Glutamate-Induced Loss of Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Cultured Rat Hippocampal Neurons

Abstract: The exposure of cultured rat hippocampal neurons to 500 µ M glutamate for 20 min induced a 55% decrease in the total Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca²⁺-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N -methyl- d -aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca²⁺ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.     

Activity of Ca2+/Calmodulin-Dependent Protein Kinase II Following Ischemia: A Comparison Between CA1 and Dentate Gyrus in a Hippocampal Slice Model

Abstract: 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. However, the microtubule-associated protein MAP2, known to be a substrate for the Ca²⁺-dependent proteases (calpains), showed extensive proteolysis evident after 90 min of reoxygenation after ischemia.     

Ca2+/Calmodulin Kinase II Translocates in a Hippocampal Slice Model of Ischemia

Abstract: Rat hippocampal slices were exposed to conditions that simulate an ischemic insult, and the subcellular distribution and the enzymatic activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase) were monitored. Semiquantitative western blots using a monoclonal antibody to the 50-kDa α subunit showed that there was a significant redistribution of the enzyme from a supernatant to a pellet fraction after 10 min of an anoxic/aglycemic insult. No significant change in the total amount of CaM kinase enzyme was detected in the homogenates for up to 20 min of exposure to the insult. Ca²⁺/CaM-dependent enzyme activity did not significantly change in the pellet during the 20-min insult. Supernatant activity decreased throughout the insult. The persistence of Ca²⁺/CaM-dependent CaM kinase activity in the pellet fraction and the detected movement of enzyme from the supernatant to the pellet indicate that redistribution may be an important mechanism in regulating the cellular location of CaM kinase activity.     

Regional and Temporal Alterations in Ca2+/Calmodulin-Dependent Protein Kinase II and Calcineurin in the Hippocampus of Rat Brain After Transient Forebrain Ischemia

We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.     

Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice

Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule, we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr(286) CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.     

Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle

The relation between CaM kinase II activity and high Ca²⁺-mediated stress responses was studied in cultured vascular smooth muscle cells. Treatment with ionomycin (1 M) for 5 min caused a significant loss of CaM kinase II activity in whole cell homegenates and prominent vesiculation of the endoplasmic reticulum (ER). Similar losses of CaM kinase II activity were observed in the soluble lysate as assessed by activity measurements and Western blotting. Examination of the post-lysate particulate fraction showed that the loss of CaM kinase II from the soluble lysate was accompanied by a redistribution of CaM kinase II to this fraction. The ionomycin-mediated response was limited to this concentration (1 M); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 M) or ATP (100 M) did not cause a shift in CaM kinase II distribution. Treatment with neither the CaM kinase II inhibitor KN-93 nor the phosphatase inhibitor okadaic acid altered the ionomycin-induced redistribution indicating that CaM kinase II activation and/or phosphorylation was not part of the mechanism. The response, however, was eliminated when the cells were treated in Ca²⁺-free medium. Washout of ionomycin led to only a partial restoration of the kinase activity in the soluble fraction after 10 min. Immunofluorescence microscopy of resting cells indicated colocalization of antibodies to CaM kinase II and an ER protein marker. ER vesiculation induced by ionomycin coincided with a parallel redistribution of CaM kinase II and ER marker proteins. These data link ionomycin-induced ER restructuring to a progressive redistribution of CaM kinase II protein to an insoluble particulate fraction and loss of cellular CaM kinase II activity. We propose that redistribution of CaM kinase II and loss of cellular activity are components of a common Ca²⁺-overload induced cellular stress response in cells.     

Ischemia-Induced Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II: Potential Role in Neuronal Damage

The activities of Ca2+/calmodulin (CaM)-dependent, Ca2+/phospholipid-dependent, and cyclic AMP-dependent protein kinases (CaM-KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM-KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM-KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM-KII 50-kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM-KII in the pellet. Thus, the loss of CaM-KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM-KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM-KII activity parallels neuronal damage associated with ischemia; down-regulation of CaM-KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM-KII phosphorylation.     

