bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Serotonin, Dopamine, or Noradrenaline with an Actin‐Like Component in Pheochromocytoma (PC12) Cells
Authors: Small David H. , Wurtman Richard J.
Published: October 2006

Abstract: A rat pheochromocytoma (PC12) cell line was used to examine the possibility that 5‐hydroxytryptamine (serotonin), 3,4‐dihydroxyphenylethylamine (dopamine), or noradrenaline may be associated with cytoplasmic actin, as was suggested by previous in vitro binding studies on an actin‐like protein from rat brain synaptosomes. When PC12 cells were incubated with [3H]serotonin, [3H]dopamine, or [3H]noradrenaline for 30 min at 37°C, approximately 2–4% of the radioactivity present in the cells was found to be associated with a high‐molecular‐weight (actin‐like) component in supernatant fractions. Evidence relating this monoamine binding component to actin filaments includes: (a) its strong absorption by myosin filaments at low ionic strength; (b) a decrease in its affinity for myosin in the presence of 1 mM ATP, which lowers the affinity of authentic actin for myosin; (c) displacement of bound [3H]serotonin from it by DNase I, which binds strongly to actin and which inhibits [3H]serotonin binding to actin in vitro; (d) an increase in its binding of each monoamine (by 25–40%) after PC12 cells were preincubated with 10 μM cytochalasin B (a drug that induces depolymerization of F‐actin). These findings suggest that serotonin, dopamine, or noradrenaline may associate with actin filaments in vivo.
Sulfate Conjugation of Monoamines in Human Brain: Purification and Some Properties of an Arylamine Sulfotransferase from Cerebral Cortex
Authors: Yu P. H. , Rozdilsky B. , Boulton A. A.
Published: October 2006

Abstract: An arylamine sulfotransferase (PST‐M) from human brain cortex that is involved in the formation of O‐sulfate esters of monoamines has been purified 272‐fold by ammonium sulfate fractionation, gel filtration, DEAE‐cellulose ion‐exchange chromatography, chromatofocussing, and hydroxyapatite chromatography. A molecular weight of 62,000, pK of pH 5.8, and an optimum pH for the reaction at 7.8–8.0 with respect to tyramines have been determined. This enzyme possesses an extremely high affinity for dopamine and m‐tyramine based on the low Km values and is moderately active toward noradrenaline and p‐tyramine. Serotonin is a poor substrate. In contrast, another sulfotransferase, PST‐P, which has been separated from PST‐M and partially purified, exhibited a very high affinity for phenol and nitrophenols but was inactive toward the amine sulfate acceptors. In the human brain the specific activity toward dopamine as well as the ratio of activity toward dopamine/phenol was considerably higher than those for rat, hog, and bovine brains.
5‐Hydroxytryptamine Uptake and Imipramine Binding Sites in Neurotumor NCB‐20 Cells
Authors: Nakaki Toshio , Roth Bryan L. , Chuang De‐Maw , Costa Erminio
Published: October 2006

Abstract: NCB‐20 cells (neuroblastoma × fetal Chinese hamster brain hybrids) are equipped with a [3H]5‐hydroxytryptamine ([3H]5‐HT) uptake system and [3H]imipramine recognition sites. Approximately 80% of the radioactivity taken up by cells incubated with [3H]5‐HT was identified with 5‐HT. [3H]5‐HT uptake was temperature‐dependent, partially sodium‐dependent, saturable (Km= 7.3 ± 0.6 μM; Vmax= 2.0 ± 0.6 pmol/min/mg), and inhibited by clomipramine, imipramine, fluoxetine, and desipramine, but not by iprindole, mianserin, or opipramol. Lineweaver‐Burk plots showed a competitive type of inhibition by imipramine and fluoxetine. [3H]5‐HT uptake was not inhibited by nisoxetine or benztropine. [3H]Imipramine binding sites had a KD of 12 ± 2 nM and a Bmax of 22 ± 7 pmol/mg protein. The binding was sodium‐sensitive although to a lesser extent than that found with brain membranes. Imipramine binding was displaced by tricyclic antidepressants with the following order of potency: clomipramine > imipramine > fluoxetine > desipramine ± iprindole = mianserin > opipramol. These results suggest that imipramine binding sites are present together with the 5‐HT uptake sites in NCB‐20 cells and that these sites interact functionally but are different biochemically.
Regulation of Dopamine Release from PC12 Pheochromocytoma Cell Cultures During Stimulation with Elevated Potassium or Carbachol
Authors: Baizer Lawrence , Weiner Norman
Published: October 2006

Abstract: To examine the role of cyclic AMP in the process of catecholamine release experiments have been performed with cultures of PC12 pheochromocytoma cells. Elevated potassium (56 mM) and carbamylcholine (carbachol, 10–4M) cause rapid increases in cyclic AMP levels in the cultures that show a time course similar to that of evoked dopamine release. These secretogogueinduced increases in cyclic AMP levels are well correlated with release in terms of relative magnitude and calcium dependence. Forskolin (a direct activator of adenylate cyclase) causes dose‐related increases in cyclic AMP levels in PC12 cell cultures that are synergistic with those caused by either elevated potassium or carbachol. At low concentrations forskolin significantly increases evoked release, whereas at higher concentrations it increases both spontaneous and evoked release. These results suggest that cyclic AMP may be involved in the process of dopamine release from PC12 cells in culture.
Effect of Reactive Oxygen Species on Myelin Membrane Proteins
Authors: Konat Gregory W. , Wiggins Richard C.
Published: October 2006

