bluuebunny/crossref_metadata_2025 huggingface dataset:
Synaptosomal Calcium Metabolism During Hypoxia and 3,4‐Diaminopyridine Treatment
Authors: Peterson Christine , Gibson Gary E.
Published: October 2006

Abstract: A decline in the calcium‐dependent release of neurotransmitters appears to underlie the decreased neuronal function that accompanies reduced oxygen tensions (hypoxia). To determine if alterations in calcium uptake are primary to these changes, synaptosomal calcium uptake was measured in the presence of 100%, 2.5%, or 0% oxygen. Calcium uptake declined 60.2 ± 0.1 and 82.4 ± 2.5% with 2.5% and 0% when compared with 100% oxygen, respectively. 3,4‐Diaminopyridine stimulated calcium uptake by synaptosomes when they were incubated in low‐potassium media. It also diminished the hypoxic‐induced decline in calcium uptake to 30.6 ± 3.1 and 33.5 ± 3.1% with 2.5% and 0% oxygen, respectively. External binding to the synaptosomal plasma membrane declined to 29.2 ± 0.3 or 11.8 ± 0.9% when the oxygen tension was reduced to 2.5% or 0% oxygen. 3,4‐Diaminopyridine increased this superficial binding from 111.7 ± 0.3 to 86.5 ± 0.9 or 23.4 ± 0.9% with 100%, 2.5%, or 0% oxygen when compared with 100% oxygen without 3,4‐diaminopyridine, respectively. Thus, the decline in neuronal processing that accompanies acute hypoxia may be due to altered calcium homeostasis, which diminishes neuro‐transmitter release.
Agonist Effects on the Intracellular Cyclic AMP Concentration of Retinal Pigment Epithelial Cells in Culture
Authors: Koh Shay‐Whey M. , Chader Gerald J.
Published: October 2006

Abstract: Accumulation of cyclic AMP in intact cultured pigment epithelial cells was rapidly enhanced by several agonists. These included vasoactive intestinal peptide (100‐fold), glucagon (fivefold), thyroid‐stimulating hormone (threefold), prostaglandin E1 (24‐fold), l‐isoproterenol (27‐fold), and histamine (fourfold). The rapidity and magnitude of these effects suggest that these agonists may regulate important retinal pigment epithelial cell functions.
Isolation and Characterization of an Enriched Golgi Fraction from Neurons of Developing Rat Brains
Authors: Gonatas Jacqueline O. , Gonatas Nicholas K. , Stieber Anna , Fleischer Becca
Published: October 2006

Abstract: We report a method for the isolation of enriched fractions of intact Golgi apparatus from neurons of 10‐ to 12‐day‐old rat brains. Neurons were prepared according to a modified method of Farooq and Norton [J. Neurochem.31, 887–894 (1978)]. Golgi‐enriched fractions were obtained after centrifugation of postmitochondrial supernatants in a discontinuous sucrose gradient. Golgi fractions 1 and 2, recovered at the interfaces of 28–34% and 34–36% sucrose densities, respectively, were examined with morphometric and enzymatic methods. Morphometric analyses showed that 21–34% of fraction 1 and 11–29% of fraction 2 consisted of intact Golgi apparatus. Lysosomes, mitochondria, ribosomes, and rough endoplasmic reticulum contaminated fraction 1 (6–10%) and fraction 2 (14–26%). Golgi fraction 1 showed a 25‐ to 65‐fold enrichment over neurons of UDP Gal:GlcNAc galactosyltransferase, CMP‐sialic acid:lactosylceramide sialyltransferase, and PAPS:cerebroside sulfotransferase activities. Golgi fraction 2 showed a 8‐to 23‐fold enrichment over neurons of the activities of the above glycolipid‐ and glycoprotein‐synthesizing enzymes. The activities of the possible marker enzymes rotenone‐insensitive NADH‐cytochrome c reductase, succinate‐cytochrome c reductase, and arylsulfatase were low or minimally elevated in the Golgi fractions. A sevenfold enrichment of Na+,K+‐ATPase activities was found in the Golgi fractions. This is consistent either with significant plasma membrane contamination or with the presence of this enzyme in the neuronal Golgi apparatus.
Elevation of RNA Coding for Tyrosine Hydroxylase in Rat Adrenal Gland by Reserpine Treatment and Exposure to Cold
Authors: Tank A. William , Lewis Elaine J. , Chikaraishi Dona M. , Weiner Norman
Published: October 2006

Abstract: When rats are treated daily with reserpine or maintained at 4°C, the level of a specific RNA coding for tyrosine hydroxylase is elevated in the adrenal gland. The increase in this specific RNA temporally precedes and is quantitatively equal to the increase in adrenal tyrosine hydroxylase enzyme activity elicited by these treatments. These results suggest that prolonged stress may lead to changes in the levels of specific RNA species in the adrenal gland.
Retrograde Effect of Muscle on Forms of Acetylcholinesterase in Peripheral Nerves
Authors: Bacou F. , Vigneron P. , Couraud J. Y.
Published: October 2006

Abstract: In the peripheral nerves of birds and mammals, acetylcholinesterase (AChE) exists in four main molecular forms (G1, G2, G4, and A12). The two heaviest forms (G4 and A12) are carried by rapid axoplasmic transport, whereas the two lightest forms (G1 and G2) are probably much more slowly transported. Here we report that nerves innervating fast‐twitch (F nerves) and slow‐twitch (S nerves) muscles of the rabbit differ both in their AChE molecular form patterns and in their anterograde and retrograde axonal transport parameters. Since we had previously shown a selective regulation of this enzyme in fast and slow parts of rabbit semimembranosus muscle, we wondered whether the differences observed in the nerve could be affected by the twitch properties of muscle. The results reported here show that in F nerves that reinnervate slow‐twitch muscles, both the AChE molecular form patterns and axonal transport parameters turn into those of the S nerve. These data suggest the existence of a retrograde specific effect exerted by the muscles on their respective motoneurons.
Characterization of [3H]Ouabain Binding Sites in Human Brain, Platelet, and Erythrocyte
Authors: Hauger R. , Luu H. M. D. , Goodwin F. K. , Paul S. M.
Published: October 2006

[3H]Ouabain binding was investigated in membranes prepared from human brain, erythrocyte, and platelet. Scatchard analysis of [3H]ouabain binding to human hypothalamic membranes revealed a single class of noninteracting binding sites with an apparent affinity constant (KD) of 21 nM. Though the number of [3H]ouabain binding sites was lower in human platelets than in erythrocytes, both tissues exhibited a single class of high‐affinity binding sites with an apparent KD similar to that found in human brain. Specific [3H]ouabain binding in basal ganglia tissue from patients with Huntington's disease was more than 50% lower than in tissue from age‐ and sex‐matched controls. These results, along with previous findings in rat brain, suggest that high‐affinity [3H]ouabain binding labels the neuronal form of Na,K‐ATPase in human brain, and may prove useful in quantitating this enzyme in postmortem brain samples.
Dopamine Inhibits Forskolin‐ and 3‐Isobutyl‐1‐Methylxanthine‐Induced Dark‐Adaptive Retinomotor Movements in Isolated Teleost Retinas
Authors: Dearry Allen , Burnside Beth
Published: October 2006

We have been investigating the mechanisms of diurnal and circadian regulation of teleost retinomotor movements. In the retinas of lower vertebrates, photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo movements at dawn and dusk. These movements continue to occur at subjective dawn and dusk in animals maintained in constant darkness. Cone myoids contract at dawn and elongate at dusk; RPE pigment disperses into the epithelial cells’ long apical processes at dawn and aggregates into the cell bodies at dusk. We report here that forskolin, an adenylate cyclase activator, and 3‐isobutyl‐1‐methylxanthine (IBMX), a phosphodiesterase inhibitor, each induces dark‐adaptive cone and RPE retinomotor movements in isolated light‐adapted green sunfish retinas cultured in constant light. Forskolin induces a 22‐fold elevation in retinal cyclic AMP content. Forskolin‐ and IBMX‐induced movements are inhibited approximately 65% and 95%, respectively, by 3,4‐dihydroxyphenylethylamine (dopamine). However, dopamine does not inhibit dark‐adaptive movements induced by dibutyryl cyclic AMP. Epinephrine is much less effective than dopamine in inhibiting forskolin‐induced movements, while phenylephrine and clonidine are totally ineffective. These results are consistent with our previous findings that treatments that increase intracellular cyclic AMP content promote darkadaptive retinomotor movement. They further suggest that dopamine inhibits adenylate cyclase activity in photoreceptors and RPE cells and thereby favors light‐adaptive retinomotor movements.
Succinic Semialdehyde as a Substrate for the Formation of γ‐Aminobutyric Acid
Authors: van Bemmelen F. J. , Schouten M. J. , Fekkes D. , Bruinvels J.
Published: October 2006

Abstract: The conversion of succinic semialdehyde into γ‐aminobutyric acid (GABA) by GABA‐transaminase was measured in rat brain homogenate in the presence of different concentrations of the cosubstrate glutamate. The calculated kinetic parameters of succinic semialdehyde for GABA‐transaminase were a limiting Km value of 168 μM and a limiting Vmax value of 38 μmol g−1 h−1. Combination with previously obtained data for the conversion of GABA into succinic semialdehyde revealed a kEq value of 0.04, indicating that equilibrium of GABAtransaminase is biased toward the formation of GABA. The increased formation of GABA in the presence of succinic semialdehyde was not due to an increased conversion of glutamate into GABA by glutamic acid decarboxylase. Therefore these results indicate that succinic semialdehyde can act as a precursor for GABA synthesis.
Glucose Transport in Astrocytes: Regulation by Thyroid Hormone
Authors: Roeder Lois M. , Williams Irene B. , Tildon J. Tyson
Published: October 2006

