bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Aging on Tyrosine Hydroxylase Protein Content and the Relative Number of Dopamine Nerve Terminals in Human Caudate
Authors: Wolf M. E. , LeWitt P. A. , Bannon M. J. , Dragovic L. J. , Kapatos G.
Published: October 2006

Abstract: This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence‐activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH‐containing nerve terminals or the concentration of TH protein were found in subjects 15–63 years of age. In light of previous studies showing an age‐related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH‐containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age‐related differences in the relative abundance of these forms were found.
Cell Damage Associated with Changing the Medium of Mesencephalic Cultures in Serum‐Free Medium Is Mediated via N‐Methyl‐D‐Aspartate Receptors
Authors: Driscoll Bernard F. , Law Mona J. , Crane Alison M.
Published: October 2006

Abstract: Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum‐free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N‐methyl‐D‐aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Ham's F‐12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbecco's modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change.
Modification of γ‐Aminobutyric AcidA Receptor Binding and Function by N‐Ethoxycarbonyl‐2‐Ethoxy‐1,2‐Dihydroquinoline In Vitro and In Vivo: Effects of Aging
Authors: Miller Lawrence G. , Lumpkin Monica , Galpern Wendy R. , Greenblatt David J. , Shader Richard I.
Published: October 2006

Abstract: The irreversible protein‐modifying reagent N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the γ‐aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration‐dependent decrease in receptor number for benzodiazepine, t‐butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10−4M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15‐1788, 1 or 10 μM, or the agonist lorazepam, 10 μM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10−4M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55‐65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15‐1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ. Administration of EEDQ to senescent mice (20 months) led to a similar decrease in benzodiazepine sites compared to young mice, but receptor density returned more slowly to control levels (calculated receptor t1/2: young, 25.3 h; senescent, 75.1 h). EEDQ modifies all three major sites on the GABAA receptor, with the greatest effect at the benzodiazepine site. This compound may be useful in assessing benzodiazepine receptor half‐life in vivo.
Synaptosomal Protein Kinase C Subspecies: A. Dynamic Changes in the Hippocampus and Cerebellar Cortex Concomitant with Synaptogenesis
Authors: Shearman Mark S. , Shinomura Tetsutaro , Oda Tomiichiro , Nishizuka Yasutomi
Published: October 2006

Abstract: The expression of protein kinase C (PKC) subspecies in synaptosomes prepared from a number of adult brain regions was compared. Cerebral cortical and thalamic/striatal synaptosomes were found to express three peaks of enzyme activity upon hydroxyapatite chromatography, corresponding to the type I(γ), type II(β), and type III(α) subspecies. Synaptosomes prepared from either the hippocampus or the cerebellar cortex, however, contained only two major peaks, corresponding to the α‐ and β‐subspecies, with barely detectable levels of the γ‐subspecies, even though these tissue areas were enriched in the latter enzyme. When the ontogenic pattern of hippocampal synaptosomal PKC subspecies was examined, it was found that at postnatal day 7, significant quantities of the γ‐subspecies were present and that this subspecies reached its peak levels at around postnatal day 14, before steadily declining to its adult level. Similar changes were observed also for the γ‐subspecies in cerebellar cortex synaptosomes. The dynamic changes in the synaptosomal PKC subspecies take place at a critical period in the development of the rat brain, concomitant with an active period of synaptogenesis, suggesting that it may play a role in synaptogenesis.
Synaptosomal Protein Kinase C Subspecies: B. Down‐Regulation Promoted by Phorbol Ester and Its Effect on Evoked Norepinephrine Release
Authors: Oda Tomiichiro , Shearman Mark S. , Nishizuka Yasutomi
Published: October 2006

Abstract: The effect of phorbol esters was investigated on the down‐regulation of protein kinase C (PKC) and on the release of [3H]norepinephrine (NE) in synaptosomes from the rat cerebrum. Treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) promoted the translocation of PKC activity in a P2 fraction from the cytosol to the membrane fraction and then its down‐regulation, in a dose‐dependent manner. TPA induced a rapid down‐regulation of the type II(β) and type III(α) subspecies, but did not change the activity of the type I(γ) subspecies in the cytosolic fraction for at least 15 min. The γ‐subspecies was subsequently decreased at a slower rate. In the synaptosomes thus having only the γ‐subspecies, a subsequent dose of TPA could not enhance K+‐evoked NE release, although, in the original synaptosomes, TPA was able to enhance K+‐evoked NE release. Pretreatment with TPA did not alter the K+‐evoked NE release itself. TPA was also found to enhance the K+‐evoked NE release from synaptosomes prepared from both hippocampus, which express the γ‐subspecies of PKC at a negligible level, and cerebral cortex, which have a significant level of the γ‐subspecies, to the same degree. These results suggest that the γ‐subspecies of PKC does not participate in the TPA‐enhanced K+‐evoked NE release from synaptosomes.
Are Several G Proteins Involved in the Different Effects of Endothelin‐1 in Mouse Striatal Astrocytes?
Authors: Marin P. , Delumeau J. C. , Durieu‐Trautmann O. , Le Nguyen D. , Prémont J. , Strosberg A. D. , Couraud P. O.
Published: October 2006

Abstract: High‐affinity specific receptors of endothelin (ET‐1) were identified on primary cultures of mouse embryo striatal astrocytes by binding experiments performed with 125I‐ET‐1. Stimulation of production of inositol phosphates, a biphasic increase of the intracellular calcium concentration, and inhibition of cyclic AMP accumulation were observed in the same cells under ET‐1 stimulation. Pretreatment of these cells with Bordetella pertussis toxin affected these effects to different extends, an observation suggesting that they are mediated by multiple transduction pathways, possibly involving several guanine nucleotide‐binding proteins.
Quinolinic Acid Stimulates Somatostatin Gene Expression in Cultured Rat Cortical Neurons
Authors: Patel Shutish C. , Papachristou Dimitrios N. , Patel Yogesh C.
Published: October 2006

Abstract: Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH‐diaphorase (NADPH‐d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three‐ to fivefold increases in SS‐like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N‐methyl‐D‐aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH‐d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH‐d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (‐)‐2‐amino‐5‐phosphon‐ovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p < 0.01) 1.6‐ and 2.5‐4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two‐ to fivefold by Quin over a 2‐ to 20‐h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose‐dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA. These increases were selective for SS mRNA and were not accompanied by any change in β‐actin mRNA. By contrast, glutamate at the single dose tested was without effect on both SSLI and SS mRNA. These results demonstrate that Quin induces a selective NMDA receptor‐mediated stimulation of SS gene expression and SS biosynthesis in vitro and suggest a similar mechanism for the augmented striatal SSLI in HD.
Long‐Term Activation of Protein Kinase C by Angiotensin II in Cultured Bovine Adrenal Medullary Cells
Authors: Tuominen Raimo K. , Hudson Pearlie M. , McMillian Michael K. , Ye Hong , Stachowiak Michal K. , Hong Jau‐Shyong
Published: October 2006

Abstract: Previous studies from our laboratory suggest that protein kinase C (PKC) is involved in the angiotensin II (AII)‐induced increase in the expression of genes encoding proen‐kephalin and catecholamine biosynthesizing enzymes in primary cultured bovine adrenal medullary (BAM) cells. The purpose of this study was to examine the effects of [Sar1]‐AII (S1‐AII), an AII agonist, on PKC activity in BAM cells. Thirty‐minute incubation with S1‐AII produced a dose‐dependent activation of PKC. The particulate PKC activity was significantly increased by 2 nM S1‐AII after both 30 min and 12 h of incubation. A high concentration of S1‐AII (200 nM) caused an increase in particulate PKC activity after 30 min of incubation and this increase was still observed after 18 h of continuous incubation. [Sar1,Thr8]‐angiotensin II (S1,T8‐AII) (100 μM), an AII antagonist, inhibited the effect of S1‐AII (20 nM) on PKC activity, suggesting a specific AII receptor‐mediated effect. An increase in BAM cell particulate PKC immunoreactivity after 18 h of S1‐AII treatment was observed in Western blot analysis of PKC‐immunoreactive protein (82 kDa). The persistent activation of PKC seen in this study is consistent with our hypothesis that PKC may mediate the S1‐AII‐induced increase in the expression of genes encoding proenkephalin and catecholamine synthesizing enzymes in BAM cells.
Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens
Authors: Checler Frédéric , Dauch Pascale , Masuo Yoshinori , Vincent Jean‐Pierre
Published: October 2006

Abstract: Whole homogenates and membrane‐bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24‐15, and endopeptidase 24‐11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24–15 mainly contributed to the catabolism of neurotensin in homogenates and membrane‐bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.
Desensitization of Metabotropic Glutamate Receptors in Neuronal Cultures
Authors: Catania M. V. , Aronica E. , Sortino M. A. , Canonico P. L. , Nicoletti F.
Published: October 2006

