bluuebunny/crossref_metadata_2025 huggingface dataset:
METABOLISM OF AMINO ACIDS AND AMMONIA IN RAT BRAIN CORTEX SLICES IN VITRO: A POSSIBLE ROLE OF AMMONIA IN BRAIN FUNCTION
Authors: Benjamin A. M. , Quastel J. H.
Published: October 2006

Abstract— (1) The sum of the values of total (tissue + medium) amino acid‐N of glutamate, glutamine, γ‐aminobutyrate, and aspartate (referred to as the glutamate system) and of ammonia‐N of incubated rat brain cortex slices is approximately constant under a variety of metabolic conditions (presence or absence of glucose or of oxygen or in the presence of metabolic inhibitors such as aminooxyacetate, malonate, methionine sulfoximine, fluoroacetate, ouabain, 2:4 dinitrophenol, or Amytal). Fluctuations in the value of one constituent are compensated by fluctuations in the values of other constituents. The same applies to infant rat brain cortex slices and to rat brain synaptosome preparations. It is suggested that the constancy of the glutamate‐ammonia system implies a coupling of neurons and glia in such a manner that glutamate released from the neurons during excitation is taken up by the glia and there converted to glutamine. The glutamine is returned to the neurons where it is hydrolysed to glutamate and ammonia. The glia, on this view, exercise an important buffering effect on the extracellular content of the excitatory amino acid, glutamate, and possibly on that of other functionally active amino acids emanating from the neurons. (2) The magnitude of the glutamate‐ammonia system in the infant rat brain cortex is about 43% of that in the adult. It is suggested that, with maturity, the development of the glutamate‐ammonia system is linked with the development of the citric acid cycle of operations. (3) The ammonia in the system is tightly linked to the activity of the ATP‐controlled glutamine synthetase. (4) Proteolytic ammonia and amino acids are formed, during the incubation, to values that seem to be independent of a wide variety of metabolic conditions. The total value is approximately 10 μmol/g in the first h of incubation. (5) As the ammonium ion is necessary for the return of glutamate to the neuron in the form of glutamine, it is inferred that the ion plays a functional role in the nervous system by helping to maintain the steady state of glutamate in the neuron.
THE INCORPORATION OF DOUBLE‐LABELLED ACETATE INTO GLUTAMATE AND RELATED AMINO ACIDS FROM ADULT MOUSE BRAIN: COMPARTMENTATION OF AMINO ACID METABOLISM IN BRAIN
Authors: Van den Berg C. J. , Ronda G.
Published: October 2006

Abstract– We have determined the incorporation of [3H]‐, [1‐14C]‐ and [2‐14C]acetate into glutamate, glutamine and aspartate of the adult mouse brain. All these three acetates were incorporated more extensively into glutamine than into glutamate. This has been reported by several authors for each of these labelled acetates in separate experiments. It was shown that [3H, 2‐14C]acetate can be used to obtain an acetate labelling ratio analogous to the previously used [2‐14C]acetate/[1‐14C]acetate labelling ratio. From these acetate labelling ratios of glutamine and glutamate conclusions can be deduced about the dynamic relationship of these amino acids with each other and with the tricarboxylic acid cycle.

A fairly large isotope effect between acetate and glutamate was observed. As this isotope effect is very likely caused by the citrate synthase reaction, it can be argued that citrate synthase involved in the conversion of labelled acetate into glutamate is far out of equilibrium in vivo. Comparing our data with literature data, the possibility can be suggested that citrate synthase in the acetate metabolizing compartment is in situ kinetically distinct from citrate synthase in other compartments of the brain.
ONTOGENESIS OF DOPAMINERGIC‐CHOLINERGIC INTERACTIONS IN THE RAT STRIATUM: A NEUROCHEMICAL STUDY
Authors: Coyle J. T. , Campochiaro P.
Published: October 2006

Abstract— In the striatum of the newborn rat, the activity of tyrosine hydroxylase, the concentration of dopamine and the activity of the synaptosomal high‐affinity uptake process for dopamine is 10% of that of the adult; there is a linear and closely associated increase in all three parameters during maturation, achieving 75% of adult levels by 4 weeks after birth. In contrast, the specific activity of choline acetyltransferase exhibits a more delayed developmental rise commencing 1 week after birth; the concentration of acetylcholine is disproportionately high in the neonatal striatum and precedes the developmental increase in the activity of choline acetyltransferase. At birth, the specific activity of dopamine‐sensitive adenylate cyclase is 20% of that of the adult striatum and achieves adult activity by 4 weeks after birth. Pretreatment with the neuroleptic, fluphenazinc. does not reduce the striatal content of acetylcholine until 8 days after birth. It is postulated that dopaminergic influences on cholinergic neuronal activity appear when the cholinergic neurons in the striatum cease dividing and start differentiating.
COMPARISON OF LEVELS OF ADENOSINE 3′,5′‐MONOPHOSPHATE IN HIGHLY PURIFIED AND MIXED PRIMARY CULTURES OF NEURONS AND NON‐NEURONAL CELLS FROM EMBRYONIC CHICK SYMPATHETIC GANGLIA
Authors: Wallace L. J. , Partlow L. M. , Ferrendelli J. A.
Published: October 2006

