bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture
Authors: Hara Masayuki , Matsuda Yoshihiro , Hirai Keisuke , Okumura Nobuaki , Nakagawa Hachiro
Published: October 2006

Abstract: Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2‐deoxy‐d‐[3H]glucose ([3H]2‐DG) into the cells. Various sugars inhibited 2‐DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Kmvalues for 2‐DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2‐DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2‐DG uptake competitively in both neuronal cells and astrocytes. The uptakes by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis after photoamnity labeling using [3H]cytochalasin B as a probe.
Effect of a Single Dose of β,β′‐Iminodipropionitrile In Vivo on the Properties of Neurofilaments In Vitro: Comparison with the Effect of Iminodipropionitrile Added Directly to Neurofilaments In Vitro
Authors: Eyer Joël , Mclean W. Graham , Leterrier Jean‐Frangois
Published: October 2006

Abstract: The biochemical properties of neurofilaments isolated from control and iminodipropionitrile‐treated rats were compared with regard to autophosphorylation capacity, hydrolysis of ATP, and the formation of a viscous gel between filaments. Both preparations exhibited a similar polypeptide composition, and no covalent cross‐Unking between neuro‐filament subunits was induced by iminodipropionitrile in vivo. An ATPase activity, systematically present in all preparations, was unaffected by the administration of iminodipropionitrile to the rats. Conversely, the autophosphorylation of neurofilament subunits in vitro was significantly higher in preparations from iminodipropionitrile‐treated rats than from control animals, with a marked increase of the phosphorylation of a high molecular weight neurofilament‐associated protein. Iminodipropionitrile provoked a higher gelation capacity of neurofilaments as measured in vitro, with a lower critical concentration for the preparation from treated animals. A similar increased interaction was obtained with millimolar concentrations of iminodipropionitrile added to bovine neurofilaments in vitro, involving likely neurofilamentassociated molecules, because the effect of the drug was lost after their extraction by 0.8 M KCl. These results support the hypothesis that iminodipropionitrile interferes with the neurofilament networks through a preferential interaction with the neurofilament‐associated proteins, resulting in a change in their properties and consequently in an increased capacity of interaction between the polymers.
Formation of Thiazolidine‐4‐Carboxylic Acid Represents a Main Metabolic Pathway of 5‐Hydroxytryptamine in Rat Brain
Authors: Susilo Rudy , Rommelspacher Hans , Höfle Gerhard
Published: October 2006

Abstract: Incubation of 5‐hydroxytryptamine (5‐HT) with rat brain homogenate resulted in the formation of (4R)‐2‐[3′‐(5′‐hydroxyindolyl)‐methyl]‐1,3‐thiazolidine‐4‐carboxylic acid (5′‐HITCA) as the major metabolite. The substance represents the condensation product of 5‐hydroxyindole‐3‐acetaldehyde with L‐cysteine. The chemical structure was confirmed by chromatographic and chemical methods as well as by fast atom bombardment mass spectrometry. Incubation of 5‐HT in the presence of L‐cysteine yielded the thiazolidine as the main metabolite up to 4 h. Under these conditions, the concentration of 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) amounted to about 20% and 57% of 5′‐HITCA (0.5 h and 4 h, respectively). In contrast to these findings, indole‐3‐acetic acid (IAA) was identified as the major metabolite when tryptamine was incubated under similar conditions. (4R)‐2‐(3′‐Indolylmethyl)‐1,3‐thiazolidine‐4‐carboxylic acid (ITCA) was found to be the main conversion product of tryptamine only during the first 30 min. To investigate the fate of the thiazolidines, radiolabelled and unlabelled ITCA was incubated with rat brain homogenate. The compound was degraded enzymatically and rapidly. Subcellular fractionation revealed that the enzyme activity was present mainly in the cytosolic fraction whereas the preparation of mitochondria showed less activity. The responsible enzyme is presumably a carbonsulfur lyase (EC 4.4.1.‐). The major metabolite was isolated by HPLC and identified by mass spectrometry as well as by comparison with reference compounds to be IAA. The present results suggest the involvement of L‐cysteine in the metabolism of biogenic aldehydes by forming thiazolidine‐4‐carboxylic acids. The biological rationale of this pathway might be a control mechanism of the chemically active aldehydes by the rapid inactivation of the compounds.
Determination of the Deoxyglucose and Glucose Phosphorylation Ratio and the Lumped Constant in Rat Brain and a Transplantable Rat Glioma
Authors: Kapoor Rajesh , Spence Alexander M. , Muzi Mark , Graham Michael M. , Abbott Gregory L. , Krohn Kenneth A.
Published: October 2006