Abstract: Fresh myelin, isolated from brainstems of adult rats, was incubated in the presence of Cu2+ and H2O2. Electrophoretic analysis of the reisolated myelin membrane revealed a gradual loss of the protein moiety from the characteristic pattern and an increase in aggregated material appearing at the origin of the gel. The aggregation of proteins was time‐dependent and was concomitant with the accumulation of lipid peroxidation products reactive with thiobarbituric acid. Furthermore, during the course of incubation, there was a gradual decrease in the amount of recovered light myelin and a quantitatively similar increase in heavier myelin subfractions. The aggregation of proteins seems not to be directly related to the buoyant densities of myelin fragments. The peroxidative damage to the myelin proteins may be an important contributor to pathochemistry of myelin sheath, in particular, and in general it implies the susceptibility of the protein moiety of cell membranes to oxygen‐induced deterioration.
Blood–Brain Barrier Protein and Phosphorylation and Dephosphorylation
Authors: Pardridge William M. , Yang Jing , Eisenberg Jody
Published: October 2006

Abstract: Capillaries in vertebrate brain have unique permeability properties that make up the blood–brain barrier (BBB). Although it is known that capillaries are innervated by nerve endings of intracerebral origin and that brain capillary function is likely acutely regulated by neuronal inputs, the possible mechanisms of neuronal regulation of capillary function are at present unknown. One possible mode of regulation is via the phosphorylation of brain capillary proteins. The present studies characterize, for the first time, the major phosphoproteins in the bovine brain capillary using both intact bovine brain capillaries and plasma membrane fractions from bovine brain capillaries. The patterns of endogenous phosphorylation of capillary proteins are compared to similar patterns obtained with synaptosomal (P2) fractions from bovine brain. The major findings of this study are: (a) The activity of protein phosphorylation in brain capillaries is localized almost exclusively to the capillary plasma membrane, and is nearly comparable to the activity of protein phosphorylation in synaptosomal membranes. (b) A major phosphoprotein doublet in the capillary fraction comigrates on a sodium dodecyl sulfate gel with a major phosphoprotein doublet of approximate molecular weight of 80K in the synaptosomal fraction, and the latter is presumed to be synapsin I; in dephosphorylation assays the synaptosomal 80K phosphoprotein doublet is not subject to measurable dephosphorylation, whereas the capillary 80K doublet is subject to rapid dephosphorylation, and is essentially completely dephosphorylated within 5 s at 0°C. (c) A prominent triplet of phosphoproteins with molecular weight of 50–55K is present in the capillary fraction, and is not present in the synaptosomal fraction; thus, this 50—55K triplet of phosphoproteins appears specific for brain capillaries. In summary, these studies provide the basis for future investigations of a protein phosphorylation paradigm in regard to the rapid control of brain capillary function by brain.
Effects of Antimycin, Glucose Deprivation, and Serum on Cultures of Neurons, Astrocytes, and Neuroblastoma Cells
Authors: Pauwels Petrus J. , Opperdoes Fred R. , Trouet André
Published: October 2006

The resistance of cultured mouse neuroblastoma cells, primary cultures of rat cerebellar neurons, and rat brain astrocytes to a block of aerobic metabolism was studied. Parameters such as lactate production and ATP content were measured in the presence of antimycin A and under various conditions of glucose, oxygen, and serum supply. The following conclusions can be drawn: (1) All cell types studied were characterized by an active production of lactate; (2) Incubation of the various cell types in the absence of glucose at normal oxygen tension did not affect ATP levels; (3) Respiration blocked by antimycin led to a Pasteur effect; (4) Neuroblastoma cells, but not the other cell types, were fully resistant to inhibition of respiration provided that sufficient glucose was supplied; (5) In the absence of glucose no stores of energy or utilizable substrate were present in the cell types studied when respiration was blocked; (6) In the presence of fetal calf serum anoxic neurons showed irreversible signs of degeneration.
Regional Distribution and Characterization of Kinin in the CNS of the Rat
Authors: Kariya K. , Yamauchi A. , Sasaki T.
Published: October 2006

