bluuebunny/crossref_metadata_2025 huggingface dataset:
Local Cerebral Glucose Utilization During Development and Aging of the Fischer‐344 Rat
Authors: London Edythe D. , Nespor Stephanie M. , Ohata Masahiro , Rapoport Stanley I.
Published: October 2006

Abstract: Local cerebral glucose utilization was measured in brain regions of awake Fischer‐344 rats. Measurements were taken in 15 regions of 1‐month‐old rats, and 19 regions of 3‐, 12‐, 24‐, and 34‐month‐old rats. Between 1 and 3 months, glucose utilization tended to increase in all brain regions; statistically significant increases occurred in seven regions. Between the ages of 3 and 12 months, glucose utilization decreased significantly in 12 regions. The greatest reductions (25% or more) occurred in the striatum, inferior colliculus, and pons, but the hypothalamus and thalamus, nucleus accumbens, and septum showed no statistically significant change. Cerebral glucose utilization did not change between 12 and 24 months or between 24 and 34 months of age. The results demonstrate a rise in cerebral glucose utilization with development from 1 to 3 months, a decline between 3 and 12 months, and a constancy in the second and third years that does not reflect reported senescence‐associated neurochemical and morphological cerebral changes.
Postmortem Stability of Dopamine‐Sensitive Adenylate Cyclase, Guanylate Cyclase, ATPase, and GTPase in Rat Striatum
Authors: Nicol Susan E. , Senogles Susan E. , Caruso Thomas P. , Hudziak James J. , McSwigan John D. , Frey William H.
Published: October 2006

Abstract: The stability of dopamine‐sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase was measured in homogenates of rat striatal tissue frozen from 0 to 24 h postmortem. ATPase, GTPase, and Mg2+‐dependent guanylate cyclase activities showed no significant change over this period. Mn2+‐dependent guanylate cyclase activity was stable for 10 h postmortem. Basal and dopamine‐stimulated adenylate cyclase activity decreased markedly during the first 5 h. However, when measured in washed membrane preparations, these adenylate cyclase activities remained stable for at least 10 h. Therefore, the postmortem loss of a soluble activator, such as GTP, may decrease the adenylate cyclase activity in homogenates. These results are not consistent with an earlier suggestion that there is a postmortem degradation of the enzyme itself. Other kinetic parameters of dopamine‐sensitive adenylate cyclase can also be measured independently of postmortem changes. Thus, it is possible to investigate kinetic parameters of dopamine‐sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in human brain obtained postmortem.
Evidence for Compartmentation of Synaptosomal Phosphate‐Activated Glutaminase
Authors: Kvamme E. , Olsen B. E.
Published: October 2006

Abstract: Phosphate‐activated glutaminase (EC 3.5.1.2) in synaptosomal preparations is inhibited 40–60% by the sulphydryl group reagent N‐ethylmaleimide (NEM), forming the basis for distinction between NEM‐sensitive and NEM‐insensitive glutaminases. The NEM effect cannot be explained by differential effects on distinct glutaminases because other glutaminases have not been detected, and the synaptosomal glutaminase activity can be fully accounted for by the activity of phosphate‐activated glutaminase. By fractionation of mitochondria isolated from synaptosomal preparations, which are preincubated with and without NEM, both NEM‐sensitive and NEM‐insensitive glutaminases are found to be localized to the inner mitochondrial membrane. Variations in pH (7.0–7.6) and the phosphate concentration (5–10 mM) affect chiefly NEM‐sensitive glutaminase, demonstrating that this glutaminase may be subject to regulation by compounds in the cytosol having restricted permeability to the inner mitochondrial membrane. Since p‐hydroxymercuribenzoate, which is known to be impermeable to the inner mitochondrial membrane, inhibits glutaminase similarly to NEM, phosphate‐activated glutaminase is assumed to be compartmentalized within the inner mitochondrial membrane. Thus, NEM‐sensitive glutaminase is localized to the outer face and NEM‐insensitive glutaminase to the inner region of this membrane and probably also to the matrix region.
22Na+ Uptake and Catecholamine Secretion by Primary Cultures of Adrenal Medulla Cells
Authors: Amy Christopher , Kirshner Norman
Published: October 2006

