bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Increased γ‐Aminobutyric Acid Levels on GAD67 Protein and mRNA Levels in Rat Cerebral Cortex
Authors: Rimvall Karin , Sheikh Sabina N. , Martin David L.
Published: October 2006

Abstract: Rats were injected with saline or the γ‐aminobutyric acid (GABA) transaminase inhibitor γ‐vinyl‐GABA for 7 days and the effects on GABA content and glutamic acid decarboxylase (GAD) activity, and the protein and mRNA levels of the two forms of GAD (GAD67 and GAD65) in the cerebral cortex were studied. γ‐Vinyl‐GABA induced a 2.3‐fold increase in GABA content, whereas total GAD activity decreased by 30%. Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75–80% decrease in GAD67 levels, whereas the levels of GAD65 remained unchanged. RNA slot‐blotting with a 32P‐labeled GAD67 cDNA probe demonstrated that the change in GAD67 protein content was not associated with a change in GAD67 mRNA levels. Our results suggest that GABA specifically controls the level of GAD67 protein. This effect may be mediated by a decreased translation of the GAD67 mRNA and/or a change in the stability of the GAD67 protein.
Microdialysis Monitoring of 3,4‐Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus
Authors: Robert Frédéric , Lambás‐Señas Laura , Ortemann Catherine , Pujol Jean‐François , Renaud Bernard
Published: October 2006

Abstract: An on‐line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4‐dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl‐DOPA, which was shown to be more stable than NSD 1015 or Ro 4‐4602 in the perfusion fluid. A 1‐h perfusion of a 10−4 mol/L of difluoromethyl‐DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α‐methyl‐p‐tyrosine. Systemic administration of the α2‐antagonist piperoxane before difluoromethyl‐DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady‐state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.
Endogenous Adenosine Regulates the Apparent Efficacy of 1‐Aminocyclopentyl‐1S,3R‐Dicarboxylate Inhibition of Forskolin‐Stimulated Cyclic AMP Accumulation in Rat Cerebral Cortical Slices
Authors: Cartmell Jayne , Kemp John A. , Alexander Stephen P. H. , Kendall David A.
Published: October 2006

Abstract: In rat cerebral cortical slices, the 1‐aminocyclopentyl‐1S,3R‐dicarboxylate (1S,3R‐ACPD) isomer of the selective metabotropic excitatory amino acid agonist ACPD inhibited forskolin‐stimulated cyclic AMP (cAMP) accumulation in a concentration‐dependent manner with a maximal inhibition of 51 ± 3% and a half‐maximally effective concentration of 8.8 ± 3.4 μM. Similarly, 1R,3S‐ACPD inhibited the forskolin response in a concentration‐dependent manner, but with an inhibition of 80 ± 5% at 3 μM. In addition to inhibiting forskolin‐stimulated cAMP levels, 1S,3R‐ACPD, but not 1R,3S‐ACPD, enhanced the cAMP response to A2b adenosine receptor activation. In the presence of 1.2 U/ml of adenosine deaminase (included to reduce the contribution of endogenous adenosine), the efficacy of 1S,3R‐ACPD was increased (88 ± 3% inhibition), but the potency was unchanged. The adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine also increased the inhibitory effect of 100 μM 1S,3R‐ACPD, from 57 ± 1 to 78 ± 5%. These results indicate that endogenous adenosine plays an important role in regulating the apparent efficacy of 1S,3R‐ACPD inhibition of forskolin‐stimulated cAMP accumulation in rat cerebral cortical slices and that previous studies in rat hippocampus and hypothalamus in the absence of added adenosine deaminase may have underestimated the efficacy of this compound.
Reduction in Microtubule Dynamics In Vitro by Brain Microtubule‐Associated Proteins and by a Microtubule‐Associated Protein‐2 Second Repeated Sequence Analogue
Authors: Yamauchi Paul S. , Flynn Gregory C. , Marsh Robert L. , Purich Daniel L.
Published: October 2006

Abstract: Microtubule‐associated protein (MAP) binding to assembled microtubules (MTs) can be reduced by the addition of polyglutamate without significant MT depolymerization or interference with MT elongation reactions. Ensuing polymer length redistribution in MAP‐depleted MTs occurs on a time scale characteristic of that observed with MAP‐free MTs. The redistribution phase occurs even in the absence of mechanical shearing and without appreciable effects from end‐to‐end annealing, as indicated by the time course of incremental changes in polymer length and MT number concentration. We also observed higher rates of MT length redistribution when the [MAP]/[tubulin] ratio was decreased. Together, these results demonstrate that MT length redistribution rates are greatly influenced by MAP content, and the data are compatible with the dynamic instability model. We also found that a peptide analogue corresponding to the second repeated sequence in the MT‐binding region of MAP‐2 can also markedly retard MT length redistribution kinetics, a finding that accords with the ability of this peptide to promote tubulin polymerization in the absence of MAPs and to displace MAP‐2 from MTs. These results provide further evidence that MAPs can modulate MT assembly/disassembly dynamics and that peptide analogues can mimic the action of intact MAPs without the need for three contiguous repeated sequences in the MT‐binding region.
Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing
Authors: Tzigaret Cezar , McIntosh Tracy K. , Okiyama Koichi , Jenkins W. L. , Prasad M. Renuka
Published: October 2006