The distribution of kinin in the CNS of the rat, which was extracted with n‐butanol from an acidified homogenate, was determined using a bradykinin (BK) radioimmunoassay system. The immunoreactive kinin was widely distributed throughout the brain. The highest content was found in the pituitary gland (4,135 fmol BK Eq/g), followed by the medulla oblongata (912 fmol/g), cerebellum (549 fmol/g), and cortex (512 fmol/g). The kinin in the posterior pituitary was concentrated 4.5 times as much as in the anterior lobe. Serial dilution of brain extracts produced binding curves parallel to the standard radioimmunoassay curve. The purified brain kinin comigrated with authentic BK during CM‐cellulose chroma tography and Sephadex LH‐20 gel chromatography. Its molecular weight was estimated to be 1,127$45 by gel filtration, which coincides well with that of BK. Chymotrypsin degraded the extracted kinin and authentic BK, but trypsin did not. These data demonstrate that a peptide indistinguishable from BK exists in the rat brain. Furthermore, pituitary kinin was separated into BK (87%), Lys‐BK (10%), and Met‐Lys‐BK (3%), using reverse phase HPLC.
Rat Brain Mast Cells: Contribution to Brain Histamine Levels
Authors: Goldschmidt Robert C. , Hough Lindsay B. , Glick Stanley D.
Published: October 2006

Recent studies have shown that mast cells (MCs) are present in rat brain, that they have a predominantly thalamic localization, and that they contain histamine (HA). However, the degree to which these cells contribute to brain HA levels has remained unclear. Our recent studies of the precise distribution of rat brain MCs permitted us to develop a method to determine both the MC numbers and HA content from the same brain. Thalamic MC numbers were highly correlated with both the amount (ng) and the concentration (ng/g) of thalamic HA in both sexes (p < 0.005). Slopes of these regression lines, suggestive of the HA content of thalamic MCs, were 2.5 and 1.3 pg/cell in males and females, respectively, substantially less than the HA levels in peritoneal MCs. Thalamic MC numbers were not correlated with HA (ng) outside of thalamus, but were significantly (p < 0.005) correlated with whole brain HA amounts (ng) and levels (ng/g). These results are direct biochemical evidence for a contribution by MCs to brain HA levels, and indicate that thalamic MCs contribute up to 90% of the HA in thalamus, and up to 50% of whole brain HA levels.
Kinetic Constants for Blood—Brain Barrier Amino Acid Transport in Conscious Rats
Authors: Miller L. P. , Pardridge W. M. , Braun L. D. , Oldendorf William H.
Published: October 2006

Abstract: The kinetic constants for large neutral amino acid (LNAA) transport across the blood–brain barrier (BBB) of conscious rats were determined in four brain regions: cortex, caudate‐putamen, hippocampus, and thalamus‐hypothalamus. Indwelling external carotid artery catheters allowed for single‐bolus (200 μ1) injections directly into the arterial system of unanesthetized and lightly restrained animals. Our results showed lower brain uptake index values for conscious rats compared to previous reports for anesthetized animals which are consistent with higher rates of cerebral blood flow in the conscious animals. Km values were lower in the conscious animals and ranged from 29% to 87% of the Km values in pentobarbital‐anesthetized animals whereas the KD values were about twofold higher in the conscious animals. No apparent regional differences were observed. Influx rates were determined which take into consideration flow rates and plasma amino acid concentrations. Our results showed an average amino acid influx value of 5.2 nmol/min/g, which is 53% higher than the average influx in pentobarbital‐anesthetized animals. The present results in conscious animals regarding the low Km of LNAA transport across the BBB lend further support to the importance of fluctuations in plasma amino acid concentrations and LNAA transport competitive effects on brain amino acid availability.
Cerebral Metabolic Changes During and Following Fluorothyl‐Induced Seizures in Ventilated Rats
Authors: Folbergrová Jaroslava , Ingvar Martin , Nevander Gunilla , Siesjö Bo K.
Published: October 2006

Abstract: The objective of the present study was to assess metabolic changes in the neocortex and hippocampus of well‐oxygenated or moderately hypoxic rats in which fluorothyl‐induced seizures were sustained for 5 or 20 min, or which were allowed recovery periods of 5, 15, or 45 min following cessation of 20‐min seizure activity by withdrawal of the convulsant gas. Sustained fluorothyl‐induced seizures were found to cause metabolic alterations qualitatively and quantitatively similar to those previously observed with other commonly used convulsants. Thus, although the phosphorylation state of the adenine nucleotide pool remained only moderately perturbed, if at all, there were decreases in tissue concentrations of phosphocreatine and glycogen, and increases in those of cyclic AMP, lactate, and pyruvate, with a calculated fall in intracellular pH of about 0.15 units and a rise in the cytoplasmic NADH/NAD+ ratio. The enhanced metabolic rate was reflected in a marked reduction in the tissue‐to‐plasma glucose concentration ratio. Induced moderate hypoxia (arterial Po2 40–50 mm Hg) had no metabolic effect after 5 min of seizures but moderately increased lactate concentrations after 20 min (from about 10 to about 15 μmol ± g−1). On cessation of seizure discharge cyclic AMP and phosphocreatine concentrations normalized already within 5 min, whereas glycogen and lactate concentrations normalized more slowly. In the neocortex (but not the hippocampus) postepileptic tissue‐to‐plasma glucose concentration ratios rose above control, probably reflecting metabolic depression. The results suggest that intracellular pH promptly returned to control, and that postepileptic alkalosis developed. They also suggest that some elevation of the NADH/NAD+ ratio persisted even after 45 min of recovery.
Benzodiazepine Binding Characteristics of Embryonic Rat Brain Neurons Grown in Culture
Authors: Syapin P. J. , Cole R. , De Vellis J. , Noble E. P.
Published: October 2006

