Long-lasting decrease in neuronal Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent seizures.
Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) activity was evaluated in a well-characterized in vitro model of epileptiform activity. Long-lasting spontaneous recurrent seizure (SRS) activity was induced in hippocampal neuronal cultures by exposure to low Mg2+ media for 3 h. Analysis of endogenous Ca2+/calmodulin-dependent phosphorylation revealed a significant long-lasting decrease in 32P incorporation into the alpha (50 kDa) and beta (60 kDa) subunits of CaM kinase II in association with the induction of SRS activity in this preparation. Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptides, Autocamtide-2 and Syntide II, was also significantly reduced following the induction of SRSs and persisted for the life of the neurons in culture. The decrement in CaM kinase II activity associated with low Mg2+ treatment remained significantly decreased when values were corrected for changes in levels of alpha subunit immunoreactivity and neuronal cell loss. Addition of the protein phosphatase inhibitors, okadaic acid and cyclosporin A, to the phosphorylation reaction did not block the SRS-associated decrease in substrate phosphorylation, indicating that enhanced phosphatase activity was not a contributing factor to the observed decrease in phosphate incorporation. The findings of this study demonstrate that CaM kinase II activity is decreased in association with epileptogenesis observed in these hippocampal cultures and may contribute to the production and maintenance of SRSs in this model.
Blair
R E
RE
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0599, USA.
Churn
S B
SB
Sombati
S
S
Lou
J K
JK
DeLorenzo
R J
RJ
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D006624:Hippocampus / Q000378:metabolism*; D015636:Magnesium Chloride; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012008:Recurrence; D012640:Seizures / Q000139:chemically induced / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*
D015636:Magnesium Chloride; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases