bluuebunny/crossref_metadata_2025 huggingface dataset:
Axonal Transport of the Molecular Forms of Acetylcholinesterase in Chick Sciatic Nerve
Authors: Couraud J. Y. , Giamberardino L. Di
Published: October 2006

Acetylcholinesterase (AChE) polymorphism was studied in the sciatic nerve of 4‐week‐old Leghorn chicks, by sucrose gradient sedimentation analysis. Four main AChE molecular forms were found with sedimentation coefficients of 5S, 7.5S, 11.5S and 20S respectively. Axonal transport of each of these forms was investigated on the basis of the enzyme accumulation kinetics measured on both sides of nerve transections and of the enzyme redistribution kinetics in nerve segments isolated in vivo. After nerve transection, 11.5S and 20S forms accumulated faster in the anterograde than in the retrograde direction and also much faster than 5S and 7.5S forms in the anterograde direction. Retrograde accumulations of 5S and 7.5S were faint or negligible. In addition, 1 h after nerve cutting, the accumulation rates for 11.5S and 20S forms (but not for 5S and 7.5S) fell, in both directions, to about one‐third of their initial values, probably owing to reversal of axonal transport at the axotomy site. Local protein synthesis inhibition by cycloheximide did not affect the accumulation of 11.5S and 20S in front of a transection, at least during the first hours, but reduced that of 5S and 7.5S by about 40%. In isolated nerve segments in vivo, the rapidly mobile fraction of AChE was estimated to constitute 23% of the total enzyme activity present in the nerve, 14% of it moving in an anterograde and 9% in a retrograde direction. A small amount of 11.5S molecules (approx. 20%) was in rapid transit (two‐thirds in the anterograde and one‐third in the retrograde direction), whereas almost all the 20S—about 90%—migrated rapidly (two‐thirds forwards and one‐third backwards). Anterograde velocities of 408 ± 94 and 411 ± 161 mm/day respectively were estimated for the 11.5S and 20S forms. Their respective retrograde velocities were 175 ± 85 and 145 ± 107 mm/day. Assuming that the totality of 5S and 7.5S molecules are moving in the anterograde direction, their accumulation rates were consistent with the average anterograde velocities of 2.9 ± 1.3 and 5.1 ± 1.4 mm/day, respectively. Couraud J. Y. and Di Giamberardino L. Axonal transport of the molecular forms of acetylcholinesterase in chick sciatic nerve. J. Neurochem. 35, 1053–1066 (1980).
Glucocorticoid Stimulation of Glutamine Synthetase Production in Cultured Rat Glioma Cells
Authors: Pishak Mark R. , Phillips Allen T.
Published: October 2006

Abstract: A sensitive radioisotopic assay has been used to examine the kinetic properties and regulation of biosynthesis of glutamine synthetase in C‐6 glioma cultures. The Km values for glutamate, MgATP, and ammonium ion were 5mM, 14 mM, and 0.042 mM, respectively, when measured at the pH optimum of 7.2. There was an absolute requirement for a divalent metal ion, with 15 mM‐ Mg2+ being the preferred ion at pH 7.2. Activity was completely inhibited after 30 min with 8 mM‐L‐methionhe sulfoximine. The addition of 1 μM‐cortisol to C‐6 cultures caused a two to threefold increase in glutamine synthetase specific activity over a 96‐h period, while dexamethasone at the same concentration elevated the level some 7‐10‐fold. This was specific for glucocorticoids, as other steroid hormones or catecholamines did not significantly affect glutamine synthetase specific activity. Cycloheximide (30 μM) or actinomycin D (0.01 μg/ml) blocked the hormone response. The continued presence of hormone was required in order to maintain an elevated enzyme level. The results suggest that glucocorticoids act to induce glutamine synthetase by stimulating new enzyme synthesis.
Histamine H1‐Receptor Binding Sites in Guinea Pig Brain Membranes: Regulation of Agonist Interactions by Guanine Nucleotides and Cations
Authors: Chang Raymond S. L. , Snyder Solomon H.
Published: October 2006

Abstract: Agonist, but not antagonist, interactions with histamine H2‐receptors labeled by [3H]mepyramine are regulated selectively by sodium, divalent cations, and guanine nucleotides. Sodium decreases the affinity of histamine and the agonist 2‐amino‐ethylpyridine for [3H]mepyramine sites in guinea pig brain membranes up to 10‐fold. The effect of sodium is exerted to a lesser extent by lithium, while potassium and rubidium are much weaker. Guanine nucleotides also decrease the affinity of histamine for H1 binding sites about twofold. GTP and its nonmetabolized analogue GMP‐PNP as well as GDP exert similar effects, while GMP, ATP, ADP, and AMP are inactive. The effects of GTP and sodium on histamine interactions with H1‐receptors are additive. By contrast, certain divalent cations enhance the potency of histamine at H1‐receptors. Manganese is most potent, while magnesium is almost as active as manganese and calcium is essentially inactive. Sodium, divalent cations, and guanine nucleotides have negligible effects on the interactions of antihistamines with H1‐receptors.
High‐Affinity Uptake of (RS)‐Nipecotic Acid in Astrocytes Cultured from Mouse Brain. Comparison with GABA Transport
Authors: Larsson O. M. , Krogsgaard‐Larsen P. , Schousboe A.
Published: October 2006

