bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Chemical Characterization of Peptide G1, an Invertebrate Neuropeptide That Stimulates Cyclic GMP Metabolism
Authors: Pavloff Michael S. , Goy Michael F.
Published: October 2006

Abstract: Bioassay analysis of extracts of the major neurose‐cretory structures of the American lobster have revealed several different agents with stimulatory effects on the cyclic GMP metabolism of various lobster tissues. The most potent of these is a peptide extracted from the sinus gland, a neurohemal organ found in the animal's eyestalk. This molecule, called peptide G1 (for its effects on cyclic GMP metabolism), can increase the cyclic GMP content of every lobster tissue tested, sometimes by as much as 200‐fold. In this article, we describe the purification and some of the chemical properties of peptide G1. Purification was accomplished by sequential anion exchange and reverse‐phase HPLC. The purified peptide is a large, extremely hydrophobic molecule. Its apparent molecular mass on a reducing sodium dodecyl sulfate‐containing gel is 6.4 kDa, and its calculated molecular mass (based on an amino acid analysis of the purified material) is 8.2 kDa. Amino acid analysis reveals a high proportion of leucine and valine residues. The amino terminus of the molecule is not susceptible to Edman degradation, but sequencing studies were successfully carried out on tryptic fragments. Based on the estimated size of the molecule, these studies provide ∼60% of the total sequence. No homologies with any previously sequenced peptide were observed, but biochemical similarities to as yet unsequenced peptides found in extracts of sinus glands from other crustaceans (hyperglycemic hormone and moult‐inhibiting hormone) are described.
Extracellular Concentrations of Cocaine and Dopamine Are Enhanced During Chronic Cocaine Administration
Authors: Pettit Hugh O. , Pan Hwai‐Tzong , Parsons Loren H. , Justice Joseph B.
Published: October 2006

Abstract: Chronic cocaine administration produces significant increases in cocaine‐induced locomotor activity and stereotypy. In vivo microdialysis procedures were used to monitor extracellular dopamine (DA) and cocaine concentrations in the nucleus accumbens (N ACC) and cocaine concentrations in plasma of animals that received chronic or acute cocaine treatments. Following a cocaine challenge injection, concentrations of both cocaine and DA increased to significantly higher levels over time in animals that had received daily cocaine injections for 10 or 30 days than in control animals that received daily injections of saline. Concentrations of cocaine and DA in the N ACC reached maximum levels in the first 30 min following a challenge injection of cocaine. The maximum cocaine concentrations of 10‐ and 30‐day chronic animals were, respectively, 186% and 156%, whereas the maximum DA concentrations were 264% and 216% above the maximum values observed in acute control animals. The results indicate that reverse tolerance effects observed following chronic cocaine administration may in part be accounted for by increased cocaine concentrations. Furthermore, chronic cocaine administration (over a 10‐ or 30‐day period) increased the concentration of cocaine detected in plasma above control levels following a challenge injection. The increase in brain concentrations of cocaine in chronic animals is apparently due to increased concentrations of cocaine in plasma. A physiological change occurs in the periphery as a result of chronic cocaine administration that increases cocaine concentrations in plasma, increases extracellular cocaine levels in the brain, and increases the extracellular concentration of DA in the N ACC.
Protein Kinase C Activity in Rat Brain Cortex
Authors: Crumrine R. Christian , LaManna Joseph C.
Published: October 2006

Abstract: The procedure used to obtain cerebral tissue for analysis of protein kinase C (PKC) activity may affect the subcellular distribution of the enzyme. We compared different methods of tissue preparation and found that the proportion of PKC activity associated with the paniculate fraction of the cerebral cortex was only 30% when the brain was frozen in situ while the animal was on life support or after decapitation followed by delayed freezing. Other methods of obtaining cerebral tissue resulted in 49–56% of the PKC activity in the particulate fraction. Freezing per se had no apparent effect on the activity or subcellular distribution of PKC. In addition, whenever the particulate PKC activity was high (>48%), there was also a significant increase in the proportion of particulate protein (from 51 to ∼63%, p > 0.05).
Ontogeny of Tyrosine Hydroxylase Concentration in Locus Coeruleus of Newborn Rats: Long‐Term Effects of RU24722
Authors: Zyzek Evelyne , Richard Francoise , Bouilloux Jean‐Pierre , Pujol Jean‐Francois
Published: October 2006

Abstract: The ontogenetic variations of tyrosine hydroxylase (TH) have been studied in locus coeruleus of developing rats. During the first 2 weeks after birth, a large increase in TH content (6.04–23.99 TH units) in the noradrenergic structure was observed, followed by a period of progressive increase of the protein concentration (42 TH units in adult rats). The expression of TH was studied in the same ontogenetic period after treatment by RU24722 (20 mg/kg, i.p.). The long‐term increase in TH concentration produced by the drug was found to follow ontogenetic variations. It becomes significant around the middle of the second week after birth and gradually increases until the 24th day of postnatal development, indicating a maturation of the mechanisms involved in the inducing effect.
Effect of Transient Cerebral Ischaemia and Cardiac Arrest on Brain Extracellular Dopamine and Serotonin as Determined by In Vivo Dialysis in the Rat
Authors: Sarna G. S. , Obrenovitch T. P. , Matsumoto T. , Symon L. , Curzon G.
Published: October 2006