Primary cultures containing ≥99% neurons, ≥99% non‐neuronal cells (glia), or both cell types were prepared from the sympathetic ganglia of 12‐day chick embryos. Levels of cyclic AMP in the non‐neuronal cells (∼14 pmol/mg protein) were approximately 3‐fold higher than levels in the neurons (∼4 pmol/mg protein). Mixed cultures had concentrations of cyclic AMP which fell between the values measured for pure neuronal and pure non‐neuronal cultures. The measured cyclic AMP values of mixed cultures were indistinguishable from values predicted by summing the expected contributions of the neurons and non‐neuronal cells. Thus, contact between the neurons and non‐neuronal cells in these mixed cultures did not appear to alter the level of cyclic AMP in either cell type. Neuronal‐glial interactions, such as the specific neuronal stimulation of non‐neuronal cell proliferation, occurred independently of any changes in the level of cyclic AMP in the mixed cultures. Cell density was varied in both pure and mixed cultures, and both cyclic AMP concentrations and amounts of [3H]thymidine incorporation into DNA were measured. The cyclic AMP content of the non‐neuronal cells varied inversely with cell density. [3H]Thymidine incorporation was independent of cell density in both neuronal and non‐neuronal cultures. Parallel density‐dependent decreases in cyclic AMP concentration and [3H]thymidine incorporation were observed in mixed cultures as cell density was increased. The data suggest that there is no relationship between changes in rate of non‐neuronal cell proliferation and cyclic AMP levels in these cultures.
COMPARATIVE STUDIES ON GLUTAMYL‐tRNA SYNTHETASES ISOLATED FROM CYTOPLASM AND MITOCHONDRIA OF CALF BRAIN
Authors: Charelziński Marian , Borkowski T.
Published: October 2006

Preparations of glutamyl‐tRNA synthetase (EC 6.1.1.17) were isolated from cytoplasm and mitochondria of calf brain and purified by 14 and 9.5 times respectively. The effect of pH and that of the concentration of basic incubation mixture components on glutamyl‐tRNA formation were estimated. The two preparations showed differences in response to optimum ration of Mg2+/ATP which is 6 for cytoplasmic system and 1 for mitochondrial system in aminoacylation reaction and a considerable difference in sensibility to concentration change of monovalent cations. Dithiotreitol not only protected both enzymatic preparations against thermal inactivation but even increased their activity.
HISTAMINE‐SENSITIVE ADENYLATE CYCLASE IN HYPOTHALAMUS OF RAT BRAIN: H1 AND H2 RECEPTORS
Authors: Portaleone P. , Pagnini G. , Crispino A. , Genazzani E.
Published: October 2006

Abstract— In in vitro experiments on rat hypothalamic homogenates the effects of biogenic amines such as histamine (HA), noradrenaline (NA), dopamine (DA), serotonin (5‐HT) and drugs such as isoprenaline (ISP), 2‐(2‐pyridyl)ethylamine (H2 stimulant—Hls), 4‐methyl‐histamine (H2 stimulant H2s), mepyramine (H1 antagonistp Hla), cimetidine (H2 antagonist—H2a) were tested on adenylate cyclase activity. HA possessed a powerful stimulating effect on hypothalamic adenylate cyclase activity, higher than that shown by the other substances.

The stimulating effect of HA was greatest in hypothalamic tissue from male rats, while tissue from females showed only a modest stimulation. H2s, induced a greater stimulation of adenylate cyclase than Hls. On the other hand, the H2a inhibited HA stimulation to a greater extent than the Hla, Hla and H2a, when used together, completely inhibited the HA stimulation. HA may have a neurotrans‐mitter role in the hypothalamus, and in this area there appears to be a mixed population of H1 and H2 receptors, with a majority of H2 receptors.
SUBCELLULAR DISTRIBUTION OF MARKER ENZYMES AND OF NEUROTOXIC ESTERASE IN ADULT HEN BRAIN1
Authors: Richardson R. J. , Davis C. S. , Johnson M. K.
Published: October 2006