Abstract: (RS)‐Nipecotic acid is taken up into cultured astrocytes by a saturable high‐affinity transport system with a Km, of 28.8 ± 2.8 μM and a Vmax of 0.294 ± 0.022 nmol × min−1× [mg cell protein]−1. The uptake which represents a net inward transport was sodium‐dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)‐nipecotic acid, (3RS,4SR)‐4‐hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC50) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)‐2,4‐Diaminobutyric acid was a more efficient inhibitor than β‐alanine of glial uptake of (RS)‐nipecotic acid. It is concluded that astroglial uptake of (RS)‐nipecotic acid and GABA is mediated by the same transport system.
Transported Enzymes in Sciatic Nerve and Sensory Ganglia of Rats Exposed to Maternal Antibodies Against Nerve Growth Factor
Authors: McDougal D. B. , Yu M. J. C. , Gorin P. D. , Johnson E. M.
Published: October 2006

Abstract: The accumulations by axoplasmic transport of selected enzyme activities proximal and distal to a ligature placed on the sciatic nerve were monitored in rats exposed in utero to maternal antibodies to nerve growth factor (NGF) and in control rats. Littermates of the animals exposed to anti‐NGF were shown elsewhere to have had a 70% reduction in the number of sensory neurons in dorsal root ganglia and a 90% reduction in number of neurons in superior cervical (sympathetic) ganglion. The accumulation of F‐‐sensitive acid phosphatase activity was depressed 75% both proximal and distal to the tie. Accumulation of F‐‐resistant acid phosphatase activity was depressed nearly 50% proximal to the tie. Distal accumulation of this activity did not occur in either group of rats. Accumulation of acetylcholinesterase activity was not affected. Proximal accumulation of glutamic dehydrogenase activity was depressed 30%. Distal accumulation of the activities of β‐glucuronidase and hexokinase was depressed 50%. In the lumbar dorsal root ganglia, dry weight was reduced 40%, and the activities of peroxide‐sensitive, F‐‐resistant acid phosphatase and of the mitochondrial enzymes hexokinase, glutamic dehydrogenase, glutamic‐oxalacetic transaminase, and NAD‐dependent isocitric dehydrogenase were all reduced a little more, 45–50% per ganglion. However, the activities of the lysosomal enzymes, F‐‐sensitive acid phosphatase and β‐glucuronidase, of the peroxide‐resistant, F‐‐resistant acid phosphatase, and of the mitochondrial enzyme glutaminase were all reduced about 60% per ganglion. The results of these measurements were interpreted to suggest that much, and perhaps all, of the F‐‐sensitive acid phosphatase activity in motion in peripheral nerve in rat is confined to sensory axons.
Distribution of Specific High‐Affinity Binding Sites for [3H]Imipramine in Human Brain
Authors: Langer S. Z. , Javoy‐Agid F. , Raisman R. , Briley M. , Agid Y.
Published: October 2006

Abstract: [3H]Imipramine binds with high affinity to membranes from different regions of the human brain. The highest density of binding sites was observed in the hypothalamus and substantia nigra and the lowest density in the white matter and cerebellum. As found in rat brain, tricyclic antidepressant drugs are potent inhibitors of [3H]imipramine binding. Atypical antidepressants are, however, much weaker at inhibiting the specific binding. The [3H]imipramine binding site in human cortex is apparently identical to the site already described in the rat brain and in human platelets.
Activation of (Na+, K+)‐ATPase by Nanomolar Concentrations of GM1 Ganglioside
Authors: Leon Alberta , Facci Laura , Toffano Gino , Sonnino Sandro , Tettamanti Guido
Published: October 2006

Abstract: GM1 ganglioside binding to the crude mitochondrial fraction of rat brain and its effect on (Na+, K+)‐ATPase were studied, the following results being obtained: (a) the binding process followed a biphasic kinetics with a break at 50 nM‐GM1; GM1 at concentrations below the break was stably associated, while over the break it was loosely associated; (b) stably bound GM1 activated (Na+, K+)‐ATPase up to a maximum of 43%; (c) the activation was dependent upon the amount of bound GM1 and was highest at the critical concentration of 20 pmol bound GM1× mg protein‐1; (d) loosely bound GM1 suppressed the activating effect on (Na+, K+)‐ATPase elicited by firmly bound GM1; (e) GM1‐activated (Na+, K+)‐ATPase had the same pH optimum and apparent Km (for ATP) as normal (Na+, K+)‐ATPase but a greater apparent Vmax; (f) under identical binding conditions (2 h, 37°C, with 40 nM substance) all tested gangliosides (GM1, GD1a, GD1b, GT1b) activated (Na+, K+)‐ATPase (from 26–43%); NeuNAc, sodium dodecylsulphate, sulphatide and cerebroside had only a very slight effect. It is suggested that the ganglioside activation of (Na+ ‐K+)‐ATPase is a specific phenomenon not related to the amphiphilic and ionic properties of gangliosides, but due to modifications of the membrane lipid environment surrounding the enzyme.
Sequestration of 125I‐Labeled p Nerve Growth Factor by Embryonic Sensory Neurons
Authors: Olender Edward J. , Wagner Barbara J. , Stach Robert W.
Published: October 2006