Abstract: Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate‐saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid‐hormone‐free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these “hypothyroid′’ cells markedly increased the net rate of 2‐deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes.
Inhibition of High‐Affinity Choline Uptake and Acetylcholine Synthesis by Quinuclidinyl and Hemicholinium Derivatives
Authors: Sterling Gerald H. , Doukas Peter H. , Ricciardi Fiori J. , Biedrzycka Diane W. , O'Neill John J.
Published: October 2006

Abstract: Choline uptake into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high‐affinity, sodium‐ and temperature‐dependent transport mechanism (HAChU). To assess the role of choline availability in regulation of ACh synthesis, the structure‐activity relationships of several hemicholinium (HC) and quinuclidinyl analogs were evaluated in a dose response manner. As confirms previous studies, the HCs, e.g., HC‐3, acetylsecohemicholinium, and HC‐15 are potent inhibitors of HAChU, HC‐3 being the most potent (I50= 6.1 × 10−8M). In the present study, the most potent quinuclidinyl derivative was the N‐methyl‐3‐quinuclidinone (I50= 5.6 × 10−7M). This compound had approximately 100‐fold greater inhibitory activity than the corresponding racemic alcohol, suggesting that the 3‐hydroxyl functional group is not absolutely essential for activity. Increasing the size of the N‐functional group from a methyl to an allyl in the alcohol led to a 10‐fold increase in activity. However, removal of the quaternizing N‐methyl group yielding the tertiary amine, 3‐quinuclidinol hydrochloride, greatly reduced its capacity to inhibit HAChU. Of the 2‐benzylidene‐3‐quinuclidinone derivatives studied, only the m‐chloro derivative significantly reduced HAChU.
Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells
Authors: Abou‐Donia M. M. , Wilson S. P. , Zimmerman T. P. , Nichol C. A. , Viveros O. H.
Published: October 2006

Abstract: Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N‐acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N‐acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three‐day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl‐ and 8‐bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP‐cyclohydrolase, the rate‐limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8‐bromo cyclic AMP. The increase in GTP‐cyclohydrolase and tetrahydrobiopterin induced by 8‐bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP‐cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP‐cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP‐dependent and cyclic AMP‐independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.
Calcium‐Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein
Authors: Israel M. , Meunier F. M. , Morel N. , Lesbats B.
Published: October 2006

Abstract: A “fatigue” of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half‐maximal at ∼30 μM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half‐maximal at ∼0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the “mediatophore.” Proteoliposomes equipped with purified mediatophore showed a calcium‐dependent activation and “fatigue” of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/ molecule) with a KD of 25 μM. The physiological implications of desensitization on the organization of release sites are discussed.
Stimulation by Bradykinin, Angiotensin II, and Carbachol of the Accumulation of Inositol Phosphates in PC‐12 Pheochromocytoma Cells: Differential Effects of Lithium Ions on Inositol Mono‐and Polyphosphates
Authors: van Calker Dietrich , Assmann Karin , Greil Waldemar
Published: October 2006

Abstract: Rat PC‐12 pheochromocytoma cells respond to stimulation with bradykinin, angiotensin II, and carbachol with an increased formation of labeled inositol phosphates after preincubation of the cells with [3H]inositol. Li+ potentiates greatly the agonist‐induced increase in amount of nositol mono‐, bis‐, and trisphosphate but not the increase in amount of inositol tetrakisphosphate. Separation of the isomers of inositol trisphosphate shows that the lithium‐induced increase in amount of inositol trisphosphate is due to potentiation evoked by lithium of the accumulation of inositol‐1,3,4‐trisphosphate.
Solubilisation of the γ‐Aminobutyric Acid/Benzodiazepine Receptor from Rat Cerebellum: Optimal Preservation of the Modulatory Responses by Natural Brain Lipids
Authors: Bristow David R. , Martin Ian L.
Published: October 2006