Abstract: Mitochondrially bound hexokinase (ATP‐D‐hexose‐6‐phosphotransferase; EC 2.7.1.1) was dissociatively extracted from normal rat brains and intracerebral and subcutaneous implants of the 36B‐10 glioma. At least 70% of the total hexokinase enzyme activity in normal and glioma tissue was associated with the mitochondrial fraction. Purification of the crude tissue extracts by ion‐exchange and affinity chromatography followed by analysis with sodium dodecyl sul‐fate‐polyacrylamide gel electrophoresis revealed a successive purification of the enzyme to homogeneity with a molecular size of 98 kilodaltons. Enzyme kinetics with glucose or 2‐deoxyglucose (2‐DG) as the substrate were measured spec‐trophotometrically by coupling the appropriate reactions to either NADPH or NAD+ formation. The Km of hexokinase with glucose as the substrate in the intracerebral glioma (0.138 m.M) and subcutaneous glioma (0.183 mM) tissues was 2.1 ‐2.7‐fold higher than that observed in normal brain tissue (0.067 mM) (p < 0.001). No significant differences were observed in the Km for hexokinase with 2‐DG as the substrate in the glioma and normal brain tissue. The phosphorylation ratio for normal brain was 0.320 and was increased in the intracerebral glioma to 0.694 and in the subcutaneous glioma to 0.519. The ratios of deoxyglucose and glucose volumes of distribution in normal brain and intracerebral glioma tissues were 1.70 and 1.85, respectively. The lumped constants calculated directly from the phosphorylation ratios and the volumes of distribution of deoxyglucose and glucose were 0.517 in normal brain and 1.168 in intracerebral glioma. Our results indicate the lumped constant is increased 2.26‐fold in intracerebral glioma compared with normal brain.
Timing of Expression of r and Its Encoding mRNAs in the Developing Cerebral Neocortex and Cerebellum of the Mouse
Authors: Mangin G. , Couchie D. , Charrière‐Bertrand C. , Nunez J.
Published: October 2006

Abstract: The expression of τ mRNA and of the corresponding encoded protein variants was studied during postnatal development in two brain regions differing in their timing of differentiation: the cerebral neocortex and the cerebellum, (a) The expression of r mRNA was different in the two regions. Maximal contents were found at early stages in the cerebral neocortex, with a 10‐fold decrease at later stages. In the cerebellum, two peaks of τ mRNA were observed soon after birth and in adulthood, with minimal values at postnatal day 6. (b) The expression of total τ proteins was similar to that of their encoding mRNAs in the cerebral neocortex, i.e., high concentrations after birth and low contents at later stages. In contrast, two peaks of τ proteins were observed in the cerebellum: the first perinatally and the second with a maximum at postnatal day 15. (c) Both in the cerebral neocortex md especially in the cerebellum, increasing concentrationsof mature τ variants were expressed at late developmental stages, i.e., when total τ protein contents were decreased. In conclusion, the fluctuations in expression of τ and of its encoding mRNA seen in the cerebellum seem to reflect differences in the timing of differentiation of the various cell types, i.e., the macroneurons and the interneurons, present in this brain region. The adult τ variants appear in both the neocortex and the cerebellum only at late developmental stages, i.e., when most of the circuitry has been established, although these two regions markedly differ in their timing of differentiation.
Glutamate Release from Guinea‐Pig Synaptosomes: Stimulation by Reuptake‐Induced Depolarization
Authors: McMahon Harvey T. , Barrie Anne P. , Lowe Martin , Nicholls David G.
Published: October 2006

Abstract: Glutamate (10–100 μM) reversibly depolarizes guinea‐pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 μMn‐methyl‐D‐aspartate (NMDA), 100 μM L‐2‐amino‐4‐phosphonobutanoate (APB), nor 100 μM quisqualate affects the Ca2+‐dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists β‐N‐ox‐alylamino‐L‐alanine and α‐amino‐3‐hydroxy‐5‐methylisox‐azole propionate cause a slow Ca2+‐independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor‐mediated neurotoxin β‐N‐methylamino‐L‐alanine do not influence Ca2+‐independent release at 100 μM. The depolarization of the plasma membrane by glutamate can be mimicked by D‐aspartate, can be blocked by the transport inhibitor dihydro‐kainate, and is accompanied by the net uptake of acidic amino acids. L‐Glutamate or D‐aspartate at 100 μM increases the cytoplasmic free Ca2+ concentration. D‐aspartate at 100 μM causes a Ca2+‐dependent release of endogenous glutamate, superimposed on the Ca2+‐independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.
Transport of Muscarinic Cholinergic Marker Protein Activities in Regenerating Sciatic Nerve of Rat
Authors: Gulya K. , Simonka J. A. , Kása P.
Published: October 2006

