bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for an Astrocyte‐Derived Vasorelaxing Factor with Properties Similar to Nitric Oxide
Authors: Murphy Sean , Minor Robert L. , Welk Greg , Harrison David G.
Published: October 2006

Abstract: To determine whether astrocytes release nonprostanoid vasodilators, cells on microcarrier beads were superfused with various agents in the presence of indomethacin, and the effluent was bioassayed and also analyzed for nitric oxide by a chemiluminescence technique. Bradykinin and A23187 induced release of a factor that relaxed arterial rings, an effect that was blocked by hemoglobin. The effluent contained either nitric oxide or a related compound that could be reduced to nitric oxide. Production of this factor was competitively inhibited by the arginine analogs NG‐nitro‐L‐arginine and NG‐methyl‐L‐arginine and could be restored with L‐arginine. Quisqualate and norepinephrine were also effective in causing the release of nitric oxide from astroglial cells. Thus, astrocyte‐derived relaxing factor has properties similar to those of an endothelium‐ and neuronderived relaxing factor.
Purification and Characterization of Neuron‐Specific Surface Antigen Defined by Monoclonal Antibody BM88
Authors: Patsavoudi E. , Hurel C. , Matsas R.
Published: October 2006

Abstract: Monoclonal antibody BM88 recognizes a neuronspecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22‐kDa polypeptide by reducing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X‐100 a monomeric structure was implied. N‐Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase‐separation experiments with Triton X‐114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3‐kDa membrane‐associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.
Dihydropyridine Modulation of Voltage‐Activated Calcium Channels in PC12 Cells: Effect of Pertussis Toxin Pretreatment
Authors: Schettini G. , Meucci O. , Grimaldi M. , Florio T. , Landolfi E. , Scorziello A. , Ventra C.
Published: October 2006

Abstract: In this study, we report the effect of pertussis toxin pretreatment on dihydropyridine modulation of voltage‐sensitive calcium channels in PC12 cells. The rise in intracellular calcium concentration caused by potassium depolarization is not affected significantly by pertussis toxin pretreatment. Nicardipine, a dihydropyridine derivative, added either before or after potassium‐induced depolarization, reduces the resultant elevation in cytosolic calcium level both in control and in pertussis toxin‐treated cells. The dihydropyridine agonist Bay K 8644, when added before potassium, is able to enhance the potassium‐induced spike of cytosolic calcium levels, an effect significantly reduced by pertussis toxin pretreatment. Moreover, the addition of Bay K 8644 after potassium holds the intracellular calcium concentration at a cytosolic sustained level during the slow inactivating phase of depolarization. This effect of Bay K 8644 is inhibited by nicardipine. Pertussis toxin pretreatment slightly weakens the effect of Bay K 8644 when added after potassium‐induced depolarization, whereas it significantly reduces the nicardipine inhibition of cytosolic calcium rise stimulated by potassium and Bay K 8644, but not by potassium alone. In conclusion, our findings suggest that a pertussis toxin‐sensitive guanine nucleotide regulatory protein could be involved in the interaction between dihydropyridine derivatives and voltage‐dependent calcium channels.
Contrasting Neurochemical Interactions of Tiletamine, a Potent Phencyclidine (PCP) Receptor Ligand, with the N‐Methyl‐D‐Aspartate‐Coupled and ‐Uncoupled PCP Recognition Sites
Authors: Rao Tadimeti S. , Contreras Patricia C. , Cler Julie A. , Mick Steve J. , Dilworth Vickie M. , Iyengar Smriti , Monahan Joseph B. , Wood Paul L.
Published: October 2006

Abstract: Neurochemical interactions of tiletamine, a potent phencyclidine (PCP) receptor ligand, with the N‐methyl‐D‐aspartate (NMDA)‐coupled and ‐uncoupled PCP recognition sites were examined. Tiletamine potently displaced the binding of [3H]1‐(2‐thienyl)cyclohexylpiperidine with an IC50 of 79 nM without affecting σ‐, glycine, glutamate, kainate, quisqualate, or dopamine (DA) receptors. Like other PCP ligands acting via the NMDA‐coupled PCP recognition sites, tiletamine decreased basal, harmaline‐, and D‐serine‐mediated increases in cyclic cGMP levels and induced stereotypy and ataxia. Tiletamine was nearly five times more potent than PCP at inhibiting the binding of 3‐hydroxy[3H]PCP to its high‐affinity NMDA‐uncoupled PCP recognition sites. However, following parenteral administration, dizocilpine maleate (MK‐801), ketamine, PCP, dexoxadrol, and 1‐(2‐thienyl)cyclohexylpiperidine HCl, but not tiletamine, increased rat pyriform cortical DA metabolism and/or release, a response modulated by the NMDA‐uncoupled PCP recognition sites. Pretreatment with tiletamine did not attenuate the MK‐801‐induced increases in rat pyriform cortical DA metabolism, a result suggesting that tiletamine is not a partial agonist of the NMDA‐uncoupled PCP recognition sites in this region. However, following intracerebroventricular administration (100–500 μg/rat), tiletamine increased pyriform cortical DA metabolism with a bell‐shaped dose‐response curve. These data indicate a differential interaction of tiletamine with the NMDA‐coupled and ‐uncoupled PCP recognition sites. The paradoxical effects of tiletamine suggest that tiletamine might activate receptor(s) or neuronal pathways of unknown pharmacology.
Excitotoxic Brain Injury Suppresses Striatal High‐Affinity Glutamate Uptake in Perinatal Rats
Authors: Hu Bernadine , McDonald John W. , Johnston Michael V. , Silverstein Faye S.
Published: October 2006