Abstract: Nerve growth factor (NGF) binds to two specific receptors on sensory nerve cells. These two receptors are characterized by different equilibrium dissociation constants. The higher affinity (type I) receptors have an equilibrium dissociation constant of 3.3 × 10‐11 M. The lower affinity (type II) receptors have an equilibrium dissociation constant of 1.7 × 10‐9M. These two receptors are not a result of negative cooperatively, but apparently are different receptors. At 22°C the rate of association is 1 × 107 M‐1 S‐1 and the rates of dissociation are 6.5 × 10‐4 s‐1 (type I) and 3.2 × 10‐2 s‐1’(type II). After binding, a time‐dependent process occurs that makes the NGF inaccessible to the external milieu (sequestered). The sequestration process is energy‐dependent, but apparently temperature‐independent. The data suggest that only the type I receptors are involved in the sequestration process. This process is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.
Characterization of Receptors for Cholecystokinin and Related Peptides in Mouse Cerebral Cortex
Authors: Saito Atsushi , Goldfine Ira D. , Williams John A.
Published: October 2006

Abstract: The characteristics of cholecystokinin (CCK) binding to its receptors in a particulate membrane fraction of mouse cerebral cortex were studied by employing biologically active radioiodinated CCK prepared by conjugation with 125I‐Bolton‐Hunter (125I‐BH) reagent. At 24°C binding was rapid, reversible, and linearly related to protein content. Binding was maximal at acidic pH (6.5) and reduced by the presence of monovalent cations. Under physiological conditions (pH 7.4, 118 mM‐NaC1, 4.7 mM‐KCl) Scatchard plots of CCK binding were linear with a KD value of 1.27 nM and binding capacity of 115 fmol/mg protein. Optimal binding required the presence of both Mg2+ and EGTA, and was inhibited by the addition of micromolar concentrations of Cu2+ (ID50= 30 μM). The cortical receptor recognized all major forms of CCK, with an order of potency of cholecystokinin octapeptide (CCK8) > CCK > cholecystokinin tetrapeptide (CCK4). Desulfated cholecystokinin octapeptide (dCCK8) had a 10‐fold lower affhity than CCK8. Dibutyryl cyclic GMP, a potent competitive inhibitor of CCK binding to receptors in pancreas, was not a specific inhibitor of CCK binding to brain receptors. These present results support the concept that CCK may function as a regulatory peptide in brain, and that the cortical CCK receptor is different from the receptors mediating the peripheral action of CCK.
High‐ and Low‐Affinity Transport of D‐Glucose from Blood to Brain
Authors: Gjedde Albert
Published: October 2006

Abstract: Measurements of the unidirectional blood‐brain glucose flux in rat were incompatible with a single set of kinetic constants for transendothelial transport. At least two transfer mechanisms were present: a high‐affinity, low‐capacity system, and a low‐affinity, high‐capacity system. The low‐affinity system did not represent passive diffusion because it distinguished between D‐and L‐glucose. The Tmax and Km, for the high‐affinity system were 0.16 mmol 100 g−1 min−1 and 1 mM; for the low‐affinity system, ∼ 5 mmol 100 g−1 min−1 and ∼ 1 M. With these values, physiological glucose concentrations were not sufficient to saturate the low‐affinity system. In normoglycemia, therefore, three independent pathways of glucose transport from blood to brain appear to exist: a high‐affinity facilitated diffusion pathway of apparent permeability 235·10−7 cm s−1, a specific but nonsaturable diffusion pathway of permeability 85·10−7 cm s−l, and a nonspecifc passive diffusion pathway of permeability 2·10−7 cm s−1.
Correlations Between Na+‐K+ ATPase Activity and Acetylcholine Release in Rat Cortical Synaptosomes
Authors: Meyer E. M. , Cooper J. R.
Published: October 2006

Abstract: [3H]Acetylcholine efflux and Na+‐K+ ATPase ion pump activity were measured concomitantly in rat cortical synaptosomes. Ouabain (500 μM), strophanthidin (500 μM). and parachloromercuribenzene sulfonate (500 μM) each inhibited ouabain‐sensitive 86Rb uptake and elevated [3H]acetylcholine release independently of the external calcium concentration. Veratridine (10 μM), electrical field stimulation (60 V, 60 Hz, 5‐ms pulse duration), or the calcium ionophore A23187 (10μg/ml) also inhibited ouabain‐sensitive 86Rb uptake and released [3H]acetylcholine, but via a calcium‐dependent process. Veratridine‐induced [3H]acetylcholine release and ion pump inhibition were correlated over a wide range of drug concentrations and both effects were blocked by pre‐treatment with tetrodotoxin (1 μM). The rate of [3H]acetyl‐choline efflux from superfused synaptosomes was increased within 15 s of exposure to ouabain, strophanthidin, veratridine, A23187, or field stimulation, while ouabain‐sensitive 86Rb uptake was significantly decreased within a similar interval. These results suggest that [3H]acetylcholine release is due at least in part to inhibition of Na+‐K+ ATPase.
Characterization and Purification from Human Brain of a Hyaluronic Acid‐Binding Glycoprotein, Hyaluronectin
Authors: Delpech Bertrand , Halavent Catherine
Published: October 2006

