bluuebunny/crossref_metadata_2025 huggingface dataset:
Microglia‐Derived Elastase Produces a Low‐Molecular‐Weight Plasminogen that Enhances Neurite Outgrowth in Rat Neocortical Explant Cultures
Authors: Nakajima Kazuyuki , Nagata Koichi , Hamanoue Makoto , Takemoto Nagisa , Kohsaka Shinichi
Published: October 2006

In the course of analysis of plasminogen in microglial conditioned medium (Mic‐CM), novel low‐molecular‐weight (LMW) zymogen with a molecular mass of ∼36 kDa was detected by casein‐urokinase zymography. Because this form was produced when rat native plasminogen was incubated with Mic‐CM, a specific protease in the Mic‐CM was thought to be responsible for the production of LMW plasminogen. The production of LMW plasminogen was strongly inhibited by elastase inhibitors. Furthermore, elastase (pancreatic or leukocyte) was also found to produce LMW zymogen from native plasminogen. These results indicate that LMW plasminogen is produced through limited proteolysis by an elastase‐like protease in Mic‐CM. To determine the biochemical characteristics of LMW plasminogen, rat native plasminogen was cleaved by pancreatic elastase, and the fragments (LMW plasminogen and nonzymogen fragments) were purified by several kinds of column chromatography. Amino acid sequence analysis revealed that LMW plasminogen is a carboxy‐terminal region that contains the fifth kringle domain and a protease active site, and the amino acid sequence is identical to that of LMW plasminogen produced by MicCM. On the other hand, the nonzymogen fragment was the amino‐terminal region containing four kringle domains. The effects of native plasminogen and the fragments on neurite outgrowth of rat brain explant were examined. LMW plasminogen promoted neurite outgrowth as well as did native plasminogen, whereas nonzymogen fragments did not. These results suggest that LMW plasminogen, which is produced from native plasminogen by elastase, may be a physiologically active molecule that mediates the intercellular interaction between microglia and neurons.
Involvement of Adenosine‐Sensitive Cyclic AMP‐Generating Systems in Cobalt‐Induced Epileptic Activity in the Rat
Authors: Hattori Yukio , Moriwaki Akiyoshi , Hayashi Yasushi , Hori Yasuo
Published: October 2006

An injection of cobalt chloride solution into the unilateral sensorimotor cortex of rats induced electrographic epileptic activity, which was followed by a peripheral motor disturbance. Brain slices were prepared from the cortical region including the injection site and from the other cortical regions of rats between 8 and 50 days after the injection. In the cortical slices, we examined cyclic AMP accumulations elicited by adenosine and its stable analogue 2‐chloroadenosine. Adenosine and 2‐chloroadenosine at their maximal dose increased cyclic AMP accumulation six‐ to 10‐fold and 10–15‐fold, respectively, and the elicitation was markedly inhibited by the adenosine antagonist 8‐phenyltheophylline. The cyclic AMP accumulation was increased in the primary epileptic region of the cortex adjacent to the injection site of cobalt chloride solution, whereas it was unchanged in the other cortical regions. The increase in cyclic AMP accumulation was observed regardless of the presence or absence of the adenosine uptake inhibitor dipyridamole, the phosphodiesterase inhibitor DL‐4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone, and adenosine deaminase. Such an increased accumulation of cyclic AMP in the primary epileptic cortex was detected as early as 8 days after the injection. The cyclic AMP accumulation continued to increase and reached a peak level 17–19 days after the injection, and it returned to the control levels after 40–50 days, in correspondence with the electrographic and behavioral findings. It is concluded that alterations in adenosine receptormediated generation of cyclic AMP in the primary epileptic cortex are closely associated with the central process of cobalt‐induced epilepsy. In general, the adenosine‐sensitive cyclic AMP‐generating systems may serve as a common mechanism in experimental models of epilepsy.
Plasma 5‐Hydroxyindoleacetic Acid as an Indicator of Monoamine Oxidase‐A Inhibition in Rat Brain and Peripheral Tissues
Authors: Celada Pau , Artigas Francesc
Published: October 2006

