bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Biochemical Characterization of a Neural Proteoglycan Expressing the L5 Carbohydrate Epitope
Authors: Streit Andrea , Faissner Andreas , Gehrig Bernd , Schachner Melitta
Published: October 2006

Abstract: The monoclonal L5 antibody reacts with an N‐glycosidically linked carbohydrate structure which is present on the neural cell adhesion molecule L1, neural chondroitin sulfate proteoglycans, and other not yet identified glycosylated proteins. Using this antibody, we isolated and characterized proteoglycans from adult mouse brain and cultured astrocytes biosynthetically labeled with Na235SO4 and a 3H‐amino acid mixture. Our data suggest that the L5 proteoglycans of both sources are identical in their biochemical properties. The apparent molecular mass of the L5 proteoglycan is approximately 500 kDa. Digestion of the iodinated L5 proteoglycan from mouse brain and of the [35S]methionine‐labeled L5 proteoglycan from cultured astrocytes with proteinase‐free chondroitinases ABC and AC revealed three major core proteins with apparent molecular masses of approximately 380, 360, and 260 kDa. These represent molecularly distinct protein cores.
Brain and Plasma Tetrahydroisoquinolines in Rats: Effects of Chronic Ethanol Intake and Diet
Authors: Collins Michael A. , Ung‐Chhun Neng , Cheng Bhe Y. , Pronger Debra
Published: October 2006

Abstract: Brain concentrations of salsolinol (SAL), a simple tetrahydroisoquinoline (sTIQ) condensation product of do‐pamine (DA) and acetaldehyde, are reported to increase in chow‐fed rats drinking ethanol/H2O ad libitum. However, our analyses showed that rat chow contains traces of SAL and, as previously reported, appreciable 3,4‐dihydroxyphe‐nylalanine (DOPA), a sTIQ precursor. To examine the effect of consumption of ethanol in a DOPA‐ and SAL‐free diet on endogenous sTIQs, we analyzed two brain regions and blood plasma of rats undergoing prolonged intake (3 weeks and 23 weeks) of liquid diet containing 6.6% ethanol or iso‐caloric carbohydrate. SAL and three other DA‐related sTIQs were quantitated using capillary gas chromatography/mass spectrometry in the selected ion mode with deuterated standards. In accord with studies on ethanol/chow‐fed rats. sTIQ concentrations in hypothalamus were elevated after 3 weeks of ethanol, although after 23 weeks, hypothalamic sTIQs were either unchanged or reduced (O‐methylated SAL). Furthermore, sTIQ concentrations in corpus triatum and, with one exception, plasma were not altered by ethanol ingestion for either duration. (However, 23 weeks of ethanol intake significantly reduced the striatal concentrations of DA and its acid metabolite, presumably reflecting neurotoxicity.) Reasoning that DOPA in diet might underlie the reported ethanol‐dependent increases in striatal sTIQs, we found that l‐DOPA supplementation (500 μg/rat/day) of EtOH/Iiquid diet‐fed rats for 13 weeks tended to increase striatal SAL. Overall, the data indicate that elevations in endogenous sTIQ concentrations due to prolonged ethanol intake depend on the brain region, duration of intake, and even associated dietary constituents. In that regard, the higher striatal SAL concentrations in rats drinking ethanol ad libitum could have been facilitated by DOPA and perhaps SAL consumed in lab chow.
Enzyme Activities in Relation to pH and Lactate in Postmortem Brain in Alzheimer‐Type and Other Dementias
Authors: Yates Celia M. , Butterworth John , Tennant Mara C. , Gordon Alexander
Published: October 2006

Abstract: Phosphate‐activated glutaminase, glutamic acid decarboxylase, pyruvate dehydrogenase, succinic dehydrogenase, pH, and lactate were measured in frontal cortex and caudate nucleus of postmortem brains from cases of Alzheimer‐type dementia (ATD), Down's syndrome, Huntington's disease, and one case of Pick's disease, as well as from sudden death and agonal controls. Lactate levels were higher and pH, phosphate‐activated glutaminase, and glutamic acid decarboxylase levels were lower in the agonal controls than in the sudden death controls. Phosphate‐activated glutaminase and glutamic acid decarboxylase were correlated with tissue pH and lactate, and also were reduced by in vitro acidification, suggesting that the low activities of these enzymes in agonal controls were related to decreased pH consequent upon lactate accumulation. Compared with control tissues at the same pH, phosphate‐activated glutaminase and glutamic acid decarboxylase were unaltered in ATD and Down's frontal cortex and reduced in Huntington's caudate nucleus, and glutamic acid decarboxylase was reduced in Huntington's frontal cortex. These data suggest that GABAergic neurons are not affected in ATD and confirm the GABAergic defect in Huntington's disease. Pyruvate dehydrogenase and succinic dehydrogenase activities were the same in agonal controls and sudden death controls and were unaffected by acid pH and lactate in vitro, and pyruvate dehydrogenase was not correlated with pH or lactate. Reduced pyruvate dehydrogenase in frontal cortex of individual ATD, Down's, and Pick's cases. and in the caudate nucleus of Huntington's and Down's cases, was accompanied by gliosis/neuron loss. We conclude that decreased pyruvate dehydrogenase reflects neuronal loss.
[3H]Thienylcyclohexylpiperidine Binding Activity in Brain Synaptic Membranes Treated with Triton X‐100
Authors: Ogita Kiyokazu , Nabeshima Toshitaka , Yoneda Yukio
Published: October 2006