Abstract: We have solubilised the γ‐aminobutyric acid/ benzodiazepine (GABA/BDZ) receptor from rat cerebellum using 3‐[(3‐cholamidopropyl)dimethylammonio] l‐propane sulphonate (CHAPS) in the presence of a natural brain lipid extract and cholesteryl hemisuccinate. The soluble material shows a homogeneous [3H]flunitrazepam ([3H]FNZ) binding population with an equilibrium dissociation constant (KD) of 4.4 × 0.2 nM compared to a KD of 2.3 × 0.2 nM in cerebellar synaptosomal membranes. The receptor complex in solution retains the characteristic facilitation of [3H]flunitrazepam binding induced by GABA, the pyrazolopyridine cartazolate, and the depressant barbiturate pentobarbital to the same extent as that observed in synaptosomal membranes. Furthermore, these responses are retained both quantitatively and qualitatively when this preparation is stored for 48 h at 4°C. This is contrary to the results obtained with a CHAPS‐soluble preparation including asolectin in which these responses are anomalous and extremely labile on storage.
Stimulation of [3H]Dopamine Release by Nicotine in Rat Nucleus Accumbens
Authors: Rowell Peter P. , Carr Laurence A. , Garner Ann C.
Published: October 2006

Abstract: The mesolimbic system of the brain has been shown to be involved in the reward properties of a number of agents. It is possible that release of monoamines by nicotine in this brain area could be related to the pleasurable aspects related to cigarette smoking. In this investigation, the effect of nicotine on the release of [3H]dopamine in the nucleus accumbens of the rat was studied. It was shown that nicotine produced a concentration‐dependent increase in [3H]dopamine release at concentrations of 0.1 μM and above. The increase in release was found to be almost completely calcium dependent. The nicotine‐induced release was only partially blocked by the nicotinic antagonists hexamethonium and d‐tubocurarine. A number of cholinergic agonists, as well as other compounds, were tested for their capacity to mimic the effect of nicotine. At equimolar concentrations there was, at most, only 50% of the activity of nicotine. The results of this study demonstrate that nicotine stimulates the release of dopamine in the nucleus accumbens at concentrations similar to those in the blood of cigarette smokers. This suggests that the release of mono‐amines in specific nuclei of the mesolimbic system may be an important determinant of the desire to smoke cigarettes.
Digitonin‐Permeabilized Cells Are Exocytosis Competent
Authors: Schäfer Theo , Karli Urs O. , Gratwohl Eugen K.‐M. , Schweizer Felix E. , Burger Max M.
Published: October 2006

Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 μg/ml of digitonin. This release is time and temperature dependent, half‐maximal at 0.3 μM Ca2+, and, after washing out of endogenous ATP, half‐ maximal at about 0.5 mA/ MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis‐competent cells. Anti‐actin and anti‐chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin‐permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane.
Modulation of In Vivo Dopamine Release by D2 but Not D1 Receptor Agonists and Antagonists
Authors: Boyar William C. , Altar C. Anthony
Published: October 2006

Abstract: The capacity of D1 and D2 agonists and antagonists to regulate the in vivo release and metabolism of dopamine (DA) in mesolimbic and nigrostriatal DA neurons of the mouse was determined using gas chromatographic and mass fragmentographic (GC‐MF) analysis. DA release was inferred from levels of 3‐methoxytyramine (3‐MT) and DA metabolism was inferred from levels of 3,4‐dihydroxyphen‐ylacetic acid (DOPAC) and homovanillic acid (HVA). DA release was increased by the D2 antagonists haloperidol and metoclopramide but not by the D1 antagonists SCH 23390 and SKF 83566. DA metabolism was increased by each of the four antagonists but to a greater extent with the D2 antagonists. The D2 agonists CGS 15855A and LY 171555 decreased DA release whereas the D1 agonist SKF 38393, at relatively high doses, only slightly affected DA release. Each of the three agonists decreased DA metabolism but again metabolism was more affected by the D2‐selective drugs. The in vivo release of DA from mesolimbic and neostriatal DA neurons appears to be modulated by D2 but not by D1 receptors, whereas both receptor types can modulate DA metabolism.
Differential Regulation by Calmodulin of Basal, GTP‐, and Dopamine‐Stimulated Adenylate Cyclase Activities in Bovine Striatum
Authors: Harrison Jeffrey K. , Mickevicius Cynthia K. , Gnegy Margaret E.
Published: October 2006

Abstract: The concentration requirements of calmodulin in altering basal, GTP‐, and dopamine‐stimulated adenylate cyclase activities in an EGTA‐washed paniculate fraction from bovine striatum were examined. In the bovine striatal paniculate fraction, calmodulin activated basal adenylate cyclase activity 3.5‐fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 ± 56 nM to 60 ± 10 nM Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50–60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP‐binding protein, Ns. Mechanisms that involve potentiation of Ns‐mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP‐stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.
Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin‐Permeabilized Chromaffin Cells
Authors: Bittner Mary A. , Holz Ronald W.
Published: October 2006

