bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Effects of Forced Locomotion, Tail‐Pinch, Immobilization, and Methyl‐β‐Carboline Carboxylate on Extracellular 3,4‐Dihydroxyphenylacetic Acid Levels in the Rat Striatum, Nucleus Accumbens, and Prefrontal Cortex: An In Vivo Voltammetric Study
Authors: Bertolucci‐D'Angio M. , Serrano A. , Scatton B.
Published: October 2006

Abstract: In vivo voltammetry with carbon fiber electrodes was used to assess extracellular 3,4‐dihydroxyphenylacetic acid (DOPAC) levels in striatum, nucleus accumbens. and anteromedial prefrontal cortex of freely moving rats subjected to altered motor activity or anxiogenic stimuli. Forced locomotion on a rotarod for 40 min caused an increase in extracellular DOPAC levels in the striatum and to a lesser extent in the nucleus accumbens but not in the prefrontal cortex. Subcutaneous injection of the anxiogenic agent methyl‐β‐carboline carboxylate (10 mg/kg) increased extracellular DOPAC levels to a similar extent in prefrontal cortex and nucleus accumbens. Immobilization for 4 min augmented dopamine (DA) metabolism preferentially in the nucleus accumbens and to a lesser extent in the prefrontal cortex. Tail‐pinch caused a selective activation of DA metabolism in the nucleus accumbens. None of these stimuli altered extracellular striatal DOPAC levels. These results confirm the involvement of dopaminergic systems projecting to the striatum and nucleus accumbens in motor function and suggest that mesolimbic and mesocortical dopaminergic systems can be specifically activated by certain kinds of anxiogenic stimuli; the relative activation of either of these latter systems could depend primarily on the nature (sensory modality, intensity) of the acute stressor.
Decrease in the Function of the γ‐Aminobutyric Acid‐Coupled Chloride Channel Produced by the Repeated Administration of Pentylenetetrazol to Rats
Authors: Corda M. G. , Giorgi O. , Longoni B. , Orlandi M. , Biggio G.
Published: October 2006

Abstract: The acute administration of pentylenetetrazol (PTZ; 25–75 mg/kg i.p.) failed to modify the specific binding of t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) to membrane preparations from the cerebral cortex of the rat. In contrast, the repeated administration of PTZ (30 mg/kg i.p., three times a week for 12 weeks) reduced by 26% the density of [33S]TBPS binding sites without modifying the dissociation constant. This effect was observed 3 days after the last PTZ administration. A parallel reduction of 7‐amino‐butyric acid (GABA)‐stimulated 36CI− uptake was measured in the cerebral cortex of PTZ‐treated rats 3 days after the last injection. The repeated administration of PTZ produced sensitization to the drug, or chemical kindling. In fact, no convulsions were observed in the first week of treatment, but all the animals became sensitized to PTZ by the 12th week. The results are consistent with the hypothesis that chronic treatment with PTZ at a subconvulsant dose causes a decrease in GABA‐coupled chloride channel activity that may be related to the chemical kindling produced by this compound.
Fish Apolipoprotein‐A‐I Has Heparin Binding Activity: Implication for Nerve Regeneration
Authors: Harel A. , Fainaru M. , Rubinstein M. , Tal N. , Schwartz M.
Published: October 2006

Abstract: This study provides evidence that apolipoprotein‐A‐I (apo‐A‐I), derived from fish plasma and nerve, has heparin binding activity. We have shown previously that injury in a regenerative CNS, such as that of fish optic nerves, leads to increased levels of apo‐A‐I in media conditioned by these nerves, as compared with media conditioned by noninjured nerves. In the present study, we have purified and characterized apo‐A‐I from both fish plasma and optic nerves. Sequence analysis of the 15 N‐terminal amino acids revealed that at least 14 amino acids are identical in these two purified apo‐A‐I samples. The purified apo‐A‐I derived from both fish plasma and optic nerves binds to heparin. Binding measurements using [3H]heparin followed by Scatchard analysis revealed that apo‐A‐I binds to heparin with relatively low affinity (KD= 2.8 × 10−6M). Results are discussed with respect to the possibility that accumulation of apo‐A‐I in the extracellular matrix of fish optic nerves is made possible via heparin binding, like that to apolipoprotein‐E in mammals.
μ‐Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine‐Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons
Authors: Van Vliet Bernard J. , Mulder Arie H. , Schoffelmeer Anton N. M.
Published: October 2006

Abstract: The receptors mediating the inhibition of D1 dopamine receptor‐stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose‐dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50= 0.05 μM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 μM). SKF 38393 (1 μM)‐stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective μ‐agonist [D‐Ala2,MePhe4,Gly‐ol5]‐enkephalin (DAGO; EC50= 0.006μM) and high concentrations of the selective δ‐agonist [d‐Ser2(O‐tert‐butyl), Leu5]‐enkephaIyl‐Thr6 (DSTBU‐LET; EC50= 0.13 μM) but not by the selective δ‐agonist [d‐penicillamine2, d‐penicillamine5]enkephalin (DPDPE). D1 dopamine receptor‐stimulated adenylate cyclase activity was also slightly reduced (by ∼20%) by high concentrations of the k‐agonist U50,488 (EC50= 0.63 μM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the μ‐opioid receptor‐selective antagonist naloxone (EC50 of ∼0.1 μM). Neither the irreversible δ‐ligand fentanyl isothiocyanate (1 μM) nor the reversible δ‐antagonist ICI 174864 (1 μM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (>0.1 μM) of the k‐opioid receptor‐selective antagonist norbinaltorphimine. The effect of DAGO (1 μM) was already detectable after 1 day in culture, whereas DPDPE (1 μM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of μ‐opioid receptors coupled as inhibitors to D1 dopamine receptor‐stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.
Intracellular Calcium Pools in Neuroblastoma × Glioma Hybrid NG108–15 Cells
Authors: Chueh Sheau‐Huei , Liu Jin‐Ann , Kao Lung‐Sen
Published: October 2006

Abstract: The intracellular nonmitochondrial calcium pools of saponin‐permeabilized NG108–15 cells were characterized using inositol 1,4,5‐trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11 % of the calcium after GTP treatment. Guanosine 5′‐O‐(3‐thio)triphosphate had little effect on IP3‐induced calcium release but completely inhibited GTP‐induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 μM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 μM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.
Calcium Movements Mediated by Proteolipid Protein and Nucleotides in Liposomes Prepared with the Endogenous Lipids from Brain White Matter
Authors: Díaz R. S. , Monreal J. , Lucas M.
Published: October 2006

Abstract: A lipid extract with a composition similar to that of myelin was used to prepare liposomes and proteoliposomes containing the Folch‐Lees proteolipid apoprotein. Freeze‐fracture replicas of the proteoliposomes were prepared to demonstrate the presence of intramembrane protein particles in the fracture faces of the lipid bilayer. Experiments with 45CaCl2 showed that a steady calcium movement occurs across liposomal membranes, approaching equilibrium between intra‐ and extravesicular spaces. The most significant finding was that Mg‐ATP, ATP analogues, and other nucleotides depressed significantly the calcium fluxes in proteoliposomes, having no effect on liposomes that lacked the proteolipid protein. It is suggested that this intrinsic protein, interacting with nucleotides and endogenous lipids, could be involved in the regulation of calcium levels in myelin by means of a conformational change mechanism. These observations could lead to implications concerning the pathophysiology of myelin.
Temperature Dependence of [3H]Ro 15–1788 Binding to Benzodiazepine Receptors in Human Postmortem Brain Homogenates
Authors: Kopp Jutta , Hall Håkan , Persson Anders , Sedvall Göran
Published: October 2006

Abstract: The temperature dependence of in vitro binding of [3H]Ro 15–1788 to benzodiazepine receptors in human postmortem neocortex and neocerebellum homogenates was studied. An increase of the equilibrium dissociation constants (KD) from 1.40 nmol/L and 1.04 nmol/L at 4°C to 6.10 nmol/L and 8.91 nmol/L at 37°C was found for neocortex and neocerebellum, respectively. In contrast, maximal binding (Bmax) remained in the range of 30–35 fmol/mg for neocortex and 24–27 fmol/mg of tissue (wet weight) for neocerebellum at all the temperatures. The KD of 6.10 nmol/L for neocortex at 37°C in vitro is of the same order as the KD of 10 nmol/L obtained by positron emission tomography for [11C]Ro 15–1788 binding to benzodiazepine receptors in the human neocortex in vivo. The differences in KD between in vitro and in vivo benzodiazepine receptor binding to human neocortex and cerebellum seem to be due at least partially to temperature differences of in vitro and in vivo studies.
Characterization of A‐2 Receptors in Postmortem Human Pineal Gland
Authors: Reynaud Denis , Gharib Abdallah , Lagarde Michel , Sarda Nicole
Published: October 2006

Abstract: We have examined the binding of the adenosine agonist radioligands [3H]N6‐cyclohexyladenosine ([3H]CHA) and [3H]5′‐N‐ethylcarboxamidoadenosine ([3H]NECA) to membranes prepared from postmortem human pineal glands. The results showed that the A‐1‐specific ligand CHA did not bind to membranes. By contrast, [3H]NECA, a nonselective A‐ 1/A‐2 ligand, gave 68% specific binding of the total binding. This specific binding was nearly insensitive to the TV‐ethylmaleimide pretreatment method. To characterize this binding, we used cyclopentyladenosine (50 nM). Under those conditions [3H]NECA binding at 30°C was rapid and reversible; the KD determined from the kinetic studies was 141 nM. In postmortem human pineal gland, the rank order of potency of adenosine analogues and drugs competing with [3H]NECA showed the specificity for an A‐2 receptor: NECA > 2‐chloroadenosine > l‐N6(2‐phenylisopropyl)adenosine > 8‐phenyltheophylline > 3‐isobutyl‐1‐methylxanthine > caffeine. Guanylylimidodiphosphate (100 μM) induced a decrease in the affinity of [3H]NECA, a result suggesting the involvement of a G protein mechanism in the coupling of the adenosine receptor to other components of the receptor complex. Scatchard analysis revealed one class of binding sites for [3H]NECA with KD and Bmax ranging from 175 to 268 nM and 11.0 to 14.1 pmol/mg protein, respectively. The binding of [3H]NECA was not affected by age, sex, or postmortem delay. [3H]NECA should be a useful tool to assess brain A‐2 receptor density in a variety of neuropsychiatric disorders.
Cholinergic Regulation of Vasoactive Intestinal Peptide Content and Release in Rat Frontal Cortex and Hippocampus
Authors: Lapchak P. A. , Beaudet A.
Published: October 2006

Abstract: The purpose of this study was to determine whether the cholinergic system might have a regulatory role on vasoactive intestinal peptide (VIP) synthesis and release in the rat hippocampus and frontal cortex. Incubation of hippocampal or frontal cortical slices with the muscarinic agonist oxotremorine or antagonist atropine did not significantly alter VIP release. The nicotinic agonist methylcarbamylcholine (MCC) and the nicotinic antagonist dihydro‐β‐erythroidine were also ineffective in altering VIP release. Chronic atropine (20 mg/kg, s.c., b.i.d., 10 days) and nicotine (0.59 mg/kg, s.c., b.i.d., 10 days) treatment significantly decreased the VIP content of the frontal cortex, by 42% and 26%, respectively. In contrast, neither treatment significantly altered the VIP content of the hippocampus. Both drug treatments decreased the amount of VIP released from tissue slices depolarized with veratridine in both cerebral cortex and hippocampus. Therefore, long‐term treatment with atropine and nicotine results in changes in the synthesis and release of VIP in the cerebral cortex, whereas in the hippocampus the effect is limited to an alteration of VIP release. These results suggest that the acetylcholine regulates VIP neurotransmission in the rat frontal cortex and hippocampus by an action on muscarinic and nicotinic receptors.
Protein Kinase C Participates in Up‐Regulation of Dihydropyridine‐Sensitive Calcium Channels by Ethanol
Authors: Messing Robert O. , Sneade Alisa B. , Savidge Beth
Published: October 2006

Abstract: Exposure to ethanol for several days increases the expression of dihydropyridine‐sensitive, voltage‐dependent Ca2+ channels in brain and in the neural cell line PC12. Since protein phosphorylation is a major mechanism by which ion channels are regulated, we used protein kinase inhibitors to investigate whether ethanol‐induced up‐regulation of Ca2+ channels involves activation of a protein kinase. Sphingosine and polymixin B, which inhibit protein kinase C and calmodulin‐dependent kinases, prevented the enhancement of 45Ca2+ uptake induced by exposure of PC12 cells to ethanol for 4 days. In addition, sphingosine blocked the ability of ethanol to increase the number of [3H]dihydropyridine binding sites in PC12 cell membranes. Sphingosine's effect was prevented by simultaneous exposure to phorbol 12,13‐dibutyrate, a potent activator of protein kinase C. Therefore, protein kinase C appears to be involved in the up‐regulation of dihydropyridine‐sensitive Ca2+ channels during prolonged exposure to ethanol.
Conservation Throughout Vertebrate Evolution of the Predicted β‐Strands in Myelin Basic Protein
Authors: Stoner Gerald L.
Published: October 2006

Abstract: To identify functionally important parts of the 18.5‐kDa myelin basic protein (MBP), the amino acid sequences from 10 species ranging from shark to human were aligned using the SEQHP computer program. The residues that are invariant or very conservatively substituted (Arg/Lys, Ser/Thr, Ile/Leu, Asp/Glu) among all 10 proteins were scored. Of the 72 conserved residues in the 170‐residue human protein (42% conserved), 32 are found within the five β‐strands previously predicted (45 residues, 71% conserved), 23 within the small‐loops region (42 residues, 55% conserved), but only 17 within the large‐loops region (83 residues, 20% conserved). Of the 22 hydrophobic residues within the predicted β‐sheet of human MBP, 20 hydrophobic residues remain in the shark protein, 19 of them in the same positions. In contrast, there are 10 hydrophobic residues elsewhere in the human protein, but only 7 remain in the shark protein and only 1 of them is in the same position. The triprolyl sequence found in all mammalian MBPs and in the chicken MBP is not conserved in the shark protein. The four alternately spliced forms of mouse MBP can be accommodated by the β‐structural model, but not the 17‐kDa human MBP, which lacks exon 5. These findings confirm the crucial role of the hydrophobic residues in the predicted β‐sheet for the structure and function of the protein. It seems likely that the conserved portions of the protein make an important contribution to the highly ordered lamellar structure of myelin.
Immunity of Fetal Mice to Prenatal Administration of the Dopaminergic Neurotoxin 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine
Authors: Melamed Eldad , Rosenthal Jutta , Youdim Moussa B. H.
Published: October 2006

Abstract: Subcutaneous injection of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) HC1 (25 mg/kg) in pregnant female mice at the 17th day of gestation markedly depleted striatal dopamine (DA) concentrations in the mothers 24 h later and at 24 h and 28 days after delivery. By contrast, in the offspring of the female mice exposed to MPTP during pregnancy, fetal brain DA concentrations at 24 h after injection and at 24 h after birth and striatal DA levels at 14 and 28 days postnatally were unaffected and identical to those in age‐matched controls. The postnatal ontogenesis of striatal DA levels was identical in offspring of control vehicle‐ and MPTP‐treated pregnant mice. Also, prenatal challenge with MPTP did not make nigrostriatal DA neurons more vulnerable to a second postnatal treatment with the toxin. Striatal DA depletions were identical in 6‐week‐old mice given MPTP, whether they were exposed to MPTP or to vehicle in utero. Monoamine oxidase (EC 1.4.3.4; MAO) type B activity was extremely low in the fetal brain and, relatively, much lower than that of MAO‐A. Prenatal MPTP administration reduced maternal striatal and also embryonal brain MAO‐B activity at 24 h post treatment but did not alter the normal postnatal development of striatal MAO‐A and ‐B activities in the offspring. Study suggests that resistance of fetal DA neurons to the DA‐depleting effect of MPTP may be due, at least in part, to an absence in the embryonal brain of adequately developed MAO‐B activity required for the conversion of MPTP to its toxic metabolite, 1‐methyl‐4‐phenylpyridinium ion.
Excitatory Amino Acid‐Induced Formation of Inositol Phosphates in Guinea‐Pig Cerebral Cortical Slices: Involvement of Ionotropic or Metabotropic Receptors?
Authors: Alexander Stephen P. H. , Hill Stephen J. , Kendall David A.
Published: October 2006

Abstract: In cerebral cortical slices from the guinea‐pig, quinoxalinedione derivatives antagonised the generation of 3H‐inositol phosphates evoked by the excitatory amino acids quisqualate and DL‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxalone propionic acid but were without effect on the trans‐DL‐1‐amino‐ 1,3‐cyclopentanedicarboxylic acid and l‐glutamate responses. Omission of calcium from the medium reduced the accumulation of 3H‐inositol phosphates induced by incubation with trans‐DL‐1‐amino‐ 1,3‐cyclopentanedicarboxylic acid (incubation for 45 min) by > 50%, whereas the responses to l‐glutamate and the two other amino acid analogues were reduced by ∼20%. Generation of inositol 1,4,5‐trisphosphate over a 30‐s period by treatment with quisqualate, trans‐DL‐1‐amino‐ 1,3‐cyclopentanedicarboxylic acid, KCI, and carbachol was abolished in the presence of nominally calcium‐free medium. l‐Glutamate induced a large, rapid increase in inositol 1,4,5‐trisphosphate mass (more than threefold), which was, however, unaffected by omission of calcium from the medium. These results indicate that of the excitatory amino acids tested, only l‐glutamate may be classed as a metabotropic receptor agonist in guinea‐pig cerebral cortical slices with respect to generation of inositol phosphates. The other agents appear to stimulate accumulation of inositol phosphates, at least in part through some mechanism requiring the presence of extracellular Ca2+, presumably Ca2+ entry.
Histamine H1‐Receptor in Heart: Unique Electrophoretic Mobility and Autoradiographic Localization
Authors: Ruat M. , Bouthenet M. L. , Schwartz J.‐C. , Ganellin C. R.
Published: October 2006

Histamine H1‐receptors, visualized in the guinea pig heart by autoradiography using [125I]iodoboipyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1‐receptor was shown to be present on a major 68‐kDa and a less abundant 54‐ to 58‐kDa protein. The 68‐kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1‐receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.
A Nerve Growth Factor‐Dependent Protein Kinase That Phosphorylates Microtubule‐Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells
Authors: Sano Mamoru , Nishiyama Kaoru , Kitajima Satoko
Published: October 2006

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF‐pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three‐ to eightfold in terms of phosphorylation of microtubule‐associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP‐dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg2+ for activity but not Mn2+ or Ca2+. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF‐dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K‐252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF‐dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF‐dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.
Interferon Suppresses Sympathetic Neuronal Cell Death Caused by Nerve Growth Factor Deprivation
Authors: Chang Jason Y. , Martin David P. , Johnson Eugene M.
Published: October 2006

Cultured rat sympathetic neurons die within 48 h after being deprived of nerve growth factor. Addition of interferons (IFN‐α/β or IFN‐γ) prevented the cell death in a dose‐dependent manner. Upon longer periods of nerve growth factor deprivation, IFNs failed to maintain survival. Thus, IFNs retarded neuronal death, but did not prevent it. Ligand binding, autoradiography, and cross‐linking experiments demonstrated the presence of specific IFN‐γ receptors on sympathetic neurons similar to those seen on other cell types. The possible relationships of the death‐suppressing actions of IFNs are compared to the mechanisms of the antiviral or antiproliferative actions of IFNs.
N‐Methyl‐D‐Aspartate Receptor Activation and Ca2+ Account for Poor Pyramidal Cell Structure in Hippocampal Slices
Authors: Feig Sherry , Lipton Peter
Published: October 2006

The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36–37°C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca2+ and much improved if it contains N‐methyl‐D‐aspartate (NMDA) receptor antagonists or 0 mM Ca2+ and 10 mM Mg2+. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca2+, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.
Characterization of a Nerve Growth Factor‐Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule‐Associated Protein 2 and pp250
Authors: Landreth Gary E. , Smith Deanna S. , McCabe Craig , Gittinger Cynthia
Published: October 2006

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250‐kDa cytoskeletal protein (pp250). We report that the microtubule‐associated protein, MAP2, is an alternative substrate for the NGF‐activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pl of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF‐stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S‐6 were not phosphorylated by this enzyme. The NGF‐stimulated kinase was distinct from A kinase, C kinase, or other NGF‐stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.
Autoradiographic Localization of Angiotensin II Receptor Binding Sites on Noradrenergic Neurons of the Locus Coeruleus of the Rat
Authors: Rowe Brian P. , Kalivas Peter W. , Speth Robert C.
Published: October 2006

The locus coeruleus (LC) of the rat was lesioned by microinjection of selective neurotoxins into the brainstem. 6‐Hydroxydopamine (6‐OHDA), 3 μg/μl, given unilaterally at two sites 0.6 mm apart on the rostro‐caudal axis of the LC, was used to lesion catecholamine‐containing neuronal elements. Ibotenic acid, 2.5 μg/0.5 μl, administered similarly was used to lesion nerve cell bodies. Two weeks after administration of the neurotoxin, lesion efficacy was determined based on the norepinephrine content of the cerebral cortex ipsi‐ and contralateral to the lesion. 6‐OHDA lesions of the LC caused a 46% reduction in ipsilateral cortical norepinephrine and a 60% reduction in specific 125I‐[Sar1, Ile8]‐angiotensin II (125I‐SIAII) binding in the LC. Ibotenic acid lesions of the LC caused a 73% reduction in ipsilateral cortical norepinephrine and a 81% reduction in specific 125I‐SIAII binding in the LC. These results indicate that All receptor binding sites in the LC are localized on noradrenergic nerve cell bodies or their dendritic and axonal ramifications within the LC.
Adenosine Transport by Rat and Guinea Pig Synaptosomes: Basis for Differential Sensitivity to Transport Inhibitors
Authors: Shank Richard P. , Baldy William J.
Published: October 2006