Abstract: Binding activity of [3H]thienylcyclohexylpiperidine was examined using rat brain synaptic membranes treated with Triton X‐100. This compound is proposed to be a noncompetitive antagonist for the N‐methyl‐d‐aspartate (NMDA)‐sensitive subclass of brain excitatory amino acid receptors. The activity decreased in proportion to increasing concentrations of the detergent up to 0.08%. In vitro addition of l‐glutamate (Glu) partially restored the decreased activity caused by this Triton treatment, whereas further addition of glycine (Gly) entirely reversed the loss of activity to the level found in membranes extensively washed but not treated with a detergent. These stimulatory effects were found to be due to the acceleration of the association of ligand. The rank order of potentiation of the activity coincided well with that of the affinity for the NMDA‐sensitive subclass among numerous Glu analogs. The potentiation by Gly as well as Glu was invariably prevented by competitive NMDA antagonists, such as dl‐2‐amino‐5‐phosphonovalerate and (×)‐3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonate, but not by strychnine. No significant difference was observed between pharmacological profiles of the activities in synaptic membranes treated and not treated with Triton X‐100, except haloperidol. The potency of this σ‐ligand to inhibit the activity was greatly reduced by the Triton treatment in the presence of both Glu and Gly. These results suggest that the regulatory properties of Triton‐treated synaptic membranes remain unchanged in terms of the interaction within the NMDA receptor complex.
Partial Purification and Characterization of the Vacuolar H+‐ATPase of Mammalian Synaptic Vesicles
Authors: Floor Erik , Leventhal Phillip S. , Schaeffer Susan F.
Published: October 2006

Abstract: Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H+‐ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57‐kDa (B) and 39‐kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H+‐ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute −20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vacuolar H+‐ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake. Moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of ∼ 1.7 nmol/mg of protein. These findings indicate that the vacuolar H+‐ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.
Nerve Growth Factor Stimulation of Arachidonic Acid Release from PC12 Cells: Independence from Phosphoinositide Turnover
Authors: Fink Donald W. , Guroff Gordon
Published: October 2006

Abstract: The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor‐stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose‐response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor‐stimulated release of [3H]‐arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor‐stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.
The Effects of Opioid Peptides on Dopamine Release in the Nucleus Accumbens: An In Vivo Microdialysis Study
Authors: Spanagel Rainer , Herz Albert , Shippenberg Toni S.
Published: October 2006

Abstract: An involvement of the mesolimbic dopamine (DA) system in mediating the motivational effects of opioids has been suggested. Accordingly, the present study employed the technique of in vivo microdialysis to examine the effects of selective μ‐, δ‐, and k‐ opioids on DA release in the nucleus accumbens (NAC) of anesthetized rats. Microdialysis probes were inserted into the NAC and perfusates were analyzed for DA and its metabolites, dihydroxyphenylacetic acid (DO‐PAC) and homovanillic acid (HVA), using a reverse‐phase HPLC system with electrochemical detection for separation and quantification. Intracerebroventricular (i.c.v.) administration of selective μ‐opioid [D‐Ala2, N‐methyl‐Phe4, Gly5‐ol]‐enkephalin (DAMGO) or δ‐opioid [d‐Pen2, d‐Pen5]‐en‐kephalin (DPDPE) agonists, at doses that function as positive reinforcers in rats, resulted in an immediate and significant increase in extracellular DA. DOPAC and HVA levels were also significantly increased. The effects of DAMGO were blocked by the selective μ‐antagonist d‐Pen‐Cys‐Tyr‐d‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) whereas those of DPDPE were blocked by the δ‐antagonist allyl2‐Tyr‐Aib‐Aib‐Phe‐Leu‐OH (ICI 174,864). In contrast to μ‐ and δ‐agonists, the k‐agonist N‐CH3‐Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐N‐CH3‐Arg‐d‐Leu‐NHC2H5 (E‐2078). a dynorphin analog that produces aversive states, decreased DA release in a biphasic manner. Norbin‐altorphimine, a selective k‐antagonist, could block this effect. These results demonstrate that μ‐, δ‐, and k‐opioid agonists differentially affect DA release in the NAC and this action is centrally mediated.
Interaction of σ‐Compounds with Receptor‐Stimulated Phosphoinositide Metabolism in the Rat Brain
Authors: Candura Stefano M. , Coccini Teresa , Manzo Luigi , Costa Lucio G.
Published: October 2006

