bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubule‐Dissociating Drugs and A23187 Reveal Differences in the Inhibition of Synaptosomal Transmitter Release by Botulinum Neurotoxins Types A and B
Authors: Ashton Anthony C. , Dolly J. Oliver
Published: October 2006

Abstract: The inhibitory effects of botulinum neurotoxins types A and B on Ca2+‐dependent evoked release of [3H]noradrenaline from rat cerebrocortical synaptosomes were compared and their molecular basis investigated. A23187, a Ca2+ ionophore, proved more efficacious in reversing the blockade produced by type A than that by B, whereas the actions of neither were changed by increasing intraterminal cyclic GMP levels using 8‐bromo‐cyclic GMP or nitroprusside. Disruption of the actin‐based cytoskeleton with cytochalasin D did not alter the inhibition seen subsequently with either toxin. However, prior disassembly of microtubules with colchicine, nocodazole, or griseofulvin reduced the potency of type B toxin, but not that of type A toxin; stabilization of the microtubules with taxol counteracted this effect of colchicine. Because colchicine treatment of synaptosomes did not interfere with the measurable binding of type B toxin or its apparent uptake, it appears to act intracellularly. Collectively, these data suggest that botulinum neurotoxins types A and B inactivate transmitter release by interaction at different sites in the process. Based on the consistent results observed with four different drugs known to affect selectively microtubules, their involvement in the action of the type B neurotoxin is proposed.
Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastoma Cells
Authors: Shea Thomas B. , Lou Beermann Mary , Nixon Ralph A.
Published: October 2006

Abstract: Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hi‐rudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N‐acetyl‐leucyl‐leucyl‐norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10‐6M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N‐acetyl‐leucyl‐leucyl‐methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell‐free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum‐free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.
Fatty Acid Composition of Human Myelin Proteolipid Protein in Peroxisomal Disorders
Authors: Bizzozero Oscar A. , Zuñiga Gonzalo , Lees Marjorie B.
Published: October 2006

Abstract: Myelin proteolipid protein (PLP) is an acylated protein which contains approximately 2 mol of ester‐bound fatty acids. In this study, the amount and composition of fatty acids covalently bound to human myelin PLP were determined during development and in peroxisomal disorders. Palmitic, oleic, and stearic acids accounted for most of the PLP acyl chains. However, in contrast to PLP in other species, human PLP contains relatively more very long chain fatty acids (VLCFA). The fatty acid composition remained essentially unchanged between 1 day and 74 years of age. The total amount of fatty acid bound to PLP was not altered in any of the pathological cases examined. However, in the peroxisomal disorder adrenoleukodystrophy, the proportions of saturated and, to a lesser extent, monounsaturated VLCFA bound to PLP were increased at the expense of oleic acid. Smaller, but significant, changes were observed in adrenomyeloneuropathy. The reduction in the levels of oleic acid was also observed in two other peroxisomal disorders, the cerebrohepatorenal (Zellweger) syndrome and neonatal adrenoleukodystrophy, as well as in the lysosomal disorder Krabbe globoid cell leukodystrophy. However, in these disorders, the decrease in oleic acid occurred at the expense of stearic acid, and not VLCFA. The results indicate that, although a characteristic PLP fatty acid pattern is normally maintained, changes in the acyl chain pool can ultimately be reflected in the fatty acid composition of the protein. The altered PLP‐acyl chain pattern in peroxisomal disorders may contribute to the pathophysiology of these devastating disorders.
Role of Dopaminergic Neurons in Denervation‐Induced α1‐Adrenergic Up‐Regulation in the Rat Cerebral Cortex
Authors: Nowak Gabriel , Zak Joanna , Superata Jerzy
Published: October 2006

Abstract: The density of [3H]prazosin binding to α1‐adrenoceptors in the rat cortex was measured after selective and mixed noradrenergic or dopaminergic lesions. DSP‐4 produced a selective noradrenergic lesion and increased the density of α1‐adrenoceptors. 6‐Hydroxydopamine produced a selective dopaminergic lesion (after desipramine protection of noradrenergic neurons) and a mixed noradrenergic and dopaminergic lesion that did not change the cortical α1‐adrenoceptor binding. On the basis of the results obtained, a hypothesis is put forward that the central dopaminergic system controls the denervation‐induced cortical α1‐adrenoceptor up‐regulation.
Cholecystokinin Modulates the Release of Dopamine from the Anterior and Posterior Nucleus Accumbens by Two Different Mechanisms
Authors: Marshall F. H. , Barnes S. , Hughes J. , Woodruff G. N. , Hunter J. C.
Published: October 2006

Abstract: The effects of various cholecystokinin (CCK)‐related peptides were investigated on 35 mM K+ ‐stimulated endogenous dopamine release from slices of either anterior or posterior nucleus accumbens of the rat. CCK sulphated octapeptide (1–10 μM), but not pentagastrin or CCK unsulphated octapeptide, was found to cause a dose‐dependent increase in the release from the posterior nucleus accumbens. This effect was blocked by low doses of the CCKA receptor antagonist L364,718 (10 nM) but not the CCKB receptor antagonist L365,260. In the anterior nucleus accumbens CCK sulphated octapeptide (1 μM) and CCK unsulphated octapeptide (0.1–1 μM) inhibited the dopamine release, and this effect was blocked by L365,260 (10–100 nM) but not by L364,718. These results suggest that CCK has a different effect on dopamine release from the anterior and posterior nucleus accumbens and that these effects are mediated by two different types of CCK receptor.
Regulation of Proenkephalin Synthesis in Adrenal Medullary Chromaffin Cells
Authors: Wilson Steven P.
Published: October 2006

Abstract: The synthesis of proenkephalin was assessed in primary cultures of bovine adrenal medullary chromaffin cells by incubation of the cells with [35S]methionine, digestion of proenkephalin‐derived peptides with trypsin and carboxy‐peptidase B, and quantitation of radioactivity incorporated into Met‐enkephalin following reversed‐phase HPLC. Nicotine, histamine, and vasoactive intestinal peptide each enhanced the rate of proenkephalin synthesis approximately 10‐fold when examined between 16 and 32 h after the drug or hormone addition. Inclusion of nifedipine (1 μM) partially blocked the stimulatory effect of nicotine, but not that of vasoactive intestinal peptide or histamine, on proenkephalin synthesis. Theophylline, tetrabenazine, and angiotensin II also increased the rate of proenkephalin synthesis (three‐ to eightfold). These increases in the apparent rate of proenkephalin synthesis were not attributable to altered [35S]methionine specific radioactivity or rates of turnover and did not reflect similar increases in total protein synthesis. The half‐life for turnover of Met‐enkephalin sequences was 3–4 days in the cultured chromaffin cell. These studies directly show that proenkephalin synthesis is the primary regulatory step in control of chromaffin cell opioid peptide content.
A Comparison of Axonal and Somatodendritic Dopamine Release Using In Vivo Dialysis
Authors: Kalivas Peter W. , Duffy Patricia
Published: October 2006

Abstract: The release of endogenous dopamine from the axon terminal field in the nucleus accumbens and from the A10 dopamine cell bodies of conscious rats was measured using intracranial dialysis. Release of dopamine from both areas was calcium‐dependent and markedly inhibited by the presence of the D2 agonist, quinpirole, in the dialysis buffer. However, the addition of tetrodotoxin to the buffer produced less of a decrease in dopamine in the A10 region than in the nucleus accumbens. When dopamine release was examined by substituting K+ for Na+ or by adding amphetamine to the buffer, the evoked release was significantly less in the A10 region than in the nucleus accumbens. Likewise, enhanced extracellular dopamine following blockade of reuptake by nomifensine addition to the dialysis buffer was not as great in the A10 region as in the nucleus accumbens. These data argue that, in general, axonal and somatodendritic dopamine release are regulated by similar factors, although somatodendritic release is less influenced by action potential generation and less responsive to some releasing agents.
Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice
Authors: Vornov James J. , Coyle Joseph T.
Published: October 2006

Abstract: In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N‐methyl‐D‐aspartate (NMDA) glutamate receptor sub‐type, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK‐801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7‐dinitroquinoxaline‐2,3‐dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non‐receptor‐mediated mechanism, or that the receptor‐mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.
Phosphorylation of Rat Tyrosine Hydroxylase and Its Model Peptides In Vitro by Cyclic AMP‐Dependent Protein Kinase
Authors: Roskoski Robert , Ritchie Patricia
Published: October 2006