Abstract: The binding of [3H]diazepam to cell homogenates of embryonic rat brain neurons grown in culture was examined. Under the conditions used to prepare and maintain these neurons, only a single, saturable, high‐affinity binding site was observed. The binding of [3H]diazepam was potently inhibited by the CNS‐specific benzodiazepine clonazepam (Ki= 0.56 ± 0.08 nM) but was not affected by the peripheral‐type receptor ligand Ro5‐4864. The KD for [3H]diazepam bound specifically to cell homogenates was 2.64 ± 0.24 nM, and the Bmax was 952 ± 43 fmol/mg of protein. [3H]Diazepam binding to cell membranes washed three times was stimulated dose‐dependently by γ‐aminobutyric acid (GABA), reaching 112 ± 7.5% above control values at 10−4M. The rank order for potency of drug binding to the benzodiazepine receptor site in cultured neurons was clonazepam > diazepam > β‐carboline‐3‐carboxylate ethyl ester > Ro15‐1788 > CL218,872 > Ro5‐4864. The binding characteristics of this site are very similar to those of the Type II benzodiazepine receptors present in rat brain. These data demonstrate that part, if not all, of the benzodiazepine‐GABA‐chloride ionophore receptor complex is being expressed by cultured embryonic rat brain neurons in the absence of accompanying glial cells and suggest that these cultures may serve as a model system for the study of Type II benzodiazepine receptor function.
Relationship Between Activation State of Pyruvate Dehydrogenase Complex and Rate of Pyruvate Oxidation in Isolated Cerebro‐Cortical Mitochondria: Effects of Potassium Ions and Adenine Nucleotides
Authors: Lai James C. K. , Sheu Kwan‐Fu Rex
Published: October 2006

Abstract: The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC;EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [I‐14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP‐dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin‐treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.
Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1‐Methyl‐4‐Phenylpyridinium Ion
Authors: Mytilineou Catherine , Cohen Gerald
Published: October 2006

Abstract: 1‐Methyl‐4‐phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) by mono‐amine oxidase (MAO). MPP+ is toxic to 3,4‐dihydroxy‐phenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 μM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 μM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.
Restrictive Transport of a Lipid‐Soluble Peptide (Cyclosporin) Through the Blood–Brain Barrier
Authors: Cefalu William T. , Pardridge William M.
Published: October 2006

Abstract: The blood–brain barrier (BBB) transport of a highly lipid‐soluble peptide, [3H]cyclosporin, was studied in ketamine‐anesthetized rats using the carotid artery injection technique. For comparison, peptide transport into rat liver was also assessed with the portal vein injection technique. Despite the high lipid solubility of this peptide (1‐octanol/Ringer's partition coefficient = 991 ± 55), the extraction by rat brain was only 2.9 ± 0.5% in the presence of 80% human serum, and this value approximated the extraction for a poorly diffusible substance such as [3H]inulin, 2.0 ± 0.1%. In contrast, the hepatic extraction of [3H]cyclosporin was high, 84 ± 2%, in the presence of 80% human serum. The BBB transport of cyclosporin is markedly restricted owing to the combined effects of binding by serum proteins and a paradoxically low permeability of the BBB to the peptide.
Dihydropyridine [methyl‐3H]PN 200–110 Binding and Myogenesis in Intact Muscle Cells In Vitro
Authors: Navarro Javier
Published: October 2006

Abstract: The radioligand dihydropyridine [methyl‐3H]PN 200–110 binds to contracting myotubes in culture derived from chick embryo pectoralis muscle. [methyl‐3H]PN 200–110 binds specifically to high‐affinity sites, with nonspecific binding only between 15 and 30% of the total binding. A Scatchard plot of the specific binding revealed a single high‐affinity binding site with a KD (dissociation constant) of 0.5 nM± 0.2 nM and Bmax (number of binding sites) of 100 fmol/106 nuclei. We employed this sensitive assay to probe the appearance of high‐affinity [methyl‐3H]PN 200–110 binding sites during myogenesis. The time course of appearance of high‐affinity binding sites lags behind that of fusion. Low‐calcium media prevented the differentiation of myoblasts and blocked the appearance of high‐affinity sites. Chelation of intracellular calcium before or after fusion of myoblasts with the calcium indicator Quin 2 prevented the appearance of dihydropyridine binding sites. These findings are consistent with the view that the expression of dihydropyridine receptors is modulated by the intracellular calcium.
Genetic Expression of Cyclic GMP Phosphodiesterase Activity Defines Abnormal Photoreceptor Differentiation in Neurological Mutants of Inherited Retinal Degeneration
Authors: Fletcher R. Theodore , Sanyal Somes , Krishna Gopal , Aguirre Gustavo , Chader Gerald J.
Published: October 2006

Abstract: We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.
Effect of Excitotoxin Lesions in the Medial Prefrontal Cortex on Cortical and Subcortical Catecholamine Turnover in the Rat
Authors: Christie M. J. , Rowe P. J. , Beart P. M.
Published: October 2006