Abstract: Preexposure of cultured cerebellar neurons to glutamate reduced the stimulation of polyphosphoinositide (PPI) hydrolysis induced by subsequent addition of glutamate without affecting the response to the muscarinic receptor agonist carbamylcholine. Desensitization of glutamate‐stimulated PPI hydrolysis developed rapidly and persisted up to 48 h after removal of glutamate from the incubation medium. Stimulation of PPI hydrolysis by quisqualate was abolished in cultures pretreated with quisqualate or glutamate, but not with N‐methyl‐D‐aspartate (NMDA). In contrast, pretreatment with NMDA reduced the stimulation of PPI hydrolysis induced by a subsequent addition of NMDA, leaving the action of quisqualate intact. The lack of cross‐desensitization between NMDA and quisqualate supports the existence of two distinct subtypes of glutamate receptors coupled to PPI hydrolysis. Desensitization induced by a 30‐min (but not by a 6‐h) exposure to glutamate was attenuated or prevented by putative protein kinase C inhibitors, including mono‐ and trisialogangliosides, sphingosine, and polymyxin B, but not by inhibitors of arachidonic acid metabolism, nor by the nonselective calpain inhibitor leupeptin, nor by the lectin concanavalin A. These results suggest that desensitization of metabotropic glutamate receptors involves, at least in its rapid component, activation of protein kinase C.
Responsiveness of Striatal Dopamine Release in Awake Animals After Chronic 1‐Methyl‐4‐Phenylpyridinium Ion‐Induced Lesions of the Substantia Nigra
Authors: Santiago M. , Westerink B. H. C. , Rollema H.
Published: October 2006

Abstract: Extracellular concentrations of dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), and homovanillic acid were measured by microdialysis in rat striatum 1 month after a unilateral infusion via a dialysis probe of a high concentration (10 mM) of 1‐methyl‐4‐phenylpyridinium ion (MPP+) into the substantia nigra. The basal extracellular DA concentration at the lesioned side was about 20% of the concentration at the nonlesioned side. However, basal DOPAC dialysate levels from the lesioned striatum represented only 2.4% of those from the contralateral side. Intrastriatal infusion with nomifensine increased the dialysate content of DA about twofold and eightfold at the lesioned and nonlesioned sides, respectively. Co‐infusion of nomifensine with (–)‐sulpiride caused an additional pronounced rise of the DA output on top of the nomifensine‐induced increase at the nonlesioned side, whereas no effect was observed at the lesioned side. Finally, MPP+ (10 mM) was infused for 45 min into both striata. The increase in the dialysate content of DA in response to MPP+ (considered as an index of the total striatal DA content) from the lesioned side was only 0.6% of the MPP+‐induced DA increase from the nonlesioned side. A strong compensatory response to increased extracellular dopamine was observed in the ipsilateral striatum. This effect was achieved by a severe suppression of reuptake mechanisms, as well as of the autoreceptor feedback response. It is concluded that infusion of MPP+ into the substantia nigra can be used as a chronic biochemical model for clinically manifest parkinsonism.
Expression of the Myelin‐Associated Glycoprotein in Cultures of Immortalized Schwann Cells
Authors: Goda Shuichiro , Hammer Jeffrey , Kobiler David , Quarles Richard H.
Published: October 2006

Abstract: Although the myelin‐associated glycoprotein (MAG) cannot be detected in primary cultures of rat Schwann cells in the absence of neurons, MAG expression was demonstrated in some lines of cultured Schwann cells that had been immortalized by repetitive passaging. Radioimmunoassay of one such Schwann cell line, S‐16, showed a remarkably high MAG concentration of about 1 ng/μg of total protein, a level that is comparable to the MAG concentration in adult sciatic nerve. The S‐16 cells divide very rapidly, are rounder than normal Schwann cells, and elaborate many processes after reaching high density. The cells are galacto‐cerebroside positive, but express little or no P0 glycoprotein or myelin basic protein. As in nerve, the MAG synthesized by the cultured cells is primarily the shorter isoform (S‐MAG). Furthermore, the posttranslational processing resembles that occurring in vivo including a similar degree of glycosylation, sulfation of oligosaccharides, and phosphorylation of the polypeptide. The sensitivity of MAG to treatment of the intact cells with trypsin or neuraminidase, as well as surface labeling with ongroup
Detection with Monoclonal Antibodies of a 15‐kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ
Authors: Morel N. , Synguelakis M. , Le Gal la Salle G.
Published: October 2006