Abstract: The effects of 20‐min transient, global, forebrain ischaemia and cardiac arrest on extracellular concentrations of dopamine (DA), serotonin (5‐HT), and their respective metabolites, homovanillic acid (HVA) and 5‐hydroxyindole‐acetic acid (5‐HIAA), were measured in vivo by dialysis of rat striatum and hippocampus. During the ischaemic period, striatal DA content increased (250‐fold basal concentrations) with parallel but much less marked increases of both striatal and hippocampal 5‐HT content (eight‐ to 10‐fold). Baseline values were restored during reperfusion. Subsequent increases of DA and 5‐HT levels on cardiac arrest were comparable after both sham operation and ischaemia. Significant decreases of HVA and 5‐HIAA levels were observed following ischaemia or cardiac arrest. The differential effects of ischaemia on DA and 5‐HT suggest selective alterations in disposition or metabolism of the two transmitters and that dopaminergic neurones may be more vulnerable to ischaemic insults.
Plant‐Derived Neurotoxic Amino Acids (β‐N‐Oxalylamino‐l‐Alanine and β‐N‐Methylamino‐l‐Alanine): Effects on Central Monoamine Neurons
Authors: Lindström Helena , Luthman Johan , Mouton Peter , Spencer Peter , Olson Lars
Published: October 2006

Abstract: In the present study the subacute effects of β‐N‐oxalylamino‐l‐alanine (BOAA) and β‐N‐methyIamino‐l‐alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high‐affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5‐HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5‐HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH‐immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P‐immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10‐‐5M) reduced high‐affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high‐affinity [3H]DA uptake and the cortical high‐affinity [3H]5‐HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.
A Comparison of the Binding of [3H]Proprionyl‐Neuropeptide Y to Rat and Human Frontal Cortical Membranes
Authors: Widdowson Peter S. , Halaris Angelos E.
Published: October 2006

Abstract: The binding characteristics of [3H]proprionyl‐neuropeptide Y ([3H]proprionyl‐NPY) were studied in frontal cortical membranes prepared from rat and human postmortem tissue. The specific binding of NPY decreased as the magnesium concentration increased from 1.05 to 10 mM. The binding was also influenced by the concentration of GTP in the buffer medium, with a resulting 45% decrease in NPY binding in the presence of 10‐‐6M GTP. Using equilibrium binding studies, [3H]proprionyl‐NPY was found to bind in both tissues with high affinity to a single class of receptors with a similar KD (0.035 nM). However, kinetic experiments in both tissues provided evidence for two components of [3H]proprionyl‐NPY binding which may be related to receptor states. Competition binding experiments showed that peptide YY (PYY) was equal to NPY in its ability to displace [3H]proprionyI‐NPY, whereas rat and human pancreatic polypeptide were without effect up to a concentration of 10‐‐6M. This suggests that, whereas PYY and NPY may compete for the same receptor(s), the pancreatic polypeptides probably act on a separate population of receptors.
Effects of Selective Monoamine Oxidase Inhibitors on the In Vivo Release and Metabolism of Dopamine in the Rat Striatum
Authors: Butcher Steven P. , Fairbrother Iain S. , Kelly John S. , Arbuthnott Gordon W.
Published: October 2006

Abstract: Brain microdialysis was used to examine the in vivo efflux and metabolism of dopamine (DA) in the rat striatum following monoamine oxidase (MAO) inhibition. Relevant catecholamines and indoleamines were quantified by HPLC coupled with a electrochemical detection system. The MAO‐B inhibitor selegiline only affected DA deamination at a dose shown to inhibit partially type A MAO. Alterations in DA and metabolite efflux were not observed when using the MAO‐B‐selective dose of 1 mg/kg of selegiline. At 10 mg/kg, selegiline reduced the efflux of DA metabolites to ∼70% of basal values without affecting DA efflux. K+‐and veratrine‐stimulated DA efflux was not affected by selegiline. Experiments using amphetamine and the DA uptake inhibitor nomifensine demonstrated that the effect of selegiline on DA metabolism was unlikely to be mediated either by inhibition of DA uptake or by an indirect effect of its metabolite amphetamine. The possibility that the effect of selegiline is mediated via a nonspecific inhibition of MAO is discussed. In contrast, the MAO‐A inhibitor clorgyline inhibited basal DA metabolism and increased basal and de‐polarisation‐induced DA efflux. A 1 mg/kg dose of clorgyline reduced basal DA metabolite efflux (40–60% of control values) without affecting DA efflux. At 10 mg/kg of clorgyline, DA efflux increased to 253 ± 19% of basal values, whereas efflux of DA metabolites was reduced to between 15 and 26% of control values. The release of DA induced by K+ and vera‐trine was not affected by 1 mg/kg of clorgyline but was increased by ∼200% following pretreatment with 10 mg/kg of clorgyline. The nonselective MAO inhibitor pargyline caused similar but more pronounced alterations in these parameters. For example, using a 75 mg/kg dose of pargyline, efflux of DA increased maximally to 310 ± 20% of basal values, and the release of DA induced by K+ and veratrine was increased by ∼300% following pargyline pretreatment. These data suggest that DA metabolism is mediated principally by MAO‐A in the rat striatum. However, under conditions of MAO‐A inhibition, a component of metabolism mediated by the type B enzyme becomes apparent.
Preparation and Characterization of Monoclonal Antibodies to Serotonin Binding Protein
Authors: Liu K. P. , Yu P.‐Y. , Hsiung S. H. , Kirchgessner A. L. , Gershon M. D. , Tamir H.
Published: October 2006

