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Perez-Alvarez S.,University of Castilla - La Mancha | Solesio M.E.,Albacete University Hospital Center | Cuenca-Lopez M.D.,University of Castilla - La Mancha | Melero-Fernndez De Mera R.M.,University of Castilla - La Mancha | And 5 more authors.
International Journal of Cell Biology | Year: 2012

Previously, we have shown that SH-SY5Y cells exposed to high concentrations of methadone died due to a necrotic-like cell death mechanism related to delayed calcium deregulation (DCD). In this study, we show that, in terms of their Ca2+ responses to 0.5mM methadone, SH-SY5Y cells can be pooled into four different groups. In a broad pharmacological survey, the relevance of different Ca2+-related mechanisms on methadone-induced DCD was investigated including extracellular calcium, L-type Ca2+ channels, -opioid receptor, mitochondrial inner membrane potential, mitochondrial ATP synthesis, mitochondrial Ca2+/2Na+-exchanger, reactive oxygen species, and mitochondrial permeability transition. Only those compounds targeting mitochondria such as oligomycin, FCCP, CGP 37157, and cyclosporine A were able to amend methadone-induced Ca2+ dyshomeostasis suggesting that methadone induces DCD by modulating the ability of mitochondria to handle Ca2+. Consistently, mitochondria became dramatically shorter and rounder in the presence of methadone. Furthermore, analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial Ca2+ uptake in a respiratory substrate-dependent way. We conclude that methadone causes failure of intracellular Ca2+ homeostasis, and this effect is associated with morphological and functional changes of mitochondria. Likely, this mechanism contributes to degenerative side effects associated with methadone treatment. Copyright © 2012 Sergio Perez-Alvarez et al. Source


Perez-Alvarez S.,University of Castilla - La Mancha | Cuenca-Lopez M.D.,University of Castilla - La Mancha | de Mera R.M.M.F.,University of Castilla - La Mancha | Puerta E.,University of Navarra | And 7 more authors.
Biochimica et Biophysica Acta - Molecular Basis of Disease | Year: 2010

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-XL and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a "bioenergetic crisis." A decreased level of cellular energy may predispose cells to necrotic-like cell death. © 2010 Elsevier B.V. Source


Baltanas A.,University of Navarra | Solesio M.E.,Albacete University Hospital Center | Zalba G.,University of Navarra | Galindo M.F.,Albacete University Hospital Center | And 3 more authors.
Journal of Physiology and Biochemistry | Year: 2013

Herein, we investigate whether the NADPH oxidase might be playing a key role in the degree of oxidative stress in the senescence-accelerated mouse prone-8 (SAM-P8). To this end, the activity and expression of the NADPH oxidase, the ratio of glutathione and glutathione disulfides (GSH/GSSG), and the levels of malonyl dialdehyde (MDA) and nitrotyrosine (NT) were determined in renal tissue from SAM-P8 mice at the age of 1 and 6 months. The senescence- accelerated-resistant mouse (SAM-R1) was used as control. At the age of 1 month, NADPH oxidase activity and Nox2 protein expression were higher in SAM-P8 than in SAM-R1 mice. However, we found no differences in the GSH/GSSG ratio, MDA, NT, and Nox4 levels between both groups of animals. At the age of 6 months, SAM-R1 mice in comparison to SAM-P8 mice showed an increase in NADPH oxidase activity, which is associated with higher levels of NT and increased Nox4 and Nox2 expression levels. Furthermore, we found oxidative stress hallmarks including depletion in GSH/GSSG ratio and increase in MDA levels in the kidney of SAM-P8 mice. Finally, NADPH oxidase activity positively correlated with Nox2 expression in all the animals (r = 0.382, P < 0.05). Taken together, our data allow us to suggest that an increase in NADPH oxidase activity might be an early hallmark to predict future oxidative stress in renal tissue during the aging process that takes place in SAM-P8 mice. © 2013 University of Navarra. Source

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