bluuebunny/crossref_metadata_2025 huggingface dataset:
Muscarinic Receptor Stimulated GTPase Activity in Synaptic Membranes from Bovine Retina
Authors: McIntosh Helen , Blazynski Christine
Published: October 2006

GTPase activity has been measured in synaptic membranes from bovine retina, with and without muscarinic receptor stimulation. Maximal stimulation above basal levels was achieved with 5 μM oxotremorine and 100μM carbachol. (4‐Hydroxy‐2‐butynyl)‐1‐trimethylammonium m‐chlorocarbanilate chloride, which is selective for the M1 muscarinic receptor, failed to stimulate GTPase activity. 4‐Diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP) inhibition of oxotremorine stimulation demonstrated the presence of two populations of receptors, a low‐affinity site (IC50±SEM, 0.63 ±0.18 μM) which accounted for 63% of the inhibition and a high‐affinity site (IC50 < 1 nM) which accounted for the remaining 37%. When carba‐chol‐stimulated GTPase activity was assayed, a single 4‐DAMP inhibitory site was apparent (IC50±SEM, 2.0 ±0.9 μM). Pirenzepine inhibited GTPase activity at a single site (IC50 values ±SEM, 46.9 ±11 and 25.4 ±6.5 μM against oxotremorine and carbachol, respectively). Methoctramine was equipotent against carbachol and oxotremorine stimulation (IC50 values, 4.2 ±1.8 and 6.2 ±1.5 μM). Inhibition of maximal carbachol and oxotremorine stimulation by muscarinic antagonists at the major site had a rank order of potency of 4‐DAMP = methoctramine > pirenzepine. Thus, the major site for muscarinic stimulation of GTPase activity in bovine retinal membranes is pharmacologically similar to M2 receptors.
Evidence for Metabotropic Excitatory Amino Acid Receptor Heterogeneity: Developmental and Brain Regional Studies
Authors: Vecil Giacomo G. , Li Peter P. , Warsh Jerry J.
Published: October 2006

To examine whether multiple subtypes of the excitatory amino acid (EAA) receptor coupled to phosphoino‐sitide (PPI) hydrolysis exist, we have pharmacologically characterized the PPI response in neonatal and adult rat brain. Activation of PPI hydrolysis was determined by the accumulation of [3H]inositol monophosphate in brain slices prelabeled with [3H]inositol. In neonatal hippocampus, D,L‐2‐amino‐3‐phosphonopropionic acid (AP3; 1 mM) inhibited the m‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (IUPAC nomenclature; ACPD; 100 μM)‐ and quisqualate (Quis; 100 μM)‐stimulated PPI hydrolysis by 73 and 66%, respectively, but had no effect in neonatal cerebellum. In adult hippocampus, AP3 stimulated PPI hydrolysis with potency and efficacy comparable to those of Quis and ACPD and completely masked the Quis concentration‐response curve. In adult cerebellum, only Quis behaved as a full agonist on the PPI response. The Quis concentration‐response curve was shifted rightward with a fourfold decrease in potency in the presence of ACPD (5 mM), whereas it was nearly additive with the PPI response induced by AP3 (5 mM). Thus, our data reveal significant developmental and brain regional differences in metabotropic EAA receptor responses and support the notion that this receptor is heterogeneous, in both a regionally specific and a develop‐mentally dependent manner.
Evidence that Axonal tRNAs Are Resistant to RNase and ATPase and Can Be Aminoacylated in the Absence of Exogenous ATP
Authors: Chakraborty G. , Nicola A. , Ingoglia N. A.
Published: October 2006

A high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein‐tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNAArg was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNALys did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNAArg and tRNALys was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme‐resistant, ATP‐tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNAArg and tRNALys were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase‐luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overho‐mogenizing the tissue. These procedures failed to render brain tRNALys susceptible to RNase. But phenol‐extracted, ethanol‐precipitated brain tRNALys was sensitive to RNase, suggesting that the protection of tRNALys may be by a protease‐ and heat‐resistant polypeptide or by a nonprotein‐aceous mechanism.
Nerve Growth Factor‐Induced Changes in the Structure of Sulfated Proteoglycans in PC 12 Pheochromocytoma Cells
Authors: Katoh‐Semba Ritsuko , Oohira Atsuhiko , Kashiwamata Shigeo
Published: October 2006

Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC 12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC 12 cells after 3 days of culture was in the range of 90–190 kDa for the intact form (Kav= 0.38 on Sepharose CL‐6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90–190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell‐associated CSPG had apparent molecular mass ranges of 120–150 kDa and 120–190 kDa (Kav= 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell‐associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell‐associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE‐cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav= 0.22 on Sephacryl S‐300 [40–84 kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE)]. The CS and core protein of this CSPG had apparent molecular masses of 26 kDa and 15–17 kDa, respectively. The peak I fraction was separated further into three fractions of different molecular sizes on Sephadex G‐200 (I‐1,I‐2,I‐3). In the presence of NGF, peak I‐1 prepared after 3 days of culture consisted of mostly CSPG (130–170 kDa by SDS‐PAGE; Kav= 0.13 on Sephacryl S‐300) with CS of 32 kDa and a core protein of 105 kDa. Peaks 1–2 and 1–3 contained, respectively, CSPGs of 38–74 kDa and 31–56 kDa for their intact forms, with CSs of 25 kDa and 15 kDa. The apparent molecular masses of the core proteins of their CSPGs were 15–17 kDa. These CSPGs had similar molecular sizes, irrespective of the presence of NGF or the duration of culture. (4) These results indicate that structural changes in PGs during the neuritogenesis in PC12 cells induced by NGF occurred mostly in cell‐associated PGs and preceded elongation of neurites.
Extracellular Concentrations of Dopamine and Metabolites in the Rat Caudate After Oral Administration of a Novel Catechol‐O‐Methyltransferase Inhibitor Ro 40–7592
Authors: Acquas E. , Carboni E. , de Ree R. H. A. , Da Prada M. , Di Chiara G.
Published: October 2006

The effect of the systemic administration of a novel, orally active, catechol‐O‐methyltransferase (COMT) inhibitor, Ro 40–7592, on the in vivo extracellular concentrations of dopamine (DA) and its metabolites, dihydroxy‐phenylacetic acid (DOPAC) and homovanillic acid (HVA), was studied by transcerebral microdialysis in the dorsal caudate of freely moving rats. Ro 40–7592 (at doses of 3.0, 7.5, and 30 mg/kg p.o.) elicited a marked and long‐lasting reduction of HVA, and at doses of 7.5 and 30 mg/kg, an increase of DOPAC output, but it failed to increase DA output. The administration of L‐β‐3,4‐dihydroxyphenylalanine (L‐DOPA, 20 and 50 mg/kg p.o.) with a DOPA decarboxylase inhibitor (benserazide) increased both HVA and DOPAC output, but failed to modify significantly extracellular DA concentrations in dialysates; in contrast, combined administration of L‐DOPA + benserazide with Ro 40–7592 (30 mg/ kg p.o.) resulted in a significant increase in DA output. Ro 40–7592 prevented the L‐DOPA‐induced increase in HVA output and markedly potentiated the increase in DOPAC output. To investigate to what extent the increase in extra cellular DA concentrations was related to an exocitotic release, tetrodotoxin (TTX) sensitivity was tested. Addition of TTX to Ringer, although abolishing DA output in the absence of L‐DOPA, partially reduced it in the presence of L‐DOPA + Ro 40–7592 and even more so after L‐DOPA without the COMT inhibitor. The results of the present study suggest that metabolism through COMT regulates extracellular concentrations of DA formed from exogenously administered L‐DOPA but not of endogenous DA. Therefore, inhibition of COMT results in a potentiation of L‐DOPA effects not only by inhibition of its peripheral metabolism (conversion to 3‐methoxy‐DOPA), but also by inhibition of the metabolism of its active metabolite, DA, in the brain.
Activation of Metabotropic Glutamate Receptors in the Hippocampus Increases Cyclic AMP Accumulation
Authors: Winder Danny G. , Conn P. Jeffrey
Published: October 2006

The selective metabotropic glutamate receptor agonist trans‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (trans‐ACPD) stimulates phosphoinositide hydrolysis and elicits several physiological responses in rat hippocampal slices. However, recent studies suggest that the physiological effects of trans‐ACPD in the hippocampus are mediated by activation of a receptor that is distinct from the phosphoinositide hydrolysis‐linked receptor. Previous experiments indicate that cyclic AMP mimics many of the physiological effects of trans‐ACPD in hippocampal slices. Furthermore, recent cloning and biochemistry experiments indicate that multiple metabotropic glutamate receptor subtypes exist, some of which are coupled to yet unidentified effector systems. Thus, we performed a series of experiments to test the hypothesis that ACPD increases cyclic AMP levels in hippocampal slices. We report that 1S,3R‐ and 1S,3S‐ACPD (but not 1R,3S‐ACPD) induce a concentration‐dependent increase in cyclic AMP accumulation in hippocampal slices. This effect was blocked by the metabotropic glutamate receptor antagonist L‐2‐amino‐3‐phosphonoproprionic acid but not by selective antagonists of ionotropic glutamate receptors. Furthermore, our results suggest that 1R,3S‐ACPD‐stimulated increases in cyclic AMP accumulation are not secondary to increases in cell firing or to activation of phosphoinositide hydrolysis.
Inositol 1,4,5‐Trisphosphate Receptor Immunoreactivity in SH‐SY5Y Human Neuroblastoma Cells Is Reduced by Chronic Muscarinic Receptor Activation
Authors: Wojcikiewicz Richard J. H. , Nakade Shinji , Mikoshiba Katsuhiko , Nahorski Stefan R.
Published: October 2006