Abstract: Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross‐reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 ± 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45‐kDa or 56‐kDa SBP. Culture supernatants were screened for the presence of anti‐SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type‐specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme‐linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44–10) was obtained and affinity‐purified. In the presence of goat anti‐mouse IgM, the partially purified 44–10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44–10 blocked the binding of [3H]serotonin ([3H]5‐HT) to 45‐kDa and 56‐kDa SBP in a concentration‐dependent manner. The 68‐kDa protein was found to bind [3H]5‐HT. Sites reacting with Mab 44–10 were located immunocytochemically in sections of rat brain. 5‐HT immunoreactivity was localized simultaneously in the same sections by using affinity‐purified rabbit anti‐5‐HT antibodies and species‐specific secondary antibodies coupled to a contrasting fluorophore. MAb 44–10 immunostaining involved neuronal cell bodies, neurites, and terminals. This immunostaining was intense within the nuclei of the median raphe and the B9 cell group. Coincident expression with 5‐HT was observed; however, MAb 44–10 also immunostained many neurons in which 5‐HT immunoreactivity was not seen. These observations may indicate that SBP is distributed more widely in the brain than 5‐HT; however, because SBP immunoreactivity is not found in nonserotonergic neurons when monospecific polyclonal antibodies are used for immunocytochemistry, it seems more likely that some nonserotonergic neurons contain another protein (such as the 68‐kDa SBP) that also contains an epitope recognized by MAb 44–10. Nevertheless, these data demonstrate that MAb 44–10 reacts with the 5‐HT binding domain of 45‐kDa and 56‐kDa SBP and will be a valuable tool for analyzing these proteins.
Isolation of Glutamyltaurine from Bovine Brains and Proof of Its γ‐Linkage by the B/E Linked Scan SIMS Technique
Authors: Nakamura Ko , Higashiura Kunihiko , Nishimura Naoharu , Yamamoto Atsushi , Tooyama Ikuo , Kimura Hiroshi , Ienaga Kazuharu
Published: October 2006

Abstract: We isolated a glutamyltaurine from bovine brains and determined its structure as γ‐glutamyltaurine (γ‐Glu‐Tau; glutaurine) by use of a new mass spectrometric technique [B/E linked scan sputtered ion mass spectrometry (SIMS)], which we have recently shown to be useful for distinguishing the γ‐ from the α‐isomer of glutamyl‐dipeptides. Neither the α‐isomer of glutamyltaurine nor any aspartyltaurines could be detected in bovine brain.
Ca2+‐Independent Stimulation of Adenylate Cyclase Activity by Muscarinic Receptors in Rat Olfactory Bulb
Authors: Olianas Maria C. , Onali Pierluigi
Published: October 2006

Abstract: In rat olfactory bulb homogenate, carbachol stimulated adenylate cyclase activity in a concentration‐dependent manner (EC50= 1.1 μM). The carbachol stimulation occurred fully in membranes that had been prepared in the presence of 1 mM EGTA and incubated in a Ca2+‐free enzyme reaction medium. Under these conditions, exogenous calmodulin (1 μM) failed to stimulate adenylate cyclase activity. In miniprisms of olfactory bulb, carbachol (1 mM) increased accumulation of inositol phosphates, but this response was markedly reduced in a Ca2+‐free medium. Moreover, the carbachol stimulation of adenylate cyclase activity was not affected by staurosporine at a concentration (1 μM) that completely blocked the stimulatory effect of phorbol 12‐myristate 13‐acetate, an activator of Ca2+/phospholipid‐dependent protein kinase. Quinacrine, a nonselective phospholipase A2 inhibitor, reduced the carbachol stimulation of adenylate cyclase activity, but this inhibition appeared to be competitive with a Ki of 0.2 μM. Nordihydroguaiaretic acid and indomethacin, two inhibitors of arachidonic acid metabolism, failed to affect the carbachol response. These results indicate that in rat olfactory bulb, muscarinic receptors stimulate adenylate cyclase activity through a mechanism that is independent of Ca2+ and phospholipid hydrolysis.
Modulation of Rat Brain Cortical α‐Adrenoceptors by Treatment with Hydrocortisone for 10 Days
Authors: Szentendrei Tibor , Fekete Márton I. K.
Published: October 2006