Abstract—

Neurotoxic esterase (NTE) is now regarded as the site of the primary biochemical lesion in the delayed neuronal degeneration produced by certain organophosphorus esters. Since hens are the species of choice in studies of this neuropathy the subcellular distribution of NTE and marker enzymes in adult hen brain was carried out. Up to 70%, of NTE was recovered in a microsomal fraction (P3) which was also enriched in 5′‐nucleotidase (5′‐ribonucleotide phosphohydrolase EC 3.1.3.5), a plasma membrane marker. The protein content of this fraction (31% of the parent homogenate) is double that of equivalent mammalian brain fractions. The LDH distribution suggests that the P3 fraction contained many small synaptosomes. Subfractionation of microsomes by rate and equilibrium centrifugation on sucrose density gradients segregated the RNA but failed to separate the NTE. 5′‐nucleotidase and glucose‐6‐phosphatase (D‐glucose‐6‐phosphate phosphohydrolase EC 3.1.3.9) from each other. NTE was considerably concentrated (2–5 times) in subfractions of the P2 fraction, which are believed to be enriched in synaptosomal membranes. A similar localization of NTE and AChE was found in subfractions of P2 from neonatal chick brain. Axon fragments contained a significant amount of NTE which was not associated with the myelin. Nuclear and mitochondrial fractions were low in NTE. Microsomes could be partitioned in biphasic aqueous polymer systems, but with little enrichment of NTE. The possible association of NTE with synaptosomal membranes suggests that early events in organophosphorus neuropathy may occur at the axonal (? synaptic) surface.
Inhibition of Histamine Synthesis in Brain by α‐Fluoromethylhistidine, a New Irreversible Inhibitor: In Vitro and In Vivo Studies
Authors: Garbarg M. , Barbin G. , Rodergas E. , Schwartz J. C.
Published: October 2006

a‐Fluoromethylhistidine (α‐FMH), a new potent inhibitor of histidine decarboxylase (HD), has been used for in vitro and in vivo studies of brain HD. Following a preincubation with (+)‐α‐FMH, brain HD activity was inhibited in a time‐dependent and concentration‐dependent manner. The enzyme activity was not restored by overnight dialysis against standard buffer. The (–) antimer of α‐FMH was ineffective. When injected intraperitoneally in a single dose of 20 mg/kg, (±)‐α‐FMH induced a complete loss in HD activity in cerebral cortex and hypothalamus as well as in peripheral tissues, such as stomach. At a dosage of 100 mg/kg (±)‐α‐FMH did not alter histamine‐N‐methyltransferase, DOPA decarboxylase, and glutamate decarboxylase activities. The maximal decrease of HD activity occurred after 2 h in both cerebral cortex and hypothalamus, but the time course of the recovery of enzyme activity was slower in the cerebral cortex. The enzyme activity reached control value within 3 days in hypothalamus and was not fully restored after 4 days in cerebral cortex. Contrasting with the diminished HD activity, a substantial concentration of histamine remained present in five regions of mouse brain. Thus, α‐FMH is a highly specific irreversible inhibitor of brain HD activity and its efficacy makes it useful to study the physiological role of brain histamine.
Synaptic Proteins After Electroconvulsive Seizures in Immature Rats
Authors: Jørgensen Ole Steen , Dwyer Barney , Wasterlain Claude G.
Published: October 2006

The forebrain content of several rat brain synaptic proteins (synaptin, D1, D2, and D3) was reduced in rats receiving electroconvulsive seizures on days 2–11, 9–18, or 19–28 and sacrificed at the age of 30 days. Forebrain weight, total protein, and the glial enzyme glutamine synthetase were also decreased, whereas the neuronal enolase 14–3–2 was unchanged. The findings suggest that seizures in the immature rat brain resulted in a parallel reduction of synaptic material and of the amount of glial cells. The increased concentration of the enolase 14–3–2 found in rats seizured on days 19–28 may reflect the high demands on the glycolytic system during the seizures.
D‐Glucose Transport in Cultured Cells of Neural Origin: The Membrane as Possible Control Point of Glucose Utilization
Authors: Keller Konrad , Lange Klaus , Noske Walter
Published: October 2006

Abstract: The function of plasma membrane as control point of glucose metabolism has been studied in confluent monolayer of C1300 neuroblastoma (N2A) and glioma (C6) cells. In neuroblastoma, steady state intracellular glucose concentration reached the extracellular levels, while intracellular contents in C6 glioma cells remained very low. In C6 glial cells the amount of glycogen as source of energy was much higher than that found in C1300 neuroblastoma cells. Influx rates of D‐glucose in C6 glioma cells were only half those found in neuroblastoma cells. During the influx period (0‐40 s) the transport of glucose in these cells did not exceed the phosphorylation rate, whereas a steady, time‐dependent increase in glucose content was observed in neuroblastoma cells. While glucose uptake in neuroblastoma cells seems to be regulated at the level of phosphorylating enzymes, the control point in C6 glioma is believed to be membrane transport.
Discrimination of Multiple [3H]5‐Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain
Authors: Pedigo N. W. , Yamamura H. I. , Nelson D. L.
Published: October 2006