Abstract: The uptake of 22Na+ and secretion of catecholamines by primary cultures of adrenal medulla cells under the influence of a variety of agonists and antagonists were determined. Veratridine, batrachotoxin, scorpion venom, and nicotine caused a parallel increase in 22Na+ uptake and Ca2+–dependent catecholamine secretion. Ba2+, depolarizing concentrations of K+, and the Ca2+ ionophore Ionomycin stimulated secretion of catecholamines but did not increase the uptake of 22Na+. Tetrodotoxin inhibited both 22Na+ uptake and catecholamine secretion evoked by veratridine, batrachotoxin, and scorpion venom, but had no effect on 22Na+ uptake and catecholamine secretion caused by nicotine. On the other hand, histrionicotoxin, which blocks the acetylcholine receptor–linked ion conductance channel, blocked nicotine–stimulated 22Na+ uptake and catecholamine secretion, but only partially inhibited veratridine–stimulated catecholamine secretion and had no effect on veratridine–stimulated 22Na+ uptake. The combination of veratridine plus tetrodotoxin, which has been shown to prevent nicotine–stimulated secretion of catecholamines by adrenal medulla cells, also prevented nicotine–stimulated 22Na+ uptake by the primary cultures. These studies demonstrate the presence of tetrodotoxin–sensitive Na+ channels in adrenal medulla cells which are functionally linked to Ca2+–dependent catecholamine secretion. However, these channels are not utilized for Na+ entry upon activation of nicotinic receptors; in this case Na+ entry occurs through the receptor‐associated ion conductance channel.
Adenosine Receptors Mediating Cyclic AMP Productioin the Rat Hippocampus
Authors: Fredholm Bertil B. , Jonzon Bror , Lindgren Eva , Lindström Karin
Published: October 2006

Abstract: In the transversely cut rat hippocampus, adenosine caused a dose‐dependent increase in the accumulation of [3H]cyclic AMP from [3H]ATP. Adenosine breakdown products were inactive. AMP was somewhat less effective than adenosine, and its effect could be partially, but not completely, abolished by α,β‐methylene‐ADP and GMP, which inhibited its metabolism by 5′‐nucleotidase. The effect of adenosine was unaffected by inhibitors of adenosine deaminase, but enhanced by several inhibitors of adenosine uptake. Some analogues of adenosine, including N6‐phenylisopropyladenosine (PIA), 2‐chloroadenosine and adenosine 5′‐ethylcarboxamide (NECA), were more active than adenosine, whereas others such as 2‐deoxyadenosine and 9‐(tetrahydro‐2‐furyl)adenine (SQ 22536) actually inhibited the response. The effect of PIA was highly stereospecific. The action of adenosine was inhibited by several alkylxanthines, the most potent of which was 8‐phenyltheophylline. [3H]Cyclohexyladenosine (CHA) bound specifically to cell membranes from the rat hippocampus. The extent of binding was similar to that found in other cortical areas. The relative potency of some adenosine analogues and alkylxanthines to displace labelled CHA was essentially similar to their potency as effectors of the cyclic AMP system. Adenosine contributed to the cyclic AMP‐elevating effect of α‐adrenoceptor‐stimulating drugs and several amino acids, but not to that seen with isoprenaline. The cyclic AMP increase seen following depolarization was only partially adenosine‐dependent. The present results demonstrate that the rat hippocampus contains adenosine receptors mediating cyclic AMP accumulation and that these receptors have similar characteristics to those mediating pyramidal cell depression. Adenosine‐induced cyclic AMP accumulation may be used as a biochemical correlate to electrophysiology and as a convenient parameter to assess the influence of drugs on adenosine mechanisms in the rat hippocampus.
Rapid Migration of Inositol Phospholipids with Axonally Transported Substances in the Rabbit Optic Pathway
Authors: Alberghina M. , Karlsson J. O. , Giuffrida A. M.
Published: October 2006