Abstract: In immature rodent brain, the glutamate receptor agonist N‐methyl‐D‐aspartate (NMDA) is a potent neurotoxin. In postnatal day (PND)‐7 rats, intrastriatal injection of 25 nmol of NMDA results in extensive ipsilateral forebrain injury. In this study, we examined alterations in high‐affinity [3H]glutamate uptake (HAGU) in NMDA‐lesioned striatum. HAGU was assayed in synaptosomes, prepared from lesioned striatum, the corresponding contralateral striatum, or unlesioned controls. Twenty‐four hours after NMDA injection (25 nmol), HAGU declined 44 ± 8% in lesioned tissue, compared with the contralateral striatum (mean ± SEM, n = 6 assays, p < 0.006, paired t test). Doses of 5–25 nmol of NMDA resulted in increasing suppression of HAGU (5 nmol, n = 3; 12.5 nmol, n = 3; and 25 nmol, n = 5 assays; p < 0.01, regression analysis). The temporal evolution of HAGU suppression was biphasic. There was an early transient suppression of HAGU (−28 ± 4% at 1 h; p < 0.03, analysis of variance, comparing changes at 0.5, 1, 2, and 3 h after lesioning); 1 or 5 days postinjury there was sustained loss of HAGU (at 5 days, −56 ± 11%, n = 3, p < 0.03, paired t test, lesioned versus contralateral striata). Treatment with the noncompetitive NMDA antagonist MK‐801 (1 mg/kg i.p.) attenuated both the early and subsequent irreversible suppression of HAGU (1 h postlesion −28 ± 4%, n = 6 assays versus −12.6 ± 5% with MK‐801, n = 4, p= 0.005; 24 h postlesion, −44 ± 8%, n = 5, versus +2.4 ± 6%, n = 3 with MK‐801, p= 0.01, Wilcoxon ranked sum tests). In immature brain excitotoxic lesions produce an acute reversible suppression of HAGU, and a delayed long‐lasting reduction in HAGU secondary to brain injury. These data suggest that accumulation of endogenous glutamate, as a consequence of the acute disruption of HAGU, could contribute to the pathogenesis of excitotoxic neuronal injury.
Selective Increase of Iron in Substantia Nigra Zona Compacta of Parkinsonian Brains
Authors: Sofic E. , Paulus W. , Jellinger K. , Riederer P. , Youdim M. B. H.
Published: October 2006

Abstract: Histochemical and biochemical determinations of total iron, iron (II), and iron (III) contents in brain regions from Parkinson's and Alzheimer's diseases have demonstrated a selective increase of total iron content in parkinsonian substantia nigra zona compacta but not in the zona reticulata. The increase of iron content is mainly in iron (III). The ratio of iron (II):iron (III) in zona compacta changes from almost 2:1 to 1:2. This change is thought to be relevant and may contribute to the selective elevation of basal lipid peroxidation in substantia nigra reported previously. Iron may be available in a free state and thus can participate in autooxidation of dopamine with the resultant generation of H2O2 and oxygen free radicals.
Flow Cytometric Analysis of Internal Calcium Mobilization via a B2‐Bradykinin Receptor on a Subclone of PC‐12 Cells
Authors: Ransom J. T. , Cherwinski H. M. , Dunne J. F. , Sharif N. A.
Published: October 2006

Abstract: Single cell Ca2+ mobilization was studied by non‐parametric, quantitative flow cytometry using a sort‐selected subclone of PC‐12 cells. The response of the parent PC‐12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2‐BK receptor agonists Lys‐BK and Met‐Lys‐BK (10 pM‐1 μM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1‐BK receptor agonist Des‐Arg9‐BK (1 nM‐1 μM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2‐receptor antagonists D‐Arg0‐Hyp3‐thienyl5,8‐D‐Phe7‐BK and D‐Arg0‐Hyp3‐D‐Phe7 (0.1 nM‐10 μM) in a concentration‐dependent manner. Des‐Arg9‐Leu8‐BK, a B1‐receptor antagonist, failed to block the BK responses at 0.1–10 μM. The agonist/antagonist profile of the BK responses indicated that the B2‐BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor‐mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor‐mediated second messenger generation at the single cell level.
Single‐Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells
Authors: Buxser Stephen , Vroegop Steven , Decker Douglas , Hinzmann Jessica , Poorman Roger , Thomsen Darrell R. , Stier Mike , Abraham Irene , Greenberg Barry D. , Hatzenbuhler Nicole T. , Shea Mary , Curry Kim A. , Tomich Che‐Shen C.
Published: October 2006

