bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Mechanism of Ouabain‐Induced Release of Acetylcholine from Synaptosomes
Authors: Satoh Eiki , Nakazato Yoshikazu
Published: October 2006

Abstract: Ouabain (5 X 10−8‐5 X 10−4 M) was confirmed to cause a dose‐dependent increase in [3H]acetylcholine (pHJACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose‐dependent decrease in membrane potential. In a low‐Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10−6 M) had no effect on the ouabain‐induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10−6‐5 X 10−4 M) inhibited the ouabain‐induced increase in both [3H]ACh release and [Ca2+]i in a dose‐dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mMMg2+ blocked ouabain‐induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.
Characterization of Mono‐ and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain
Authors: Ostermann‐Latif Christel , Mäder Michael , Unger Jürgen W. , Bartke Ilse , Naujoks Kurt , Peters Johann H. , Felgenhauer Klaus
Published: October 2006

Abstract: Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme‐linked immu‐nosorbent assay (ELISA) with immunoaffinity‐purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgGl subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immu‐nohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.
Ouabain Binding, ATP Hydrolysis, and Na+,K+‐Pump Activity During Chemical Modification of Brain and Muscle Na+,K+‐ATPase
Authors: Teisinger J. , Zemková H. , Svoboda P. , Amler E. , Vyskočil F.
Published: October 2006

Abstract: The effects of 16 group‐specific, amino acid‐modifying agents were tested on ouabain binding, catalytical activity of membrane‐bound (rat brain microsomal), sodium dodecyl sulfate‐treated Na+,K+‐ATPase, and Na+,K+‐pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group‐oriented drugs to suppress ouabain binding and Na+,K+‐ATPase activity correlated with inhibition of the Na+,K*‐pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane‐bound or isolated enzyme) and than Na+,K+‐pump activity. The efficiency of various drugs belonging to the same “specificity’ group differed markedly. Tyrosine‐oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like di‐ethyl pyrocarbonate (histidine groups) or butanedione (ar‐ginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the ‐Glu‐Tyr‐Thr‐Trp‐Leu‐Glu‐ sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long‐distance regulation of Na+,K+‐ATPase active sites.
Ischemia‐Induced Loss of Brain Calcium/Calmodulin‐Dependent Protein Kinase II
Authors: Yamamoto Hideyuki , Fukunaga Koji , Lee Kevin , Soderling Thomas R.
Published: October 2006

Abstract: Forebrain ischemia in gerbils, produced by brief bilateral carotid occlusion, induced the dramatic loss of Ca2+/ calmodulin‐dependent protein kinase II (CaM‐kinase II) as determined by both kinase activity assays and western blot analysis. In cortex and hippocampus, cytosolic CaM‐kinase II was completely lost within 2–5 min of ischemia. Particulate CaM‐kinase II was more stable and decreased in level ~40% after 10 min of ischemia followed by 2 h of reperfusion. CaM‐kinase II in cerebellum, which does not become ischemic, was not affected. The rapid loss of CaM‐kinase II within 2‐5 min was quite specific because cytosolic cyclic AMP kinase and protein kinase C in hippocampus were not affected. These data indicate that cytosolic CaM‐kinase II is one of the most rapidly degraded proteins after brief ischemia. Because the multifunctional CaM‐kinase II has been implicated in the regulation of numerous neuronal functions, its loss may destine the neuronal cell for death.
Normal and Heat‐Induced Patterns of Expression of Heme Oxygenase‐1 (HSP32) in Rat Brain: Hyperthermia Causes Rapid Induction of mRNA and Protein
Authors: Ewing J. F. , Haber S. N. , Maines M. D.
Published: October 2006