Inositol 1,4,5‐trisphosphate (InsP3) receptor immunoreactivity in SH‐SY5 Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, ∼275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half‐maximal and maximal at ∼2 and ∼6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12‐myristate 13‐acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol‐induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH‐SY5 Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.
Neuraminidase Activities in Oligodendroglial Cells of the Rat Brain
Authors: Saito Megumi , Sato‐Bigbee Carmen , Yu Robert K.
Published: October 2006

Abstract: Neuraminidase activities in oligodendroglial cells were characterized using rats of different ages. Rat oligodendroglial cells had intrinsic neuraminidase activities directed toward GM3 and N‐acetylneuramin(2–3)lactitol (NL). Developmental profiles of the neuraminidase activities toward the two substrates in oligodendroglial cells were different from each other. The neuraminidase activity toward GM3 increased rapidly with the onset of active myelination and, after 26 days of development, reached the adult level which was about 18 times higher than that in myelin. At the adult age, oligodendroglial cells had the highest neuraminidase activity toward GM3 among the individual brain cell types examined. The activity of NL‐neuraminidase showed a less remarkable developmental profile, with a peak value at 26 days. The UDP‐galactose:ceramide galactosyltransferase activity in oligodendroglial cells increased during the period of active myelination and, afterward, returned to the basal level. The enrichment and unique developmental profile in oligodendroglial cells of the neuraminidase activity toward GM3 suggest that this enzyme may play an important role in the formation and maintenance of the myelin sheath.
Role of Myelin‐Associated Neuraminidase in the Ganglioside Metabolism of Rat Brain Myelin
Authors: Saito Megumi , Yu Robert K.
Published: October 2006

Abstract: The role of myelin‐associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di‐and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di‐and polysialo‐species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin‐associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat brain myelin.
Effect of Hydroperoxy Fatty Acids on Acylation and Deacylation of Arachidonoyl Groups in Synaptic Phospholipids
Authors: Zaleska Malgorzata M. , Wilson David F.
Published: October 2006

Abstract: The effect of hydroperoxy fatty acids on reactions involved in the acylation‐deacylation cycle of synaptic phospholipids was studied in vitro, using nerve ending fraction isolated from rat forebrain. 15‐Hydroperoxyeicosatetraenoic acid (15‐HPETE), 13‐hydroperoxylinoleic acid (13‐HP 18: 2), and hydroperoxydocosahexaenoic acid (22:6 Hpx), at 25 μM final concentration, all inhibited the incorporation of [1‐14C]arachidonate into synaptosomal phosphatidylinositol (PI), phosphatidylcholine (PC), and triacylglycerides by 50–80%. The lowest effective concentration of 15‐HPETE and 13‐HP 18:2 resulting in significant inhibition of the reacylation of PI was 5 μM, whereas the inhibition of [1‐14C]arachidonate incorporation into PC required 10 and 5 μM hydroperoxy fatty acids, respectively. Cumene hydroperoxide and tert‐butyl hydroperoxide at concentrations of 100 μM did not inhibit reacylation of PI and PC. Synthesis of labeled arachidonoyl‐CoA from [1‐14C]arachidonate was decreased by about 50% by 25 μM hydroperoxy fatty acids both in synaptosomes and in the microsomal fraction. Use of [1‐14C]arachidonoyl‐CoA as a substrate, to bypass the fatty acid activation reaction, revealed that activity of acyltransferase was not affected significantly by 25 μM 15‐HPETE and 13‐HP 18:2. At the same time, however, the hydrolysis of labeled arachidonoyl‐CoA was substantially enhanced. Exposure of synaptosomes to 25 μM fatty acid hydroperoxides did not affect significantly the endogenous concentrations of five major free fatty acids. It is concluded that (1) among synaptic phospholipids, reacylation of PI and PC is the most susceptible to the inhibitory action of fatty acid hydroperoxides, and (2) the enzymes affected by these compounds in nerve endings are arachidonoyl‐CoA synthetase and hydrolase.
Determination of Acidic Metabolites of Biogenic Amines in Human Aqueous Humour by Gas Chromatography‐Negative Ion Chemical Ionisation Mass Spectrometry
Authors: Watson David G. , McGhee Charles N. J. , Midgley John M. , Zhou Ping , Doig William M.
Published: October 2006

Abstract: The concentrations of acidic metabolites derived from the biogenic amines o‐, m‐, and p‐tyramines (o‐, m‐, and p‐hydroxyphenylacetic acids), p‐octopamine/p‐synephrine (p‐hydroxymandelic acid), and dopamine (homovanillic acid and 3,4‐dihydroxyphenylacetic acid) were measured in human aqueous humour obtained from patients undergoing elective surgery for cataract removal or for trabeculectomy as a treatment for chronic open‐angle glaucoma. There were no clear differences in the pattern of metabolism of neurotransmitters between the two groups. An unexpected finding was that the o‐tyramine metabolite, o‐hydroxyphenylacetic acid, was present in aqueous humour.
Purification of Neuropathy Target Esterase from Avian Brain After Prelabelling with [3H]Diisopropyl Phosphorofluoridate
Authors: Rüffer‐Turner Marianne E. , Read David J. , Johnson Martin K.
Published: October 2006