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [3H]5‐hydroxytryptamine ([3H]5‐HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000‐fold difference between its dissociation constants (2‐12 nM versus 35,000 nM) for the high‐ and low‐affinity sites, respectively. Inhibition of [3H]5‐HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two‐site model in each brain region. In the frontal cortex the high‐affinity neuroleptic sites comprised about 60% of the total [3/H]5‐HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [3H]5‐HT binding assayed in the absence or presence of 1 μM‐spiperone (a concentration that completely blocks the high‐affinity site while having minimal activity at the low‐affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [3H]5‐HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [3H]5‐HT binding sites having a differential regional distribution in rat brain.
Partial Inactivation of Na,K‐ATPase in Cortical Brain Slices Incubated in Normal Krebs‐Ringer Phosphate Medium at 1 and at 10 Atm Oxygen Pressures
Authors: Kovachich G. B. , Mishra O. P.
Published: October 2006

Na, K‐ATPase (ATP phosphohydrolase EC 3.6.1.3) activity was determined in homogenates of cortical brain slices after incubation in normal Krebs‐Ringer phosphate medium at 1 atm oxygen pressure. After 10 min of incubation Na,K‐ATPase activity was reduced by approximately 50%. Longer incubation did not cause further change in activity. The presence of 0.1 mM‐MnCl2 in the medium offered significant protection. while an excursion to 10 atm oxygen pressure caused further inactivation. Measurements of malonaldehyde levels suggest that the inhibition of Na,K‐ATPase is a result of lipid peroxidation. The evidence indicates that brain slices incubated under standard conditions suffer considerable oxidative damage.
β‐Hydroxybutyrate as a Precursor to the Acetyl Moiety of Acetylcholine
Authors: Sterling Gerald H. , McCafferty Mary R. , O'Neill John J.
Published: October 2006

Abstract— Rat brain cortex slices were incubated with 10 mm‐glucose and trace amounts of [6‐3H]glucose and [3‐14C]β‐hydroxybutyrate. The effects of (‐)‐hydroxycitrate, an inhibitor of ATP‐citrate lyase; methylmalonate, an inhibitor of β‐hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and 3H:14C ratios calculated. Incorporation of [14C]β‐hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of 14C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [3H‐,14C]‐glucose. (–)‐Hydroxycitrate (5.0 mM) reduced incorporation of [3H]glucose and [14C]β‐hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm) showed a decrease in 14C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm) also produced a decreased 14C incorporation into ACh and its precursors. At 10 mm, acetoacetate reduced 3H and 14C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β‐hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.
Localisation of Tricyclic Antidepressant Binding Sites on Serotonin Nerve Terminals
Authors: Sette M. , Raisman R. , Briley M. , Langer S. Z.
Published: October 2006

Abstract: High‐affinity specific [3H]imipramine binding has been demonstrated in the brain and platelets of various species including man. Electrolytic lesion of the rat dorsal raphe, which resulted in a significant decrease in the endogenous levels of serotonin produced a reduction in the density of [3H]imipramine binding sites in the hypothalamus and cortex. The affinity constants were unchanged. These results suggest that [3H]imipramine binding sites are located on serotonin nerve terminals.
Phosphoryl Group Transfer by a Fraction of the Soluble Proteins of Catecholamine Storage Vesicles
Authors: Taugner G. , Wunderlich I.
Published: October 2006

Abstract: The terminal phosphate group of ATP was transferred to ADP by an enzyme present in the soluble core proteins of adrenal medulla catecholamine storage vesicles. It was purified 10–30‐fold by DEAE Sephadex chromatography (Fraction I). The enzyme required divalent metal ions for activation; Mn2+ was almost as effective as Mg2+, but Ca2+ was only a weak activator. Activation by Mg2+ took place over a very narrow concentration range (0.5–3 mm). The specificity of the enzyme activity to nucleoside triphosphates was broad, to the nucleoside diphosphates narrow, favouring adenosine diphosphate. In dependence on the pH the activity increased from pH 4 to pH 7 and remained constantly high between pH 7 and 9. The Arrhenius plot was linear between 5 and 70°C, with an activation energy of 11.1 kcal/mol. The phosphoryl group transfer reaction depended on the function of thiol groups; p‐hydroxymercuribenzoate inhibited 50% of the enzyme activity; dithioerythritol reactivated it completely. Gel electrophoresis revealed that in Fraction I, a protein of molecular weight about 45,000, was enriched compared with the total soluble proteins. The enzyme‐enriched Fraction I differed significantly in its relative amino acid composition from that of the total soluble proteins; in general, the acidic amino acids were reduced and the more basic acids enhanced.
Susceptibility of the Myelin‐Associated Glycoprotein and Basic Protein to a Neutral Protease in Highly Purified Myelin from Human and Rat Brain
Authors: Sato Shuzo , Quarles Richard H. , Brady Roscoe O.
Published: October 2006