Abstract Catecholamine turnover in brain areas innervated by dopaminergic neurons was examined 2, 6, and 12 days after bilateral, N‐methyl‐D‐aspartate lesions confined to the rat medial prefrontal cortex. The lesion produced a significant regional increase in the concentration of 3,4‐dihydroxyphenylethylamine (DA, dopamine) in both the medial prefrontal cortex and the ventral tegmental area. DA concentrations were increased in the nucleus accumbens on day 6 (128% of control), in the ventral tegmental area on day 2 (130% of control), and in the medial prefrontal cortex on days 2 (145% of control) and 6 (127% of control). The only significant changes in the concentration of 3,4‐dihydroxyphenylacetic acid (DOPAC) (197% of control), and in the ratio DOPAC/DA (163% of control) were found in the medial prefrontal cortex on day 6 post‐lesion. All parameters had returned to control levels by day 12. DA depletion after the administration of α‐methyl‐p‐tyrosine (AMPT) was not significantly different between excitotoxin‐lesioned and sham animals on day 6 in all brain regions. Noradrenaline (NA) and 3,4‐dihydroxyphenylethyleneglycol concentrations and their ratios, and the depletion of noradrenaline after AMPT were also determined, and the lesion resulted in a significant regional increase in NA in both the nucleus accumbens and the ventral tegmental area. An elevation of NA (147% of control) in the nucleus accumbens was found on day 12. Since the excitotoxin lesion destroys corticofugal efferents from medial prefrontal cortex to the nucleus accumbens, the anterior corpus striatum and the ventral tegmental area, our results provide no evidence for a role of these cortical projections in the regulation of subcortical DA metabolism. Corticofugal efferents from the medial prefrontal cortex do appear to exert a modulatory effect over NA stores of the nucleus accumbens.
Monoamine Oxidase Activity and Monoamine Metabolism in Brains of Parkinsonian Patients Treated with l‐Deprenyl
Authors: Riederer Peter , Youdim Moussa B. H.
Published: October 2006

Abstract: Monoamine oxidase (MAO) type A and type B were measured using kynuramine, 3,4‐dihydroxyphenyl‐ethylamine (dopamine, DA), and 5‐hydroxytryptamine (5‐HT, serotonin) in 20 brain areas. The highest activities were found in the striatum (caudate nucleus, putamen, globus pallidus, and substantia nigra), hypothalamus, and c‐mammilare. The ratio of DA to 5‐HT deamination varied in the different regions, being in favor of DA in the striatum. With kynuramine as the substrate IC50 values of a number of inhibitors indicated that l‐deprenyl was far more potent an inhibitor of human brain MAO than clorgyline or harmaline. N‐Desmethylpropargylindane hydrochloride (AGN 1135) was also shown to have MAO‐B inhibitory selectivity similar to that of l‐deprenyl. Brains obtained at autopsy from l‐deprenyl‐treated Parkinsonian patients showed that, whereas MAO‐B was fully inhibited by the therapeutic doses of l‐deprenyl, substantial MAO‐A activity was still evident. These results are matched by the significant increases of DA noted in caudate nucleus, globus pallidus, putamen, and substantia nigra and the unaltered 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) in the same regions. These data indicate that the therapeutic actions of l‐deprenyl may lie in its selective inhibition of MAO‐B resulting in increased brain levels of DA formed from L‐dihydroxyphenylacetic acid (L‐DOPA).
Blood‐Brain Barrier Monoamine Oxidase: Enzyme Characterization in Cerebral Microvessels and Other Tissues from Six Mammalian Species, Including Human
Authors: Kalaria Rajesh N. , Harik Sami I.
Published: October 2006

Abstract: We studied the enzyme monoamine oxidase (MAO) in isolated cerebral microvessels, and in mitochondria‐enriched brain and liver preparations from six mammalian species, including human. We also studied MAO distribution in various tissues and in discrete brain regions of the rat. MAO was assessed by measuring the specific binding of [3H]pargyline, an irreversible MAO inhibitor, and the rates of oxidation of known MAO substrates: benzylamine, tyramine, tryptamine, and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetra‐hydropyridine (MPTP). Molecular forms of MAO were examined by using specific MAO inhibitors, and by polyacrylamide gel electrophoresis after [3H]pargylihe binding. In general, the liver from all species had higher MAO levels than the brain, with minor variation among species in their brain and liver MAO content. However, there were remarkable species differences in brain microvessel MAO, with rat microvessels having one of the highest MAO activity among all tissues, whereas MAO activities in brain microvessels from humans, mice, and guinea pigs were very low. In most rat tissues, including the brain, there was a preponderance of MAO‐B over MAO‐A. The only exceptions were the heart and skeletal muscle. Estimates of MAO half‐life in rat brain microvessels, rat brain, and rat liver indicated that microvessel MAO had a higher turnover rate. The reasons underlying the remarkable enrichment of rat cerebral microvessels with MAO‐B are unknown, but it is evident that there are marked species differences in brain capillary endothelium MAO activity. The biological significance of these findings vis a vis the role of MAO as a “biochemical blood‐brain barrier” that protects the brain from circulating neurotoxins and biogenic amines should be investigated.
Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites
Authors: Awad M. , Gavish M.
Published: October 2006