Abstract: Most cells possess a variety of mechanisms, such as high levels of glutathione, that guard against cytotoxic free radicals, which are suspected in the etiology of various neurological deficits. Neurons, however, are deficient in this an‐tioxidant source. The list of other potent antioxidants includes the bile pigments biliverdin and bilirubin. Heme oxygenase (HO) isozymes, HO‐1 (HSP32) and HO‐2, catalyze the rate‐limiting step in the only biological pathway by which bile pigments are produced. In this study, heat shock is identified as the only stimulus reported to date that can alter expression in brain HO‐1 of protein and mRNA in vivo. Using a HO‐1 cDNA probe, we examined the level of HO‐1 mRNA in normal rat brain and in brain 1 and 6 h following heat shock. Exposure of male rats to 42°C for 20 min caused a 20‐fold increase in brain HO‐1 1.8‐kb mRNA within 1 h after treatment. Quantification of brain HO‐1 protein by HO‐1 ra‐dioimmunoassay revealed a fourfold increase at 6 h posttreatment. In normal brain, HO‐1 protein was sparsely expressed in few select neuronal and nonneuronal cell populations in forebrain, diencephalon, cerebellum, and brainstem regions. Six hours following heat shock, an intense increase in HO‐1 protein in glia throughout the brain, epen‐dyma lining the ventricles of the brain, paraventricular nucleus, Purkinje cell layer of the cerebellum, and cochlear nucleus of brainstem was observed. We suggest that increases in HO‐1 transcript and protein reflect a means to elevate levels of antioxidants in cells with compromised defense mechanisms caused by stress.
N‐Methylation of Dopamine‐Derived 6,7‐Dihydroxy‐1,2,3,4‐Tetrahydroisoquinoline, (R)‐Salsolinol, in Rat Brains: In Vivo Microdialysis Study
Authors: Maruyama Wakako , Nakahara Daiichiro , Ota Miyuki , Takahashi Tsutomu , Takahashi Akira , Nagatsu Toshiharu , Naoi Makoto
Published: October 2006

Abstract: N‐Methylation of (R)‐1‐methyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline [(R)‐salsolinol] derived from dopamine was proved by in vivo microdialysis study in the rat brain. The striatum was perfused with (R)‐salsolinol and N‐methylated compound was identified in the dialysate using HPLC and electrochemical detection with multichanneled electrodes. N‐Methylation of (R)‐salsolinol was confirmed in three other regions of the brain, the substantia nigra, hypothalamus, and hippocampus. In the substantia nigra, the amount of N‐methylated (R)‐salsolinol was significantly larger than in the other three regions. These results indicate that around dopaminergic neurons, particularly in the substantia nigra, (R)‐salsolinol was methylated into N‐methyl‐(R)‐salsolinol, which has a chemical structure similar to that of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine, the selective dopaminergic neurotoxin. N‐Methylation of tetrahydroisoquinolines and β‐carbolines have already been proven to increase their toxicity to dopaminergic neurons and N‐methylation might be an essential step for these alkaloids to increase their toxicity. On the other hand, after perfusion of (R)‐salsolinol, release of dopamine and 5‐hydroxytryptamine was observed and inhibition of monoamine oxidase was indicated. (R)‐Salsolinol and its derivatives may be candidates for being dopaminergic neurotoxins.
Amitriptyline Prevents N‐Methyl‐D‐Aspartate (NMDA)‐Induced Toxicity, Does Not Prevent NMDA‐Induced Elevations of Extracellular Glutamate, but Augments Kainate‐Induced Elevations of Glutamate
Authors: McCaslin P. P. , Yu X. Z. , Ho I. K. , Smith T. G.
Published: October 2006

Abstract: The effect of amitriptyline on kainate‐ and N‐methyl‐D‐aspartate (NMDA)‐induced toxicity and release of amino acids from cerebellar granule neurons was studied. The ED50 for amitriptyline, imipramine, and nortriptyline protection against NMDA‐induced toxicity was 6.9, 6.5, and 1.3 μM, respectively. None of these compounds protected against kainate‐induced toxicity. Even though amitriptyline was protective against NMDA‐induced toxicity, it had no effect on the NMDA‐induced increase in extracellular levels of glutamate or aspartate from these cells, indicating a dissociation between NMDA receptor activation (as indicated by glutamate content elevations) and NMDA‐induced toxicity. However, kainate and quisqualate treatment resulted in elevations of glutamate and taurine levels that were further augmented in the presence of 25 μM amitriptyline. These findings confirm the reports of others that tricyclic antidepressants have neuroprotective effects related to the NMDA receptor and expand on these reports by showing that even though there is protection against toxicity, the NMDA receptor is nevertheless activated, suggesting an involvement of these compounds at sites removed from the receptor. Furthermore, this is the first report showing an interaction of tricyclic antidepressants with the function of non‐NMDA receptors.
Tachykinin‐Stimulated Inositol Phospholipid Hydrolysis and Taurine Release from Human Astrocytoma Cells
Authors: Lee C. M. , Tung W. L. , Young J. D.
Published: October 2006