Abstract: The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0°C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than −40°C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane‐ and pentobarbital‐anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5‐trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0°C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5‐trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0°C from in situ frozen brains (p < 0.05). These results indicate that dissection of cortex and hippocampus at 0°C following in situ freezing will eliminate decapitation‐induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5‐trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.
Characterization of a Human NK1 Tachykinin Receptor in the Astrocytoma Cell Line U 373 MG
Authors: Heuillet E. , Ménager J. , Fardin V. , Flamand O. , Bock M. , Garret C. , Crespo A. , Fallourd A. M. , Doble A.
Published: October 2006

Abstract: The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]‐senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L‐Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was ∼ 1,000‐fold less potent. The most potent peptide antagonist was trans‐4‐hydroxy‐1‐(1 H‐indol‐3‐ylcarbonyl)‐L‐prolyl‐N‐methyl‐N‐(phenylmethyl)‐L‐tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)‐CP 96,345 > (±)‐CP 96,345 > (±)‐2‐chlorobenzylquinuclidinone > (R,R)‐CP 96,345 > RP 67580 > RP 68651. In [3H]‐inositol‐labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.
Induction of Primary Response Genes by Excitatory Amino Acid Receptor Agonists in Primary Astroglial Cultures
Authors: Condorelli Daniele F. , Dell'Albani Paola , Amico Carla , Kaczmarek Leszek , Nicoletti Ferdinando , Lukasiuk Katarzyna , Stella Anna Maria Giuffrida
Published: October 2006

Abstract: We have characterized the genomic response of astroglial cells to excitatory amino acids by using selective agonists and antagonists for the various receptor subtypes and by analyzing different primary response genes, such as members of the Fos (c‐fos and fosB) and Jun (c‐jun, junB, and junD) families, zif/268, and c‐myc. A rapid and transient elevation of mRNA levels for c‐fos, fosB, c‐jun, junB, and zif/268 was observed after addition of glutamate to cultured astrocytes, whereas junD and c‐myc expression was not affected. The level of AP‐1 DNA binding activity, as measured by the electrophoretic mobility shift assay, also increased after addition of glutamate to cultured astrocytes. Glutamate‐induced c‐fos expression was not affected by the N‐methyl‐d‐aspartate receptor antagonists MK‐801 and D‐2‐amino‐5‐phosphonopentanoate, by the kainate/α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX), or by the broad‐spectrum antagonist kynurenate. Kainate and AMPA were also effective in inducing primary response gene expression, and their actions were antagonized by kynurenate and DNQX but not by MK‐801. 1S,3R‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid, a selective agonist for the metabotropic glutamate receptor, induced primary response gene expression, but its action was not antagonized by different glutamate antagonists, including L‐2‐amino‐3‐phosphonopropionate. In conclusion, our data suggest that cultured astrocytes express both kainate/AMPA ionotropic receptors and metabotropic receptors coupled to the rapid and coordinated activation of different classes of transcriptional factor genes.
Involvement of Calcium Channels in Depolarization‐Evoked Release of Adenosine from Spinal Cord Synaptosomes
Authors: Cahill Catherine M. , White Thomas D. , Sawynok Jana
Published: October 2006

Abstract: The potential involvement of L‐ and N‐type voltage‐sensitive calcium (Ca2+) channels and a voltage‐independent receptor‐operated Ca2+ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K+ and capsaicin was examined. Bay K 8644 (10 nM) augmented release of adenosine in the presence of a partial depolarization with K+ (addition of 6 mM) but not capsaicin (1 and 10 μM). This augmentation was dose dependent from 1 to 10 nM and was followed by inhibition of release from 30 to 100 nM. Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose‐dependent manner, but neither antagonist had any effect on release of adenosine produced by K+ (24 mM) or capsaicin (1 and 10 μM) ω‐Conotoxin inhibited K+‐evoked release of adenosine in a dose‐dependent manner but had no effect on capsaicin‐evoked release. Ruthenium red blocked capsaicin‐induced release of adenosine but had no effect on K+‐evoked release. Although L‐type voltage‐sensitive Ca2+ channels can modulate release of adenosine when synaptosomes are partially depolarized with K+, N‐type voltage‐sensitive Ca2+ channels are primarily involved in K+‐evoked release of adenosine. Capsaicin‐evoked release of adenosine does not involve either L‐ or N‐type Ca2+ channels, but is dependent on a mechanism that is sensitive to ruthenium red.
A Toxin Fraction (FTX) from the Funnel‐Web Spider Poison Inhibits Dihydropyridine‐Insensitive Ca2+ Channels Coupled to Catecholamine Release in Bovine Adrenal Chromaffin Cells
Authors: Duarte Carlos B. , Rosario Luis M. , Sena Cristina M. , Carvalho Arsélio P.
Published: October 2006

