EFFECT OF CEREBRAL-ISCHEMIA ON CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE-II ACTIVITY AND PHOSPHORYLATION

The effects of cerebral ischemia on calcium/ calmodulin-dependent kina se II (CaM kinase II) were investigated using the rat four-vessel occl usion model. In agreement with previous results using rat or gerbil mo dels of cerebral ischemia or a rabbit model of spinal cord ischemia, t his report demonstrates that transient forebrain ischemia leads to a r eduction in CaM kinase II activity within 5 min of occlusion onset. Lo ss of activity from the cytosol fractions of homogenates from the neoc ortex, striatum, and hippocampus correlated with a decrease in the amo unt of CaM kinase alpha and beta isoforms detected by immunoblotting. In contrast, there was an apparent increase in the amount of CaM kinas e alpha and beta in the particulate fractions. The decrease in the amo unt of CaM kinase isoforms from the cytosol but not the particulate fr actions was confirmed by autophosphorylation of CaM kinase II after de naturation and renaturation in situ of the blotted proteins. These res ults indicate that ischemia causes a rapid inhibition of CaM kinase II activity and a change in the partitioning of the enzyme between the c ytosol and particulate fractions. CaM kinase II is a multifunctional p rotein kinase, and the loss of activity may play a critical role in in itiating the changes leading to ischemia-induced cell death. To identi fy a structural basis for the decrease in enzyme activity, tryptic pep tide maps of CaM kinase II phosphorylated in vitro were compared. Phos phopeptide maps of CaM kinase alpha from particulate fractions of cont rol and ischemic samples revealed not only reduced incorporation of ph osphate into the protein but also the absence of a limited number of p eptides in the ischemic samples. This suggested that certain sites are inaccessible, possibly due to a conformational change, a covalent mod ification of CaM kinase II, or steric hindrance by an associated molec ule. Verifying one of these possibilities should help to elucidate the mechanism of ischemia-induced modulation of CaM kinase II.