We have examined the changes induced by the monoamine oxidase (MAO; EC 1.4.3.4) inhibitors tranylcypromine, clorgyline, and deprenyl on MAO activity and 5‐hydroxytryptamine (serotonin, 5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) content in rat brain and blood (plasma and whole blood). The decreases of MAO‐A activity observed in the liver and lungs after different doses of clorgyline or tranylcypromine correlated significantly (r > 0.80 in all cases) with the decline of plasma 5‐HIAA. This was unaffected by 0.25 and 5 mg kg−1 of deprenyl, indicating that 5‐HT was deaminated exclusively in the periphery by MAO‐A. It is interesting that very potent and significant correlations (r > 0.75) were found between plasma 5‐HIAA and MAO‐A activity, 5‐HIAA and 5‐HT content in brain tissue. These results suggest that plasma 5‐HIAA can be used confidently as a peripheral indicator of the inhibition of MAO‐A in brain. This may represent a favorable alternative to the analysis of 5‐HIAA in CSF in psychiatric patients undergoing antidepressant treatment with nonspecific MAO inhibitors or with the new selective MAO‐A inhibitors.
Chronic Cocaine Administration Increases CNS Tyrosine Hydroxylase Enzyme Activity and mRNA Levels and Tryptophan Hydroxylase Enzyme Activity Levels
Authors: Vrana Sheila L. , Vrana Kent E. , Koves Timothy R. , Smith James E. , Dworkin Steven I.
Published: October 2006

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate‐limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self‐administration by rats when given ad libitum access to this dose. This chronic, response‐independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striaturn and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response‐independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.
Determination of Kainic Acid‐Induced Release of Nitric Oxide Using a Novel Hemoglobin Trapping Technique with Microdialysis
Authors: Balcioglu Aygul , Maher Timothy J.
Published: October 2006

We have demonstrated the usefulness of a novel hemoglobin‐trapping technique to quantify nitric oxide (NO) concentrations in vivo. Concentric microdialysis probes were implanted into the hippocampus of rats under urethane anesthesia and perfused with 1 μM oxyhemoglobin in artificial CSF to sequester NO in extracellular fluid. The concentration of methemoglobin was then determined spectrophotometrically. The basal level of NO in hippocampus was 2.2 ± 0.5 nM(in vitro sensitivity of the probe was 0.2 nM). Administration of 13 mg/kg, i.p., of kainic acid (KA) produced a maximal 5.3‐fold increase at 100 min in NO levels (11.8 ± 0.2 nM). This response was significantly attenuated by pretreatment with the NO synthase inhibitor N‐monomethyl‐L‐arginine (50 mg/kg, 30 min before KA). These results demonstrate that a microdialysis probe using a novel hemoglobin‐trapping technique possesses adequate sensitivity to determine the basal levels of NO and document the ability of KA to increase these levels via a NO synthase‐mediated mechanism.
Neurotoxicity of Human Amylin in Rat Primary Hippocampal Cultures: Similarity to Alzheimer's Disease Amyloid‐β Neurotoxicity
Authors: May Patrick C. , Boggs Leonard N. , Fuson Kimberly S.
Published: October 2006

Amylin, a 37‐amino‐acid amyloidogenic peptide, bears biophysical similarities to the amyloid‐β peptide (Aβ) deposited in Alzheimer's disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with Aβ(1–40). Treatment with human amylin (1–37) resulted in prominent toxicity as assessed by phasecontrast microscopy and quantification of lactate dehydrogenase in the medium. Amylin‐induced neurotoxicity was morphologically similar to that induced by Aβ(1–40). In contrast, the nonamyloidogenic rat amylin showed negligible neurotoxicity despite having 95% sequence similarity to human amylin. Only full‐length human amylin was toxic; various amylin peptide fragments including amino acid residues 20–29 were nontoxic at similar concentrations. These studies suggest that unrelated amyloidogenic peptides like human amylin and Aβ can adopt a similar neurotoxic conformation in vitro. Similar conformation‐dependent neurotoxicity may drive the prominent neurite degeneration around compacted but not diffuse deposits of Aβ in Alzheimer's disease.
Differential Effects of the Alkylating Agent N‐Ethoxycarbonyl‐2‐Ethoxy‐1,2‐Dihydroquinoline on Brain α2‐Adrenoceptors and I2‐Imidazoline Sites In Vitro and In Vivo
Authors: Miralles Antonio , Ribas Catalina , Olmos Gabriel , García‐Sevilla Jesús A.
Published: October 2006