Abstract: The binding of [3H]PK 11195 and [3H]Ro 5–4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05–10 nM) bound with high affinity to rat and alf cerebral cortical and kidney membranes. [3H]Ro 5–4864 (0.05–30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5–4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf, tissues. The potency of unlabeled Ro 5–4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig < cat = dog < rabbit < calf. Analysis of these displacement curves revealed that Ro 5–4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat < calf < guinea pig < rabbit < dog < rat, whereas when [3H]Ro 5–4864 was used, the rank order of binding capacity was cat < guinea pig < rat < rabbit < calf < dog. These results further demonstrate species differences of “peripheral‐type” benzodiazepine binding sites and also provide evidence of their heterogeneity in the kidney of rat and calf and in the cerebral cortex of rat, guinea pig, rabbit, and calf.
Biochemical Basis of Type AB GM2 Gangliosidosis in a Japanese Spaniel
Authors: Ishikawa Yukitoshi , Li Su‐Chen , Wood Philip A. , Li Yu‐Teh
Published: October 2006

Abstract: The biochemical basis of a case of GM2 gangliosidosis in a Japanese Spaniel was studied. This dog had a massive accumulation of GM2 ganglioside in the brain. The β‐hexosaminidase activity in this affected dog brain was ∼ 12 times higher than that of normal brain. However, the activity toward p‐nitrophenyl‐6‐sulfo‐2‐acetamido‐2‐de‐oxyglucopyranoside was only four times higher in the affected brain than in normal brain. The GM2 activator preparation obtained from the normal dog brain could stimulate the hydrolysis of GM2 ganglioside by β‐hexosaminidase isolated from the affected dog. However, the corresponding activator fraction from the affected dog could not stimulate such a reaction. It was concluded that the biochemical basis of the Gm2 gangliosidosis in this Japanese Spaniel was due to the attenuation in the stimulatory activity of GM2 activator. This case represents the first animal form similar to the activator deficiency (or defect) of Type AB GM2 gangliosidosis in humans.
Sodium‐Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral‐Commissural Projections of CA3 Pyramidal
Authors: Nadler Cells J. Victor
Published: October 2006

Abstract: Na+‐dependent uptake of L‐[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippo‐campal regions. Among hippocampal regions, Na+‐depen‐dent proline uptake was significantly greater in areas CA1 and CA2‐CA3‐CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+‐dependent γ‐[14C]aminobutyric acid ([14C] GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+‐de‐pendent proline uptake by an average of 41% in area CA1 and 52% in area CA2‐CA3‐CA4, without affecting the Na+‐dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high‐affinity (KT=6.7 μM) and a low‐affinity (KT= 290 μM) Na+‐dependent process, whereas L‐[14C]glutamate is taken up predominantly by a high‐affinity (KT=6.1 μM) process. A bilateral kainic acid lesion reduced the Vmax of high‐affinity proline uptake by an average of 72%, the Vmax of low‐affinity proline uptake by 44%, and the Vmax of high affinity glutamate Uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral‐commissural projections of CA3 hippocampal pyramidal cells Appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3‐derived Schaffer collateral, commissural, and ipsilateral associational fibers.
Monoamine Oxidase in Rat and Bovine Endocrine
Authors: Lenzen Tissues Sigurd , Freisinger‐Treichel Marion , Panten Uwe
Published: October 2006

Monoamine oxidase (MAO) was characterized in tissue homogenates from rat pancreatic islets, rat neurohy‐pophysis and adenohypophysis, and rat and bovine adrenal medulla and adrenal cortex. Phenylethylamine was preferentially deaminated by rat pancreatic islet and bovine adrenal medulla MAO and with slight preference by rat neurohypophysis MAO, whereas 5‐hydroxytryptamine was preferentially deaminated by MAO from all other endocrine tissues. Tyramine was a good substrate for all tissues. Clor‐gyline, a selective inhibitor of MAO‐A, preferentially inhibited deamination of 5‐hydroxytryptamine by all tissue homogenates, whereas deprenyl, a selective inhibitor of MAO‐B, preferentially inhibited deamination of phenyl‐ethylamine. Km values for 5‐hydroxytryptamine and tyra‐mine were higher by one to two decimal powers than for phenylethylamine in homogenates from all endocrine tissues. Km values were significantly lower for 5‐hydroxytryptamine and significantly higher for phenylethylamine in rat and bovine adrenal cortex than in adrenal medulla. According to these results, the contributions of MAO‐B to total enzyme activity were 70% for rat pancreatic islets, 45% for rat neurohypophysis, 15% for rat adenohypophysis, 20% for rat adrenal medulla, 10% for rat adrenal cortex, 60% for bovine adrenal medulla, and 20% for bovine adrenal cortex. PC 12 cells also contained predominantly MAO‐A (90%); however, an increased Km for phenylethylamine and a sensitivity of deamination of this MAO‐B substrate to inhibition by clorgyline are indicators of abnormal behavior of MAO in this clonal rat pheochromocytoma cell line.
Differences Between Vascular and Plaque Core Amyloid in Alzheimer's Disease
Authors: Prelli Frances , Castano Eduardo , Glenner George G. , Frangione Blas
Published: October 2006