Abstract: The activation of NK1 receptors on U373 MG human astrocytoma cells by substance P (SP) and related tachykinins was accompanied by an increase in taurine release and an accumulation of inositol phosphates. Both of these effects could be inhibited by spantide, a SP receptor antagonist. The relative potency of tachykinins in stimulating 3H‐inositol phosphate accumulation correlated very well with their effects in stimulating the release of [3H]‐taurine and inhibiting 125I‐Bolton‐Hunter reagent‐conjugated SP binding. The effect on [3H]taurine release was mimicked by a protein kinase C (PKC) activator, phorbol 12‐myristate 13‐acetate (PMA). The inactive phorbol ester analogue 4‐α‐phorbol 12,13‐didecanoate, however, was without effect. Both SP‐ and PMA‐induced releases of [3H]‐taurine were markedly inhibited by staurosporine, a potent PKC inhibitor. Pretreatment of U373 MG cells with 10 μM PMA for 19 h to down‐regulate PKC activity also markedly inhibited both SP‐ and PMA‐induced releases of [3H]‐taurine. Treatment of cells with 100 nM SP induced a time‐dependent translocation of PKC from the cytosolic fraction to the membrane fraction. These findings are consistent with the hypothesis that an activation of NK1 receptors on U373 MG cells results in the release of inositol phosphates and activation of PKC, which in turn may regulate the release of taurine.
Differential Modulation by Divalent Cations of [3H]MK‐801 Binding in Brain Synaptic Membranes
Authors: Enomoto Riyo , Ogita Kiyokazu , Han Daiken , Yoneda Yukio
Published: October 2006

Abstract: Endogenous divalent cations, such as Mg2+, Ca2+, and Zn2+, differentially affected the binding of (+)‐[3H]5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate ([3H]MK‐801) to an ion channel associated with an N‐methyl‐D‐aspartate‐sensitive subclass of excitatory amino acid receptors in different preparations of brain synaptic membranes. Both Mg2+ and Ca2+ were weak inhibitors of the binding in membranes which had not been extensively washed (nonwashed membranes), over a concentration range effective in markedly potentiating the binding in the absence of any added stimulants in membranes which had been extensively washed, but not treated with a detergent (untreated membranes). In membranes extensively washed and treated with Triton X‐100 (Triton‐treated membranes), both cations significantly potentiated the binding in the presence of added glutamate alone. In contrast, Zn2+ was invariably active as a potent inhibitor of the binding irrespective of the membrane preparations used. In untreated membranes, Ca2+ markedly accelerated the initial association rate of [3H]MK‐801 binding without affecting the binding at equilibrium in a manner similar to that found with glycine, as well as with glutamate; Mg2+, however, facilitated the initial association rate with a concomitant reduction of the binding at equilibrium. Zn2+ was effective in accelerating the initial rapid phase of association, with the initial slow phase being delayed, and in markedly reducing the binding at equilibrium. Both Mg2+ and Ca2+ also facilitated dissociation of the bound [3H]MK‐801 and Zn2+ slowed the dissociation in untreated membranes. In the presence of Mg2+ or Ca2+, glycine was ineffective in potentiating [3H]MK‐801 binding irrespective of the membrane preparations used, whereas glutamate was an effective stimulator in Triton‐treated membranes, but not in untreated membranes. However, spermidine was still active at potentiating the binding in both membrane preparations in the presence of Mg2+ or Ca2+. These results suggest that the three different divalent cations differentially modulate the opening processes of an ion channel associated with the N‐methyl‐naspartate‐sensitive receptor through the respective mechanisms in the brain.
Lipid Composition in Synaptic and Nonsynaptic Mitochondria from Rat Brains and Effect of Aging
Authors: Ruggiero F. M. , Cafagna F. , Petruzzella V. , Gadaleta M. N. , Quagliariello E.
Published: October 2006

Abstract: The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mitochondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 21 % aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.
Novel S‐Adenosylmethionine‐Dependent Indole‐N‐Methylation of β‐Carbolines in Brain Particulate Fractions
Authors: Matsubara Kazuo , Neafsey Edward J. , Collins Michael A.
Published: October 2006

Abstract: Guinea pig brain S‐adenosylmethionine (SAM)‐dependent N‐methyltransferase activity toward physiologically relevant β‐carboline (BC) substrates was examined with reverse‐phase HPLC and radiochemical detection. Representative BCs, norharman and harmine, were enzymatically methylated on the 2[β]‐nitrogen by [3H]CH3‐SAM in undialyzed homogenates to yield 2[β]‐methylated BCs and subsequently on the 9[indole]‐nitrogen to generate 2,9‐dimethylated BC products. This may be the first account of mammalian indole N‐methyl transfer. There was no HPLC evidence for 9‐methyl BC or (from carbon methylation) 2,6‐dimethyl BC products. Capillary gas chromatography‐mass spectrometry analysis confirmed the structures of the 2,9‐dimethyl and 2‐methyl products of norharman. The 2[β]‐ and 9[indole]‐N‐methylation activities were mainly in the nuclear fractions and were negligible in undialyzed cytosol. This differs from the cytosolic SAM‐dependent N‐methylations reported with other azaheterocyclics, including 1,2,3,4‐tetrahydro‐BCs. The involvement of a single enzyme was suggested because the two N‐methyl transfers with BC substrate had similar subcellular activity patterns, regional brain distributions, and Km and Vmax values. Sequential N‐methylation of various BCs that have been observed in vivo may be a unique route to centrally retained N2,N9‐dimethylated β‐carbolinium ions. Because they resemble the synthetic parkinsonian toxicant, N‐methyl‐4‐phenylpyridinium, with respect to structure and neurotoxic activity, such “bioactivated” carbolinium ions could be endogenous causative factors in Parkinson's disease.
Identification of a GM1‐Binding Protein on the Surface of Murine Neuroblastoma Cells
Authors: Fueshko Susan M. , Schengrund Cara‐Lynne
Published: October 2006