Changes in Phosphorylation of τ During Ischemia and Reperfusion in the Rabbit Spinal Cord

Abstract: The microtubule-associated protein τ plays an important role in the dynamics of microtubule assembly necessary for axonal growth and neurite plasticity. Ischemia disrupts the neuronal cytoskeleton both by promoting proteolysis of its components and by affecting kinase and phosphatase activities that alter its assembly. In this study the effect of ischemia and reperfusion on the expression and phosphorylation of τ was examined in a reversible model of spinal cord ischemia in rabbits. τ was found to be dephosphorylated in response to ischemia with a time course that closely matched the production of permanent paraplegia. Dephosphorylation of τ was limited to the caudal lumbar spinal cord. In a similar manner, Ca²⁺/calmodulin-dependent kinase II activity was reduced only in the ischemic region. Thus, dephosphorylation of τ is an early marker of ischemia as is the rapid loss of Ca²⁺/calmodulin-dependent kinase II activity, τ, however, was rephosphorylated rapidly during reperfusion at site(s) that cause a reduction in its electrophoretic mobility regardless of the neurological outcome. Alterations in phosphorylation or degradation of τ may affect microtubule stability, possibly contributing to disruption of axonal transport but also facilitating neurite plasticity in a regenerative response.     

Persistent Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia

Abstract: The influence of brain ischemia on the subcellular distribution and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca²⁺/calmodulin-dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.     

Effect of ischemia in vivo and oxygen-glucose deprivation in vitro on NOS pools in the spinal cord: comparative study

1. This study was performed to compare both the Ca(2+)-dependent nitric oxide synthase (NOS) activity and the neuronal nitric oxide synthase immunoreactivity (nNOS-IR) in the rabbit lumbosacral spinal cord after 15 min abdominal aorta occlusion (ischemia in vivo) and oxygen-glucose deprivation of the spinal cord slices for 45 and 60 min (ischemia in vitro). All ischemic periods were followed by 15, 30 and 60 min reoxygenation in vitro. 2. Catalytic nitric oxide synthase activity was determined by the conversion of (L)-[(14)C]arginine to (L)-[(14)C]citrulline. Neuronal nitric oxide synthase immunoreactivity in the spinal cord was detected by incubation of sections with polyclonal sheep-nNOS-primary antibody and biotinylated anti-sheep secondary antibody. 3. Our results show that ischemia in vivo and the oxygen-glucose deprivation of spinal cord slices in vitro result in a time-dependent loss of constitutive NOS activity with a partial restoration of enzyme activity during 15 and 45 min ischemia followed by 30 min of reoxygenation. A significant decrease of enzyme activity was found during 60 min ischemia alone, which persisted up to 1 h of oxygen-glucose restoration. The upregulation of neuronal nitric oxide synthase was observed in the ventral horn motoneurons after all ischemic periods. The remarkable changes in optical density of neuronal nitric oxide synthase immunoreactive motoneurons were observed after 45 and 60 min ischemia in vitro followed by 30 and 60 min reoxygenation. 4. Our results suggest that the oxygen-glucose deprivation followed by reoxygenation in the spinal cord is adequately sensitive to monitor ischemia/reperfusion changes. It seems that 15 min ischemia in vivo and 45 min ischemia in vitro cause reversible changes, while the decline of Ca(2+)-dependent nitric oxide synthase activity after 60 min ischemic insult suggests irreversible alterations.     

cDNA cloning and sequencing of Ca2+ calmodulin-dependent protein kinase IIα subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity, known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, se.) produces first mild ataxia followed by paralysis in 7-14 days in hens. DFP treatment also increases in vitro autophosphorylation of Ca²⁺ calmodulin-dependent protein kinase II (CaM kinase II) and the phosphorylation of several cytoslceletal proteins in the hen brain. To investigate whether increase in CaM kinase II activity is associated with increased expression of its mRNA, we cloned and sequenced CaM kinase II a subunit cDNA, and used it to study CaM kinase II expression in brain regions and spinal cord. Hen CaM kinase II subunit differs in 7 amino acids from that of rat CaM kinase II. Its mRNA occurs predominantly as a 6.7 kb message, which is very close to that of human CaM kinase II a subunit. Northern blot analysis showed a transient increase in CaM kinase II subunit mRNA in the cerebellum and spinal cord of DFP-treated chickens. The increase in CaM kinase II mRNA expression is consistent with the previously reported increase in its activity in brain and spinal cord, and its increased expression only in cerebellum and spinal cord, which are sensitive to the Wallerian-type degeneration characteristic of OPIDN, suggests the probable role of this enzyme in delayed neurotoxicity.     