Abstract: The predominant protein of cerebrovascular and plaque core amyloid in Alzheimer's disease, Down's syndrome, hereditary hemorrhage with amyloidosis—Dutch type, sporadic cerebral amyloid angiopathy, and age‐related amyloidosis is a unique polypeptide, called β protein. The length of the plaque amyloid protein was reported to be 42–43 residues, but the complete length of the cerebral vascular amyloid is not known. To clarify this issue, amyloid fibrils from the leptomeninges of an Alzheimer's disease patient were isolated and the primary structure determined. The complete sequence of cerebrovascular β‐amyloid protein, although homologous to the plaque core amyloid protein previously reported, has 39 residues instead of 42. Amino terminal heterogeneity is present but minimal, and it is three residues shorter at the carboxy terminus. These differences are similar to those found in two cases of hereditary hemorrhage with amyloidosis—Dutch type. The differences between vascular and plaque β‐amyloid may reflect diverse processing of the β protein precursor in the vessel wall and brain parenchyma due to tissue‐specific endopeptidases.
Isatin: Identity with the Purified Endogenous Monoamine Oxidase Inhibitor Tribulin
Authors: Glover Vivette , Halket J. M. , Watkins P. J. , Clow Angela , Goodwin B. L. , Sandier M.
Published: October 2006

Abstract: Purified tribulin, an endogenous monoamine oxidase (MAO) inhibitor, has been identified by direct probe insertion mass spectrometry as the indole‐2,3‐dione, isatin. A gas chromatographic‐mass spectrometric assay for isatin has been developed and used to measure its relatively high concentrations in unpurified human urine, and in rat heart and brain. Isatin is a known compound with a broad range of biological activity; this is the first report of its presence in the animal body. Isatin is a potent inhibitor of MAO, particularly of MAO B (IC50, 3 μM), and also binds to central benzodiazepine receptors (IC50 against clonazepam, 123 μM).
Glycosaminoglycans in Cortical Autopsy Samples from Alzheimer Brain
Authors: Jenkins H. G. , Bachelard H. S.
Published: October 2006

Abstract: Each of the known classes of mammalian glycosaminoglycans, with the exception of keratan sulphate, was found in cerebral cortex samples from patients with Alzheimer‐type dementia and age‐matched controls. These molecules were quantitated, after electrophoresis and staining with Alcian Blue dye, by scanning densitometry. No significant differences were found between the mean levels of each of the above glycosaminoglycans in frontal cortex from patients with dementia compared with controls. An increase (26%; p < 0.05) in the mean level of hyaluronate, but not of other glycosaminoglycans, was found in temporal cortex samples. On the other hand, the uronic acid content of hyaluronate degradation products following Streptomyces hyaluronidase treatment of brain glycosaminoglycans did not reveal any statistically significant changes in Alzheimer's disease. HPLC of disaccharide products from Arthrobacter chondroitinase AC digests did not reveal any significant changes in sulphate substitution of chondroitin sulphate in Alzheimer brain.
Acetylcholine and Dopamine Promote the Production of Platelet Activating Factor in Immature Cells of Chick Embryonic Retina
Authors: Bussolino Federico , Pescarmona Gianpiero , Camussi Giovanni , Gremo Fulvia
Published: October 2006

Abstract: We have previously shown that platelet‐activating factor (PAF), a naturally occurring lipid mediator of cell‐to‐cell communication, was produced by 3‐day‐old chick retina stimulated with acetylcholine (ACh) and dopamine (DA), but not with other neurotransmitters. ACh and DA stimulated PAF synthesis via a dithiothreitol (DTT)‐insensitive choline‐phosphotransferase, without affecting the acetyltransferase pathway, which was stimulated only by the calcium ionophore A23187. Therefore, we attempted to study the effects of neurotransmitters on PAF production and on the activities of the DTT‐insensitive cholinephosphotransferase and acetyltransferase in the developing chick embryo retina up to hatching. Our results show that PAF was produced already at 8 days of development, when retinal cells are still rather immature and ganglion and Mueller cells are the only differentiated cells. The stimulation of PAF production occurred with ACh and not with other neurotransmitters. In older stages, DA also stimulated PAF production, as already described in the chick after hatching. DTT‐insensitive cholinephosphotransferase and acetyltransferase activities were present in 8‐day‐old embryos, the earliest stage analyzed. Both enzymatic activities increased with age; DTT‐insensitive cholinephosphotransferase increased rapidly from day 12 up to day 18, whereas acetyltransferase activity increased linearly up to the time of hatching. To promote PAF production, ACh and DA activate DTT‐insensitive cholinephosphotransferase, but not acetyltransferase. The developmental sequence of PAF stimulation suggests that PAF production occurs through stimulation of ACh and D2‐dopamine receptors, which are mostly present in ganglion and amacrine cells. Synaptic maturation enhances the effects of ACh and DA. Our data taken together suggest that PAF may play a physiological role after the time of hatching, since light stimulation induces neurotransmitter release which can eventually lead to PAF production.
Xanthine and Uric Acid Levels in Rat Brain Following Focal Ischemia
Authors: Kanemitsu Hideaki , Tamura Akira , Kirino Takaaki , Karasawa Shinji , Sano Keiji , Iwamoto Takeo , Yoshiura Masahiko , Iriyama Keiji
Published: October 2006

