Cytochemical demonstration of aspartate aminotransferase in the mossy-fibre system of the rat hippocampus

A lead-salt procedure was used for the cytochemical demonstration of aspartate aminotransferase (AT) in the CA3 of the rat hippocampus. Cytoplasmic- and mitochondrial-AT isoenzymes were demonstrated in different neuronal structures, but not in astrocytes. Of special interest was the localization of cytoplasmic AT in most mossy-fibre boutons. This might indicate that cytoplasmic AT is responsible for the production of releasable glutamate in the glutamatergic/aspartatergic mossy fibre system.     

Cytochemical demonstration of aspartate aminotransferase in the mossy-fibre system of the rat hippocampus

Summary A lead-salt procedure was used for the cytochemical demonstration of aspartate aminotransferase (AT) in the CA3 of the rat hippocampus. Cytoplasmic- and mitochondrial-AT isoenzymes were demonstrated in different neuronal structures, but not in astrocytes. Of special interest was the localization of cytoplasmic AT in most mossy-fibre boutons. This might indicate that cytoplasmic AT is responsible for the production of releasable glutamate in the glutamatergic/aspartatergic mossy fibre system.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1)     

Distributions of aspartate aminotransferase and malate dehydrogenase activities in rat retinal layers

Aspartate aminotransferase (AAT), an enzyme interconverting glutamate and aspartate, has been suggested to be a marker for glutamatergic and/or aspartatergic neurons. However, AAT, glutamate, and aspartate are also involved in cellular metabolism, e.g., the malate-aspartate shuttle. To investigate the extent to which AAT might be involved in these several functions in retina, the distribution of AAT activity in rat retinal layers was compared to that of malate dehydrogenase (MDH), an enzyme of aerobic metabolism proposed to be physically complexed with AAT in the malate-aspartate shuttle mechanism. The distribution of AAT activity in retinal layers closely paralleled that of MDH (correlation coefficient AAT versus MDH = 0.93). AAT activity was proportionately higher than MDH in the photoreceptor inner segments, containing a high density of mitochondria, and in the outer plexiform layer (OPL), containing photoreceptor terminals and bipolar and horizontal cell processes. The amount of total AAT activity in the inner segments related to the mitochondrial isoenzyme is almost twice that in the other layers tested, including the OPL. The correlation between AAT and MDH activities is consistent with AAT involvement in retinal energy metabolism, although other functions, such as neurotransmission, are possible.     

Dementia of the Alzheimer's Type: Changes in Hippocampal L-[3H]Glutamate Binding

Abstract: Glutamate or a related excitatory amino acid is thought to be the major excitatory neurotransmitter of hippocampal afferents, intrinsic neurons, and efferents. We have used an autoradiographic technique to investigate the status of excitatory amino acid receptors in the hippocampal formation of patients dying with dementia of the Alzheimer type (DAT). We examined l-[³H]glutamate binding to sections from the hippocampal formation of six patients dying of DAT and six patients without DAT and found marked reductions in total [³H]glutamate binding in all regions of hippocampus and adjacent parahippocampal cortex in DAT brains as compared to controls. When subtypes of excitatory amino acid receptors were assayed, it was found that binding to the N -methyl-d-aspartate (NMDA)-sensitive receptor was reduced by 75–87%, with the greatest loss found in stratum moleculare and stratum pyramidale of CA1. Binding to quisqualate (QA)-sensitive receptors was reduced by 45–69%. There were smaller reductions (21–46%) in GABA_A receptors in DAT cases. Muscarinic cholinergic receptors assayed in adjacent sections of hippocampal formation were unchanged in DAT. Benzodiazepine receptors were reduced significantly only in parahippocampal cortex by 44%. These results suggest that glutamatergic neurotransmission within the hippocampal formation is likely to be severely impaired in Alzheimer's disease. Such impairment may account for some of the cognitive decline and memory deficits that characterize DAT.     