Abstract: Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus‐infected Spodoptera frugiperda (SF‐9) insect cell system. Culture supernatants contained 2–3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single‐step affinity chromatography procedure using a high‐affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1–2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transacted into the SF‐9 cells coded for pro‐NGF, the NGF recovered after purification was > 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 ± 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 ± 20 pM and 9.6 ± 1.5 pM for a 3‐day primary response and a 1‐day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 ± 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from sub‐maxillary glands.
Changes in Inositol 1,4,5‐Trisphosphate and Inositol 1,3,4,5‐Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine
Authors: Challiss R. A. John , Jones J. Alison , Owen P. Jane , Boarder Michael R.
Published: October 2006

Abstract: In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H‐labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol‐labelled cells to observe a bradykinin‐mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5–10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin‐elicited Ins(1,3,4,5)P4 response increased to a maximum at 30–60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol‐loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin‐stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.
Effect of Presynaptic P2 Receptor Stimulation on Transmitter Release
Authors: Sperlagh B. , Vizi E. S.
Published: October 2006

Abstract: Because ATP is degraded to adenosine, its effect could be mediated by both P] and P2 receptors. Hence, the actions of an ATP analogue, resistant to enzymatic breakdown (α,β‐methylene ATP), were studied on the resting and electrically evoked release of radioactivity from longitudinal muscle strips of guinea pig ileum, preloaded either with [3H]choline or with [3H]noradrenaline. Their effects were compared with the actions of adenosine and ATP. Although adenosine and ATP markedly decreased the [3H]acetylcholine release evoked by field stimulation, α,β‐methylene‐ATP, a potent and selective agonist of P2x receptors, enhanced’ this release. However, 2‐methyl‐2‐thio‐ATP, an agonist of the P2y receptors, neither enhanced nor inhibited the [3H]‐acetylcholine release. 8‐Phenyltheophylline, an antagonist of PI receptors, increased the stimulation‐evoked release of acetylcholine, indicating that the release of acetylcholine is ton‐ically controlled by endogenous adenosine via PI receptors. When α,β‐methylene‐ATP and 8‐phenyltheophylline were added together, their potentiating effect on the acetylcholine release proved to be additive. Because α,β‐methylene‐ATP failed to antagonize the presynaptic effect of adenosine on PI purinoceptors, it seems very likely that its effect to enhance transmitter release is mediated via separate receptors, i.e., via P2x receptors, located on the axon terminals. Similarly, the stimulation‐evoked release of [3H]noradrenaline was enhanced slightly by α,β‐methylene‐ATP. Our results suggest that both cholinergic and noradrenergic axon terminals are equipped with P2 receptors through which the stimulation‐evoked release of transmitter can be modulated by ATP in a positive manner. Because α,β‐methylene‐ATP had an effect on it and 2‐methyl‐2‐thio‐ATP was without action, it is suggested that presynaptic P2x purinoceptors are involved. Therefore, it is concluded that if ATP is coreleased with a transmitter or released from another source, it might exert per se a positive influence on transmitter release evoked by axonal firing. Subsequently, adenosine, its breakdown product, might reduce transmitter release via presynaptic PI receptors.
Synapsins Contain O‐Linked N‐Acetylglucosamine
Authors: Liithi T. , Haltiwanger R. S. , Greengard P. , Bähler M.
Published: October 2006

Abstract: The neuron‐specific synaptic vesicle‐associated phosphoproteins synapsin I and synapsin II were shown to contain terminal 7V‐acetylglucosamine (GlcNAc) residues as determined by specific labeling with bovine galactosyltrans‐ferase and UDP‐[3H]galactose. The β‐elimination of galac‐tosyltransferase radiolabeled synapsin I and subsequent analysis of released saccharide on high‐voltage paper electrophoresis confirmed the presence of monosaccharidic GlcNAc moieties in O‐linkage to the protein. Partial cleavage of synapsin I by collagenase, 2‐nitro‐5‐thiocyanobenzoic acid, and Staphylococcus aureus V8 protease suggests that at least three glycosylation sites exist along the molecule. Taken together these data present the first evidence that a neuron‐specific protein contains 0‐glycosidically bound GlcNAc.
Dopamine‐Dependent Postnatal Development of Enkephalin and Tachykinin Neurons of Rat Basal Ganglia
Authors: Sivam S. P. , Krause J. E. , Breese G. R. , Hong J.‐S.
Published: October 2006

