Catalytic enantioselective [3+2] and [4+2]-annulation of cyclic N-sulfonyl ketimines with γ- or δ-hydroxy-α,ß-unsaturated ketones.

A highly efficient asymmetric [3+2] and [4+2]-annulation of cyclic N-sulfonyl ketimines with γ- or δ-hydroxy-α,ß-unsaturated ketones has been developed. This innovative reaction employs an organocatalytic approach, utilizing a hydrogen-bonding bifunctional squaramide-based catalyst. The process enables precise synthesis of chiral polyheterotricyclic oxazolidines and 1,3-oxazinane derivatives, revealing intricate structures with incorporated chiral quaternary centers. Remarkably, this method delivers high yields and exceptional enantioselectivities and diastereoselectivities, achieving up to 99% yield, >20 : 1 dr, and >99% ee.

Group 1 metabotropic glutamate receptor 5 is involved in synaptically-induced Ca2+-spikes and cell death in cultured rat hippocampal neurons.

Group 1 metabotropic glutamate receptors (mGluRs) can positively affect postsynaptic neuronal excitability and epileptogenesis. The objective of the present study was to determine whether group 1 mGluRs might be involved in synaptically-induced intracellular free Ca2+ concentration ([Ca2+]i) spikes and neuronal cell death induced by 0.1 mM Mg2+ and 10 µM glycine in cultured rat hippocampal neurons from embryonic day 17 fetal Sprague-Dawley rats using imaging methods for Ca2+ and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays for cell survival. Reduction of extracellular Mg2+ concentration ([Mg2+]o) to 0.1 mM induced repetitive [Ca2+]i spikes within 30 sec at day 11.5. The mGluR5 antagonist 6-Methyl-2-(phenylethynyl) pyridine (MPEP) almost completely inhibited the [Ca2+]i spikes, but the mGluR1 antagonist LY367385 did not. The group 1 mGluRs agonist, 3,5-dihydroxyphenylglycine (DHPG), significantly increased the [Ca2+]i spikes. The phospholipase C inhibitor U73122 significantly inhibited the [Ca2+]i spikes in the absence or presence of DHPG. The IP3 receptor antagonist 2-aminoethoxydiphenyl borate or the ryanodine receptor antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate also significantly inhibited the [Ca2+]i spikes in the absence or presence of DHPG. The TRPC channel inhibitors SKF96365 and flufenamic acid significantly inhibited the [Ca2+]i spikes in the absence or presence of DHPG. The mGluR5 antagonist MPEP significantly increased the neuronal cell survival, but mGluR1 antagonist LY367385 did not. These results suggest a possibility that mGluR5 is involved in synaptically-induced [Ca2+]i spikes and neuronal cell death in cultured rat hippocampal neurons by releasing Ca2+ from IP3 and ryanodine-sensitive intracellular stores and activating TRPC channels.

Cyanidin-3-glucoside inhibits amyloid ß25-35-induced neuronal cell death in cultured rat hippocampal neurons.

Increasing evidence implicates changes in [Ca2+]i and oxidative stress as causative factors in amyloid beta (Aß)-induced neuronal cell death. Cyanidin-3-glucoside (C3G), a component of anthocyanin, has been reported to protect against glutamate-induced neuronal cell death by inhibiting Ca2+ and Zn2+ signaling. The present study aimed to determine whether C3G exerts a protective effect against Aß25-35-induced neuronal cell death in cultured rat hippocampal neurons from embryonic day 17 fetal Sprague-Dawley rats using MTT assay for cell survival, and caspase-3 assay and digital imaging methods for Ca2+, Zn2+, MMP and ROS. Treatment with Aß25-35 (20 µM) for 48 h induced neuronal cell death in cultured rat pure hippocampal neurons. Treatment with C3G for 48 h significantly increased cell survival. Pretreatment with C3G for 30 min significantly inhibited Aß25-35-induced [Zn2+]i increases as well as [Ca2+]i increases in the cultured rat hippocampal neurons. C3G also significantly inhibited Aß25-35-induced mitochondrial depolarization. C3G also blocked the Aß25-35-induced formation of ROS. In addition, C3G significantly inhibited the Aß25-35-induced activation of caspase-3. These results suggest that cyanidin-3-glucoside protects against amyloid ß-induced neuronal cell death by reducing multiple apoptotic signals.

