bluuebunny/crossref_metadata_2025 huggingface dataset:
3‐[(±)‐2‐Carboxypiperazin‐4‐yl]propyl‐1‐Phosphonic Acid Recognizes Two N‐Methyl‐D‐Aspartate Binding Sites in Rat Cerebral Cortex Membranes
Authors: van Amsterdam Frank Th. M. , Giberti Alessandra , Mugnaini Manolo , Ratti Emiliangelo
Published: October 2006

Abstract: Binding of 3‐[(pmn)‐2‐carboxypiperazin‐4‐yl][3H]‐propyl‐1‐phosphonic acid ([3H]CPP), a competitive inhibitor of N‐methyl‐D‐aspartate (NMDA), has been studied in synaptic plasma membranes from rat cerebral cortex. Computer analysis of saturation and homologous displacement isotherms deriving from these plasma membranes indicated the existence of two binding sites: a specific, saturable, high‐affinity binding site with a pKD value of 7.53 pmn 0.03 (29.5 nM) and a maximum binding value (Bmax) of 2.25 pmn 0.36 pmol/mg of protein, and a low‐affinity site with a KD of approximately 600 nM and a Bmax of 7.0 pmol/mg of protein. It is argued that, in the light of current literature evidence, the low‐affinity binding site may represent an agonist‐dependent receptor, linked to physiological processes such as neurotransmitter release and channel regulation, whereas the high‐affinity binding site may be linked to an antagonist‐preferred receptor, for which no function has yet been reported.
The Effect of Cyclohexyladenosine on the Penischemic Increases of Hippocampal Glutamate and Glycine in the Rabbit
Authors: Cantor Stacey L. , Zornow Mark H. , Miller Leonard P. , Yaksh Tony L.
Published: October 2006

Abstract: We investigated the ability of N6‐cyclohexyladenosine (CHA), a potent and selective agonist of the adenosine A1 receptor, to attenuate elevations of levels of extracellular hippocampal glutamate and glycine that result from episodes of transient global cerebral ischemia (TGCI). A total of 30 New Zealand white rabbits were randomly assigned to receive O (n = 5), 0.1 (n = 8), 1.0 (n = 6), 10 (n = 6), or 100 (n = 5) γM CHA. The drug was dissolved in artificial CSF (vehicle) and administered via a microdialysis probe placed stereotactically into the dorsal hippocampus. A second microdialysis probe placed into the contralateral hippocampus of each animal was perfused with vehicle alone. Ten minutes of TGCI was induced by neck tourniquet inflation and deliberate hypotension from O to 10 min. Microdialysissamples were collected as follows: every 20 min preischemia (at ‐80, ‐60, ‐40, ‐20, and 0 min); every 5 min during ischemia and in the immediate reperfusion period (at 5, 10, 15, and 20 min); and every 20 min for the remainder of the reperfusion period (at 40, 60, and 80 min). Samples were then analyzed for their concentration of glutamate and glycine by HPLC. Following 10 min of ischemia, glutamate levels increased to a peak of 3.28 pmn 0.55 times baseline and returned to preischemic levels by 40 min, i.e., during reperfusion. Glycine concentrations increased to 5.41 pmn 0.91 times over baseline and remained elevated for the duration of the study. Glutamate and glycine levels were converted to a release index, which was defined as the fractional increase (relative to levels just before ischemia) in amino acid concentration multiplied by time during ischemia and reperfusion. Regression analysis revealed a significant dose‐dependent reduction in the glycine release index (p= 0.03) on the CHA‐treated side. The contralateral side showed no such attenuation (p= 0.5). For glutamate, there was a nonsignificant tendency for CHA to reduce dose‐dependently glutamate levels (p= 0.054), whereas the contralateral (vehicletreated) side showed no such tendency (p= 0.2). These results indicate that CHA (0.1–100 μM) has a dose‐dependent attenuating effect on the periischemic increase in extracellular glycine levels. Elevated glycine levels may contribute to glutamate's neurotoxicity. It is uncertain to what degree this decrease in glycine levels by CHA may contribute to previously reported cerebral protection.
Formation and Clearance of Interstitial Metabolites of Dopamine and Serotonin in the Rat Striatum: An In Vivo Microdialysis Study
Authors: Cumming Paul , Brown Erin , Damsma Geert , Fibiger Hans
Published: October 2006

Abstract: In vivo microdialysis was employed in order to characterize the steady‐state kinetics of the turnover of specific dopamine and serotonin metabolites in the rat striaturn 48 h after surgery. Inhibitors of monoamine oxidase (MAO; pargyline) and catechol‐O‐methyltransferase (COMT; Ro 40‐7592) were administered, either separately or in conjunction, at doses sufficient to block these enzymes in the CNS. In some experiments, the acid metabolite carrier was blocked with probenecid. Temporal changes were then observed in the efflux of interstitial dopamine, 3‐methoxytyramine (3‐MT), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA). The fractional rate constants for the accumulation or disappearance of the metabolites could be determined after pharmacological blockade of catabolic enzymes or the acid metabolite carrier. Interstitial 5‐HIAA was found to be cleared with a half‐life of approximately 2 h. After blockade of either MAO or COMT, HVA disappeared with a half‐life of 17 min. Experiments employing probenecid suggested that some of the interstitial HVA was cleared by the acid metabolite carrier, the remainder being cleared by a probenecid‐insensitive process, possibly conjugation. After MAO inhibition, DOPAC disappeared with an apparent half‐life of 11.3 min. The rate of 3‐MT accumulation after pargyline indicated that the majority of interstitial HVA (>95%) is formed from DOPAC rather than 3‐MT. The formation of 3‐MT from interstitial dopamine, calculated from the accumulation rate of 3‐MT after pargyline, appeared to follow first‐order kinetics (k = 0.1 min−1).
Glucose Metabolism Assessed with 2‐Deoxyglucose and the Effect of Glutamate in Subdivisions of Rat Hippocampal Slices
Authors: McDougal David B. , Carter Joyce G. , Pusateri Mary Ellen , Manchester Jill K. , Lowry Oliver H.
Published: October 2006

Abstract: A new approach to the study of glucose phosphorylation in brain slices is described. It is based on timed incubation with nonradioactive 2‐deoxyglucose (DG), after which the tissue levels of DG and 2‐deoxyglucose‐6‐phosphate (DG6P) are measured separately with sensitive enzymatic methods applied to specific small subregions. The smallest samples had dry weights of sip0.5 mug. Direct measurements in different regions of hippocampal slices showed that within 6 min after exposure to DG, the ratios of DG to glucose in the tissue were almost the same as in the incubation medium, which simplifies the calculation of glucose phosphorylation rates and increases their reliability. Data are given for ATP, phosphocreatine, sucrose space, and K+ in specific subregions of the slices. DG6P accumulation proceeded at a constant rate for at least 10 min, even when stimulated by 10 mM glutamate in the medium. The calculated control rate of glucose phosphorylation was 2 mmol/ kg (dry weight)/min. In the presence of 10 mM glutamate it was twice as great. The response to 10 mM glutamate of different regions of the slice was not uniform, ranging from 164% of control values in the molecular layer of CA1 to 256% in the stratum radiatum of CA1. There was a profound fall in phosphocreatine levels (75%) in response to 10 mM glutamate despite a 2.4‐fold increase in glucose phosphorylation. Even in the presence of 1 mM glutamate, the increase in glucose phosphorylation (50%) was not great enough to prevent a significant drop in phosphocreatine content.
Rat Brain Creatine Kinase Messenger RNA Levels Are High in Primary Cultures of Brain Astrocytes and Oligodendrocytes and Low in Neurons
Authors: Molloy George R. , Wilson Charlie D. , Benfield Pamela , deVellis Jean , Kumar Shalini
Published: October 2006

Abstract: Rat brain creatine kinase (CKB) gene expression is highest in the brain but is also detectable at lower levels in some other tissues. In the brain, the CKB enzyme is thought to be involved in the regeneration of ATP necessary for transport of ions and neurotransmitters. To understand the molecular events that lead to high CKB expression in the brain, we have determined the steady‐state levels of CKB mRNA in homogeneous cultures of primary rat brain astrocytes, oligodendrocytes, and neurons. Northern blot analysis showed that whereas the 1.4‐kb CKB mRNA was detectable in neurons, the level was about 17‐fold higher in oligodendrocytes and 15‐fold higher in astrocytes. The blots were hybridized with a CKB‐specific 32P‐antisense RNA probe, complementary to the 3’untranslated sequence of CKB, which hybridizes to CKB mRNA but not CKM mRNA. Also, the 5’and 3’ends of CKB mRNA from the glial cells were mapped, using exon‐specific antisense probes in the RNase‐protection assay, and were found to be the same in astrocytes and oligodendrocytes. This indicated that (a) the site of in vivo transcription initiation in astrocytes and oligodendrocytes was directed exclusively by the downstream, nonconcensus TTAA sequence at ‐25 bp in the CKB promoter that is also utilized by all other cell types that express CKB and (b) the 3’end of mature CKB mRNA was the same in astrocytes and oligodendrocytes. In addition, there was no detectable alternate splicing in exon 1, 2, or 8 of CKB mRNA in rat astrocytes and oligodendrocytes. Also, our studies showed that 1.4‐kb CKB mRNA is expressed in established C6 glioma cells at an intermediate level about threefold higher than that in primary neurons.
Measurement of Lithium‐Induced Changes in Mouse Inositol(1)Phosphate Levels In Vivo
Authors: Atack John R. , Cook Susan M. , Watt Alan P. , Ragan C. Ian
Published: October 2006