Abstract— The alkylating agent N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) is a peptide‐coupling agent that is being used to inactivate irreversibly α2‐adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l2‐imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10−8‐10−5M) markedly decreased (20–90%) the specific binding of the selective antagonist [3H]R821002 to α2‐adrenoceptors without affecting that of [3H]idazoxan (in the presence of adrenaline) to l2‐imidazoline sites. In EEDQ‐pretreated membranes (10−5M, 30 min at 25°c), the density of l2‐imidazoline sites (Bmax= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10−6M (‐)‐adrenaline (Bmax= 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [3H]idazoxan binding sites (Bmax= 107 ± 6 fmol/mg of protein) (l2‐imidazoline sites plus a2‐adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 ± 1%) the specific binding of [3H]‐R821002 to α2‐adrenoceptors, but also the binding of [3H]idazoxan to l2‐imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ‐induced α2‐adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ‐induced inactivation of l2‐imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase‐A sites labeled by [3H]Ro 41–1049 or that of monoamine oxidase‐B sites labeled by [3H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [3H]idazoxan to l2‐imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ‐pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l2‐imidazoline sites when using [3H]‐imidazoline ligands that also recognize α2‐adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l2‐imidazoline sites probably by an indirect mechanism. Key Words: Brain l2‐imidazoline sites—[3H]‐Idazoxan—α2‐Adrenoceptors—[3H] R821002—N ‐Ethoxycarbonyl‐2‐ethoxy‐li2‐dihydroquinoline—Monoamine oxidase‐A—[3H]Ro 41–1049—Monoamine oxidase‐B—[3H]Ro 19–6327.
Antibodies Specific for GABAA Receptor α Subunits Reveal that Chronic Alcohol Treatment Down‐Regulates α‐Subunit Expression in Rat Brain Regions
Authors: Mhatre Molina C. , Pena Gina , Sieghart Werner , Ticku Maharaj K.
Published: October 2006

Abstract— Chronic administration of ethanol results in the development of tolerance and dependence. The molecular mechanism underlying these behavioral actions of ethanol is poorly understood. Several lines of evidence have suggested that some of the pharmacological actions of ethanol are mediated via a potentiation of GABAergic transmission. Chronic ethanol administration results in a reduction in the GABAA receptor‐mediated 36Cl− uptake in cortical synaptoneurosomes and primary cultured neurons. We and others have shown that it also results in a 40‐50% reduction in GABAA receptor α‐subunit mRNA levels in the rat cerebral cortex. In the present study, we investigated the expression of α1, α2, and α3 subunits of the GABAA receptor in the cerebral cortex and the α1 subunit in the cerebellum by immunoblotting using polyclonal antibodies raised against α1‐, α2‐, and α3‐subunit polypeptides following chronic ethanol treatment. These results reveal that chronic ethanol administration to rats results in a 61 ± 4% reduction in level of the GABAA receptor α1subunit (51 kDa), 47 ± 8% reduction in level of the α2subunit (53 kDa), and 30 ± 7% reduction in level of the α3subunit (59 kDa) in the cerebral cortex and a 56 ± 5% reduction in content of the α1 subunit in the cerebellum. In summary, this ethanol‐induced reduction in content of the GABAA receptor α subunits may underlie alterations in the GABAA receptor function and could be related to cellular adaptation to the functional disturbance caused by ethanol.
Interacting Presynaptic k‐Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes
Authors: Ronken E. , Mulder A. H. , Schoffelmeer A. N. M.
Published: October 2006

Abstract— The presynaptic regulation of stimulated dopa‐mine release from superfused rat striatal synaptosomes by opioids and γ‐aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D2 autoreceptors, calcium‐dependent K+‐stimulated [3H]dopamine release was inhibited through activation of a homogeneous population of k‐opioid receptors in view of the potent inhibitory effect of the k‐selective agonist U69.593 (EC50 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ‐nor δ‐selective receptor agonists affected release of [3H]‐dopamine. In addition, GABA potently inhibited the evoked [3H]dopamine release (EC50 0.4 nM) through activation of GABAA receptors in view of the GABA‐mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro‐toxin and bicuculline, and the absence of an effect of the GABAB receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release‐inhibitory effect, indicating that these receptors and the presynaptic D2 receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N‐methyl‐d‐aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [3H]dopamine release, which was subject to k‐opioid receptor‐mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.
Cystine Uptake and Glutathione Level in Fetal Brain Cells in Primary Culture and in Suspension
Authors: Sagara Jun‐ichi , Miura Kenji , Bannai Shiro
Published: October 2006

Abstract— The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine‐free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na+‐independent, glutamate‐sensitive route previously found in various types of cells and designated as system x−c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.
Interaction of Guanine Nucleotides with [3H]Kainate and 6‐[3H]Cyano‐7‐Nitroquinoxaline‐2,3‐dione Binding in Goldfish Brain
Authors: Barnes Janine M. , Murphy Paul A. , Kirkhara David , Henley Jeremy M.
Published: October 2006