Abstract: Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE). The volatilizable counts assay and analytical SDS‐PAGE were used to monitor the protein. The 150‐kDa subunit polypeptide appears as a single band on analytical SDS‐PAGE.
Purification of a Synaptic Membrane Na+/Ca2+ Antiporter and Immunoextraction with Antibodies to a 36‐kDa Protein
Authors: Michaelis M. L. , Nunley E. W. , Jayawickreme C. , Hurlbert M. , Schueler S. , Guilly C.
Published: October 2006

Abstract: The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low‐pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S‐400 HR, ion exchange on diethylaminoethyl‐Sephacel, and metal chelate chromatography on tris‐(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36‐kDa protein permitted immunoextraction of >95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross‐reacted with the electroeluted 50‐kDa protein on enzyme‐linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36‐kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.
Lithium Effects on Inositol Phospholipids and Inositol Phosphates: Evaluation of an In Vivo Model for Assessing Polyphosphoinositide Turnover in Brain
Authors: Sun Grace Y. , Navidi Meena , Yoa Fu‐Gen , Lin Teng‐Nan , Orth Oliver E. , Stubbs Evan B. , MacQuarrie Ronald A.
Published: October 2006

Abstract: Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time‐ and dose‐dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37‐ and 20‐fold increases in the mass of myo‐inositol 1‐phosphate and 4‐phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo‐inositol 1,4‐bisphosphate in brain. The lithium‐induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium‐treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine‐induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.
Differential Effects of Presynaptic Phospholipase A2 Neurotoxins on Torpedo Synaptosomes
Authors: Délot Emmanuèle , Bon Cassian
Published: October 2006

Abstract: The effects of several phospholipase A2 neurotoxins from snake venoms were examined on purely cholinergic synaptosomes from Torpedo electric organ. The noncatalytic component A of crotoxin had no effect, whereas its phospholipase component B, used alone or complexed to component A, elicited a rapid and dose‐dependent acetylcholine (ACh) release and a depolarization of the preparation. Subsequent ACh release evoked by high K+ levels or calcium ionophore was identical to the control after the action of component A but reduced after the action of crotoxin or of component B. These effects were not observed when the phospholipase A2 activity of the toxin was blocked either by replacing Ca2+ by Ba2+ (respectively, activator and inhibitor of phospholipase A2) or by alkylation of component B with p‐bromophenacyl bromide. β‐Bungarotoxin, another very potent phospholipase A2 neurotoxin, induced release of little ACh, did not affect ionophore‐evoked ACh release, but significantly reduced depolarization‐induced ACh release. The single‐chain phospholipase A2 neurotoxin agkistrodotoxin behaved like crotoxin component B. A nonneurotoxic phospholipase A2 from mammalian pancreas induced release of an amount of ACh similar to that released by crotoxin but did not affect the evoked responses. The obvious differences in effect of the various neurotoxins suggest that they exert their specific actions on the excitation‐secretion coupling process at different sites or by different mechanisms.
Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells
Authors: Kiedrowski Lech , Costa Erminio , Wroblewski Jarda T.
Published: October 2006

Abstract: The glutamate receptor agonist N‐methyl‐D‐aspartate (NMDA) stimulated a rapid, extracellular Ca2+‐dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor‐mediated activation of nitric oxide (NO) synthase. The NMDA‐induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled l‐arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose‐dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK‐801 and by the inhibitors of NO synthase, NG‐monomethyl‐L‐arginine (MeArg) and NG‐nitro‐L‐arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 μM) stimulated 45Ca2+ influx to the same extent as 100 μM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.
An Activation of Synaptosomal Na+, K+‐ATPase by a Novel Dibenzoxazepine Derivative (BY‐1949) in the Rat Brain: Its Functional Role in the Neurotransmitter Uptake Systems
Authors: Nagafuji Toshiaki , Koide Tohru , Miyauchi Tatsuo , Takato Michiaki
Published: October 2006

Abstract: In search of factors mitigating the final outcome of ischemic and epileptic brain damage, we tested a novel dibenzoxazepine derivative (BY‐1949), as the compound has been shown to be effective under these two conditions. First, using rat brain, we assessed whether or not BY‐1949 affects the Na+,K+‐ATPase activity. Although in vitro applications of either BY‐1949 or its three major metabolites did not cause any apparent effects, both acute and chronic oral administrations of the compound (10 mg/kg) invariably increased the Na+,K+‐ATPase activity in the synaptosomal plasma membranes by increasing Vmax values. Second, it was shown by this study that the drug treatment caused marked increases in the uptake of both glutamic acid and γ‐aminobutyric acid into the synaptosomes. These results suggest that the activity against ischemic/epileptic brain damage by BY‐1949 is explicable, at least partly, in terms of improvement of ionic derangements across the neural membranes via Na+,K+‐ATPase activation.
Inhibition of Brain Glutamate Decarboxylase Activity Is Related to Febrile Seizures in Rat Pups
Authors: Arias Clorinda , Valero Hilda , Tapia Ricardo
Published: October 2006