Abstract: The transport characteristics of choline acetyl‐transferase (ChAT; EC 2.3.1.6), acetylcholinesterase (AChE; EC 3.1.1.7), and the muscarinic acetylcholine receptors (mAChR) were studied in perineurally sutured, regenerating rat sciatic nerve. At different times after repair, the sciatic nerve was ligated for 24 h, and the activities of the cholinergic marker proteins, as well as the binding capacity, were measured proximally and distally from the ligature. The number of bidirectionally transported receptors increased linearly up to 5 months postoperatively (6.1–33.6% and 5.6–25.6% of the control level proximal and distal to the ligature, respectively). The quantity of anterogradely transported ChAT reached a plateau 3 months postoperatively (74.9% of the control level), whereas the retrogradely transported enzyme was then only 34.7% of the control value. The activity of AChE increased linearly during nerve regeneration, and exceeded the control level after 4 months (121.0% and 63.7% proximally and distally, respectively). The data indicate that the altered bidirectional transport of cholinergic marker proteins may be monitored quantitatively during nerve regeneration. This method might be suitable for studies of the nerve regeneration process.
Effects of Oxygen Depletion on Norepinephrine‐ and Carbachol‐Stimulated Phosphoinositide Turnover in Rat Brain Slices
Authors: Ninomiya Haruaki , Taniguchi Takashi , Fujiwara Motohatsu , Kameyama Masakuni
Published: October 2006

Abstract: We examined the effects of in vitro anoxia and in vivo hypoxia (8% 02/92% N2) on norepinephrine (NE)‐ and carbachol‐stimulated phosphoinositide (PI) turnover in rat brain slices. The formation of 3H‐labeled polyPI in cortical slices was impaired by in vitro anoxia and fully restored by reoxygenation. Accumulation of 3H‐labeled myo‐inositol phosphates (3H‐IPs) stimulated by 10−5M NE was significantly reduced by anoxia (control at 60 min, 1,217 ± 86 cpm/mg of protein; anoxia for 60 min, 651 ±82 cpm/mg; mean ± SEM; n =5;p > 0.01), and reoxygenation following anoxia resulted in overshooting of the accumulation (control at 120 min, 1,302 ± 70 cpm/mg; anoxia for 50 min plus oxygenation for 70 min, 1,790 ± 126 cpm/mg; n = 5; p > 0.01). The underlying mechanisms for the two phenomena—the decrease caused by anoxia and the overshooting caused by reoxygenation following anoxia—seemed to be completely different because of the following observations, (a) Although the suppression of NE‐stimulated accumulation at low 02 tensions was also observed in Ca2+‐free medium, the overshooting in response to reoxygenation was not. (b) Carbachol‐stimulated accumulation was significantly reduced by anoxia and was restored by reoxygenation only to control levels. Thus, the postanoxic overshooting in accumulation of 3H‐IPs seems to be a specific response to NE. (c) The decrease observed at low 02 tensions was due to a decrease in Emax value, whereas the postanoxic overshooting was due to a decrease in EC50 value. There was also a significant increase in NE‐stimulated accumulation of 3H‐IPs in cortical slices from rats exposed to in vivo hypoxia (8% 02/92% N2) for <6 h, compared with those from rats exposed to room air. These results indicate that depletion of oxygen causes significant changes in receptor‐mediated PI metabolism in brain slices and may provide a clue for understanding biochemical mechanisms for the electrophysiologically demonstrable effects of hypoxia/anoxia on synaptic transmission.
Muscarinic Agonists Cause Calcium Influx and Calcium Mobilization in Forebrain Neurons In Vitro
Authors: Reynolds Ian J. , Miller Richard J.
Published: October 2006

Abstract: We have examined the effects of the muscarinic agonist carbachol on the intracellular free Ca2+ concentration ([Ca2+]i) in primary cultures of neurons from rat forebrain using the Ca2+‐sensitive fluorescent dye fura‐2. Addition of carbachol increased the [Ca2+]i in ∼60% of cells studied. Oxotremorine‐M, but not pilocarpine, mimicked the effects of carbachol. The response was reduced by 60% on removal of extracellular Ca2+, a finding suggesting that muscarinic receptor activation causes Ca2+ influx in addition to intracellular Ca2+ mobilization. Tetrodotoxin and nitrendipine also significantly reduced the response to carbachol. These studies suggest that the changes in [Ca2+]i produced by activation of muscarinic receptors result in part from mobilization of intracellular Ca2+ and that influx through voltage‐sensitive Ca2+ channels also provides a significant contribution to the net [Ca2+]i change observed
Brain Microvessels Produce 12‐Hydroxyeicosatetraenoic Acid
Authors: Moore Steven A. , Figard Paul H. , Spector Arthur A. , Hart Michael N.
Published: October 2006

Abstract: Cerebral microvessels isolated from perfused, adult murine brain produce a compound with the chromatographic properties of a monohydroxyeicosatetraenoic acid when incubated with arachidonic acid or stimulated with calcium ionophore A23187. The formation of this arachidonic acid metabolite is not reduced in the presence of the cyclooxy‐genase inhibitor ibuprofen, but it is abolished by the lipoxygenase inhibitor nordihydroguaiaretic acid. Analysis by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonre‐duced derivatives of the metabolite indicate that the compound is 12‐hydroxyeicosatetraenoic acid. Fractions of isolated microvessels enriched with capillaries produce 2.1 times more 12‐hydroxyeicosatetraenoic acid per microgram of protein than do fractions of microvessels enriched with arterioles. These studies confirm that brain microvessels can produce 12‐hydroxyeicosatetraenoic acid and strongly suggest that cerebral endothelia are the primary source of microvessel‐derived 12‐hydroxyeicosatetraenoic acid. They further suggest that in brain injury, the liberation and accumulation of arachidonic acid in cerebral tissues may lead to the production of 12‐hydroxyeicosatetraenoic acid within microvessels. The 12‐hydroxyeicosatetraenoic acid formed in this way may mediate some of the blood‐brain barrier and cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.
Purification and Characterization of a Central Cholinergic Enhancing Factor from Rat Brain: Its Identity as Phosphoethanolamine
Authors: Bostwick J. Robert , Landers Diane W. , Crawford Garrett , Lau Kenneth , Appel Stanley H.
Published: October 2006