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin‐associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower‐molecular‐weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin‐related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.
Alanine Aminotransferase in Bovine Brain: Purification and Properties
Authors: Ruščák Michal , Orlický Jozef , Žúbor Vladimir , Hager Hermann
Published: October 2006

Abstract: Mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) were partially purified (140‐ and 180‐fold, respectively) from bovine brain cortex by means of (NH4)2SO4 precipitation, gel filtration on Sephadex G‐150, and ion‐exchange chromatography on DEAE A‐50 and characterized. The enzymes exhibited identical molecular weights (110,000 ± 10,000) and pH optima (7.8), but were eluted from CM Sephadex C‐50 at different ionic strengths. Isoelectric focusing of the enzymes indicated a pi value of 5.2 for the cytosolic enzyme and 7.2 for the mitochondrial enzyme. The Km values of the mitochondrial enzyme were 5.1 mM, 6.6 mM, 0.7 mM, and 0.4 mM and of the cytosolic isozyme were 30.3 mM, 4.3 mM, 0.7 mM, and 0.5 mM for alanine, glutamate, 2‐oxoglutarate, and pyruvate, respectively. The results indicated that two forms of alanine aminotransferase exist in nerve tissue, which suggests that they may play different roles in the cellular metabolism of nerve tissue.
Inhibition, by 2‐Oxo Acids That Accumulate in Maple‐Syrup‐Urine Disease, of Lactate, Pyruvate, and 3‐Hydroxybutyrate Transport Across the Blood‐Brain Barrier
Authors: Cremer Jill E. , Teal Hazel M. , Cunningham Vincent J.
Published: October 2006

Abstract: Data are presented in support of the transport of (‐)‐d‐3‐hydroxybutyrate across the blood‐brain barrier (BBB) being a carrier‐mediated process. The kinetic parameters in 21‐day‐old pentobarbital‐anaesthetized rats were Vmax 2.0 μmol.g−1.min−1, Km 29 mM, and KD 0.024 ml.g−1.min−1. The value for Vmax was the same as that for l‐lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2‐oxo‐3‐methylbutanoate or 2‐0x0‐4‐methylpentanoate, the 2‐oxo acids that can accumulate in patients with maple‐syrup‐urine disease. The Km values for the 2‐oxo acids were severalfold lower than the respective Km values. 2‐oxo‐3‐phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites‐tumour cells.
Simultaneous Separation and Purification of Neurofilament and Glial Filament Proteins from Brain
Authors: Liem Ronald
Published: October 2006

Abstract: Neurofilaments (NF) and glial filaments (GF) were purified from bovine brain by the axonal flotation method, followed by hydroxyl‐apatite chromatography in 8 m‐urea. The proteins were shown to be competent to reassemble into intermediate filaments with removal of the denaturant, and reassembly was used as the final step in the purification of the filament proteins. The reassembly was found to be dependent on ionic strength and pH. This dependence was greater for neurofilaments than for the glial filaments. The NF and GF preparations were found not to be contaminated with each other by their gel electrophoretic profile and their immunological distinctness. The filament proteins can be obtained in high yield, and remain in solution if the urea is removed by dialysis against a low‐ionic‐strength buffer. Hence, they can provide a source for further biochemical studies.
An Improved Method for the Purification of “Light” Noradrenaline Vesicles from Rat Vas Deferens: Some Biochemical Characteristics
Authors: Willems M. F. , De Potter W. P.
Published: October 2006

Abstract: “Light” noradrenaline storage vesicles from nerve endings have been isolated by differential centrifugation and differential gradient centrifugation. They have been further purified by isopycnic sucrose/D2O centrifugation. By using these centrifugation techniques, we obtained an isopycnic gradient fraction in which noradrenaline was enriched about 41 times versus a total homogenate. This factor could be raised to 61 by using seminal ducts of castrated rats. Comparison of the distribution patterns in sucrose/D2O isopycnic gradients indicated that light noradrenaline vesicles of nerve endings contain Mg2+‐stimulated ATPase and ATP, but that only a minor part of the dopamine β‐hydroxylase can be associated with these vesicles.
GABA, Glutamic Acid Decarboxylase, and GABA Transaminase Levels in the Myenteric Plexus in the Intestine of Humans and Other Mammals
Authors: Miki Yukiko , Taniyama Kohtaro , Tanaka Chikako , Tobe Takayoshi
Published: October 2006