Abstract: An anion‐exchange HPLC mass assay was used to characterize Swiss‐Webster mouse brain and peripheral tissue inositol(1)phosphate [Ins(1)P] levels. Ins(1)P was identified in all tissues studied but Ins(4)P could be identified only in brain, and then only as a part of a peak containing an additional, unidentified component. As a result, it was not possible to quantify Ins(4)P levels. Following a single subcutaneous dose of lithium (10 mmol/kg), brain Ins(1)P levels were maximally elevated after 6 h (corresponding to peak brain lithium concentrations) and were increased to levels 35‐ and 20‐fold higher than in saline‐treated animals in cholinergic agonist (pilocarpine)‐stimulated and unstimulated animals, respectively. The ED50 for the lithium‐induced accumulation of brain Ins(1)P 6 h after administration was 4–6 mmol/kg. The pilocarpine stimulation of lithium‐induced brain Ins(1)P accumulation had an ED50 of 22 mg/kg, with maximal accumulation occurring 120 min after pilocarpine administration. Atropine reduced Ins(1)P levels, in both the absence and the presence of lithium, by 40%, indicating that cholinergic systems contribute a large (40%) component of basal brain phosphatidylinositol (PI) cycle activity. In peripheral tissues, there were lithium‐induced accumulations of Ins(1)P in kidney, heart, and liver (but not testes) but these were less than that seen in the brain, suggesting that under basal (and pilocarpine‐stimulated) conditions, the brain has a higher turnover of the PI cycle than the various peripheral tissues studied. These data support the hypothesis that lithium exerts its effects in vivo via modulation of the PI cycle. In addition, the susceptibility of brain rather than peripheral tissue to the pharmacological effects of lithium may be a consequence of higher PI cycle turnover in brain.
Expression of an Inwardly Rectifying Potassium Channel in Xenopus Oocytes
Authors: Périer François , Coulter Kathryn L. , Radeke Carolyn M. , Vandenberg Carol A.
Published: October 2006

Abstract: The murine macrophage‐like cell line J774.1 was used as a source of mRNA for the expression of inwardly rectifying potassium channels in Xenopus oocytes. RNA was isolated, poly(A)+‐selected, and size‐fractionated by sucrose density gradient centrifugation. Oocytes injected with J774.1 RNA expressed large‐amplitude current (0.9 pmn 0.07 μA; mean pmn SEM, n = 31) at ‐100 mV in 96 mM extracellular K+ showing prominent inward rectification. The inwardly rectifying currents were most strongly expressed by an mRNA size class of 4–5 kb. The expressed current displayed selectivity, conductance, and rectification properties of inwardly rectifying potassium channels in their native membranes. The current was potassium selective and was specifically blocked by Ba2+. The conductance and the voltage dependence of the current rectification depended on the extracellular potassium concentration, with the midpoint in peak conductance following the potassium equilibrium potential. This high level of expression of inward rectifier current and the absence of other expressed currents suggest that J774.1 mRNA represents an excellent starting material for expression cloning of the inward rectifier potassium channel cDNA.
Inhibition of Protein Kinase C Restores Na+, K+‐ATPase Activity in Sciatic Nerve of Diabetic Mice
Authors: Hermenegildo Carlos , Felipo Vicente , Miñana María‐Dolores , Grisolía Santiago
Published: October 2006

Abstract: We have tested if inhibition of protein kinase C is able to prevent and/or to restore the decrease of Na+, K+‐ATPase activity in the sciatic nerve of alloxan‐induced diabetic mice. Mice were made diabetic by subcutaneous injection of 200 mg of alloxan/kg of body weight. The activity of Na+, K+‐ATPase decreased rapidly (43% after 3 days) and slightly thereafter (58% at 11 days). We show that intraperitoneal injection of 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), an inhibitor of protein kinase C, prevents completely the loss of Na+, K+‐ATPase activity produced by alloxan. Also, H7 injected into diabetic mice, 4–9 days after the injection of alloxan, restores the activity of the enzyme. The amount of activity recovered depends on the dose of H7 administered; complete recovery was reached with injection of 15 mg of H7/kg of body weight. The effect of H7 is transient, with a half‐life of ∼ 1 h.
Detection of mRNAs Encoding Distinct Isoenzymes of Type II Calcium/Calmodulin‐Dependent Protein Kinase Using the Polymerase Chain Reaction
Authors: Beaman‐Hall Carol M. , Hozza Mark J. , Vallano Mary Lou
Published: October 2006

Abstract: A modification of the polymerase chain reaction (PCR) was used to amplify nucleotide sequences encoding the 50‐kDa (α) or 58‐ to 60‐kDa (β′,β) subunits of a brain‐specific type II calcium/calmodulin‐dependent protein kinase (CaM kinase II). Rat brain RNA from different regions and at different postnatal ages was purified, and reverse transcriptase was used to produce cDNA templates. Oligonucleotide primer pairs flanking a unique sequence in the coding region of the β′,β subunit‐specific cDNA or a unique sequence in the 3′ noncoding region of the α subunit‐specific cDNA were used to amplify sequences encoding portions of these subunits by PCR. Adult rat forebrain contained approximately three times as much α subunit mRNA as β′,β subunit mRNA, whereas adult rat cerebellum contained a molar ratio of 1 α: 5 β′,β. Intermediate levels of α and β′,β subunit mRNAs were observed in adult pons/medulla, and in 4‐ and 8‐day neonatal forebrain. This amplification assay was also used to demonstrate the presence of α subunit mRNA in cerebellar granule cells and 4‐day neonatal forebrain, which was reported to be undetectable by other methods. Cerebellar granule cells contained less α subunit RNA relative to whole cerebellum, suggesting that this cell type expresses an isoform of CaM kinase II containing less α subunit protein in the holoenzyme. The observed levels of subunit‐specific mRNAs were shown to parallel the levels of expressed protein subunits, suggesting that expression of kinase isoforms is transcriptionally regulated. The data also indicate that the conditions used for amplification of CaM kinase II mRNAs are semi‐quantitative.
Angiotensin II Receptors Are Coupled to ω‐Conotoxin‐Sensitive Calcium Influx in Bovine Adrenal Medullary Chromaffin Cells
Authors: McMillian M. K. , Tuominen R. K. , Hudson P. M. , Suh H. H. , Hong J. S.
Published: October 2006