Abstract: Following an intraocular injection of myo‐[2‐3H]inositol, the axonal transport of labelled water‐soluble substances and inositol phospholipids was investigated. Evidence was obtained for a rapid axonal transport of a relatively small amount of labelled inositol phospholipids. In contrast to other axonally transported phospholipids, there was no significant accumulation of labelled, rapidly transported inositol phsopholipids in the nerve terminal region at later time intervals following the isotope administration.
Myelin Gangliosides in Vertebrates
Authors: Cochran Frederick B. , Yu Robert K. , Ledeen Robert W.
Published: October 2006

Abstract: A phylogenetic survey of brain myelin ganglioside patterns and concentrations has been carried out on 16 vertebrate species. Gangliosides were isolated from purified myelin and found to vary in concentration from 25 μg of sialic acid per 100 mg of myeh for goldfish to a value of 395 for turkey. The latter species had approximately equivalent amounts of GM1 and GM4 as the two major gangliosides. The 11 mammals studied all had GM1 as the major ganglioside, with variable amounts of GM4; rhesus monkey and human had 20‐25% GM4, whereas the others had less than 10%. Amphibia and fish myelin contained the least total ganglioside, with patterns that showed relatively little GM1 and no detectable GM4. Alligator myelin was unique in having a total concentration as high as the avian species, but a pattern with predominantly diand trisialo gangliosides.
The Influence of Bicuculline‐Induced Seizures on Free Fatty Acid Concentrations in Cerebral Cortex, Hippocampus, and Cerebellum
Authors: Siesjö Bo K. , Ingvar Martin , Westerberg Eva
Published: October 2006

Abstract: Using ventilated rats maintained on N2O‐O2 (70:30, vol/vol) we induced continuous seizures with i.v. bicuculline and analysed free fatty acids (FFA) in cerebral cortex, hippocampus, and cerebellum after seizure durations of 1–120 min. In the cerebral cortex, peak FFA concentrations were observed after 5 min, with a threefold increase in total FFA content. The values then remained unchanged for the next 15‐20 min, but decreased thereafter. At 60 and 120 min, total FFA contents were only moderately increased above control. In the initial period, arachidonic acid increased about 10‐fold and stearic acid 2‐ to 3‐fold, with little change in palmitic acid and linoleic acid concentrations. At all times, the docosahexenoic acid concentration was markedly increased. Following its massive accumulation at 1 min, arachidonic acid gradually decreased in concentration. Pretreatment of animals with indomethacin did not alter this behaviour. After 20 and 120 min of seizure activity, changes in total and individual FFA concentrations in the hippocampus were similar to those observed in the cerebral cortex. The cerebellum behaved differently. Thus, at 20 min the only significant change was a 5‐ to 10‐fold increase in arachidonic acid concentration and, after 120 min, total and individual FFA concentrations were similar to control values. Furthermore, since the control values for arachidonic acid were much lower in the cerebellum, the 20‐min values were only about 20% of those observed in the cerebral cortex and the hippocampus.
Inhibition of Human Brain Aldose Reductase and Hexonate Dehydrogenase by Alrestatin and Sorbinil
Authors: O'Brien Margaret M. , Schofield Philip J. , Edwards Michael R.
Published: October 2006

Abstract: Human brain aldose reductase and hexonate dehydrogenase are inhibited by alrestatin (AY 22,284) and sorbinil (CP 45,634). Inhibition by alrestatin is noncompetitive for both enzymes, and slightly stronger for hexonate dehydrogenase (KI values 52‐250 μM) than for aldose reductase (KI values 170‐320 μM). Sorbinil inhibits hexonate dehydrogenase far more potently than aldose reductase, KI values being 5 μM for hexonate dehydrogenase and 150 μM for aldose reductase. The inhibition of hexonate dehydrogenase by sorbinil is noncompetitive with respect to both aldehyde and NADPH substrates, and is thus kinetically similar to the inhibition by alrestatin. However, sorbinil inhibition of aldose reductase is uncompetitive with respect to glyceraldehyde and noncompetitive with NADPH as the varied substrate. Inhibition of human brain aldose reductase by these two inhibitors is much less potent than that reported for the enzyme from other sources.
Active Transport of Nicotine by the Isolated Choroid Plexus In Vitro
Authors: Spector Reynold , Goldberg Mark J.
Published: October 2006