Abstract: A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two‐ to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5′‐di‐phosphoethanolamine at concentrations above 100 μM. This effect appears to result from an increase in the accumulation of choline via the high‐affinity, sodium‐dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium‐dependent high‐affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.
Effects of Lithium on Inositol Phospholipid Hydrolysis and Inhibition of Dopamine D1 Receptor‐Mediated Cyclic AMP Formation by Carbachol in Rat Brain Slices
Authors: Whitworth Paul , Kendall David A.
Published: October 2006

Abstract: The in vitro and ex vivo effects of lithium on muscarinic cholinergic inositol phospholipid hydrolysis and muscarinic cholinergic inhibition of dopamine D1‐receptor‐stimulated cyclic AMP formation were examined in rat brain slices. Following chronic lithium feeding, carbachol‐stimu‐lated inositol phosphate accumulation was reduced ex vivo in slices of cerebral cortex but not in striatal slices. Lithium (1 mM) in vitro had no direct effect on dopamine D1‐receptor‐stimulated cyclic AMP formation, but enhanced the inhibitory effect of carbachol on the D1 response, in striatal slices, and this was not significantly altered by prior lithium feeding. Lithium therefore has effects on two discrete muscarinic responses in rat brain which are apparently maintained after chronic exposure to the ion and might be relevant to its an‐timanic actions.
No Role for Phospholipase A2 and Protein Kinase C in the Potentiation by α‐Adrenoceptors of β‐Adrenoceptor‐Mediated Cyclic AMP Formation in Rat Brain
Authors: Robinson Jean P. , Kendall David A.
Published: October 2006

Abstract: This study was undertaken to examine the role of phospholipase A2 and protein kinase C in the potentiation of β‐adrenoceptor‐mediated cyclic AMP formation by α‐ad‐renoceptors in rat cerebral cortical slices. Inhibition of ara‐chidonic acid metabolism by a range of cyclooxygenase and lipoxygenase inhibitors had no effect on the potentiation of isoprenaline‐stimulated cyclic AMP. Conversely, stimulation of leukotriene formation had no effect on the response to isoprenaline. The phospholipase A2 activator, melittin, stimulated cyclic AMP and potentiated the effect of isoprenaline, but these responses were not influenced by cyclooxygenase or lipoxygenase inhibitors. Indomethacin was also ineffective against the potentiation of vasoactive intestinal peptide‐stimulated cyclic AMP by noradrenaline. Phorbol ester potentiated the cyclic AMP response to isoprenaline, and this potentiation was antagonized by three different putative protein kinase C inhibitors. However, the same inhibitors did not affect the α‐adrenoceptor‐stimulated enhancement of the response to isoprenaline. We have found no evidence, therefore, to support the suggestion that arachidonic acid and its metabolites and/or protein kinase C mediate the α‐adreno‐ceptor modulation of β‐adrenoceptor function.
Ionic Composition of Microdialysis Perfusing Solution Alters the Pharmacological Responsiveness and Basal Outflow of Striatal Dopamine
Authors: Moghaddam Bita , Bunney Benjamin S.
Published: October 2006

Abstract: While using the technique of in vivo microdialysis, we have assessed the effect of the ionic composition of the perfusing solution on extracellular dopamine levels during resting conditions and following a pharmacological manipulation. Our results indicate that perfusion with solutions containing the ionic composition of commercially available Ringer's solution, which mimic the ionic composition of plasma as opposed to brain extracellular fluid, alters the turnover rate and basal release of dopamine. Moreover, perfusion with solutions containing higher calcium levels, i.e., 3.4 mM, than the amount we have determined to be present in the extracellular fluid of striatum (1.2 mM) alters the pharmacological responsiveness of the nigrostriatal dopamine system to synthesis inhibition.
Glutamate‐Induced Increase in Intracellular Ca2+ in Cerebral Cortex Neurons Is Transient in Immature Cells but Permanent in Mature Cells
Authors: Wahl Philip , Schousboe Arne , Honoré Tage , Drejer Jorgen
Published: October 2006

