Excessive elevation of intracellular Ca2+ levels and, subsequently, hyperactivation of Ca2+/
calmodulin-dependent processes might play an important role in the pathologic events following
cerebral ischemia. PEP-19 is a neuronally expressed
polypeptide that acts as an endogenous negative regulator of
calmodulin by inhibiting the association of
calmodulin with
enzymes and other
proteins. The aims of the present study were to investigate the effect of PEP-19 overexpression on cell death triggered by Ca2+ overload and how the
polypeptide levels are affected by
glutamate-induced excitotoxicity and
cerebral ischemia. Expression of PEP-19 in HEK293T cells suppressed
calmodulin-dependent signaling and protected against cell death elicited by Ca2+
ionophore. Likewise, primary cortical neurons overexpressing PEP-19 became resistant to
glutamate-induced cell death. In immunoprecipitation assay, wild type PEP-19 associated with
calmodulin, whereas mutated PEP-19, which contains mutations within the
calmodulin binding site of PEP-19, failed to associate with
calmodulin. We found that the mutation abrogates both the ability to suppress
calmodulin-dependent signaling and to protect cells from death. Additionally, the endogenous PEP-19 levels in neurons were significantly reduced following
glutamate exposure, this reduction precedes neuronal cell death and can be blocked by treatment with
calpain inhibitors. These data suggest that PEP-19 is a substrate for
calpain, and that the decreased PEP-19 levels result from its degradation by
calpain. A similar reduction of PEP-19 also occurred in the hippocampus of gerbils subjected to transient global
ischemia. In contrast to the reduction in PEP-19, no changes in
calmodulin occurred following excitotoxicity, suggesting the loss of negative regulation of
calmodulin by PEP-19. Taken together, these results provide evidence that PEP-19 overexpression enhances resistance to Ca2+-mediated cytotoxicity, which might be mediated through
calmodulin inhibition, and also raises the possibility that PEP-19 degradation by
calpain might produce an aberrant activation of
calmodulin functions, which in turn causes neuronal cell death.