Abstract: Using affinity chromatography and enzyme‐labelled immunological assays combined with aftinity adsorption, we have obtained evidence for the binding of a brain glycoprotein to hyaluronic acid, and on this basis named it hyaluronectin. This binding was inhibited by hyaluronic acid and by the products of its hydrolysis by hyaluronidase from bovine testis, but was not inhibited by other glycosaminoglycans or by monosaccharides. Preparative affinity chromatography of brain acid‐soluble proteins produced hyaluronectin in a good degree of purity. Contamination by albumin was less than 1% and the yield was as high as 80%.
Production and Purification of Antibody to Bovine White Matter Proteolipid Apoprotein
Authors: Macklin Wendy B. , Schwarting Gerald A. , Lees Marjorie B. , Cohen Steven R.
Published: October 2006

Circulating antibody to the bovine white matter proteolipid apoprotein was detected in rabbits 1 month after a single injection of the water‐soluble form of the apoprotein. By double immunodiffusion, the antiserum reacted specifically with the delipidated proteolipid apoprotein and the crude proteolipid fraction containing complex lipids; after exposure of the proteolipid apoprotein to sodium dodecyl sulfate (SDS), no reactivity was observed. The antiserum did not react with other myelin components, i.e., basic protein, cerebroside or GM1 ganglioside, nor was there reactivity with non‐neural proteolipids. The anti‐apoprotein antibody was purified by affinity chromatography. The antibody‐antigen interaction is apparently very hydrophobic, since elution of the antibody from the affinity column requires buffer containing 0.5% Triton X‐100‐4 M‐urea.
Effect of Homo‐β‐Proline and Other Heterocyclic GAB A Analogues on GABA Uptake in Neurons and Astroglial Cells and on GABA Receptor Binding
Authors: Larsson O. M. , Thorbek P. , Krogsgaard‐Larsen P. , Schousboe A.
Published: October 2006

Abstract: Two groups of GABA (γ‐aminobutyric acid) analogues, one comprising derivatives of β‐proline and the other compounds structurally related to nipecotic acid, were investigated as potential inhibitors of high‐affinity GABA transport in neurons and glial cells, as well as displacers of GABA receptor binding. In addition to cis‐4‐hydroxynipecotic acid, which is known as a potent inhibitor of GABA uptake, homo‐β‐proline was the only compound which proved to be a potent inhibitor of glial as well as neuronal GABA uptake. IC50 values for GABA uptake into glial cells and brain cortex “prisms” were 20 and 75 μM, respectively, and the IC50 value obtained for GABA uptake into cultured neurons was 10 μM. A kinetic analysis of the action of homo‐β‐proline on GABA uptake into cultured astrocytes and neurons showed that this compound acts as a competitive inhibitor of GABA uptake in both cell types. From the apparent Km values, Ki values for homo‐β‐proline of 16 and 6 μM could be calculated for glial and neuronal uptake, respectively. This mechanism of action strongly suggests that homo‐β‐proline interacts with the GABA carriers. Furthermore, homo‐β‐proline also displaced GABA from its receptor with an IC50 value of 0.3 μM. The cis‐4‐hydroxynipecotic acid analogues, cis‐ and trans‐4‐mercaptonipecotic acid, had no inhibitory effect on glial or neuronal GABA uptake. Other SH reagents, PCMB, NEM and DTNB, were shown to be relatively weak inhibitors of GABA uptake into cultured astrocytes, suggesting that SH groups are not directly involved in the interaction between GABA and its transport carrier.
Sciatin Is a Transferrin‐Like Polypeptide
Authors: Markelonis George J. , Bradshaw Ralph A. , Oh Tae H. , Johnson Jeri L. , Bates Owen J.
Published: October 2006

Abstract: Sciatin, an acidic glycoprotein from chicken sciatic nerve, has myotrophic effects on avian skeletal muscle cells in culture. As sciatin was found to have certain structural similarities to transferrin, we further investigated the physicochemical characteristics of sciatin in order to determine the relationship between these two proteins. Sciatin was found to be strikingly similar to ovotransferrin in amino acid composition. In addition, amino acid sequence analysis revealed that sciatin and ovotransferrin had identical amino‐terminal sequences for at least the first 20 amino acid residues. Chicken ovotransferrin, but not human serum transferrin, cross‐reacted with rabbit antisciatin antibodies upon rocket immunoelectrophoresis and double immunodiffusion in agar. In addition, in the presence of bicarbonate, sciatin bound approximately 2 mol ferrous iron/mol protein. Using the purification procedure developed for sciatin, we purified a protein from chicken serum that cross‐reacted with antisciatin serum, migrated at a position identical to that of sciatin or ovotransferrin on two‐dimensional gel electrophoresis, had an amino composition very similar to ovotransferrin and sciatin, and had myotrophic effects on cultured muscle cells. From these data, we conclude that sciatin is a growth‐promoting polypeptide closely related in structure to transferrin.
Influence of Calcium on Dopamine Synthesis and Tyrosine Hydroxylase Activity in Rat Striatum
Authors: Kapatos Gregory , Zigmond Michael J.
Published: October 2006

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium‐induced activation of tyrosine hydroxylase. Calcium (12.5–200 μM) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 mM) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μM) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μM) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM) decreased cell‐free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.
Inhibitory Effects of Ascorbic Acid on the Binding of [3H]Dopamine Antagonists to Neostriatal Membrane Preparations : Relationship to Lipid Peroxidation
Authors: Heikkila Richard E. , Cabbat Felicitas S. , Manzino Lawrence
Published: October 2006