Abstract: Tetanus exotoxin inhibited Ca2+‐dependent cate‐cholamine secretion in a dose‐dependent manner in digito‐nin‐permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca2+. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca2+ concentrations. Tetanus toxin also inhibited catechol‐amine secretion enhanced by phorbol ester‐induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin‐permeabilized cells to interact with a component of the Ca2+‐dependent exocytotic pathway to inhibit secretion.
Chaotropic Ions Affect the Conformation of Quisqualate Receptors in Rat Cortical Membranes
Authors: Honoré Tage , Drejer Jørgen
Published: October 2006

Abstract: Binding of [3H](R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid ([3H]AMPA) to quisqualate receptors in the presence of SCN ions produced curvilinear Scatchard plots. Kinetic investigations of [3H]‐AMPA binding showed that the curvilinearity cannot be explained by assuming binding to two separate binding sites or by considering it due to cooperative interaction. A more likely explanation is that the quisqualate receptors exist in two states, one with high and one with low affinity for [3H]AMPA. Chaotropic ions change the relaxation constant between the two states.
Studies on the Tissue Distribution of the Puromycin‐Sensitive Enkephalin‐Degrading Aminopeptidases
Authors: McLellan Stacey , Dyer Simon H. , Rodriguez Gerry , Hersh Louis B.
Published: October 2006

Abstract: An antiserum generated to the soluble form of the rat brain puromycin‐sensitive enkephalin‐degrading amino‐peptidase was used to determine the tissue distribution of the soluble and membrane‐associated forms of this enzyme. All tissues examined contained significant levels of the soluble enzyme form, with this enzyme accounting for >90% of the arylamidase activity in brain, heart, and skeletal muscle. Native gel electrophoresis coupled with activity staining as well as inhibition studies were used to confirm the presence of this enzyme in various tissues. Serum was found not to contain this particular aminopeptidase. In contrast to the results obtained with the soluble enzyme form, brain was the only tissue found to contain the membrane‐associated enzyme form. Although all tissues contained membrane‐associated aminopeptidase activity only the brain enzyme could be maintained in solution in the absence of detergent. In addition, the brain membrane‐associated enzyme could be distinguished from the membrane‐associated aminopeptidase activity in other tissues on the basis of its sensitivity to inhibition by puromycin.
Myelin‐Associated Calpain II
Authors: Yanagisawa Katsuhiko , Sato Shuzo , O'Shannessy Daniel J. , Quarles Richard H. , Suzuki Koichi , Miyatake Tadashi
Published: October 2006

Abstract: Anti‐chicken muscle calpain (calcium‐activated neutral protease) antibody (ACAb) was found to be absorbed by purified human brain myelin when titrated by enzyme‐linked immunosorbent assay, suggesting the close association of the protease with myelin. To confirm this, calcium‐dependent protease was extracted from myelin membrane and purified on a phenyl Sepharose CL 4B column. It was activated by calcium ion in the millimolar range, and therefore was determined to be calpain II. This enzyme fraction was electrophoresed and immunostained with ACAb, resulting in staining as a single band with apparent molecular weight of 80K. This protease degraded exogenous myelin‐associated glycoprotein. From the present results, it is suggested that calpain is bound to myelin membrane and involved in the turnover of myelin proteins.
Expression of the mRNA for τ Proteins During Brain Development and in Cultured Neurons and Astroglial Cells
Authors: Couchie D. , Charrière‐Bertrand C. , Nunez J.
Published: October 2006

Abstract: Two τ cDNA probes of 1.6 and 0.3 kilobases (kb) have been used to study the expression of the τ mRNAs during mouse brain development and in highly homogeneous primary cultures of neurons and astrocytes. (1) Whatever the stage, a 6‐kb mRNA was detected with the two probes. In the astrocytes a 6‐kb mRNA hybridized clearly only with the 1.6‐kb probe. (2) During brain development the abundance of τ mRNA increases from a late fetal stage (— 4 days) until birth, remains high until 6 days postnatal, and then markedly decreases to reach very low values in adulthood. Such a marked decrease in the abundance of τ mRNA parallels that of α‐tubulin mRNA. These data suggest that: (1) depending on the stage of development and on the cell type (neurons or astrocytes) τ mRNAs of the same size encode several τ proteins differing in molecular weight: several τ proteins are expressed either during early stages of development (juvenile τ proteins of 48 kilodaltons) or in adulthood (mature τ proteins of 50–70 kilodaltons) or are specific of the astrocyte (83 kilodaltons). (2) The expression of the two major components of axonal microtubules, tubulin and τ proteins, seems to be developmentally coordinated.
Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes
Authors: Mills A. , Demoliou‐Mason C. D. , Barnard E. A.
Published: October 2006

Analysis of the equilibrium binding of [3H]‐neurotensin(1–13) at 25°C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH= 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising <10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association‐dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin > neurotensin(8–13) > neurotensin(1–13). Smaller neurotensin analogues and neurotensin‐like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (KD= 5.5 nM, Bmax= 250 fmol/mg protein, nH= 1.0) was obtained in 2% digitonin/l mM Mg2+ extracts of membranes which had been preincubated (25°C, 1 h) with 1 mM Mg2+ prior to solubilization. Association‐dissociation kinetic studies then revealed the presence of two classes of sites (KD1= 0.5 nM, KD2= 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]‐neurotensin activity retained its sensitivity to cations and guanine nucleotide.
Neurotransmitter‐Induced Inositol Phosphate Formation in Neurons in Primary Culture
Authors: Weiss Samuel , Schmidt Bernard H. , Sebben Michèle , Kemp Dorothy E. , Bockaert Joël , Sladeczek Fritz
Published: October 2006

Abstract: Inositol‐1,4,5‐trisphosphate, produced in cells as a breakdown product of phosphatidylinositol‐4,5‐bisphosphate, induces, in many cell types, release of calcium from intracellular stores. In murine striatal neurons, differentiated in primary culture, carbachol, norepinephrine, glutamate, and neurotensin stimulate 3H‐labeled inositol phosphate (3H‐IP) production. The glutamate response was recently characterized as being mediated primarily by receptors of the quisqualate subtype. In the present study, we found that major differences exist between glutamate‐stimulated 3H‐IP formation and those stimulated by the other neuromediators. The maximal response to glutamate occurred before and during synaptogenesis and declined thereafter, whereas the maximal response to either carbachol or norepinephrine required complete neuronal differentiation. Although the glutamate response appears to be mediated exclusively by direct interaction with the neuro‐transmitter receptors, responses to carbachol, norepinephrine, and neurotensin were partially or completely blocked by tetrodotoxin.
Characterization of Microtubule‐Associated Protein 2 from Mouse Brain and Its Localization in the Cerebellar Cortex
Authors: Niinobe Michio , Maeda Nobuaki , Ino Hidetoshi , Mikoshiba Katsuhiko
Published: October 2006

Abstract: Microtubule‐associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A‐5m gel nitration. The purified preparation showed a single protein band on sodium dodecyl sul‐fate‐polyacrylamide gel electrophoresis using both a gradient gel (3.75–12.5%) and a low‐percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to close of porcine brain MAP2. Im‐munoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.
Localization of the Novel Neuropolypeptide h3 in Subsets of Tissues from Different Species
Authors: Bollengier F. , Mahler A.
Published: October 2006

Abstract: Recently we reported the isolation and partial biochemical characterization of a novel polypeptide, h3, from the human brain and liver. Thin‐layer isoelectric focusing showed that the polypeptide was ubiquitously distributed throughout the human brain. Immunophosphatase transfer electrophoresis showed that this protein was localized in several mammalian species and different tissues. In addition, h3 or h3‐like protein was demonstrated in subsets of tissues from one avian species. Protein h3 was present in epithelial and muscular tissue, as well as in nervous tissue; however, for all species investigated, it was most abundant in CNS and muscle.
Targeting and Processing of Pro‐Opiomelanocortin in Neuronal Cell Lines
Authors: Noël Gilles , Zollinger Lyze , Laliberté France , Rassart Eric , Crine Philippe , Boileau Guy
Published: October 2006

Abstract Pro‐opiomelanocortin (POMC) is the precursor to several pituitary hormones including adrenocorticotropic hormone and β‐endorphin (β‐END). POMC is also expressed in the brain, predominantly in discrete neuronal cell populations of the hypothalamus. In the pituitary and brain, POMC undergoes tissue‐specific proteolysis to release different bioactive peptides. POMC processing in neuronal cell lines was studied after infection of PC 12 and Neuro2A cells with a recombinant retrovirus carrying the porcine POMC cDNA. Our results indicate that both cell lines synthesize and target POMC to the regulated secretory pathway. Only the Neuro2A cells, however, can achieve proteolytic processing of POMC. Chromatographic and immunological characterization of the POMC‐related material showed that β‐lipotropin (β‐LPH) and nonacetylated β‐END(1‐31) are major maturation products of POMC in these cells. Release of both β‐LPH and β‐END(1‐31) from infected Neuro2A cells can be stimulated by secretagogues in a calcium‐dependent manner. Taken together, our results suggest that the cellular machinery of Neuro2A cells can recognize a foreign prohormone, target it to neurosecretory vesicles, process it into biologically active peptides, and secrete it in a manner characteristic to peptidergic neurons.
Increased Nigral Iron Content and Alterations in Other Metal Ions Occurring in Brain in Parkinson's Disease
Authors: Dexter D. T. , Wells F. R. , Lee A. J. , Agid F. , Agid Y. , Jenner P. , Marsden C. D.
Published: October 2006