Abstract: The free cytosolic Ca2+ concentration ([Ca2+],) of cultured cerebral cortex neurons was determined using a fluorescent Ca2+chelator (Fluo‐3) after exposure of the neurons to glutamate. Mature neurons (8 days in culture) responded within 45 s to 100 μM glutamate by an increase in [Ca2+]j from 75 to 340 nM, an increase that during the following 6 min of exposure reached 400 nM. This increase in [Ca2+]j could not be reversed by removal of glutamate. In the absence of extracellular CaCl2, only part of the initial, rapid, glutamate‐induced increase in [Ca2+]j was observed in these neurons. In contrast to these findings, neurons cultured for only 2 days (immature neurons) exhibited only a small (from 75 to 173 nM) increase in [Ca2+]j after exposure to 100 μM glutamate, and this rapid increase in [Ca2+]j tended to decline on prolonged exposure to glutamate. Moreover, after removal of glutamate, the increase in [Ca2+]j was fully reversible. Pharmacological characterization of the response to glutamate in mature neurons showed that the yN‐methyl‐D‐aspartate (NMDA) receptor antagonists phencyclidine and D‐2‐amino‐5‐phosphonovalerate blocked 75 and 90%, respectively, of the response, whereas the non‐NMDA antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione had little effect.
Late‐Phase Accumulation of Inositol Phosphates Stimulated by Prostaglandins D2 and F2α in Neuroblastoma × Glioma Hybrid NG108‐15 Cells
Authors: Hatanaka Michiyo , Yumoto Noboru , Miwa Naoto , Morii Hiroshi , Tanemura Mihoko , Ueno Ryuji , Watanabe Yasuyoshi , Hayaishi Osamu
Published: October 2006

Abstract: The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108‐15 cells preincubated with myo‐[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15‐60 min) of incubation in the cells. PGD2 and PGF2adid not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP1) formation at a late phase of incubation. The maximum stimulation was obtained at <10−7M concentrations of these PGs. the levels being three‐and twofold for IP2 and IP1 respectively. 9α, 11 β‐PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2α have no effect up to 10−6M. The effects of PGD2 and PGF2α were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation.

Inositol 1‐monophosphate was mainly identified in the stimulation by 10−5 M PGD2 and 10−5MPGF2a, whereas both inositol 1‐monophosphate and inositol 4‐monophosphate were produced in the stimulation by 10−5M bradykinin. Depletion of extracellular Ca2+ diminished the stimulatory effect of PGD2 and PGF2α and late‐phase effect of bradykinin, but simple Ca2+ influx into the cells by high K+, ionomycin, or A23187 failed to cause such late‐phase effects. These results suggest that PGD2 and PGF2α specifically stimulate hydrolysis of inositol phospholipids.
Sialidase in Cerebellar Granule Cells Differentiating in Culture
Authors: Pitto Marina , Chigorno Vanna , Giglioni Antonella , Valsecchi Manuela , Tettamanti Guido
Published: October 2006

Abstract: The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using f3H]GDla and 2′‐(4‐methylumbelliferyl)‐α‐D‐N‐acetyl‐neuraminic acid (MUB‐NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3HJGDla and 0.1 M for MUB‐NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB‐NeuNAc and 0.1 mM for [3H]GDla; enzyme activity linear with time up to 30 min with MUB‐NeuNAc and up to 90 min with f3HJGDla; and enzyme activity linear with enzyme protein content up to 80 μg with MUB‐NeuNAc and up to 20 μg with f3H]GDla. The assay with [3H]GDla required the presence of Triton X‐100 in a molar ratio to GDla of 15:1. Poly‐L‐lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GDla/ Triton X‐100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB‐NeuNAc. Using no more than 20 μg of cellular protein, the contamination, if any, by poly‐L‐lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7–8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB‐NeuNAc and from 1 to 100 nmol of product released/ h/mg of protein with [3H]GDla. The values of enzyme activity in differentiated granule cells are the highest ever reported for mammalian sialidases in isolated cells or tissue homogenates. In fully differentiated cells, the sialidase activity against endogenous substrates was 4.2 nmol of liberated N‐acetylneuraminic acid/h/mg of protein. The marked increase of sialidase activity in cerebellar granule cells during the process of differentiation with formation of functional synapses suggests that sialidase enrichment is a marker for the same process.
A Comparison of the Distribution of Neurotransmitters in Brain Regions of the Rat and Guinea‐Pig Using a Chemiluminescent Method and HPLC with Electrochemical Detection
Authors: Wetherell Janet R. , Fosbraey Paul , French Mary C.
Published: October 2006

Abstract: Six brain areas of rats and guinea‐pigs, killed by microwave irradiation, were used for the concomitant measurement of the levels and regional distribution of cholinergic, biogenic amine, and amino acid neurotransmitters and metabolites. Acetylcholine (ACh) and choline (Ch) were quantified by chemiluminescence; noradrenaline (NA), dopamine (DA), 5‐hydroxytryptamine (5‐HT), and their metabolites by HPLC with electrochemical detection (HPLC‐EC); and six putative amino acid neurotransmitters by HPLC‐EC following derivatisation. The levels and regional distribution of these transmitters and their metabolites in the rat were similar to those reported in previous studies, except that biogenic amine transmitter levels were higher and metabolite concentrations were lower. The guinea‐pig showed a similar regional distribution, but the absolute levels of ACh were lower in striatum and higher in hippocampus, midbrain‐hypothalamus, and medulla‐pons. In all areas, the levels of Ch were higher and those of NA, 5‐HT, and taurine were lower than in the rat. The most marked differences between the rat and guinea‐pig were in the relative proportion of DA metabolites and 5‐HT turnover, as estimated by metabolite/transmitter ratios. This study can be used as a basis for a comprehensive understanding of the central effects of drugs on the major neurotransmitter systems.
Substance P‐Induced Reduction in the Initial Accumulation of Cytosolic myo‐[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor
Authors: Dietl M. M. , Torrens Y. , Beaujouan J.‐C. , Glowinski J.
Published: October 2006