Abstract: In vitro, the transport of [14C]nicotine into the isolated choroid plexus, the anatomical locus of the blood–CSF barrier, was studied. The isolated rabbit choroid plexus accumulated [14C]nicotine by two processes: an active saturable transport process and a nonsaturable process. The [14C]nicotine accumulation process by choroid plexus was not due to binding or intracellular metabolism of the [14C]nicotine. The [14C]nicotine accumulation process in isolated choroid plexus was inhibited by weak bases, including tolazoline and lidocaine, but not by the weak acid probenecid. The accumulation process was decreased 60% by iodoacetate and dinitrophenol and by low temperatures. These results are consistent with previous autoradiographic evidence showing the choroid plexus concentrated [14C]nicotine in vivo, and suggest that the choroid plexus may transfer nicotine between blood and CSF in vivo.
In Vitro Studies of 5,10‐Methylenetetrahydrofolate Reductase: Inhibition by Folate Derivatives, Folate Antagonists, and Monoamine Derivatives
Authors: Hollinger J. L. , Hommes O. R. , van de Wiel Th. J. J. M. , Kok J. C. N. , Jansen M. J. T.
Published: October 2006

Abstract: Folate monoglutamates and folate antagonists have an inhibitory action on the activity of 5, 10‐methylenetetrahydrofolate reductase in rat brain. The type of inhibition was studied for dihydrofolic acid using the Lineweaver–Burk transformation. Some of the monoamine alkaloids, the in vitro products of 5, 10‐methylenetetrahydrofolate reductase, have either a stimulatory or inhibitory effect on the enzyme activity.
Choline Uptake in Cultured Human Y79 Retinoblastoma Cells: Effect of Polyunsaturated Fatty Acid Compositional Modifications
Authors: Hyman Bradley T. , Spector Arthur A.
Published: October 2006

Abstract: Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high‐affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA: choline O‐acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high‐affinity system has a K′m= 2.16 ± 0.13 μm and V′max= 27.0 ± 2.9 pmol min−1 mg−1, whereas the low‐affinity system has K′m= 20.4 ± 1.3 μm and V′max= 402 ± 49 pmol min−1 mg−1. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n‐3 polyunsaturated fatty acids) or arachidonic acid (n‐6 polyunsaturated fatty acid), the K′m of the high‐affinity choline transport system was reduced by 40–60%. The V′max also was reduced by 20–40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high‐affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n‐3 and n‐6 classes of polyunsaturated fatty acids.
Human CSF GABA Concentrations: Revised Dowd for Controls, but Not Decreased in Huntington's Chorea
Authors: Perry Thomas L. , Hansen Shirley , Wall Richard A. , Gauthier Serge G.
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) concentrations were measured in CSF specimens from two large groups of control subjects, one without neurological or psychiatric disease, and one with a variety of neurological disorders not known to involve altered GABAergic function in brain. CSF GABA was also measured in patients with Huntington's chorea and in patients with other choreiform disorders. GABA was measured in CSF by a modification of the ion exchange‐fluorometric method that featured use of a relatively large cation exchange column, and a markedly decreased quantity of sulfosalicylic acid for deproteinization of CSF. Mean GABA concentrations in CSF were 87 and 77 nmol/liter for neurologically normal and abnormal control subjects, 82 nmol/liter for the Huntington's chorea patients, and 105 nmol/liter for patients with other forms of chorea. The mean concentration of homocarnosine was not reduced in CSF of Huntington's chorea patients as compared with controls. Mean CSF GABA concentrations found in control subjects were less than half the lowest control means previously reported. These low values are attributable in part to a reduction in on‐column hydrolysis of conjugated forms of GABA in CSF, which can be produced by excessive sulfosalicylic acid, and in part to improved chromatographic resolution of GABA from other unknown o‐phthalaldehyde‐reactive compounds in CSF. Analysis of free GABA in CSF does not appear useful for diagnosis of suspected Huntington's chorea, nor as a possible predictive test for persons genetically at risk for Huntington's chorea.
Peripheral Nervous System Myelin and Schwann Cell Glycoproteins: Identification by Lectin Binding and Partial Purification of a Peripheral Nervous System Myelin‐Specific 170,000 Molecular Weight Glycoprotein
Authors: Shuman Sandra , Hardy Mattie , Pleasure David
Published: October 2006