Abstract: The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP‐dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromo‐cytoma tyrosine hydroxylase for cyclic AMP‐dependent protein kinase and obtained values of 136 μM and 7.1 μmol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP‐dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36–46), for example, exhibited a Km of 108 μM and a Vmax of 6.93 μmol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.
Phosphoglycerates and Protein Phosphorylation: Identification of a Protein Substrate as Glucose‐1,6‐Bisphosphate Synthetase
Authors: Morino Hideo , Fischer‐Bovenkerk Carolyn , Kish Phillip E. , Ueda Tetsufumi
Published: October 2006

Abstract: We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3‐phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72‐kDa protein (3PG‐PP72). This system was separated into the substrate, 3PG‐PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1‐32P]1,3‐bisphosphoglycerate ([1‐32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [γ‐32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG‐PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1‐32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG‐PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose‐1‐phosphate or glucose‐6‐phosphate. Finally, 3PG‐PP72 showed chromatographic behaviors identical to those of glucose‐1,6‐bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG‐PP72 is G1,6P2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG‐PP72 preparation.
Brain Tryptophan Hydroxylation in the Portacaval Shunted Rat: A Hypothesis for the Regulation of Serotonin Turnover In Vivo
Authors: Bengtsson Finn , Bugge Mogens , Johansen Kaj H. , Butterworth Roger F.
Published: October 2006

Abstract: Regional and whole‐brain tryptophan‐hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5‐hydroxytryptophan, serotonin, and 5‐hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5‐hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan‐loaded PCS rats showed increased brain serotonin turnover compared with tryptophan‐loaded sham rats. Regionally, this supranormal tryptophan‐hydroxylating activity was most pronounced in the mesencephalon‐pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan‐hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo.
Effect of Portacaval Anastomosis on Electrically Stimulated Release of Glutamate from Rat Hippocampal Slices
Authors: Butterworth Roger F. , Le Oanh , Lavoie Joel , Szerb John C.
Published: October 2006

Abstract: To evaluate the effects of chronic liver failure on release of the excitatory transmitter glutamate, electrically stimulated Ca2+‐dependent and Ca2+‐independent release of glutamate in the absence or presence of NH4+ was studied in superfused slices of hippocampus from portacaval‐shunted or sham‐operated rats 4 weeks after surgery. Spontaneous and stimulation‐evoked release of glutamate was higher in shunted rats in the presence of normal or low Ca2+ concentrations, and this release was depressed by 5 mM ammonium chloride. These findings suggest that portacaval shunting results in increased levels of extracellular glutamate in brain, probably due to a decreased reuptake of glutamate into perineuronal astrocytes, shown in previous studies to undergo neurqpathological changes following portacaval shunting. Changes in the inactivation of transmitter glutamate could be responsible, at least in part, for the neurological dysfunction resulting from sustained hyperammonemia and portal‐systemic shunting resulting from chronic liver failure.
Increased Stimulated Release and Uptake of Dopamine in Nucleus Accumbens After Repeated Cocaine Administration as Measured by In Vivo Voltammetry
Authors: Ng Jeffrey P. , Hubert George W. , Justice Joseph B.
Published: October 2006

Abstract: Electrically stimulated dopamine (DA) release (overflow) and uptake were measured with in vivo voltam‐metry in the nucleus accumbens (N ACC) of anesthetized rats that had previously received repeated cocaine treatments. Electrically stimulated DA release was induced by a 10‐s stimulation in the medial forebrain bundle (2‐ms, 200‐/iA, biphasic pulses at 100 Hz). DA overflow and uptake were measured with fast chronoamperometry using a Nafion‐plated, carbon fiber electrode. Animals given repeated doses of cocaine (10 mg/kg s.c. from day 1 to 5, 20 mg/kg s.c. from day 6 to 10) showed marked increases in DA uptake (5.47±0.28 vs. 2.93±0.26 μM/s) and in stimulated DA overflow (27.3±1.1 vs. 18.9±1.3 nM) compared with DA uptake and stimulated overflow in saline control animals. The increased uptake was shown to be independent of the increased overflow. Uptake was monitored as a function of stimulation current, and the data were extrapolated to zero stimulation, resulting in calculated rates of uptake of 2.43 and 3.71 μM/s in the control and cocaine‐treated groups, respectively. These effects were found to be temporary, as there were no significant differences in stimulated release or uptake between saline control animals and animals given 10 days of cocaine followed by a 10‐day abstinence period. These alterations in the N ACC produced by repeated cocaine administration may be a compensatory response to prolonged uptake blockade of synaptic DA.
Mechanism of Inhibition of N‐Methyl‐d‐Aspartate‐Stimulated Increases in Free Intracellular Ca2+ Concentration by Ethanol
Authors: Dildy‐Mayfield Jo Ellen , Leslie Steven W.
Published: October 2006