Abstract: The influence of deprivation of the neurotransmitter dopamine (DA) on the development of [Met5]‐enkephalin (ME) and substance P (SP) neuropeptide systems of the stria‐turn was investigated in Sprague‐Dawley rats. The neurotoxin 6‐hydroxydopamine (6‐OHDA) was used to induce DA deficiency on postnatal day 3 in rats, and the animals were killed at different postnatal time points until 35 days of age. The levels of ME and SP were determined by radioimmu‐noassay, and the abundance of preproenkephalin (PPE) and preprotachykinin (PPT) mRNA in the striatum was assessed by Northern blot hybridization analysis. The concentrations of DA, 5‐hydroxytryptamine (5‐HT), and their acid metabolites were determined by HPLC with electrochemical detection. The postnatal development of the PPE‐derived pep‐tide ME and the PPT‐derived peptide SP closely paralleled the appearance of the respective mRNAs coding for these peptides. The dopaminergic lesion with 6‐OHDA led to a marked depletion of DA and its metabolites but produced an increase in content of 5‐HT and its metabolite in the striatum. The lesion did not affect the ME and PPE mRNA levels in the striatum up to 25 days but increased the levels at 35 days. In contrast, a decreased developmental expression in SP and PPT mRNA was observed throughout the observation period. The lesion failed to influence the development of the mRNA coding for the structural protein β‐actin. The results indicate that the normal development of enkephalin, tachy‐kinin, and 5‐HT systems of the striatum is dependent on the availability of DA, the integrity of dopaminergic neurons, or both. The studies provide evidence for an interrelationship and interdependence between the development of neurotransmitter and neuropeptide systems. It is suggested that an early developmental abnormality in the DA system could permanently alter the neuropeptide systems, which in turn could influence the progression and expression of the DA‐deficiency state parkinsonism, Lesch‐Nyhan disease, or both.
Two Types of Clathrin‐Coated Vesicles Isolated from Rat Brain: Analysis of Biochemical Properties and Cellular Origin
Authors: Uriu Toshiko , Omori Kyoko , Omori Koichiro , Yamamoto Akitsugu , Inoue Masafumi , Inagaki Chiyoko
Published: October 2006

Abstract: Two major fractions rich in clathrin‐coated vesicles (CVs) (fraction I, ρ= 1.140 g/cm3; fraction II, ρ= 1.113 g/ cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl‐ translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85±9.5 nm in diameter; fraction II, 72±6.8 nm in diameter). Fraction II contained potent ouabain‐sensitive ATPase activity, whereas fraction I contained only a little activity. Im‐munoblot analysis for the Na+,K+‐ATPase catalytic subunit, a and α(+), demonstrated that fraction II exhibited predominantly α(+), whose proportion to α was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling‐chase experiments using 125I‐transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface‐bound ligand in the fraction with lower density (ρ= 1.090 g/cm3), a result suggesting that the involvement of Na+,K+‐ATPase in fraction II is attributable to endocytic vesicles. Cl‐ uptake in fraction II was approximately threefold higher than that in fraction I. ALEthylmaleimide (100 μM) completely inhibited the CI‐ uptake in fraction I but partially (∼50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types—glial and neuronal cells—and differ in size distribution of CVs, content of Na+,K+‐ATPase, and mechanism for Cl‐ uptake.
Modulation of [3H]Paroxetine Binding to the 5‐Hydroxytryptamine Uptake Site in an Animal Model of Depression
Authors: Edwards Emmeline , Harkins Kelly , Wright George , Henn Fritz
Published: October 2006

Abstract: The effects of learned helplessness on the 5‐hydroxytryptamine (5‐HT) uptake site were studied in rats using [3H]paroxetine binding. This ligand was chosen because it was demonstrated to label directly the 5‐HT uptake site whereas the [3H]imipramine binding site has been demonstrated to be heterogeneous in nature. Moreover, [3H]imipramine appears to bind to a presynaptic recognition site different from the uptake site. Exposure to uncontrollable shock training and testing resulted in an overall increase in [3H]paroxetine binding in all the groups studied [nonhelpless (NLH), learned helpless (LH), spontaneously helpless (SPLH)] as compared to naive controls (NC). However, the increase in [3H]paroxetine binding was significantly higher in the LH and SPLH groups. The maximum number of [3H]paroxetine binding sites in the rat hippocampus was increased significantly in learned helpless rats (LH and SPLH) at day 4 and day 30 after the shock escape test as compared to NC and NLH rats. By contrast, in the rat hypothalamus the maximum number of [3H]paroxetine binding sites was reduced significantly in the LH rats as compared to naive controls and NLH rats during the same time course. There was no change in [3H]paroxetine binding sites in any other brain regions examined in LH, NLH, and NC rats. The results suggest that a hippocampal hypothalamic connection might play a role in the serotonergic mediation of learned helpless behavior.
Neuronal Localization of Pyroglutamate Aminopeptidase II in Primary Cultures of Fetal Mouse Brain
Authors: Cruz Carlos , Charli Jean‐Louis , Vargas Miguel Angel , Joseph‐Bravo Patricia
Published: October 2006