Abstract: S2OY murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo‐GM1). To identify proteins with which the oligo‐GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo‐GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo‐GM1 to 1‐deoxy‐1‐aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2‐(p‐azidosalicylamido)ethyl‐1,3′‐dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of ∼ 71 kDa. In competition experiments, as little asa 10‐fold molar excess of oligo‐GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200‐fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the ∼ 71‐kDa protein specifically associates with oligo‐GM1. Cell surface location of the oligo‐GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.
Excitatory Amino Acid‐Induced Phosphoinositide Turnover in Guinea Pig Cerebral Cortical Slices: Selective Enhancement by Spermine of the Response to DL‐1‐Aminocyclopentane‐ trans‐1, 3‐Dicarboxylate
Authors: Alexander Stephen P. H. , Kendall David A. , Hill Stephen J.
Published: October 2006

Abstract: In the presence of 1 mM spermine, accumulations of 3H labelled inositol phosphates elicited by quisqualate (100 μM) and 1 ‐aminocyclopentane‐trans‐ 1, 3‐dicarboxylate (t‐ACPD, 300 μM) were significantly enhanced by 21 and 26%, respectively, without a significant alteration in the accumulation elicited by l‐glutamate (10 mM) or DL‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxalone propionate (10 μM). Analysis of concentration‐response data indicated that the presence of spermine led to an increase in the maximal response to t‐ACPD without altering the EC50 value. The stimulatory effect of spermine on the accumulation of t‐ACPD‐elicited 3H‐inositol phosphates was not reversed by ifenprodil or diethylenetriamine (putative polyamine site antagonists), by agents that activate or inhibit protein kinase C, or by calcium channel blockade, but was abolished in the presence of elevated extracellular calcium ion concentration. We conclude that spermine enhances the phosphoinositide turnover in guinea pig cerebral cortical slices elicited by the “metabotropic” excitatory amino acid receptor. The site through which the action of spermine is mediated remains to be defined, but it is apparently distinct from that suggested to modulate N‐methyl‐D‐aspartate receptor activity.
Abnormalities in Amino Acid Neurotransmitters in Discrete Brain Regions of Genetically Dystonic Hamsters
Authors: Löscher Wolfgang , Hörstermann Dirk
Published: October 2006

Abstract: The concentrations of 11 amino acids, including the neurotransmitters γ‐aminobutync acid, glutamate, aspartate, glycine, and taurine, were determined by HPLC in 12 brain regions of genetically dystonic (dtsz) hamsters and age‐matched nondystonic controls. Since dystonia in mutant dtsz hamsters is transient and disappears after about 70 days of age, amino acids were determined at the age of maximum severity of dystonia (30‐40 days) and after disappearance of the disease, to examine which neurochemical changes were related to dystonia. In dtsz hamsters with the maximum severity of dystonia, significant changes in concentrations of the neurotransmitters γ‐aminobutyric acid, glutamate, aspartate, and taurine were found in several regions involved in motor functions, e.g., cerebellum, thalamus, and corpus striatum. Most of these changes were not permanent but disappeared in parallel with dystonia, implicating a causal relationship between altered aminoacidergic neurotransmission and dystonia in mutant dtsz hamsters.
MK‐801 Prevents 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐Induced Parkinsonism in Primates
Authors: Zuddas Alessandro , Oberto Germano , Vaglini Francesca , Fascetti Flavia , Fornai Francesco , Corsini Giovanni U.
Published: October 2006

Abstract: In cynomologus monkeys, systemic administration of MK‐801, a noncompetitive antagonist for the N‐methyl‐4‐aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). MK‐801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK‐801, the levels of toxic l‐methyl‐4‐phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK‐801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.
Brain Levels of Microtubule‐Associated Protein τ Are Elevated in Alzheimer's Disease: A Radioimmuno‐Slot‐Blot Assay for Nanograms of the Protein
Authors: Khatoon Sabiha , Grundke‐Iqbal Inge , Iqbal Khalid
Published: October 2006

