The Physiological Behaviour of IMR-32 Neuroblastoma Cells is Affected by a 12-h Hypoxia/24-h Reoxygenation Period

Abstract

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.

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

“…Indeed, relatively long term incubation with DFO (and also ⅐ NO) elicits an "iron starvation" response that interrupts iron homeostasis (53). To avoid this prolonged incubation, we applied a method involving disruption of cell membrane, thereby allowing DFO to access iron rapidly and avoiding effects on cellular iron homeostasis (24). We detected only 0.04 Ϯ 0.02 nmol/mg protein of DFO-chelatable iron in RAW 264.7 cells using this method.…”

“…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.…”

“…Moreover, a rapid measurement is needed because, as described below, anoxia-induced cheatable iron disappears during reoxygenation (which is inevitable during the incubation with DFO), and DFO is only slowly taken up by cells (46). Therefore, we employed a modified method involving disrupting cell membranes in the presence of DFO to ensure the immediate access of DFO to cellular iron (24). As shown in Fig.…”

“…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.…”