Alanine aminotransferase in the rat nervous system during the postnatal development referring to the glutamate transmitter metabolism

Alanine aminotransferase has been studied in various nervous tissues during the postnatal development of the rat. At birth the enzyme activity was low and showed similar levels in all tissues studied. In the hippocampal formation and in the cerebellum which are supposed to be endowed with glutamatergic structures, the enzyme activity increased significantly during the postnatal development. These results contrast markedly with dorsal root ganglia and superior cervical ganglia, in which glutamatergic transmission processes are obviously absent. In these peripheral ganglia the time course of the enzyme activity persisted on a very low level after birth. The participation of alanine aminotransferase in forming of transmitter glutamate is discussed.     

Release of Glutamate and Aspartate from CA1 Synaptosomes: Selective Modulation of Aspartate Release by Ionotropic Glutamate Receptor Ligands

Abstract: Synaptosomes prepared from area CA1 of the rat hippocampus were used to determine (a) whether Schaffer collateral-commissural-ipsilateral associational terminals release both aspartate and glutamate in a Ca²⁺-dependent manner when reuptake of released glutamate is minimal and (b) whether autoreceptor mechanisms described in CA1 or hippocampal slices could reflect direct actions of glutamate receptor ligands on the synaptic terminal. When challenged for 1 min with either 25 m M K⁺ or 300 µ M 4-aminopyridine, CA1 synaptosomes released both glutamate and aspartate in a Ca²⁺-dependent manner. The glutamate/aspartate ratio was ∼5:1 in each case. K⁺-evoked glutamate release was unaffected by ligands active at NMDA or ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Unlike glutamate release, the release of aspartate was enhanced by NMDA, and this effect was blocked by d -2-amino-5-phosphonovalerate ( d -AP5). Kainate selectively depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) selectively increased the K⁺-evoked release of aspartate. AMPA enhanced aspartate release, like the antagonist CNQX. When applied in the presence of diazoxide, which blocks the desensitization of AMPA receptors, AMPA and kainate both depressed aspartate release. These findings support the view that Schaffer collateral-commissural-ipsilateral associational terminals release aspartate as well as glutamate and that these two release processes are regulated by different autoreceptor mechanisms.     

Motor Axon Synapses on Renshaw Cells Contain Higher Levels of Aspartate than Glutamate

Motoneuron synapses on spinal cord interneurons known as Renshaw cells activate nicotinic, AMPA and NMDA receptors consistent with co-release of acetylcholine and excitatory amino acids (EAA). However, whether these synapses express vesicular glutamate transporters (VGLUTs) capable of accumulating glutamate into synaptic vesicles is controversial. An alternative possibility is that these synapses release other EAAs, like aspartate, not dependent on VGLUTs. To clarify the exact EAA concentrated at motor axon synapses we performed a quantitative postembedding colloidal gold immunoelectron analysis for aspartate and glutamate on motor axon synapses (identified by immunoreactivity to the vesicular acetylcholine transporter; VAChT) contacting calbindin-immunoreactive (-IR) Renshaw cell dendrites. The results show that 71% to 80% of motor axon synaptic boutons on Renshaw cells contained aspartate immunolabeling two standard deviations above average neuropil labeling. Moreover, VAChT-IR synapses on Renshaw cells contained, on average, aspartate immunolabeling at 2.5 to 2.8 times above the average neuropil level. In contrast, glutamate enrichment was lower; 21% to 44% of VAChT-IR synapses showed glutamate-IR two standard deviations above average neuropil labeling and average glutamate immunogold density was 1.7 to 2.0 times the neuropil level. The results were not influenced by antibody affinities because glutamate antibodies detected glutamate-enriched brain homogenates more efficiently than aspartate antibodies detecting aspartate-enriched brain homogenates. Furthermore, synaptic boutons with ultrastructural features of Type I excitatory synapses were always labeled by glutamate antibodies at higher density than motor axon synapses. We conclude that motor axon synapses co-express aspartate and glutamate, but aspartate is concentrated at higher levels than glutamate.     