Abstract: A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15‐kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15‐kDa proteolipid band on Western blots of purified mediatophore were selected. A 15‐kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15‐kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti‐15‐kDa proteolipid monoclonal antibodies.
Reduced Striatal [3H]Inositol 1,4,5‐Trisphosphate Binding in Huntington's Disease
Authors: Warsh Jerry J. , Politsky Jeffrey M. , Li Peter P. , Kish Stephen J. , Hornykiewicz O.
Published: October 2006

Abstract: Specific [3H]inositol 1,4,5‐trisphosphate ([3H]InsP3) binding was studied in regions of postmortem brain from 15 patients with Huntington's disease (HD) and 13 nonneurological controls. Single‐point binding analyses, using 5.0 nM InsP3, showed statistically significant reductions in specific [3H]InsP3 binding in the caudate (–71%) and putamen (–75%) of HD patients compared with controls. Frontal and occipital cortical [3H]InsP3 binding was not significantly different between HD and controls, a finding suggesting that the reduced [3H]InsP3 binding parallels the brain regional specificity of the neuropathological changes in HD. Scatchard analyses of data from [3H]InsP3 competition binding assays performed on caudate nucleus revealed that the reductions found using single‐point binding assays were due to a decrease in both binding density (–57%) and affinity (–50%) in HD brain compared with controls. The concomitant changes in InsP3 receptor density and affinity in HD brain suggest that these alterations may be produced by processes in addition to cell loss. These results suggest the possibility that disturbances in InsP3 receptor function, possibly resulting in altered intracellular calcium flux and homeostasis, occur in HD and may participate in the pathogenesis of this neurodegenerative disorder.
Incubation of Tissue Slices in the Absence of Ca2+ and Mg2+ Can Cause Nonspecific Damage
Authors: Lonart Gyorgy , Zigmond Michael J.
Published: October 2006

Abstract: Supervision of striatal slices with a medium deficient in Ca2+ and Mg2+ caused a large and sustained increase in release of lactate dehydrogenase, a finding indicative of the disruption of plasma membranes. This was associated with an efflux of dopamine (DA) and the depletion of DA from the tissue. In addition, whereas DA efflux was stimulated by either D‐amphetamine (10 μM) or L‐glutamate (10 mM) in the absence of Ca2+, these effects were greatly reduced when Mg2+ also was withdrawn from the buffer. These results suggest that (a) incubation in a Ca2+/Mg2+‐free buffer disrupts plasma membranes, (b) this disruption affects dopaminergic neurons as well as those of other striatal elements, and (c) the failure of a treatment to stimulate DA release in a Ca2+/Mg2+‐free buffer cannot be used as a test of Ca2+ dependence.
Characterization of Nicotinic Receptor‐Mediated [3H]Dopamine Release from Synaptosomes Prepared from Mouse Striatum
Authors: Grady Sharon , Marks Michael J. , Wonnacott Susan , Collins Allan C.
Published: October 2006

Abstract: This study establishes that presynaptic nicotinic receptors modulate dopamine release in the mouse striatum. Nicotinic agonists elicit a dose‐dependent increase in the release of [3H]dopamine from synaptosomes prepared from mouse striatum. At low concentrations, this release is Ca2+ dependent, whereas at higher concentrations Ca2+‐independent, mecamylamine‐insensitive release was also observed. The Ca2+‐dependent nicotine‐evoked release was not blocked by α‐bungarotoxin but was effectively blocked by neuronal bungarotoxin as well as several other nicotinic receptor antagonists. The relationship between potency for stimulation of release for agonists and potency for inhibition of release for antagonists was compared to the affinity of these compounds for the [3H]nicotine binding site. The overall correlation between release and binding potency was not high, but the drugs may be classified into separate groups, each of which has a high correlation with binding. This finding suggests either that more than one nicotinic receptor regulates dopamine release or that not all agonists interact with the same receptor in an identical fashion.
Cleavage of Bovine Brain Microtubule‐Associated Protein‐2 by Human Immunodeficiency Virus Proteinase
Authors: Ainsztein Alexandra M. , Purich Daniel L.
Published: October 2006

Abstract: The high‐molecular‐weight dendritic cytoskeletal protein known as microtubule‐associated protein (MAP)‐2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP‐2 into a C‐terminal Mr 28,000–35,000 microtubule‐binding fragment and a larger N‐terminal Mr 240,000 projection‐arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP‐2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an Mr 72,000 species that retains the ability to associate with taxol‐stabilized microtubules. Treatment of this Mr 72,000 species with thrombin releases the same Mr 28,000 component as that derived from thrombin action on intact high‐molecular‐weight MAP‐2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N‐terminus. The association of the Mr 72,000 component with microtubules can be disrupted by the presence of a 21 ‐amino acid peptide analogue of the second repeated sequence in the MAP‐2 microtubule‐binding region. We also studied HIV proteinase action on MAP‐2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self‐assembly behavior. Cleavage of isolated MAP‐2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.
Endogenous Noradrenaline and Dopamine in Nerve Terminals of the Hippocampus: Differences in Levels and Release Kinetics
Authors: Verhage Matthijs , Ghijsen Wim E. J. M. , Boomsma Frans , Lopes da Silva Fernando H.
Published: October 2006

