Background::It has been reported that activation of glutamate kainate receptor subunit 2
(GluK2) subunit-containing glutamate receptors and the following Fas ligand(FasL) up-regulation,
caspase-3 activation, result in delayed apoptosis-like neuronal death in hippocampus CA1 subfield
after cerebral ischemia and reperfusion. Nitric oxide-mediated S-nitrosylation might inhibit the
procaspase activation, whereas denitrosylation might contribute to cleavage and activation of
procaspases.
Objectives::The study aimed to elucidate the molecular mechanisms underlying procaspase-3
denitrosylation and activation following kainic acid (KA)-induced excitotoxicity in rat
hippocampus.
Methods::S-nitrosylation of procaspase-3 was detected by biotin-switch method. Activation of
procaspase-3 was shown as cleavage of procaspase-3 detected by immunoblotting. FasL expression
was detected by immunoblotting. Cresyl violets and TdT-mediated dUTP Nick-End Labeling
(TUNEL) staining were used to detect apoptosis-like neuronal death in rat hippocampal CA1 and
CA3 subfields.
Results::KA led to the activation of procaspase-3 in a dose- and time-dependent manner, and the
activation was inhibited by KA receptor antagonist NS102. Procaspase-3 was denitrosylated at 3 h
after kainic acid administration, and the denitrosylation was reversed by SNP and GSNO. FasL ASODNs
inhibited the procaspase-3 denitrosylation and activation. Moreover, thioredoxin reductase
(TrxR) inhibitor auranofin prevented the denitrosylation and activation of procaspase-3 in rat
hippocampal CA1 and CA3 subfields. NS102, FasL AS-ODNs, and auranofin reversed the KAinduced
apoptosis and cell death in hippocampal CA1 and CA3 subfields.
Conclusions::KA led to denitrosylation and activation of procaspase-3 via FasL and TrxR.
Inhibition of procaspase-3 denitrosylation by auranofin, SNP, and GSNO played protective effects
against KA-induced apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields.
These investigations revealed that the procaspase-3 undergoes an initial denitrosylation process
before becoming activated, providing valuable insights into the underlying mechanisms and
possible treatment of excitotoxicity.