Abstract: Dissociated brain cells were isolated from newborn rat pups and loaded with fura‐2. These cells were sensitive to low N‐methyl‐d‐aspartate (NMDA) concentrations with EC50 values for NMDA‐induced intracellular Ca2+ concentration ([Ca2+]i) increases of approximately 7–16 μM measured in the absence of Mg2+. NMDA‐stimulated [Ca2+]j increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mMKQ prior to NMDA addition. Under these predepolarized conditions, 100 mMethanol inhibited 25 μM NMDA responses by approximately 50%, which was similar to the ethanol inhibition observed in buffer without added Mg2+. Ethanol did not alter [Ca2+]i prior to NMDA addition. In the absence of Mg2+, 50 and 100 mM ethanol did not significantly alter the EC50 value for NMDA, but did inhibit NMDA‐induced increases in [Ca2+]i in a concentration‐dependent manner at 4, 16, 64, and 256 μM NMDA. Whereas NMDA‐induced increases in [Ca2+]i were dependent on extracellular Ca2+ and were inhibited by Mg2+, the ability of 100 mM ethanol to inhibit 25 μM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 μM) enhanced 25 μM NMDA‐induced increases in [Ca2+]i by approximately 50%. Glycine (1‐100 μM) prevented the 100 mM ethanol inhibition of NMDA‐stimulated [Ca2+]i observed in the absence of exogenous glycine. MK‐801 (25‐400 μM) inhibited 25 μM NMDA‐stimulated rises in [Ca2+]i in a concentration‐dependent manner. Unlike the additive inhibition observed with Mg2+ plus ethanol, as the concentration of MK‐801 increased above 50 μM, ethanol (at 100 mM) did not produce further inhibition of NMDA responses compared with MK‐801 alone. These results suggest that ethanol may produce a noncompetitive inhibition of NMDA‐stimulated Ca2+ influx in dissociated brain cells, with interactions at the glycine and possibly the phencyclidine site on the NMDA‐receptor complex.
Isolation of Monoaminergic Synaptosomes from Rat Brain by Immunomagnetophoresis
Authors: Docherty Maureen , Bradford H. F. , Cash C. D. , Ehret M. , Maitre M. , Job Tong H.
Published: October 2006

Abstract: Monoaminergic synaptosomes have been isolated and purified from rat brain by immunomagnetophoresis. This novel technique uses magnetic beads to which Protein A is bound. Noradrenergic, dopaminergic, and serotonergic synaptosomes (previously cell‐surface labelled with anti‐dopamine‐β‐hydroxylase, anti‐tyrosine hydroxylase, and anti‐tryptophan hydroxylase, respectively) may be isolated in a highly purified state. The synaptosomal subpopulations are recovered in a viable metabolic state and show glucose‐stimulated respiration and Ca2+‐dependent neurotransmitter release. A novel subtype of dopamine‐β‐hydroxylase was found in dopaminergic terminals. No evidence for glutamate core‐lease from monoaminergic synaptosomes was obtained.
Immunocytochemical Localization of Chondroitin and Chondroitin 4‐ and 6‐Sulfates in Developing Rat Cerebellum
Authors: Flaccus A. , Janetzko A. , Tekotte H. , Margolis R. K. , Margolis R. U.
Published: October 2006