Effect of diabetes on calcium/calmodulin dependent protein kinase-II from rat brain.

Changes in the protein levels and activity of Ca2+/Calmodulin dependent protein kinase II (CaM kinase II) level were studied in cytosolic and particulate fractions from cerebral hemisphere, cerebellum, brain stem, thalamus and hypothalamus regions of rat brain after 4 and 12 weeks of induction of diabetes. Streptozotocin induced diabetes, resulted in pronounced increase of CaM kinase II activity as determined by the kinase activity assay. The total amount of enzyme protein (alpha-subunit specific) also showed increase as revealed by western blotting. Parallel studies were also made in age matched control rats and insulin treated diabetic rats. The increase in CaM kinase II activity was more pronounced in the 12 weeks diabetic group. Insulin treatment of diabetic rats, resulted in recovery of enzyme activity near to control values from majority of the brain regions studied. The expression of alpha-subunit specific CaM kinase II correlates with the enzyme activity in the diabetic rat brain.     

Time Course of the Translocation and Inhibition of Protein kinase C During Complete Cerebral Ischemia in the Rat

Abstract: The time course for the ischemia-induced changes in the subcellular distribution of protein kinase C (PKC) (α), (β311). and (γ) and the activity of PKC were studied in the neocortex of rats subjected to 1, 2, 3, 5, 10, and 15 min of global cerebral ischemia. In the particulate fraction, a 14-fold increase in PKC (γ) levels was seen at 3 min of ischemia, which further increased at 5–15 min of ischemia. At 15 min of ischemia, PKC (γ) and (βll) levels had increased two- and six-fold, respectively. In the cytosolic fraction, a transient early 1.4-fold increase in PKC (βll) and PKC (γ) levels was seen, whereas no change in the levels PKC (α) was noted. PKC (γ) levels then progressively declined, reaching 50% at 15 min of ischemia. At 5 min of ischemia, a 43% decrease in PKC activity was seen in the particulate fraction, reaching 50% at 15 min of ischemia concomitant with a 27% decrease in the cytosolic fraction. There was no change in the activator-independent PKC activity. Pretreatment with the ganglioside AGF2 prevented the redistribution of PKC (γ) in the particulate fraction at 5 min. but not at 10 min of ischemia. The observed time course for the translocation of PKC (γ) parallels the ischemia-induced release of neurotransmitters and increased levels of diacylglycerols, arachidonate, and intra-cellular calcium and delineates this subspecies as especially ischemia-sensitive. Ganglioside pretreatment delayed the translocation of PKC (γ), possibly by counteracting the effects of ischemia-induced factors that favor PKC binding to cell membranes.     

Regulatory Properties of Calcium/Calmodulin-Dependent Protein Kinase II in Rat Brain Postsynaptic Densities

Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-independent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon autophosphorylation of both subunits of the enzyme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 +/- 5% of the total activity within 1-2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophosphorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the alpha- and beta-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281-309 corresponding to a portion of the regulatory domain (residues 281-314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50 = 3.6 +/- 0.8 microM). Binding of Ca2+/CaM to peptide 281-309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281-309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.     

Global Forebrain Ischemia Induces a Posttranslational Modification of Multifunctional Calcium- and Calmodulin-Dependent Kinase II

The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.     

Protein kinase C alterations in the fetal rat brain after global ischemia

Marked changes in the intracellular localization of brain protein kinase C are evident after global ischemia generated by the restriction of the placental blood flow in the near-term rat embryo. A rapid (5 min) ischemia-dependent translocation of the enzyme from the cytosol to the particulate membrane fraction, which is completely reversible upon reperfusion, is observed. After 30 min of ischemia, substantial losses in protein kinase C activity and content as measured by [3H]phorbol dibutyrate binding are apparent. This is accompanied by a marked increase of a Ca2+-phosphatidylserine-independent kinase activity, already evident after 5 min of ischemia. By 15 or 30 min the total activity of the latter enzyme is equally distributed between the particulate and the cytosol fractions and is more than 3-fold higher in ischemic in comparison to naive animals. Activation and possible deregulation of protein kinase C are proposed to represent an initial step in the pathophysiology of brain ischemia.     