Abstract: The presence and release of endogenous catechol‐amines in rat and guinea pig hippocampal nerve terminals was studied by fluorimetric HPLC analysis. In isolated nerve terminals (synaptosomes) the levels and breakdown of endogenous catecholamines were determined and the release process was characterized with respect to its kinetics and Ca2+ and ATP dependence. Endogenous noradrenaline and dopamine, but not adrenaline, were detected in isolated hippocampal nerve terminals. For dopamine both the levels and the amounts released were more than 100‐fold lower than those for noradrenaline. In suspension, released endogenous catecholamines were rapidly broken down. This could effectively be blocked by monoamine oxidase inhibitors, Ca2+‐free conditions, and gluthatione. The release of both noradrenaline and dopamine was highly Ca2+ and ATP dependent. Marked differences were observed in the kinetics of release between the two catecholamines. Noradrenaline showed an initial burst of release within 10 s after K+ depolarization. The release of noradrenaline was terminated after approximately 3 min of K+ depolarization. In contrast, dopamine release was more gradual, without an initial burst and without clear termination of release within 5 min. It is concluded that both catecholamines are present in nerve terminals in the rat hippocampus and that their release from (isolated) nerve terminals is exocytotic. The characteristics of noradrenaline release show several similarities with those of other classical transmitters, whereas dopamine release characteristics resemble those of neuropeptide release in the hippocampus but not those of dopamine release in other brain areas. It is hypothesized that in the hippocampus dopamine is released from large, dense‐cored vesicles, probably colocalized with neuropeptides.
Effects of Hypoxia on Choline Exchange Among Organs
Authors: Scremin O. U. , Jenden D. J.
Published: October 2006

Abstract: Cerebral blood flow (CBF) and the arteriovenous (A‐V) difference for choline (Ch) across brain, lung, splanchnic territory, liver, kidney, and lower limb were studied in anesthetized, mechanically ventilated rats subjected to 10–20‐min periods of hypoxia induced by lowering the inspired O2 concentration to 13%. A large, time‐dependent increase in arterial blood Ch concentration occurred during hypoxia. This phenomenon coincided with a net rate of uptake of Ch by the brain during hypoxia (0.81 ± 0.24 nmol/min, n = 10; p < 0.05), which contrasted with a net rate of loss of Ch by this organ during the control period that preceded hypoxia (‐0.20 ± 0.08 nmol/min, n = 10; p < 0.05). During hypoxia, lungs and splanchnic territory showed negative A‐V differences for Ch levels (net Ch loss), whereas brain, liver, kidney, and lower limb showed positive A‐V differences for Ch levels (net Ch uptake). Ch output from lungs was already detected at 5 min within the period of hypoxia and reversed rapidly after restoration of normal oxygenation. On the other hand, Ch output from the splanchnic territory became evident only 10 min after commencement of hypoxia and outlasted this experimental condition. It is concluded that extracerebral production of Ch during hypocapnic hypoxia raises the arterial concentration of this molecule and, by reversing the gradient across cerebral capillaries, prevents the cerebral loss of Ch in this condition.
Noradrenergic Inhibition and α2‐Adrenergic Stimulation of Melatonin Secretion in the Pigeon
Authors: Vakkuri O. , Rintamäki H. , Leppäluoto J.
Published: October 2006

Abstract: Adrenergic regulatory mechanisms of melatonin synthesis and secretion were studied in the pigeon in vivo. Late‐afternoon intraperitoneal injection of noradrenaline (NA; 1 mg/kg) resulted in a significant decrease in plasma melatonin levels in 3 h. The same effect was seen after phenylephrine treatment (1 mg/kg i.p.), indicating that an α1‐adrenergic mechanism may mediate the inhibition. Propranolol treatment had no effect on plasma melatonin levels, supporting this concept. Detomidine (1 mg/kg i.p.), an α2‐adrenergic agonist, increased melatonin levels. This stimulatory effect was blocked by yohimbine, an α2‐adrenergic antagonist. However, yohimbine alone had no effect on the plasma melatonin levels, suggesting that α2‐adrenergic transmission is not primarily responsible for the nocturnal stimulation of melatonin synthesis and secretion in the pigeon.
Regulatory Interactions Among Axon Terminals Affecting the Release of Different Transmitters from Rat Striatal Slices Under Hypoxic and Hypoglycemic Conditions
Authors: Milusheva E. , Doda M. , Pasztor E. , Lajtha A. , Sershen H. , Vizi E. S.
Published: October 2006