Abstract: In adrenal chromaffin cells, depolarization‐evoked Ca2+ influx and catecholamine release are partially blocked by blockers of L‐type voltage‐sensitive Ca2+ channels. We have now evaluated the sensitivity of the dihydropyridine‐resistant components of Ca2+ influx and catecholamine release to a toxin fraction (FTX) from the funnel‐web spider poison, which is known to block P‐type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K+‐depolarization‐induced Ca2+ influx by 50%, as monitored with fura‐2, whereas nitrendipine (0.1–1 μM) and FTX (3:3), a synthetic FTX analogue (1 mM), blocked the [Ca2+]i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca2+]i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by ∼80 and 70%, respectively, but both substances together abolished the K+‐evoked catecholamine release, as measured by HPLC. The ω‐conotoxin GVIA (0.5 μM) was without effect on K+‐stimulated 45Ca2+ uptake. Our results indicate that FTX blocks dihydropyridine‐ and ω‐conotoxin‐insensitive Ca2+ channels that, together with L‐type voltage‐sensitive Ca2+ channels, are coupled to catecholamine release.
Labelling of 5‐Hydroxytryptamine3 Receptors with a Novel 5‐HT3 Receptor Ligand, [3H]RS‐42358–197
Authors: Wong Erik H. F. , Bonhaus Douglas W. , Wu Irene , Stefanich Eric , Eglen Richard M.
Published: October 2006

Abstract: RS‐42358–197{(S)‐N‐(1‐azabicyclo[2.2.2]oct‐3‐yl)‐2,4,5,6‐tetrahydro‐1H‐benzo[de]isoquinolin‐1‐one hydrochloride} displaced the prototypic 5‐hydroxytryptamine3 (5‐HT3) receptor ligand [3H]quipazine in rat cerebral cortical membranes with an affinity (pKi) of 9.8 ± 0.1, while having weak affinity (pKi < 6.0) in 23 other receptor binding assays. [3H]RS‐42358–197 was then utilized to label 5‐HT3 receptors in a variety of tissues. [3H]RS‐42358–197 labelled high‐affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (KD) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (Bmax) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [3H]RS‐42358–197 was similar to that labelled with [3H]GR 65630, but was significantly less than that found with [3H]‐quipazine. The binding of [3H]RS‐42358–197 had a pharmacological profile similar to that of [3H]quipazine, as indicated by the rank order of displacement potencies: RS‐42358–197 > (S)‐zacopride > tropisetron > (R)‐zacopride > ondansetron > MDL72222 > 5‐HT. However, differences in 5‐HT3 receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1‐(m‐chlorophenyl)biguanide when displacing [3H]RS‐42358‐197 binding. [3H]RS‐42358–197 also labelled a population (Bmax= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (KD= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5‐HT3 receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [3H]RS‐42358–197 have detected differences in 5‐HT3 receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5‐HT3 receptor.
Tachykinins Potentiate N‐Methyl‐D‐Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn
Authors: Rusin K. I. , Bleakman D. , Chard P. S. , Randic M. , Miller R. J.
Published: October 2006

Abstract: Substance P and neurokinin A both potentiated N‐methyl‐d‐aspartate (NMDA)‐induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca2+]i in these cells. Substance P, but not neurokinin A, increased [Ca2+]i in a subpopulation of neurons. The increase in [Ca2+]i was found to be due to Ca2+ influx through voltage‐sensitive Ca2+ channels. Substance P and neurokinin A also potentiated the increase in [Ca2+]i produced by NMDA, but not by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, kainate, or 50 mM K+. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca2+]i can be dissociated from their effects on NMDA receptors.
Solubilization and Biochemical Characterization of the Melatonin Deacetylase from Xenopus laevis Retina
Authors: Grace Michael S. , Besharse Joseph C.
Published: October 2006

Abstract: Melatonin deacetylase, an enzyme activity recently discovered in the Xenopus laevis retina, regulates local melatonin levels. The deacetylase occurs in retina, retinal pigment epithelium, and skin, all sites of melatonin action, and is widely distributed among vertebrates. We have solubilized the enzyme from Xenopus retina and pigment epithelium using nonionic detergents, and have developed a specific enzyme assay. We have characterized the enzyme and now report that the deacetylase is relatively specific for melatonin and is inhibited by the melatonin precursor N‐acetylserotonin and the product of the deacetylase, 5‐methoxytryptamine. Inhibition of deacetylase activity by eserine (physostigmine) suggests a relationship between deacetylase and cholinesterase activities. However, among a variety of cholinesterase inhibitors tested, only eserine inhibits the deacetylase. Furthermore, eserine is much less potent as an inhibitor of the deacetylase than the cholinesterases, and purified cholinesterases failed to deacetylate melatonin. We also show that melatonin deacetylase and aryl acylamidase (an enzyme related to cholinesterases) activities are differentially extractable from Xenopus ocular tissues, and that they exhibit different pH optima and inhibition profiles. Our results provide an initial characterization of the Xenopus retinal melatonin deacetylase, and indicate that deacetylase activity is distinct from cholinesterase and aryl acylamidase activities.
Role of Extracellular Adenosine in Ethanol‐Induced Desensitization of Cyclic AMP Production
Authors: Rabin Richard A. , Fiorella David , Van Wylen David G. L.
Published: October 2006