Abstract: Monoclonal antibodies specific for unsulfated, 4‐sulfated, and 6‐sulfated disaccharide “stubs’ that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of Chondroitin and the two isomeric Chondroitin sulfates in developing rat cerebellum. At 1–2 weeks postnatal, unsulfated Chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of Chondroitin 4‐sulfate is similar to that for Chondroitin at 1–2 weeks postnatal, but in contrast to Chondroitin, Chondroitin 4‐sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6‐sulfate is present predominantly in the prospective white matter of 1–2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6‐sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain. HPLC of chondroitin sulfate proteoglycan disaccharides demonstrated that the significant amounts of chondroitin (29%) and chondroitin 6‐sulfate (19%) present at 7 days are replaced almost entirely by chondroitin 4‐sulfate in adult brain, and immu‐noblots of chondroitinase‐treated proteoglycans from 7‐day and adult brain revealed a distinctive pattern of major core proteins that stained with each of the three antibodies. The different localizations and developmental changes of chondroitin, chondroitin 4‐sulfate, and chondroitin 6‐sulfate suggest that sulfation may be an important factor in specifying the biological functions of chondroitin sulfate proteoglycans in nervous tissue.
Degradation of Microtubule‐Associated Protein 2 and Brain Spectrin by Calpain: A Comparative Study
Authors: Johnson Gail V. W. , Litersky Joel M. , Jope Richard S.
Published: October 2006

Abstract: The in vitro degradation of microtubule‐associated protein 2 (MAP‐2) and spectrin by the calcium‐dependent neutral protease calpain was studied. Five major results are reported. First, MAP‐2 isolated from twice‐cycled microtubules (2XMT MAP‐2) was extremely sensitive to calpain‐induced hydrolysis. Even at an enzyme‐to‐substrate ratio (wt/wt) of 1:200, 2XMT MAP‐2 was significantly degraded by calpain. Second, MAP‐2 purified from the total brain heat‐stable fraction (total MAP‐2) was significantly more resistant to calpain‐induced hydrolysis compared with 2XMT MAP‐2. Third, MAP‐2a and MAP‐2b were proteolyzed similarly by calpain, although some relative resistance of MAP‐2b was observed. Fourth, the presence of calmodulin significantly increased the extent of calpain‐induced hydrolysis of the α‐subunit of spectrin. Fifth, the two neuronal isoforms of brain spectrin (240/235 and 240/235E, referred to as α/βN and α/βE, respectively) showed different sensitivities to calpain. αN‐spectrin was significantly more sensitive to calpain‐induced degradation compared to αE‐spectrin. Among other things, these results suggest a role for the calpain‐induced degradation of MAP‐2, as well as spectrin, in such physiological processes as alterations in synaptic efficacy, dendritic remodeling, and in pathological processes associated with neurodegeneration.
Autoreceptor Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area
Authors: Martin David , Bustos Gonzalo A. , Bowe Mark A. , Bray Sherrilynn D. , Nadler J. Victor
Published: October 2006

Abstract: Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N‐methyl‐D‐aspartate (NMDA)‐receptor antagonists depressed the release of glutamate, aspartate, and γ‐aminobutyrate evoked by 50 mMK+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3‐[(±)‐2‐carboxypiperazin‐4‐yl]‐propyl‐1‐phosphonic acid (CPP) and D‐2‐amino‐5‐phosphonovalerate (D‐AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 μM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D‐AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)‐α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K+‐evoked release of aspartate. Conversely, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.
Characteristics of [3H]GBR 12935 Binding in the Human and Rat Frontal Cortex
Authors: Hitri Ana , Venable Diane , Nguyen Hung Q. , Casanova Manuel F. , Kleinman Joel E. , Wyatt Richard J.
Published: October 2006

Abstract: Binding characteristics of the selective dopamine uptake inhibitor [3H]GBR 12935 have been described for the striatum but not for the frontal cortex. We have developed assay conditions for quantifying [3H]GBR 12935 binding in the frontal cortex. In both the rat and human frontal cortex, the assay required four times more tissue (8 mg/ml) than in the striatum (2 mg/ml).[3H]GBR 12935 binding in the frontal is complex, as it involves multiple binding sites. The high‐affinity binding site is sodium dependent and is inhibited by sodium. In human but not in rat frontal cortex, addition of K+ reversed the sodium inhibition. The pharmacological profile of the high‐affinity [3H]GBR 12935 binding site is consistent with that of the dopamine transporter, because drugs with the most selective dopamine reuptake blocking activities are the most potent displacers of [3H]GBR 12935 binding. There is a positive correlation between the rat and human inhibitory constants, a finding indicating that there are similar pharmacological profiles across at least these two species. Rats with a 6‐hydroxydopamine lesion had a 47% decrease in number of [3H]GBR 12935 binding sites, a result indicating that at least a portion of these sites had been on presynaptic dopamine terminals.
Comparison of Freeze‐Blowing and Funnel‐Freezing of Rat Brain for the Measurement of Cerebral Glucose Concentration In Vivo
Authors: Mies Günter , Cruz Nancy , Sokoloff Louis
Published: October 2006

