Autonomous activity and autophosphorylation of CAMPK-II in rat hippocampal slices: effects of tissue preparation.
Measurement of the proportion of calcium/calmodulin-stimulated protein kinase II (CaMPK-II) that is autonomously active or phosphorylated on Thr(286) is thought to provide an index of the degree to which CaMPK-II in a tissue has been activated. We have examined how various ways of handling hippocampal tissue can alter these properties. Both autonomous activity and phospho-Thr(286) content was high in freshly dissected hippocampus or freshly cut hippocampal slices. After incubation of hippocampal slices in artificial cerebrospinal fluid for 120 min, both properties of CaMPK-II decreased to a steady state level. Freeze-thaw or cutting the equilibrated slices could rapidly increase both autonomous activity and phospho-Thr(286) immunoreactivity of CaMPK-II. These increases were comparable to changes induced by experimental treatment. Therefore, our results suggest that considerable care needs to be taken over the way in which hippocampal slices are handled.
Lengyel
I
I
The Neuroscience Group, Faculty of Medicine and Health Sciences, University of Newcastle, Callaghan, NSW, Australia.
Cammarota
M
M
Brent
V A
VA
Rostas
J A
JA
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005615:Freezing; D006624:Hippocampus / Q000378:metabolism*; D066298:In Vitro Techniques; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D020858:Tissue and Organ Harvesting / Q000379:methods*
D004791:Enzyme Inhibitors; D010455:Peptides; C085903:autocamtide-2; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases