The Effects of Hypoxia/Reoxygenation on the Physiological Behaviour of U373-Mg Astrocytes

Abstract

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.

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

“…Many studies have indicated the increase of chelatable iron during hypoxia/anoxia/ischemia (23)(24)(25)(26)(27)(28)(29)(30)(31)(32); however, quantitation has only been reported by two groups. One group used molar concentrations of nitrite in an unbuffered solution in order to form DNIC for quantitation (54), a treatment that is likely to perturb and liberate iron from normally masked pools.…”

“…Similar to our method, another group lysed cells in the presence of DFO and measured the Fe 3ϩ -DFO adduct by high performance liquid chromatography (23)(24)(25)(26)(27)(28). Using this method, up to a 5-fold increase of chelatable iron was reported, depending on the cell type and the length of hypoxia/anoxia/ischemia (23)(24)(25)(26)(27)(28). Consistently, we find that DFO-chelatable iron increases about 5 times to 0.19 Ϯ 0.06 nmol/mg protein after 1 h of anoxia and more than 7 times to 0.29 Ϯ 0.05 nmol/mg protein after 2 h of anoxia.…”

“…It has also been reported that chelatable iron decreases during reoxygenation following the increase during hypoxia/anoxia (23,24,26,27), although the time course has not been determined. Knowing how rapidly it decreases is important for defining the time frame of sample processing after hypoxia/ anoxia.…”

“…Reoxygenation-It has been reported that chelatable iron increases dramatically during hypoxia/anoxia (23)(24)(25)(26)(27)(28)(29)(30)(31)(32). To test under our conditions whether the increase during anoxia of DNIC is due to the increase of chelatable iron, we simultaneously quantified the cellular level of chelatable iron and of DNIC from brief ⅐ NO exposure after anoxia pretreatment.…”

“…To our surprise (and dismay), we initially found that treatment of murine macrophage RAW 264.7 cells for 2 min with disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate⅐methanol (PROLI/NO), a donor releasing ⅐ NO with a half-life of 2 s at 37°C and pH 7.4, resulted in no significant amount of DNIC. A biologically and clinically relevant condition that displays elevated levels of chelatable iron is hypoxia/anoxia (23)(24)(25)(26)(27)(28)(29)(30)(31)(32), and then we discovered that the incubation of cells in a sealed vial (or in a hypoxic chamber) resulted in a dramatic increase in the DNIC signal upon subsequent brief ⅐ NO exposure. Here we utilize this method to selectively manipulate the levels of DNIC and probe their cellular functions.…”