Abstract: The microtubule‐associated protein τ which stimulates the assembly of α‐β tubulin heterodimers into microtubules, is abnormally phosphorylated in Alzheimer's disease (AD) brain and is the major component of paired helical filaments. In the present study, the levels of τ and abnormally phosphorylated τ were determined in brain homogenates of AD and age‐matched control cases. A radioimmuno‐slot‐blot assay was developed, using a primary monoclonal antibody, Tau‐1, and a secondary antibody, antimouse 125I‐immunoglobulin G. To assay the abnormally phosphorylated τ, the blots were treated with alkaline phosphatase before immunolabeling. The levels of total τ were about eightfold higher in AD (7.3 ± 2.7 ng/μg of protein) than in control cases (0.9 ± 0.2 ng/μg), and this increase was in the form of the abnormally phosphorylated protein. These studies indicate that the abnormal phosphorylation—not a decrease in the level of τ—is a likely cause of neurofibrillary degeneration in AD.
Potent Inhibition of Scrapie‐Associated PrP Accumulation by Congo Red
Authors: Caughey Byron , Race Richard E.
Published: October 2006

Abstract: Transmissible spongiform encephalopathies (prion diseases), Alzheimer's disease, and other amyloidoses result in the accumulation of certain abnormally stable proteins that are thought by many to play central roles in disease pathogenesis. Using serapieinfected neuroblastoma cells as a model system, we found that Congo red, an amyloid‐binding dye, potently inhibits the accumulation of the scrapie‐associated, protease‐resistant isoform of protein PrP without affecting the metabolism of the normal isoform. Growth of the cells with submicromolar concentrations of Congo red for 5 days reduced the amount of protease‐resistant PrP detected in the cultures by >90%. This activity of Congo red suggests that it selectively disrupts the conversion of PrP to the protease‐resistant isoform or destabilizes this isoform once it is made. Potential therapeutic applications of Congo red are discussed.
Inhibition of the Phospholipase C‐Linked Metabotropic Glutamate Receptor by 2‐Amino‐3‐Phosphonopropionate Is Dependent on Extracellular Calcium
Authors: Lonart Gyorgy , Alagarsamy Sudarkodi , Ravula Radhika , Wang Jia , Johnson Kenneth M.
Published: October 2006

Abstract: D,L‐2‐Amino‐3‐phosphonopropionate (AP‐3), a proposed metabotropic receptor antagonist, produced a concentration‐dependent increase in the formation of inositol 1,4,5‐trisphosphate in rat hippocampal slices. The response was maximal at 1 mM and completely due to the l‐isomer. d,l‐AP‐3 was half as efficacious as (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD), a selective agonist of this receptor. The response produced by maximally effective concentrations of l‐AP‐3 and 1S,3R‐ACPD together for 5 min was not significantly different from that produced by 1S,3R‐ACPD alone. However, pretreatment for 40 min with either 1 mM l‐AP‐3 or D,L‐AP‐3 completely inhibited the response to 1S,3R‐ACPD. This inhibition was long‐lasting (wash‐resistant) and was reversed by reduction of the extracellular Ca2+ concentration. Also, pretreatment for 40 min with 1S,3R‐ACPD reduced, but did not completely block, the response to readdition of 1S,3R‐ACPD. l‐AP‐3 (1 mM) also produced a stereoselective 2.3‐fold increase in the efflux of glutamate from the hippocampal slices. These data suggest that incubation of hippocampal slices with AP‐3 induces a time‐dependent desensitization of the metabotropic response by a mechanism that is dependent on extracellular Ca2+. The possible roles of receptor occupancy and inhibition of glutamate uptake by AP‐3 are also discussed.
Nonopioid Effect of Morphine on Electrically Evoked Acetylcholine Release from Torpedo Electromotor Neurons
Authors: Oron Lea , Sarne Yosef , Michaelson Daniel M.
Published: October 2006

Abstract: The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed.
Posttranslational Processing of Proenkephalin in SK‐N‐MC Cells: Evidence for Phosphorylation
Authors: Lindberg I. , Shaw E.
Published: October 2006

