A non-radioactive assay for the cAMP-dependent protein kinase activity in rat brain homogenates and age-related changes in hippocampus and cortex

Cyclic AMP-dependent protein kinase (PKA) activity was involved in a number of brain functions such us cognitive process or aging. The measurement of PKA activity is traditionally based on the use of [P-32]ATP in phosphorylat ion of specific protein. Recently non-isotopic PKA assays have been develop ed, but none has been tested on brain homogenates. This work aimed to adapt a fluorimetric method of PKA activity into a novel assay never applied bef ore in brain homogenate, and to characterize the enzyme activity and ratio in hippocampus and cortex from rats of different ages. Optimal conditions o f homogenization and enzyme protections were determined. The method was sen sitive and reproducible (intra-assay and interassay variation was 5.0% and 9.0%, respectively). In hippocampal cytosol, PKA activity was 27 +/-8 and 8 0 +/-9 nmol/min per mg protein in basal and cAMP-stimulated activity, respe ctively. and accounted for 80% of total cell PKA activity. The non-PKA acti vity, assessed by the use of the PKA specific inhibitor (PKI) accounted for 49.0% and 65.0% of endogenous levels in cytosol and membrane, respectively , cAMP-augmenting drugs effects were measured and increase of 53%, 273% and 118% over basal by 10 muM isoproterenol, 100 muM forskolin, 1 muM SP-AMP, respectively, was observed. With respect to the changes in animal age, PKA activity increased from newborn to the mature rats but decreased in older r ats. The PKA ratio was higher in cytosol than in particulate fraction, and was decreased in hippocampal sample from old rats (P<0.05). This last resul t was interpreted as related to the loss of cognitive capacities in old ani mals. (C) 2001 Published by Elsevier Science B.V.