Abstract: Pyroglutamate aminopeptidase II is a highly specific membrane‐bound ectopeptidase proposed to inactivate thyrotropin releasing hormone (TRH) in brain extracellular space. Its activity was measured in primary cell cultures of fetal brain in an attempt to define its cellular localization. Enzyme activity was detected in hypothalamic or cortical cell membrane fractions from 4‐ to 12‐day‐old cultures. When proliferation of nonneuronal cells was abolished by cytosine arabinoside treatment, pyroglutamate aminopeptidase II specific activity was increased as compared to untreated cultures, the opposite was observed for pyroglutamate aminopeptidase I activity. Treatment of cortical cells with the neu‐rotoxic agent glutamate reduced simultaneously pyroglutamate aminopeptidase II and glutamate decarboxylase activities. Glial cell cultures expressed pyroglutamate aminopeptidase I or glutamate synthase activities but not pyroglutamate aminopeptidase II. The data suggest that pyroglutamate aminopeptidase II is predominantly localized in neu‐ronal cells. This is consistent with a role for pyroglutamate aminopeptidase II in TRH‐ergic synaptic transmission. Key Words: Pyroglutamate aminopeptidase II—Thyrotropin releasing hormone—Peptidase—Neuronal cultures—Pyroglutamate aminopeptidase I—Synapsis. Cruz C. et al. Neuronal localization of pyroglutamate aminopeptidase II in primary cultures of fetal mouse brain. J. Neurochem. 56, 1594–1601 (1991).
Mechanism of Accumulation of the 1‐methyl‐4‐Phenylpyridinium Species into Mouse Brain Synaptosomes
Authors: Scotcher Kevin P. , Irwin Ian , DeLanney Louis E. , Langston J. William , Di Monte Donate
Published: October 2006

Abstract: The mechanism of accumulation of 1‐methyl‐4‐phenylpyridinium ion (MPP+), the toxic metabolite of 1‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine, into neuronal terminals was studied using mouse brain synaptosomes as an in vitro model. Addition of MPP+ to synaptosomal preparations, essentially devoid of contamination by extrasynap‐tosomal mitochondria, resulted in its time‐ and concentration‐dependent accumulation. Intrasynaptosomal concentrations of 79 and 106 μM were reached 10 and 30 min, respectively, after addition of 50 μM MPP+. The accumulation of 50 μM MPP+ into synaptosomes was only slightly affected by the catecholamine uptake blockers mazindol and nomifensine; in contrast, it was markedly enhanced by tetra‐phenylborate, a lipophilic anion that increases the rate of accumulation of permeant cations via a Nernstian concentration gradient. MPP+ accumulation was significantly increased or decreased as a consequence of hyperpolarization or depolarization, respectively, of the plasma membrane of synaptosomes. This effect was evident after incubation for 10 min. Changes in mitochondrial membrane potential also affected MPP+ accumulation, although only after 30 min of incubation. Data indicate that polarization of neuronal membranes may significantly contribute to the accumulation of MPP+ into nerve terminals.
Muscarinic Cholinergic Stimulation of Exogenous Phosphatidylinositol Hydrolysis Is Regulated by Guanine Nucleotides in Rabbit Brain Cortical Membranes
Authors: Carter Helen R. , Fain John N.
Published: October 2006

Abstract: Rabbit brain cortical membranes, which have been extracted with 2 M KCl, hydrolyze exogenously added [3H]phosphatidylinositol ([3H]PI) in a guanine nucleotide‐and carbachol‐dependent manner. Both oxotremorine‐M and carbachol are full agonists with EC50 values of 8 and 73 μM, respectively. Pirenzepine and atropine inhibit carbacholstimulated [3H]PI hydrolysis. The hydrolysis‐resistant guanine nucleotide analog guanosine 5′‐O(3‐thiotriphosphate) (GTPγS) is the most potent in supporting carbachol‐stimu‐lated hydrolysis of PI. There is no effect of carbachol in the absence of guanine nucleotides or in the presence of 100 μM adenosine 5′‐O(3‐thiotriphosphate), adenosine‐5′‐(β,γ‐imido)triphosphate, or sodium pyrophosphate. Guanylyl‐5′‐(β,γ‐imido)triphosphate [Gpp(NH)p] in the presence of carbachol also stimulates PI hydrolysis although much less than that seen with GTP‐γS. GDP and Gpp(NH)p are potent antagonists of the GTPγS‐dependent carbachol response. Optimal stimulation by carbachol and GTP7S was observed at 0.3–1 μM free Ca2+ and 6mM MgCl2. Limited trypsinization resulted in loss of receptor‐regulated PI breakdown and a slight decrease in basal activity. These results demonstrate that phospholipase C hydrolysis of exogenous PI by rabbit cortical membranes may be stimulated by carbachol in a guanine nucleotide‐dependent manner.
Potassium Cotransport with Sodium and Chloride in the Choroid Plexus
Authors: Bairamian Dikran , Johanson Conrad E. , Parmelee Judith T. , Epstein Mel H.
Published: October 2006