Abstract:Because previous work showed that in the newborn brain, but not in the adult brain, glutamate decarboxylase (GAD) is notably susceptible to heat, we have studied the possible involvement of GAD inhibition in febrile convulsions and the related changes in γ‐aminobutyric acid (GABA) content. Rats of different ages were subjected to hyperthermia, and GAD activity was determined in brain homogenates by measuring the release of14CO2from labeled glutamate and by measuring the formation of GABA. The latter method gave considerably lower values than the former in the youngest rats, and was considered more reliable. With this method, we found a 37–48% inhibition of GAD activity in rat pups 2–5 days old, which showed febrile seizures at progressively higher body temperatures, whereas in 10‐ and 15‐day‐old animals, which did not show convulsions, GAD activity was not affected by hyperthermia. Whole‐brain GABA levels, however, did not change at any age. In contrast to GAD, choline acetyltransferase and lactic dehydrogenase activities were not altered by hyperthermia at any of the ages studied. These results suggest that a decreased efficiency of the inhibitory neurotransmission mediated by GABA, consequent to the inhibition of GAD activity, may be a factor related to febrile convulsions.
Potassium‐Induced Depolarization of Rat Telencephalic Synaptoneurosomes Increases [3H]Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionate Receptor Binding
Authors: Bernard Julie , Massicotte Guy , Baudry Michel
Published: October 2006

Abstract: Potassium‐induced depolarization of synaptoneurosomes prepared from rat telencephalon was found to increase [3H]amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate ([3H]AMPA) binding to the AMPA receptor. The effect required the presence of calcium because it was blocked by the calcium chelator EGTA but was not blocked by an antagonist of the N‐methyl‐D‐aspartate receptor, aminophosphonopentanoate. The depolarization‐induced increase in [3H]AMPA binding was markedly reduced by a blocker of voltage‐dependent calcium channels, verapamil. Saturation kinetic experiments revealed that the increase in [3H]AMPA binding produced by potassium depolarization was due to an increase in the affinity of the AMPA receptor. These results provide additional support for a critical role of calcium in the regulation of the AMPA receptors. The syn‐aptoneurosome preparation might represent an interesting tool to determine the role of different calcium‐dependent enzymes involved in the regulation of the AMPA receptor.
Vesamicol Binding to Subcellular Membranes That Are Distinct from Catecholaminergic Vesicles in PC 12 Cells
Authors: Blumberg Deborah , Schweitzer Erik S.
Published: October 2006

Abstract: We have examined PC12 cells for the localization of binding sites for vesamicol [l‐2‐(4‐phenylpiperidino) cyclohexanol], a compound that has previously been shown to bind to cholinergic vesicles and to inhibit the uptake of acetylcholine. Initial studies presented in this article demonstrate the existence of a specific, saturable vesamicol binding site in PC12 cells. Subsequent experiments show that these binding sites reside in a membrane population that is distinct from catecholamine‐containing compartments with respect to density and antigenic composition. In particular, vesamicol binding compartments have a lower density than Catecholaminergic vesicles and, unlike these latter vesicles, do not appear to contain the vesicle‐specific proteins syn‐aptophysin and SV2 as part of the same membrane. These results suggest that vesicular transport proteins for acetylcholine and catecholamines are differentially sorted to distinct membrane compartments in PC12 cells.
Pertussis Toxin in the A10 Region Increases Dopamine Synthesis and Metabolism
Authors: Steketee Jeffery D. , Striplin Caryn D. , Murray Thomas F. , Kalivas Peter W.
Published: October 2006

Abstract: Inhibitory regulation of dopamine neurons is mediated by dopamine autoreceptor and γ‐aminobutyric acidB receptor opening of potassium channels. Increased potassium conductance by either receptor is G protein dependent. To evaluate the role of G proteins in vivo, pertussis toxin (PTX) was microinjected into the A10 dopamine region and changes in dopamine metabolism and synthesis measured. PTX produced an elevation in dopamine metabolism and synthesis in the A10 region and nucleus accumbens for up to 4 days after injection. By day 7 the levels of the dopamine precursor and metabolites had returned to normal. A less consistent increase was also measured in the A9 dopamine region and the prefrontal cortex. Although dopamine synthesis and metabolism had returned to normal by day 7, the in vitro ADP‐ribosylation of G proteins in the A10 region by PTX remained depressed by approximately 50% from day 1 to day 14 after administration, returning to normal by day 30. The data suggest that in vivo ribosylation of G proteins may lead to a short‐term attenuation of the tonic inhibitory control of dopamine neurons, which can be compensated for by PTX‐insensitive mechanisms.
Characterization of Substance P‐Like Immunoreactivity and Tachykinin‐Encoding mRNAs in Rat Medullary Thyroid Carcinoma Cell Lines
Authors: Cremins Jean D. , Michel Jacquelyn , Farah John M. , Krause James E.
Published: October 2006