Abstract: The decrease in receptor‐stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol‐induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol‐induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4‐day exposure to ethanol decreased basal as well as 2‐chloroadenosine‐ and forskolin‐stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2‐day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2‐chloroadenosine‐ and forskolin‐stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum‐free defined media. The ethanol‐induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24‐h exposure of PC12 cells to 2‐chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin‐stimulated cyclic AMP levels were increased. Similar results were obtained after a 4‐day exposure of PC12 cells to 2‐chloroadenosine or 5′‐N‐ethylcarboxamido‐adenosine. The present results indicate that the ethanol‐induced decrease in receptor‐stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.
Specific In Vitro Biological Activity of Snake Venom Myotoxins
Authors: Brusés Juan L. , Capaso Juan , Katz Eleonora , Pilar Guillermo
Published: October 2006

Abstract: Some snake venoms contain toxins that are reported to be selective for damaging muscle. This specificity can be used to design experiments intended to eliminate muscle. We studied the small myotoxins and fractions IV and V of Bothrops nummifer venom to evaluate their direct effect on cultured muscle cells, neurons, macrophages, and a fibroblast cell line. The small myotoxins, at 100 μg/ml for 2 h, had no effect in vitro, contrary to the in vivo applications. Fractions IV and V were both myotoxic and, at 100 μg/ml, destroyed all cell types. However, at 10 μg/ml the effects of fraction IV were more selective for muscle. Vacuolation of the sarcoplasmic reticulum and T‐tubules was first seen in the poisoned muscles, without initial lesions in the nuclei, sarcolemma, mitochondria, and rough endoplasmic reticulum. Fractions IV and V have different toxic activity in cells other than muscles and are a mixture of two basic proteins (i and ii). Protein ii is predominant in fraction IV and protein i is predominant in fraction V. The toxic effects may be mediated by the formation of nonspecific ionic pores in the sarcolemma and/or T‐tubule muscle membrane.
Cerebral Cortex Ammonia and Glutamine Metabolism in Two Rat Models of Chronic Liver Insufficiency‐Induced Hyperammonemia: Influence of Pair‐Feeding
Authors: Dejong Cornelis H. C. , Deutz Nicolaas E. P. , Soeters Peter B.
Published: October 2006

Abstract: Enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis, and glutamine release into the bloodstream have been hypothesized to deplete cerebral cortex glutamate pools. We investigated this hypothesis in rats with chronic liver insufficiency‐induced hyperammonemia and in pair‐fed controls to rule out effects of differences in food intake. Cerebral cortex plasma flow and venous‐arterial concentration differences of ammonia and amino acids, as well as cerebral cortex tissue concentrations, were studied 7 and 14 days after surgery in portacaval‐shunted/bile duct‐ligated, portacaval‐shunted, and sham‐operated rats, while the latter two were pair‐fed to the first group, and in normal unoperated ad libitum‐fed control rats. At both time points, arterial ammonia was elevated in the chronic liver insufficiency groups and arterial glutamine was elevated in portacaval shunt/biliary obstruction rats compared to the other groups. In the chronic liver insufficiency groups net cerebral cortex ammonia uptake was observed at both time points and was accompanied by net glutamine release. Also in these groups, cerebral cortex tissue glutamine, many other amino acid, and ammonia levels were elevated. Tissue glutamate levels were decreased to a similar level in all operated groups compared with normal unoperated rats, irrespective of plasma and tissue ammonia and glutamine levels. These results demonstrate that during chronic liver insufficiency‐induced hyperammonemia, the rat cerebral cortex enhances net ammonia uptake and glutamine release. However, the decrease in tissue glutamate concentrations in these chronic liver insufficiency models seems to be related primarily to nutritional status and/or surgical trauma.
Glial and Neuronal Marker Proteins in the Silicone Chamber Model for Nerve Regeneration
Authors: Karlsson J.‐E. , Rosengren L. E. , Wang S. , Danielsen N. , Haglid K. G.
Published: October 2006

