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

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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% N2: 2% O2: 5% CO2, v:v) by evaluating cell viability, modifications of NO, intracellular Ca2+ concentration [Ca2+]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 [Ca2+]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% O2 hypoxia induces variations of NO and [Ca2+]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.

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Acknowledgments

Work supported in part by MIUR, PRIN 2005 PROT.2005 06 9071-005 and in part by PAR servizi.

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Authors and Affiliations

  1. Department of Physiology, University of Siena, via A. Moro, 53100, Siena, Italy

    Carlo Aldinucci, Silvia Maria Maiorca, Paola De Rosa & Gian Paolo Pessina

  2. Department of Biomedical Sciences, University of Siena, 53100, Siena, Italy

    Mitri Palmi, Claudia Sticozzi & Giuseppe Valacchi

  3. Department of Pathophysiology, Experimental Medicine and Public Health, University of Siena, 53100, Siena, Italy

    Lucia Ciccoli, Silvia Leoncini, Cinzia Signorini & Giuseppe Valacchi

  4. Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea

    Giuseppe Valacchi

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  1. Carlo Aldinucci
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  2. Silvia Maria Maiorca
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  3. Paola De Rosa
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  4. Mitri Palmi
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  6. Lucia Ciccoli
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  7. Silvia Leoncini
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  9. Giuseppe Valacchi
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  10. Gian Paolo Pessina
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Correspondence to Gian Paolo Pessina.

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Aldinucci, C., Maiorca, S.M., De Rosa, P. et al. The Physiological Behaviour of IMR-32 Neuroblastoma Cells is Affected by a 12-h Hypoxia/24-h Reoxygenation Period. Neurochem Res 35, 1691–1699 (2010). https://doi.org/10.1007/s11064-010-0231-2

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  • DOI: https://doi.org/10.1007/s11064-010-0231-2

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