Abstract: Rat thyroid tissue and three rat medullary thyroid carcinoma cell lines, 6‐23, WE4/2, and CA77, have been examined for substance P (SP) and SP‐like peptide expression. Analysis by combined HPLC and radioimmunoassay revealed the presence of SP in thyroid and 6‐23 cell extracts. The presence of SP‐encoding mRNAs was also detected in 6‐23 cells by solution hybridization‐nuclease protection analysis. SP‐encoding mRNA expression was increased (fourfold) by maintaining the 6‐23 cells in low serum (2%) for 4 or 10 days. The 6‐23 cells also expressed other SP‐like immunoreactive species, which were chromatographically and immunologically distinct from established tachykinin peptides. WE4/2 cells did not contain SP but did display SP‐like immunoreactivity (SPLI), which migrated like the unidentified SPLI in 6‐23 cells. CA77 cells did not contain SP or SP‐encoding mRNA but did contain SPLI that migrated identically to the unidentified SPLI in the other cell lines. This novel SPLI was detected with an antiserum directed against the SP carboxyl terminus and to a lesser extent with an antiserum directed against the neurokinin A carboxyl terminus, but it showed minimal cross‐reactivity using an anti‐serum directed against the midportion of SP. Treatment with 50 mM KC1 resulted in secretion of this SPLI from CA77 cells. Gel filtration analysis demonstrated that this novel SPLI had an apparent molecular weight of ∼ 1,000. These results are discussed in terms of cell lines that express tachykinin peptides and in terms of the molecular nature of the new SPLI detected in CA77 cells.
Rat Hippocampal Lactate Efflux During Electroconvulsive Shock or Stress Is Differently Dependent on Entorhinal Cortex and Adrenal Integrity
Authors: Krugers Harm J. , Jaarsma Dick , Korf Jakob
Published: October 2006

Abstract: The role of the entorhinal cortex and the adrenal gland in rat hippocampal lactate formation was assessed during and after a short‐lasting immobilization stress and elec‐troconvulsive shock (ECS). Extracellular lactate was measured on‐line using microdialysis and enzyme reactions (a technique named lactography); in some rats, unilateral lesions of the entorhinal cortex were made or the bilateral adrenal glands were removed. The stress‐evoked increase in hippocampus lactate was not altered either ipsi‐ or contralateral to an entorhinal cortex lesion. The response to ECS was attenuated only in the hippocampus ipsilateral to the entorhinal cortex lesion. Removal of bilateral adrenal glands caused some delay in the increase in hippocampal lactate after ECS and a major reduction in the stress‐evoked lactate response. These results indicate that (1) the entorhinal cortex is activated by ECS, thereby activating hippocampal lactate efflux and presumably metabolism, and (2) the adrenal gland is essential in the response to stress and, to a minor extent, in the ECS‐altered hippocampal metabolism.
Activation of Microtubule‐Associated Protein Kinase in PC12D Cells in Response to Both Fibroblast Growth Factor and Epidermal Growth Factor and Concomitant Stimulation of the Outgrowth of Neurites
Authors: Sano Mamoru , Kitajima Satoko
Published: October 2006

Abstract: When PC12D cells, a subline of PC12 cells, were cultured with nerve growth factor (NGF), outgrowth of neurites was promoted even when RNA synthesis was blocked. This property of PC12D cells may enable us to resolve the mechanism of the outgrowth of neurites that is induced in a transcription‐independent manner. The outgrowth of neurites from PC12D cells was also stimulated in response to fibroblast growth factor (FGF) and was slightly stimulated in response to epidermal growth factor (EGF). The brief exposure of intact PC12D cells not only to NGF but also to FGF or to EGF stimulated a protein kinase activity in extracts of such cells that catalyzed phosphorylation of microtubule‐associated protein 1 (MAP‐1) and MAP‐2 in vitro. Similar dose‐response relationships for the effects of NGF and of FGF on the activation of the kinase and on the outgrowth of neurites were observed. The effects of combinations of NGF and FGF or EGF were not additive in terms of either the outgrowth of neurites or the increase in the kinase activity. Treatment of cells with phorbol 12‐myristate 13‐acetate (PMA) also stimulated the kinase activity that phosphorylated MAPs in vitro. However, the level of the enzymatic activity that resulted from the combined treatment of cells with PMA and NGF was additive, as is the case with dibutyryl cyclic AMP and NGF. These findings suggest that NGF, FGF, and EGF may stimulate the activity of the same MAP kinase. The close relationship between the activation of the kinase and the outgrowth of neurites from PC12D cells in response to various agents suggests that activation of the kinase may play an important role in the outgrowth of neurites.
Comparison of the N‐Linked Oligosaccharide Structures of the Two Major Human Myelin Glycoproteins MAG and P0: Assessment and Relative Occurrence of Oligosaccharide Structures by Serial Lectin Affinity Chromatography of 14C‐Glycopeptides
Authors: Burger Danielle , Perruisseau Geneviève , Simon Maryline , Steck Andreas J.
Published: October 2006