Adenosine transport by rat and guinea pig synaptosomes was studied to establish the basis for the marked differences in the potency of some transport inhibitors in these species. An analysis of transport kinetics in the presence and absence of nitrobenzylthioinosine (NBTI) using synaptosomes derived from several areas of rat and guinea pig brain indicated that at least three systems contributed to adenosine uptake, the Km values of which were ˜0.4, 3, and 15 μM in both species. In both species, the system with the Km of 3 μM was potently (IC50 of ˜0.3 nM) and selectively inhibited by NBTI. This NBTI‐sensitive system accounted for a greater proportion of the total uptake in the guinea pig than in the rat and was inhibited by dipyridamole, mioflazine, and related compounds more potently in the guinea pig. Preliminary experiments with other species indicate that adenosine transport in the mouse is similar to that in the rat, whereas in the dog and rabbit, it is more like that in the guinea pig. In the rat, none of the systems appeared to require Na+, but the two systems possessing the higher affinities for adenosine were inhibited by veratridine‐ and K+‐induced depolarization. The transport systems were active over a broad pH range, with maximal activity between pH 6.5 and 7.0. Our results are consistent with the possibility that adenosine transport systems may be differentiated into uptake and release systems.
Protein Phosphorylation in Intact Superior Cervical Ganglion During Regeneration
Authors: Watterson J. G. , Good R. , Hearn M. T. W. , Austin L.
Published: October 2006

The incorporation of radioactive phosphate into proteins of both normal and regenerating superior cervical ganglion nerve of the rat is reported. Incorporation studies carried out by in vitro and in vivo methods are compared. In the in vitro method, excised intact ganglia or their homogenates were incubated in the presence of inorganic phosphate or ATP, respectively, under various conditions. Proteins were analyzed by gel electrophoresis followed by autoradiography, in which quantitative but not qualitative differences between regenerating and control cases were apparent. In the in vivo procedure, inorganic phosphate was injected into the living animal 4 h before removal of ganglia. At least fivefold more proteins became labeled in vivo than in vitro, whereas no similarity in the pattern of labeling between the two methods was observed. For example, the most heavily labeled protein in the in vivo method, tentatively identified as micro tubule‐associated protein‐2, was not detected on autoradiograms of proteins labeled by the in vitro method. In this latter method, an 85‐kDa species and growth‐associated pro‐tein‐43 were always labeled, and the extent of their phosphorylation was enhanced by the additional presence of phosphatidylserine and Ca2+, a result indicating that these labeled species are substrates of protein kinase C. The in vitro conditions also led to the labeling of proteins identified as a‐and β‐tubulin. Comparison of the methods suggests that removal of the ganglion interferes with the function of protein phosphorylation systems and that this effect involves elements of the cytoskeleton.
Presence of the Plasma Membrane Proteolipid (Plasmolipin) in Myelin
Authors: Cochary Elizabeth F. , Bizzozero Oscar A. , Sapirstein Victor S. , Nolan Charles E. , Fischer Itzhak
Published: October 2006

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+, K+‐ATPase and a 70‐kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2–4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross‐reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.
An Inhibitor of Endopeptidase‐24.15 Blocks the Degradation of Intraventricularly Administered Dynorphins
Authors: Molineaux Christopher J. , Ayala Julia M.
Published: October 2006