Abstract: An in vitro model of ischemia was utilized to study the effects of both oxygen and glucose depletion on transmitter release from rat striatal slices. The spontaneous and stimulation‐evoked releases of tritiated dopamine, γ‐aminobutyric acid, glutamate, and acetylcholine were measured. Hypoxia increased the evoked release of glutamate and dopamine without effect on the resting release. In contrast, hypoglycemia itself increased the resting release of dopamine. Hypoxia in combination with hypoglycemia provoked a massive release of glutamate, dopamine, and γ‐aminobutyric acid. The effect on acetylcholine release was less pronounced. Ca2+ withdrawal partly reduced the effect of hypoxia combined with hypoglycemia on dopamine release and application of tetrodotoxin (1 μM) abolished it. MK‐801 (3 μM), an N‐methyl‐d‐aspartate receptor antagonist, attenuated the effect of hypoxia and hypoglycemia on [3H]dopamine release. ω‐Conotoxin (0.1 μM) had a similar effect on stimulation‐evoked release under a hypoxic condition. The D2 receptor antagonist sulpiride (100 μM) failed to enhance the release of [3H]acetylcholine in hypoxia combined with hypoglycemia. It was suggested that in response to hypoxia combined with hypoglycemia there is a massive release of glutamate due to the increased firing rate which in turn releases dopamine from the axon terminals through stimulation of presynaptic N‐methyl‐d‐aspartate receptors. Dopaminergic inhibitory control on ACh release seems not to be operative under conditions of hypoxia combined with hypoglycemia.
Magnesium‐Dependent Inhibition of Agonist‐Stimulated Phosphoinositide Breakdown in Rat Cortical Slices by Excitatory Amino Acids
Authors: Lee Horng‐Mo , Fain John N.
Published: October 2006

Abstract: The excitatory amino acid agonists kainate, N‐methyl‐D‐aspartate (NMDA), and quisqualate inhibited ligand‐stimulated phosphoinositide hydrolysis in rat cortical slices. The NMDA channel blocker MK‐801 antagonized the inhibition by NMDA but had no effect on the inhibition due to kainate or quisqualate. The antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione blocked the effects of quisqualate and kainate but not the effect of NMDA. These data indicate that activation of the NMDA, α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid, and kainate types of ionotropic receptors has the same effect. In membranes prepared from cortical slices, there was no inhibition of carbachol‐stimulated phosphoinositidase C activity by excitatory amino acids, suggesting that excitatory amino acids indirectly affect carbachol‐stimulated phosphoinositide hydrolysis. The inhibition by excitatory amino acids of carbachol‐stimulated phosphoinositide breakdown was dependent on extracellular Mg2+ and was abolished by procedures that increase intracellular Ca2+. Veratridine inhibition of carbachol‐stimulated phosphoinositide hydrolysis was reversed by ouabain but not by other procedures that increase intracellular Ca2+. In contrast to excitatory amino acids, veratridine potentiated carbachol‐stimulated phosphoinositide breakdown in the presence of 10 mM extracellular Mg2+. These data suggest that excitatory amino acids inhibit carbachol‐stimulated phosphoinositide breakdown in rat cortex by lowering intracellular Ca2+ through a mechanism dependent on extracellular Mg2+.
Local Blood‐Brain Barrier in the Newborn Rabbit: Postnatal Changes in α‐Aminoisobutyric Acid Transfer Within Medulla, Cortex, and Selected Brain Areas
Authors: Tuor U. I. , Simone C. , Bascaramurty S.
Published: October 2006