Abstract: Levels of iron, copper, zinc, manganese, and lead were measured by inductively coupled plasma spectroscopy in parkinsonian and age‐matched control brain tissue. There was 31‐35% increase in the total iron content of the parkinsonian substantia nigra when compared to control tissue. In contrast, in the globus pallidus total iron levels were decreased by 29% in Parkinson's disease. There was no change in the total iron levels in any other region of the parkinsonian brain. Total copper levels were reduced by 34–45% in the substantia nigra in Parkinson's disease; no difference was found in the other brain areas examined. Zinc levels were increased in substantia nigra in Parkinson's disease by 50–54%, and the zinc content of the caudate nucleus and lateral putamen was also raised by 18–35%. Levels of manganese and lead were unchanged in all areas of the parkinsonian brain studied when compared to control brains, except for a small decrease (20%) in manganese content of the medial putamen. Increased levels of total iron in the substantia nigra may cause the excessive formation of toxic oxygen radicals, leading to dopamine cell death.
Enhanced Brain Cell Proliferation Following Early Adrenalectomy in Rats
Authors: Yehuda Rachel , Fairman Kenneth R. , Meyer Jerrold S.
Published: October 2006

Abstract: We have previously demonstrated an increase in adult brain DNA content in rats adrenalectomized on postnatal day 11. The present studies examined cell proliferation in cerebral cortex, cerebellum, hippocampus, and midbrain‐diencephalon following adrenalectomy at this age. Compared to sham‐operated controls, adrenalectomized animals showed increased [3H]thymidine incorporation into DNA (measured at 1 h following a pulse injection) in all brain regions at 7 and 14 days postsurgery. In some areas, the effect was already present as early as 2 days following adrenalectomy. Chronic replacement with corticosterone prevented this increase in DNA labelling in a dose‐dependent manner. When cell proliferation in the cerebral cortex and cerebellum was independently assessed by measuring changes in thymidine kinase activity, enzyme activity was significantly elevated in both areas at 7 and 14 days postsurgery. Finally, histological ex amination of the cerebellar cortex suggested a delayed disappearance of the external granular layer in several cerebellar lobules of adrenalectomized animals. Overall, these findings indicate that day‐11 adrenalectomy leads to a prolonged stimulation of mitotic activity in areas where cell formation at this time is exclusively glial (i.e., cerebral cortex and mid‐brain‐diencephalon) as well as in areas where postnatal neurogenesis is also occurring (cerebellum and hippocampus). It is hypothesized that this stimulation results from the removal of a tonic inhibitory effect exerted by circulating glucocorticoids in the normal intact animal.
Effects of Pregnancy on the Contents of Water, Taurine, and Total Amino Nitrogen in Rat Cerebral Cortex
Authors: Law R. O.
Published: October 2006

Abstract: Plasma osmolality and the levels of water, taurine, and total amino nitrogen (detected as ninhydrin‐positive substances) have been measured in the cerebral cortices of nonpregnant and 19‐day pregnant Wistar rats. Plasma osmolality fell by 11 mosmol/kg during pregnancy. Brain water content remained unaltered, but levels of taurine and ninhydrin‐positive substances fell by 48.5 and 21.9%, respectively. It is suggested that one way in which brain cells are prevented from swelling during the mild hypoosmolality of pregnancy is through loss of cellular amino nitrogen, particularly taurine.
Predominant Low‐Molecular‐Weight Proteins in Isolated Brain Capillaries Are Histones
Authors: Pardridge William M. , Nowlin Dawn M. , Calaycay Jimmy , Shively John E.
Published: October 2006

Abstract: Blood‐brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB‐specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16‐18K triplet of low‐molecular‐weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary‐free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16‐18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) and C4 reverse‐phase HPLC. Amino acid sequencing of the N‐terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are α‐globin, histone 2B, histone 3, and histone 2A, respectively. SDS‐PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16‐18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14‐18K proteins seen on SDS‐PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS‐PAGE.
Kinetic Analysis of Leucine‐Enkephalin Cellular Uptake at the Luminal Side of the Blood‐Brain Barrier of an In Situ Perfused Guinea‐Pig Brain
Authors: Zlokovic Berislav V. , Mackic Jasmina B. , Djuricic Bogdan , Davson Hugh
Published: October 2006