Abstract:Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate‐polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A,Helix pomatia, Limulus polyphemus, Madura pomifera, peanut, soybean,Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2M) inhibited lectin binding to the nitrocellulose‐fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high‐molecular‐weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mrand lectinbinding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mrglycoprotein is apparently unique to PNS myelin.
Biosynthesis of N‐Acylethanolamine Phospholipids by Dog Brain Preparations
Authors: Natarajan V. , Schmid Patricia C. , Reddy Padala V. , Zuzarte‐Augustin Mary Lou , Schmid Harald H. O.
Published: October 2006

Abstract: Dog brain homogenates and subcellular preparations incubated in the presence of Ca2+ produced a new phospholipid that was isolated and identified by its infrared spectrum and by chemical degrActation as a mixture of 1,2‐diacyl, alkenylacyl, and alkylacyl sn‐glycero‐3‐phospho(N‐acyl)ethanolamines, 50, 45, and 5%, respectively. The TV‐acyl groups consisted almost exclusively of 16:0, 18:0, and 18:1 fatty acids. Formation of Nacylethanolamine phospholipids from endogenous substrates was linear for about 90 min at approximately 4.5 nmol/h/mg protein and exhibited a pH optimum of 10. Biosynthetic activity was associated with paniculate fractions, primarily microsomes, synaptosomes, and mitochondria, but not with myelin. In each case, small amounts (‐0.5 nmol/h/mg protein) of long‐chain N‐acylethanolamines were also produced. Incubation of dog brain microsomes with 1,2‐di[1′‐14C]palmitoyl glycerophosphocholine yielded N‐acylethanolamine phospholipids labeled at both N‐acyl (55%) and O‐acyl (45%) moieties. It appears that dog brain organelles may contain a phosphatidylethanolamine N‐acyl transferase (transacylase) analogous to that recently demonstrated in the myocardial tissue.
Reaction of Muscimol with 4‐Aminobutyrate Aminotransferase
Authors: Fowler Leslie J. , Lovell David H. , John Robert A.
Published: October 2006

Abstract: The reaction of muscimol as amino donor substrate for GABA transaminase (GABA‐T) has been studied using enzyme purified from rabbit brain. Enzyme activity was assayed by measuring the glutamate produced using glutamate dehydrogenase. Kinetic parameters determined at 37°C were for GABA, Km (app) = 1.92 ± 0.24 mM, specific activity = 7.33 ± 0.27 μmol/min/mg (kcat= 13.7s−1), and for muscimol, Km (app) = 1.27 ± 0.15 mM, specific activity = 0.101 ± 0.009 μmol/min/mg (kcat= 0.19s−1). Addition of muscimol to the enzyme caused the spectral changes associated with conversion of the pyridoxaldimine form to the pyridoxamine form, and the first‐order rate constant for the reaction showed a dependence on muscimol concentration that followed saturation kinetics, with a K= 1.1 ±0.18 mM and kmax= 0.065 ± 0.004 s−1 (19°C). The rate of spectral change observed on addition of muscimol to ornithine transaminase was extremely slow—at least an order of magnitude slower than that seen with GABA‐T.
Differential Binding Properties of Adenosine Receptor Agonists and Antagonists in Brain
Authors: Marangos Paul J. , Patel Jitendra , Martino Andrea M. , Dilli Mark , Boulenger Jean Philippe
Published: October 2006