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo‐[3H]inositol. This effect was rapid, because a reduction of ∼15% could be seen already at 30 s, with the maximal effect (∼45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo‐[3H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP‐induced reduct on in cytosolic ravo[3H]inositol accumulation was concentration dependent; the EC50 obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg1, D‐Trp79, Leu]SP), a SP antagonist, used at a concentration oif 105 A/, gave a competitive shift of the dose‐response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo‐[3H]inositol. [L‐Pro9]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo‐H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C‐terminal fragments were more potent than shorter ones. SP N‐terminal fragments and SP free acid were ‐without effect. [Pro7]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC50 values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw‐[3H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP‐induced reduction in cytosolic myo‐[3H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo‐[3H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo‐[3H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.
Regulation of the γ‐Aminobutyric AcidA Receptor by γ‐Aminobutyric Acid Levels Within the Postsynaptic Cell
Authors: Wood J. D. , Davies M.
Published: October 2006

Abstract: Synaptosomes and synaptoneurosomes were prepared from rat cerebral cortex. Comparison of the amino acid levels in the two types of organelles and of the effects of gabaculine thereon indicated that the neurosome portion of synaptoneurosomes constituted the major influencing component of the organelles. Administration to rats of inhibitors of γ‐aminobutyric acid (GABA) degradation, such as gabaculine and L‐cycloserine, resulted in elevated GABA levels in synaptoneurosomes and a decrease in muscimol‐stimulated C1 uptake by the organelles. Addition of gabaculine directly to the incubation medium for the uptake assay had no effect on the C1 transport. In contrast, administration to rats of isonicotinic acid hydrazide, an inhibitor of GABA synthesis, decreased the GABA level in synaptoneurosomes and increased the muscimol‐stimulated Cl− uptake by the organelles. Although the evidence is not unequivocal, it does support the concept of GABA released from nerve endings being taken up by the postsynaptic cell, from where it exerts a regulatory influence on the functioning of the GABA receptor/ion channel complex.
Elevation by Atrial Natriuretic Factors of Cyclic GMP Levels in Astroglia‐Rich Cultures from Murine Brain
Authors: Friedl Arno , Harmening Christine , Schmalz Felicitas , Schuricht Barbara , Schiller Max , Hamprecht Bernd
Published: October 2006

Abstract: Atrial natriuretic factors, peptide hormones originally found in the heart, slowly but strongly elevate the level of cyclic GMP in primary astrocyte‐rich cultures derived from brains of newborn rats or mice but not in neuron‐rich cultures prepared from embryonic rat brain. In the absence of a phosphodiesterase inhibitor, a plateau level of cyclic GMP is obtained within 10 min. In the presence of the inhibitor 3‐isobutyl‐1‐methylxanthine, the concentration of cyclic GMP continues to rise, even after 30 min. The elevation of the level of cyclic GMP in response to atrial natriuretic factor is much more pronounced in the rat cultures than the mouse cultures. Even at peptide concentrations of 1 μM, plateaus of the concentration–response curves are not yet reached. The potencies of the active peptides vary over a range of ∼1.5 orders of magnitude, with atriopeptins II and III and auriculin A being the most potent ones. These results suggest (a) that atrial natriuretic factors may regulate functions of glial cells, most likely of astrocytes, in brain and (b) that such cultures may be useful tools in defining such astroglial functions.
Choline Acetyltransferase Activities in Single Spinal Motor Neurons from Patients with Amyotrophic Lateral Sclerosis
Authors: Kato Takahiko
Published: October 2006

Abstract: Activities of choline acetyltransferase (ChAT) were microassayed in individual cell bodies of motor neurons, isolated from freeze‐dried sections after autopsy of lumbar spinal cords from four patients with sporadic amyotrophic lateral sclerosis (ALS) and four control patients with nonneurological diseases. Numerous large neurons were found in the anterior horn at the early degeneration stage of ALS, but the cell bodies atrophied and decreased in number at the late advanced stage. The small, atrophied neurons were very fragile and were easily destroyed during the isolation procedure with a microknife. The average activity, expressed on a dry weight basis, of 58 ALS neurons was lower than that of 67 control neurons. The large, well‐preserved neurons at the early nonadvanced stage had markedly lower ChAT activities than control neurons. The specific activity gradually increased with the progress of atrophy but did not return to the control level.
Pertussis Toxin Attenuates 5‐Hydroxytryptamine1A Receptor‐Mediated Inhibition of Forskolin‐Stimulated Adenylate Cyclase Activity in Rat Hippocampal Membranes
Authors: Okada Fumihiko , Tokumitsu Yukiko , Nomura Yasuyuki
Published: October 2006