Abstract: Regional distribution of endogenous γ‐ aminobutyric acid (GABA), its synthesizing enzyme, glutamic acid decarboxylase (GAD), and metabolic enzyme, GABA transaminase (GABA‐T), were determined in the intestinal tract of guinea pigs and cats and the findings compared with the number of ganglion cells in Auerbach's plexus. There were positive correlations among the GABA contents and the numbers of neural cells of the plexus. The precise localization of GABA and GAD in individual layers (mucosa, circular and longitudinal muscles, and Auerbach's plexus) in the human and cat colon was also determined. The endogenous GABA contents and GAD activity were the highest in Auerbach's plexus in tissues of both species. These results indicate that GABA is synthesized and localized in Auerbach's plexus and probably plays a significant role in the enteric nervous system.
Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia
Authors: Shiu Gerald K. , Nemmer Joseph P. , Nemoto Edwin M.
Published: October 2006

Abstract: We previously reported that whole‐brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di‐ and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital‐anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one‐half to one‐ fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9‐fold for total FFA, and between 7 and 12‐fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.
Relative Levels of Hexokinase in Isolated Neuronal, Astrocytic, and Oligodendroglial Fractions from Rat Brain
Authors: Snyder Cynthia D. , Wilson John E.
Published: October 2006

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligoden‐droglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron‐microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.
Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors
Authors: Lowry Oliver H. , Berger Sosamma J. , Carter Joyce G. , Chi Maggie M.‐Y. , Manchester Jill K. , Knor Joseph , Pusateri Mary E.
Published: October 2006

Abstract: Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01–0.25 μg dry weight, were dissected from freeze‐dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose‐6‐phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6‐phosphogluconate dehydrogenase, and β‐hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose‐6‐phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6‐phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol‐1‐phosphate, and malate averages ranged from 104% to 127% of brain; and fructose‐1,6‐bisphosphate and glucose‐1,6‐bis‐phosphate levels were on the average 50% and 70% those of brain, respectively.
Sodium Transport in Capillaries Isolated from Rat Brain
Authors: Betz A. Lorris
Published: October 2006

Abstract: Brain capillary endothelial cells form a bloodbrain barrier (BBB) that appears to play a role in fluid and ion homeostasis in brain. One important transport system that may be involved in this regulatory function is the Na+,K+‐ATPase that was previously demonstrated to be present in isolated brain capillaries. The goal of the present study was to identify additional Na+ transport systems in brain capillaries that might contribute to BBB function. Microvessels were isolated from rat brains and 22Na + uptake by and efflux from the cells were studied. Total 22Na + uptake was increased and the rate of 22Na + efflux was decreased by ouabain, confirming the presence of Na+,K+‐ATPase in capillary cells. After inhibition of Na+,K+‐ATPase activity, another saturable Na + transport mechanism became apparent. Capillary uptake of 22Na + was stimulated by an elevated concentration of Na +or H+ inside the cells and inhibited by extracellular Na+, H+, Li+, and NH4+. Amiloride inhibited 22Na + uptake with a Ki between 10−5 and 10−6M but there was no effect of 1 mM furosemide on 22Na+ uptake by the isolated microvessels. These results indicate the presence in brain capillaries of a transport system capable of mediating Na +/ Na + and Na +/H + exchange. As a similar transport system does not appear to be present on the luminal membrane of the brain capillary endothelial cell, it is proposed that Na +/H + exchange occurs primarily across the antiluminal membrane.
Development and Regional Distribution of Deoxyuridine 5′‐Triphosphatase in Rabbit Brain
Authors: Spector Reynold , Boose Barbara
Published: October 2006

Abstract: The development and regional distribution of deoxyuridine 5′‐triphosphatase (dUTPase) in rabbit brain was studied. After partial purification of the dUTPase activity, newborn brain and liver activities fell from Vmax values of 1.0 and 6.9 nmol/mg protein/min, respectively, to adult levels of 0.1 (brain) and <0.01 (liver). The dUTPase activity was evenly distributed throughout the entire adult rabbit brain, and there was no difference in the Km of dUTPase in newborn and adult brain (0.1 μM). The low level of dUTPase in adult brain and the lack of activity in adult liver were not due to inhibitors or phenylmethylsulfonyl fluoride‐sensitive proteases. dUTPase in brain, as in other tissues, probably inhibits the misincorporation of uracil into DNA.
Effects of ATP and Taurine on Calcium Uptake by Membrane Preparations of the Rat Retina
Authors: Lombardini J. B.
Published: October 2006