Abstract: The effects of loop diuretics and ion substitution on the 2‐min uptake of K (86Rb as marker) were analyzed to obtain evidence for K cotransport with Na and Cl in the choroid plexus epithelium. The isolated plexuses, which were excised from lateral ventricles of adult rats, were bathed in artificial cerebrospinal fluid (aCSF). At concentrations of 10‐6 to 10‐4M, the specific cotransport inhibitors, bumetanide and piretanide, suppressed uptake of K in a dose‐dependent manner. Ouabain‐insensitive K uptake was stimulated by preincubating the choroid plexus in aCSF very low in [Na] and [K], then incubating it in much higher concentrations of these cations; bumetanide (10‐4M) blocked this stimulated uptake by 52%. Moreover, tissue preincubation in Na‐ or Cl‐free medium, followed by incubation with normal concentrations of both ions, stimulated the ouabain‐insensitive uptake of K from 15 (baseline) to 35 nmol/mg dry weight. This stimulation of K transport depended on the simultaneous presence of both Na and Cl in aCSF, and replacing either ion alone did not stimulate the ouabain‐insensitive K uptake. Collectively, these findings, together with those from a previous pharmacological study of 22Na and 36Cl transport, constitute strong evidence for the cotransport of Na, K, and Cl in rat choroid plexus.
Cerebral Metabolic Effects of Monoamine Oxidase Inhibition in Normal and 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Acutely Treated Monkeys
Authors: Palombo Ernesta , Porrino Linda J. , Crane Alison M. , Bankiewicz Krzysztof S. , Kopin Irwin J. , Sokoloff Louis
Published: October 2006

Abstract: The neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) induces dopaminergic cell death in the substantia nigra pars compacta (SNpc) and clinical parkinsonism in humans and experimental animals. Pretreatment with monoamine oxidase inhibitors prevents this cell death and associated parkinsonism by blocking the oxidation of MPTP to a toxic intermediate. The 2‐deoxyglucose method was used to study the acute effects of MPTP in the monkey brain and the effects of monoamine oxidase inhibition on local cerebral glucose utilization in both normal and MPTP‐treated monkeys. MPTP administration alone caused a major increase in glucose utilization in the SNpc and smaller increases in some subnuclei within the ventral tegmental area in which eventual dopaminergic cell loss also occurs. Pretreatment with pargyline abolished these metabolic increases, a finding suggesting both that the oxidized product of MPTP generates the metabolic increases and that the increased glucose consumption may contribute to cell toxicity. On the other hand, in most cortical, thalamic, striatal, brainstem, and cerebellar areas MPTP alone caused reductions in glucose utilization, and pargyline failed to prevent these effects. Pargyline alone depressed metabolism in the locus coeruleus and a few other monoaminergic structures.
N‐Methyl‐D‐Aspartate Releases Excitatory Amino Acids in Rat Corpus Striatum In Vivo
Authors: Young Andrew M. J. , Bradford H. F.
Published: October 2006

Abstract: There is a considerable amount of conflicting evidence from several studies as to the action of applied N‐methyl‐D‐aspartate (NMDA) on the release of glutamate and aspartate in the brain. In the present study the effect of NMDA on extracellular levels of endogenous amino acids was investigated in conscious, unrestrained rats using intracerebral microdialysis. NMDA caused dose‐related increases in extracellular levels of glutamate and aspartate; threonine and glutamine were unaffected. The NMDA‐evoked release of glutamate and aspartate was significantly decreased by the specific NMDA receptor antagonist 3‐[(±)‐2‐carboxypiperazin‐4‐yl]‐propyl‐1‐phosphonic acid. In addition, increasing the perfusate concentration (and therefore the extracellular concentration) of Ca2+ significantly enhanced the NMDA‐evoked release of glutamate and aspartate, whereas removal of Ca2+ and addition of a high Mg2+ concentration to the perfusate caused a significant reduction in their NMDA‐evoked release. Moreover, the NMDA‐evoked release of glutamate and aspartate was reduced in decorticate animals. These results demonstrate that, in the striatum in vivo, NMDA causes selective release of endogenous glutamate and aspartate from neurone terminals and that this action occurs through an NMDA receptor‐mediated mechanism. The ability of NMDA receptor activation to induce release of glutamate and aspartate, perhaps by a positive feedback mechanism, may be relevant to the pathologies underlying epilepsy and ischaemic and hypoglycaemic brain damage.
myo‐Inositol Transport in Mouse Astroglia‐Rich Primary Cultures
Authors: Wiesinger Heinrich
Published: October 2006

Abstract: Uptake of radiolabeled myo‐inositol was studied in astroglia‐rich primary cultures derived from neonatal mouse brains. The uptake was saturable in the presence of Na− with a Km of 25 μM and a Vmax of 60 pmol·min−· (mg protein)−1, suggesting a high‐affinity transport system for myo‐ inositol in astroglial cells. In addition, a Na+ ‐independent, nonsaturable component was found. Camer‐mediated uptake was not inhibited by cytochalasin B (50 μM), but was reduced by depolarizing concentrations of K+ and, to different extents, in the presence of phloretin, ouabain, or amiloride (1 mM each). scyllo‐Inositol, glucose, and galactose also reduced myo‐inositol uptake; inhibition by the tmo hexoses was not reversed in the presence of 0.4 Wsorbinil. On the other hand, uptake of 2‐deoxyglucose was not inhibited by high concentrations of myo‐inositol. Preincubation of the cells with glucose‐free or inositol‐free medium stimulated uptake of myo‐inositol and preincubation with 25 mM glucose in the presence of 0.4 mM sorbinil had no effect on the rate of uptake. The results suggest that myo‐inositol is taken up into the astroglial cells by a transport mechanism that is distinct from that of glucose and probably is an active one. Sorbitol pathway activity does not interfere with myo‐inositol uptake.
Phorbol Myristate Acetate and 8‐Bromo‐Cyclic AMP‐Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites
Authors: Harrison Beth C. , Mobley Philip L.
Published: October 2006