Abstract: Ascorbic acid, sodium ascorbate, and isoascorbic acid (the stereo‐isomer of ascorbic acid) inhibited the stereospecific binding of [3H]spiroperidol to neostriatal membrane preparations. Greater inhibitory effects were obtained at intermediate concentrations of the three ascorbic acid analogs (i.e., 0.06 and 0.6 mM) than at higher (6 mM) or lower (0.006 mM) concentrations. In parallel experiments, the three ascorbic acid analogs induced lipid peroxidation, which was also greater at the two intermediate than at higher or lower concentrations. Several known inhibitors of lipid peroxidation, including propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, α‐naphthol, and cobalt chloride, as well as the iron chelating agents EDTA and DETAPAC (diethylenetriaminepentaacetic acid) were able to counteract the effects of the ascorbic acid analogs on both lipid peroxidation and on [3H]spiroperidol binding. These data strongly suggest that an iron‐catalyzed lipid peroxidation is responsible for the observed inhibitory effects on binding. In other experiments, neostriatal membrane preparations that were preincubated with ascorbic acid (0.6 mM) and subsequently washed still had greatly diminished capacity to bind [3H]spiroperidol, indicating that ascorbic acid need not be physically present during the binding assay in order to affect binding. This experimental procedure also appears to be a way in which [3H]spiroperidol binding sites can be inactivated and washed free of the inactivating agent.
On the Significance of Endogenous 3‐Methoxytyramine for the Effects of Centrally Acting Drugs on Dopamine Release in the Rat Brain
Authors: Westerink B. H. C. , Spaan S. J.
Published: October 2006

Abstract: 3‐Methoxytyramine (3‐MT) was measured in the striata of rats killed by microwave radiation. Apomorphine, γ‐butyrolactone (GBL), and reserpine decreased the 3‐MT content. A slight but transient increase in 3‐MT was observed after haloperidol. The turnover rate of 3‐MT was unchanged 60 min after haloperidol treatment. (+)‐Amphetamine induced a pronounced rise in the 3‐MT content, which was potentiated after combined treatment with haloperidol. The increased 3‐MT turnover rate that was observed after amphetamine treatment suggests that monoamine oxidase (MAO) inhibition is no explanation for the mechanism of interaction of this drug with dopamine (DA) metabolism. The central stimulants amphonelic acid and nomifensine in‐creased 3‐MT levels; no substantial change was seen after benztropine, morphine, or oxotremorine. It is concluded that a decreased release of DA is closely and rapidly reflected by decreased formation of 3‐MT. 3‐MT seems to be a much better indicator for decreased DA release than 3,4‐dihydroxyphenylacetic acid or homovanillic acid.
Studies on the Presence of Angiotensin II in Rat Brain
Authors: Meyer Dieter K. , Phillips M. Ian , Eiden Lee
Published: October 2006

Abstract: Angiotensin II‐like immunoreactivity was extracted from brains of bilaterally nephrectomized rats with several different extraction procedures (90% methanol, distilled water, 6 M urea, 0.1 N HCI, and 2 M acetic acid). The activity was measured with radioimmunoassays using three different antisera, two of which had been used previously for immunocytochemical studies. With none of the extraction procedures or antisera employed was more than 80 pg/g wet weight of angiotensin II‐like immunoreactivity found. Analysis was undertaken with two different reverse‐phase high‐pressure liquid chromatography systems; in one of these the immunoreactivity did not coelute with angiotensin II or III. On the basis of its elution pattern from a molecular sieving column, the immunoreactivity seems to have a higher molecular weight than angiotensin II. It is concluded that neurons in the brain do not synthesize and store angiotensin II.
γ‐Aminobutyric Acid Can Both Inhibit and Facilitate Dopamine Release in the Caudate Nucleus of the Rabbit
Authors: Reimann W. , Zumstein A. , Starke K.
Published: October 2006

Abstract: Slices from rabbit caudate nucleus were preincubated with [3II]dopamine and then superfused and stimulated electrically. y‐Aminobutyric acid IW4 and moYL increased both the basal and the stimulation‐evoked overflow of tritium. The effects were not changed by picrotoxin and were only slightly reduced by bicuculline. In the presence of nipecotate mol/L, y‐aminobutyric acid decreased rather than enhanced the basal and the evoked overflow. The inhibition persisted in the presence of bicuculline. Muscimol did not affect, whereas baclofen decreased, the evoked overflow of tritium. Similar results were obtained with synaptosomes that were stimulated by 30 mmoVL K′. The results indicate that y‐aminobutyric acid can both facilitate and depress the release of dopamine. Facilitation occurs after entry of y‐aminobutyric acid into the dopaminergic terminal axons, whereas inhibition is probably mediated by a receptor site located in the membrane of these terminals.
The Rate of Utilization of Glucose Via Hexosemonophosphate Shunt in Brain
Authors: Gaitonde M. K. , Evison E. , Evans G. M.
Published: October 2006