Abstract: The effects of ATP and taurine on the kinetics of calcium uptake in rat retinal membrane preparations were determined. ATP increased calcium uptake at low calcium ion concentrations. Addition of ATP plus taurine further increased calcium uptake. Cooperative relationships were observed for calcium uptake in the absence of ATP and taurine. In the presence of phosphate ions reciprocal plots demonstrated upward deflections from linear ty, while in the absence of phosphate ions downward deflections were noted. Addition of ATP plus taurine to the incubation system appeared to obliterate the cooperativity. Two uptake systems for calcium were observed.
Vitamin E Concentrations in the Brains and Some Selected Peripheral Tissues of Selenium‐Deficient and Vitamin E‐Deficient Mice
Authors: Vatassery G. T. , Angerhofer C. K. , Peterson F. J.
Published: October 2006

Abstract: Weanling male CD‐1 mice were fed control, vitamin E‐deficient or selenium‐deficient diets for periods of 12 to 20 weeks. α‐Tocopherol concentrations in plasma, liver, and testes, as well as in three specific areas in the brain (cerebral hemisphere, cerebellum, and medulla plus pons) were determined by high performance liquid chromatography. Significant concentrations of α‐tocopherol were found in all brain samples from vitamin E‐deficient animals long after the peripheral tissues were depleted, indicating that brain is more resistant to vitamin E deficiency than peripheral tissues. Cerebellar concentrations of α‐tocopherol were consistently lower than those of cerebral hemisphere and medulla‐pons. Further more, the cerebellar α‐tocopherol concentration sustained a larger decline than the other two brain areas within 6 weeks of vitamin E deficiency treatment. These and other data suggest that cerebellum may be more susceptible to damage from vitamin E deficiency than other parts of the brain. Selenium deficiency did not affect brain a‐tocopherol concentrations during the 12 weeks of the study.
[3H]Nitrobenzylthioinosine Binding to the Guinea Pig CNS Nucleoside Transport System: A Pharmacological Characterization
Authors: Hammond James R. , Clanachan Alexander S.
Published: October 2006

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10–0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site‐specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane‐located nucleoside transport system in CNS membranes.
Binding of [3H]Serotonin to Skeletal Muscle Actin
Authors: Small David H. , Wurtman Richard J.
Published: October 2006

Abstract: We previously observed that the neurotransmitter 5‐hydroxytryptamine (5‐HT, serotonin) binds with high‐ and low‐affinity interactions to an actin‐like protein prepared from rat brain synaptosomes. In this study, we examined its binding to highly purified actin obtained from rabbit skeletal muscle. Monomeric G‐actin bound serotonin with high and low affinities, exhibiting equilibrium dissociation constants (KD values) of 5 × 10−5M and 4 × 10−3M, respectively. The serotonin binding site on actin was distinct from those sites previously characterized for divalent cations, nucleotides, and cytochalasin alkaloids. The binding of serotonin (1 μM) to G‐actin was increased as much as 26‐fold by divalent cations. Potassium iodine (KI) increased the affinity of G‐actin for serotonin, KD values for this binding being 3 × 10−7M and 6 × 10−5M. Serotonin bound with even higher affinity to polymerized F‐actin, with KD values of 2 × 10−8M and 2 × 10−5M. However, the total number of binding sites on F‐actin was only about 4% of the number of G‐actin. The binding of serotonin (0.1 μM) to G‐actin could be inhibited by phenothiazines (1 μM) or reserpine (10 μM), but not by classical antagonists of serotonin receptors or by drugs that release serotonin or inhibit its uptake. The binding of serotonin to actin in vivo may participate in a contractile process related to neurotransmitter release.
Effect of Glutamine on Glutamate Release from Hippocampal Slices Induced by High K+ or by Electrical Stimulation: Interaction with Different Ca2+ Concentrations
Authors: Szerb John C. , O'Regan Patrick A.
Published: October 2006

To characterize the effect of glutamine on the release of glutamate, aspartate, and γ‐aminobutyric acid (GABA), rat hippocampal slices were superfused with different concentrations of glutamine or Ca2+. Amino acids released and retained were analyzed by HPLC. Glutamine (0.5 mmol/L) increased more than threefold the release of glutamate evoked by 50 mmol/L K+ in the presence of 2.6 mmol/L Ca2+ without a corresponding increase in glutamate content, while the release of aspartate was increased less and that of GABA not at all by glutamine. The evoked release of all three amino acids, including the enhanced release of glutamate in the presence of glutamine, was strongly dependent on Ca2+ concentrations between 0.1 and 2.6 mmol/L. The potentiation of glutamate release by glutamine reached a plateau at 0.25 mmol/L glutamine. Intermittent electrical field stimulation increased the release of only glutamate and this release was nearly doubled by glutamine. The increased release was Ca2+ dependent and tetrodotoxin (TTX) sensitive. Results suggest that extracellular glutamine promotes primarily the formation of releasable glutamate and this enhancement is dependent on extracellular Ca2+.
Purine Nucleotide Synthesis in Adrenal Chromaffin Cells
Authors: Rotllán P. , Miras‐Portugal M. T.
Published: October 2006

