Global forebrain ischemia results in decreased immunoreactivity of calcium/calmodulin-dependent protein kinase II.
Previous studies utilizing crude brain homogenates have shown that forebrain ischemia results in inhibition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (1C3-3D6) directed against the beta- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia-induced inhibition of CaM kinase II. Immunohistochemical investigations showed that the ischemia-induced decreased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia-sensitive cells such as pyramidal cells of the hippocampus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, autophosphorylation of CaM kinase II in the presence of low (7 microM) or high (500 microM) ATP did not alter immunoreactivity of the enzyme with 1C3-3D6. The data demonstrate the production of a monoclonal antibody that recognizes the beta-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. Together with previous studies, the data support the hypothesis that rapid, ischemia-induced inhibition of CaM kinase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke.
Churn
S B
SB
Department of Neurology, Medical College of Virginia, Richmond 23298.
Yaghmai
A
A
Povlishock
J
J
Rafiq
A
A
DeLorenzo
R J
RJ
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002545:Brain Ischemia / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004797:Enzyme-Linked Immunosorbent Assay; D005849:Gerbillinae; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D016548:Prosencephalon / Q000098:blood supply / Q000201:enzymology; D011494:Protein Kinases / Q000378:metabolism*; D012044:Regression Analysis
D000911:Antibodies, Monoclonal; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases