bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Stability of Rat Brain Transketolase After Conversion to the Apo Form
Authors: Jeyasingham Marina D. , Pratt Oliver E. , Thomson Allan D. , Shaw George K.
Published: July 2010

The stability of rat brain transketolase, whether measured at 37 or 0°C, was reduced after conversion to the apo form by removal of thiamine diphosphate, as shown by a decline in the activity recovered when assayed in the presence of thiamine diphosphate. Both the shape of the breakdown curve and the failure to recover the full activity, even after incubation with thiamine diphosphate, showed that the breakdown of the apotrans‐ketolase was complex. The initial rate of breakdown of the apoenzyme was sharply pH dependent, being minimal at 37°C at a pH value of 7.6, close to that likely to exist in vivo. The rate rose sharply with deviation of the pH in either direction. The stability of the enzyme on storage at 0°C showed a similar pattern of pH dependence, provided that allowance is made for temperature effects on dissociation constants. These findings provide further support for the hypothesis that differences in brain transketolase may play a part in the etiology of Wernicke‐Korsakoff's syndrome.
Detection and Characterization of Some New Basic Proteins in Chicken Postsynaptic Densities
Authors: Yang Zan Wei , Babitch Joseph A.
Published: July 2010

Chicken brain postsynaptic density (PSD) polypeptides, obtained by treating synaptosomes with 0.5% Triton X‐100 and then further purified on a sucrose gradient, are demonstrated to contain four basic proteins of 76K (pi > 9.2), 58K (pi 8.1–8.8, heterogeneous), 40K (pI 9.0), and 24K (pi 8.9). Nonequilibrium pH gradient‐sodium dodecyl sulfate two‐dimensional gels further reveal six more basic proteins with pI values higher than 9.2: 76K, 52K, 47K, 45K, 36K, and 34K. These basic proteins are a major part of the total chicken PSD polypeptides appearing on the gels. Some of these basic proteins (58K, 52K, 47K, 36K, 24K, and two at 76K) are distinguishable from those of brain mitochondria, the major contaminant. The 40K and 34K proteins may be common mitochondrial polypeptides. The 45K protein is probably a mitochondrial contaminant. A number of proteins including 76K (synapsin I‐like protein) and 58K, along with some other minor ones, can be phosphorylated by endogenous protein kinase(s) in the presence of Ca2+, Mg2 +, and [γ‐32P]ATP. No PSD basic proteins bind Ca2+.
A Protein Modulator Stimulates C Kinase‐Dependent Phosphorylation of a 90K Substrate in Synaptic Membranes
Authors: Kligman Douglas , Patel Jitendra
Published: July 2010

We have identified and partially purified an acidic, heat‐stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+‐dependent phosphorylation of an Mr90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+‐ and phospholipid‐dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mrform of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+‐dependent phosphorylation of both substrates is inhibited by the Ca2+‐binding proteins S‐100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr9K and 13K after limited proteolysis with V8 protease. Two‐dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic iso‐electric points (pI 5.0). MOP enhances Ca2+‐dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate‐polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase‐mediated phosphorylation in the nervous system.
Enhancement of γ‐Aminobutyric Acid Binding by Quazepam, a Benzodiazepine Derivative with Preferential Affinity for Type I Benzodiazepine Receptors
Authors: Corda M. G. , Sanna E. , Concas A. , Giorgi O. , Ongini E. , Nurchi V. , Pintori T. , Crisponi G. , Biggio G.
Published: July 2010

Abstract: We evaluated the effect of the two N‐trifluoroethyl benzodiazepines, quazepam and its 2‐oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H]γ‐aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N‐desalkyl‐2‐oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen‐thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4°C in the absence and presence of quazepam or its metabolites. Addition of 10−‐6M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding‐enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 × 10−‐6M Ro15–1788.
Effect of Nerve Growth Factor on Glucose Utilization and Nucleotide Content of Pheochromocytoma Cells (Clone PC12)
Authors: Morelli Alessandro , Grasso Marina , Calissano Pietro
Published: July 2010

Abstract: The effect of nerve growth factor (NGF) on the utilization and fate of uniformly labeled 14C glucose and on the content of several pyridine and purine nucleotides has been tested in the clonal cell line PC12. After incubation for 72 h with NGF, PC12 cells exhibit a 2.7‐fold increase in glucose utilization and a 4.7‐fold increase in CO2 release. During the same incubation period, all the nucleotides tested (NAD+, AMP, GMP, UDP‐glucose, UDP‐galactose, UDP, ADP, GDP, UTP, CTP, ATP, and GTP) underwent significant increments, varying from a minimum of 27% for ADP to a maximum of 90–120% for AMP, GMP, UDP‐glucose, and UDP‐galactose. These findings are discussed in connection with the trophic and differentiative effects of NGF in PC12 cells, which, in the presence of this factor, shifted from a neoplastic to a neu‐ronal‐like cell population.
Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium‐Dependent Acetylcholine Release from Proteoliposomes
Authors: Birman Serge , Israël Maurice , Lesbats Bernard , Morel Nicolas
Published: July 2010

Abstract: In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium‐dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium‐dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein‐lipid complex of large size and low density which was partially purified. The specificity of the calcium‐dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium‐dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.
Presence of Vasoactive Intestinal Polypeptide‐Like Immunoreactivity in the Cholinergic Electromotor System of Torpedo marmorata
Authors: Agoston D. V. , Conlon J. M.
Published: July 2010

Abstract: Vasoactive intestinal polypeptide (VIP)‐like immunoreactivity was detected in the cholinergic electromotor system of Torpedo marmorata using a combination of immunohistochemical assays, radioimmunoassay, and HPLC. The immunohistochemical assays revealed that the distribution of VIP‐like immunoreactivity in the electric lobes, electromotor nerves, and electric organ is comparable to that of the stable cholinergic synaptic vesicle marker vesicle‐specific proteoglycan. Ligation of the electromotor nerves caused a marked accumulation of VIP‐like immunoreactivity in the lobes (180%) and the proximal portions of the electromotor nerves (130%) and a decrease in the electric organ (‐50%), when measured by radioimmunoassay using synthetic VIP (porcine sequence) as the standard. VIP‐like immunoreactivity in extracts of electric lobes electromotor nerves, and electric organ was eluted from a semipreparative reverse‐phase HPLC column as a single peak with a retention time similar to that of porcine VIP. Rechromatography at higher resolution on an analytical column indicated diversity between the molecular forms of VIP‐like immunoreactivity extracted from electric lobe and electric organ, suggesting the possibility of posttranslational processing.
Desensitization of β‐Adrenergic Receptor‐Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine
Authors: Okada Fumihiko , Tokumitsu Yukiko , Ui Michio
Published: July 2010

Abstract:Continuous treatment (1–10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the β‐adrenergic receptor‐coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol‐stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of β‐adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl‐5′‐yl‐β‐γ‐imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p‐induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug‐treated rats. The lag was shortened by the addition of isoproterenol, indicating that β–receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of β‐adrenergic receptor‐mediated adenylate cyclase in the cerebral cortex during desipramine therapy.
Uptake of γ‐Aminobutyric Acid and Glycine by Synaptosomes from Postmortem Human Brain
Authors: Hardy J. A. , Barton A. , Lofdahl E. , Cheetham S. C. , Johnston G. A. R. , Dodd P. R.
Published: July 2010

Abstract:Synaptosomes prepared from frozen postmortem human brain accumulated the neurotransmitter γ‐aminobutyric acid (GABA) and the conformationally restricted GABA analoguecis‐3‐aminocyclohexanecarboxylic acid (ACHC) by a sodium‐dependent, temperature‐sensitive, high‐affinity transport process into an osmotically sensitive compartment. This transport process could be inhibited by GABA analogues (ACHC, 2,4‐di‐aminobutyric acid, nipecotic acid, arecaidine, guvacine) that have been shown in studies on other species to be relatively selective for neuronal rather than glial uptake systems, whereas the glial uptake inhibitor β‐alanine was ineffective. Synaptosomes prepared from frozen postmortem human medulla and spinal cord, but not cerebral cortex, took up the neurotransmitter glycine by a sodium‐dependent high‐affinity transport process. The kinetic parameters for the high‐affinity uptake of GABA, ACHC. and glycine wereKm= 10 ± 3, 49 ± 19, and 35 ± 19 μM;and Vmax= 98 ± 15, 84 ± 25, and 5.5 ± 2.5 nmol/min/100 mg protein, respectively. These results demonstrate the feasibility of using human CNS preparations for studying GABA and glycine uptake, and suggest that such studies may be useful neurochemical markers for transmitter‐specific presynaptic terminals in health and disease.
Mobilization of Storage Pool Dopamine and Late Ipsilateral Augmentation of Striatal Dopamine Synthesis in the Trained Circling Rat
Authors: Bennett Barbara A. , Freed Curt R.
Published: July 2010

