Ischemia Enhances Activation by Ca2 and Redox Modification of Ryanodine Receptor Channels from Rat Brain CortexReport as inadecuate

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Cerebral ischemia stimulates Ca2+influx and thus increases neuronal intracellular free Ca2+. Using a rat model of cerebral ischemiawithout recirculation, we tested whether ischemia enhances the activation by Ca2+ of ryanodine receptor RyR channels, a requisitefeature of RyR-mediated Ca2+-induced Ca2+ release CICR. To this aim, we evaluated how single RyR channels from endoplasmicreticulum vesicles, fused into planar lipid bilayers, responded to cytoplasmic Ca2+ changes. Endoplasmic reticulum vesicles wereisolated from the cortex of rat brains incubated without blood flow for 5 min at 37°C ischemic or at 4°C control. Ischemic brainsdisplayed increased oxidative intracellular conditions, as evidenced by a lower ratio ~130:1 of reduced-oxidized glutathione thancontrols ~200:1. Single RyR channels from ischemic or control brains displayed the same three responses to Ca2+ reported previously,characterized by low, moderate, or high maximal activity. Relative to controls, RyR channels from ischemic brains displayed withincreased frequency the high activity response and with lower frequency the low activity response. Both control and ischemic corticalvesicles contained the RyR2 and RyR3 isoforms in a 3:1 proportion, with undetectable amounts of RyR1. Ischemia reduced ~3Hryanodinebinding and total RyR protein content by 35%, and increased at least twofold endogenous RyR2 S-nitrosylation and S-glutathionylationwithout affecting the corresponding RyR3 endogenous levels. In vitro RyR S-glutathionylation but not S-nitrosylation favored the emergenceof high activity channels. We propose that ischemia, by enhancing RyR2 S-glutathionylation, allows RyR2 to sustain CICR; theresulting amplification of Ca2+ entry signals may contribute to cortical neuronal death.

Author: Finkelstein, José Pablo; - Donoso Laurent, Paulina; - Gálvez, Jorge; - Sánchez, Gina; - Behrens Pellegrino, María Isabel; - B



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