Abstract: The efficacy of funnel‐freezing of rat brain to inactivate metabolic processes and preserve in vivo tissue glucose concentration was validated by comparing the results obtained by funnel‐freezing with those obtained with freeze‐blowing of brain. The arterial plasma glucose level was clamped at 9 mM in halothane‐anesthetized rats to produce identical glucose levels in brain tissue prior to freeze fixation. In funnel‐frozen and freeze‐blown brains, tissue glucose concentrations were 2.47 ± 0.05 and 2.47 ± 0.06 μMol/g (means ± SEM), respectively. Lactate levels in funnel‐frozen brains were slightly but significantly higher than those in freeze‐blown brains, i.e., 1.56 ± 0.05 μMol/g versus 1.30 ± 0.05 μMol/g (means ± SEM; p < 0.05). Regional analysis in funnel‐frozen brains revealed that glucose concentrations in superficial and basal brain areas remained approximately equal at 2.30 ± 0.1 μMol/g and 2.31 ± 0.09 μMol/g (means ± SEM), respectively. Our findings indicate that in the anesthetized rat, funnel‐freezing of brain is suitable for the measurement of regional in vivo glucose concentrations.
Gangliosides in Human Fetal Brain
Authors: Svennerholm Lars , Rynmark Britt‐Marie , Vilbergsson Gudjon , Fredman Pam , Gottfries Johan , Månsson Jan‐Eric , Percy Alan
Published: October 2006

Abstract: The ganglioside concentration and composition were determined in 42 human fetal brains from gestational week 10 to 22, a period that is morphologically characterized by rapid neuroblast proliferation and migration. The ganglioside concentration was constant during this period, ∼1 μMol of ganglioside sialic acid/g of fresh tissue weight. At gestational week 10 the ganglioside pattern was dominated by gangliosides of the ganglio b series, with the major ganglioside being GT1b, contributing 40% of total ganglioside sialic acid, whereas GD1b and GD3 contributed only 15 and 10%, respectively. The proportion of b series ganglioside decreased to gestational week 22, with the most pronounced relative reduction affecting GD3, but also GT1b and GD1b to a lesser extent. The ganglioside GQ1b increased in content from gestational week 10 and peaked around week 16. The proportion of GD1a increased markedly between gestational week 12 and 14 and slowly between week 14 and 18 and then increased rapidly from week 20. Ganglioside GM1 underwent a similar change. Gangliosides of the lacto series contributed 6–10% of ganglioside sialic acid between gestational week 10 and 15, and thereafter the proportion slowly decreased. 3′‐isoLM1 decreased rapidly in content from gestational week 10 (20 nmol/g of fresh weight) to week 22 (<0.5 nmol/g of fresh weight), whereas the gangliosides of the neolacto series (3′‐LM1 and 3′,8′‐LD1) showed a slower and less marked decline in level. The biological significance of the ganglioside changes is discussed.
Two 68‐kDa Proteins in Slow Axonal Transport Belong to the 70‐kDa Heat Shock Protein Family and the Annexin Family
Authors: Sekimoto Sumito , Tashiro Tomoko , Komiya Yoshiaki
Published: October 2006