Moderately Different NADPH-Diaphorase Positivity in the Selected Peripheral Nerves after Ischemia/ Reperfusion Injury of the Spinal Cord in Rabbit

1. To vicariously investigate the nitric oxide synthase (NOS) production after spinal cord injury, NADPH-d histochemistry was performed on the selected peripheral nerves of adult rabbits 7 days after ischemia. The effect of transient spinal cord ischemia (15 min) on possible degenerative changes in the motor and mixed peripheral nerves of Chinchilla rabbits was evaluated.2. The NADPH-diaphorase histochemistry was used to determine NADPH-diaphorase activity after ischemia/reperfusion injury in radial nerve and mediane nerve isolated from the fore-limb and femoral nerve, saphenous nerve and sciatic nerve separated from the hind-limb of rabbits. The qualitative analysis of the optical density of NADPH-diaphorase in selected peripheral nerves demonstrated different frequency of staining intensity (attained by UTHSCSA Image Tool 2 analysis for each determined nerve).3. On the seventh postsurgery day, the ischemic spinal cord injury resulted in an extensive increase of NADPH-d positivity in isolated nerves. The transient ischemia caused neurological disorders related to the neurological injury—a partial paraplegia. The sciatic, femoral, and saphenous nerves of paraplegic animals presented the noticeable increase of NADPH-d activity. The mean of NADPH-diaphorase intensity staining per unit area ranged from 134.87 (±32.81) pixels to 141.65 (±35.06) pixels (using a 256-unit gray scale where 0 denotes black, 256 denotes white) depending on the determined nerve as the consequence of spinal cord ischemia. The obtained data were compared to the mean values of staining intensity in the same nerves in the limbs of control animals (163.69 (±25.66) pixels/unit area in the femoral nerve, 173.00 (±32.93) pixels/unit area in saphenous nerve, 186.01 (±29.65) pixels/unit area in sciatic nerve). Based on the statistical analysis of the data (two-way unpaired Mann–Whitney test), a significant increase (p≤0.05) of NADPH-d activity in femoral and saphenous nerve, and also in sciatic nerve (p≤0.001) has been found. On the other hand, there was no significant difference between the histochemically stained nerves of fore-limbs after ischemia/reperfusion injury and the same histochemically stained nerves of fore-limbs in control animals.4. The neurodegenerative changes of the hind-limbs, characterized by damage of their motor function exhibiting a partial paraplegia after 15 min spinal cord ischemia and subsequent 7 days of reperfusions resulted in the different sensitivity of peripheral nerves to transient ischemia. Finally, we suppose that activation of NOS indirectly demonstrable through the NADPH-d study may contribute to the explanation of neurodegenerative processes and the production of nitric oxide could be involved in the pathophysiology of spinal cord injury by transient ischemia.     

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Neurite Outgrowth of PC12 Cells

Abstract: To investigate the physiological role of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca²⁺/calmodulin-independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.     

Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.     

Increased protein kinase C activity in the central nervous system of the newt during limb regeneration.

Protein kinase C (PKC) activity was examined in the CNS of the newt Pleurodeles waltlii undergoing regeneration after limb amputation. In the spinal cord and brain of control newts, the level of PKC activity was virtually the same for the cytosolic and the particulate fractions. At days 7 and 14 after amputation of two limbs, a twofold increase in overall PKC activity occurred in the spinal cord and accounted for increased membrane-bound activity, while cytosolic activity was not significantly impaired. In contrast, overall PKC activity was not affected in brain. However, a twofold increase in the brain particulate fraction occurred at day 14 while cytosolic activity decreased proportionately. Similar alterations were observed in newts undergoing one or multiple limb amputations. Such changes in PKC activity neither occurred in the CNS of newt after limb denervation nor in the CNS of limb amputated frog Rana temporaria, an Amphibian which is unable to regenerate. Taken together, these results provide evidence that PKC of the CNS is involved in the regeneration process of newts. Changes in activation-associated PKC distribution proceeded through different mechanisms: long-lasting increase in membrane bound activity with a net increase of overall activity in the spinal cord, and long-term redistribution of enzyme activity to the particulate fraction in brain.