Abstract: The N‐linked Oligosaccharide structures of human myelin‐associated glycoprotein (MAG) and P0 have been characterized by serial lectin affinity chromatography (SLAC) of 14C‐glycopeptides. 14C‐Glycopeptides were prepared from purified MAG derivative and P0 by extensive proteolytic digestion and N‐14C‐acetylation. Assuming that all the 14C‐glycopeptides were radiolabelled to the same specific radioactivity, the relative occurrence of the Oligosaccharide structures was correlated to the amount of incorporated radioactivity. Sixteen and 15 fractions were generated by SLAC of MAG and P014C‐glycopeptides, respectively. Despite this tremendous structural heterogeneity, the Oligosaccharide “fingerprints’ of MAG and P0 obtained by SLAC displayed similarities: (a) of the three types of N‐linked oligosaccharides, the complex type accounted for 80.4% and 94.9% of MAG and P0 radioactivity, respectively; (b) biantennary complex oligosaccharides were the major structures present on MAG and P0; (c)60% of MAG and P0 oligosaccharides possessed a bisecting N‐acetylglucosamine residue; and (d) large amounts of oligosaccharides with an α(l‐6)fucose residue were found in both MAG and P0 and, noticeably, 25% of the tri‐ and/or tetraantennary and 90% of the bisected biantennary oligosaccharides of both glycoproteins contained α(l‐6)fucose residues in the core. This study demonstrates that MAG and P0, both belonging to the immunoglobulin superfamily, display structural similarities in their N‐linked Oligosaccharide contents.
Comparison of the N‐Linked Oligosaccharide Structures of the Two Major Human Myelin Glycoproteins MAG and P0: Assessment of the Structures Bearing the Epitope for HNK‐1 and Human Monoclonal Immunoglobulin M Found in Demyelinating Neuropathy
Authors: Burger Danielle , Perruisseau Geneviève , Simon Maryline , Steck Andreas J.
Published: October 2006

Abstract: The epitope for HNK‐1 and patient's monoclonal autoantibodies in demyelinating polyneuropathy associated with immunoglobulin M gammopathy is borne by different types of N‐linked Oligosaccharide structures in human PO and myelin‐associated glycoprotein (MAG). Fourteen glycopep‐tide fractions bearing different Oligosaccharide structures were obtained from either MAG or P0 glycopeptides by serial lectin affinity chromatography on concanavalin A‐Sepharose, Phaseolus vulgaris erythrophytohemagglutinin‐agarose, Pisum sativum agglutinin‐agarose, and Phaseolus vulgaris leucophytohemagglutinin‐agarose. As shown by dot‐TLC plate immunostaining, the same MAG and P0 glycopeptide fractions were recognized by HNK‐1 and patient's immunoglobulin M, confirming that these antibodies display similar specificities. The antigenic carbohydrate was present in glycopeptide fractions that either interact with Pisum sativum agglutinin‐agarose or were bound by Aleuria aurantia agglutinin‐digoxigenin, indicating that these structures contained α(l‐6)fucose residues. This study demonstrates that the L2/ HNK‐1 epitope is borne mainly or even exclusively by N‐linked Oligosaccharide structures α(l‐6)fucosylated in the core.
Reserpine‐Induced Processing of Chromogranin A in Cultured Bovine Adrenal Chromaffin Cells
Authors: Watkinson Allan , Robinson Iain
Published: October 2006

Abstract: The effect of reserpine on the processing of the secretory granule protein chromogranin A (CgA) in isolated bovine adrenal chromaffin cells was investigated using two radioimmunoassays employing site‐specific antisera. The two antisera were directed against closely associated regions of the CgA molecule which would be exposed by specific processing: antiserum L331 was raised against the C‐terminus of the regulatory peptide pancreastatin, and the second anti‐serum, L300, was raised against the synthetic peptide [Tyr0]CgA306–313 (YLSKEWEDA), a sequence that lies immediately C‐terminal to pancreastatin and adjacent to a dibasic amino acid cleavage site. Chronic reserpine treatment of chromaffin cells produced a time‐ and dose‐dependent increase in processing, as demonstrated by an increase in pancreastatin‐ and YLSKEWEDA‐immunoreactivity (ir). The reserpine‐induced rise in pancreastatin‐ir was due predominantly to an increase in pancreastatin 1–47, whereas the rise in YLSKEWEDA‐ir was due to increases in three polypep‐tides: a 51‐kDa YLSKEWEDA‐ir polypeptide, CgA297–313, and CgA248–313. The latter predominated. The action of reserpine on both pancreastatin‐ and YLSKEWEDA‐ir was found to be largely inhibited by the protein synthesis inhibitor cycloheximide. The results show that treatment of isolated chromaffin cells with reserpine induces both the selective proteolytic processing and peptidyl‐glycine amidation of CgA and its derived fragments. As reserpine has a similar effect on proenkephalin in chromaffin cells, the results suggest that reserpine induces a general increase in the activity of the processing enzymes, partially by an increase in protein synthesis.
Gene Expression and Receptor Binding of Insulin‐Like Growth Factor‐II in Pig Choroid Plexus Epithelial Cells
Authors: Nilsson Christer , Blay Pilar , Nielsen Finn C. , Gammeltoft Steen
Published: October 2006