Abstract: The role of glucocorticoids in the modulation of central α2‐receptor mechanisms was investigated by in vitro receptor binding studies. [3H]Clonidine and [3H]idazoxan were used as radioligands. The α2‐receptor subtypes and guanine nucleotide sensitivity were studied in homologue and heterologue displacement experiments following hydrocortisone treatment (25 mg/kg s.c.) for 10 days. High and low agonist affinity states of the α2‐receptor could be identified in 3H‐antagonist‐agonist and 3H‐agonist‐antagonist displacement experiments, which may correspond to different regulatory protein‐nucleotide associated forms of the receptor. In the presence of 10 μM GTP, the high‐affinity binding was depressed. Following hydrocortisone treatment, there was no detectable change either in the affinity or the binding site concentration of clonidine in homologue displacement (“cold saturation”) experiments. The affinity of idazoxan, however, was depressed. The effect of GTP was similar to the controls in this experimental arrangement. In contrast, in heterologue binding studies the high‐affinity binding site was not demonstrable and the amount of low‐affinity binding increased following the hydrocortisone treatment. The high‐affinity site reappeared in the presence of GTP. The change in GTP sensitivity suggests that the nucleotide regulatory system may be involved in the action of adrenal steroids on central α2‐receptoral mechanisms.
Effects of Creatine on Synthesis and Release of γ‐[3H]Aminobutyric Acid
Authors: Schultheiss Karl , Thate Anja , Meyer Dieter K.
Published: October 2006

Abstract: Creatine has been used previously to alter the energy balance of neurons in brain slices. In the present experiments, it was found to reduce the accumulation of γ‐[3H]aminobutyric acid [3H]GABA) as synthesized from [3H]glutamic or [3H]glutamic acid in slices of rat neostriatum. The lowest effective concentration was 5 mM. Creatine (25 mM) was also effective when the degrading enzyme of GABA, i.e., GABA‐α‐oxoglutarate transaminase, was blocked by gabaculine. Creatine (25 mM) did not inhibit the uptake and subsequent accumulation of [3H]GABA. Thus, indirect evidence was obtained that creatine decreased the activity of the synthesizing enzyme of GABA, i.e., glutamate decarboxylase. When the direct effect of creatine (25 mM) on glutamate decarboxylase was studied in vitro, the agent indeed decreased the activity of the enzyme. Creatine (25 mM) also diminished the release of [3H]GABA (expressed as dpm/mg wet weight) from rat neostriatal slices, probably by reducing its synthesis and thus its readily releasable pool. These data are of importance for studies with creatine in complex neuronal systems, because they show that the agent changes not only neuronal energy balance, but also synthesis and release of the ubiquitous transmitter GABA.
Involvement of Dopamine Neurons in the Regulation of β‐Adrenergic Receptor Sensitivity in Rat Prefrontal Cortex
Authors: Hervé Denis , Trovero Fabrice , Blanc Gérard , Vezina Paul , Glowinski Jacques , Tassin Jean‐Pol
Published: October 2006

Abstract: The contribution of dopamine (DA) afferents to the regulation of β‐adrenergic receptor sensitivity (isoproterenol‐stimulated adenylate cyclase activity) in the rat prefrontal cortex was investigated by comparing the effects of lesions affecting either both DA and noradrenaline (NA) or NA fibers alone. Bilateral 6‐hydroxydopamine (6‐OHDA) lesions made in the ventral tegmental area destroyed ascending DA and to a variable extent ascending NA fibers innervating the prefrontal cortex. Two opposite effects were observed depending on the extent of cortical NA denervation: (a) When NA denervation was complete (<4% of controls), a marked increase in the isoproterenol‐sensitive adenylate cyclase activity (+78%) was found. The amplitude of this denervation supersensitivity was similar to that occurring following complete and selective destruction of NA innervation induced by bilateral 6‐OHDA injections made into the pedunculus cerebellaris superior. (b) When 6‐OHDA injections into the ventral tegmental area led to a partial destruction of cortical NA afferents (10–40% of control values), a hyposensitivity of the isoproterenol‐induced adenylate cyclase activity (–30%) was observed. This effect contrasted with the moderate supersensitivity seen in rats with partial, but selective, destruction of NA innervation (pedunculus cerebellaris superior lesions). The hyposensitivity of β‐adrenergic receptors obtained in rats with partial lesions of cortical NA fibers, but devoid of cortical DA innervation, suggests that DA neurons may regulate, under certain conditions, the denervation supersensitivity of β‐adrenergic receptors.
Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C
Authors: Uchiyama Haruaki , Ohara Koichi , Haga Kazuko , Haga Tatsuya , Ichiyama Arata
Published: October 2006

Abstract: Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM‐binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM‐binding and glycosylation sites was ∼ 16 kDa. Cerebral receptors were 32P‐phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C‐terminal segment including a part of the third intracellular loop, because a 32P‐labeled peptide of 12–14 kDa reacted with anti‐(m 1 C‐terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of ∼ 17 kDa containing the [3H]PrBCM‐binding site, but not the glycosylation sites, was partly converted to a peptide of ∼ 12 kDa on treatment with β‐mercaptoethanol.
In Vivo Striatal Dopamine Release by M1 Muscarinic Receptors Is Induced by Activation of Protein Kinase C
Authors: Xu Mann , Yamamoto Toshifumi , Kato Takeshi
Published: October 2006

