In the CNS, where Ca(2+) overload has been established as a mechanism contributing to neuronal damage associated with excitotoxicity,
stroke and
ischemia, there is interest in understanding the role of
calpain inhibition in rescuing neurons from death. In these settings, the activation of large stores of latent
calpain may rapidly lead to the demise of the neuron within hours. The activity of
calpain is strictly regulated by
calcium concentrations and interactions with
calpastatin (endogenous
calpain inhibitor). The interaction between calpains and
calpastatin is
calcium dependent, and little is known about the regulation of the neuronal
calpain-
calpastatin system in vivo. It has been postulated that
calpastatin can be modulated by
nerve growth factors (NGFs). We have demonstrated in vitro as well as in vivo a
neuroprotective effect of the beta(2)-adrenoceptor agonist
clenbuterol (CLN) mediated through an increased
NGF expression. In this study we attempt to find out whether CLN is capable (1) of modulating proteolysis regulated by the
calpain-
calpastatin system and (2) of attenuating DNA-fragmentation induced by
cerebral ischemia. Rats received CLN daily for 1 week, were then subjected to
ischemia and finally perfused at different times post-
ischemia. The proteolytic activity of
calpain was measured by the immunolocalisation of
calpastatin and
spectrin-breakdown products (SBP). The time course of apoptosis was assessed by terminal dUTP nick end-labeling (TUNEL)-staining. CLN reduced CA1-hippocampal cell damage by 23%, attenuated
DNA-laddering and decreased proteolysis of
spectrin by enhancing
calpastatin activity. These results provide evidence that CLN is a potent neuroprotective substance, which through the enhancement of
calpastatin synthesis attenuates the apoptotic machinery and modulates proteolysis.