Ca2+/calmodulin-dependent protein kinase II in the rat cerebellum: an immunohistochemical study with monoclonal antibodies specific to either alpha or beta subunit.

Monoclonal antibodies specific to either alpha or beta subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for alpha subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these alpha subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. beta Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of alpha and beta subunits of CaM kinase II in the cerebellum were demonstrated.     

Ca/calmodulin-dependent protein kinase II in the rat cerebellum: An immunohistochemical study with monoclonal antibodies specific to either α or β subunit

Monoclonal antibodies specific to either α or β subunit of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for α subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these α subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. β Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of α and β subunits of CaM kinase II in the cerebellum were demonstrated.     

Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.     

Expression and phosphorylation of delta-CaM kinase II in cultured Alzheimer fibroblasts

Dysregulation of calcium homeostasis is among the major cellular alterations in Alzheimer's disease (AD). We studied Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), one of the major effectors regulating neuronal responses to changes in calcium fluxes, in cultured skin fibroblasts from subjects with sporadic AD. We found, by using PCR and Western analysis, that human fibroblasts express the delta-isoform of this kinase, and that CaM kinase II is the major Ca(2+)/calmodulin-dependent kinase in these cells. Protein expression level of the kinase was not significantly different in AD fibroblasts. However, the total activity of the kinase (stimulated by Ca(2+)/calmodulin) was significantly reduced in AD cell lines, whereas Ca(2+)-independent activity was significantly enhanced. The percent autonomy of the kinase (%Ca(2+)-independent/Ca(2+)-dependent activity) in AD cell lines was 62.8%, three-fold the corresponding percentage in control fibroblasts. The abnormal calcium-independent activity was not due to enhanced basal autophosphorylation of Thr(287). The observed abnormalities, if present in brain tissue, may be implicated either in dysfunction of neuroplasticity and cognitive functions or in dysregulation of cell cycle.     

Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.     

Contrasting patterns in the localization of glutamic acid decarboxylase and Ca2+/calmodulin protein kinase gene expression in the rat central nervous system.

The expression of the genes encoding the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) and the 67,000 mol. wt form of glutamic acid decarboxylase was examined throughout the rat central nervous system. In situ hybridization histochemistry, using cRNA probes, revealed a dense population of CaM II kinase alpha-expressing cells throughout the telencephalon and diencephalon. CaM II kinase alpha mRNA was also expressed in the midbrain, cerebellum and medulla oblongata, but at greatly reduced levels. No CaM II kinase alpha gene expression was detected in nuclei producing monoamines or acetylcholine. By contrast, the glutamic acid decarboxylase gene was moderately to highly expressed throughout the central nervous system. In several regions there was a complementarity in the distributions of cells expressing the glutamic acid decarboxylase or CaM II kinase alpha genes. Cells in certain nuclei such as the thalamic reticular nucleus or globus pallidus showed glutamic acid decarboxylase gene expression only; others such as the majority of the dorsal thalamic nuclei showed CaM II kinase alpha gene expression only. Several regions contained both glutamic acid decarboxylase and CaM II kinase alpha expressing cells. However, simultaneous immunostaining for both proteins revealed only two regions where CaM II kinase alpha and glutamic acid decarboxylase immunoreactivity were colocalized: the cerebellar Purkinje cells and the commissural nucleus of the stria terminalis. The results imply that CaM II kinase alpha is primarily expressed in non-GABAergic neurons. In several regions CaM II kinase alpha mRNA is concentrated in nuclei known to contain populations of neurons that use excitatory amino acid transmitters.     

Calcium/calmodulin dependent protein kinase II mRNA in the gerbil brain after cerebral ischemia.

The effect of transient cerebral ischemia on the expression of Ca2+/calmodulin dependent protein kinase II (CaM kinase II) mRNA in the gerbil brain was analyzed by Northern blots using cDNA clones for CaM kinase II. Ten minutes of bilateral carotid occlusion and 30 min of reperfusion resulted in reduced protein levels for alpha and beta subunits of the CaM kinase II, decreasing to 35% of control levels at 24 h. Recovery of immunoreactivity was detected in the cortex after 48 h. Eight to twelve hours after ischemia, the cortex showed a decrease in alpha and beta CaM kinase II mRNA levels. By 12-24 h of reperfusion the level of CaM kinase II mRNA was reduced to 26% of the control mRNA levels. CaM kinase II mRNA levels recovered by 48 h after ischemia, coinciding with the increase in CaM kinase II protein immunoreactivity. These results suggest that CaM kinase II is involved in neuronal survival through the reorganization of the neuroarchitecture and that the regulation of this role is controlled at the level of gene expression.     