Black Rice (Oryza sativa L., Poaceae) Extract Reduces Hippocampal Neuronal Cell Death Induced by Transient Global Cerebral Ischemia in Mice.

Rice is the most commonly consumed grain in the world. Black rice has been suggested to contain various bioactive compounds including anthocyanin antioxidants. There is currently little information about the nutritional benefits of black rice on brain pathology. Here, we investigated the effects of black rice (Oryza sativa L., Poaceae) extract (BRE) on the hippocampal neuronal damage induced by ischemic insult. BRE (300 mg/kg) was orally administered to adult male C57BL/6 mice once a day for 21 days. Bilateral common carotid artery occlusion (BCCAO) was performed for 23 min on the 8th day of BRE or vehicle administration. Histological analyses conducted on the 22nd day of BRE or vehicle administration revealed that administering BRE profoundly attenuated neuronal cell death, inhibited reactive astrogliosis, and prevented loss of glutathione peroxidase expression in the hippocampus when compared to vehicle treatment. In addition, BRE considerably ameliorated BCCAO-induced memory impairment on the Morris water maze test from the 15th day to the 22nd day of BRE or vehicle administration. These results indicate that chronic administration of BRE is potentially beneficial in cerebral ischemia.

Dapoxetine induces neuroprotective effects against glutamate-induced neuronal cell death by inhibiting calcium signaling and mitochondrial depolarization in cultured rat hippocampal neurons.

Selective serotonin reuptake inhibitors (SSRIs) have an inhibitory effect on various ion channels including Ca2+ channels. We used fluorescent dye-based digital imaging, whole-cell patch clamping and cytotoxicity assays to examine whether dapoxetine, a novel rapid-acting SSRI, affect glutamate-induced calcium signaling, mitochondrial depolarization and neuronal cell death in cultured rat hippocampal neurons. Pretreatment with dapoxetine for 10min inhibited glutamate-induced intracellular free Ca2+ concentration ([Ca2+]i) increases in a concentration-dependent manner (Half maximal inhibitory concentration=4.79µM). Dapoxetine (5µM) markedly inhibited glutamate-induced [Ca2+]i increases, whereas other SSRIs such as fluoxetine and citalopram only slightly inhibited them. Dapoxetine significantly inhibited the glutamate-induced [Ca2+]i responses following depletion of intracellular Ca2+ stores by treatment with thapsigargin. Dapoxetine markedly inhibited the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine-induced [Ca2+]i increases. Dapoxetine significantly inhibited the glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i responses in either the presence or absence of nimodipine. Dapoxetine also significantly inhibited AMPA-evoked currents. However, dapoxetine slightly inhibited N-methyl-D-aspartate (NMDA)-induced [Ca2+]i increases. Dapoxetine markedly inhibited 50mMK+-induced [Ca2+]i increases. Dapoxetine significantly inhibited glutamate-induced mitochondrial depolarization. In addition, dapoxetine significantly inhibited glutamate-induced neuronal cell death and its neuroprotective effect was significantly greater than fluoxetine. These data suggest that dapoxetine reduces glutamate-induced [Ca2+]i increases by inhibiting multiple pathways mainly through AMPA receptors, voltage-gated L-type Ca2+ channels and metabotropic glutamate receptors, which are involved in neuroprotection against glutamate-induced cell death through mitochondrial depolarization.

Brief low [Mg(2+)]o-induced Ca(2+) spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons.

