Ischemia enhances activation by Ca2+ and redox modification of ryanodine receptor channels from rat brain cortex.
Cerebral ischemia stimulates Ca2+ influx and thus increases neuronal intracellular free [Ca2+]. Using a rat model of cerebral ischemia without recirculation, we tested whether ischemia enhances the activation by Ca2+ of ryanodine receptor (RyR) channels, a requisite feature of RyR-mediated Ca2+-induced Ca2+ release (CICR). To this aim, we evaluated how single RyR channels from endoplasmic reticulum vesicles, fused into planar lipid bilayers, responded to cytoplasmic [Ca2+] changes. Endoplasmic reticulum vesicles were isolated from the cortex of rat brains incubated without blood flow for 5 min at 37 degrees C (ischemic) or at 4 degrees C (control). Ischemic brains displayed increased oxidative intracellular conditions, as evidenced by a lower ratio (approximately 130:1) of reduced/oxidized glutathione than controls (approximately 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 with increased frequency the high activity response and with lower frequency the low activity response. Both control and ischemic cortical vesicles contained the RyR2 and RyR3 isoforms in a 3:1 proportion, with undetectable amounts of RyR1. Ischemia reduced [3H]ryanodine binding and total RyR protein content by 35%, and increased at least twofold endogenous RyR2 S-nitrosylation and S-glutathionylation without affecting the corresponding RyR3 endogenous levels. In vitro RyR S-glutathionylation but not S-nitrosylation favored the emergence of high activity channels. We propose that ischemia, by enhancing RyR2 S-glutathionylation, allows RyR2 to sustain CICR; the resulting amplification of Ca2+ entry signals may contribute to cortical neuronal death.
Bull
Ricardo
R
Centro de Estudios Moleculares de la Célula, Fondo de Investigación Avanzada en Areas Prioritarias, Universidad de Chile, Santiago 7, Chile. rbull@med.uchile.cl
Finkelstein
José Pablo
JP
Gálvez
Jorge
J
Sánchez
Gina
G
Donoso
Paulina
P
Behrens
María Isabel
MI
Hidalgo
Cecilia
C
D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002545:Brain Ischemia / Q000378:metabolism* / Q000503:physiopathology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D016923:Cell Death / Q000502:physiology; D002540:Cerebral Cortex / Q000378:metabolism* / Q000503:physiopathology; D003593:Cytoplasm / Q000378:metabolism; D004195:Disease Models, Animal; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D008297:Male; D009410:Nerve Degeneration / Q000378:metabolism* / Q000503:physiopathology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D013347:Subcellular Fractions; D022161:Transport Vesicles / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
D019837:Ryanodine Receptor Calcium Release Channel; D005978:Glutathione; D002118:Calcium
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