Abstract: The concentration of 6‐phosphogluconate in the brain increased from 0–24 nmol/g in the controls to 1430 and 1506 nmol/g in rats treated with 50 mg of 6‐aminonicotinamide/kg of body weight. A dose‐dependent increase in the concentrations of glucose and glucose 6 phosphate as well as of 6‐phosphogluconate was found in the brains of 6‐aminonicotinamide‐treated rats. The biochemical changes and symptoms of neurological disorder in 6‐aminonicotinamide‐treated rats were not due to hypothermia. The rate of utilization of glucose via the hexosemonophosphate shunt was determined by isolation of gluconate from 6‐phosphogluconate and measurement of its [14C]content at short time intervals after injection of [U‐14C]glucose into 6‐aminonicotinamide‐treated rats; it was 16.5 nmol of glucose utilized/min per g of brain, and represented approximately 2.3% of the overall utilization of glucose in the brain. A highly significant correlation was observed between the concentration of 6‐phosphogluconate and the concentration of glucose 6‐phosphate and free glucose. The validity of this correlation was supported by the results of previous investigations involving several other treatments.
Phosphate‐Activated Glutaminase in Relation to Huntington's Disease and Agonal State
Authors: Butterworth J. , Yates C. M. , Simpson J.
Published: October 2006

Abstract: Involvement of phosphate‐activated glutaminase in Huntington's disease and agonal state was investigated in caudate nucleus and frontal cortex from postmortem brains. In Huntington's disease the activities of phosphate‐activated glutaminase, glutamic acid decarboxylase, succinic dehydrogenase, choline acetyl‐transferase, and acetylcholinesterase were significantly reduced in the caudate nucleus, but not in the frontal cortex. The activity of phosphate‐activated glutaminase, and to a lesser extent of glutamic acid decarboxylase, was reduced in cases of terminal illness, as compared with cases of sudden death. Succinic dehydrogenase and choline acetyltransferase were reduced only in the few cases of prolonged and severe terminal illness. Enzyme activities of the caudate nucleus were more affected by agonal state than were those of frontal cortex. Results indicate that phosphate‐activated glutaminase could be a useful marker of neuronal damage due to agonal state, and that phosphate‐activated glutaminase and succinic dehydrogenase are reduced in Huntington's disease.
Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP‐Dependent Ca2+ Transport in Synaptic Plasma Membranes
Authors: Coutinho O. P. , Carvalho A. P. , Carvalho C. A. M.
Published: October 2006

Abstract: Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in→out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i‐loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP‐dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP‐dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP‐dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP‐dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.
Desmethylimipramine Enhances the Release of Endogenous GABA and Other Neuro trans mitter Amino Acids from the Rat Thalamus
Authors: Korf Jakob , Venema Kor
Published: October 2006

The influence of desmethylimipramine (DMI) on the release of endogenous γ‐aminobutyric acid (GABA) and some other amino acids from the rat thalamus was studied with a push‐pull perfusion technique. Following HPLC the amino acids were fluorimetrically estimated. Added to the perfusion medium at a concentration of 10 μmol L‐1, DMI caused a 5‐ to 10‐fold increase in the release of GABA. Similar effects were found with imipramine, trimeprimine, haloperidol, and propranolol. The elevation of GABA release induced by DMI was Ca dependent. The release of aspartate and glutamate was also increased by DMI, but in contrast to K ions, DMI did not reduce the thalamic output of glutamine.
Asymmetric and Globular Forms of AChE in Slow and Fast Muscles of 129/ReJ Normal and Dystrophic Mice
Authors: Gisiger Victor , Stephens Heather R.
Published: October 2006

Abstract: Acetylcholinesterase activities and molecular forms were studied in normal and dystrophic 129/ReJ mice, focusing on four predominantly fast‐twitch muscles and the slow‐twitch soleus. The asymmetric and globular forms were analyzed separately so that the effect of dystrophy on each form could be determined. This comparative study showed the following. (1) In the normal condition, each muscle exhibited a distinct distribution of the molecular forms. (2) The diversity among the fast muscles resulted mainly from variations in the proportions of the three globular forms; in contrast, these muscles showed a constant and precise A12/A8/A4 ratio. (3) The slow‐twitch soleus clearly differed from the other muscles in its low acetylcholinesterase activity and distinct distribution of the molecular forms, characterized by a low level of G4 and a peculiar ratio among its asymmetric forms, resulting from a relative increase of the A8 and A4 forms. (4) In dystrophic mice, the diversity of the acetylcholin esterase distribution was lost; all the fast muscles displayed profiles exhibiting the characteristics typical of the soleus. The fast‐twitch extensor digitorum longus, sternomastoid, and plantaris converged towards an identical set of acetylcholinesterase molecules. (5) In contrast, the acetylcholinesterase activity and molecular forms of the soleus were only slightly affected by the disease. These results reveal that the dystrophy modifies both categories of molecular forms of acetylcholinesterase in a very precise manner. Such complex changes, which are highly reproducible in a variety of different muscles, are unlikely to result from nonspecific reactions secondary to the disease.
Evidence for the Localization of Hydrogen Peroxide‐Stimulated Cyclooxygenase Activity in Rat Brain Mitochondria: A Possible Coupling with Monoamine Oxidase
Authors: Seregi András , Serfózó Péter , Mergl Zsuzsanna
Published: October 2006

Abstract: The distribution of basal and of H2 O2‐ stimulated cyclooxygenase activity in the primary fractions of rat brain homogenates and in the subfractions of crude mitochondrial fraction was studied. For comparison, the localization of H2 O2‐ generating monoamine oxidase (MAO) as well as that of the mitochondrial marker succinate dehydrogenase (SDH) was also examined. H2 O2 was generated by MAO using 5 × 10−4M noradrenaline (NA) or 2 × 10−4M 2‐phenylethylamine (PEA) as substrates, or by 25 μg glucose oxidase (GOD) per ml in the presence of 1 mM glucose. For nonstimulated (basal) cyclooxygenase, the relative specific activity (RSA) was high in microsomes (1.79) and in the free mitochondria‐containing subfraction of the crude mitochondrial fraction (1.94). Parallel distribution of MAO and H2 O2‐ stimulated cyclooxygenase was observed in all fractions studied in the presence of NA. The highest RSA was found in the purified mitochondria for both enzymes (1.85 for MAO and 1.97 for H2 O2‐ stimulated cyclooxygenase). The enrichment of SDH (RSA = 2.21) indicated a high concentration of mitochondria in this fraction. The same distribution of H2 O2‐ stimulated cyclooxygenase was obtained when, instead of the MAO‐NA system, hydrogen peroxide was generated by GOD in the presence of glucose. H2 O2 generated by deamination of NA or PEA by MAO, or during the enzymatic oxydation of glucose by GOD, caused a threefold increase in mitochondrial endoperoxide formation. Indomethacin (2 × 104M), catalase (50 μg/ml) and pargyline (2 × 10−4M) eliminated the MAO‐dependent mitochondrial synthesis of PG endoperoxides. The GOD‐dependent cyclooxygenase activity of this fraction was abolished by in domethacin or catalase, but not by pargyline. The results show the existence of a mitochondrial cyclooxygenase in brain tissue. The enzyme is sensitive to H2 O2 and produces prostaglandin endoperoxides from an endogenous source of arachidonic acid. The identical localization of H2 O2‐ producing MAO and H2 O2‐ sensitive cyclooxygenase suggests a possible coupling between monoamine and arachidonic acid metabolism.
Identification of a Proteoglycan Antigen Characteristic of Cholinergic Synaptic Vesicles
Authors: Walker J. H. , Obrocki J. , Zimmermann C. W.
Published: October 2006

Abstract: An antiserum to cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata was purified by adsorption with fractions containing unwanted antigens. The adsorbed antiserum responds to the proteoglycan core material of the cholinergic synaptic vesicles. The major antigen migrates in an anomalous fashion on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), forming a broad band with an apparent molecular weight of approximately 120,000–300,000. The distribution of this antigen after sucrose density gradient centrifugation of synaptic vesicles is the same as that of vesicular ATP. The antigen comigrates with a substance that can be stained with Alcian‐Blue after SDS‐PAGE of highly purified synaptic vesicles. This substance is related to the low‐molecular‐weight, Alcian‐Blue‐positive glycosaminoglycan vesicu‐lin, which is formed from the high‐molecular‐weight proteoglycan by prolonged dialysis against water or by protease treatment. No antibodies were detected against vesiculin itself, indicating that the antigenic determinants are restricted to the proteoglycan.
Ammonia Inhibits Protein Synthesis in Slices from Young Rat Brain
Authors: Schott Klaus , Poetter Ulla , Neuhoff Volker
Published: October 2006

Abstract: Ammonium chloride, widely used as an inhibitor of lysosomal protein degradation, was shown to inhibit strongly protein synthesis in neocortical slices from 10‐day‐old rats at a concentration of 10 mmol/L. Its usefulness in experiments with brain tissue is doubted. The possible meaning of the finding with respect to ammonia toxicity is briefly discussed.
Passive Transfer of Lambert‐Eaton Myasthenic Syndrome in Mice: Decreased Rates of Resting and Evoked Release of Acetylcholine from Skeletal Muscle
Authors: Lang B. , Molenaar P. C. , Newsom‐Davis J. , Vincent A.
Published: October 2006

Abstract: Mice were injected for 1–2 months daily with 10 mg immunoglobulin G (IgG) from four patients with Lambert‐Eaton myasthenic syndrome (LEMS); control mice were injected with pooled human IgG from normal donors. Gastrocnemius muscles were homogenised for the assay of acetylcholine (ACh), choline acetyltrans‐ferase (ChAT), and cholinesterase (ChE). The ACh, ChAT, and ChE contents of gastrocnemius muscles from “LEMS mice” were about the same as the control values, which were 180 pmol, 40 nmol * h−1 (37°C), and 15 μmol * h−1 (37°C), respectively. Hemidiaphragms were treated with an irreversible ChE inhibitor (Soman) and incubated at 20°C for estimation of ACh release. Resting ACh release from experimental muscles was reduced by about 25% (P2 < 0.05) and the release evoked by 3 s−1 nervous stimulation by 50% (P2 < 0.05). On the other hand, 50 mM KCl‐induced transmitter release was not abnormal in LEMS mice. The findings indicate that IgG antibody from patients with LEMS may bind to nerve terminal determinants that are involved in quantal and nonquantal ACh release.
An Improved, Automated Adenylate Cyclase Assay Utilizing Preparative HPLC: Effects of Phosphodiesterase Inhibitors
Authors: Schulz David W. , Mailman Richard B.
Published: October 2006