Abstract: The contribution of an ω‐conotoxin GVIA (ωCgtx)‐sensitive Ca2+ influx pathway to the effects of angiotensin II (AII) receptor activation was examined in bovine adrenal medullary (BAM) cells. Pretreatment of BAM cells with 10–6MωCgtx blocked stimulation of exocytosis by the degradation‐resistant analogue, sarcosine1–angiotensin II (S1‐AII). In contrast, ωCgtx had no effect on basal secretion, nor did it inhibit [3H]norepinephrine and [32P]ATP release in response to bradykinin, another phospholipase C‐linked receptor agonist. Similarly, ωCgtx pretreatment inhibited the stimulation of 45Ca2+ uptake by S1‐AII, but did not affect the response to bradykinin. This selective inhibition did not appear to be due to blockade of AII receptors by ωCgtx, as the accumulation of 3H‐labeled inositol phosphates in response to S1‐AII was not inhibited. The peak S1‐AII‐stimulated increase in the intracellular free Ca2+ concentration (Cai) in fura 2‐loaded BAM cells also was not significantly reduced by ωCgtx (or by stimulating in nominally Ca2+‐free buffer), indicating that this response is dependent on intracellular Ca2+ pools. However, a small ωCgtx‐sensitive Cai response was detected after depletion of intracellular Ca2+ pools with ionomycin. This study shows that AII receptors, but not bradykinin receptors, are linked to an ωCgtx‐sensitive Ca2+ influx pathway in BAM cells.
Identification of Ascorbate as an Endogenous Substance That Irreversibly Inhibits Binding of Dihydropyridine Calcium Channel Blockers
Authors: Ebersole Barbara J. , Molinoff Perry B.
Published: October 2006

Abstract: Endogenous material present in heat‐denatured extracts of rat brain that inhibited the binding of [3H]‐isopropyl‐4‐(2,1,3‐benzoxadiazol‐4‐yl)‐1,4‐dihydro‐5‐methoxycarbonyl‐2,6‐dimethyl‐3‐pyridinecarboxylate ([3H]‐PN200‐110) to calcium channels in brain membranes was purified. Spectrophotometric analysis of material purified by strong anion‐exchange and reverse‐phase chromatography showed an absorption maximum at 266 nm at pH 7.0 that shifted to 245 nm at pH 2.0. This pH‐dependent spectral shift was indistinguishable from that of ascorbic acid. Samples of the purified extract contained ascorbic acid; however, the inhibition of binding by purified material was always greater than the inhibition seen with equivalent concentrations of ascorbate, implying the presence of additional inhibitory factors. Attempts to detect and identify such inhibitory substances by chromatography showed that inhibitory activity was coincident with the presence of ascorbate, and the inhibitory activity of purified material was abolished after treatment with ascorbic acid oxidase. Iron enhanced the inhibition produced by ascorbate, and chemical analysis of purified preparations revealed the presence of iron. Studies comparing the potency of the purified material with that of a mixture of ascorbate plus iron showed that the content of ascorbate and iron in the purified brain extract is sufficient to explain the observed inhibition of binding of [3H]PN200‐110.
Perfusion of Immobilized Isolated Nerve Terminals as a Model for the Regulation of Transmitter Release: Release of Different, Endogenous Transmitters, Repeated Stimulation, and High Time Resolution
Authors: Verhage Matthijs , Sandman Hugo , Mosselveld Fred , van de Velde Marco , Hengst Pieter A. , da Silva Fernando H. Lopes , Ghijsen Wim E. J. M.
Published: October 2006

Abstract: To study the release of neurotransmitters, i.e., the recruitment of transmitters for release and the regulation of the release process, isolated nerve terminals (synaptosomes) of the rat forebrain were immobilized in Sephadex gel inside a perfusion chamber. In this way, the following were achieved: (a) A very limited pressure stress was exerted on the synaptosomes, so that these remained viable for long periods (>30 min) inside the chamber and did not elute from the chamber, which allowed long‐term experiments with repeated stimulations; (b) estimation of the release of various endogenous transmitters, both in a Ca2+‐dependent (exocytotic) and Ca2+‐independent manner; (c) a step‐like stimulation with depolarizing agents (rise time, 3–4 s) and a high time resolution (600‐ms sampling); and (d) negligible reuptake of transmitter into the terminals or extracellular breakdown. It is concluded that this perfusion setup helps to provide new insights in the presynaptic stimulus–secretion coupling, co‐transmission, and the exo–endocytosis cycle.
Differentiation Between Alterations in Plasma and Mitochondrial Membrane Potentials in Synaptosomes Using a Carbocyanine Dye
Authors: Hare Michael F. , Atchison William D.
Published: October 2006