Abstract: σ‐Receptors are nonopioid, nondopaminergic receptors that bind with high affinity several antipsychotic drugs and appear to be involved in regulation of posture and movement. The second messenger system coupled to these receptors is still unknown. Recently, an inhibitory effect of various σ‐compounds on carbachol‐stimulated phosphoinositide metabolism has been reported. We have investigated the effect of six σ‐compounds on carbachol‐ and norepinephrine‐stimulated 3H‐inositol phosphate accumulation in rat cerebral cortex slices. All compounds tested had a dose‐dependent inhibitory effect on both systems, although their order of potency differed between neurotransmitters. Pentazocine and 1,3‐di‐o‐tolylguanidine were the most potent inhibitors of carbachol‐stimulated phosphoinositide turnover (IC50= 31.5 and 45.7 μM, respectively), while haloperidol showed the greatest potency on the norepinephrine‐coupled system (IC50= 3.5 μM). In the presence of IC50 concentrations of these inhibitors, the dose‐response curves for the agonists were shifted to the right and the EC50 values were significantly increased. σ‐Compounds also inhibited the binding of [3H]quinuclidinyl benzilate to muscarinic receptors and of [3H]prazosin to α1‐adrenoceptors in cortical membranes. In the presence of IC50 concentration (11 μM) of 1,3‐di‐o‐tolylguanidine, no differences were found in the maximal number of muscarinic binding sites, whereas the dissociation constant increased approximately fivefold, indicating a decrease of the radioligand's affinity for the receptor. These results indicate that σ‐compounds, at micromolar concentrations, inhibit muscarinic and α1‐adrenergic receptor‐coupled phosphoinositide metabolism, probably through an interaction with the neurotransmitter recognition sites.
Changes in Glycosaminoglycans During the Neuritogenesis in PC12 Pheochromocytoma Cells Induced by Nerve Growth Factor
Authors: Katoh‐Semba Ritsuko , Oohira Atsuhiko , Kashiwamata Shigeo
Published: October 2006

Abstract: Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat‐shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat‐shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in ∼30% of cells) and 9 (in 60–70%) days of culture, respectively, (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF‐treated cultures, the amounts of cell‐associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [35S]sulfate during the last 48 h of the culture were 1.5‐to twofold higher in the NGF‐treated cultures than in the respective controls at any time. (e) The amount of cell‐associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [35S]sulfate‐labeled CS associated with the cells and released into the medium, (f) NGF enhanced the amount of N‐sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4‐sulfate units to 6‐sulfate units in CS. (g) At 3 days in culture, the uptake of [35S]sulfate by PC12 cells was lower in the NGF‐treated culture than in the nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF‐treated culture than in the non‐treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural change of HS induced by NGF occur preceding the neurite elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.
Acetylcholine Translocating Protein: Mediatophore at Rat Neuromuscular Synapses
Authors: Israël Maurice , Lesbats Bernard , Sbia Mohamed , Morel Nicolas
Published: October 2006

Abstract: The neuromuscular synapses of the rat sternomastoid muscles contain a membrane protein, mediatophore, that endows artificial membranes with a calcium‐dependent acetylcholine release mechanism. Mediatophore and choline acetylase had similar distributions along the muscle. Sciatic nerve membranes contain mediatophore, and a purified preparation was obtained from the nerve.
Binding of the Adenosine A2 Receptor Ligand [3H]CGS 21680 to Human and Rat Brain: Evidence for Multiple Affinity Sites
Authors: Wan W. , Sutherland G. R. , Geiger J. D.
Published: October 2006

Abstract: A new radiolabeled adenosine receptor agonist, 2‐[p‐(2‐carboxyethyl) phenethylamino]‐5′‐.N‐ethylcarboxamidoadenosine (CGS 21680), apparently specific for high‐affinity binding sites of the A2 subtype in rat brain, was used to identify and pharmacologically characterize adenosine receptors in human brain. The binding of [3H]CGS 21680, as determined by standard radioligand binding technique in the presence of exogenously added adenosine deaminase, reached equilibrium after 40 min at 25°C. In saturation studies, a single class of high‐affinity binding sites with values for KD of 22 ± 0.5 nM and Bmax of 444 ± 63 fmol/mg of protein were observed. Similar binding characteristics were observed regardless of whether rapid filtration or centrifugation was used to separate bound versus free ligand. Of the 14 brain regions examined, [3H]CGS 21680 binding was highest in putamen, followed by globus pallidus and caudate nucleus. The level of [3H]CGS 21680 binding in these areas of basal ganglia was identical to 5′‐N‐[3H]ethylcarboxamidoadenosine ([3H]NECA) binding in the presence of 50 nM N6‐cydopentyladenosine (CPA). The rank order of agonist potencies as determined by a series of competition experiments was NECA > CGS 21680 > 2‐chloroadenosine > N6‐(R)‐phenylisopropyladenosine > N6‐cyclohexyladenosine > N6‐(S)‐phenylisopropyladenosine. This potency order was the same for the binding of [3H]CGS 21680 to rat, and of [3H]NECA in the presence of 50 nM CPA to rat and human, brain membranes. Data from competition studies with labeled and unlabeled CGS 21680 when analyzed by nonlinear regression demon strated the presence of three sites in human basal ganglia with mean values for KD of 16, 1,232, and 15,202 nM, and for Bmax of 305, 3,045, and 30,45 1 fmol/mg of protein. When similar competition experiments were conducted in the absence or presence of 50 nM CPA to determine if the lower‐affinity binding components corresponded to labeling of A1 sites, we found that the binding data conformed best to a two‐component binding model with KD values of 28 and 2,468 nM and Bmax values of 392 and 3,536 fmol/mg of protein for the high‐ and low‐affinity sites, respectively. For rat striatum, high‐ and low‐affinity sites were observed with kinetic values similar to those of the two higher‐affinity sites in human brain. The high‐affinity [3H]CGS 21680 binding sites most likely represent A2a sites. The low‐affinity sites appear to be composed of a combination of both A1‐like and A2b‐like adenosine receptors. [3H]CGS 21680 has the potential to become a useful probe in determining the relative importance of A2 sites in mediating the actions of adenosine in structures comprising the basal ganglia and in other discrete regions of mammalian CNS, and may represent the first radioligand available for the study of the previously proposed A2b receptor sites.
Characterization in Mammalian Brain of a DARPP‐32 Serine Kinase Identical to Casein Kinase II
Authors: Girault Jean‐Antoine , Hemmings Hugh C. , Zorn Stevin H. , Gustafson Eric L. , Greengard Paul
Published: October 2006