Abstract: Analysis of adenylate cyclase (ACase) activity in broken cell preparations usually involves conversion of [α‐32P]ATP to [32P]cyclic AMP (cAMP) followed by purification of cAMP by liquid chromatographic methods. An automated, preparative reverse‐phase HPLC procedure was developed that purifies cAMP rapidly and decreases variability and background. It permits the separation procedure to be validated rapidly prior to use with actual samples, and is readily adaptable for assaying guanylate cyclase, phosphodiesterases (PDE), or a variety of other related nucleotide‐metabolizing enzymes. For ACase assays, 4.5% ZnSO4‐10% Ba(OH)2 is added to the incubation mixture, and following centrifugation, the supernatant is injected on an HPLC apparatus fitted with a Waters Z‐Module containing a 10‐μ C18 reverse‐phase cartridge. Using a mobile phase of 0.15 M sodium acetate‐20% methanol (pH 5.0) at a flow rate of 4 ml/min, cAMP is eluted at k′ > 1.25, whereas k′ < 0.5 for all other adenine nucleotides, permitting collection of the cAMP fraction after running the other nucleotides to waste. The method was validated by characterizing dopamine‐sensitive ACase in homogenates of striatum from Sprague‐Dawley rats. Basal activity (177 ± 16 pmol/mg protein/min), the stimulation by dopamine (186 ± 19 pmol/mg/min), the apparent Km for dopamine (5.0 ± 1.5 μM), and expected effects of varying magnesium, EGTA, and GTP were similar to available data. However, it was found that isobutylmethylxanthine (IBMX) or theophylline, usually included in the incubation mixture as PDE inhibitors, markedly inhibited the synthesis of cAMP in both the presence and absence of dopamine. A consequence of this inhibition was a marked change in the apparent Km of dopamine calculated from a Lineweaver‐Burk plot. The use of IBMX to inhibit PDEs was compared with an alternate strategy, the addition of excess exogenous cAMP. Simultaneous analysis of PDE and ACase activity was accomplished by including [3H]cAMP in the incubation and quantifying the amounts of [3H]cAMP hydrolyzed and [32P]cAMP synthesized. Without IBMX, a concentration of 1 mM exogenous cAMP was sufficient to prevent significant loss of [3H]cAMP. In the absence of exogenous cAMP, 0.5 mM IBMX did not completely prevent the breakdown of [3H]cAMP, whereas 2.5 mM IBMX did. Although there was 25% less [3H]cAMP recovered in the presence of 0.5 mM IBMX than with 2.5 mM IBMX, there was no difference in the amount of [32P]cAMP formed (either with or without dopamine). Moreover, in the presence of IBMX, there was a 20–30% lower synthesis of [32P]cAMP compared with incubations in which only 1 mM cAMP was used to prevent breakdown of [32P]cAMP. These data suggest that alkylxanthines, possibly through effects on adenosine receptors, may cause unexpected effects on estimations of dopamine‐stimulated ACase. The use of exogenous cAMP as an alternate substrate for PDEs may be one way to obviate these problems.
Differences and Similarities in Muscarinic Receptors of Rat Heart and Retina: Effects of Agonists, Guanine Nucleotides, and N‐Ethylmaleimide
Authors: Dunlap Janet , Brown Joan Heller
Published: October 2006

Abstract: Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N‐ethylmaleimide decreases agonist affinity for the high‐affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of highand low‐affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low‐affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high‐affinity binding sites in retina. Third, the “partial agonist” pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high‐ and low‐affinity sites and to effect a biological response.
Impulse Conduction Regulates Myelin Basic Protein Phosphorylation in Rat Optic Nerve
Authors: Murray Norman , Steck Andreas J.
Published: October 2006

Abstract: The influence of action potential conduction in myelinated axons on the state of phosphorylation of myelin basic protein was studied in rat optic nerve incubated in vitro. For this purpose we used a technique that permits continuous recording of the responses of nerves to electrical stimulation together with the “back‐phosphorylation” assay. Our results indicate that action potential conduction, but not electrical stimulation, increased the state of phosphorylation of myelin basic protein. The increment in basic protein phosphorylation was related to the number of impulses conducted, up to a maximal change which occurred after 12 × 103 impulses. Also, the effect of action potential conduction was reversible, since the state of myelin basic protein phosphorylation returned to control levels within 5 min of stopping stimulation. These findings raise the interesting possibility that myelin basic protein phosphorylation plays a role in some dynamic function of myelin, perhaps related to ion transport or to the process of recovery of ionic gradients.
Changes in the Amino Acid Content of Nerve Endings (Synaptosomes) Induced by Drugs that Alter the Metabolism of Glutamate and γ‐Aminobutyric Acid
Authors: Geddes J. W. , Wood J. D.
Published: October 2006

Abstract: The study was centered on the changes in the amino acid content of nerve endings (synaptosomes) induced by drugs that alter the metabolism of glutamate or γ‐aminobutyric acid (GABA), and that possess convulsant or anticonvulsant properties. The onset of seizures induced by various convulsant agents was associated with a decreased content of GABA and an increased content of glutamate in synaptosomes. The concurrent administration of pyridoxine prevented both the biochemical changes and the convulsions. The administration of gabaculine to mice resulted in large increases in the GABA content of synaptosomes that were counteracted by decreases in glutamate, glutamine, and aspartate levels such that the total content of the four amino acids remained unchanged. The administration of aminooxyacetic acid (0.91 mmol/kg) resulted initially in seizure activity, but subsequently in an anticonvulsant action. No simple relationship existed between the excitable state of the brain induced by aminooxyacetic acid and the changes in the synaptosomal levels of any of the amino acid transmitters. A hypothesis was, however, formulated that explained the convulsant‐cum‐anticonvulsant action of aminooxyacetic acid on the basis of compartmentation of GABA within the nerve endings.