GluR6-FasL-Trx2 mediates denitrosylation and activation of procaspase-3 in cerebral ischemia/reperfusion in rats.

Global cerebral ischemia/reperfusion (I/R) facilitates the activation of procaspase-3 and promotes apoptosis in hippocampus. But the mechanisms have remained uncharacterized. Protein S-nitrosylation and denitrosylation is an important reversible posttranslational modification, which is a common mechanism in signal transduction and affects numerous physiological and pathophysiological events. However, it is not known whether S-nitrosylation/denitrosylation modification of procaspase-3 serves as a component of apoptosis and cell death induced by cerebral I/R. Here we show that procaspase-3 is significantly denitrosylated and activated after I/R in rat hippocampus. NS102, a glutamate receptor 6 (GluR6) antagonist, can inhibit the denitrosylation of procaspase-3 and diminish the increased Fas ligand (FasL) and thioredoxin (Trx)-2 expression induced by cerebral I/R. Moreover, downregulation of FasL expression by antisense oligodeoxynucleotides inhibits the denitrosylation and activation of procaspase-3. Auranofin, a TrxR inhibitor or TrxR2 antisense oligodeoxynucleotide, has similar effects. In primary hippocampal cultures, Lentiviral-mediated knockdown of FasL and TrxR2 before the oxygen and glucose deprivation/reoxygenation further verifies that FasL and TrxR2 are involved in the denitrosylation of procaspase-3. In situ TUNEL staining and cresyl violet staining validate that inhibiting denitrosylation of procaspase-3 may exert neuroprotective effect on apoptosis and cell death induced by cerebral I/R in hippocampal CA1 pyramidal neurons. This is the first evidence that cerebral I/R mediates procaspase-3 denitrosylation and activation through GluR6-FasL-Trx2 pathway, which leads to neuronal apoptosis and cell death.

Exogenous nitric oxide negatively regulates the S-nitrosylation p38 mitogen-activated protein kinase activation during cerebral ischaemia and reperfusion.

AIMS: A number of studies have suggested that nitric oxide (NO) plays an important role in the reactive phosphorylation of p38MAPKα (p38). However, whether S-nitrosylation of p38 is activated by NO and the details remain unclear. The aim of the present work was to assess the activation of p38, the S-nitrosylation site and the p38 signalling pathway in rat hippocampus and in HEK293 cell induced by exogenous NO. METHODS: Primary hippocampal cultures, HEK293 cells and rat model of cerebral ischaemia/reperfusion (brain ischaemia was induced by four-vessel occlusion procedure) were used in this study. Biotin-switch method and immunoblotting were performed to study the S-nitrosylation and phosphorylation of p38, and neuronal loss was observed by histology. RESULTS: Endogenous NO increased p38 phosphorylation and S-nitrosylation, and the activation of p38 was dependent on the S-nitrosylation of Cys-211, which was critical for the NO-mediated activation of p38. The exogenous NO donor sodium nitroprusside, S-nitrosoglutathione, 7-nitroindazole, the inhibitor of the neuronal nitric oxide synthase, inhibited the activation of p38 signal pathway induced by cerebral ischaemia/reperfusion and attenuated the damage in rat hippocampal neurones. Moreover, the N-methyl-D-aspartate receptor (NMDAR) is probably involved in the p38 activation process of S-nitrosylation and phosphorylation. CONCLUSION: Endogenous NO induces the S-nitrosylation and phosphorylation of p38 and mediates p38 signalling pathway by NMDAR, and as exogenous NO inhibits this process and is neuroprotective in rat cerebral ischaemia/reperfusion, it may make a contribution to stroke therapy.

Endogenous nitric oxide induces activation of apoptosis signal-regulating kinase 1 via S-nitrosylation in rat hippocampus during cerebral ischemia-reperfusion.

Apoptosis signal-regulating kinase 1 (ASK1) is a general mediator of cell death in response to a variety of stimuli, including reactive oxygen species, tumor necrosis factor α, lipopolysaccharide, endoplasmic reticulum stress, calcium influx and ischemia. Here we reported ASK1 was activated by nitric oxide (NO) through S-nitrosylation during cerebral ischemia-reperfusion. The reagents that abrogate neuronal nitric oxide synthase (nNOS) activity such as nNOS inhibitor 7NI and N-methyl-D-aspartate receptor antagonist MK801 prevented ASK1 activation via decreasing ASK1 S-nitrosylation. In HEK293 cells, over-expressed ASK1 could be S-nitrosylated by both exogenous and endogenous NO and Cys869 was identified as the site of ASK1 S-nitrosylation. S-nitrosylation increased the level of ASK1 phosphorylation at Thr845, which represents ASK1 activation. Our results further confirmed that S-nitrosylation led to the increment of ASK1 dimerization. S-nitrosylation of ASK1 also activated the downstream JNK signaling and JNK-mediated nucleic pathway. The exogenous NO (SNP and GSNO) reversed the effect of endogenous NO by suppressing S-nitrosylation of ASK1 and exerted neuroprotection during ischemia-reperfusion. These results suggest that inhibiting ASK1 S-nitrosylation may be a novel approach for stroke therapy.

Dual inhibitory roles of geldanamycin on the c-Jun NH2-terminal kinase 3 signal pathway through suppressing the expression of mixed-lineage kinase 3 and attenuating the activation of apoptosis signal-regulating kinase 1 via facilitating the activation of Akt in ischemic brain injury.