Abstract: To elucidate the function of insulin‐like growth factor‐II (IGF‐II) in the choroid plexus, the gene expression and receptor binding of IGF‐II were studied in isolated epithelial cells from the porcine choroid plexus. The choroid plexus expressed multiple IGF‐II transcripts of 1.2, 1.6, 2.4, and 4.4 kb, at levels higher than those found in porcine liver and kidney. These data suggest that IGF‐II is synthesized by the choroid plexus. Choroid plexus epithelial cells contained high levels of IGF‐I receptors on the cell surface whereas very low levels of receptor binding were found for 125I‐IGF‐II and I25l‐insulin. Solubilization of epithelial cells showed that a large proportion of the IGF‐I receptors were present in the detergent‐insoluble fraction whereas IGF‐II receptors and insulin receptors were concentrated in the detergent‐soluble fraction. These results suggest that IGF‐I receptors are located in clathrin‐coated pits of the plasma membrane whereas IGF‐II receptors and insulin receptors are present in endosomal vesicles. The tyrosine kinase activity of the IGF‐I receptor β‐subunit was stimulated by IGF‐I, IGF‐II, and insulin, in order of potency, suggesting that these peptides exert a regulatory function in the chdroid plexus epithelium. In conclusion, we propose that the IGF‐I receptor tyrosine kinase on the surface of the epithelial cells in the pig choroid plexus mediates effects of IGF‐I and IGF‐II, whereas IGF‐II receptors are down‐regulated due to the synthesis and secretion of IGF‐II in these cells.
Magnesium‐Dependent Enhancement of Endogenous Agonist Binding to A1 Adenosine Receptors: A Complicating Factor in Quantitative Autoradiography
Authors: Parkinson Fiona E. , Fredholm Bertil B.
Published: October 2006

Abstract: Quantitative autoradiography was used to investigate the effects of Mg2+ on agonist and antagonist binding to A1 receptors in rat striatum. A1 receptors were labelled with the selective agonist N6‐[3H]cyclohexyladenosine ([3H]CHA) or the selective antagonist 1,3‐[3H]dipropyl‐8‐cy‐clopentylxanthine ([3HJDPCPX). Mg2+ had no significant effect on equilibrium binding constants for [3H]CHA [control: KD (95% confidence interval) of 0.34 (0.15‐0.80) nM and Bmax of 267 ± 8 fmol/mg of gray matter; with 10 mA/Mg2+: KD of 0.8 (0.13‐4.9) nM and Bmax of 313 ± 8.9 fmol/mg of gray matter] or [3H]DPCPX [control: KD of 0.54 (0.30‐0.99) nM and Bmax of 256 ± 2.3 fmol/mg of gray matter; with 10 mMMg2+: KD of 1.54 (0.2‐11.0) nM and Bmax of 269 ± 35.7 fmol/mg of gray matter]. In contrast, Mg2+ slowed the apparent association rate for both ligands; this was observed as a shift from a one‐component to a two‐component model for [3H]DPCPX. Mg2+ also affected the dissociation rates of both ligands; for [3H]CHA, dissociation in the presence of Mg2+ was not detected. Mg2+ produced a concentration‐dependent inhibition of [3H]CHA binding only prior to equilibrium. HPLC was performed on untreated sections, sections preincubated with adenosine deaminase (ADA), and sections preincubated with ADA and incubated with ADA in the absence or presence of Mg2+ Adenosine was found in measurable quantities under all conditions, and the concentration was not influenced by Mg2+ or by the inclusion of GTP in the preincubation medium. From these data, we conclude the following: (a) adenosine is present and may be produced continuously in brain sections; (b) ADA is not capable of completely eliminating the produced adenosine; (c) Mg2+ apparently does not influence adenosine production or elimination; (d) A1 receptor‐guanine nucleotide binding protein coupling is maximal in this preparation; and (e) Mg2+ decreases the dissociation rate of bound endogenous adenosine from A1 receptors, thus limiting the access of [3H]CHA and [3H]DPCPX to the receptors. Thus, enhancement of endogenous adenosine binding to A1 receptors by Mg2+ is a complicating factor in receptor autoradiography and may be so in other preparations as well.