Abstract: Islet‐activating protein was unilaterally microinjected into rat stratum, and a dialysis cannula was implanted into the same area under anesthesia. After 2 days, various agents were perfused continuously into the striatum through the dialysis membrane, under freely moving conditions. Isletactivating protein (2 μg/2 μ1) treatment alone did not change in vivo striatal dopamine (DA) release and metabolism, but completely abolished the increase of striatal DA release evoked in vivo by the M1‐selective agonist McN‐A‐343 (10−7M). Forskolin (10−5M), an adenylate cyclase activator, increased DA release and showed an additive effect on the DA release evoked by McN‐A‐343. Polymyxin B, a rather selective inhibitor of protein kinase C, decreased DA release and completely blocked the effect of McN‐A‐343. These results suggest that in vivo striatal DA release elicited by M1 muscarinic receptors is coupled with interaction with a G protein and is induced by activation of protein kinase C.
Effect of 5′‐(N‐Ethylcarboxamido)adenosine on Adenosine Transport in Cultured Chromaffin Cells
Authors: Delicado Esmerilda G. , Rodrigues Alexandra , Sen Raquel P. , Sebastiao Ana M. , Ribeiro J. Alexandre , Miras‐Portugal M. Teresa
Published: October 2006

Abstract: Extracellular adenosine is transported into chromaffin cells by a high‐affinity transport system. The action of adenosine receptor ligands was studied in this cellular model 5‐(N‐Ethylcarboxamido)adenosine (NECA), an agonist receptors, activated adenosine transport. Km values for adenosine were 4.6 ± 1.0 (n = 5) and 10.2 ± 3.0 μM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 ± 23.5 and 170.2 ± 30 pmol/106 cells/min for control and 100 nM NECA, respectively. The A1 agonist N6‐cyclohexyladenosine, the A1 antagonist 8‐cyclopentyl‐1, 3‐dipropylxanthine, and the A1‐A2 antagonist 1,3‐dipropyl‐8‐{4‐[(2‐aminoethyl)amino]‐carbonylmethyloxyphenyl}‐xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]‐nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 ± 800 and 23,200 ± 700 transporters/cell for controls and 100 nM NECA, repectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.
Differential Calcium Dependence Between the Release of Endogenous Dopamine and Noradrenaline from Rat Brain Synaptosomes
Authors: Okada Mitsuko , Mine Kazunori , Fujiwara Michihiro
Published: October 2006

Abstract: The characteristics of the release of endogenous dopamine and noradrenaline from rat brain synaptosomes were studied using HPLC with an electrochemical detector. The spontaneous release of dopamine and noradrenaline was inhibited by ∼50–60% in a Ca2+‐free medium or a 100 μM La3+‐containing medium. Also, the high‐K+ (30 mM)‐evoked release of dopamine and noradrenaline was inhibited by ∼50–60% in a Ca2+‐free medium or a 100 μM La3+‐containing medium. From these results, the ratio of the Ca2+‐dependent component to the total release of noradrenaline seemed to be similar to that of dopamine. On the other hand, 20 μM La3+ or 1 μM diltiazem inhibited both the spontaneous and 30 mM K+‐evoked release of dopamine by ∼50–60% but inhibited neither the spontaneous nor the 30 mM K+‐evoked release of noradrenaline. The K+‐evoked rise in intrasynaptosomal Ca2+ concentration was mostly blocked in Ca2+‐free medium or 100 μM La3+‐containing medium but was only partially blocked by 20 μM La3+ or 1 μM diltiazem. These data indicate alternative possibilities in that the Ca2+‐dependent release of noradrenaline might be less sensitive to a change of intracellular Ca2+ concentration than that of dopamine and that the calcium channels directly involved in the noradrenaline release may be more resistant to diltiazem and La3+ than those involved in the dopamine release.
Developmental Expression of Go in Neuronal Cultures from Rat Mesencephalon and Hypothalamus
Authors: Granneman James G. , Kapatos Gregory
Published: October 2006

Abstract: The developmental expression of the α‐subunit of Go was examined in neuronal cultures derived from rat mesencephalon (MES) and hypothalamus (HYP). These cultures were essentially free of contaminating glia and were maintained as a stable population for periods up to 3 weeks. Immunoblotting utilizing specific antisera against Go indicated that in neurons from both brain regions, membrane concentrations of Go increased dramatically during the first 2 weeks in vitro. Thereafter, increases in the amount of Go per neuron kept pace with increasing process (axons and dendrites) formation. Multiple forms of immunoreactive Go were detected in MES and HYP neurons, and the proportions of these forms changed between 4 and 14 days in culture. Finally, increasing neuron density significantly increased membrane levels of Go in MES but not HYP cultures.
Expression of a Novel Transcript of the Myelin Basic Protein Gene
Authors: Kitamura K. , Newman S. L. , Campagnoni C. W. , Verdi J. M. , Mohandas T. , Handley V. W. , Campagnoni A. T.
Published: October 2006