Conversion of the octapeptide dynorphin (Dyn) A‐(1–8) to Leu5‐enkephalin (LE) by endopeptidase EC 3.4.24.15 (EP‐24,15) in vivo was examined using the technique of ventriculocisternal perfusion. Peptides were administered intra‐cerebroventricularly in the presence or absence of the EP‐24.15 inhibitor N‐[1‐(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Phe‐p‐aminobenzoate (cFPAAF‐pAB) via cannulae placed into the lateral ventricle of urethane‐anesthetized rats. The concentration of Dyn‐like peptides and LE within the CSF was monitored by radioimmunoassay in samples of CSF taken from a second cannula placed in the cisterna magna. In the absence of inhibitor, <5% of the Dyn A‐(1–8) administered was recovered in CSF. Immunoreactive LE, which is normally not found in CSF, increased rapidly in content following Dyn A‐(1–8) infusion, an observation suggesting that the larger peptide is converted to LE. When the inhibitor cFPAAF‐pAB was coadministered with Dyn A‐(1–8), the concentration of immunoreactive Dyn A‐(1–8) after 5 min was 40 times higher than that found in the absence of inhibitor. The angiotensin converting enzyme inhibitor captopril reduced the degradation of Dyn A‐(1–8) to a much lesser degree. The inhibitor of EP‐24.15 also afforded some protection of other Dyn‐like peptides. No EP‐24.15 activity was found in rat CSF, whereas high activity was found in the choroid plexus. Taken together, these data clearly indicate that an ectoenzyme form of EP‐24.15 rapidly converts intra‐cerebroventricularly administered Dyn‐like peptides to LE. Key Words: Dynorphin—Enkephalin—Enkephalinase—Ectoenzyme—Ventriculocisternal—Angiotensin converting enzyme. Molíneaux C. J. and Ayala J. M. An inhibitor of endopeptidase‐24.15 blocks the degradation of intraventricularly administered dynorphins. J. Neurochem. 55, 611–618 (1990).
Imaging the Dopamine Uptake Site with Ex Vivo [18F]GBR 13119 Binding Autoradiography in Rat Brain
Authors: Ciliax Brian J. , Kilbourn Michael R. , Haka Michael S. , Penney John B.
Published: October 2006

We studied the binding of [18F]GBR 13119 {1‐[[(4‐[18F]fluorophenyl) (phenyl)methoxy]ethyl]‐4‐(3‐phenylpropyl)piperazine} to rat brain with autoradiography after intravenous injection. The rank order of binding was dorsal striatum > nucleus accumbens = olfactory tubercle > sub‐stantia nigra = ventral tegmental area > other areas. Binding was blocked by prior injection of dopamine uptake blockers but not by injection of dopamine receptor antagonists or drugs that bind to the dialkylpiperazine site. Unilateral 6‐hydroxy dopamine lesions of dopamine neurons caused a marked decrease in striatal and nigral binding on the side of the lesion. We conclude that intravenous injection of [18F]GBR 13119 provides a useful marker of presynaptic dopamine uptake sites.
Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes
Authors: Pittel Zipora , Heldman Eliahu , Rubinstein Rachel , Cohen Sasson
Published: October 2006

The effect of McN‐A‐343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope‐labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN‐A‐343 enhanced the release of isotope‐labeled ACh, whereas oxotremorine inhibited the K+‐evoked release but had no effect on the basal release. The enhancing effect of McN‐A‐343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN‐A‐343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN‐A‐343 was 3.6‐fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6‐fold more potent in the cerebellum. The displacements of radio‐labeled pirenzepine and m‐dioxolane confirmed the M1 subtype preference of McN‐A‐343 and the M2 subtype preference of oxotremorine. These observations suggest that (a) activation of one subpopulation of muscarinic receptors by McN‐A‐343 enhances ACh release, whereas activation of another subpopulation by oxotremorine decreases this process (these subpopulations may correspond to the M1 and M2 subtypes, respectively), (b) the effect of the stimulatory receptors is pronounced at the resting state and that of the inhibitory receptors during depolarization, and (c) activation of the stimulatory receptors may be inversely related to the activity of the Ch transporter.
Posttetanic Long‐Term Potentiation in Rat Dentate Area Increases Postsynaptic 411B Immunoreactivity
Authors: Bullock Sarah , Lössner Bernd , Krug Manfred , Frey Sabine , Rose Steven P. R. , Matthies Hansjürgen
Published: October 2006