CaM kinase II-alpha activity,levels and Ca/calmodulin dependent phosphorylation of substrate proteins in mice brain during fatal murine cerebral malaria

The activity and levels of CaM kinase II-alpha was investigated in the cytosolic and membrane fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). In parallel, Ca(2+)/Calmodulin dependent phosphorylation of target substrate proteins was studied using syntide-2 as substrate. Pathology of FMCM resulted in decreased CaM kinase-II activity in both cortex and cerebellum though western analysis revealed no appreciable changes in the levels of CaM kinase-II alpha in cytosol and membrane fractions from control and cerebral malaria infected brain. Given the abundant expression of Cam kinase-II in neuronal tissue, its significance in neurotransmitter release and synthesis and signal transduction during apoptosis, decreased levels of enzyme activity and altered phosphorylation of substrate proteins by CaM kinase II may serve as important cues in understanding the CaM kinase signal transduction events central to neurological disorders during FMCM.     

Immunohistochemical localization of Ca(2+)/calmodulin-dependent protein kinase kinase beta in the rat central nervous system

We examined regional and intracellular distribution of Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaM-KK beta), which activated Ca(2+)/calmodulin-dependent protein kinase I and IV (CaM-K I and IV) immunohistochemically in the central nervous system of the rat by light and electron microscopy. Although most neurons in the brain and spinal cord exhibited the immunoreactivity, no labeled neurons were observed in the globus pallidus or entopeduncular nucleus, and only a small number of neurons showed weak immunoreactivity in the substantia nigra pars reticulata. In general, the immunoreactivity was observed both in the cytoplasm and cellular nucleus, although the immunoreactivity was not found in the cellular nucleus in some large neurons such as in the mesencephalic trigeminal nucleus, lateral vestibular nucleus or gigant cellular reticular formation. As to motoneurons of the cranial nerve nuclei and the anterior horn of the spinal cord, they revealed the immunoreactivity both in the cytoplasm and nucleus. The reaction product appeared as fine granules in the cytoplasm and nucleus under light microscopy. Electron microscopic observations confirmed that the reaction product was localized mainly on the Golgi apparatus or on the nuclear chromatin. Immunolabeling for antibody against CaM-KK beta was discussed with the distribution of CaM-K I, IV and another CaM-KK, CaM-KK alpha, in the central nervous system.     

Heparin inhibits phosphorylation and autonomous activity of Ca(2+)/calmodulin-dependent protein kinase II in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) hyperproliferation is a characteristic feature of both atherosclerosis and restenosis seen after vascular surgery. A number of studies have shown that heparin inhibits VSMC proliferation in vivo and in culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering Ca(2+) regulated pathways involved in mitogenic signaling in VSMC, we analyzed the effect of heparin on multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II) which is abundantly expressed in VSMC. Using activity assays, radioactive labeling, and immunoprecipitation it was found that heparin inhibits the overall phosphorylation of the delta-subunit of CaM kinase II which is consistent with inhibition of autophosphorylation-dependent, Ca(2+)/calmodulin-independent CaM kinase II activity. This effect was less evident in heparin-resistant cells, consistent with a role for CaM kinase II in mediating the antiproliferative effect of heparin. Finally, the effects of pharmacological inhibitors of phosphatases like okadaic acid, calyculin, and tautomycin suggest that heparin inhibits CaM kinase II phosphorylation by activating protein phosphatases 1 and 2A. These findings support the hypothesis that alterations in calcium-mediated mitogenic signaling pathways may be involved in the antiproliferative mechanism of action of heparin.     

CaMKII in the cardiovascular system: sensing redox states

The multifunctional Ca(2+)- and calmodulin-dependent protein kinase II (CaMKII) is now recognized to play a central role in pathological events in the cardiovascular system. CaMKII has diverse downstream targets that promote vascular disease, heart failure, and arrhythmias, so improved understanding of CaMKII signaling has the potential to lead to new therapies for cardiovascular disease. CaMKII is a multimeric serine-threonine kinase that is initially activated by binding calcified calmodulin (Ca(2+)/CaM). Under conditions of sustained exposure to elevated Ca(2+)/CaM, CaMKII transitions into a Ca(2+)/CaM-autonomous enzyme by two distinct but parallel processes. Autophosphorylation of threonine-287 in the CaMKII regulatory domain "traps" CaMKII into an open configuration even after Ca(2+)/CaM unbinding. More recently, our group identified a pair of methionines (281/282) in the CaMKII regulatory domain that undergo a partially reversible oxidation which, like autophosphorylation, prevents CaMKII from inactivating after Ca(2+)/CaM unbinding. Here we review roles of CaMKII in cardiovascular disease with an eye to understanding how CaMKII may act as a transduction signal to connect pro-oxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of cardiovascular disease.     