Abstract: A cDNA (M41) corresponding to a mouse myelin basic protein (MBP) mRNA with a longer 5′‐untranslated region than predicted from earlier studies of MBP gene structure has been isolated and characterized. The additional 5′‐untranslated region is encoded by two previously unidentified exons upstream of the major transcription start site of the gene. Using a DNA probe specific for M41‐MBP mRNAs, Northern blot analysis indicated that expression of this transcript follows a developmental course in mouse brain similar to that of the majority of MBP mRNAs, but that the level of expression varies between brain and spinal cord. Expression of MBP mRNAs similar to the mouse M41‐MBP also was identified in rat brain. The results suggest that the structure of the MBP gene is more complex than originally thought, containing at least two more exons. There appears to be at least one more MBP gene promoter that directs the synthesis of a subset of MBP mRNAs with a unique 5′‐untranslated region.
Strychnine‐Sensitive, Glycine‐Induced Release of [3H]Norepinephrine from Rat Hippocampal Slices
Authors: Schmidt Christopher J. , Taylor Vicki L.
Published: October 2006

Abstract: The amino acid glycine is the primary inhibitory neurotransmitter of the mammalian spinal cord, Glycine has also been shown to facilitate the excitatory actions of glutamate at the N‐methyl‐D‐aspartic acid receptor subtype. In this article, glycine is shown to increase the Ca2+‐dependent release of [3H]norepinephrine from preloaded slices of the rat hippocampus. This effect was inhibited noncompetitively by nanomolar concentrations of strychnine, which differentiates it from the glycine site associated with the N‐methyl‐D‐aspartate receptor. Glycine also released [3H]acetylcholine, but was without effect on the efflux of [3H]serotonin or γ‐[3H]aminobutyric acid from the same tissue preparation. The release of [3H]norepinephrine was reversibly blocked by tetrodotoxin, indicating the effect is not initiated at the noradrenergic terminals, but requires propagation of an action potential. The results suggest that a glycine site that is pharmacologically similar to that found in the spinal cord exists in the rat hippocampus. We suggest that this site may participate in modulating the release of specific neurotransmitters in the brain.
Solubilization of Spermidine‐Sensitive (+)‐[3H]5‐Methyl‐10,11 ‐Dihydro‐5H‐Dibenzo[a,d]cyclohepten‐5,10‐Imine ([3H]MK‐801) Binding Activity from Rat Brain
Authors: Ogita Kiyokazu , Yoneda Yukio
Published: October 2006

Abstract: The receptor‐ionophore complex of the N‐methyl‐d‐aspartate (NMDA)‐sensitive receptor was solubilized by deoxycholic acid from rat brain using (+)‐[3H]5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,b]cyclohepten‐5,10‐imine ([3H]MK‐801) binding as a marker for the receptor. Gel filtration of the solubilized preparations on a Sephadex G‐25 column revealed significant [3H]MK‐801 binding sensitive to potentiation by glutamate and glutamate/glycine, which was prevented by competitive antagonists for the NMDA and strychnine‐insensitive glycine (GlyB) sites. In contrast to NMDA and glycine, spermidine markedly potentiated the amount of [3H]MK‐801 binding in solubilized preparations by increasing the apparent affinity of the ligand. In the presence of all three stimulants, the solubilized preparations exhibited pharmacological profiles similar to those in the membrane preparations. These results clearly indicate that the whole macromolecular NMDA receptor‐ionophore complex is solubilized under the experimental conditions used.
Physicochemical Properties of Serotonin 5‐HT3 Binding Sites Solubilized from Membranes of NG 108‐15 Neuroblastoma‐Glioma Cells
Authors: Miquel M.‐C. , Emerit M. B. , Bolaños F. J. , Schechter L. E. , Gozlan H. , Hamon M.
Published: October 2006

Abstract: Specific binding sites with pharmacological properties typical of serotonin 5‐HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108‐15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield. 50%) could be achieved using the detergent 3–[3–(cholamidopropyl)dimethylammonio]‐1‐propane sul‐fonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris‐HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000‐g CHAPS extract was saturable and showed characteristics (Bmax= 425 ± 81 fmol/mg of protein; KD= 0.19 ± 0.02 nM) closely related to those of membrane‐bound sites (Bmax= 932 ± 183 fmol/mg of protein; KD= 0.60 ± 0.03 nM). Determination of association (K+1=0.17 nM min−1) and dissociation (k‐1= 0.02 min−1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r= 0.99) in the CHAPS‐soluble extract and in cell membranes, although some increase in the affinity for 5‐HT3 antagonists (zacopride, ICS 205–930, and MDL 72222) and decrease in the affinity for 5‐HT3 agonists (2‐methyl‐5‐hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS‐soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S‐400 and wheat germ agglutinin‐agarose columns indicated marked enrichment (by 2.5– and 10‐fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5‐HT3 receptors. Two soluble materials with apparent molecular masses of ∼600 and ∼36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S‐400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108–15 cells gave a value of ∼35 kDa.
[3H]WIN 35,065–2: A Ligand for Cocaine Receptors in Striatum
Authors: Ritz Mary C. , Boja John W. , Grigoriadis D. , Zaczek R. , Carroll F. Ivy , Lewis Anita H. , Kuhar Michael J.
Published: October 2006