411B is a monoclonal antibody raised to chick forebrain postsynaptic densities (PSDs) which also recognises an antigen in brain tissue from adult Wistar rats but not liver, heart, or lung. This antigen is enriched in the PSD fraction and appears to be a useful biochemical marker for plastic changes of postsynaptic structures in the rat brain. The aim of this study was to investigate whether 411B immunoreactivity is changed in various hippocampal subregions by post‐tetanic long‐term potentiation (LTP). LTP was elicited in freely moving rats by applying four trains of 300 square‐wave pulses (frequency 200 Hz, pulse duration 0.2 ms, and intensity 300 mA) into the right perforant path; this included an increase in transmission efficacy at the ipsilateral perforant path‐granular cell synapse of the dentate gyrus lasting several days. Eight hours after tetanisation, antigens recognised by monoclonal 411B and a polyclonal anti‐actin antiserum were assayed in lysed homogenates of ipsi‐ and contralateral CA1, CA3, and CA4/dentate area hippocampal subfields as well as in visual cortex, cerebellum, and olfactory bulb dissected from LTP rats, and compared to passive controls. Under these experimental conditions, tetanisation of the perforant path resulted in a significant increase in the titre of 411B in the ipsilateral CA4/dentate area subfield (+34.0%; p < 0.001) compared with passive controls, whereas in all other brain regions studied no differences between experimental and control rats were observed. In no region were anti‐actin titres significantly different from controls. Our results support the hypothesis that the “late phase” of posttetanic LTP is accompanied by, or even based on, macromolecular changes elicited at the postsynaptic site of perforant path‐granular cell synapses.
Induction of Nerve Growth Factor Gene Expression by 12‐O‐Tetradecanoyl Phorbol 13‐Acetate
Authors: D'Mello Santosh R. , Heinrich Gerhard
Published: October 2006

Nerve injury leads to activation of fibroblasts, including stimulation of nerve growth factor (NGF) gene expression. Although interleukin‐1 has been implicated as a mediator of NGF gene induction, the underlying mechanisms are not known. We investigated whether 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA), also a known stimulator of protein kinase C, regulates NGF gene expression. We show here that TPA stimulates NGF mRNA in mouse kidney and L929 fibroblasts but not in dispersed salivary cells. NGF mRNA stimulation in L929 cells is delayed by 2h, is transient, and is followed by a parallel increase in NGF secretion. The induction of NGF mRNA is inhibited by cycloheximide. NGF mRNA levels decrease to similar values after 4 h of incubation with actinomycin D alone or in combination with TPA. These results indicate that the TPA response is cell specific and suggest that it is mediated at the transcriptional level via newly synthesized protein.
Anterograde Axonal Transport of Peptidylglycine α‐Amidating Monooxygenase in Rat Sciatic Nerves
Authors: Tozawa Kaeko , Arakawa Eiji , Chikuma Toshiyuki , Oh‐hashi Yoshihiro , Yajima Ryuichi , Takeda Katsumichi , Shinozaki Hiroshi , Kato Takeshi
Published: October 2006

Abstract: Axonal transport of peptidylglycine α‐amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel nitration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy‐terminal‐amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.
Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase
Authors: Checler F. , Grassi J. , Masson P. , Vincent J. P.
Published: October 2006

Abstract: Commercially available and affinity‐purified bu‐tyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine‐enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucineenkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90‐ and 180‐kDa protein bands by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG‐purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide‐hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.
Detection of Several Novel γ‐Aminobutyric Acid‐Containing Compounds in Human CSF
Authors: Miller Joanne M. , Ferraro Thomas N. , Hare Theodore A.
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) concentrations in human CSF are known to increase significantly after hydrolysis; however, the source of this increase has been unknown. Using either ion‐exchange or reverse‐phase chromatography coupled with on‐line alkaline hydrolysis, we have shown 2‐pyrrolidinone, the lactam of GABA, to be present in insufficient quantity to account for this increase. Subsequent experiments involving fraction collection of column eluents followed by acid hydrolysis and rechromatography demonstrated the presence of several previously undetected GABA‐containing compounds.
Induction of Ornithine Decarboxylase in Cerebral Cortex by Excitotoxin Lesion of Nucleus Basalis: Association with Postsynaptic Responsiveness and N‐Methyl‐d‐Aspartate Receptor Activation
Authors: Reed L. J. , Belleroche J. de
Published: October 2006

Abstract: The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal fore‐brain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injecton results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the poly‐amines spermidine and spermine were unaffected by lesioning. Increases in ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK‐801, a result indicating the involvement of N‐Methyl‐d‐Aspartate (NMDA) receptors. The effect of MK‐801 was dose dependent, with the maximal effect being obtained at a dose of 1 mg/kg, and also critically dependent on the time of administration, with its effectiveness being significantly reduced if given as late as 4 h after lesioning. The anticonvulsant barbiturate pentobarbital produced an effect similar to that of MK‐801. The potentiation of cortical cholinergic responsiveness (assessed by measuring carbachol‐stimulated phosphoinositide turnover in cortical slices) was also prevented by MK‐801. Thus, both the induction of ODC and the enhanced postsynaptic responsiveness appear to be mediated by the involvement of NMDA receptors, in view of the efficacy of MK‐801.