Abstract:Rats were infused intraventricularly with [3H]tyrosine over a 20–min period during various times while circling. 3,4–Dihydroxyphenylethylamine (dopamine) and dihydroxyphenylacetic acid (DOPAC) levels were measured using HPLC with electrochemical detection and fractions were collected for tritium monitoring. During the first 20 min of circling, the specific activity of dopamine was increased by 290% in striatum contralateral to the circling direction whereas DOPAC specific activity was increased 50% on the same side. This differential change in relative specific activity suggests that unlabeled storage pool dopamine was mobilized to DOPAC during circling. Synthesis of dopamine and DOPAC in contralateral striatum returned to baseline levels as turning slowed (50–70 min). When turning ceased, there was an increase in ipsilateral striatal dopamine synthesis during the 20–min period following circling. We hypothesize that this ipsilateral increase represents either a “stop” signal following circling or a release of inhibition of ipsilateral nigral neurons.
Pharmacological Differentiation and Characterization of 5‐HT1A, 5‐HT1B, and 5‐HT1CBinding Sites in Rat Frontal Cortex
Authors: Peroutka Stephen J.
Published: July 2010

Abstract:Drug interactions with 5‐HT1(5‐hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8‐Hydroxy‐N, N‐dipropyl‐2‐aminotetralin (8‐OH‐DPAT) displays high affinity (Ki3.3 ± 1 nM) for 29 ± 3% of total [3H]5‐HT binding in rat frontal cortex and low affinity (K, 9,300 ± 1,000) for 71 ± 4% of the remaining 5‐HT1sites. Therefore, non‐5‐HT1Abinding in rat frontal cortex was defined as specific [3H]5‐HT binding observed in the presence of 100 nM8‐OH‐DPAT. 5‐Methoxy 3‐(1,2,3,6‐tetrahydro‐4‐pyridinyl) 1Hindole (RU 24969), 1–(m‐trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5‐HT binding from non‐5‐HT1Asites. Addition of 10−‐3MGTP does not increase the apparent Hill slopes of these competition curves. Computer‐assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non‐5‐HT1Abinding sites, each representing ∼35% of the total [3H]5‐HT binding in the rat frontal cortex. All three 5‐HT, binding site subtypes display nanomolar affinity for 5‐HT and 5‐methoxytryptamine. A homogeneous population of 5‐HT1Asites can be directly labeled using [3H]8‐OH‐DPAT. These sites display nanomolar affinity for 8‐OH‐DPAT, WB 4101, RU 24969, 2‐{4‐(4‐(2‐pyrimidinyl)‐1‐piperazinyl]butyl}‐1,2‐benzisothiazol‐3‐(2H)one‐1,1‐dioxidehydrochloride (TVX Q 7821), 5‐methoxydimethyltryptamine, andN‐lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5‐HT binding are increased by addition of 100 nM8‐OH‐DPAT and 3,000 nMmianserin to the [3H]5‐HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5‐HT binding is designated 5‐HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5‐HT binding to non‐5‐HT1Asites in the presence of 100 nM8‐OH‐DPAT and 10 nMRU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5‐HT1C. Drug competition studies using a series of 24 agents reveals that each 5‐HT, subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5‐HT, binding sites labeled by [3H]5‐HT in rat frontal cortex.
An Investigation of the Low Intrinsic Activity of Adenosine and Its Analogs at Low Affinity (A2) Adenosine Receptors in Rat Cerebral Cortex
Authors: Bazil Carl W. , Minneman Kenneth P.
Published: July 2010

Abstract:The potencies and intrinsic activities of adenosine analogs for stimulating cyclic AMP accumulation in slices of rat cerebral cortex were examined. 5′‐N‐Ethylcarboxamidoadenosine (NECA) caused the greatest increase in cyclic AMP accumulation (19.2‐fold). 2‐Chloroadenosine (2‐CAD) induced a similar increase, but adenosine and six other analogs caused much smaller increases. All agonists tested had similar potencies in activating this response. Inhibition of adenosine uptake with 10 μMdipyridamole did not affect the maximal response to any agonist, although the potency of adenosine was increased approximately threefold. Each analog was also able to block partially the stimulation of cyclic AMP accumulation caused by NECA. Levels of cyclic AMP accumulation in the presence of NECA plus another analog were similar to those observed when the analog alone was present, as expected for partial agonists. Furthermore, the EC50value forR‐(‐)‐N6(2‐phenylisopropyl)adenosine in increasing cyclic AMP accumulation was similar to theK1value for inhibiting the response to NECA. The EC50value for adenosine was substantially higher than theK1value for inhibiting the response to NECA; however, in the presence of dipyridamole, the two values were more closely correlated. The response to NECA was blocked by 8‐phenyltheophylline, 1,3‐diethyl‐8‐phenylxanthine, and 8‐p‐sulfophenyltheophylline, withK1values from 1 to 10 μM. The results suggest that adenosine analogs stimulate cyclic AMP accumulation in cerebral cortex through low‐affinity receptors, but that some analogs only partially activate these receptors. Adenosine itself may also be a partial agonist, or its actions may be obscured by simultaneous activation of another receptor.
A and B Forms of Monoamine Oxidase Within the Monoaminergic Neurons of the Rat Brain
Authors: Fagervall Ingrid , Ross Svante B.
Published: July 2010

Abstract:The inhibition of the A and B forms of monoamine oxidase (MAO) inside and outside serotonergic, noradrenergic, and dopaminergic synaptosomes in homogenates of rat hypothalamus or striatum by clorgyline, a selective and irreversible MAO‐A inhibitor, and selegiline, a selective and irreversible MAO‐B inhibitor, was examined. Intrasynaptosomal deamination at low concentrations of the substrates [14C]5‐hydroxytryptamine ([14C]5‐HT; 0.1 μM), [14C]noradrenaline (0.25 μM), [14C]3,4‐dihydroxyphenylethylamine ([14C]dopamine; 0.25 μM), and [14C]tyramine (0.25 μM), was hindered by selective uptake inhibitors (citalopram, maprotiline. and amfonelic acid) in the incubation media. Thus, the difference between the deamination of14C‐amine in the absence and presence of the appropriate selective uptake inhibitor provided a measure of deamination in the specific aminergic synaptosomes. This was verified by determining the loss of MAO activity within noradrenergic and serotonergic systems after degeneration of the nerve terminals by the neurotoxinsN‐chloroethyl‐N‐ethyl‐2‐bromobenzylamine andp‐chloroamphetamine. Results with the two inhibitors revealed that the A and B forms were responsible for 80 and 20%, respectively, of the deamination of [14C]5‐HT within serotonergic synaptosomes from the hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes from the hypothalamus and that of [14C]dopamine and [14C]tyramine within the striatal dopaminergic synaptosomes were due to MAO‐A. About 10% of the deamination of [14C]noradrenaline, [14C]dopamine, and [14C]tyramine outside the noradrenergic or dopaminergic synaptosomes was brought about by the B form, with the remainder being deaminated by MAO‐A.
Developmental Change in the Glycosaminoglycan Composition of the Rat Brain
Authors: Oohira Atsuhiko , Matsui Fumiko , Matsuda Motoko , Shoji Ryujiro
Published: July 2010

Abstract: Glycosaminoglycans (GAGs) were isolated from the brains of pre‐ and postnatal rats. The GAG content of the brain, based on the amount of DNA, was constant during the period from day 13 to day 15 of gestation. After day 15, the GAG content began to increase and reached a plateau by 10 days after birth. Hyaluronate (HA) was the main GAG (> 60% of the total) in the fetal rat brain, and the relative amount of HA decreased after birth. Conversely, the relative amount of chondroitin sulfate increased with development and reached the adult level by 20 days after birth. Heparan sulfate (HS) was the major sulfated GAG in the fetal rat brain at early developmental stages, but HS accounted for approximately 10% of the total GAG in the postnatal brains. In addition to these GAGs, a polysialosyl glycoconjugate was isolated from rapidly growing brains of the rat. These three GAGs could be isolated either from the cerebellum, cerebrum, or brainstem of the newborn rat. A closely similar age‐related change in the GAG composition was observed in each of these different regions of the brain. The developmental change could be implicated in morphogenesis or maturation of the brain.
Role of Adenylate Cyclase in Presynaptic α2‐Adrenoceptor‐ and μ‐Opioid Receptor‐Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices
Authors: Schoffelmeer Anton N. M. , Wierenga Eddy A. , Mulder Arie H.
Published: July 2010

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ionophore A 23187 (10 μM) in the presence of 1.2 mMCa2+. Forskolin (10 μM), 8‐bromo‐cyclic AMP (300 μM), and dibutyryl‐cyclic AMP (300 μM) facilitated both the electrically evoked and A 23187‐induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3‐isobutyl‐1‐methylxanthine (IBMX, 300 μM) and 4‐(3‐cyclopentyloxy‐4‐methoxyphenyl)‐2‐pyrrolidone (ZK 62771, 30 μM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM–1 μM) and the facilitatory effect of phentolamine (0.01–10 μM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8‐bromo‐cyclic AMP. Clonidine (1 μM) reduced and phentolamine (3 μM) enhanced A 23187‐induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 μM) and [D‐Ala2‐D‐Leu5]enkephalin (DADLE, 0.3 μM), like that of the Ca2+antagonist Cd2+(15 μM), on the electrically evoked release of [3H]noradrenaline were not affected by 8‐bromo‐cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187‐induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic μ‐opioid receptors, α2‐adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed‐forward action of cyclic AMP on the secretion process.
Cerebrovascular Permeability Coefficients to Sodium, Potassium, and Chloride
Authors: Smith Quentin R. , Rapoport Stanley I.
Published: July 2010

CSF and regional brain concentrations of42K,22Na,36Cl, and [14C]mannitol were determined 3–45 min after intravenous injection of the tracers in pentobarbitalanesthetized rats. Rapid influx of36Cl and22Na into ventricular CSF immediately established concentration gradients from CSF to brain extracellular fluid. The CSF contribution to brain uptake of tracers was greatest in periventricular brain regions, where brain36Cl concentrations were up to ninefold higher than concentrations in regions distant from ventricular CSF. Acetazolamide (20 mg kg−1i.p.), an inhibitor of CSF formation, decreased36Cl uptake into CSF and into periventricular brain regions but not into frontal cortex.36Cl uptake into brain was unidirectional for 10 min after intravenous injection, and, during that period, diffusion from ventricular CSF did not contribute to uptake in the frontal cortex. Therefore, cerebrovascular permeability coefficients could be calculated from tracer concentrations in frontal cortex at 10 min and equaled, in cm s−1, 13.5 × 10−1for42K, 1.4 × 10−7for22Na, 0.9 × 10−7for36Cl, and 1.5 × 10−7for [14C]mannitol. The low cerebrovascular permeabilities to K, Na, and Cl, comparable to those of some cell membranes, and the permselectivity (K >> Na > Cl) suggest that a signficant fraction of ion transport across cerebral capillaries is transcellular, i.e., across the endothelial cell membrane.
Activation of the Voltage‐Sensitive Sodium Channel by a β‐Scorpion Toxin in Rat Brain Nerve‐Ending Particles
Authors: Bablito Joseph , Jover Emmanuel , Couraud François
Published: July 2010

Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of β‐scorpion toxin (β‐ScTx) on this preparation has not yet been described. In this report we show that a β‐ScTx induced an increase in22Na+uptake through synaptosomal voltage‐sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between β‐ScTx and veratridine, as is the case for α‐scorpion toxin (α‐ScTx) and veratridine. The effects of α‐ and β‐ScTx were additive and the concentration‐effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. β‐ScTx was shown to inhibit the uptake and to stimulate the release of [3H]γ‐aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltagesensitive sodium channels induced by the binding of β‐ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.
Highly Sensitive Immunoassay for Rat Brain‐Type Creatine Kinase: Determination in Isolated Purkinje Cells
Authors: Kato Kanefusa , Suzuki Fujiko , Shimizu Atsuko , Shinohara Haruo , Semba Reiji
Published: July 2010

Ultrasensitive enzyme immunoassay method for the measurement of rat brain‐type creatine kinase BB (CK‐BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK‐BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab′)2 fragments and the same antibody Fab' fragments labeled with β‐D‐galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK‐B), showing about 10% cross‐reactivity with CK‐MB, but it did not cross‐react with CK‐MM and neuron‐specific γγ enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK‐BB, being sufficiently sensitive for the measurement of CK‐B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK‐B in a single Purkinje cell was 1.64 pg. The CK‐B concentration in rat cerebellum (about 22 μg/mg protein) was about twofold higher than that (about 13 μg/mg protein) in the cerebrum. High levels (> 5 μ/mg protein) of CK‐B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK‐B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.
Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain
Authors: Colon Arlene D. , Plaitakis Andreas , Perakis Antonis , Berl Soll , Clarke Donald D.
Published: July 2010

Glutamate dehydrogenase (GDH) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low‐speed pellet (480gfor 10 min). Approximately 60% of the high‐speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similarKmvalues for the two GDH fractions for the substratesμ‐ketoglutarate, ammonia, and glutamate; however, there were small but significant differences inKmvalues for NADH and NADPH. Although the allosteric stimulation by ADP and L‐leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mMADP, with apparentKivalues of 18.5 and 9.0 μMGTP for the soluble and particulate fractions, respectively. The HIll plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45°C and pH 7.4 in 50 mMphosphate buffer.
Cross‐Linking of Neurofilament Proteins of Rat Spinal Cord In Vivo After Administration of 2,5‐Hexanedione
Authors: Lapadula Daniel M. , Irwin Richard D. , Suwita Elizabeth , Abou‐Donia Mohamed B.
Published: July 2010

The aliphatic hexacarbonsn‐hexane, methyl‐nbutyl ketone, and 2,5‐hexanedione are known to produce a peripheral neuropathy that involves an accumulation of 10‐nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. In this study, rats were treated with 0.5% 2,5‐hexanedione in drinking water for 180 days, and their spinal cord neurofilaments were isolated after development of the neuropathy. Visualization by sodium dodecyl sulfate‐polyacrylamide gel electro phoresis revealed a significant reduction in content of the neurofilament triplet proteins in treated animals and the presence of bands migrating at 138K and 260K that were not present in control animals. Analysis of the lanes using immunoblotting procedures and anti‐70K, anti‐160K, and anti‐210K neurofilament antibodies revealed many crosslinked peptides. The 138K band cross‐reacted with the anti‐160K neurofilament antibody. This suggests that the 138K band is an intramolecular cross‐link of the 160K neurofilament subunit. In addition to this peptide, there were numerous high‐molecular‐weight peptides immuno reactive with all three neurofilament protein antibodies. In addition to cross‐linking, there was also a diminished amount of immunoreactive breakdown product of all three neurofilament proteins. This report demonstrates direct evidence of 2,5‐hexanedione‐induced cross‐linking of neurofilament proteins in vivo, which maybe responsible for the accumulation of neurofilament proteins pathognomic of this neuropathy.
An Assessment of the Role of Opioid Receptors in the Response to Cannabimimetic Drugs
Authors: Devane William A. , Spain James W. , Coscia Carmine J. , Howlett Allyn C.
Published: July 2010

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in NI8TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the δ subtype were found on N18TG2 membranes using [3H]D‐Ala2‐D‐Leu5‐enkephalin. No δ or K receptors were detected using selective ligands for these sites. The δ binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs Δ9‐tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D‐Ala2‐Met5‐enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108‐15 neuroblastoma x glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.
Secretin Stimulates Cyclic AMP Formation in the Rat Brain
Authors: Fremeau Robert T. , Korman Louis Y. , Moody Terry W.
Published: July 2010

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mMtheophylline resulted in a dose‐dependent increase in the cyclic AMP levels. The half‐maximal increase in cyclic AMP occurred using a 1 μM dose of secretin or a 2 μMdose of PHI or VIP. Preincubation of slices with secretin‐(5–27) produced a dose‐dependent inhibition of the secretin but not VIP‐ or PHI‐stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin‐(5–27) produced a dose‐dependent inhibition (Ki= 400 nM) of125I‐secretin but not of125I‐VIP binding to rat brain membranes. Guanyl‐5′‐yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound125I‐secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin‐(5–27) may function as a central secretin receptor antagonist.
Catecholamine‐Metabolizing Enzymes of Bovine Brain Microvessel Endothelial Cell Monolayers
Authors: Baranczyk‐Kuzma Anna , Audus Kenneth L. , Borchardt Ronald T.
Published: July 2010

The activities of monoamine oxidase (MAO), catechol‐O‐methyltransferase (COMT), phenol sulfotransferase (PST), alkaline phosphatase (AP), γ‐glutamyl transpeptidase (GT), and angiotensin converting enzyme (ACE) were quantitated in primary cultures of bovine brain microvessel endothelial cell monolayers and cerebral gray matter. Significant MAO‐A and ‐B, cytosolic and membrane‐bound COMT, PST, AP, GT, and ACE activities are demonstrated in bovine gray matter. By comparison, enzyme activities of the monolayers vary with the age of the monolayer and are generally higher in complete monolayers. Relative to gray matter enzyme activities, the monolayers are enriched with AP, GT, and ACE, enzymes considered to be markers for brain endothelium. Results also indicate that the activities of MAO‐A and PST in the monolayers approach those found in the gray matter. Conversely, cytosolic COMT and MAO‐B activities in the monolayers are negligible and much lower, respectively, compared to activities in gray matter. Additional studies with both tissues suggest that the PST of both tissues is the thermostable form of the enzyme.
Evidence that High‐ and Low‐Affinity DL‐α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid (AMPA) Binding Sites Reflect Membrane‐Dependent States of a Single Receptor
Authors: Hall Randy A. , Kessler Markus , Lynch Gary
Published: July 2010

Abstract:Binding of DL‐α‐[3H]amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid ([3H]AMPA) to lysed rat brain membranes in the presence of potassium thiocyanate resulted in curvilinear Scatchard plots that could be resolved by regression analysis into a large low‐affinity component and a small high‐affinity component. Solubilization with Triton X‐100 resulted in solubilized and nonsolubilized fractions that were considerably enriched in the high‐affinity component and correspondingly reduced in the low‐affinity component. It thus appears that solubilization converts low‐affinity AMPA receptors into high‐affinity receptors. Also, synaptic plasma membranes were found to be greatly enriched in the low‐affinity form and deficient in the high‐affinity form of the AMPA receptor. These experiments provide evidence for the hypothesis that the high‐ and low‐affinity components of AMPA binding are inter‐convertible states of the same receptor rather than separate binding sites and that the conversion of these receptors from their native high‐affinity state to the low‐affinity state occurs on insertion of the receptors into synapses.
Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+
Authors: Mayer Bernd , Klatt Peter , Böhme Eycke , Schmidt Kurt
Published: July 2010

Abstract:Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+in a concentration‐dependent manner with half‐maximal effects observed at 0.3 μMand 0.2 μMwhen its activity was assayed as formation of NO and L‐citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+concentrations required for the activation of NO synthase, indicating a down‐regulation of the signal in NO‐producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3‐isobutyl‐1‐methylxanthine did not block this reaction effectively. The effects of Ca2+on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin‐dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO‐producing neurons.
Inhibition of Phosphorylation of Synapsin I and Other Synaptosomal Proteins by β‐Bungarotoxin, a Phospholipase A2Neurotoxin
Authors: Ueno Eiko , Rosenberg Philip
Published: July 2010

Abstract:Some snake venom neurotoxins, such as β‐bungarotoxin (β‐BuTX), which possess relatively low phospholipase A2(PLA2) activity, act presynaptically to alter acetylcholine (ACh) release both in the periphery and in the CNS. In investigating the mechanism of this action, we found that β‐BuTX (5 and 15 nM) inhibited phosphorylation, in both resting and depolarized synaptosomes, of a wide range of proteins, including synapsin I.Naja naja atraPLA2, which has higher PLA2activity, also inhibited phosphorylation but was less potent than β‐BuTX. At 1 nM, β‐BuTX andN. n. atraPLA2inhibited phosphorylation of synapsin I only in depolarized synaptosomes. Synaptosomal ATP levels were not affected by 5 or 15 nMβ‐BuTX or by 5 nM N. n. atraPLA2. Limited proteolysis, usingStaphylococcus aureusV‐8 protease, indicated that β‐BuTX inhibited phosphorylation of synapsin I in both the head and the tail regions. The inhibition of phosphorylation was not antagonized by nordihydroguaiaretic acid or indomethacin, suggesting that arachidonic acid derivatives do not mediate this inhibition. Furthermore, inhibition of phosphorylation by β‐BuTX andN. n. atraPLA2was not altered in the presence of the phosphatase inhibitor okadaic acid, suggesting that stimulation of phosphatase activity is not responsible for this inhibition. Inhibition of protein phosphorylation by PLA2neurotoxins and enzymes may be associated with an inhibition of ACh release.
Regulation of Expression of Dopamine β‐Hydroxylase in PC12 Cells by Glucocorticoids and Cyclic AMP Analogues
Authors: McMahon Anne , Sabban Esther L.
Published: July 2010

Abstract:Regulation of catecholamine biosynthesis is crucial in the adaptation to various physiological conditions, such as stress, and in several disorders, including hypertension and depression. In this study we have found that in PC12 cells, the mRNA levels of dopamine β‐hydroxylase (DBH), the enzyme that catalyzes the formation of norepinephrine from dopamine, can be regulated by glucocorticoids and cyclic AMP (cAMP) analogues. Treatment with dexamethasone increased DBH mRNA levels by 6 h, with maximal elevation (four‐ to fivefold) obtained after 1 day of exposure, and these levels were maintained for up to 4 days. DBH mRNA levels were also elevated on treatment of PC12 cells with 8‐bromo cAMP for 8 h to 1 day. The response to 8‐bromo cAMP, however, was bimodal, because DBH mRNA levels declined below control values on treatment for > 1 day. In combined treatments with 8‐bromo cAMP and dexamethasone, the cAMP effect was dominant. To begin to characterize the regulation of DBH mRNA, genomic clones for rat DBH were isolated, and 1 kb of the 5’flanking region was sequenced. Several putative regulatory elements, which may be involved in cAMP and glucocorticoid regulation, were identified, including two adjacent cAMP response elements, another element that can also bind members of the ATF/CREB family of transcription factors, a NF‐kB‐like sequence, several AP‐2 sites, and three core glucocorticoid receptor binding sequences.
Biphasic 24‐Hour Variations in Cyclic GMP Accumulation in the Rat Pineal Gland Are Due to Corresponding Changes in the Activity of Cytosolic and Particulate Guanylate Cyclase
Authors: Spessert Rainer , Heil Katja , Gupta Braj B. P. , Vollrath Lutz
Published: July 2010

Abstract:Various parameters of the rat pineal gland display a 24‐h rhythm. However, nothing is known about possible 24‐h variations in cyclic GMP (cGMP) metabolism. In the present study, 24‐h variations in pineal gland cGMP accumulation were investigated by determining the increase in cGMP level with and without inhibitors of phosphodiesterase at different time points over a light/dark cycle (12/12 h). Furthermore, the activity of guanylate cyclase (GC) was determined under substrate‐saturated conditions regarding the cytosolic and particulate forms of the enzyme. It has been found that cGMP accumulation and GC activity display biphasic 24‐h variations with two peaks—one ∼7 h after lights “on” and the other ∼7 h after lights “off.” The activity of cytosolic GC remains unchanged in the presence of the nitric oxide (NO) synthesis inhibitorN‐monomethyl‐L‐arginine, indicating that 24‐h variations in the activity do not reflect changes in the synthesis of the GC stimulator NO.
Recovery of Cholinergic Phenotype in the Injured Rat Neostriatum: Roles for Endogenous and Exogenous Nerve Growth Factor
Authors: Altar C. Anthony , Armanini Mark , Dugich‐Djordjevic Millicent , Bennett Gregory L. , Williams Regina , Feinglass Shamiram , Anicetti Vince , Sinicropi Dominick , Bakhit Charles
Published: July 2010

Abstract: Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high‐affinity 125I‐rhNGF binding but not its receptor, and cross‐reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain‐derived neurotrophic factor, neurotrophin‐3, ciliary neurotrophic factor, insulin‐like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF‐positive neurons in the rat neostriatum. The NGF‐positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF‐ and glial fibrillary acidic protein‐positive astrocytes. Enzyme‐linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high‐affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 μg of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23–29% above intact neostriatal levels and by 53–65% relative to HACU at both survival times. Thus, lesion‐induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.
Ethanol and Excitotoxicity in Cultured Cortical Neurons: Differential Sensitivity ofN‐Methyl‐D‐Aspartate and Sodium Nitroprusside Toxicity
Authors: Lustig Heather S. , von Brauchitsch Kristine L. , Chan Jane , Greenberg David A.
Published: July 2010

Abstract:Neural injury due to ischemia and related insults is thought to involve the action of excitatory amino acids atN‐methyl‐D‐aspartate receptors, which results in the influx of extracellular Ca2+and the generation of nitric oxide. Because ethanol inhibits physiologic responses to excitatory amino acids, we examined its effect on toxicity induced byN‐methyl‐D‐aspartate and by the nitric oxide donor sodium nitroprusside in neuron‐enriched cultures prepared from rat cerebral cortex. BothN‐methyl‐D‐aspartate and sodium nitroprusside were cytotoxic, as measured by the release of lactate dehydrogenase and by microfluorescent determination of cell viability. Ethanol (3–1,000 mM) protected cultures fromN‐methyl‐D‐aspartate but not sodium nitroprusside toxicity, and the ability of a series ofn‐alkanols to reproduce the effect of ethanol was related to carbon‐chain length. Neuroprotection by ethanol was accompanied by a decrease in theN‐methyl‐D‐aspartate‐evoked elevation of free intracellular Ca2+and did not appear to involve γ‐aminobutyric acid‐ or cyclic GMP‐mediated mechanisms. These findings suggest that ethanol inhibits excitotoxicity at an early step in theN‐methyl‐D‐aspartate signaling pathway, probably by reducing Ca2+influx, and not by interfering with the action of nitric oxide.