Abstract: DARPP‐32, a dopamine‐ and cyclic AMP‐regulated phosphoprotein of Mr 32,000, is phosphorylated in vitro by casein kinase II at a site which is also phosphorylated in intact cells. In the present study, we show that a protein kinase activity, present in caudate‐putamen cytosol, phosphorylates DARPP‐32 on a seryl residue located on the same thermolytic peptide that is phosphorylated by purified casein kinase II. This DARPP‐32 serine kinase was indistinguishable from casein kinase II on the basis of a number of biochemical criteria. Excitotoxic lesions of the caudate‐putamen and immunocytochemistry revealed the presence of casein kinase II in the medium‐sized striatonigral neurons which are known to contain DARPP‐32. Casein kinase II activity was high in all rat brain regions studied, and casein kinase II‐like immunoreactivity was detected in most brain neurons, although some neuronal populations (e.g., cortical pyramidal cells and large striatal neurons) were stained more intensely than others. In rat caudate‐putamen, 45% of the total casein kinase II activity was in the cytosol and 20% in the synaptosomal fraction. In mouse cerebral cortex and caudate‐putamen, casein kinase II activity was high at embryonic day 16, and remained elevated during development. In addition to DARPP‐32, several major substrates for casein kinase II were observed specifically in brain, but not in liver extracts. The high activity of casein kinase II in brain from the embryonic period to adult age and the existence of a number of specific substrates suggest that this enzyme may play an important role in both developing and mature brain, possibly in modulating the responsiveness of target proteins to various extracellular signals.
Corticosterone Regulates the Expression of ADP‐Ribosylation Factor Messenger RNA and Protein in Rat Cerebral Cortex
Authors: Duman R. S. , Winston S. M. , Clark J. A. , Nestler E. J.
Published: October 2006

Abstract: ADP‐ribosylation factors (ARFs) comprise a family of small GTP‐binding proteins found in brain and other tissues. Recent studies have demonstrated that the expression of the larger heterotrimeric guanine nucleotide binding proteins is under control by steroid hormones. Therefore, in the present study, we examined the influence of glucocorticoids on the expression of ARF raRNA and protein, using specific cDNA probes and antisera, respectively. Chronic administration of corticosterone (7 days) significantly increased levels of mRNA for ARF1 and ARF3, two subtypes of ARF, in rat cerebral cortex. Chronic administration of corticosterone was also found to increase levels of ARF immunoreactivity in this brain region. However, 1‐day administration of corticosterone did not influence levels of mRNA for either ARF1 or ARF3. In contrast to corticosterone, bilateral adrenalectomy (7 days after surgery) was found to decrease ARF1 and ARF3 message relative to sham controls; this effect of adrenalectomy was reversed by corticosterone treatment. These results demonstrate that the expression of ARF is under hormonal control and may underlie aspects of glucocorticoid action on neuronal function.
Expression of the Ganglioside GD3 in Human Meningiomas Is Associated with Monosomy of Chromosome 22
Authors: Fredman P. , Dumanski J. , Davidsson P. , Svennerholm L. , Collins V. P.
Published: October 2006

Abstract: The ganglioside composition of 59 meningiomas has been compared with a molecular genetic analysis of chromosome 22 in the same specimens. Major gangliosides were GM3 (II3NeuAc‐LacCer) and/orGD3 [II3(NeuAc)2‐LacCer]. In specimens with no or partial deletions of chromosome 22, the GM3 ganglioside predominated, and the mean value for GM3, 61% of total sialic acid, was around four times higher than that for GD3. A loss of chromosome 22, found in 56% of the specimens, was shown to be associated with an increase in the proportion of ganglioside GD3, with the ratio between mean values of GM3 and GD3 being ∼ 1:1. Logistic regression revealed that the probability of predicting monosomy of chromosome 22 by the GD3 proportion was 66%.
Rat Brain Synaptosomes Prepared by Phase Partition
Authors: Enriquez J. A. , Sánchez‐Prieto J. , Blanco M. T. Muiño , Hernandez‐Yago J. , López‐Pérez M. J.
Published: October 2006