Abstract: The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8‐14C]adenine and [8‐14C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [14C]Formate and [U‐14C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver. The enzymes responsible for adenine and adenosine salvage, adenine phosphoribosyltransferase and adenosine kinase, were purified about 1,500‐fold. Both enzymes are mainly cytosolic and exhibit a similar molecular weight of around 42,000. The results suggest that chromaffin cells can replenish their intracellular nucleotides lost during the secretory event by an active synthesis from salvage and de novo precursors.
Differences in Binding Properties of μ and δ Opioid Receptor Subtypes from Rat Brain: Kinetic Analysis and Effects of Ions and Nucleotides
Authors: Zajac Jean‐Marie , Roques Bernard P.
Published: October 2006

Abstract: Differences in binding properties of μ and δ opioid receptors were investigated using DAGO (Tyr‐d‐Ala‐Gly‐MePhe‐Gly‐ol) and DTLET (Tyr‐d‐Thr‐Gly‐Phe‐Leu‐Thr), which occur, respectively, as the most selective μ and δ radioligands available. At high concentration, each agonist is able to interact with its nonspecific sites. Competition experiments indicated that a two‐site competitive model was adequate to explain the interactions of DAGO and DTLET with [3H]DTLET and [3H]DAGO binding sites, respectively. The weak cross‐reactivity (≃10%) of DTLET for μ sites was taken into account in these experiments. On the other hand, DAGO and DTLET exhibit differential binding kinetics. Thus, at 35°C, the lifetime of DTLET within its receptor site about 14 times longer than that of the μ agonist. Sodium and manganese ions decrease the maximal number of high affinity μ and δ sites, but the sensitivity of μ receptors is three times higher towards Na+ and 20‐fold higher towards Mn2+ than that of δ receptors. GTP reduces similarly the μ and δ binding whereas only the DAGO binding was modified by the nonhydrolyzable analogue guanylylimidodiphosphate [GMP‐P(NH)P]. However, in the presence of Na+ ions, GMP‐P(NH)P inhibits the DTLET binding in a concentration‐dependent manner. The effects of Na+ and GMP‐P(NH)P could be explained by a sequential transformation of δ receptors to low‐affinity states. This model predicts that Na+, by lowering the affinity of a fraction of sites, produces a decrease in the maximal number of high‐affinity δ receptors and that GMP‐P(NH)P enhances the Na+, effect. Moreover, the binding kinetic to this high‐affinity state was also modified by Na+ and nucleotides. All of these data support the existence of two independent μ and δ binding sites, the properties of which are differentially regulated by these endogenous effectors.
Possible Role of Gangliosides in Regulating an Adenylate Cyclase‐Linked 5‐Hydroxytryptamine (5‐HT1) Receptor
Authors: Berry‐Kravis Elizabeth , Dawson Glyn
Published: October 2006

Abstract: Cultured NCB‐20 hybrid cells express adenylate cyclase‐coupled receptors for 5‐hydroxytryptamine (5‐HT) that correspond biochemically and pharmacologically to 5‐HT1 receptors in rodent brain membrane preparations, apart from a much‐reduced affinity for 5‐HT (160 nM compared to < 5 nM in brain). Since NCB‐20 cells also differ from rodent brain both qualitatively and quantitatively in their ganglioside composition, the effects of exogenously added gangliosides on the affinity of the 5‐HT1 receptor for 5‐HT were tested. Both GM1 ganglioside (the cholera toxin receptor) and tetrasialoganglioside GQ1b produced a 10‐fold increase in receptor affinity for [3H]5‐HT, measured by binding studies. All gangliosides, at submicromolar concentrations, resulted in significantly reduced EC50 values for 5‐HT‐mediated elevation of intracellular cyclic AMP levels. GQ1b had the capacity to most dramatically enhance the potency of 5‐HT in mediating increases in cyclic AMP levels. Gangliosides had no effect on the potency of DADLE or 3,4‐dihydroxyphenylethylamine (dopamine)‐mediated depression of cyclic AMP levels, suggesting some specificity for 5‐HT. Our data are interpreted as implying a specific role for polysialogangliosides in modulating the affinity of the 5‐HT1 receptor and the coupling of the 5‐HT1 receptor‐guanine nucleotide binding protein adenylate cyclase complex.