Abstract: Both the protein kinase C (PK‐C) activator, phorbol 12‐myristate 13‐acetate (PMA), and the cyclic AMP‐dependent protein kinase (PK‐A) activator, 8‐bromo‐cyclic AMP (8‐BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8‐BR results in the morphological transformation of flat, polygonal‐shaped cells into stellate, process‐bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8‐BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8‐BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8‐BR treatment was restricted to serine residues in the N‐terminal domain of these proteins. Studies were also conducted to compare the two‐dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8‐BR with those produced when the proteins were phosphorylated with purified PK‐C or PK‐A. PK‐C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK‐C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells. The increased phosphorylation of vimentin observed in intact cells treated with 8‐BR and with PK‐A using purified vimentin as a substrate resulted in similar phosphopeptide maps. A common major phosphorylated peptide fragment was observed following phosphorylation of GFAP by PK‐A and in intact cells treated with 8‐BR; however, an additional phosphorylated fragment was observed with the purified kinase that was not observed in intact cells. These studies indicate that phosphorylation events mediated by PMA and 8‐BR converge to phosphorylate several of the same regions of GFAP and vimentin, and suggest that these effects are the direct result of the activation of PK‐C and PK‐A.
A Microdialysis Study of the Regulation of Endogenous Noradrenaline Release in the Rat Hippocampus
Authors: Thomas D. N. , Holman R. B.
Published: October 2006

Abstract: In vivo microdialysis was used to investigate the regulation of noradrenaline release in rat hippocampus. Idazoxan, an α2‐adrenoreceptor antagonist (1–10 mg/kg), increased noradrenaline release in a dose‐dependent manner. Inhibition of noradrenaline uptake by desipramine (0.05–20 μM; via the probe) also increased the extracellular content of the transmitter. In the presence of this increased noradrenaline content (desipramine via the probe), the effect of a low dose of idazoxan (1 mg/kg) was potentiated. Local perfusion of idazoxan (1–500 μM) in the hippocampus also increased noradrenaline release but not to the same extent as following systemic administration. In the presence of desipramine, unlike the systemic injection of idazoxan, local perfusion did not potentiate noradrenaline release. The data are consistent with the regulation of extracellular noradrenaline content in the hippocampus by neuronal uptake and to a lesser extent by presynaptic autoreceptors.
Taurine Inhibits Protein Kinase C‐Catalyzed Phosphorylation of Specific Proteins in a Rat Cortical P2 Fraction
Authors: Li Yi‐Ping , Lombardini John B.
Published: October 2006

Abstract: We previously reported that taurine inhibits the phosphorylation of specific proteins in a P2 synaptosomal fraction prepared from the rat cortex. In the present study, the regulation of the phosphorylation of an ∼20K Mr protein whose phosphorylation is inhibited by taurine was further investigated. The phosphorylation of the ∼20K Mr protein in a hypo‐osmotically shocked P2 fraction from rat cortex was dependent on the free Ca2+ in the reaction medium. Depolarization induced by 30 mM K+ stimulated the phosphorylation of the ∼20K Mr protein in an intact synaptosomal P2 preparation by 30‐fold. This stimulation was inhibited 35% by taurine, whereas guanidinoethanesulfonic acid, a taurine analogue, did not have any effect, thereby indicating the specificity of taurine. Addition of phorbol 12‐myristate 13‐acetate, a phorbol ester, together with phosphatidylserine, stimulated the phosphorylation of the ∼20K Mr protein in the hypo‐osmotically shocked P2 synaptosomal fraction by fivefold, whereas cyclic AMP, cyclic GMP, and calmodulin did not have any effect on the phosphorylation of this particular protein. Phorbol 12‐myristate 13‐acetate–stimulated phosphorylation of the ∼20K Mr protein is blocked 30% by taurine. Taurine also inhibited phorbol 12‐myristate 13‐acetate‐activated phosphorylation of two other proteins that were similar in molecular weight and isoelectric point to the ∼20K Mr protein on two‐dimensional gels. These results suggest that taurine modulates the phosphorylation of specific proteins regulated by the signal transduction system in the brain. Thus, taurine may modulate neuroactivity by inhibiting the phosphorylation of specific proteins involved in regulatory function.
Effect of Adenosine on 45Ca2+ Uptake by Electrically Stimulatad Rat Brain Synapatosomes
Authors: Gongalves M. L. , Pinto F. , Ribeiro J. A.
Published: October 2006