Abstract: Synaptosomes from rat forebrain can easily be isolated by combining centrifugation with partition in an aqueous two‐phase system composed of dextran T500 and polyethylene glycol 4000 in which synaptosomes have an extreme affinity for the upper phase. The fraction thus obtained has been characterized by electron microscopy and biochemical markers for synaptosomes and some other cell components. The contamination by microsomes, free mitochondria, and myelin was 4.4, 3.2, and 0.1%, respectively. The morphometric analysis of the electron micrographs shows that >60% of the structures are synaptosomes. This preparation of the isolation procedure is remarkably short (<1 h), formance as assayed by their respiratory activities and ATP level in the absence and presence of depolarizing agents. Synaptosomes prepared by phase partition release the neurotransmitter glutamate in a Ca2+‐dependent manner. The duration of the isolation procedure is remarkably short (<1 h), no ultracentrifuge is required, and the method can be applied for small‐ or large‐scale preparations.
Presynaptic Glutamate/Quisqualate Receptors: Effects on Synaptosomal Free Calcium Concentrations
Authors: Adamson P. , Hajimohammadreza I. , Brammer M. J. , Campbell I. C. , Meldrum B. S.
Published: October 2006

Abstract: Intracellular free [Ca2+]j was measured using fura‐2 in synaptosomes prepared from cerebral cortices of adult male rats (12 weeks). L‐(+)‐Glutamate, d‐(‐)‐glutamate, and quisqualate produced similar dose‐dependent increases in [Ca2+]i, with EC50 values of 0.38 μM, 0.74 μM, and 0.1 μM, respectively, and maximum increases of approximately 40%. Ibotenate showed less affinity (EC50 4.4 μM) but had a greater maximum effect (57%). N‐methyl‐d‐aspartate (NMDA) and α‐ amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) did not increase [Ca2+]i‐ The increases in [Ca2+]i induced by quisqualate and ibotenate were not diminished in the absence of extrasynaptosomal Ca2+. l‐2‐Amino‐4‐phosphonobutyrate (L‐AP4) (1 μM) completely blocked the changes in [Ca2+]i induced by l‐(+)‐glutamate, d‐(‐)‐glutamate, quisqualate, or ibotenate. The effects of quisqualate and ibotenate on [Ca2+]i were also blocked by coincubation of synaptosomes with l‐(+)‐serine‐O‐phosphate (L‐SP) (1 mM) (which, like L‐AP4, blocks the effects of quisqualate and ibotenate on inositol phospholipid metabolism). 6‐Cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) had no effect on agonist‐mediated increases in [Ca2+]i when coincubated with either quisqualate or ibotenate. These data are consistent with the existence of presynaptic glutamate receptors (of the excitatory amino acid metabotropic type) which activate phospholipase C leading to the elevation of inositol 1,4,5‐trisphosphate and release of Ca2+ from intracellular stores.
Administration of Vigabatrin (γ‐Vinyl‐γ‐Aminobutyric Acid) Affects the Levels of Both Inhibitory and Excitatory Amino Acids in Rat Cerebrospinal Fluid
Authors: Halonen Toivo , Pitkänen Asla , Riekkinen Paavo J.
Published: October 2006

Abstract: The effect of vigabatrin (γ‐vinyl‐γ‐aminobutyric acid), a new anticonvulsant drug, on the transmitter amino acids in rat cisternal CSF was studied. CSF was collected through a permanently implanted polyethylene cannula from freely moving rats at 5, 24, 48, and 96 h after administration of 1,000 mg/kg of vigabatrin. The free γ‐aminobutyric acid (GABA) level was elevated maximally (13.5‐fold; p < 0.01) at 24 h after injection, The homocarnosine (GABA‐histidine) level also was increased (123%; p < 0.01) at 24 h after injection, and its concentration remained at the same level for the next 3 days. Glycine and taurine concentrations had increased [31% (p < 0.05) and 63% (p < 0.01), respectively] at 5 h after injection. It is interesting that the levels of glutamate and aspartate increased [330% (p < 0.05) and 421% (p < 0.01), respectively] at 96 h after injection, the time when the free GABA level had returned to the baseline concentration and the vigabatrin level was 3% of the maximal concentration. The present study indicates that a single dose of vigabatrin in rats elevates levels of both the inhibitory and excitatory amino acids in CSF. However, the temporal profile of observed changes in relation to vigabatrin injection shows that neither the long‐lasting elevation of GABA content nor the increase in glutamate and aspartate levels correlates with the level of vigabatrin in CSF. These findings suggest that the excitatory mechanisms are also augmented following acute administration of vigabatrin, especially when the content of GABA had decreased to the baseline level and the level of vigabatrin was low.
Substance P Enhances Desensitization of the Nicotinic Response in Bovine Chromaffin Cells but Enhances Secretion upon Removal
Authors: Lyford L. K. , Kent‐Braun J. A. , Westhead E. W.
Published: October 2006