Abstract: SK‐N‐MC cells have recently been shown to be a rich source of proenkephalin and/or the proenkephalin‐derived peptide, peptide B. We have investigated the synthesis and the posttranslational processing of proenkephalin in these cells. SK‐N‐MC cells retain very little of the proenkephalin synthesized; >99% of the immunoreactive enkephalin synthesized within a 48‐h period is secreted into the medium rather than contained intracellularly. When medium samples were subjected to gel filtration and assayed for the various enkephalins present within proenkephalin, only two major molecular‐weight classes of peptides, with molecular weights and immunoreactive profiles consistent with those of proenkephalin and the 3.6‐kDa carboxyl‐terminal fragment peptide B, were observed. The proenkephalin‐like peptide present in medium samples was shown by western blot procedures to consist of a 32‐kDa protein with a slight amount of a higher‐molecular‐weight immunoreactive component above it. Only proenkephalin‐sized peptides were present within cell extracts. Radiolabeled proenkephalin added to cell cultures was also cleaved to products similarly sized to those found in medium extracts; radiolabeled proenkephalin incubated in the absence of cells was not cleaved. Cleavage of exogenous proenkephalin thus probably at least partially occurs following secretion. Cell radiolabeling experiments with [32P]orthophosphate demonstrated that SK‐N‐MC proenkephalin is phosphorylated. Microheterogeneity of proenkephalin was also observed using isoelectric focusing coupled with western blotting. Our results suggest that the SK‐N‐MC cell line represents a useful model to study the earliest steps of the posttranslational processing of human proenkephalin in a neuronal cell type.
Coordinate Regulation of the Cyclic AMP System with Firing Rate and Expression of Tyrosine Hydroxylase in the Rat Locus Coeruleus: Effects of Chronic Stress and Drug Treatments
Authors: Melia Kathleen R. , Rasmussen Kurt , Terwilliger Rose Z. , Haycock John W. , Nestler Eric J. , Duman Ronald S.
Published: October 2006

Abstract: Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP‐dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP‐ and forskolin‐stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP‐dependent protein kinase activity in the LC. 6‐Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP‐dependent protein kinase activity in this brain region. The results demonstrate the coordinate regulation of LC firing rate, TH expression, and the cAMP system by chronic stress, catecholamine depletion, and various drug and hormone treatments and raise the possibility that adaptations in the cAMP system in response to these treatments contribute to regulation of LC neuronal firing and TH expression.
Brain Synthesis and Cerebrovascular Action of Epoxygenase Metabolites of Arachidonic Acid
Authors: Amruthesh Shivachar C. , Falck J. R. , Ellis Earl F.
Published: October 2006

Abstract: The purpose of this study was to determine if whole brain makes epoxygenase metabolites of arachidonic acid and, if so, whether they are vasoactive on the cerebral microcirculation. Blood‐free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, and the extracted metabolites were resolved by HPLC. Metabolite structures were confirmed by gas chromatography/mass spectrometry. In addition to prostaglandins, leukotriene B4, and hydroxyeicosatetraenoic acids, mouse brain metabolized arachidonic acid into several other compounds. Among them, we identified 5,6‐ and 14,15‐epoxyeicosatrienoic acid. Next, we tested the effect of topical application of brain‐synthesized 5,6‐epoxyeicosatrienoic acid and synthetic epoxyeicosatrienoic acids on in vivo rabbit cerebral arteriolar diameter using the cranial window technique and in vivo microscopy. Brain‐synthesized 5,6‐epoxyeicosatrienoic acid caused a transient 28% arteriolar dilation, similar to that produced by 5 μg/ml of synthetic 5,6‐epoxyeicosatrienoic acid. A concentration of synthetic 14,15‐ and 11,12‐epoxyeicosatrienoic acid of 5 μg/ml CSF had little or no effect on diameter, whereas 8,9‐epoxyeicosatrienoic acid caused a maximum dilation of 8%. These studies suggest that brain‐synthesized 5,6‐epoxyeicosatrienoic acid may play a role in the normal or pathophysiological regulation of the cerebral microcirculation.
Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme‐Linked Immunosorbent Assay for Human τ Detection
Authors: Mercken Marc , Vandermeeren Mark , Lübke Ursula , Six Jan , Boons Jef , Vanmechelen Eugène , Van De Voorde Andre , Gheuens Jan
Published: October 2006

Abstract: Immunoaffinity chromatography with a monoclonal antibody produced against bovine τ protein was used to purify τ proteins from human brain. Fifty grams of brain tissue yielded τ 2 mg of pure τ proteins. The affinity‐purified human τ was used to produce a high‐titered rabbit anti‐human τ serum. The monoclonal anti‐τ antibody and the polyclonal rabbit anti‐τ serum were then used to construct a sandwich enzyme‐linked immunosorbent assay for detection of human τ proteins, with a sensitivity of 1 ng/ml.
Involvement of Putrescine in the Development of Kindled Seizure in Rats
Authors: Hayashi Yasushi , Hattori Yukio , Hori Yasuo
Published: October 2006