Abstract: The effect of adenosine on 45Ca2+ uptake by rat brain synaptosomes stimulated by electrical pulses was investigated. 45Ca2+ uptake was voltage dependent. Adenosine (1 μM‐1 μM) decreased the uptake of 45Ca2+ induced by electrical stimulation (amplitude, 20 V; duration, 400 pμs; frequency, 10 pulses/s) in a concentration‐dependent manner. At a concentration of 1 μM, adenosine almost abolished the 45Ca2+ uptake induced by electrical stimulation (92.9 ± 5.3% inhibition), but when the calcium uptake was induced by high‐K+ (60 mM) medium, the effect of adenosine (1 μM) was smaller (43.8 ± 5.2% inhibition). The inhibitory effect of 1 μM adenosine on calcium uptake induced by electrical stimulation was antagonized by 1,3‐dipropyl‐8‐p‐sulfophenylxanthine (5 μM). The possibility that adenosine interacts with the calcium channels opened by electrical stimulation is discussed.
Potassium‐ and Carbachol‐Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH‐SY5Y
Authors: Murphy Nuala P. , Ball Stephen G. , Vaughan Peter F. T.
Published: October 2006

Abstract: The human neuroblastoma clone SH‐SY5Y expresses potassium‐, carbachol‐, and calcium ionophore A23187‐evoked, calcium‐dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki= 0.33 nM), hexahydrosiladifenidol (Ki= 18 nM), and pirenzepine (Ki= 1,183 nM) completely inhibited the carbachol‐evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2‐selective antagonists meth‐octramine (10‐4M) and AFDX‐116 (10‐4M), by ∼14 and 46%, respectively. The nicotinic antagonist d‐tubocurarine (10‐4M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH‐SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.
Identification of Intact Neurotensin in the Substantia Nigra After Its Retrograde Axonal Transport in Dopaminergic Neurons
Authors: Castel M. N. , Faucher D. , Cuiné F. , Dubédat P. , Boireau A. , Laduron P. M.
Published: October 2006

Abstract: Two hours after the injection of (3‐[125I]iodotyrosyl3) neurotensin into the striatum, a labeling was observed in the ipsilateral substantia nigra. In the present study, we demonstrated by HPLC that this radioactivity corresponded to intact neurotensin and to degradation products of this peptide. This finding provides the first clear‐cut evidence that a neuropeptide can be internalized and retrogradely transported in brain neurons. Therefore, the fact that intact neurotensin can be seen to exist over a long period of time in the cell body suggests that the retrograde transport process could perhaps be involved in the long‐term effects of neuropeptides.
Acute Effects of 1‐Methyl‐4‐Phenyl‐1, 2, 3, 6‐Tetrahydropyridine in a Model of Rat Designated a Poor Metabolizer of Debrisoquine
Authors: Jiménez‐Jiménez Félix Javier , Tabernero César , Mena María Angeles , García de Yébenes Justo , Jesús García de Yébenes María , Casarejos María José , Pardo Beatriz , García‐Agundez José Augusto , Benítez Julio , Martínez Armando , García‐Asenjo José Antonio López
Published: October 2006

Abstract: The relationship between oxidative polymorphisms and the cause of Parkinson's disease is controversial. The drug l‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP), which induces parkinsonism in humans and in some animal models, is metabolized by cytochrome P450 db1 isozyme (the same enzymatic system implicated in 4‐hydroxylation of debrisoquine). In this study, we treated females of three rat species, which differ in their ability to hydroxylate debrisoquine, with MPTP (three doses of 30 mg/kg s.c. at 12‐h intervals), and we measured their motor activity and brain monoamine levels. Female dark‐adapted rats (poor metabolizers of debrisoquine) showed a more pronounced and more maintained reduction of their motor activity after treatment with MPTP. MPTP‐treated, dark‐adapted rats also had a depletion of noradrenaline in the diencephalon and a depletion of dopamine and serotonine and their respective metabolites in the limbic system when compared with the other two species. These results suggest that oxidative polymorphism of debrisoquine plays a role in the acute effects of MPTP.
Protection of Ischaemic Synaptosomes from Calcium Overload by Addition of Exogenous Lactate
Authors: Boakye Pearl , White Elizabeth J. , Clark John B.
Published: October 2006

Abstract: In depolarised anoxic synaptosomes, in which lactate production was significantly raised compared with normoxic conditions, calcium uptake, net acetylcholine release, and the intrasynaptosomal calcium concentration were all significantly lowered. In contrast, lactate production in synaptosomes incubated under aglycaemic‐and ischaemic‐type conditions was significantly lower and basal calcium uptake, acetylcholine release, and intrasynaptosomal calcium concentration were elevated compared with normoxia. In addition, the increase in intrasynaptosomal calcium concentration under the ischaemic‐type condition appeared to be greater than could be accounted for by the rise in calcium uptake alone. Intrasynaptosomal pH reflected the lactate production under each condition investigated. Addition of exogenous lactate to normoxic synaptosomes mimicked the effects observed in anoxia, suggesting that lactate itself may have blocked the calcium uptake, inhibiting the rise in intrasynaptosomal calcium and acetylcholine release occurring in depolarised anoxic synaptosomes. When lactate was added to ischaemic synaptosomes, the large rise in intrasynaptosomal calcium concentration, calcium uptake, and acetylcholine release were decreased, suggesting that lactate may have a protective role in preventing cell death by calcium overload under ischaemic‐type conditions. Evidence is presented to suggest that the effect of l‐lactate was due to the lactate moiety itself rather than the associated acidosis.