Kohji Fukunaga

Among the molecular mechanisms that have been proposed to contribute to long-term potentiation in hippocampus are the activation and autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Here we report that high, but not low frequency stimulation applied to two groups of CA1 afferents resulted in a long lasting increase in the Ca(2+)-independent and total activities of the enzyme as well as an increase in the ratio of Ca(2+)-independent to total activity. The effect

A Ca2+- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest t

Induction of long-term potentiation in the CA1 region of hippocampal slices is associated with increased activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (Fukunaga, K., Stoppini, L., Miyamoto, E., and Muller, D. (1993) J. Biol. Chem. 268, 7863-7867). Here we report that application of high but not low frequency stimulation to two groups of afferents in the CA1 region of 32P-labeled slices resulted in the phosphorylation of two major substrates of this enzyme, synapsin I an

In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA

Polyclonal antibodies against Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both subunits of the enzyme with Mrs 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neuronal somata and dendrites and weak immunore

Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor ag

We here investigated endothelial nitric oxide synthase (eNOS) expression after 10 minutes of forebrain ischemia. Real-time polymerase chain reaction, immunoblots and immunohistochemical studies revealed up-regulation of eNOS expression in the hippocampal CA1 subfield of gerbil. Immunoreactivity of eNOS significantly increased in endothelium but neither in neurons nor astrocytes after 6 to 168 hours of reperfusion. An increased Akt activity preceded the postischemic eNOS up-regulation. Intracereb

Growth factors including insulin-like growth factor-1 (IGF-1) promote cell survival in ischemic brain injury. Stimulation of IGF-1 receptor coupled with tyrosine kinase activates phosphatidylinositol 3-kinase and subsequently, protein kinase B (Akt) in hippocampal neurons. Here we introduce a new approach of signal transduction therapy for brain damage occurring in ischemic insult. As has been shown for IGF-1, intracerebroventricular injection of sodium orthovanadate, a protein tyrosine phosphat

Conditions that regulate the generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in cultured rat cerebellar granule cells have been investigated. Under basal conditions, 4-5% of total CaM-kinase II activity, assayed in the presence of Ca2+/CaM, was the Ca2(+)-independent form active in the presence of EGTA. Depolarization with 56 mM K+ produced a transient increase to 9% Ca2+ independence within 15 s followed by a decline to 5-6% at 10 min. Th

Using autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) as substrate, we now find that long-term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A-sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+-dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with c