Abstract: During the development of kindling by daily electrical stimulations applied to the left amygdala of rats, concentrations of the polyamines putrescine, spermidine, and spermine were measured in the left amygdala and the remainder of the cerebrum. A significant increase of putrescine concentration appeared first at the left amygdala in prekindled rats and then propagated to the remainder of the cerebrum with the development of kindling. This increase in putrescine concentration in the left amygdala was higher in prekindled rats than in fully kindled rats and lasted for at least 24 h after the final kindling stimulation. The concentrations of spermidine and spermine were slightly increased in a fully kindled state. To clarify the role of putrescine in kindling, the development of amygdaloid kindling was examined in rats after microinjections of α‐difluoromethylornithine, a specific inhibitor of polyamine synthesis, and putrescine into the ipsilateral amygdala. Pretreatment with α‐difluoromethylornithine (50 nmol) for 10 days accelerated both the development of behavioral kindling and the propagation of the afterdischarge from the left amygdala to the frontal cortex. In contrast, pretreatment with putrescine (200 nmol) for 10 days retarded the development of kindling. These results suggest that the increase in putrescine concentration in the kindled brain has an inhibitory effect on the development of kindling.
Specificity of Neurotensin Metabolism by Regional Rat Brain Slices
Authors: Davis Thomas P. , Gillespie Terrence J. , Konings Pierre N. M.
Published: October 2006

Abstract: Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm X 230 μm) prepared from nucleus accumbens, caudate‐putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N‐[1(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Phe‐p‐ aminobenzoate, and o‐Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin‐converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin‐degrading proteolytic activity varied by brain region. Significantly less (35.0 ± 1.6%) neurotensin was lost from hippocampus than from caudateputamen (45.4 ± 1.0%) or nucleus accumbens (47.8 ± 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin‐converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate‐putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.
Endothelin Receptors on Cultured Fetal Rat Diencephalic Glia
Authors: Levin Ellis R. , Frank Harrison J. L. , Pedram Ali
Published: October 2006

Abstract: The recently described family of proteins, the endothelins, are produced in neurons and bind to extravascular sites in the CNS. To characterize these receptors, we carried out studies on cultures of fetal rat diencephalic glia. Scatchard analysis of saturation binding studies was done for astrocytes (>95% glial fibrillary acidic protein positive). For endothelin 3 (ET‐3) and ET‐1, respectively, a single receptor class of KD 0.41 ± 0.05 and 0.62 ± 0.04 nM and a receptor density of 42 ± 0.8 and 58 ± 1.1 fmol/mg of glial protein was found. Bound and cross‐linked 125I‐ET‐3 or ET‐1 showed a single predominant receptor band at M, 52,000 on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis; a minor band at 50,000 was also seen. At concentrations equal to the receptor KD, the major brain form of ET, ET‐3, stimulated a nearly 200% increase in the incorporation of tritiated thymidine into glia. ET‐3 and ET‐1 significantly impaired the ability of atrial natriuretic peptide (ANP) to generate cyclic GMP, and isoproterenol to generate cyclic AMP. The ability of ET to inhibit ANP‐induced cyclic GMP generation was reversed by cycloheximide and actinomycin‐D, whereas the inhibition of isoproterenol‐induced cyclic AMP generation was partially and significantly blocked by inhibitors of calcium influx, protein kinase C action, or G protein activation, as well. Astrocytes from this part of the brain are a potential target cell for endothelin, assuming these findings are present in vivo. This neuropeptide may serve as a growth stimulator for astrocytes and modulator of the actions of catecholamines or ANP on glia by inhibiting second messenger generation.
Manganese Uptake and Efflux in Cultured Rat Astrocytes
Authors: Aschner Michael , Gannon Maureen , Kimelberg Harold K.
Published: October 2006

Abstract: Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific highaffinity transport system for Mn2+. Saturation kinetics are clearly indicated by both l/s versus l/s plots (Km= 0.30 ± 0.03 μMM; Vmax= 0.30 ± 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 μM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn2+‐free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 ± 2.2% of 54Mn2+ transported extracellularly, and 37.2 ± 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system.
Potentiation of γ‐Aminobutyric Acid‐Mediated Chloride Flux by Pentobarbital and Diazepam but Not Ethanol
Authors: Mihic S. J. , Wu P. H. , Kalant H.
Published: October 2006