Abstract: [3H]WIN 35,065–2 binding to striatal membranes was characterized, primarily by centrifugation assay. Like [3H]cocaine, [3H]WIN 35,065–2 binds to both high‐ and low‐affinity sites. [3H]WIN 35,065–2, however, exhibits consistently higher affinities than [3H]cocaine. Saturation experiments indicate a low‐affinity binding site with an apparent KD of ∼ 160 nM and a Bmaxof 135 fmol/mg of tissue. A high‐affinity site has also been identified with an apparent KD of 5.6 nM and a Bmax of 5.2 fmol/mg of tissue. The specific‐to‐nonspecific binding ratios with [3H]WIN 35,065–2 were higher than with [3H]cocaine in both centrifugation and filtration assays. Pharmacological characterization suggests that [3H]WIN 35,065–2 binds to the dopamine transporter. Mazindol, GBR 12909, nomifensine, and (‐)‐cocaine are potent inhibitors of [3H]WIN 35,065–2 binding. In contrast, the norepinephrine transporter ligand desipramine is a weak inhibitor, and the serotonin transporter ligand citalopram does not inhibit binding. The effect of sodium on binding was examined under conditions in which (a) the low‐affinity site was primarily (87%) occupied and (b) ∼50% of both sites were occupied. The results indicate that both sites are sodium dependent. Injection of 6‐hydroxydopamine into the striatum results in a significant loss of both high‐ and low‐affinity sites, a finding suggesting that both sites are on dopaminergic nerve terminals. Taken together, these data are consistent with the presence of multiple cocaine binding sites associated with the dopamine transporter.
ATP‐Evoked Arachidonic Acid Mobilization in Astrocytes Is via a P2Y‐Purinergic Receptor
Authors: Bruner Gretchen , Murphy Sean
Published: October 2006

Abstract: To reveal more of the mechanism whereby ATP induces arachidonic acid (AA) mobilization in astrocytes, primary cell cultures prelabeled with [3H]AA were exposed to ATP and various analogs. Release of 3H was dose and time dependent and was inhibited by blocking ATP binding. The potencies of a range of ATP analogs in mobilizing AA were consistent with that predicted for the involvement of a P2Y‐purinergic receptor. Mobilization of AA was not due to nonspecific cell permeabilization, as assessed by leakage of cytoplasmic lactate dehydrogenase. AA mobilization by ATP was reduced when mobilization of intracellular calcium was inhibited and in the absence of extracellular calcium. Thap sigargin, which induces release of intracellular calcium, evoked mobilization of AA and thromboxane formation, findings similar to the effects of ATP. These results suggest that ATP stimulates AA mobilization via a P2Y‐purinergic receptor and that, although extracellular calcium is involved, mobilization of intracellular calcium activates phospholipase A2.
Detection of Cystathionine Ketimine in Bovine Cerebellum
Authors: Ricci G. , Vesci L. , Matarese R. M. , Antonucci A. , Maggio A. , Pecci L. , Cavallini D.
Published: October 2006

Abstract: A new sulfur‐containing cyclic imino acid, cystathionine ketimine, has been detected in bovine cerebellum by gas chromatography, gas chromatography‐mass spectrometry, and high pressure liquid chromatography procedures. Gas chromatography and gas‐mass analyses are based on derivatization of endogenous cystathionine ketimine with diazomethane after a simple enrichment procedure. The high pressure liquid chromatography procedure takes advantage of the selective absorbance at 380 nm of the phenyl isothio‐cyanate‐ketimine interaction product. The concentration of this new sulfur imino acid found in a pool of four bovine cerebella is ∼0.5 nmol/g.
Endogenous Release of γ‐Aminobutyric Acid from the Medial Preoptic Area Measured by Microdialysis in the Anaesthetised Rat
Authors: Herbison Allan E. , Heavens Robert P. , Dyer Richard G.
Published: October 2006

Abstract: The characteristics of γ‐aminobutyric acid (GABA) release as monitored by microdialysis have been investigated in the chloral hydrate anaesthetised rat. The high outflow of GABA following insertion of the microdialysis probe (membrane 2 mm in length, 0.5 mm in diameter) into the medial preoptic area was found to decline to a stable baseline level after 2 h. After this time, perfusion with a medium containing 100 mM potassium ions evoked a 56‐fold increase in GABA outflow. The addition of the calcium channel blocker verapamil (100 nM) to the perfusion medium induced significant 25 and 50% reductions in basal and potassium‐stimulated GABA outflow, respectively. In the same animals, verapamil caused an 80% decrease in potassium‐stimulated noradrenaline outflow. The glutamic acid decarboxylase inhibitors 3‐mercaptopropionic acid and l‐allylglycine added to the perfusion medium at a concentration of 10 mM reduced basal GABA release by approximately 50% with different lime‐courses of action. Ethanolamine‐O‐suIfate, a GABA‐transaminase inhibitor, induced significant increases in basal GABA outflow 90 min after inclusion in the perfusion medium. These results demonstrate that microdialysis is a suitable technique with which to monitor extracellular levels of GABA and provide in vivo data on GABA release and degradation mechanisms.
Possible Involvement of Pertussis Toxin‐Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor‐Adenylate Cyclase System in Rat Cerebral Cortex
Authors: Murayama Toshihiko , Itahashi Yuriko , Nomura Yasuyuki
Published: October 2006