Abstract: The cationic potentiometric fluorescent probe 3,3′‐diethylthiadicarbocyanine iodide [DiS‐C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (Ψp and Ψm, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 μM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. Ψp was calculated to be –54 mV and was not altered significantly by prior depolarization of Ψm with cyanide and oligomycin (hereafter referred to as “poisoned”synaptosomes). Similarly, the linear relationship between dye fluorescence and Ψp was not altered by depolarization of Ψm. Valinomycin, a K+ ionophore, caused a Ψp‐dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized Ψp by about 5 mV. The poreforming antibiotic gramicidin depolarized both Ψp and Ψm maximally. Under these conditions, Triton X‐100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of Ψp). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage‐dependent effects on Ψp and Ψm. The magnitude of the fluorescence response of synaptosomes to depolarization with sufficient K+ to increase [K+]e by 40 mM was dependent on the initial Ψp and was not altered by prior depolarization of Ψm. Thus, K+‐induced depolarization may be useful for estimating Ψp in the face of fluorescence change associated with depolarization of both Ψp and Ψm. Thus, carbocyanine dyes may be used to differentiate effects of experimental treatments on synaptosomal Ψp as compared with intrasynaptosomal Ψm.
Solubilization and Purification of an α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid Binding Protein from Bovine Brain
Authors: Hunter Chyren , Wenthold Robert J.
Published: October 2006

Abstract: α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X‐100 in 0.5 M phosphate buffer and 20% glycerol at 37°C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high‐ and low‐affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr∼ 500,000. With sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr= 110,000. Previously, we have shown that a monoclonal antibody (KAR‐B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr∼ 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.
Localization of Arachidonate 12‐Lipoxygenase in Canine Brain Tissues
Authors: Nishiyama Makoto , Okamoto Hisayo , Watanabe Takashi , Hori Tomokatsu , Hada Takahiko , Ueda Natsuo , Yamamoto Shozo , Tsukamoto Hideo , Watanabe Keiichi , Kirino Takaaki
Published: October 2006

Abstract: The cytosol fraction from a thoroughly imgated canine cerebrum was subjected to immunoaffinity chromatography using a monoclonal antibody against porcine leukocyte 12‐lipoxygenase. Arachidonate 12‐lipoxygenase eluted from the column with some retardation. The enzyme, with a specific activity of 9 nmol/min/mg of protein, converted arachidonic acid to 12(S)‐hydroperoxy‐5,8,10,14‐eicosatet‐raenoic acid. The enzyme was active not only with arachidonic acid, but also with linoleic and α‐linolenic acids. In contrast, 12‐lipoxygenase of canine platelets was almost inactive with linoleic and α‐linolenic acids, and the platelet enzyme was also distinguished from the cerebral enzyme in terms of reactivity with the anti‐12‐lipoxygenase antibody. 12‐Lipoxygenase activity was also detected in the cytosol fractions of other parts of canine brain: basal ganglia, hippocampus, cerebellum, olfactory bulb, and medulla oblongata.
Clonidine Inhibition of Norepinephrine Release from Normal and Morphine‐Tolerant Guinea Pig Cortical Slices
Authors: Tomasini C. , Guidorzi R. , Bianchi C. , Beani L.
Published: October 2006

Abstract: Endogenous norepinephrine (NE) release in cerebral cortex slices taken from normal and morphine‐tolerant guinea pigs was measured by HPLC. In normal slices, a linear relationship was found between electrically evoked NE release and the log of the frequency of stimulation in the range of 1–20 Hz. The efficiency of the α2‐mediated autofeedback was tested by adding the α2 agonist clonidine and the α2 antagonist idazoxan. NE release was dose‐dependently reduced by clonidine (1 nmol/L–1 μmol/L) and increased by idazoxan (10–100 nmol/L). The inhibition by clonidine was significantly greater at 1 Hz than at 3 Hz, whereas the absolute increase in NE release induced by idazoxan was greater at 3 Hz than at 1 Hz. Morphine at 1 μmol/L (a concentration per se ineffective) shifted to the left the clonidine concentrations able to inhibit NE release at 3 and 1 Hz (1–10 nmol/L), but at both frequencies, the opiate reduced the maximal inhibition induced by clonidine at 1 μmol/L. In slices taken from morphine‐tolerant guinea pigs (in the presence of morphine at 1 μmol/L), clonidine (1 nmol/L–1 μmol/L) was ineffective at the stimulation rate of 3 Hz, but it was more active than in normal slices at 1 Hz. Such a response pattern suggests a reduced availability of α2 receptors and an increase in their sensitivity to clonidine. However, chronic morphine treatment did not influence the physiological autoinhibition because the increase in NE release elicited by idazoxan (10–100 nmol/L) at 1 and 3 Hz was the same in normal and in “morphine‐tolerant”slices. Therefore, morphine tolerance affects only the clonidine responsiveness of some α2 receptors, possibly located far from the hot spots of NE release. In contrast, the autofeedback exerted by synaptically released transmitter, as evaluated by testing idazoxan, remains unchanged.
High‐Level Synthesis and Fate of Acetylcholine in Baculovirus‐Infected Cells: Characterization and Purification of Recombinant Rat Choline Acetyltransferase
Authors: Habert Estelle , Birman Serge , Mallet Jacques
Published: October 2006

Abstract: Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild‐type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for ∼24% of the cell cytosolic proteins as being ChAT. The apparent Km, values of the enzyme for choline and acetyl‐CoA were 299 and 221 μM, respectively, whereas the respective Vmax values were 10.6 and 11.4 μmol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one‐step purification procedure starting with 108 infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore‐mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level.
Putative Neurotoxicity of SKF 38393 and Other D1 Dopaminergic Drugs Investigated in Rat Striatum
Authors: Isaac L. , Mills R. , Fowler L. J. , Starr B. S. , Starr M. S.
Published: October 2006