Abstract: The inhibition of forskolin‐stimulated adenylate cyclase activity by 5‐hydroxytryptamine (5‐HT) receptor agonists was measured in rat hippocampal membranes isolated from animals treated with vehicle or islet‐activating protein (IAP; pertussis toxin). In vehicle‐treated animals, 5‐HT, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, buspirone, and gepirone were potent in inhibiting forskolin‐stimulated adenylate cyclase activity with EC50 values of 60, 76, 376, and 530 nM, respectively. IAP treatment reduced by 30–55% the 5‐HT1A agonist inhibition of adenylate cyclase activity via 5‐HT1A receptors. The data indicate that the inhibitory guanine nucleotide‐binding protein or Go (a similar GTP‐binding protein of unknown function purified from brain) mediates the 5‐HT1A agonist inhibition of hippocampal adenylate cyclase.
Calmodulin Antagonist W‐7 Inhibits Lysosomal Sphingomyelinase Activity in C6 Glioma Cells
Authors: Masson Martial , Albouz Samia , Boutry Jeanne‐Marie , Spezzatti Bettina , Castagna Monique , Baumann Nicole
Published: October 2006

Abstract: N‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7) is known to be a potent calmodulin antagonist and inhibitor of calmodulin‐dependent protein kinases. W‐7 and 1‐(5‐isoquinolinyl‐sulfonyl)‐2‐methylpiperazine (H‐7) are inhibitors of protein kinase C and cyclic nucleotide‐dependent protein kinases. In C6 glioma cells, W‐7 and not H‐7 inhibited dose‐dependently acid sphingomyelinase, a result indicating the modulation of this lysosomal enzyme by a calmodulin‐dependent system. Other lysosomal enzymes, such as β‐glucosidase, α‐galactosidase, and arylsulfatase A, were unaffected by W‐7 and H‐7, a finding indicating a selective effect of W‐7 on sphingomyelinase.
Analysis of Biogenic Amines in Bovine Retina by Gas Chromatography‐Negative Ion Chemical Ionisation Mass Spectrometry
Authors: Macfarlane R. G. , Macleod S. C. , Midgley J. M. , Watson D. G.
Published: October 2006

Abstract: Biogenic amines in bovine retina have been identified and quantified by an extraction‐derivatisation procedure involving their reaction with 3,5‐di(trifluoromethyl)benzoyl chloride (DTFMBCl) in the aqueous phase followed by extraction into an organic solvent, hydrolysis of phenolic esters, and conversion of free hydroxyl groups to trimethylsilyl ethers. Subsequent analysis of these DTFMB‐trimethylsilyl derivatives by gas chromatography‐negative ion chemical ionisation mass spectrometry revealed that the molecular ion carried most (>60%) of the ion current, which made the method highly specific and gave a potential limit of detection below the picogram level. This method establishes unequivocally that the principal amines in bovine retina are p‐tyra‐mine, dopamine, and 5‐hydroxytryptamine
Multiple Neurotrophic Factors from Skeletal Muscle: Demonstration of Effects of Basic Fibroblast Growth Factor and Comparisons with the 22‐Kilodalton Choline Acetyltransferase Development Factor
Authors: McManaman James , Crawford Frances , Clark Robin , Richker Jeffrey , Fuller Forrest
Published: October 2006

Abstract: Extracts of skeletal muscle contain chromato‐graphically distinct molecules that enhance the cholinergic development of cultured embryonic rat spinal cord neurons. We have recently purified a 20–22 kilodalton anionic polypeptide choline acetyltransferase (ChAT) development factor (CDF) from rat skeletal muscle extracts that stimulates the development of ChAT activity in rat spinal cord cultures. The maximum increase in the level of ChAT activity achieved by this factor, however, is less than that achieved by the addition of the crude extract. We now show that muscle extract also contains mitogenic activity that is immunologically related to basic fibroblast growth factor (bFGF) and also that recombinant bFGF stimulates ChAT development in rat spinal cord cultures. bFGF, however, differs from CDF in its physicochemical, chromatographic, and immunological properties and by its action on nonneuronal cells. Individually, CDF and bFGF each enhance the level of ChAT activityin rat spinal cord cultures two‐ to threefold after 2 days of treatment. However, their combined actions result in a five‐to sixfold enhancement of ChAT activity, suggesting that they are affecting cholinergic development through different means. The demonstration that extracts of rat skeletal muscle contain two biochemically and immunologically distinct polypeptides, with additive effects on cultured embryonic spinal cord neurons, provides additional evidence for the involvement of multiple target‐derived neurotrophic factors in the regulation of cholinergic development
Glutamate‐Induced 45Ca2+ Uptake into Immature Cerebral Cortex Neurons Shows a Distinct Pharmacological Profile
Authors: Frandsen Aase , Drejer Jørgen , Schousboe Arne
Published: October 2006