Abstract: Changes of the xanthine and uric acid (UA) levels in rat forebrain following focal cerebral ischemia were studied by reversed‐phase HPLC with electrochemical detection. Focal ischemia was induced by occluding the left middle cerebral artery in the rat. The xanthine level in the normal group was 11.50 nmol/g tissue. In the ischemic group, the xanthine concentration in the ischemic hemisphere progressively increased after occlusion and reached a maximum value of 59.42 nmol/g tissue 4 h after operation. The UA level in the normal group was 2.20 nmol/g tissue, whereas in the ischemic group the UA concentration in the ischemic hemisphere gradually increased after occlusion, reaching a value of 38.53 nmol/g tissue 24 h after ischemia. The concentration of UA remained elevated in the ischemic hemisphere until 48 h after occlusion, and reached a maximum value of 38.98 nmol/g tissue. The xanthine and UA levels in the contralateral hemisphere remained unchanged. The xanthine and UA concentrations in the sham‐operated group did not show a significant increase after operation. The time course of the xanthine and UA levels suggests that in ischemic forebrain UA is formed from xanthine as a product of purine metabolism.
Spatial Learning Potentiates the Stimulation of Phosphoinositide Hydrolysis by Excitatory Amino Acids in Rat Hippocampal Slices
Authors: Nicoletti F. , Valerio C. , Pellegrino C. , Drago F. , Scapagnini U. , Canonico P. L.
Published: October 2006

Abstract: Stimulation of phosphoinositide (PI) hydrolysis by excitatory amino acids (glutamate and ibotenate) or nor‐epinephrine was potentiated in hippocampal slices from rats trained in an eight‐arm radial maze, used as a test of spatial learning. No difference in basal or carbamylcho‐line‐stimulated PI hydrolysis was found between control and trained animals. An increased PI response to excitatory amino acids and norepinephrine was not found in hippocampal slices prepared from animals trained in a shock conditioning avoidance test. These results suggest a possible involvement of specific glutamate receptors coupled with PI hydrolysis in the synaptic mechanisms underlying formation and/or storage of spatial memory.
A Possible Role for Taurine in Osmoregulation Within the Brain
Authors: Wade J. V. , Olson J. P. , Samson F. E. , Nelson S. R. , Pazdernik T. L.
Published: October 2006

Abstract: Intracranial microdialysis was used to measure changes in extracellular amino acids within the rat brain during local osmotic alteration of the extracellular micro‐environment or during systemic water intoxication. Increased cellular hydration produced by either of these methods was accompanied by a marked increase in extracellular taurine levels without affecting the other amino acids measured. With local osmotic alteration, this increase was osmolarity dependent and reversible. The specificity, sensitivity, and reversibility of the increase in extracellular taurine strongly suggest a functional role in osmoregulation in the brain under normal as well as pathological conditions.
Chronic Stress Increases Serotonin and Noradrenaline in Rat Brain and Sensitizes Their Responses to a Further Acute Stress
Authors: Adell Albert , Garcia‐Marquez Cristina , Armario Antonio , Gelpi Emilio
Published: October 2006

Abstract: The effects of 1 h/day restraint in plastic tubes for 24 days on the levels of serotonin (5‐HT), 5‐hydroxyindole‐acetic acid (5‐HIAA), tryptophan (TP), and noradrenaline (NA) in six regions of rat brain 20 h after the last restraint period were investigated. The levels of 5‐HT, 5‐HIAA, and NA but not TP increased in several regions. The effects of 1 h of immobilization on both control and chronically restrained rats were also studied. Immobilization per se did not alter brain 5‐HT, 5‐HIAA, and TP levels, but decreased NA in the pons plus medulla oblongata and hypothalamus. However, immobilization after chronic restraint decreased 5‐HT, increased 5‐HIAA, and decreased NA in most brain regions in comparison with values for the chronically restrained rats. We suggest that chronic restraint leads to compensatory increases of brain 5‐HT and NA synthesis and sensitizes both monoaminergic systems to an additional acute stress. These changes may affect coping with stress demands.
Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain
Authors: Yu Robert K. , Macala Lawrence J. , Taki Takao , Weinfeld Henry M. , Yu Franklin S.
Published: October 2006

Abstract: Developmental changes in ganglioside composition and biosynthesis were studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, “b” series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, “a” series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N‐acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3 → GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N‐acetylgalacto‐saminyltransferase activity (GM3 ± GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the “a” and “b” pathways to synthesis of the more complex gangliosides.