It is well documented that heat-shock protein (hsp90) plays an essential role in maintaining stability and activity of its clients. Recent studies have shown that geldanamycin (GA), an inhibitor of hsp90, could decrease the protein of mixed-lineage kinase (MLK) 3 and activate Akt; our previous research documented that MLK3 and Akt and subsequent c-Jun N-terminal kinase (JNK) were involved in neuronal cell death in ischemic brain injury. Here, we investigated whether GA could decrease the protein of MLK3 and activate Akt in rat four-vessel occlusion ischemic model. Our results showed that global cerebral ischemia followed by reperfusion could enhance the association of hsp90 with MLK3, the association of hsp90 with Src, and JNK3 activation. As a result, GA decreased the protein of MLK3 and down-regulated JNK activation. On the other hand, Src kinase was activated and phosphorylated Cbl, which then recruited the p85 subunit of phosphatidylinositol 3-kinase (PI-3K), resulting in PI-3K activation, and as a consequence increased Akt activation, which inhibited ASK1 activation and down-regulated JNK3 activation. In summary, our results indicated that GA showed a dual inhibitory role on JNK3 activation and exerted strong neuroprotection in vivo and in vitro, which provides a new possible approach for stroke therapy.

Neuroprotection against ischemic brain injury by a small peptide inhibitor of c-Jun N-terminal kinase (JNK) via nuclear and non-nuclear pathways.

Our previous studies and the others have strongly suggested that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Here we reported that Tat-JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), a smaller 11-mer peptide corresponding to residues 153-163 of murine JIP-1 conjugated to Tat peptide, perturbed the assembly of JIP-1-JNK3 complexes, thus inhibiting the activation of JNK3 induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. As a result, Tat-JBD diminished the increased phosphorylation of c-Jun (a nuclear substrate of JNK) and the increased expression of Fas ligand induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and the release of Bax from Bcl-2/Bax dimers, Tat-JBD attenuated Bax translocation to mitochondria and the release of cytochrome c induced by ischemia/reperfusion. Furthermore, the activation of caspase3 and hydrolyzation of poly-ADP-ribose-polymerase induced by brain ischemia/reperfusion were also significantly suppressed by preinfusion of the peptide Tat-JBD. Importantly, Tat-JBD showed neuroprotective effects on ischemic brain damage in vivo, and administration of the peptide after ischemia also achieved the same effects as preinfusion of the peptide did. Thus, our findings imply that Tat-JBD induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via inhibiting nuclear and non-nuclear pathways of JNK signaling. Taken together, these results indicate that Tat-JBD peptide provides a promising therapeutic approach for ischemic brain injury.

[Studies on mechanism of the inhibition of Ca2+/CaM PKII activity induced by hypoxia in rat hippocampal slices].

The effects of Ca2+ and ketamine on Ca2+/CaM PK II activity in rat hippocampal slices under in vitro hypoxic states have been studied and the effect of hypoxia on glutamate accumulation was also investigated. The results were as follows: (1) Ca2+/CaM PK II activity decreased gradually with increasing hypoxic time in culture medium with or without 1.3 Ca2- mmol/L, with the former case being much more pronounced. (2) Extracellular accumulation of GLU increased about two-fold after hypoxia for 30 min in vitro. (3) When slices were incubated for 30 min under conditions in excess of exogenous GLU the enzyme activity markedly decreased, a finding suggesting that the inhibition of enzyme activity induced by hypoxia may result from excitotoxicity. (4) The inhibition of the enzyme activity induced by either hypoxia or excess exogenous glutamate alone could be antagonized markedly by pretreatment with ketamine, suggesting that the hypoxia induced inhibition of the enzyme activity is mediated by NMDA receptor.

[Ketamine and nifedipine protect cultured cortical neurons against injurious effect of glutamate].

The effects of glutamate on cultured cortical neurons and the protective effect of ketamine and nifedipine were studied. On day 10 after plating of the cortical cells from 16-18 day-old fetal rats, the cultures were exposed to 50 mumol.L-1 glutamate and low glucose (1 g.L-1) for 10 min-24 h. The results showed that a release of lactate dehydrogenase (LDH) into the culture supernatant was observed as a function of time. The values of LDH efflux in culture medium was significantly lower than those of controls when the cells were pretreated with ketamine or nifedipine 10 min prior to addition of glutamate. More significant decrease of LDH activity in culture medium was observed when the two drugs were used in combination. These results demonstrate that the dissociated cultured cortical neurons from fetal rat are seriously damaged by glutamate. Such damage could be attenuated by ketamine and nifedipine, suggesting that ketamine and nifedipine may protect neurons from the glutamate toxicity, and the effect of combining ketamine and nifedipine was greater than either ketamine or nifedipine alone.

Determination of strychnine and brucine by capillary zone electrophoresis.

A procedure for quantitative estimation of strychnine and brucine in the extracts of Strychnos nux-vomica seeds by capillary zone electrophoresis (CZE) was developed. The buffer solution used was 10mM phosphate buffer-MeOH (9:1), pH 2.5. The linear calibration range was 0.01-0.15 mg/ml. This method is useful for the qualitative and quantitative determination of strychnine and brucine in plant drug samples, as well as in human plasma.