Abstract— Recent reports have suggested that a major proportion of [3H]kainate binding in goldfish brain is to a novel form of G‐protein‐linked glutamate receptor. Here we confirm that guanine nucleotides decrease [3H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G‐protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [3H]kainate binding and a 50% reduction in the apparent Bmax values in both Mg2+/Na+ and Mg2+/Na+‐free buffer when assayed at 0°c. The pharmacology of [3H]kainate binding is similar to that of well‐characterised ionotropic kainate receptors but unlike that of known me‐tabotropic glutamate receptors, with neither 1S,3R‐amino‐1,3‐cyclopentanedicarboxylic acid (1S,3R‐ACPD) nor ibo‐tenic acid being effective competitors. The molecular mass of the [3H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand‐gated ion channels. Furthermore, GTP‐γS also inhibited the binding of the non‐NMDA receptor‐selective antagonist 6‐[3H]cyano‐7‐ni‐troquinoxaline‐2,3‐dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [3H]kainate and 6‐[3H]cyano‐7‐nitroquinoxaline‐2,3‐dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G‐protein‐mediated modulation.
Dopaminergic Regulation of Cannabinoid Receptor mRNA Levels in the Rat Caudate‐Plitamen: An In Situ Hybridization Study
Authors: Mailleux Pierre , Vanderhaeghen Jean‐Jacques
Published: October 2006

Abstract: By quantitative in situ hybridization, we examined in vivo in the rat caudate‐putamen the effects on levels of cannabinoid receptor mRNA of an interruption of dopamine neurotransmission for up to 1 month, by either 6‐hydroxydopamine lesioning of the medial forebrain bundle or dopamine receptor blockade. We found, in a first set of experiments, that unilateral 6‐hydroxydopamine dopa‐minergic deafferentation of the striatum (characterized by a contralateral turning behavior in response to apomor‐phine, the almost complete disappearance of the tyrosine hydroxylase hybridization signal in the substantia nigra, and an increase of preproenkephalin A mRNA level in the striatum) was associated with significantly increased (45%) cannabinoid receptor mRNA levels in the homolateral caudate‐putamen. In a second set of experiments, treatments with the dopamine D1 receptor antagonist SCH‐23390, haloperidol, and the D2 receptor antagonist sulpiride induced significantly higher cannabinoid receptor mRNA levels (respectively, 67, 34, and 27%) in the caudate‐putamen. These observations suggest for the first time that, in vivo, cannabinoid receptor gene expression in the caudate‐putamen is under the negative control of dopamine receptor‐mediated events.
Role of Nerve Growth Factor in Oxidanl Homeostasis: Glutathione Metabolism
Authors: Pan Zhaohui , Perez‐Polo Regino
Published: October 2006

Abstract— Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin‐like NGF‐responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased γ‐glutamylcysteine synthetase (GCS) activity, the rate‐limiting enzyme for GSH synthesis, by 50% after 9h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6‐phosphate dehydroge‐nase by 50% after 2 days. Treatment with NGF and cyclo‐heximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6‐phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide‐induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 μMl‐buthionine‐(S,R)‐sulfoximine, an inhibitor of GCS. However, treatment with 10 μMl‐buthionine‐(S,R)‐sulfoximine alone did not affect PC12 cell viability, NGF stimulation of neurite extension, and NGF induction of GCS, GSH peroxidase, and glucose 6‐phosphate dehydrogenase activity. When GSH levels were measured in PC12 cells that were treated for 24 h with other neurotrophins and growth factors, such as brain‐derived neurotrophic factor, neurotro‐phin‐3, epidermal growth factor, insulin‐like growth factor‐I, and basic fibroblast growth factor, only epidermal growth factor was found to increase GSH levels by 30%. Whereas NGF increased GSH levels in the human neuro‐blastoma SK‐N‐SH‐SY5Y and the human melanoma A‐875 in serum‐free medium, addition of fetal calf serum to the medium abolished the NGF effects on GSH levels in the NGF‐responsive cell lines, SK‐N‐SH‐SY5Y, A‐875, and the CNS C6 rat glioma subclone 2BD.
Lithium Modulation of Phosphoinositide Signaling System in Rat Cortex: Selective Effect on Phorbol Ester Binding
Authors: Li Peter P. , Sibony David , Green Marty A. , Warsh Jerry J.
Published: October 2006