Abstract: Postnatal changes in local permeability of the blood‐brain barrier to an inert neutral amino acid (α‐[14C]‐aminoisobutyric acid) were investigated in 25 rabbits. The local transfer constant (K) for this tracer was measured with quantitative autoradiographic techniques at postnatal ages of 1, 3, 8, and 17 days, and adult. In adults, the amino acid penetrated the blood‐brain barrier poorly in most regions examined (K < 1 μ1 g−1 min−1) except within and in proximity to structures with a relatively leaky blood‐brain barrier such as area postrema and choroid plexus. The rate of tracer entry into “impermeable” regions was seven‐ to 10‐fold greater in 1‐day‐old rabbits than adults and not dependent on active transport. In young animals, there was a pronounced regional variation in K with the lowest values occurring in white matter and the highest in gray matter such as cerebral cortex, posterior thalamus, and hippocampus. During postnatal development, K decreased (p < 0.01) with most regions having values near those of adults by 17 days of age. The results indicate that the blood‐brain barrier of the newborn rabbit is relatively leaky to a small hydrophilic nonelectrolyte with a distribution that is heterogeneous regionally. Irrespective of age, such blood‐borne substances can accumulate in certain brain areas considered to have impermeable vessels (e.g., nucleus tractus solitarii).
Effect of pH on Glutamine Content Derived from Exogenous Glutamate in Astrocytes
Authors: Brookes Neville
Published: October 2006

Abstract: A shift in pH from 7.4 to 7.8 in the incubation solution caused a 3.4‐fold increase in the free glutamine content of mouse cerebral astrocytes that were incubated with glutamate (100 μM) and ammonium (100 μM). This large and reversible steady‐state increase in glutamine content was accompanied by smaller transient increases in the following: (a) net formation of glutamine; (b) clearance of glutamate from the incubation solution; and (c) glutamate content. The content of glutamine was reduced markedly by omission of either glutamate or ammonium from the incubation solution, or by inhibition of glutamine synthetase activity with methionine sulfoximine. The rate at which glutamine was exported from the astrocytes was unaffected by the pH change. The effects of pH on the concentration of free ammonia or on glutamate uptake do not appear to mediate the increase in glutamine content. Uptake of exogenous glutamine was little affected by the pH change. Therefore, possible mediation of the effect by an increase in intracellular pH must be considered. The response to altered pH described here may provide a cellular basis for the increased level of brain glutamine observed in hyperammonemia.
Corticosterone Enhances Kainic Acid‐Induced Calcium Elevation in Cultured Hippocampal Neurons
Authors: Elliott Elicia M. , Sapolsky Robert M.
Published: October 2006

Abstract: Corticosterone, a steroid secreted during stress, increases hippocampal neuronal vulnerability to excitotoxins, hypoxia‐ischemia, and antimetabolites. Energy supplementation and N‐methyl‐d‐aspartate receptor antagonists prevent this corticosterone‐enhanced neurotoxicity. Because neuronal calcium regulation is energy dependent and a large calcium influx accompanies N‐methyl‐d‐aspartate receptor activation, we investigated whether corticosterone exacerbates the elevation of hippocampal neuronal calcium induced by the glutamatergic excitotoxin kainic acid. Corticosterone caused a 23‐fold increase in the magnitude of the calcium response to kainic acid, a sevenfold increase in the peak magnitude of the calcium response, and a twofold increase in calcium recovery time. This corticosterone effect may be energetic in nature as corticosterone decreases hippocampal neuronal glucose transport. Glucose supplementation reduced the corticosterone effect on the magnitude and peak magnitude of the calcium response to kainic acid. Glucose reduction, by the approximate magnitude by which corticosterone inhibits glucose transport, mimicked the corticosterone effect on the peak magnitude of the calcium response to kainic acid. Thus, corticosterone increases calcium after kainic acid exposure in hippocampal neurons in an energy‐dependent manner. Elevated calcium is strongly implicated in stimulating neurotoxic cascades during other energetic insults and may be the mechanism for the corticosterone‐induced hippocampal neuronal vulnerability and toxicity.
Cerebral Cortex Ammonia and Glutamine Metabolism During Liver Insufficiency‐Induced Hyperammonemia in the Rat
Authors: Dejong Cornells H. C. , Kampman Margitta T. , Deutz Nicolaas E. P. , Soeters Peter B.
Published: October 2006