Abstract: The influx of 36Cl‐ into cerebral cortical and cerebellar microsacs from ICR mice and Sprague‐Dawley rats was studied in incubations lasting 3 s, 500 ms, or 21 ms. In the 3‐s assay, 10‐40 mM ethanol did not affect either basal or γ‐arninobutyric acid (GABA)‐mediated Cl‐ flux, at any GABA concentration tested. Only at a concentration of 600 mM did ethanol potentiate Cl‐ flux in both mouse and rat preparations. Ethanol (20 mM) also did not affect the significant potentiation of GABA‐mediated flux produced by 50 γM pentobarbital or 2 γM diazepam in ICR mouse microsacs. In 21‐ and 500‐ms incubations (quench‐flow method), 50 γM pentobarbital significantly potentiated GABA‐mediated Cl‐ flux in rat cortical microsacs, but 10‐50 mM ethanol did not. These studies suggest that some as yet unrecognized factor is essential for ethanol enhancement of GABA‐mediated Cl‐ flux, as reported by others in brain homogenates and in tissue culture.
Biochemical Correlates of Epilepsy in the El Mouse: Analysis of Glial Fibrillary Acidic Protein and Gangliosides
Authors: Brigande John V. , Wieraszko Andrzej , Albert Mary D. , Balkema Grant W. , Seyfried Thomas N.
Published: October 2006

Abstract: The El (epileptic) mouse is considered a model for complex partial seizures in humans. Seizures in El mice begin around 7–8 weeks of age and persist throughout life. To determine if astrocytic gliosis was present in adult seizing El mice, the distribution of glial fibrillary acidic protein (GFAP) was studied in the hippocampus using an antibody to GFAP. The mean number of GFAP‐positive cells per square millimeter of hippocampus was approximately 15‐ to 40‐fold higher in adult El mice than in nonseizing control C57BL/ 6J (B6) mice or in young nonseizing El mice. Relative GFAP concentration (expressed per milligram of total tissue protein) in hippocampus and cerebellum was estimated by densitometric scanning of peroxidase‐stained western blots. GFAP concentration was 2.7‐fold greater in hippocampus of adult seizing El mice than in the control B6 mice. No differences in GFAP content were detected between the strains in the cerebellum. Because gangliosides can serve as cell surface markers for changes in neuronal cytoarchitecture, they were analyzed to determine if the gliotic response in El mice was associated with changes in neural composition. Although the total ganglioside concentration of hippocampus, cerebral cortex, and cerebellum was similar in adult El and control B6 mice, a synaptic membrane enriched ganglioside, GD1a, was elevated in the adult El cerebral cortex and hippocampus. The findings indicate that El mice express a type of gliosis that is not accompanied by obvious neuronal loss.
Distinct Cellular Expression of Calcineurin Aα and Aβ in Rat Brain
Authors: Kuno Takayoshi , Mukai Hideyuki , Ito Akira , Chang Chang‐Duk , Kishima Koji , Saito Naoaki , Tanaka Chikako
Published: October 2006

Abstract: Specific polyclonal antibodies that distinguish the two distinct isoforms of the catalytic subunit of calmodulin‐dependent protein phosphatase, calcineurin Aα and Aβ, were prepared, and the distribution of calcineurin Aα and Aβ in rat brain was studied using immunochemical and immunocytochemical techniques. Immunochemical measurement revealed that the regional distributions of the two isoforms differed and that Aα was more abundant than Aβ in the rat brain. The subcellular distribution patterns of both isoforms were similar. Both isoforms were highly enriched in cytosolic fractions, including the synaptosomal cytosol. Immunocytochemical analysis revealed that both Aα and Aβ immunoreactivities differed in regional and cellular localizations. These different patterns of expression suggest that the two isoforms of calcineurin A may each have specific functions in modulating neuronal activity in particular cell types.
Fetal‐Type Phosphorylation of the τ in Paired Helical Filaments
Authors: Kanemaru Kazutomi , Takio Koji , Miura Reiko , Titani Koiti , Ihara Yasuo
Published: October 2006

Abstract: To determine the phosphorylation sites of the τ in paired helical filaments (PHF), two types of PHF antisera with different specificities were used: One was a conventional anti‐PHF, and the other was an antiserum to formic acid‐denatured PHF (anti‐HFoPHF). Phosphorylated τ‐specific antibodies, anti‐ptau 1 and anti‐ptau 2, were prepared from anti‐PHF and anti‐HFoPHF, respectively. We found that both anti‐ptau 1 and anti‐ptau 2 labeled fetal or juvenile τ but not adult τ. The anti‐ptau 1‐ and anti‐ptau 2‐recognition sites were immunochemically localized to the fragment Asp313 to Ile328 in the most COOH‐terminal portion of τ. Furthermore, Ser315 was determined as the anti‐ptau 2 recognition site. The sequence surrounding Ser315 was not found in the canonical sequences phosphorylated with known kinases.