Abstract— Recent work indicates that the therapeutic action of lithium may be mediated through perturbation of postreceptor second messenger systems. To elucidate further the postreceptor cellular sites of action(s) of lithium, the effect of chronic lithium treatment on various components of the receptor‐activated phosphoinositide pathway was investigated. We found that chronic administration of lithium (0.2% LiCI, 21 days) to adult male rats did not significantly affect phosphoinositide hydrolysis in cerebral cortical slices induced by carbachol (1 mM) or NaF (10 mM). Nor did the same treatment alter the carbachol (1 mM) potentiation of guanosine 5′‐(γ‐thio)triphosphate (30 μM) stimulation of phosphoinositide hydrolysis (an index of receptor/G protein coupling) in cortical membranes. Immunoblotting studies revealed no changes in the levels of Gαq/11 immunoreactivity in the cortex after chronic lithium treatment. The levels of protein kinase C, as revealed by specific binding of [3H]phorbol dibutyrate ([3H]PDBu), were significantly reduced in the cytosolic fraction and increased in the particulate fraction of rat cortex after chronic lithium, whereas the KD of [3H]PDBu binding remained relatively constant. A small and insignificant decrease in the density of [3H]inositol 1,4,5‐trisphosphate binding was also found in the cortex. The above data suggest that chronic lithium treatment affects neither the muscarinic cholinergic‐linked phosphoinositide turnover nor the putative G protein α subunit (Gαq/11) responsible for phospholipase C activation. However, a possible translocation and activation of protein kinase C activity may be significant in the therapeutic effect of this mood‐stabilizing agent.
High Activities of Glutamine Transaminase K (Dichlorovinylcysteine β‐Lyase) and ω‐Amidase in the Choroid Plexus of Rat Brain
Authors: Cooper Arthur J. L. , Abraham Dicky G. , Gelbard Alan S. , Lai James C. K. , Petito Carol K.
Published: October 2006

Abstract: Certain halogenated hydrocarbons, e.g., dichlo‐roacetylene, are nephrotoxic to experimental animals and neurotoxic to humans; cysteine‐S‐conjugate β‐lyases may play a role in the nephrotoxicity. We now show that with dichlorovinylcysteine as substrate the only detectable cysteine‐S‐conjugate β‐lyase in rat brain homogenates is identical to glutamine transaminase K. The predominant (mitochondrial) form of glutamine transaminase K in rat brain was shown to be immunologically distinct from the predominant (cytosolic) form of the enzyme in rat kidney. Glutamine transaminase K and ω‐amidase (constituents of the glutaminase II pathway) activities were shown to be widespread throughout the rat brain. However, the highest specific activities of these enzymes were found in the choroid plexus. The high activity of glutamine transaminase K in choroid plexus was also demonstrated by means of an immunohistochemical staining procedure. Glutamine transaminase K has a broad specificity toward amino acid and α‐keto acid substrates. The ω‐amidase also has a broad specificity; presumably, however, the natural substrates are α‐ketoglutaramate and α‐ketosuccinamate, the α‐keto acid analogues of glutamine and aspara‐gine, respectively. The high activities of both glutamine transaminase K and ω‐amidase in the choroid plexus suggest that the two enzymes are linked metabolically and perhaps are coordinately expressed in that organ. The data suggest that the natural substrate of glutamine transaminase K in rat brain is indeed glutamine and that the metabolism of glutamine through the glutaminase II pathway (i.e., l‐glutamine and α‐keto acid α‐ketoglutarate and l‐amino acid + ammonia) is an important function of the choroid plexus. Moreover, the present findings also suggest that any explanation of the neurotoxicity of halogenated xenobiotics must take into account the role of glutamine transaminase K and its presence in the choroid plexus.
Role of the Cellular Stress Response in the Biogenesis of Cysteamine‐Induced Astrocytic Inclusions in Primary Culture
Authors: Mydlarski Marc B. , Liang Jin‐Jun , Schipper Hyman M.
Published: October 2006

Abstract— Cysteamine (CSH; 2‐mercaptoethylamine) stimulates the accumulation of peroxidase‐positive inclusions in cultured astroglia akin to those observed in the aging periventricular brain. Because CSH induces the synthesis of a stress protein (heme oxygenase) in rat liver, we hypothesized that aspects of the cellular stress response may play a role in the biogenesis of CSH‐induced astro‐cyte granules. In the present study, we performed indirect immunofluorescent staining and immunoblotting for various stress proteins in rat neuroglial cultures. Exposure of astrocyte cultures to CSH enhanced immunostaining for heme oxygenase‐1 (HO‐1) and heat‐shock proteins 27, 72, and 90, but not glucose‐regulated protein 94, relative to untreated cultures. CSH‐pretreated astrocytes exhibited enhanced tolerance to H2O2 toxicity relative to untreated cells, providing physiological evidence of an antecedent stress response in the former. In addition, exposure for 12 days to H2O2, a known inducer of the stress response, elicited astrocyte granulation similar to that observed with CSH. Chronic induction of HO‐1 and other stress proteins may participate in the biogenesis of metal‐loporphyrin‐rich inclusions in CSH‐treated astroglial cultures and in astrocytes of the aging periventricular brain.
Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity
Authors: Schmidt Bonnie M. , Rylett R. Jane
Published: October 2006

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl‐CoA in cholin‐ergic neurons. The present study examined conditions for modulation of kinase‐mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti‐human choline acetyltransferase antibody to immunoprecipi‐tate cytosolic and membrane‐associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with 32Pi incorporation into the enzyme in nerve terminals incubated in nominally calcium‐free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane‐associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μM veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane‐associated choline acetyltransferase, although the specific activity of the membrane‐associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.
Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells
Authors: Chueh Sheau‐Huei , Kao Lung‐Sen
Published: October 2006