Abstract: The recently alleged neurotoxicity of the D1 receptor agonist, SKF 38393, was investigated in rat striatum by measuring the enzymes acetylcholinesterase (AChE) and glutamate decarboxylase (GAD). First, unilateral intrastriatal microinjection of the excitotoxin kainic acid (2 μg in 1 μ1) was shown to evoke vigorous contraversive circling, followed 1 or 2 weeks later by profound decreases in striatal AChE (24 and 54%), GAD (51 and 75%), and protein (36 and 47%), as well as loss of GAD (45% at 2 weeks) in the ipsilateral substantia nigra. Similar striatal treatments with SKF 38393 (30 μg in 0.5–1 μ), the related benzazepines SKF 82526 (D1 agonist, 30 μg in 1 μ1) and SCH 23390 (D1 antagonist, 5 μg in 1 μ1), or the phenanthridine D1 agonist CY 208‐243 (5 μg in 1 μ1) failed to affect the rats' behaviour or their striatal levels of AChE, GAD, and protein. Intrastriatal SKF 38393 (30 μg in 0.5 μ1) also had no influence on these enzymes in the substantia nigra. It is concluded that none of the D1 dopaminergic compounds examined here was neurotoxic toward the many different cell groups that contain AChE and/or GAD in the striatum.
Increased Phosphorylation of a 17‐kDa Protein Kinase C Substrate (P17) in Long‐Term Potentiation
Authors: Klann Eric , Chen Shu‐Jen , Sweatt J. David
Published: October 2006

Abstract: Hippocampal long‐term potentiation (LTP) is a persistent increase in the efficacy of synaptic transmission, which is widely thought to be a cellular mechanism that could contribute to learning and memory. Studies on the biochemical mechanisms underlying LTP suggest the involvement of protein kinases in both LTP induction and maintenance. In this report we describe an LTP‐associated increase in the phosphorylation in vitro of a 17‐kDa protein kinase C (PKC) substrate protein, which we have termed P17, in homogenates from the CA1 region of rat hippocampal slices. This LTP‐associated increase in phosphorylation was expressed independent of significant levels of free Ca2+, as phosphorylation reactions were performed in the presence of 500 μM EGTA. The increased phosphorylation of P17 was substantially inhibited by PKC(19–36), a selective inhibitor of PKC. These data support the model that persistent PKC activation contributes to the maintenance of LTP and implicate P17 as a potential target for PKC in the CA1 region of the hippocampus.
Promoter Elements of the Rat Choline Acetyltransferase Gene Allowing Nerve Growth Factor Inducibility in Transfected Primary Cultured Cells
Authors: Bejanin Stephane , Habert Estelle , Berrard Sylvie , Edwards Jean‐Baptiste Dumas Milne , Loeffler Jean‐Philippe , Mallet Jacques
Published: October 2006

Abstract: Choline acetyltransferase, the enzyme responsible for the synthesis of acetylcholine, provides a convenient index for cholinergic neurons. Using a previously identified rat cDNA clone, we have isolated several corresponding genomic clones and have characterized a 1,902‐bp fragment that contains part of the first noncoding exon as well as promoter sequences. The promoter activity of this fragment was tested, taking advantage of the recently developed lipopolyamine‐mediated DNA transfer method, which allows transfection of primary neurons. The 1,902‐bp sequence drives the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in a culture of dissociated cells prepared from the septal area of fetal (embryonic day 17) rats, a structure rich in cholinergic neurons. Moreover, addition of nerve growth factor to the culture increases CAT expression by ∼56‐fold, indicating that our DNA fragment contains sequences required for NGF induction. In addition, it contains consensus sequences for various transcription factors, including those of the basic helix–loop–helix family. Finally, experiments to characterize the transcription start site are presented.
Regulation of γ‐Aminobutyric Acid Synthesis in the Brain
Authors: Martin David L. , Rimvall Karin
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) is synthesized in brain in at least two compartments, commonly called the transmitter and metabolic compartments, and because reglatory processes must serve the physiologic function of each compartment, the regulation of GABA synthesis presents a complex problem. Brain contains at least two molecular forms of glutamate decarboxylase (GAD), the principal synthetic enzyme for GABA. Two forms, termed GAD65 and GAD67, are the products of two genes and differ in sequence, molecular weight, interaction with the cofactor, pyridoxal 5′‐phosphate (pyridoxal‐P), and level of expression among brain regions. GAD65 appears to be localized in nerve terminals to a greater degree than GAD67, which appears to be more uniformly distributed throughout the cell. The interaction of GAD with pyridoxal‐P is a major factor in the short‐term regulation of GAD activity. At least 50% of GAD is present in brain as apoenzyme (GAD without bound cofactor; apoGAD), which serves as a reservoir of inactive GAD that can be drawn on when additional GABA synthesis is needed. A substantial majority of apoGAD in brain is accounted for by GAD65, but GAD67 also contributes to the pool of apoGAD. The apparent localization of GAD65 in nerve terminals and the large reserve of apo‐GAD65 suggest that GAD65 is specialized to respond to short‐term changes in demand for transmitter GABA. The levels of apoGAD and the holoenzyme of GAD (holoGAD) are controlled by a cycle of reactions that is regulated by physiologically relevant concentrations of ATP and other polyanions and by inorganic phosphate, and it appears possible that GAD activity is linked to neuronal activity through energy metabolism. GAD is not saturated by glutamate in synaptosomes or cortical slices, but there is no evidence that GABA synthesis in vivo is regulated physiologically by the availability of glutamate. GABA competitively inhibits GAD and converts holo‐ to apoGAD, but it is not clear if intracellular GABA levels are high enough to regulate GAD. There is no evidence of short‐term regulation by second messengers. The syntheses of GAD65 and GAD67 proteins are regulated separately. GAD67 regulation is complex; it not only is present as apoGAD67, but the expression of GAD67 protein is regulated by two mechanisms: (a) by control of mRNA levels and (b) at the level of translation or protein stability. The latter mechanism appears to be mediated by intracellular GABA levels.
Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats
Authors: Valchar Martin , Hanbauer Ingeborg
Published: October 2006