Abstract: A fundamental process in neurosecretion is desensitization, or a declining response to a stimulus. The response of chromaffin cells to continuous nicotinic stimulation, secretion of catecholamines, desensitizes within a few minutes. The neuropeptide substance P (SP) has been reported to prevent desensitization in culture dish experiments and to enhance desensitization in patch clamp studies. In the present study, these contradictory responses have been demonstrated and the apparent contradictions resolved. We have measured catecholamine secretion by on‐line electrochemical detection in a constant‐pressure flow system. Isolated chromaffin cells cultured on quartz plates were stimulated with the nicotinic agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) in the presence and absence of SP. SP inhibited secretion and increased the rate of desensitization compared with stimulation by DMPP alone. However, when the cells were stimulated a second time with DMPP alone immediately after 5‐min stimulation with SP + DMPP, the rate of desensitization was markedly lower than the control. Removal of SP after a desensitizing stimulation with SP + DMPP caused a slow secondary release of catecholamine in response to the continued stimulation with DMPP. The kinetic analysis of the secretory response shows that the primary response to SP is enhanced desensitization, but that upon removal of SP the response to DMPP desensitizes less rapidly. We suggest that SP protects some receptors from nicotinic desensitization while holding them in an inactive state, and that upon removal of SP these receptors can slowly respond to DMPP.
Characterization and Distribution of Transferrin Receptors in the Rat Brain
Authors: Mash Deborah C. , Pablo John , Flynn Donna D. , Efange Simon M. N. , Weiner William J.
Published: October 2006

Abstract: Transferrin receptors were characterized with 125Iferrotransferrin on membrane fractions prepared from the rodent forebrain. The distribution of transferrin receptors in the rat brain was investigated further by in vitro autoradiography. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 2 nM and a binding site density of 15 pmol/g. The Hill coefficient derived from these data was 1.05. indicating the absence of cooperativity and that 125I‐ferrotransferrin binds to a single class of sites. Estimates of the kinetically determined Kd for forebrain membranes were within the 2–4 nM range, in agreement with the equilibrium measurements. Apotransferrin and ferrotransferrin competitively displaced the binding of 125I‐ferrotransferrin, while ferritin, albumin, and cytochrome c failed to compete for the binding site. Ceruloplasmin, the copper transport protein, was a weak inhibitor of 125I‐ferrotransferrin binding. Autoradiographic localization studies demonstrate a heterogeneous distribution of transferrin receptors in the rat brain. Transferrin receptor densities were markedly elevated over the cerebral cortex and the hippocampus. Moderate to high 125I‐ferrotransferrin binding was also apparent throughout areas involved in motor functions, including the caudate‐putamen, the nucleus accumbens, the substantia nigra, the red nucleus, and the cerebellum.
Myelin Proteolipid Protein Contains Thioester‐Linked Fatty Acids
Authors: Bizzozero Oscar A. , Good Louise K.
Published: October 2006

Abstract: Myelin proteolipid protein (PLP) is known to contain long‐chain, covalently bound fatty acids. Previous studies, including our own, have suggested the occurrence of an oxyester type of linkage between fatty acids and PLP. However, we found that protein‐SH groups are required in the acylation reaction, suggesting the possible presence of thioesters. In the present study, we have examined the nature of the acyl‐PLP linkages by determining whether free thiol groups are generated on removal of fatty acids. Incubation of reduced and carboxyamidomethylated proteolipid apoprotein (RCM‐APL) with 0.2 M hydroxylamine and [14C]iodoacetamide at pH 7.5 and 37°C resulted in the release of fatty acids and the concomitant labeling of newly formed thiol groups. Incubation with Tris or methylamine at pH 7.5 failed to remove fatty acids and generate free ‐SH groups. The possibility that on treatment buried thiol groups became exposed was essentially excluded because (1) similar results were obtained in 2‐chloroethanol, a solvent in which acylated and deacylated PLP have the same conformation, and (2) small PLP peptides were labeled only in the presence of hydroxylamine. On incubation with [14C]methylamine at pH 9.0. RCM‐APL was not labeled, thus excluding the occurrence of intramolecular thiol esters. On the other hand, fatty acids were released as radioactive N‐methyl fatty acylamide, indicating the presence of intermolecular thioesters between fatty acids and protein. These results demonstrate that a large proportion of fatty acids covalently bound to PLP are linked to ‐SH groups.
Serotonin Metabolism by Monoamine Oxidase in Rat Primary Astrocyte Cultures
Authors: Fitzgerald Lawrence W. , Kaplinsky Lynne , Kimelberg Harold K.
Published: October 2006

Abstract: The oxidative deamination of serotonin (5‐HT) to 5‐hydroxyindoleacetic acid (5‐HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5‐HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5‐HT concentration over the tested range of 5 ± 10−7 to 10−4M. At 5 ± 10−7M 5‐HT, intracellular 5‐HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5‐HT taken up into astrocytes is not stored for re‐release, but is rapidly metabolized to 5‐HIAA, which is then extruded from the cell. At 5 ± 10−7M 5‐HT, 5‐HIAA formation in intact cells was blocked 63% by the selective high‐affinity 5‐HT uptake inhibitor fluoxetine. 5‐HT oxidation to 5‐HIAA is carried out principally by MAO‐A, because clorgyline was more effective at inhibiting the production of 5‐HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000‐fold more effective than pargyline at inhibiting MAO activity toward 14C‐labelled 5‐HT. However, the relatively selective MAO‐B substrate β‐phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO‐B activity; the Km values for MAO‐A oxidation of 5‐HT and MAO‐B oxidation of PEA were 135 and 45 μM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (>20 γM) were oxidized by both MAO‐A and MAO‐B isozymes. These data support the idea that astrocytes in situ might take up 5‐HT and metabolize it to 5‐HIAA using the MAO‐A isozyme. However, sufficient activity of the MAO‐B isozyme appears to be present in astrocytes for metabolism of amines which also show MAO‐B selectivity.
Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy
Authors: Olasmaa Marjut , Rothstein Jeffrey D. , Guidotti Alessandro , Weber Richard J. , Paul Steven M. , Spector Sydney , Zeneroli Maria L. , Baraldi Mario , Costa Erminio
Published: October 2006

Abstract: The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti‐benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti‐benzodiazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained ∼ 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased ∼ 10‐fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.
Blood‐Brain Transfer of l‐Phenylalanine Declines After Peripheral but Not Central Nervous Administration of Vasopressin
Authors: Brust Peter , Diemer Nils Henrik
Published: October 2006

Abstract: To determine whether a previously reported effect of vasopressin on blood‐brain transfer of leucine extends to other large neutral amino acids, we measured the regional blood‐brain transfer of l‐phenylalanine with the integral technique. Intravenous co‐injection of l‐phenylalanine and arginine vasopressin (30 nmol to 10 pmol) resulted in a decrease of the permeability‐surface area (PaS) product of phenylalanine of between 11 and 39%. In addition, the peptide elicited a decrease of the cerebral blood flow of between 11 and 56% combined with a drastic decrease of the cardiac output (32–64%) and an elevation of the blood pressure to ∼150% of control values. However, we found no changes of the cardiac output, the blood pressure, or the PaS product of phenylalanine after microdialysis (30 min, 5 μl min−1) of arginine vasopressin (15 μmol l−1) into the dorsal hippocampus, but cerebral blood flow was decreased. The results support the hypothesis that arginine vasopressin receptors at the blood‐brain barrier are involved in the regulation of large neutral amino acid transfer from blood to brain and indicate that these receptors are located at the luminal membrane of the endothelial cells.
Homologous Desensitization of the D1 Dopamine Receptor
Authors: Balmforth Anthony J. , Warburton Philip , Ball Stephen G.
Published: October 2006

Abstract: Preincubation of D384 cells, derived from the human astrocytoma cell line G‐CCM, with dopamine resulted in a time‐dependent attenuation of cyclic AMP responsiveness to subsequent dopamine stimulation. This effect was agonist specific because the prostaglandin E1 (PGE,) stimulation of cyclic AMP of similarly treated cells remained unchanged. The attenuation by dopamine was concentration dependent with a maximum observed at 100 μM. A comparison of dopamine concentration‐response curves of control and dopamine‐preincubated cells revealed no change in the Ka apparent value, but a marked attenuation of the maximal response. Preincubation of cells with dopamine in the presence of D1 but not D2 selective antagonists partially prevented the observed attenuation. Attenuations in dopamine responsiveness were also obtained when D384 cells were preincubated with D1 but not D2 receptor agonists. The level of attenuation attained related to agonist efficiency in stimulating cyclic AMP: SKF38393 < 3,4‐dihydroxynomifensine < fenoldopam < 2‐amino‐6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene = dopamine. However, increasing the efficiency of 3,4‐dihydroxynomifensine stimulation of cyclic AMP, using the synergistic effect of adding a low concentration of forskolin, produced no further change in the attenuation of the subsequent response to dopamine. Thus, the D1 dopamine receptors expressed by D384 cells undergo homologous desensitization. Uncoupling of the D1 dopamine receptor appears to be independent of cyclic AMP formation, analogous to a mechanism proposed for the β‐adrenergic receptor.
Turnover Rate of Brain Acetylcholine Using HPLC Separation of the Transmitter
Authors: Bertrand N. , Bralet J. , Beley A.
Published: October 2006

Abstract: A simple, reliable method was developed for measuring brain acetylcholine (ACh) turnover using HPLC methodology. Mice were injected intravenously with [3H]choline ([3H]Ch), and the turnover rate of ACh was calculated from the formation of [3H]ACh. Ch and ACh were separated from phosphorylcholine and from other radioactive compounds using tetraphenylboron extraction and counterion/reverse‐phase chromatography. Endogenous Ch and ACh were quantified electrochemically through hydrogen peroxide production in a postcolumn reactor containing covalently bonded ACh esterase and Ch oxidase. Labeled Ch and ACh were quantified in the same sample by collecting the chromatographic fractions for radioactive content determinations. The method is rapid, well adapted to large series, and highly reproducible, with recoveries of 72.1% for Ch and 79.3% for ACh. The turnover value in mouse cerebral hemispheres was 16.02 nmol g‐1 min‐1 and decreased to 9.94 nmol g‐1 min‐1 in mice treated with oxotremorine.
Changes in α‐Tubulin and Actin Gene Expression During Optic Nerve Regeneration in Frog Retina
Authors: Mizobuchi Tokihide , Yagi Yasuyuki , Mizuno Aritake
Published: October 2006