Abstract: Glutamate‐induced 45Ca2+ uptake was studied in cerebral cortex neurons cultured for 4 days, i.e., at a developmental stage where the neurons are sensitive to the mixed agonist glutamate but not to the actions of N‐methyl‐D‐aspartate or other excitatory amino acids. Using this experimental approach, allowing the investigation of effects elicited only by glutamate, it was demonstrated that the glutamate‐stimulated Ca2+ influx could be completely antagonized by MK‐801, phencyclidine, and cyclazocine in the nanomolar range, and by 3‐(2‐carboxypiperazin‐4‐yl)propyl‐l‐phosphonate and D‐2‐amino‐5‐phosphonopentanoate (APV) in the low micromolar range. However, the glutamate response was unaffected by variations in the Mg2+ concentration in the exposure media. In addition, the two qui‐noxalinediones 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and 6,7‐dini‐troquinoxaline‐2,3‐dione were equipotent with APV in blocking the glutamate‐stimulated Ca2+ uptake. PK 26124 blocked the response in the high micromolar concentration range. Ketamine and γ‐glu‐tamylaminomethylsulfonate were essentially without effect at concentrations up to 10 μM and 300 μM, respectively. These results may suggest the existence of a glutamate receptor with a pharmacological profile not compatible with the existent classification of glutamate receptor subtypes.
Axolinin Localization in the Nervous Tissue of Squid Revealed by Monoclonal Antibodies Specific for Axolinin: Cellular and Subcellular Localization of Axolinin in the Squid Neuron
Authors: Arai Takao , Matsumoto Gen
Published: October 2006

Abstract: Cellular and subcellular distributions of axolinin, the 260‐kilodalton (kD) microtubule‐associated glycoprotein originally purified from squid axons, in various squid tissues such as optical lobes, bundles of small nerve fibers (fin nerves), giant stellate ganglia, skin, muscle, liver, and gill, were immunologicaly studied using monoclonal antibodies specifically recognizing the polypeptide chain of axolinin. The following results were obtained: (1) Axolinin is confined to squid neurons and skin; (2) axolinin is localized in the axon whereas another 260‐kD microtubule‐associated protein, MAP B, is localized in the cell bodies; and (3) axolinin is localized mainly in the peripheral part of the axoplasm of the squid giant axon. The last result has confirmed our previous conclusion obtained using polyclonal antisera against axolinin, which contain antibodies recognizing not only axolinin‐specific epitopes but also nonspecific epitopes. The physiological importance of the localization of axolinin in axons and the skin is discussed based on its possible relationship to excitability function.
Characterisation, Density, and Distribution of Kainate Receptors in Normal and Alzheimer's Diseased Human Brain
Authors: Cowburn Richard F. , Hardy John A. , Briggs Roger S. , Roberts Peter J.
Published: October 2006

Abstract: The specific binding of [3H]kainic acid was investigated in membrane preparations from human parietal cortex obtained postmortem. Saturation studies revealed that binding occurred to a single population of sites with a KD of 15 nM and a Bmax of 110 fmol/mg of protein. The kinetically determined dissociation constant for these sites agreed well with that obtained from saturation analyses. Pharmacological characterisation of these sites gave a profile consistent with those reported for kainate receptor sites in animal brain. The integrity of kainate receptors was studied in several brain regions from six patients who had died of Alzheimer's disease and from six closely matched control subjects. No change in either the affinity or the number of kainate receptors was seen in any of the regions studied, despite the loss of neo‐cortical and hippocampal glutamatergic terminals in the Alzheimer's diseased brains, as previously reported.
Occurrence in Brain Lysosomes of a Sialidase Active on Ganglioside
Authors: Fiorilli Amelia , Venerando Bruno , Siniscalco Carola , Monti Eugenio , Bresciani Roberto , Caimi Luigi , Preti Augusto , Tettamanti Guido
Published: October 2006

A lysosomal preparation, obtained from brain ho‐mogenate of 17‐day‐old C57BL mice by centrifugation on a self‐generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40–120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GDla and fluorimetrically with 4‐methylumbelliferyl‐α‐D‐N‐acetylneuraminic acid (MUB‐NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane‐bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma Membrane‐bound enzyme. The apparent Km values for GDl a and MUB‐NeuAc were 1.5 × 10‐5 and 4.2 × 10‐5M, respectively, for the lysosomal enzyme and 2.7 × 10‐4 and 6.3 × 10‐5M for the plasma membrane‐bound one. Triton ×‐ 100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane‐bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane‐bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane‐bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers. It is thus evident that the sialidase present in the lysosomal fraction is an authentic lysosomal enzyme distinct and different from the sialidase contained in the plasma membrane. The lysosomal sialidase affected other ganglio‐sides, like GDlb and GM3. These data constitute the first direct evidence for the presence in brain lysosomes of a sialidase activity on gangliosides and contribute to a better knowledge of ganglioside breakdown and turnover in the brain.