Aspartate Aminotransferase and Glutamate Dehydrogenase Activities in the Squid Giant Nerve

Abstract: The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea . Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.     

Glutamate dehydrogenase from apodachlya (oomycetes)

A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine-dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya.     

Atorvastatin and Fluoxetine Prevent Oxidative Stress and Mitochondrial Dysfunction Evoked by Glutamate Toxicity in Hippocampal Slices

Atorvastatin has been shown to exert a neuroprotective action by counteracting glutamatergic toxicity. Recently, we have shown atorvastatin also exerts an antidepressant-like effect that depends on both glutamatergic and serotonergic systems modulation. Excitotoxicity is involved in several brain disorders including depression; thus, it is suggested that antidepressants may target glutamatergic system as a final common pathway. In this study, a comparison of the mechanisms involved in the putative neuroprotective effect of a repetitive atorvastatin or fluoxetine treatment against glutamate toxicity in hippocampal slices was performed. Adult Swiss mice were treated with atorvastatin (10 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.), once a day during seven consecutive days. On the eighth day, animals were killed and hippocampal slices were obtained and subjected to an in vitro protocol of glutamate toxicity. An acute treatment of atorvastatin or fluoxetine was not neuroprotective; however, the repeated atorvastatin or fluoxetine treatment prevented the decrease in cellular viability induced by glutamate in hippocampal slices. The loss of cellular viability induced by glutamate was accompanied by increased D-aspartate release, increased reactive oxygen species (ROS) and nitric oxide (NO) production, and impaired mitochondrial membrane potential. Atorvastatin or fluoxetine repeated treatment also presented an antidepressant-like effect in the tail suspension test. Atorvastatin or fluoxetine treatment was effective in protecting mice hippocampal slices from glutamate toxicity by preventing the oxidative stress and mitochondrial dysfunction.     

Glutamate producing aspartate aminotransferase in glutamatergic perforant path terminals of the rat hippocampus

Summary The enzyme aspartate aminotransferase was demonstrated cytochemically in the rat hippocampus 4, 7, and 14 days after unilateral entorhinal cortex lesion. At the light microscopic level the enzyme showed a significant activity decrease in the ipsilateral entorhinal terminal field which was similar at all postlesion times investigated. Non-denervated areas, i.e. the inner one-third of the dentate gyrus molecular layer and the radiatum layer of CA2/3, showed an increase of aminotransferase activities. At the electron microscopic level in the entorhinal terminal field of the control (unoperated) side aspartate aminotransferase was localized preferentially in a great number of boutons, containing the cytoplasmic and mitochondrial isoenzymes. Following entorhinal lesion a significant loss of these positively reacting boutons was seen. Most of the degenerating boutons contained reaction product but a small number was negative for aspartate aminotransferase. From 4 to 14 postlesion days the positively reacting boutons of the non-denervated supragranular zone expanded outward into the denervated area according to the known terminal proliferation of the commissural and associational systems. The remaining denervated entorhinal terminal field was reinnervated predominantly by negatively reacting boutons (probably terminal proliferations of septal afferents) and by a small number of positively reacting boutons (probably terminal proliferations of the crossed temporodentate pathway). The presence of cytoplasmic aspartate aminotransferase in the terminals of a well-known glutamatergic system is discussed in relation to the possible importance of this enzyme for the production of releasable glutamate.     

Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area: Effects of Adenosine and Baclofen

Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA1 area were employed to test the effects of adenosine- and gamma-aminobutyrate (GABA)-related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+-dependent and originated predominantly from the CA3-derived pathways. Adenosine reduced the K+-evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 microM 8-phenyltheophylline. The order of potency for adenosine analogues was as follows: L-N6-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than D-N6-phenylisopropyladenosine approximately equal to 2-chloroadenosine greater than adenosine much greater than 5'-N-ethylcarboxamidoadenosine. 8-Phenyltheophylline (10 microM) by itself enhanced glutamate/aspartate release, whereas dipyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer collateral-commissural-ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, L-N6-phenylisopropyladenosine, nor 8-phenyltheophylline affected the release of glutamate or aspartate evoked by 10 microM veratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage-sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage-sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.     

Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi.

1. The cultured, epimastigote-form of Trypanosoma cruzi contains NADP-linked glutamate dehydrogenase (EC 1.4.1.4), with a molecular weight of about 280,000, similar to the enzyme from Plasmodium chabaudi and different from the enzymes from higher animal sources. 2. T. cruzi also contains aspartate aminotransferase (EC 2.6.1.1), with properties similar to those of the enzyme from mammals. 3. The concerted action of the transaminase and glutamate dehydrogenase might be responsible for the production of NH3 which characterizes the protein catabolism in T. cruzi.     

Evidence that Aspartate Aminotransferase Activity and Ketodicarboxylate Carrier Function Are Essential for Biosynthesis of Transmitter Glutamate

Based on the selective inhibition of glutamate release in cerebellar granule cells in primary cultures by the aspartate aminotransferase inhibitor, aminooxyacetic acid, and by the ketodicarboxylate carrier inhibitor, phenylsuccinate, a novel model for synthesis of transmitter glutamate is suggested: Glutamate is formed from glutamine in the mitochondrial intramembrane space by phosphate-activated glutaminase, transported across the inner membrane in exchange with aspartate, transaminated in the matrix to alpha-ketoglutarate, which via the ketodicarboxylate carrier is transferred to the cytoplasm, and transaminated to form transmitter glutamate. Such a mechanism would explain the functional role of aspartate aminotransferase in glutamatergic neurons.     

Glutamate producing aspartate aminotransferase in glutamatergic perforant path terminals of the rat hippocampus. Cytochemical and lesion studies

The enzyme aspartate aminotransferase was demonstrated cytochemically in the rat hippocampus 4, 7, and 14 days after unilateral entorhinal cortex lesion. At the light microscopic level the enzyme showed a significant activity decrease in the ipsilateral entorhinal terminal field which was similar at all postlesion times investigated. Non-denervated areas, i.e. the inner one-third of the dentate gyrus molecular layer and the radiatum layer of CA2/3, showed an increase of aminotransferase activities. At the electron microscopic level in the entorhinal terminal field of the control (unoperated) side aspartate aminotransferase was localized preferentially in a great number of boutons, containing the cytoplasmic and mitochondrial isoenzymes. Following entorhinal lesion a significant loss of these positively reacting boutons was seen. Most of the degenerating boutons contained reaction product but a small number was negative for aspartate aminotransferase. From 4 to 14 postlesion days the positively reacting boutons of the non-denervated supragranular zone expanded outward into the denervated area according to the known terminal proliferation of the commissural and associational systems. The remaining denervated entorhinal terminal field was reinnervated predominantly by negatively reacting boutons (probably terminal proliferations of septal afferents) and by a small number of positively reacting boutons (probably terminal proliferations of the crossed temporo-dentate pathway). The presence of cytoplasmic aspartate aminotransferase in the terminals of a well-known glutamatergic system is discussed in relation to the possible importance of this enzyme for the production of releasable glutamate.     

Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

Modulation of Glutamate and Aspartate Release from Slices of Hippocampal Area CA1 by Inhibitors of Arachidonic Acid Metabolism