Abstract: In the present study, neuronal and Schwann cell marker proteins were used to biochemically characterize the spatiotemporal progress of degeneration/regeneration in the silicone chamber model for nerve regeneration. Rat sciatic nerves were transected and the proximal and distal stumps were inserted into a bridging silicone chamber with a 10‐mm interstump gap. Using dot immunobinding assays, S‐100 protein and neuronal intermediate filament polypeptides were measured in different parts of the nerve 0–30 days after transaction. In the most proximal nerve segment, all the measured proteins were transiently increased. In the proximal and distal stumps adjacent to the transection, the studied proteins were decreased indicating degeneration of the nerve. Within the silicone chamber, the regenerating nerve expressed the Schwann cell S‐100 protein already at 7 days, whereas the neurofilament polypeptides appeared later. These observations are corroborated by previous morphological studies. The biochemical method described provides a new and fast approach to the study of nerve regeneration.
Arachidonic Acid Turnover and Phospholipase A2 Activity in Neuronal Growth Cones
Authors: Negre‐Aminou Pascale , Pfenninger Karl H.
Published: October 2006

Abstract: We analyzed de novo synthesis and local turnover of phospholipids in the growing neuron and the isolated nerve growth cone. The metabolism of phosphatidylinositol (PI) was studied with regard to the incorporation of saturated and unsaturated fatty acids and inositol. A comparison of de novo phospholipid synthesis in the intact neuron (whole brain, cell cultures) versus local turnover in isolated growth cone particles (GCPs) from fetal rat brain revealed different incorporation patterns and, in particular, high arachidonic acid (AA) turnover in PI of GCPs. These observations, together with elevated levels of free AA (2.5% of total AA content) in GCPs, demonstrate the predominance of acylation/deacylation in the sn‐2 position of PI. GCP phospholipase A2 (PLA2) activity was demonstrated using [3H]‐or [14C]AA‐phosphatidylcholine (PC) or ‐PI as the substrate in vitro and GCPs or a cytosolic GCP extract as the source of enzyme. In contrast to PC, which is hydrolyzed very slowly, PI is a very good GCP PLA2 substrate. PLA2 activity is much higher in GCPs than that of phospholipase C, as demonstrated by the comparison of AA and inositol turnover, by the low levels of 1,2‐diacylglycerol generated by GCPs, and by the resistance of AA release to treatment of GCPs with RHC‐80267, a specific inhibitor of diacylglycerol lipase. The predominance of PLA2 activity in GCPs raises questions regarding its regulation and the functional roles of PI metabolites, especially lysocompounds, in growth cones.
Identification of Tryptophan 2,3‐Dioxygenase RNA in Rodent Brain
Authors: Haber Roberta , Bessette Daniel , Hulihan‐Giblin Bridget , Durcan Michael J. , Goldman David
Published: October 2006

Abstract: The gene for tryptophan 2,3‐dioxygenase (TDO) heretofore was believed to be expressed only in liver. The data presented here demonstrate that RNA encoding TDO is present in rodent brain. Oligonucleotide primers based on the rat liver TDO cDNA sequence were synthesized and used to amplify RNA derived from mouse whole brain and liver and rat brain regions by the RNA‐PCR. Reaction products were purified and subjected to DNA sequencing. Identical sequences were obtained when mouse whole brain and liver RNAs were amplified, and these sequences were shown to be 96% identical to the published rat liver tryptophan TDO cDNA sequence. In addition, TDO sequences were found in RNA derived from rat brainstem, cerebellum, cortex, hypothalamus, and the remainder of the brain.
Sustained Depletion of Cortical and Hippocampal Serotonin and Norepinephrine but Not Striatal Dopamine by 1‐Methyl‐4‐(2′‐Aminophenyl)‐1,2,3,6‐Tetrahydropyridine (2′‐NH2‐MPTP): A Comparative Study with 2′‐CH3‐MPTP and MPTP
Authors: Andrews Anne M. , Murphy Dennis L.
Published: October 2006

Abstract: Unlike 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), which produces consistent decreases in levels of striatal dopamine (DA) with considerably smaller and more variable effects on mouse brain levels of serotonin (5‐HT) and norepinephrine (NE), a novel amine‐substituted MPTP analogue, 1‐methyl‐4‐(2′‐aminophenyl)‐1,2,3,6‐tetrahydropyridine (2′‐NH2‐MPTP), administered in a standard mouse dosing paradigm for MPTP (20 mg/kg X 4) did not affect striatal DA but led to marked reductions (60–70%) in levels of 5‐HT, 5‐hydroxyindoleacetic acid (5‐HIAA), and NE measured in frontal cortex and hippocampus 1 week after treatment. Another 2′‐substituted MPTP analogue, 1‐methyl‐4‐(2′‐methylphenyl)‐1,2,3,6‐tetrahydropyridine, affected cortical and hippocampal 5‐HT, 5‐HIAA, and NE only minimally, while markedly reducing the DA content in striatum (90%), thus indicating that the substituent (‐NH2 versus ‐CH3) at the 2’position is important for the differential effects of these MPTP analogues. In a replication study with a 3‐week end point, hippocampal and cortical 5‐HT, 5‐HIAA, and NE levels remained depressed with no indication of recovery. These results suggest that 2′‐NH2‐MPTP may be a novel, regionally selective neurotoxin for serotonergic and norad‐renergic nerve terminals.
The Protooncogene bcl‐2 Inhibits Apoptosis in PC12 Cells
Authors: Mah S. P. , Zhong L. T. , Liu Y. , Roghani A. , Edwards R. H. , Bredesen D. E.
Published: October 2006

