Cerebral ischemia stimulates] changes. Endoplasmic reticulum vesicles were isolated from the cortex of rat brains incubated without blood flow for 5 min at 37°C (ischemic) or at 4°C (control). Ischemic brains displayed increased oxidative intracellular conditions, as evidenced by a lower ratio (ϳ130:1) of reduced/oxidized glutathione than controls (ϳ200:1). Single RyR channels from ischemic or control brains displayed the same three responses to Ca 2ϩ 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 [ 3 H]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 Ca 2ϩ entry signals may contribute to cortical neuronal death.

We have previously reported that primary hippocampal neurons exposed to synaptotoxic amyloid beta oligomers (AβOs), which are likely causative agents of Alzheimer’s disease (AD), exhibit abnormal Ca2+ signals, mitochondrial dysfunction and defective structural plasticity. Additionally, AβOs-exposed neurons exhibit a decrease in the protein content of type-2 ryanodine receptor (RyR2) Ca2+ channels, which exert critical roles in hippocampal synaptic plasticity and spatial memory processes. The antioxidant N-acetylcysteine (NAC) prevents these deleterious effects of AβOs in vitro. The main contribution of the present work is to show that AβOs injections directly into the hippocampus, by engaging oxidation-mediated reversible pathways significantly decreased RyR2 protein content but increased single RyR2 channel activation by Ca2+ and caused considerable spatial memory deficits. AβOs injections into the CA3 hippocampal region impaired rat performance in the Oasis maze spatial memory task, decreased hippocampal glutathione levels and overall content of plasticity-related proteins (c-Fos, Arc, and RyR2) and increased ERK1/2 phosphorylation. In contrast, in hippocampus-derived mitochondria-associated membranes (MAM) AβOs injections increased RyR2 levels. Rats fed with NAC for 3-weeks prior to AβOs injections displayed comparable redox potential, RyR2 and Arc protein contents, similar ERK1/2 phosphorylation and RyR2 single channel activation by Ca2+ as saline-injected (control) rats. NAC-fed rats subsequently injected with AβOs displayed the same behavior in the spatial memory task as control rats. Based on the present in vivo results, we propose that redox-sensitive neuronal RyR2 channels partake in the mechanism underlying AβOs-induced memory disruption in rodents.

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca2+ concentration ([Ca2+]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263-1277, 1998), with low, moderate, and high maximal fractional open times (Po). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 microM cytoplasmic [Ca2+]. At 10 microM [Ca2+], low-Po channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (KaATP) = 422 microM] than moderate-Po channels (KaATP = 82 microM). Oxidation of low-Po channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-Po channels and decreased KaATP from 422 to 82 microM. At 0.1 microM cytoplasmic [Ca2+], ATP induced an almost negligible activation of low-Po channels. After oxidation to high-Po behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-Po channels at 10 microM [Ca2+] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode.

Despite their relevance for neuronal Ca(2+)-induced Ca(2+) release (CICR), activation by Ca(2+) of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg(2+)], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg(2+)], and RyR redox state on the Ca(2+) dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg(2+)] up to 1 mM inhibited vesicular [(3)H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg(2+) inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca(2+) dependencies, defined by low, moderate, or high maximal fractional open time (P(o)), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca(2+)] for activation, increased maximal P(o), and shifted channel inhibition to higher [Ca(2+)]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg(2+)] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg(2+)] induced a right shift in Ca(2+) dependence for all channels so that [Ca(2+)] <30 microM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca(2+) at physiological [ATP] and [Mg(2+)]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR.

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