Reducing [Mg(2+)]o to 0.1 mM can evoke repetitive [Ca(2+)]i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg(2+)]o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca(2+) spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg(2+)]o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca(2+) channel antagonist nimodipine, which blocked 0.1 mM [Mg(2+)]o-induced [Ca(2+)]i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca(2+)]i spikes. The intracellular Ca(2+) chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca(2+)]i spikes. While Gö6976, a specific inhibitor of PKCα had no effect on the tolerance, both the PKCε translocation inhibitor and the PKCζ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca(2+)]i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg(2+)]o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca(2+)]i spike-induced activation of PKCε and PKCξ, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.

Cyanidin-3-glucoside inhibits glutamate-induced Zn2+ signaling and neuronal cell death in cultured rat hippocampal neurons by inhibiting Ca2+-induced mitochondrial depolarization and formation of reactive oxygen species.

Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is a potent natural antioxidant. However, effects of C3G on glutamate-induced [Zn(2+)]i increase and neuronal cell death remain unknown. We studied the effects of C3G on glutamate-induced [Zn(2+)]i increase and cell death in cultured rat hippocampal neurons from embryonic day 17 maternal Sprague-Dawley rats using digital imaging methods for Zn(2+), Ca(2+), reactive oxygen species (ROS), mitochondrial membrane potential and a MTT assay for cell survival. Treatment with glutamate (100 µM) for 7 min induces reproducible [Zn(2+)]i increase at 35 min interval in cultured rat hippocampal neurons. The intracellular Zn(2+)-chelator TPEN markedly blocked glutamate-induced [Zn(2+)]i increase, but the extracellular Zn(2+) chelator CaEDTA did not affect glutamate-induced [Zn(2+)]i increase. C3G inhibited the glutamate-induced [Zn(2+)]i response in a concentration-dependent manner (IC50 of 14.1 ± 1.1 µg/ml). C3G also significantly inhibited glutamate-induced [Ca(2+)]i increase. Two antioxidants such as Trolox and DTT significantly inhibited the glutamate-induced [Zn(2+)]i response, but they did not affect the [Ca(2+)]i responses. C3G blocked glutamate-induced formation of ROS. Trolox and DTT also inhibited the formation of ROS. C3G significantly inhibited glutamate-induced mitochondrial depolarization. However, TPEN, Trolox and DTT did not affect the mitochondrial depolarization. C3G, Trolox and DTT attenuated glutamate-induced neuronal cell death in cultured rat hippocampal neurons, respectively. Taken together, all these results suggest that cyanidin-3-glucoside inhibits glutamate-induced [Zn(2+)]i increase through a release of Zn(2+) from intracellular sources in cultured rat hippocampal neurons by inhibiting Ca(2+)-induced mitochondrial depolarization and formation of ROS, which is involved in neuroprotection against glutamate-induced cell death.

Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca(2+) Concentration, ROS Formation and Mitochondrial Depolarization in PC12 Cells.

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5'-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free Ca(2+) concentration ([Ca(2+)]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP (100µM) for 90 sec induced [Ca(2+)]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1µ g/ml to 100µg/ml) for 30 min inhibited the ATP-induced [Ca(2+)]i increases in a concentration-dependent manner (IC50=15.3µg/ml). Pretreatment with cyanidin-3-glucoside (15µg/ml) for 30 min significantly inhibited the ATP-induced [Ca(2+)]i responses following removal of extracellular Ca(2+) or depletion of intracellular [Ca(2+)]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [Ca(2+)]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [Ca(2+)]i responses in the presence of nimodipine and ω-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [Ca(2+)]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular Ca(2+) chelator BAPTA-AM or the mitochondrial Ca(2+) uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular Ca(2+) through the nimodipine and ω-conotoxin-sensitive and -insensitive pathways and the release of Ca(2+) from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting Ca(2+)-induced mitochondrial depolarization.

Inhibitory effects of acorn extract on glutamate-induced calcium signaling in cultured rat hippocampal neurons.