Abstract: The uptake of enkephalin‐(5‐L‐leucine) (Leu‐en‐kephalin) at the luminal side of the blood‐brain barrier was measured by means of an in situ vascular brain perfusion technique in the anaesthetized guinea pig. This method allows measurements of cerebrovascular peptide uptake over periods of up to 20 min, and excludes the solute under study from the general circulation and systemic metabolic influences. A capillary unidirectional transfer constant, Kin, for [tyrosyl‐3,5‐3H]Leu‐enkephalin was estimated graphically from the multiple‐time brain uptake data in the presence of different concentrations of unlabelled peptide, and dose‐dependent self‐inhibition was demonstrated. Analysis of unidirectional influx of blood‐borne Leu‐en kephalin into the brain revealed Michaelis‐Menten saturation kinetics in the parietal cortex, caudate nucleus, and hippocampus, with Vmax between 0.14 and 0.16 nmol min−1 g−1 and Km ranging from 34 to 41 μM, for the saturable component, whereas the estimated diffusion constant, Kd, was not significantly different from zero. Entry of [3H]Leu‐enkephalin was not inhibited in the presence of either a 5 mM concentration of unlabelled L‐tyrosine, tyro‐sylglycine, and tyrosylglycylglycine, or aminopeptidase inhibitor, bestatin (0.5 mM), suggesting that the saturable mechanism of the tracer at the luminal side of the blood‐brain barrier does not involve uptake of the peptide's N‐terminal amino acid and/or its tyrosine‐containing fragments. The specific δ‐opioid antagonist, allyl2‐Tyr‐AIB‐Phe‐OH, and μ‐opioid receptor agonist, Tyr‐D‐Ala‐Gly‐Me‐Phe‐NH(CH2)20H, at concentrations in the perfusate above the Km value for the saturable transport of Leu‐enkephalin, did not affect significantly uptake of [3H]Leu‐enkephalin. The present study provides, for the first time, a characterization of the kinetic parameters of the unidirectional uptake of a peptide from the luminal side of the blood‐brain barrier
Release of Glutamate, Aspartate, and γ‐Aminobutyric Acid from Isolated Nerve Terminals
Authors: Nicholls David G.
Published: October 2006

Abstract: With the advent of cloning, sequencing, and patchclamping techniques, knowledge of the postsynaptic actions of amino acid neurotransmitters has undergone a dramatic advance. The primary sequences of the inhibitory receptors for γ‐aminobutyric acid (GABA) (Schofield et al., 1987) and glycine (Grenningloh et al., 1987) are now established, and patch‐clamp analysis has elucidated many of the factors that regulate the opening of their ion channels. The excitatory glutamate receptors are being extensively characterized at both the pharmacological (reviewed by Foster and Fagg, 1984) and the electrophysiological (reviewed by Cull‐Candy and Usowicz, 1987) level. In this climate, it is perhaps surprising that the fundamental presynaptic release mechanism for the amino acid neurotransmitters remains controversial.
Utilization of High‐Density Lipoprotein Sphingomyelin by the Developing and Mature Brain in the Rat
Authors: Bentejac Marc , Bugaut Maurice , Delachambre Marie Claire , Lecerf Jean
Published: October 2006

Abstract:Utilization of very long chain saturated fatty acids by brain was studied by injecting 20‐day‐old and adult rats with high‐density lipoprotein containing [stearic or lignoceric acid‐14C, (methyl‐3H)choline]sphingomyelin. Labeling was followed for 24 h. Very small amounts of14C were recovered in the brain of all rats, and there was no preferential uptake of lignoceric acid. Approximately 20% of the entrapped14C was located in the form of unchanged sphingomyelin 24 h after injection. This result shows that the rat brain utilizes very little very long chain fatty acids (≥20 C atoms) from high‐density lipoprotein sphingomyelin, even during the myelinating period. The [3H]choline moiety from sphingomyelin was recovered in brain phosphatidylcholine in a higher proportion in comparison with the14C uptake. The brain3H increased throughout the studied period in all experiments, but was much higher in the myelinating brain than in the mature brain. From the radioactivity distribution in liver and plasma lipids, it is clear that the choline3H in the brain originates from either double‐labeled phosphatidylcholine of lipoproteins or tritiated lysophosphatidylcholine bound to albumin, both synthesized by the liver.
Development and Ageing of the Rat Nigrostriatal Dopamine System Studied with Fast Cyclic Voltammetry
Authors: Stamford Jonathan A.
Published: October 2006

Abstract: Fast cyclic voltammetry at carbon fibre microelectrodes was used to measure electrically stimulated dopamine release in the striatum of anaesthetised young, adult, and senescent Wistar rats. By alteration of stimulus parameters and by use of nomifensine, investigation of dopamine release, uptake, and compartmentalisation within the striatum was possible. The rate of dopamine release was highest in adult rats. No difference was observed between young and old animals. The size of the releasable (newly synthesised) dopamine pool was also largest in the adult group, again with no significant difference occurring between young and aged rats. The rate of dopamine uptake was highest in adult rats, although, when expressed as a function of dopamine release, young and aged rats showed proportionally greater uptake. Nomifensine (10 mg/kg i.p.) increased dopamine release by mobilising the inert storage pool to a greater extent in young than in adult rats, whereas the effect of the drug on uptake was similar in all age groups. The functional significance and possible explanation of these results are discussed.