Abstract: To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor‐G protein‐adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet‐activating protein, IAP) and apomorphine on A1 adenosine agonist (‐)N6‐R‐[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 ± 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 ± 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high‐affinity state (KD= 2.30 ±1.16 nM) and a low‐affinity state (KD= 1,220 ± 230 nM). Guanosine 5′‐(3‐O‐thio)triphosphate or IAP treatment reduced the number of the high‐affinity state binding sites without altering the KD for PIA. Apomorphine (100 μM) increased the KD value 10‐fold and decreased Bmax by ∼20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high‐affinity to low‐affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.
Neuronal Intermediate Filament Expression During Neurite Outgrowth from Explanted Goldfish Retina: Effect of Retinoic Acid
Authors: Hall Christina M. , Else Cindy , Schechter Nisson
Published: October 2006

Abstract: Regulation of the goldfish neuronal intermediate filament proteins ON1 and ON2 was investigated in a retinal explant system. The synthesis of these proteins in explanted retina decreased with increasing time in culture, despite continuing neurite outgrowth. Thus, ON1/ON2 neurofilament expression is regulated independently from neurite outgrowth. During regeneration of the goldfish optic nerve in vivo, the expression of these proteins increased during the later phase of the process, when growing axons make contact with the optic tectum. The declining synthesis of ON1 and ON2 during neurite outgrowth in culture suggests that factors extrinsic to the retina are necessary to support synthesis of these proteins.

Treating retinal explants with retinoic acid stimulated the synthesis of the ON1/ON2 proteins in a dose‐dependent manner. This stimulation was effective during a period of declining synthesis of the ON1/ON2 proteins, restoring their synthesis towards initial levels of expression. These results show that retinoic acid serves as a modulator of neurofilament expression in this in vitro model of nerve regeneration.
Interaction of the Two Structural Domains of Calmodulin with Mature and Immature Rat Brain Microtubules
Authors: Passareiro Heloisa , Erneux Christophe , Nunez Jacques
Published: October 2006

Abstract: The inhibitory effect of calmodulin on the assembly of mature and immature rat brain microtubules was compared with that of the two major structural domains of this protein, the COOH‐terminal fragment (amino acids 78–148) and the NH2‐terminal fragment (amino acids 1–77), to determine the calmodulin structural domain responsible for the inhibitory effect on microtubule assembly. Microtubules prepared during the early stages of brain development, i.e., during intensive neurite outgrowth, are more sensitive to inhibition by the Ca2+‐calmodulin complex than those obtained from adult brain. Significant inhibition of immature microtubule assembly was observed with both fragments in the absence of Ca2+, but the effects were more important when Ca2+ was present. With adult brain microtubules, the two fragments remained without effect on assembly in the absence of Ca2+, whereas some inhibition was seen in its presence but only with the COOH‐terminal polypeptide. Under all these conditions, the COOH‐terminal fragment was always more active than the NH2‐terminal fragment on microtubule polymerization, albeit to a lesser extent than native calmodulin.
Phosphorylase a and Labile Metabolites During Anoxia: Correlation to Membrane Fluxes of K+ and Ca2+
Authors: Folbergrová Jaroslava , Minamisawa Hiroaki , Ekholm Anders , Siesjö Bo K.
Published: October 2006

Abstract: The objective of the present study was to explore mechanisms responsible for activation of ion conductances in the initial phases of brain ischemia, particularly for the early release of K+ that precedes massive cell depolarization, and rapid downhill fluxes of K+, Na+, Cl−, and Ca2+. As it has been speculated that a K+ conductance can be activated either by an increase in the free cytosolic calcium concentration (Ca2+i) or by a fall in ATP concentration, the question arises whether the early increase in extracellular K+ concentration (K+e) is preceded by a rise in Ca2+i and/or a fall in ATP content. In the present experiments, ischemia was induced in rats by cardiac arrest, the time courses of the rise in K+e and cellular depolarization were determined by microelectrodes, and the tissue was frozen in situ through the exposed dura for measurements of levels of labile metabolites, including adenine nucleotides and cyclic AMP (cAMP), after ischemic periods of 15, 30, 60, and 120 s. Conversion of phosphorylase b to a was assessed, because it depends, among other things, on changes in Ca2+i. The K+e value rose within a few seconds following induction of ischemia, but massive depolarization (which is accompanied by influx of calcium) did not occur until after ∼65 s. Activation of phosphorylase was observed already after 15 s and before glycogenolysis had begun. At that time, 3′,5′‐cAMP concentrations were unchanged, and total 5′‐AMP concentrations were only moderately increased. The results demonstrate that a K+ conductance is activated at a time when the overall ATP concentration remains at 95% of control values. If major compartmentation can be excluded, the results fail to demonstrate that an ATP‐activated K+ conductance is involved. In view of the early activation of phosphorylase, one may speculate that the triggering event is a rise in Ca2+i.