Abstract— ATP‐induced changes in the intracellular Ca2+concentration ([Ca2+]i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca2+indicator fura‐2, we have shown that the [Ca2+]i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Caz+]i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′‐thiotriphosphate and 5′‐adenylyl‐β, γ‐imidodiphosphate, could not trigger significant [Ca2+]i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β‐methylene‐ATP, β,γ‐methylene‐ATP, and 2‐methylthio‐ATP, as well as UTP and adenosine, all had no effect on [Ca2+]i at 3 mM. In the absence of extracellular Ca2+, the effect of ATP was inhibited totally, but could be restored by the addition of Ca2+ to the cells. Upon removal of Mg2+, the maximum increase in [Ca2+]i induced by ATP was enhanced by about 42%. Ca2+‐channel blockers partially inhibited the ATP‐induced [Ca2+]i rise. The ATP‐induced [Ca2+]i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin‐induced [Ca2+]i rise completely. No heterologous desensitization of [Ca2+]i rise was observed between ATP and bradykinin. The magnitude of the [Ca2+]i rise induced by ATP increased between 1.5 and 3.1 times when external Na+was replaced with Tris, N‐methyl‐d‐glucamine, choline, or Li+. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca2+]i to the basal level in Na+‐containing or Na+‐free Tris solution. Our results suggest that ATP stimulates Ca2+influx via at least two pathways: ion channels that are permeable to Ca2+ and Na+, and pores formed by ATP4‐.
Damage to Neurons in Culture Following Medium Change: Role of Glutamine and Extracellular Generation of Glutamate
Authors: Driscoll Bernard F. , Deibler Gladys E. , Law Mona J. , Crane Alison M.
Published: October 2006

Abstract— Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N‐methyl‐d‐aspartate (NMDA)‐type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10–20 μM) of 2‐amino‐5‐phos‐phonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 μM) of 2‐amino‐5‐phosphonovaleric acid can protect cultures when added at much later times (30–60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 μrn pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6‐Diazo‐5‐oxo‐l‐norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.
Effect of Global Ischaemia, Under Simulated Penumbral Conditions, on Brain Monoamine Neurochemistry and Subsequent Neurological and Histological Deficits
Authors: Richards D. A. , Obrenovitch T. P. , Johonson‐Mora A. , Islekel S. , Symon L. , Curzon G.
Published: October 2006

Abstract— We have measured changes in the levels of do‐pamine (DA), 5‐hydroxytryptamine (5‐HT), and their metabolites in striatal dialysates during 30 min of global ischaemia under simulated penumbral conditions, and compared these with neurological assessments over the following 7 days and histological damage at the end of this period. On the basis of dialysate DA levels during ischaemia, the animals fell into two subgroups; group I, with little or no DA increase (less than three times basal); and group II, with a much larger increase (greater than 30 times basal). Changes in 5‐HT, though of lesser magnitude, showed a similar pattern. These findings may indicate that the amine changes depend on a critical reduction of blood flow within the range obtained by our experimental procedure. Levels of deaminated metabolites fell in all ischaemic animals, with comparable decreases of 3, 4‐dihydroxyphenylacetic acid plus homovanillic acid in both groups. Decreases of 5‐hydroxyindoleacetic acid were greater in group II than in group I, but the relative differences between the groups were much less marked than those of 5‐HT. These neuro‐chemical findings suggest that moderate ischaemia affects extracellular amine and deaminated metabolite levels by different mechanisms. Only one of the ischaemic rats (a member of group II) showed a marked neurological deficit, but histological damage, as indicated by neuronal loss and gliosis in vulnerable structures, was apparent in all ischaemic animals. Although damage tended to be greater in animals with marked increases in extracellular monoamines, differences were not significant. These findings suggest that the large increases of extracellular DA and 5‐HT that sometimes occur in ischaemia may play a relatively small part in the genesis of neuronal damage, though these transmitters may well have a permissive role.
Free Fatty Acid and Diacylglycerol Accumulation in the Rat Brain During Recurrent Seizures Is Related to Cortical Oxygenation
Authors: Visioli F. , Rihn L. L. , Rodriguez de Turco E. B. , Kreisman N. R. , Bazan N. G.
Published: October 2006

