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- Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentrationPublication . Tiago, Teresa; Marques-da-Silva, Dorinda; Samhan-Arias, A. K.; Aureliano, M.; Gutiérrez-Merino, CarlosCytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidantinduced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594- DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure ofCGNto a flux of peroxynitrite as low as 0.5–1 M/min during 30 min (achieved with 0.1mMSIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca2+ channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca2+ dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca2+ concentration response to the L-VOCC’s agonist FPL-64176 and to the L-VOCC’s blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca2+ influx through L-VOCC. Combined analysis of cytosolic Ca2+ concentration and G-actin/Factin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.
- Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and functionPublication . Tiago, Teresa; Palma, Pedro; Gutiérrez-Merino, Carlos; Aureliano, M.The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the effi ciency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modifi cation in the peroxynitrite-induced myosin functional impairment.