Abstract: During development, many neuronal populations undergo a process of normal, programmed cell death, or apoptosis. Trophic factors regulate this process, but the mechanism by which they suppress apoptosis remains unclear. In the immune system, recent studies have implicated the protooncogene bcl‐2 in the lymphocyte survival response to growth factors. To determine whether a similar survival pathway exists in a neuroendocrine cell type, we have expressed bcl‐2 in the rat pheochromocytoma PC12 cell line and found that it abrogates the requirement for stimulation by growth factors to survive. bcl‐2 expression also substantially delays the onset of injury by the calcium ionophore A23187.
Studies on the Role of B‐50 (GAP‐43) in the Mechanism of Ca2+‐Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger
Authors: Hens Jacques J. H. , De Wit Marina , Dekker Lodewijk V. , Boomsma Frans , Oestreicher A. Beate , Margolis Frank , Gispen Willem Hendrik , De Graan Pierre N. E.
Published: October 2006

Abstract: The involvement of B‐50, protein kinase C (PKC), and PKC‐mediated B‐50 phosphorylation in the mechanism of Ca2+‐induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin‐O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP‐dependent manner as a result of an elevation of the free Ca2+ concentration from 10−8 to 10−5M Ca2+ The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca2+‐induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca2+‐induced NA release was inhibited by either N‐ or C‐terminal‐directed anti‐B‐50 antibodies, confirming a role of B‐50 in the process of exocytosis. In addition, both anti‐B‐50 antibodies inhibited PKC‐mediated B‐50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC‐mediated B‐50 phosphorylation in Ca2+‐induced NA release. PKC pseudosubstrate PKC19‐36, which inhibited B‐50 phosphorylation (IC50 value, 10−5M), failed to inhibit Ca2+‐induced NA release, even when added before the Ca2+ trigger. Similar results were obtained with PKC inhibitor H‐7, whereas polymyxin B inhibited B‐50 phosphorylation as well as Ca2+‐induced NA release. Concerning the Ca2+ sensitivity, we demonstrate that PKC‐mediated B‐50 phosphorylation is initiated at a slightly higher Ca2+ concentration than NA release. Moreover, phorbol ester‐induced PKC down‐regulation was not paralleled by a decrease in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Finally, the Ca2+‐ and phorbol ester‐induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B‐50 is involved in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Evidence is presented challenging a role of PKC‐mediated B‐50 phosphorylation in the mechanism of NA exocytosis after Ca2+ influx. An involvement of PKC or PKC‐mediated B‐50 phosphorylation before the Ca2+ trigger is not ruled out. We suggest that the degree of B‐50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca2+ influx resulting in exocytosis of NA.
Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH‐SY5Y
Authors: Vaughan Peter F. T. , Murphy Mary G. , Ball Stephen G.
Published: October 2006

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY‐270766 (an inhibitor of 5‐lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium‐evoked release of [3H]noradrenaline ([3H]NA) from the human neuroblastoma clone SH‐SY5Y with IC50 values of 10, 15, and 30 μM, respectively. In contrast, these inhibitors only partially inhibited carbachol‐evoked release and had little effect on the calcium ionophore A23187‐evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium‐ and A23187‐evoked release but did not reverse the inhibition of potassium‐evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH‐SY5Y. This conclusion is strengthened by our studies in which SH‐SY5Y cells were grown in medium supplemented with bovine serum albumin‐linoleic acid (50 μM). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium‐, veratridine‐, carbachol‐, or calcium ionophoreevoked release of [3H]NA.
Transcriptional Regulation of Choline Acetyltransferase Gene by Cyclic AMP
Authors: Misawa Hidemi , Takahashi Ryosuke , Deguchi Takeo
Published: October 2006

Abstract: The effect of cyclic AMP on the gene expression of choline acetyltransferase (ChAT) was studied in NG108‐15, mouse neuroblastoma and rat glioma hybrid cell lines. Addition of dibutyryl cyclic AMP to the culture medium increased both the ChAT mRNA level and ChAT activity twofold. Polymerase chain reaction analysis of the ChAT mRNA indicated that, among the multiple mRNA species, M‐type mRNA was transcribed most efficiently, with or without the addition of dibutyryl cyclic AMP. The 5’region of the mouse ChAT gene was ligated to the bacterial chloramphenicol acetyltransferase gene, and the expression of chloramphenicol acetyltransferase activity was determined by transfection analysis. Cyclic AMP derivatives enhanced the reporter gene expression in both transiently and stably transfected cells. DNA deletion analysis indicated that the intron region downstream of the M‐type exon is necessary for the cyclic AMP responsiveness, and that cyclic AMP derivatives increase ChAT gene transcription mainly from M‐type promoter. These results suggest that a cis‐acting DNA element that confers the cyclic AMP responsiveness of the ChAT gene is present in the intron downstream of the M‐type exon.
Triiodothyronine Is a High‐Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes
Authors: Blondeau Jean‐Paul , Beslin Aline , Chantoux Françoise , Francon Jacques
Published: October 2006