Abstract: Slices of hippocampal area CA1 were used to test inhibitors of arachidonic acid metabolism for their effects on glutamate/aspartate release from the CA3-derived Schaffer collateral, commissural, and ipsilateral associational terminals. Test compounds [3 µ M nordihydroguaiaretic acid (NDGA) and 1 µ M 3-[3-(4-chlorobenzyl)-3- tert -butylthio-5-isopropylindol-2-yl]-2,2-dimethyl-propanoic acid (MK-886)] that reduced the production and release of 5-lipoxygenase metabolites also selectively reduced the K⁺-evoked release of aspartate. In contrast, the cyclooxygenase inhibitor indomethacin (100 µ M ) selectively enhanced the release of glutamate. At a concentration (100 µ M ) that nonselectively depressed the release of arachidonic acid and its metabolites, NDGA markedly depressed the release of aspartate, glutamate, and GABA. An inhibitor of the 12-lipoxygenase and an inhibitor of nitric oxide synthase did not affect the K⁺-evoked release of any transmitter amino acid. These results suggest that a 5-lipoxygenase product selectively enhances aspartate release and a cyclooxygenase product selectively depresses glutamate release. They are also consistent with previous evidence that arachidonic acid and/or platelet-activating factor enhances the release and depresses the uptake of glutamate and aspartate. The K⁺-evoked release of excitatory amino acids is much more sensitive to modulation by lipid mediators than is GABA release. Activation of NMDA receptors may enhance the K⁺-evoked release of glutamate and aspartate from CA1 slices by stimulating the production and release of lipid modulators.     

Cellular Origin of Ischemia-Induced Glutamate Release from Brain Tissue In Vivo and In Vitro

The uptake and release of D-[3H]aspartate (used as a tracer for endogenous glutamate and aspartate) were studied in cultured glutamatergic neurons (cerebellar granule cells) and astrocytes at normal (5 mM) or high (55 mM) potassium and under conditions of hypoglycemia, anoxia or "ischemia" (combined hypoglycemia and anoxia). In glutamatergic neurons it was found that "ischemic" conditions led to a 2.4-fold increase in the potassium-induced release of D-[3H]aspartate as compared to normal conditions. Hypoglycemia or anoxia alone affected the release only marginally. The ischemia-induced induced increase in the evoked D-[3H]aspartate release was shown to be calcium-dependent. In astrocytes no difference was found in the potassium-induced release between the four conditions and the K+-induced release was not calcium-dependent. The uptake of D-[3H]aspartate was found to be stimulated at high potassium in both glutamatergic neurons (98%) and in astrocytes (70%). This stimulation of D-aspartate uptake, however, was significantly reduced under conditions of anoxia or "ischemia" in both cell types. In glutamatergic neurons (but not in astrocytes) hypoglycemia also decreased the potassium stimulation of D-aspartate uptake. In a previous report it was shown, using the microdialysis technique, that during transient cerebral ischemia in vivo the extracellular glutamate content in hippocampus was increased eightfold. In the present paper it is shown that essentially no increase in extracellular glutamate is seen under ischemia when the perfusion is performed using calcium-free, cobalt-containing perfusion media. The results from the in vitro and in vivo experiments indicate that the glutamate accumulated extracellularly under ischemia in vivo originates from transmitter pools in glutamatergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epilepsy, Neurodegeneration, and Extracellular Glutamate in the Hippocampus of Awake and Anesthetized Rats Treated with Okadaic Acid

We have previously shown that the intrahippocampal microinjection of okadaic acid (OKA), a potent inhibitor of serine/threonine protein phosphatases, induces epileptic seizures, neuronal death, and the hyperphosphorylation of the NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor. We administered OKA by reverse microdialysis in the hippocampus of awake and halothane-anesthetized rats, with simultaneous collection of microdialysis fractions and recording of the EEG activity, and subsequent histological analysis. OKA produced intense behavioral and persistent EEG seizure activity in the awake rats but not in the anesthetized animals, and did not significantly alter the extracellular concentration of glutamate and aspartate detected in the microdialysis fractions. One day after the experiment a remarkable neurodegeneration of CA1 hippocampal region was observed in both the awake and the anesthetized rats. We conclude that the OKA-induced epilepsy cannot be ascribed to increased extracellular glutamate, but to an increased sensitivity of NMDA receptor. We propose that halothane protected against the epilepsy because it blocks NMDA receptor overactivation, and that the neurodegeneration of CA1 region is independent of this overactivation and due probably to alterations of cytoskeletal proteins consequent to the OKA-induced hyperphosphorylation.