Dendritic localization of type II calcium calmodulin-dependent protein kinase mRNA in normal and reinnervated rat hippocampus.

In situ hybridization histochemistry has revealed a diffuse distribution of the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) mRNA in the neuropil of regions containing CaM II kinase alpha-expressing cells and has led some to propose that it may be expressed in dendrites. In order to determine if CaM II kinase alpha mRNA is expressed in dendrites and if the gene encoding CaM II kinase alpha is regulated in response to synaptic reinnervation, we examined its expression in the hippocampus of normal rats, of rats that had received a unilateral injection of kainic acid and of rats with a unilateral entorhinal cortex lesion. The relatively specific elimination of the CA3 pyramidal cells by kainate lesions precisely correlated with the loss of CaM II kinase alpha cRNA hybridization in the stratum radiatum as well as the stratum pyramidale. Following entorhinal cortex lesions, during the period of new synapse formation in the dentate gyrus, there was no detectable change in the level of CaM II kinase alpha gene expression. These data suggest that CaM II kinase alpha mRNA is expressed in the dendrites of hippocampal pyramidal cells and, therefore, is likely to be expressed in dendrites in other regions of the central nervous system exhibiting CaM II kinase alpha cRNA labeling in the neuropil. However, changes in expression were not found to accompany new synapse formation.     

Calcium/calmodulin-dependent kinase IV in immune and inflammatory responses: novel routes for an ancient traveller

Ca(2+) is a pivotal second messenger controlling the activation of lymphocytes. Crucial events in the social life of immunocytes are regulated by the calcium/calmodulin complex (Ca(2+)/CaM), which controls the activation status of many enzymes, including the Ca(2+)/CaM-dependent Ser-Thr kinases (CaMK) I, II and IV. Although CaMKI and CaMKII are expressed ubiquitously, CaMKIV is found predominately in cells of the nervous and immune systems. To be active, CaMKIV requires binding of Ca(2+)/CaM and phosphorylation by CaMKKalpha or beta. The requirement of two CaM kinases in the same signalling pathway led to the concept of a CaM kinase cascade. In this review, we focus on the roles of CaMKK and CaMKIV cascades in immune and inflammatory responses.     

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca²⁺ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca²⁺-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca²⁺-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca²⁺ (Ca_0.5 = 780 nM) and a low sensitivity to vanadate (IC₅₀ = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca²⁺ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca²⁺ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca²⁺ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca²⁺ pumping activity.     

Possible role for Ca(2+) calmodulin-dependent protein kinase II as an effector of the fertilization Ca(2+) signal in mouse oocyte activation

The present study shows that Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) is physiologically activated in fertilized mouse oocytes and is involved in the Ca(2+) response pathways that link the fertilization Ca(2+) signal to meiosis resumption and cortical granule (CG) exocytosis. After 10 min of insemination, CaM kinase II activity increased transiently, then peaked at 1 h and remained elevated 30 min later when most of the oocytes had completed the emission of the second polar body. In contrast, in ethanol-activated oocytes the early transient activation of CaM kinase II in response to a monotonic Ca(2+) rise was not followed by any subsequent increase. Inhibition of CaM kinase II by 20 micromol/l myristoylated-AIP (autocamtide-2-related inhibitory peptide) negatively affected MPF (maturing promoting factor) inactivation, cell cycle resumption and CG exocytosis in both fertilized and ethanol-activated oocytes. These results indicate that the activation of CaM kinase II in mouse oocytes is differently modulated by a monotonic or repetitive Ca(2+) rise and that it is essential for triggering regular oocyte activation.     

Expression of Ca2+/calmodulin-dependent protein kinase (CaMK) Ibeta2 in developing rat CNS

We observed the onset time and distribution pattern of beta2 isoform of Ca2+/calmodulin-dependent protein kinase I (CaMKIbeta2) in the CNS of the rat during the embryonic period until birth using an immunohistochemical method. The expression of CaMKIbeta2 started at embryological day 10 when the three primary brain vesicles and neural tube are generated from the neural plate. During the embryonic period, highly immunoreactive products were ubiquitously detected in neurons in the CNS, although neurons in the caudate-putamen and globus pallidus were faintly immunostained or immunonegative. High expression of CaMKIbeta2 persisted in the olfactory bulb, lymbic system, neocortex, septal nuclei, amygdala complex, some hypothalamic nuclei, pontine nuclei, Purkinje cells and granule cells in the cerebellar cortex through the developing period. At the subcellular level, CaMKIbeta2 was strongly expressed in nuclei of neurons but faintly in their cytoplasm, suggesting that this protein has an important role in the nuclear signaling pathway.This study demonstrates that expression of CaMKIbeta2 begins at the earliest developmental stage of the rat CNS and persists through the developing period.