Abstract: The major 68‐kDa protein found selectively in the faster of the two subcomponents of slow axonal transport [group IV or slow component b (SCb)] in the rat sciatic nerve has been characterized. It was found to contain two distinct classes of proteins, S1 and S2, both of which have isoelectric points of 5.7, but differ in their solubility in the presence of calcium. The S1 protein, which contributes up to 70% of the 68‐kDa component, was soluble in the presence or absence of calcium, whereas the S2 protein was bound to the cytoskeleton in a calcium‐dependent manner. Further characterization of the two proteins by peptide mapping and immunological methods revealed that the S1 protein belonged to a family of proteins related to the 70‐kDa heat shock protein, whereas the S2 protein was identical to 68‐kDa calelec‐trin (annexin VI). Selective occurrence in SCb of these proteins with potential abilities to regulate protein‐protein or protein‐membrane interactions suggests that they may play important roles in the control of cytoskeletal organization in the axon, because SCb contains mainly cytoskeletal proteins in a more dynamic form compared with the slowest rate component, slow component a, which is enriched in the stably polymerized form of these proteins.
Characterization of Extracellular Histamine in the Striatum and Bed Nucleus of the Stria Terminalis of the Rat: An In Vivo Microdialysis Study
Authors: Cumming P. , Damsma G. , Fibiger H. C. , Vincent S. R.
Published: October 2006

Abstract: The intracerebral microdialysis technique, coupled with a sensitive radioenzymatic assay, was employed to study histamine release in the striatum and in the bed nucleus of the stria terminalis (BNST) in conscious, freely moving rats. In these brain regions, extracellular histamine concentrations decreased by 20% when calcium was omitted from the perfusion solution. Extracellular histamine was insensitive to the addition of tetrodotoxin to the perfusion medium. In striatum, extracellular histamine concentrations declined in an apparent biexponential manner after the administration of α‐fluoromethylhistidine, an inhibitor of histamine synthesis. The half‐lives for the disappearance of histamine were 32 min and 7.7 h, indicating the presence of at least two histamine pools. Histidine loading resulted in a nearly twofold increase in histamine outflow in striatum. In the BNST, yohimbine increased the extracellular histamine content by 50%, suggesting that histamine release is subject to α2‐adrenergic regulation in vivo. The extent to which histamine detected in cerebral microdialysis samples is of neurogenic origin remains to be established.
Robustness of G Proteins in Alzheimer's Disease: An Immunoblot Study
Authors: McLaughlin Mark , Ross Brian M. , Milligan Graeme , McCulloch James , Knowler John T.
Published: October 2006

Abstract: Many of the neurotransmitter systems that are altered in senile dementia of the Alzheimer type are known to mediate their effects via G proteins, yet the integrity of guanine nucleotide‐binding proteins (G proteins) in Alzheimer's diseased brains has received minimal investigation. The aim of this study was to establish whether the level of Gα subunits of five G proteins was altered in Alzheimer's disease. We used immunoblotting (Western blotting) to compare the amounts of Gi1, Gi2, GsH (heavy molecular weight), GSL (light molecular weight), and Go in the frontal cortex and hippocampus, two regions severely affected by the disease, and the cerebellum, which is less severely affected. The number of senile plaques was also quantified. We report that there was no significant difference in the level of these Gα subunits between Alzheimer's diseased and age‐matched postmortem brains. These results suggest that alterations in the amount of G protein α subunits are not a feature of Alzheimer's disease.
Purification, Properties, and Phosphorylation by Protein Kinase C of Two Phosphoinositidase C Isozymes from Rat Brain
Authors: Blank Jonathan L. , Foster Keith A. , Hawthorne John N.
Published: October 2006

Abstract: Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl‐Sepharose, and Mono Q. Gradient sodium dodecyl sulphate‐polyacrylamide gel elec‐trophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC‐β‐1 and PLC‐γ of bovine brain. With both enzymes phosphatidylinositol 4,5‐bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2‐cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC‐β‐1 and PLC‐γ on Mono Q. Purified rat brain protein kinase C phosphorylated PLO‐γ but not PLC‐β‐1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphory‐lation of another protein in the preparations.
Detection of G Proteins in Purified Bovine Brain Myelin
Authors: Larocca J. N. , Golly F. , Ledeen R. W.
Published: October 2006