Abstract— Cerebral blood flow and oxygenation increase during the early seizures of a series, but the increase in cerebral blood flow attenuates during late seizures, sometimes resulting in decreased cortical oxygenation. Cortical free fatty acids (FFA) and diacylglycerols also increase during early seizures and the increase attenuates during late seizures. We analyzed the correlation between lipid accumulation and cortical O2 during periodic pentylenetet‐razol‐induced seizures. During early seizures, both FFA and diacylglycerols increased in the cerebral cortex, particularly arachidonate (20:4) and stearate (18:0). Changes in lipids were different during late seizures, depending on cortical O2 levels. An increase in cortical O2 during late seizures was associated with lower FFA levels compared with early seizures, and FFA levels recovered to basal levels during interictal periods. A decline in cortical O2 was associated with a further increase in FFA, which remained elevated during interictal periods. Our results indicate that periseizure lipid accumulation is related to cortical oxygenation.
Altered Composition of Cerebral Microvessel Membrane Phosphoglycerides from Senescent Mouse
Authors: Williams Wesley M. , Rapoport Stanley I.
Published: October 2006

Abstract— Phosphoglyceride and fatty acid composition was determined in the cellular membranes of isolated cerebral microvessels and brain parenchymal cells (neurons and glia) taken from 10‐, 20‐, and 27–30‐month‐old C57BL6/NNIA mice. Lipids were extracted from each fraction and the fatty acid profiles of ethanolamine, cho‐line, serine, and inositol phosphoglycerides analyzed by gas chromatography. The results suggest that membrane phosphoglycerides from cerebral microvessels are significantly more affected by the aging process than are those of the brain parenchyma. Relative percentage for fatty acids in cerebral microvessels indicate an overall decline in membrane unsaturation with a concomitant elevation in the level of saturation. The decline in unsaturation is reflected primarily in the loss of precursor fatty acids for arachidonic (18:2n‐6 and 20:3n‐6) and docosahexaenoic (20:5n‐3 and 22:5n‐3) acids. Levels of arachidonic (20:4n‐6) and docosahexaenoic (22:6n‐3) acids in each phos‐phoglyceride remained unchanged with age; however, mol% for ethanolamine plasmalogen, a major source of these fatty acids, was significantly reduced in 27–30‐month‐old mice. Conversely, mol% for choline phospho‐glyceride increased with age. The age‐related changes in fatty acid profile for microvessel membrane phosphoglycerides are reflected by increased saturation/unsaturation ratios and decreased unsaturation indices. These parameters were not affected by aging in parenchymal membranes.
Substance P‐Evoked Calcium Mobilization and Ionic Current Activation in the Human Astrocytoma Cell Line U 373 MG: Pharmacological Characterization
Authors: Pradier L. , Heuillet E. , Hubert J. P. , Laville M. , Le Guern S. , Doble A.
Published: October 2006

Abstract— In the human astrocytoma cell line U 373 MG, application of substance P (SP) leads to a transient increase in cytosolic calcium concentration and to a biphasic current response in voltage‐clamped cells. Using these two functional assays we have characterized pharmacologically the SP response in U 373 MG cells. SP and [l‐Pro9]SP displayed high potencies in both assays with EC50values of 2.5 ± 10−9M and 1 ± 10−9M on calcium responses and 110−9M and 510−9M on ion current responses, respectively. The high potency of SP and [l‐Pro9]SP as well as the low potency of [Lys5,MeLeu9,N‐Leu10]neurokinin A(4‐10) and the inactivity of senktide demonstrate the NK1‐type pharmacology of these responses. Furthermore, the NK1 antagonists (±)‐CP 96,345, its chloro analogue, (±)‐cis‐3‐(2‐chlorobenzylamino)‐2‐benz‐hydrylquinuclidine, and RP 67580 were potent antagonists of both SP responses. For the calcium mobilization induced by SP (1 (10−7M), the IC50 values for the three antagonists were 4 ± 10−10M, 4 ± 10−9M, and 9 ± 10−9M, respectively, whereas on the current response evoked by SP 10−8M), the IC50 values were 8 ± 10−9M, 2.4 ± 10−8M, and 1.2 10−7M, respectively. Despite differences in the absolute IC50 values obtained with both techniques, the relative potencies of the three antagonists correlate fairly well. The U 373 MG cell line provides a useful model system for studies of the pharmacology of the human NK1receptor and its transduction mechanisms at the level of second messengers and modulation of ion currents.
Further Evidence for Multiple Forms of an N‐Methyl‐d‐Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques
Authors: Zuo Pingping , Ogita Kiyokazu , Suzuki Takeo , Han Daiken , Yoneda Yukio
Published: October 2006