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3′,5‐triiodo‐L‐thyronine (T3) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na+‐independent, and efficiently inhibited by T3 (half‐inhibition at ∼ 2 μM). Two Na+‐independent L‐like systems (L1 and L2), common to leucine and aromatic amino acids, were characterized kinetically. System L2 had a low affinity for leucine and tryptophan (Km= 0.3–0.9 mM). The high‐affinity system L1 (Km∼ 10 μM for both amino acids) was competitively inhibited by T3 with a Ki of 2–3 μM (close to the T3 transport Km). Several T3 analogues inhibited system L1 and the T3 transport system similarly. Glutamine uptake and α‐(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T3 had little effect on the uptakes of glutamine and α‐(methylamino)isobutyric acid. The results indicate that the T3 transport system and system L1 are related.
Cocaine and Cocaethylene: Microdialysis Comparison of Brain Drug Levels and Effects on Dopamine and Serotonin
Authors: Bradberry Charles W. , Nobiletti J. B. , Elsworth J. D. , Murphy B. , Jatlow P. , Roth R. H.
Published: October 2006

Abstract: Cocaethylene is a pharmacologically active metabolite resulting from concurrent cocaine and ethanol consumption. The effects of cocaine and cocaethylene on extracellular levels of dopamine in the nucleus accumbens, and serotonin in the striatum were characterized in vivo in the anesthetized rat. Both intravenous (3 μmol/kg) and intraperitoneal (44 μmol/kg) routes of administration were used. In addition to monitoring neurotransmitter levels, microdialysate levels of cocaine and cocaethylene were determined at 4‐min intervals after intravenous administration, and at 20‐min intervals after intraperitoneal administration. Extracellular levels of dopamine in the nucleus accumbens were increased to ∼400% of preinjection value by both cocaine and cocaethylene when administered intravenously. Cocaine caused a significant increase of striatal serotonin to 200% preinjection value, whereas cocaethylene had no effect. Brain levels of cocaine and cocaethylene after intravenous administration did not differ. After intraperitoneal administration, extracellular levels of dopamine in the nucleus accumbens were increased to 400% of preinjection levels by cocaine, but were only increased to 200% of preinjection levels by cocaethylene, the difference being statistically significant. Serotonin levels were increased to 360% of preinjection levels by cocaine, but only to 175% of preinjection value by cocaethylene. Levels of cocaine attained in brain were significantly higher than those for cocaethylene, suggesting pharmacokinetic differences with the intraperitoneal route. These results confirm in vivo that cocaethylene is more selective in its actions than cocaine with respect to dopamine and serotonin uptake. In addition, route‐dependent differences in attainment of brain drug levels have been observed that may impact on interpretations of the relative potency of the reinforcement value of these compounds.
Transcription‐Independent Activation of Ornithine Decarboxylase Activity by Heparin in Cloned Cerebral Endothelial Cells
Authors: Bauer H. , Amberger A. , Bauer H. C. , Webersinke G. , Zach O. , Olson J. W. , Hennig B.
Published: October 2006

Abstract: Heparin, a highly sulfated glycosaminoglycan, is known to be obligatory for long‐term endothelial cell cultures; it potentiates the mitogenic activities of endothelial cell growth factors and prolongs the replicative life span of the cells. Here we have shown that besides its growth factor‐supportive role, heparin exerts a specific action on cerebral capillary endothelial cells (cECs), unrelated to serum or growth factors, by increasing activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in these cells. For our experiments we have used two different types of cloned cECs: type I cECs, grown in the presence of endothelial cell growth factor and heparin, and type II cECs, usually cultivated without growth factors. Heparin action on ODC activity was shown to be dose dependent within the range of 1–100 μg/ml. Increasing concentrations of or depletion of endothelial cell growth factor from type I cultures had no effect on ODC activity. The increase in enzyme activity was highest after 30 min to 1 h of heparin treatment. As evidenced by northern analysis, the heparin‐mediated enhancement of ODC activity was not accompanied by changes of ODC mRNA levels. Studies of DNA replication revealed that in the absence of heparin‐binding growth factors, heparin did not affect the proliferative activity of cloned cECs.
The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells
Authors: Mutoh Tatsuro , Tokuda Akira , Guroff Gordon , Fujiki Norio
Published: October 2006