Abstract: In contrast to striatal membranes of adult rats, where high‐ (KD1= 34 nM) and low‐ (KD2= 48,400 nM) affinity binding sites for [3H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low‐affinity binding sites were found (KD= 336,000 nM). The binding of [3H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [3H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [3H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100‐8,000 times higher concentrations than were needed to displace [3H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR‐12909, GBR‐12935, and rimcazole, inhibited [3H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation (r= 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [3H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC50 values of drugs that inhibited [3H]WIN 35,428 binding and [3H]DA uptake in CVMNs. The cytosolic [3H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.
N‐Methyl‐d‐Aspartate Releases γ‐Aminobutyric Acid from Rat Striatum In Vivo: A Microdialysis Study Using a Novel Preloading Method
Authors: Young Andrew M. J. , Bradford H. F.
Published: October 2006

Abstract: In vivo microdialysis was used in conjunction with a novel dual‐label preloading method to monitor changes in extracellular levels of γ‐aminobutyric acid (GABA) and glutamate due to N‐methyl‐d‐aspartate (NMDA) infusion in the striatum of conscious, unrestrained rats. [14C]GABA and [3H]glutamate were applied in the dialysis stream for a preloading period of 30 min, after which dialysis perfusion was continued for up to 6 h and dialysate samples were collected for analysis by liquid scintillation spectrometry. NMDA (300 μM in the dialysate) caused significant rises in both 14C and 3H content measured in the dialysates, the majority of which remained associated with the preloaded GABA and glutamate, respectively. The NMDA‐evoked release of both GABA and glutamate was blocked by the specific NMDA receptor antagonist 3‐[(±)‐2‐carboxypiperazin‐4‐yl]propyl‐1‐phosphonic acid (CPP), indicating that the response was receptor mediated. The NMDA‐stimulated release of glutamate was also totally abolished by concomitant application of the adenosine agonist 2‐chloroadenosine or by prior frontal decortication. However, these two treatments caused little change in NMDA‐evoked GABA release. These results show that NMDA causes release of GABA from the striatum in vivo by an NMDA receptor‐mediated mechanism and that the majority of this release is not secondary to glutamate release from terminals of the corticostriate pathway. In addition, they confirm the results of previous studies investigating the effect of NMDA on endogenous glutamate release.
Metabotropic Glutamate Receptors in Cultured Cerebellar Granule Cells: Developmental Profile
Authors: Aronica E. , Condorelli D. F. , Nicoletti F. , Dell'Albani P. , Amico C. , Balázs R.
Published: October 2006

Abstract: Excitatory amino acid (EAA)‐induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L‐Glu, quisqualate, and trans‐(±)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid: The stimulation of [3H]inositol monophosphate ([3H]‐InsP) formation was low at 2 days in vitro (DIV), but the response increased steeply, reaching a peak at 4 DIV, followed by a progressive decline. In contrast, carbamylcholine‐induced PPI hydrolysis exhibited a plateau after a pronounced increase during the first week in vitro. At 6 DIV, but not at 4 DIV, when the activity peaked, PPI hydrolysis elicited by Glu was reduced by the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801, indicating that in cultured granule cells, NMDA receptors contribute to [3H]‐InsP formation and that this component of the response develops relatively late. Accordingly, NMDA‐induced [3H]‐InsP formation, estimated under Mg2+‐free conditions, increased markedly from very low values at 2 DIV to a plateau at 8–10 DIV. The developmental pattern of EAA‐induced PPI hydrolysis was paralleled by changes in the level of an mRNA for a specific mGluR subtype (mGluR1 mRNA). RNA blot analysis performed with the pmGR1 cDNA probe revealed that the hybridization signal in RNA extracts from cultures at 1 DIV was very weak, but mGluR mRNA levels increased dramatically between 1 and 3 DIV, followed by a progressive decrease, so that by 15 DIV the mRNA levels were only ∼10% of the values at 3 DIV. These observations indicate that the functional expression of the mGluR is subject to developmental regulation, which critically involves receptor mRNA levels.
Ca2+ Release from Inositol 1,4,5‐Trisphosphate‐Sensitive Ca2+ Store by Antidepressant Drugs in Cultured Neurons of Rat Frontal Cortex
Authors: Shimizu Masami , Nishida Akira , Hayakawa Hiroshi , Yamawaki Shigeto
Published: October 2006

