NEURONAL NITRIC-OXIDE SYNTHASE AND CALMODULIN-DEPENDENT PROTEIN-KINASE II-ALPHA UNDERGO NEUROTOXIN-INDUCED PROTEOLYSIS

Calpain (calcium-activated neutral protease) has been implicated as pl aying a role of neuronal injury in cerebral ischemia and excitotoxicit y. Here we report that, in addition to extreme excitotoxic conditions [N-methyl-D-aspartate (NMDA), lpha-amino-3-hydroxy-5-methyl-4-isoxazol epropionic acid (AMPA), and kainate challenges], other neurotoxins suc h as maitotoxin, A23187, and okadaic acid also induce calpain activati on, as detected by m-calpain autolytic fragmentation and nonerythroid alpha-spectrin breakdown, Under the same conditions, calmodulin-depend ent protein kinase II-alpha (CaMPK-II alpha) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fra gmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CH O and PD151746). In vitro digestion of protein extract from cortical c ultures with purified mu- and m-calpain produced fragmentation pattern s far CaMPK-II alpha and nNOS similar to those produced in situ, Also, several other calpain-sensitive calmodulin-binding proteins (plasma m embrane calcium pump, microtubule-associated protein 2, and calcineuri n A) and protein kinase C-alpha are also degraded in neurotoxin-treate d cultures. Lastly, in a rat pup model of acute excitotoxicity, intras triatal injection of NMDA resulted in breakdown of CaMPK-II alpha and nNOS, The degradation of CaMPK-II alpha, nNOS, and other endogenous ca lpain substrates may contribute to the neuronal injury associated with Various neurotoxins.