Abstract: Hyperammonemia has been suggested to induce enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis and accumulation, and finally net glutamine release into the blood stream, but this has never been confirmed in liver insufficiency models. Therefore, cerebral cortex ammonia‐ and glutamine‐related metabolism was studied during liver insufficiency‐induced hyperammonemia by measuring plasma flow and venous‐arterial concentration differences of ammonia and amino acids across the cerebral cortex (enabling estimation of net metabolite exchange), 1 day after portacaval shunting and 2, 4, and 6 h after hepatic artery ligation (or in controls). The intra‐organ effects were investigated by measuring cerebral cortex tissue ammonia and amino acids 6 h after liver ischemia induction or in controls. Arterial ammonia and glutamine increased in portacaval‐shunted rats versus controls, and further increased during liver ischemia. Cerebral cortex net ammonia uptake, observed in portacaval‐shunted rats, increased progressively during liver ischemia, but net glutamine release was only observed after 6 h of liver ischemia. Cerebral cortex tissue glutamine, γ‐aminobutyric acid, most other amino acids, and ammonia levels were increased during liver ischemia. Glutamate was equally decreased in portacaval‐shunted and liver‐ischemia rats. The observed net cerebral cortex ammonia uptake, cerebral cortex tissue ammonia and glutamine accumulation, and finally glutamine release into the blood suggest that the rat cerebral cortex initially contributes to net ammonia removal from the blood during liver insufficiency‐induced hyperammonemia by augmenting tissue glutamine and ammonia pools, and later by net glutamine release into the blood. The changes in cerebral cortex glutamate and γ‐aminobutyric acid could be related to altered ammonia metabolism.
Possible Existence of Two Subsets of Platelet‐Activating Factor Receptor to Mediate Polyphosphoinositide Breakdown and Calcium Influx in Neuroblastoma × Glioma Hybrid NG 108–15 Cells
Authors: Chau Lee‐Young , Hu Chia‐Yuan , Chang Wen‐Tsan , Hsu Yu‐Shen
Published: October 2006

Abstract: Platelet‐activating factor (PAF) initiated polyphosphoinositide (polyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma × glioma hybrid NG 108–15 cells. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [3H]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8–12 s and returned to basal levels within 1 min as measured in fura 2‐loaded cells. When cells were suspended in Ca2+‐depleted medium, the PAF‐induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF‐induced accumulation of 3H‐labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of ∼1 × 10−10 and 5 × 10−8M, respectively. The PAF analogs 1‐O‐hexadecyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine and 1‐O‐hexadecyl‐2‐O‐methyl‐rac‐glycerol‐3‐phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF‐induced accumulation of 3H‐inositol phosphates. In contrast, the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration. SRI‐63675, a PAF antagonist, blocked both PAF‐induced responses effectively, whereas another antagonist, BN‐52021, at the same dose inhibited substantially the accumulation of 3H‐inositol phosphates but had a negligible effect on the Ca2+ response. Scatchard analysis of radioligand saturation binding data revealed the existence of two PAF binding sites in NG 108–15 cell membranes: one was high affinity with a dissociation constant (KD) of 6.1 ± 1.8 × 10−11M and a receptor density (Bmax) of 54.0 ± 2.8 fmol/mg of protein; another was low affinity with a KD of 1.7 ± 0.4 × 10−8M and a Bmax of 5.6 ± 0.5 pmol/mg of protein. Displacement of specific [3H]PAF binding by unlabeled PAF also demonstrated two binding sites with inhibitory constant (Ki) values of 7.5 ± 2.5 × 10−11 and 2.7 ± 1.7 × 10−7M, respectively. BN‐52021 and SRI‐63675 displayed differential affinities to these two binding sites. Taken together, these data suggest the existence of two subtypes of PAF receptor to mediate polyPI hydrolysis and Ca2+ influx in NG 108–15 cells.
Evidence for the Implication of Phosphoinositol Signal Transduction in μ‐Opioid Inhibition of DNA Synthesis
Authors: Barg Jacob , Belcheva Mariana M. , Coscia Carmine J.
Published: October 2006

Abstract: An opioid receptor agonist, [D‐Ala2, Me‐Phe4, Glyol5]enkephalin (DAMGE), decreased [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the μ‐specific antagonist cyclic D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr amide (CTOP) reversed the DAMGE effect, arguing for a receptor‐mediated mechanism. The μ‐opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly μ‐selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis, whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H]inositol trisphosphate and glutamate stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also in hibited thymidine incorporation dose‐dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.
Developmental Changes in the Modulation of Cyclic AMP Formation by the Metabotropic Glutamate Receptor Agonist 1S,3R‐Aminocyclopentane‐1,3‐Dicarboxylic Acid in Brain Slices
Authors: Casabona G. , Genazzani A. A. , Di Stefano M. , Sortino M. A. , Nicoletti F.
Published: October 2006

Abstract: Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide‐binding regulatory protein‐coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (1S, 1R‐ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin‐stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R‐ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N‐methyl‐d‐aspartate, kainate, and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate and was antagonized by l‐2‐amino‐3‐phosphonopro‐pionate (l‐AP‐3). l‐AP‐3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R‐ACPD on forskolin‐stimulated cAMP formation. In hippocampal slices from 1‐, 8‐, or 15‐day‐old rats, 1S,3R‐ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar developmental pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R‐ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin‐stimulated cAMP formation by 1S,3R‐ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.