Various effects of acorn extract have been reported including antioxidant activity, cytotoxicity against cancer cells, and the levels of acetylcholine and its related enzyme activities in the dementia mouse models. However, it is unclear whether acorn extract inhibits glutamate-induced calcium signaling in hippocampal neurons. This study was an investigation into the effect of acorn extract on intracellular free Ca concentrations ([Ca]) in cultured rat hippocampal neurons using fura-2-based digital calcium imaging and photometry. Hippocampal neurons were used between 10 and 14 d in culture from embryonic day-18 rats. Treatment with acorn extract (1 µg/mL to 1 mg/mL) for 30 min inhibited glutamate (100 µM)-induced [Ca] increases in a dose-dependent manner (IC=46.9 µg/mL). After depletion of intracellular Ca stores by treatment with the inhibitor endoplasmic reticulum Ca-ATPase, thapsigargin (1 µM), treatment with acorn extract (50 µg/mL) for 30 min decreased the subsequent glutamate-induced [Ca] increases. Acorn extract significantly inhibited (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (30 µM)-induced [Ca] increases. In addition, acorn extract inhibited the AMPA-induced [Ca] responses in the presence of 1 µM nimodipine. Acorn extract also significantly inhibited N-methyl-D-aspartate (100 µM)-induced [Ca] increases. Acorn extract significantly inhibited 50 mM KCl -induced [Ca] increases. Acorn extract significantly inhibited (S)-3,5-dihydroxyphenylglycine-induced [Ca] responses. Moreover, acorn extract almost completely blocked synaptically mediated [Ca] spikes induced by decreasing extracellular Mg concentration to 0.1 mM. These results suggest that acorn extract inhibits synaptically induced frequent [Ca] spikes through multiple pathways such as ionotropic glutamate receptors, voltage-gated Ca channels and metabotropic glutamate receptors in cultured rat hippocampal neurons.

Fluoxetine suppresses synaptically induced [Ca²âº]i spikes and excitotoxicity in cultured rat hippocampal neurons.

Fluoxetine is a widely used antidepressant with an action that is primarily attributed to the inhibition of serotonin re-uptake into the synaptic terminals of the central nervous system. Fluoxetine also has blocking effects on various ion channels, including Ca(2+) channels. It remains unclear, however, how fluoxetine may affect synaptically induced [Ca(2+)](i) spikes. We investigated the effects of fluoxetine on [Ca(2+)](i) spikes, along with the subsequent neurotoxicity that is synaptically evoked by lowering extracellular Mg(2+) in cultured rat hippocampal neurons. Fluoxetine inhibited the synaptically induced [Ca(2+)](i) spikes in p-chloroamphetamine-treated and non-treated neurons, in a concentration-dependent manner. However, other selective serotonin reuptake inhibitors, such as paroxetine and citalopram, did not significantly affect the spikes. Pretreatment with fluoxetine for 5 min inhibited [Ca(2+)](i) increases induced by glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and N-methyl-d-aspartate. Fluoxetine also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. In addition, fluoxetine decreased the [Ca(2+)](i) responses induced by the metabotrophic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine or the ryanodine receptor agonist caffeine. Fluoxetine inhibited [Ca(2+)](i) responses induced by 20mM KCl. Fluoxetine decreased the release of FM1-43 induced by electric field stimulation. Furthermore, fluoxetine inhibited 0.1mM [Mg(2+)](o)-induced cell death. Collectively, our results suggest that fluoxetine suppresses the spikes and protects neurons against excitotoxicity, particularly in cultured rat hippocampal neurons, presumably due to both direct inhibition of presynaptic glutamate release and postsynaptic glutamate receptor-mediated [Ca(2+)](i) signaling. In addition to an indirect inhibitory effect via 5-HT levels, these data suggest a new, possibly direct inhibitory action of fluoxetine on synaptically induced [Ca(2+)](i) spikes and neuronal cell death.