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF‐induced neurite outgrowth from PC12 cells, NGF‐induced activation of ribosomal protein S6 kinase, and NGF‐induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [3H]‐thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca2+ in the medium. 125I‐NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.
Cholesterol Synthesis in Regenerating Peripheral Nerve Is Not Influenced by Serum Cholesterol Levels
Authors: Goodrum Jeffry F.
Published: October 2006

Abstract: Following a nerve crush, cholesterol from degenerating myelin is retained within the nerve and reutilized for new myelin synthesis during nerve regeneration, apparently via a lipoprotein‐mediated process. Because at least some serum components have access to the endoneurium of injured nerve, it has been suggested that serum lipoproteins are also significant contributors of cholesterol to Schwann cells during nerve regeneration. To test this hypothesis, serum cholesterol levels were reduced by >90% with 4‐aminopyrazolopyrimidine, followed by measurement of the activity of the key regulatory enzyme in cholesterol synthesis, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase. Treatment with 4‐aminopyrazolopyrimidine caused a sevenfold increase in 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity in kidney but had no effect on the activity of this enzyme in either intact or regenerating sciatic nerve. These data indicate that serum‐derived cholesterol is neither necessary for nor contributes significantly to myelin synthesis in regenerating nerve.
Increased Acetylcholine Content Induced by Adenosine in a Sympathetic Ganglion and Its Subsequent Mobilization by Electrical Stimulation
Authors: Tandon A. , Collier B.
Published: October 2006

Abstract: The present study was initiated to examine the effects of ATP on acetylcholine (ACh) synthesis. The exposure of superior cervical ganglia to ATP increased ACh stores by 25%, but this effect was also evident with ADP, AMP, and adenosine, but not with βγ‐methylene ATP, a nonhydrolyzable analogue of ATP, or with inosine, the deaminated product of adenosine. Thus, we attribute the enhanced ACh content caused by ATP to the presence of adenosine derived from its hydrolysis by 5′‐nucleotidase. The adenosine‐induced increase of tissue ACh was not the consequence of an adenosine‐induced decrease of ACh release. The extra ACh remained in the tissue for more than 15 min after the removal of adenosine, but it was not apparent when ganglia were exposed to adenosine in a Ca2+‐free medium. Incorporation of radiolabelled choline into [3H]ACh was also enhanced in the presence of adenosine, suggesting an extracellular source of precursor. Moreover, the synthesis of radiolabelled forms of phosphorylcholine and phospholipid was not reduced in adenosine's presence, suggesting that the extra ACh was not likely derived from choline destined for phospholipid synthesis. Aminophylline did not prevent the adenosine effect to increase ACh content; this effect was blocked by dipyridamole, but not by nitrobenzylthioinosine (NBTI). In addition, two benzodiazepine stereoisomers known to inhibit stereoselectively the NBTI‐resistant nucleoside transporter displayed a similar stereoselective ability to block the effect of adenosine. Together, these results argue that adenosine is transported through an NBTI‐resistant nucleoside transporter to exert an effect on ACh synthesis. The extra ACh accumulated as a result of adenosine's action was releasable during subsequent preganglionic nerve stimulation, but not in the presence of vesamicol, a vesicular ACh transporter inhibitor. We conclude that the mobilization of ACh is enhanced as a result of adenosine pretreatment.
Acrylamide and Glycidamide Impair Neurite Outgrowth in Differentiating N1E.115 Neuroblastoma Without Disturbing Rapid Bidirectional Transport of Organelles Observed by Video Microscopy
Authors: Brat Daniel J. , Brimijoin Stephen
Published: October 2006

Abstract: The nature of the pathogenic insult in acrylamide neuropathy is unknown, but axonal transport disturbances are suspected. Using N1E.115 neuroblastoma in vitro, we examined acrylamide and related compounds in terms of general cytotoxicity, ability to block neurite outgrowth, and effects on neurite integrity and fast axonal transport. Acrylamide, glycidamide, and methylene‐bisacrylamide were weakly cytotoxic in a 51Cr‐release assay, but only at ≥10 mM (order of efficacy: methylene‐bis‐acrylamide > glycidamide > acrylamide). Neurite outgrowth by differentiating cells was inhibited at 100‐fold lower concentrations, with similar EC50 values for all three toxicants, i.e., acrylamide, 70 ± 15 μM; methylene‐bis‐acrylamide, 92 ± 31 μM; glycidamide, 120 ± 30 μM. Only glycidamide (1 mM) caused degeneration of established neurites within a period of 48 h. Video‐enhanced contrast differential interference contrast microscopy was used to test the effect of acrylamide and glycidamide on organelle transport in the neurites. In exposures of ≤48 h at 1 mM, neither toxicant altered bidirectional organelle flux, measured as organelles transported per minute per micrometer of neurite diameter. Anterograde and retrograde organelle speeds were also undisturbed. These results suggest that mechanisms other than direct inhibition of organellar motility are responsible for acrylamide's neurotoxicity in vivo.