Abstract: The optic nerve of the bullfrog was transected and the regeneration process was investigated. We previously reported that α‐tubulin mRNA in the retina increased to a maximum 1–2 h after optic nerve transection with no specific change in actin mRNA. In the present investigation, we examined the long‐term effect of optic nerve transection. Northern blot analysis revealed that α‐tubulin mRNA increased again gradually after the rapid and transient increase and actin mRNA increased to a maximum at 7 days (more than twofold compared to the control retinas). The period during which actin mRNA reaches a maximal increase almost corresponds to the time lag between the axotomy and the initiation of axonal outgrowth. The main cytoskeletons of neuronal growth cones have been shown to consist of actin containing microfilaments. Therefore, the transient increase of actin mRNA may have a relationship to the initial out growth of axons. On the other hand, the rapid and transient increase of α‐tubulin mRNA observed in our previous studies is probably one of the initial responses of retinal ganglion cells to the axotomy, and the gradual increase in α‐tubulin mRNA observed in this study can probably be interpreted as provision of the structural materials necessary for axonal elongation.
Activation of Melatonin Receptor Sites Retarded the Depletion of Norepinephrine Following Inhibition of Synthesis in the C3H/HeN Mouse Hypothalamus
Authors: Fang Jun‐Ming , Dubocovich Margarita L.
Published: October 2006

Abstract: The aim of the present study was to determine the effect of activation of melatonin receptor sites on the activity of noradrenergic neurons in the C3H/HeN mouse brain. Changes in noradrenergic activity were assessed by measuring norepinephrine (NE) levels in the hypothalamus, frontal cortex, and hippocampus following inhibition of NE synthesis with α‐methyl‐p‐tyrosine (α‐MpT) (300 mg/kg, i.p., 2 h). 6 Chloromelatonin (1–30 mg/kg, i.p.) significantly retarded the α‐MpT‐induced decrease in NE levels in the hypothalamus, but not in hippocampus and frontal cortex. This effect was observed at 30 min and 60 min after 6‐chloromelatonin administration and was dose dependent. At noon, when the levels of endogenous melatonin are low, the melatonin receptor antagonist luzindole (30 mg/kg, i.p., 30 min) did not affect the depletion of NE by α‐MpT; however, it (1–30 mg/ kg) completely antagonized the 6‐chloromelatonin‐induced reduction of NE depletion elicited by α‐MpT in hypothalamus. These results suggest that activation of melatonin receptor sites in brain of C3H/HeN mouse retarded the depletion of NE elicited by α‐MpT. At midnight, when the levels of melatonin are high, luzindole (30 mg/kg) significantly accelerated the depletion of NE by α‐MpT in hypothalamus, but not in frontal cortex or hippocampus, suggesting activation of melatonin receptor sites by endogenous melatonin. We conclude that activation of melatonin receptor sites in C3H/HeN mouse brain by endogenous melatonin inhibits the activity of noradrenergic neurons innervating the hypothalamus.
Heterotropic Cooperativity Between Putative Recognition Sites for Progesterone Metabolites and the Atypical Benzodiazepine Ro 5–4864
Authors: Belelli Delia , McCauley Linda , Gee Kelvin W.
Published: October 2006

Abstract: The binding of the cage convulsant t‐butylbicyclo phosphorothionate (TBPS) and 36CI‐ uptake by synaptoneurosomes were used to test the ability of progesterone metabolites to modulate allosterically the Ro 5–4864 (4′‐chlorodiazepam) binding site that is functionally coupled to the γ‐aminobutyric acid (GABA)/benzodiazepine receptor complex (GBRC) in rat brain. Dose‐dependent enhancement of [35S]TBPS binding by Ro 5–4864 occurs in rat cerebral cortex in the presence of the progesterone metabolites 5αpregnan‐3α‐ol‐20‐one (3α‐OH‐DHP) and 5α‐pregnan‐3α, 20α‐diol(pregnanediol). The pregnanediol effect is completely GABA dependent, whereas that of 3α‐OH‐DHP is not. Conversely, Ro 5–4864 opposed the action of 3α‐OH‐DHP by increasing the IC50 for 3α‐OH‐DHP inhibition of [35S]TBPS binding. In cortical snaptoneurosomes, Ro 5–4864 antag onized both 3α‐OH‐DHP and pregnanediol enhancement of GABA‐stimulated 36CI‐ uptake. In both binding and functional studies, pregnanediol showed limited efficacy relative to 3α‐OH‐DHP, as previously reported. These findings provide the initial evidence that the GBRC‐linked Ro 5–4864 binding site is allosterically coupled to the putative progesterone metabolite recognition site and confirm the GABA mimetic properties of 3α‐OH‐DHP and pregnanediol.
Bovine Brain Coated Vesicles Contain Adenosine A1 Receptors. Presence of Adenylate Cyclase Coupled to the Receptor
Authors: Gonzalez‐Calero Gloria , Martin Mairena , Cubero Ana , Andrés Antonio
Published: October 2006

Abstract: Clathrin‐coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6‐Cyclohexyl[3H]adenosine ([3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists N6CHA, N‐cyclopentyladenosine, 5′‐(N‐ethylcarboxamido)adenosine, and N6‐[(R)‐ and N6‐[(S)‐phenylisopropyl]adenosine and antagonists (theophylline, 3‐isobutyl‐1‐methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6‐phenylisopropyladenosine, showing 33 times more affinity for N6‐[(R)‐ than for N6‐[(S)‐phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis‐resistant GTP analogue guanyl‐5‐ylimidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.