Abstract— Pretreatment with sulfhydryl‐reactive agents, such as N‐ethylmaleimide and p‐chloromercuriphenylsul‐fonic acid, invariably resulted in marked inhibition of the binding of dl‐(E)‐2‐amino‐4‐[3H]propyl‐5‐phosphono‐3‐pentenoic acid ([3H]CGP 39653), a competitive antagonist at an N‐methyl‐d‐aspartate (NMDA)‐sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X‐100, but did not significantly affect the binding of L‐[3H]‐glutamic acid ([3H]Glu), an endogenous agonist. The pre‐treatment was effective in reducing the binding of [3H]‐CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl‐reactive agents also enhanced the potencies of NMDA agonists to displace [3H]‐CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [3H]Glu binding. The binding of both [3H]CGP 39653 and [3H]Glu was similarly sensitive to pretreatment with four different proteases in Tritontreated membranes, whereas pretreatment with phospho‐lipase A2 or C markedly inhibited [3H]CGP 39653 binding without altering [3H]Glu binding. Moreover, both phospho‐lipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [3H]CGP 39653 and [3H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl‐reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [3H]CGP 39653, with concomitant facilitation of that in an agonist‐preferring form by [3H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.
Pharmacological Characterization of 5‐Hydroxytryptamine3Receptors in Murine Brain and Ileum Using the Novel Radioligand [3H]RS‐42358–197: Evidence for Receptor Heterogeneity
Authors: Bonhaus Douglas W. , Wong Erik H. F. , Stefanich Eric , Kunysz Elizabeth A. , Eglen Richard M.
Published: October 2006

Abstract: Previous studies have demonstrated species‐specific differences in 5‐hydroxytryptamine3 (5‐HT3) receptors, but unequivocal evidence of 5‐HT3 receptor subtypes, within a species, has not yet been obtained. The purpose of the current study was to test for heterogeneity in 5‐HT3 receptors in murine tissues. 5‐HT3 receptors in membranes derived from brain cerebral cortex of CD‐1, C57BI/6, and Swiss Webster mice and ileum of CD‐1 mice were labeled with the 5‐HT3 receptor antagonist [3H]RS‐42358–197. Structurally diverse competing ligands were then used to characterize the binding site. [3H]RS‐42358‐197 bound with similar affinity in each of the cortical tissues (mean KD= 0.14 nM; range, 0.06–0.32 nM) but bound with lower affinity in ileal tissue (2.5 nM). The density of sites labeled with [3H]RS‐42358–197 ranged from 10.4 fmol/mg of protein in Swiss Webster mouse cortex to 44.2 fmol/mg of protein in Sprague‐Dawley rat cortex. Displacing ligands produced a pharmacologic profile of the [3H]RS‐42358–197 binding site consistent with it being a 5‐HT3 receptor: (R)‐YM060 > (S)‐zacopride > (R)‐zaco‐pride > MDL 72222 > 2‐methyl‐5‐HT. However, 10‐fold differences in the affinity of certain ligands were found when comparing 5‐HT3 binding sites in membranes from cerebral cortex of the different strains of mice and when comparing 5‐HT3 binding sites in brain and ileal membranes prepared from the CD‐1 mouse strain. Ligands demonstrating selectivity included RS‐42358–197, (R)‐za‐copride, 1‐(m‐chlorophenyl) biguanide, (R)‐YM060, and MDL 72222. These studies demonstrate tissue‐and strain‐dependent differences in murine 5‐HT3 binding sites. This suggests that 5‐HT3 receptors exist as multiple subtypes within species and that subtype‐selective 5‐HT3 ligands may be identified.
Rapid Communication: Adrenalectomy Attenuates Stress‐Induced Elevations in Extracellular Glutamate Concentrations in the Hippocampus
Authors: Lowy Martin T. , Gault Laura , Yamamoto Bryan K.
Published: October 2006

Abstract— Glucocorticoids and stress have deleterious effects on hippocampal cell morphology and survival. It has been hypothesized that these effects are mediated via an excitatory amino acid mechanism. The present study was designed to evaluate the effects of acute stress on the extracellular levels of glutamate in the hippocampus and to determine if adrenalectomy modifies this response. Rats were adrenalectomized or sham‐adrenalectomized and implanted with microdialysis probes in the CAS region of the hippocampus. Three days later rats were subjected to an acute 1 ‐h period of immobilization stress. Stress significantly increased extracellular glutamate levels in the sham‐operated rats, which peaked at 20 min following the initiation of stress. Extracellular glutamate levels also increased immediately following the termination of stress. In the adrenalectomized rats there was a 30% decrease in basal extracellular concentrations of glutamate and a marked attenuation (‐70%) of the stress‐induced increase in extracellular glutamate levels. Extracellular concentrations of taurine were not modified by adrenalectomy and did not change in response to stress. These results suggest that glucocorticoid‐in‐duced elevations in extracellular glutamate concentrations may contribute to the deleterious effects of stress on hippocampal neurons.