Abstract: The ability of antidepressant drugs (ADs) to increase the concentration of intracellular Ca2+ ([Ca2+]i) was examined in primary cultured neurons from rat frontal cortices using the Ca2+‐sensitive fluorescent indicator fura‐2. Amitriptyline, imipramine, desipramine, and mianserin elicited transient increases in [Ca2+]i in a concentration‐dependent manner (100 μM to 1 mM). These four AD‐induced [Ca2+]i increases were not altered by the absence of external Ca2+ or by the presence of La3+ (30 μM), suggesting that these ADs provoked intracellular Ca2+ mobilization rather than Ca2+ influx. All four ADs increased inositol 1,4,5‐trisphosphate (IP3) contents by 20–60% in the cultured cells. The potency of the IP3 production by these ADs closely correlated with the AD‐induced [Ca2+]i responses. Pretreatment with neomycin, an inhibitor of IP3 generation, significantly inhibited amitriptyline‐ and imipramine‐induced [Ca2+]i increases. In addition, by initially perfusing with bradykinin (10 μM) or acetylcholine (10 μM), which can stimulate the IP3 generation and mobilize the intracellular Ca2+, the amitriptyline responses were decreased by 76% and 69%, respectively. The amitriptyline‐induced [Ca2+]i increases were unaffected by treatment with pertussis toxin. We conclude that high concentrations of amitriptyline and three other ADs mobilize Ca2+ from IP3‐sensitive Ca2+ stores and that the responses are pertussis toxin‐insensitive. However, it seems unlikely that the effects requiring high concentrations of ADs are related to the therapeutic action.
Corticotropin‐Releasing Factor Stimulates Catecholamine Release in Hypothalamus and Prefrontal Cortex in Freely Moving Rats as Assessed by Microdialysis
Authors: Lavicky Jan , Dunn Adrian J.
Published: October 2006

Abstract: In vivo microdialysis was used to measure changes in extracellular concentrations of catecholamines and indoleamines in freely moving rats in response to administration of corticotropin‐releasing factor (CRF). Dialysis probes were placed stereotaxically in either the medial hypothalamus or the medial prefrontal cortex. We used a repeated‐measures design in which each rat received artificial CSF or one dose of CRF 3–4 h apart, and each subject was retested with the same treatments in the reverse order 5–7 days later. With the dialysis probe in the hypothalamus, intracerebroventricular administration of CRF (17 or 330 pmol) dose‐dependently increased dialysate concentrations of norepinephrine (NE), dopamine (DA), and all their measurable catabolites except normetanephrine. The effects on NE were substantially greater than those on DA. Dialysate concentrations of serotonin could not be measured reliably, but those of its catabolite, 5‐hydroxyindoleacetic acid, were also elevated. Concentrations of NE and DA were elevated within the first one or two (20 min) collection periods, with a peak response at ∼ 1–2 h. Dialysate concentrations of catecholamines and metabolites normally returned to baseline within 3 h. Similar data were obtained with dialysis probes in the medial prefrontal cortex after intracerebroventricular administration of 17 or 167 pmol of CRF, except that the increases in DA exceeded those of NE in this region. Intraperitoneal administration of CRF (1 nmol) similarly elevated dialysate concentrations of NE, DA, 5‐hydroxyindoleacetic acid, and all catecholamine catabolites except normetanephrine in both medial hypothalamus and medial prefrontal cortex. These results support earlier neurochemical data suggesting that CRF administered both centrally and peripherally stimulates the release of both DA and NE in the brain.
Effect of P2Y Agonists on Adenosine Transport in Cultured Chromaffin Cells
Authors: Sen Raquel P. , Delicado Esmerilda G. , Castro Enrique , Miras‐Portugal M. Teresa
Published: October 2006

Abstract: Adenosine transport in cultured chromaffin cells was inhibited by purinergic P2y‐receptor agonists without significant changes in the affinity constant, the values being between 1 ± 0.4 and 1.6 ± 0.6 μM. The Vmax parameter was modified significantly, being 40 ± 1.0, 26 ± 5.0, 32 ± 3.0, and 22 ± 4.7 pmol/106 cells/min for control, adenosine‐5′‐O‐(2‐thiodiphosphate), 5′‐adenylylimidodiphosphate, and P1,P4‐di(adenosine‐5′‐) tetraphosphate (Ap4A) (100 μM for every effector), respectively. Ap4A, a physiological ligand for P2y receptors in chromaffin cells, showed the highest inhibitory effect (45%). This transport inhibition is explained by an increase in the cytosolic Ca2+ concentration ([Ca2+]i) and the activation of protein kinase C (PKC). Experiments of [Ca2+]i measurement with the fura‐2 technique showed that P2y agonists, as well as bradykinin, were able to increase [Ca2+]i, this effect being independent of the presence of extracellular Ca2+. The peptide bradykinin, determined to be coupled to phosphatidylinositol hydrolysis and internal Ca2+ mobilization in chromaffin cells, exhibited a behavior similar to that of P2y agonists in adenosine transport inhibition (39%). P2y agonists and bradykinin increased PKC activity associated with the membrane fraction (about 50% increase in particulate PKC activity with respect to controls). The present studies suggest that adenosine transport is regulated by P2y‐purinergic receptors mediated via Ca2+ mobilization and PKC activation.