Abstract: Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5′‐O‐(3‐thiotriphosphate) ([35S]GTPγS) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 ± 9 vs. 271 ± 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5′‐adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [α‐32P]GTP bound to three bands in the 21–27‐kDa range in a manner inhibited by GTP and GTPγS but not App(NH)p. ADP‐ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholatc extract of myelin subjected to chromatography on a column of phenyl‐Sepharose gave at least three major peaks of [35S]GTPγS binding activity. SDS‐PAGE and immunoblot analyses of peak I indicated the presence of Goα, Giα, and Gsα. Further fractionation of peak II by diethyl‐aminoethyl‐Sephacel chromatography gave one [35S]GTPγS binding peak with the low‐molecular‐mass (21–27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS‐PAGE. Immunoblot analysis of this material identified the 36‐kDa protein as the β subunit, whereas the fraction containing 40‐kDa polypeptides reacted with specific antibodies to Goα and Giα. Thus, purified myelin from bovine brain has been shown to contain several GTP‐binding proteins resembling in broad outline the G proteins of whole brain and potentially able to transduce signals received by muscarinic (and perhaps other) receptors in this membrane.
[3H]MK‐801 Binding to N‐Methyl‐d‐Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine
Authors: Bakker Margot H. M. , McKernan Ruth M. , Wong Erik H. F. , Foster Alan C.
Published: October 2006

Abstract: The N‐methyl‐d‐aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK‐801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small‐molecular‐weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in μM): glycinc, 0.31; d‐serine, 0.20; d‐cycloserine, 1.46; (+)‐HA‐966, 4.06; and 7‐chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK‐801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 μM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK‐801 binding approached equilibrium, their ability to enhance [3H]MK‐801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK‐801 binding under both equilibrium and nonequilibrium conditions, although the high‐affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specifc [3H]MK‐801 binding under nonequilibrium conditions with an IC50 of 4 μM, and this value was unaltered when [3H] MK‐801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines, and desipramine are integral components of the NMDA receptor protein.
2‐Amino‐4‐Phosphonobutyric Acid Exerts a Light‐Dependent Effect on Post‐Gabaculine Levels of Retinal γ‐Aminobutyric Acid (GABA): Evidence that ON Synaptic Pathways Regulate Retinal GABAergic Transmission
Authors: Cubells Joseph F. , Ndubuka Christopher , Makman Maynard H.
Published: October 2006

Abstract: The effects of light, 2‐amino‐4‐phosphonobutyric acid (APB), and kainic acid on rat retinal γ‐aminobutyric acid (GABA)‐ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA‐transaminase. Post‐gabaculine levels of retinal GABA in light‐exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post‐gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post‐gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light‐dependent increment in post‐gabacu line accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post‐gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either ON or ON‐OFF amacrine cells.
Structure of the Mouse Myelin P2 Protein Gene
Authors: Narayanan Vinodh , Kaestner Klaus H. , Tennekoon Gihan I.
Published: October 2006

Abstract: Myelin P2 protein is a small (14,800 Da) protein found in peripheral and central nervous system myelin. To investigate the regulation of expression of this myelin protein, a mouse genomic library was screened with a rabbit P2 cDNA (pSN2.2–2), and a single positive phage clone containing a 20‐kb insert was obtained. This insert contained a single internal Sail restriction site and several EcoRI sites. The EcoRI fragments from this insert were subcloned into Bluescript. The rabbit P2 cDNA (pSN2.2–2) hybridized with a 4‐kb EcoRI fragment, and this 4‐kb fragment was then sequenced after the creation of nested deletions. The mouse gene contained four exons: exon 1 coded for amino acids 1–24, exon 2 for amino acids 24–81, exon 3 for amino acids 82–115, and exon 4 for amino acids 116–131. The three introns were 1.2 kb, 150 bp, and 1.3 kb in length. Primer extension analysis revealed two transcription start sites at +1 and +47, consistent with the presence of two P2mRNAs, with the larger transcript appearing more abundant. The amino acid sequences predicted from the mouse DNA indicate that the mouse protein differs from the rabbit protein at 16 different positions, with most of the differences located in exon 3. When the gene structure of fatty acid binding protein (FABP) genes were compared, the P2 gene had the same overall structure as others in the FABP family, suggesting a common ancestral gene for members of this family. The 5′‐flanking region contains candidate TATA and CAAT sequences, as well as two AP‐l binding sites that may be important in modulation of the expression of this gene.
Solubilization of Myelin Membranes by Detergents
Authors: Aveldaño M. I. , Díaz R. S. , Regueiro P. , Monreal J.
Published: October 2006

Abstract: The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS), Triton X‐100, and Zwittergent 3–14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS‐polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.