Visit our Expo - Redox and Inflammation signaling 2012

Session III : Protein kinase cascades as therapeutic targets Poster III, 55
Effects of chemical ischemia in cerebral cortex slices. Focus on mitogen activated
protein kinase cascade.
Anna Siniscalchi1, Sabrina Cavallini1, Silvia Marino1 Sofia Falzarano2, Lara
Franceschetti2,
and Rita Selvatici2
1Department of Clinical and Experimental Medicine, Section of Pharmacology,
2Department of Experimental and Diagnostic Medicine, Section of Medical Genetics;
University of Ferrara, Italy. E-mail: snn@unife.it
Mitochondrial toxins could model, in vitro, the energy failure that occurs during transient
brain ischemia in vivo, in alternative to either oxygen and glucose deprivation, or to high
glutamate treatment. In our laboratory, sodium azide (NaN3), combined with the glycolysis
blocker, 2-deoxyglucose (2-DOG), has been used to induce chemical ischemia (CI) in rat
cerebral cortex slices (Cavallini, Neurochem Int 2005, 47:482), and the involvement of
glutamate overflow, calcium overload and nitric oxide efflux in its mechanism of action has
been shown. In the present work, the downstream events following CI have been addressed:
superfused rat cerebral cortex slices, continuously electrically (5 Hz) stimulated, were treated
with 10 mM NaN3 plus 2 mM 2-DOG for 5 min, then reperfused with normal medium for
one hour (REP). For Ras/MAP kinases analysis by Western blotting, membranes were
incubated with rabbit polyclonal antibodies against p21Ras, ERK1/2 (p44/42), phospho-
ERK1/2, p38, phospho-p38, SAPK/JNK and phospho-SAPK/JNK. Densitometric analysis of
autoradiographic bands was performed with a Bio-Rad densitometer. The level of p21 Ras
was increased by 40 % immediately after CI, and did not recover to control values following
REP. The total levels of both ERK1 and ERK2 were reduced by CI and partially recovered to
control values in REP; their phosphorylation degree (phosphorylated to total protein level
ratio, about 50 % in the controls) did not significantly change either under CI or under REP
conditions. The phosphorylation degree of MAPK p38, very low in control slices (17%), was
not modified by either CI or REP. The activation of SAPK/JNK was full in the controls and
was reduced to 70% after CI and to 64% following REP. All these effects were prevented by
the NMDA receptor antagonist MK801, 10 µM, suggesting the involvement of glutamate.
The present findings show that CI reduces the MAPK levels, possibly because of acute energy
depletion, although the p21Ras protein is increased. The decrease in SAPK/JNK, observed
under CI/REP conditions, may lead to necrotic neuronal death, since its activation has been
related to both apoptosis and neuroprotection mechanisms. These results could be of interest
in view of preventive treatments of ischemia/reperfusion-induced brain damages.
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