Activity of Ca2+/calmodulin-dependent protein kinase II following ischemia: a comparison between CA1 and dentate gyrus in a hippocampal slice model.

Both CA1 and dentate gyrus regions of the hippocampal slice exhibit an irreversible loss of synaptic transmission after exposure to in vitro ischemic conditions (buffer without oxygen and glucose). However, after shorter durations of ischemia (8-10 min) the CA1 region shows an irreversible loss of synaptic responses, whereas the dentate gyrus region completely recovers synaptic responses upon reoxygenation. To determine biochemical mechanisms underlying this differential susceptibility, we have examined changes in Ca2+/calmodulin-dependent protein kinase II (CaM-KII) and cyclic AMP-dependent protein kinase activities in homogenates from CA1 and dentate gyrus regions of the hippocampal slice after increasing durations of in vitro ischemia. Time-dependent changes in CaM-KII activities were correlated with changes in electrophysiological responses. CA1 homogenates from slices exposed to 1 min of ischemia showed significant increases in CaM-KII activity, whereas there was no significant change in kinase activity in dentate homogenates after 1 min of ischemia. However, after longer durations of ischemia (5, 10, and 20 min) we found a time-dependent reduction in CaM-KII activity in both CA1 and dentate gyrus regions, whereas no change was detected in cyclic AMP-dependent protein kinase activity. Irreversible depression of CaM-KII activity was seen at shorter durations of ischemia (10 min) in the CA1 region than in dentate region (20 min), which correlated with irreversible effects on synaptic responses. Immunoblot analysis showed that the decrease in CaM-KII activity was not due to degradation of CaM-KII protein.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain.

Oligonucleotide DNA probes were used to determine the distribution of mRNAs encoding the alpha- and beta-subunits of Ca2+/calmodulin-dependent protein kinase type II (CaM-KII) in developing rat brain. The regional and temporal distribution of these mRNAs closely paralleled the distribution and developmental appearance previously reported for their respective protein subunits. alpha-Subunit mRNA was barely detectable in sagittal sections at 4 d postnatal but increased as much as 10-fold in frontal cortex by day 16. beta-Subunit mRNA, on the other hand, was readily detected at 4 d postnatal and changed only slightly during development. Telencephalic structures exhibited the highest levels of CaM-KII mRNA and the brain stem displayed the least. alpha-Subunit mRNA was not observed in cerebellar granule cells and was barely detectable in Purkinje cells, while the beta-mRNA was easily detected in both neuronal types. mRNAs for both alpha- and beta-subunits were present in many neuronal cell bodies; however, only the alpha-subunit mRNA was localized to molecular layers of the hippocampus and lamina I of the frontal cortex. These layers of neuropil are relatively cell sparse and contain extensive dendritic arborizations and synaptic contacts. Since polyribosomes have been observed near hippocampal dendritic spines, the localization of alpha-subunit mRNA to dendrites of pyramidal and dentate granule cells suggests that this subunit is synthesized in situ at postsynaptic sites. The co-localization of translational machinery and high concentrations of CaM-KII in postsynaptic elements suggests an important relationship between alpha-subunit synthesis and the maintenance and plasticity of postsynaptic structures.

Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity.

We have examined the role of Thr-286 autophosphorylation in the autoregulation of Ca2+/calmodulin-dependent protein kinase II. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-286 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.

The influence of estrous cycle and intrastriatal estradiol on sensorimotor performance in the female rat.

The influence of estrous cycle and intrastriatal implants of 17 beta-estradiol (17 beta-estradiol). 17 alpha-estradiol (17 alpha-estradiol) or cholesterol on the number of footfaults made by female rats traversing a narrow suspended beam was investigated. Female rats made fewer footfaults on estrus than on other days of the cycle. This was true when testing occurred during either the light or dark phase of the light:dark cycle. Intrastriatal implants of 30% 17 beta-estradiol for 6 hours resulted in a significant improvement in sensorimotor performance as soon as 4 hours after hormone implant and persisting for days. In contrast, intrastriatal implants of either 30% 17 alpha-estradiol or cholesterol had no influence on performance. The extent of hormone diffusion away from the implant cannula was minimal, and the resulting concentration of 17 beta-estradiol in the striatum was less than 10 pg/mg. It is concluded that estradiol has a direct, stereospecific effect in the striatum that influences performance of a skilled motor act in the female rat.