-isoprostanes levels increased significantly after hypoxia. Finally ERK proteins increased slowly and not significantly after hypoxia and the same trend was observed after the re-oxygenation period.On the whole, our results indicate that 2% O 2 hypoxia induces a moderate oxidative stress, well tolerated by U-373 MG cells, remaining the ATP production, mitochondrial membrane potential and activated ERK proteins, similar to the values of controls.

The present study examined the effect on rat cortical synaptosomes of a 2 h exposure to 50-Hz electromagnetic fields (EMFs) with a peak magnetic field of 2 mT. We measured modifications of synaptosomal mitochondrial respiration rate, ATP production, membrane potential, intrasynaptosomal Ca(2+) concentration and free iron release. The O(2) consumption remained unvaried in exposed synaptosomes at about 2 nM O(2)/min/mg proteins; ATP production was also unchanged. The intrasynaptosomal Ca(2+) concentration decreased slowly and there was a slight, but non-significant, depolarisation of the synaptosomal membrane. Finally, the free iron release by synaptosomal suspensions, a useful predictor of neuro-developmental outcome, remained unchanged after EMF exposure. On the whole, our results indicate that the physiological behaviour of cortical synaptosomes is not affected by weak pulsed EMFs.