Abstract: The binding properties of N6‐cydohexyl [3H]adenosine ([3H]CHA) and 1, 3‐diethyl‐8‐[3H]phenyl‐xanthine ([3H]DPX) in rat forebrain membrane are compared. The kinetic parameters of binding for each ligand are quite distinct, with [3H]CHA displaying two populations of binding sites (KD= 0.4 ± 0.05 nM and 4.2 ± 0.3 nM; Bmax= 159 ± 17 and 326 ± 21 fmol/mg protein), whereas [3H]DPX yielded monophasic Scatchard plots (KD= 13.9 ±1.1 nM;Bmax= 634 ± 27 fmol/mg protein). The metals copper, zinc, and cadmium are potent inhibitors of [3H]CHA binding, with respective IC50 concentrations of 36 [μM, 250 )μM, and 70 μM. Copper is a much less potent inhibitor of [3H]DPX binding (IC50= 350 μM). The inhibitory effect of copper on both [3H]CHA and [3H]DPX binding is apparently irreversible, as membranes pretreated with copper cannot be washed free of its inhibitory effect. The inhibitory effect of both copper and zinc on [3H]CHA binding was reversed by the guanine nucleotide Gpp(NH)p. [3H]DPX binding is only partially inhibited by zinc and cadmium (60% of specific binding remains unaffected), suggesting that this adenosine receptor ligand binds to two separate sites. Guanine nucleotides had no effect on the inhibition of [3H]DPX binding by either copper or zinc. Differential thermal and proteolytic denaturation profiles are also observed for [3H]CHA and [3H]DPX binding, with the former ligand binding site being more labile in both cases. Stereospecificity is observed in the inhibition of both [3H]CHA and [3H]DPX binding, with l‐N‐phenylisopropyladenosine (PIA) being 50‐fold more potent than d‐PIA in both cases. Evidence is therefore provided that adenosine receptor agonists and antagonists have markedly different binding properties to brain adenosine receptors.
Degradation of Neurotensin by Rat Brain Synaptic Membranes: Involvement of a Thermolysin‐Like Metalloendopeptidase (Enkephalinase), Angiotensin‐Converting Enzyme, and Other Unidentified Peptidases
Authors: Checler Frederic , Vincent Jean‐Pierre , Kitabgi Patrick
Published: October 2006

Abstract: Neurotensin was inactivated by membrane‐bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8‐Arg9, Prol0‐Tyr11, and Tyr11‐Ile12 peptide bonds with the membrane‐bound activity and the Arg8‐Arg9 and Pro10‐Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8‐Arg9 bond by the membrane‐bound activity resulted mainly from the conversion of neurotensin1–10 to neurotensin1–8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin‐converting enzyme. Thiorphan inhibited the cleavage at the Tyr11‐−Ile12 bond by the membrane‐bound activity with an IC50 (17 nM) similar to its Ks (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10‐phenanthroline. These and other data suggested that angiotensin‐converting enzyme and a thermolysin‐like metalloendopeptidase (enkephalinase) were the membrane‐bound peptidases responsible for cleavages at the Arg8‐Arg9 and Tyr11‐Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8‐Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin‐converting enzyme. The cleavage at the Pro10‐Tyr11 bond by both the membrane‐bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.
Chromatin Structure in Neuronal and Neuroglial Cell Nuclei as a Function of Age
Authors: Berkowitz Ellen M. , Sanborn Anne C. , Vaughan Deborah W.
Published: October 2006

Abstract: Nuclei from the cerebral cortices of animals of different ages were separated into neuronal and neuroglial populations. Nuclei from cerebellar neurons were also studied. Using the enzyme micrococcal nuclease as a probe for chromatin structure, we found that the DNA from both neuronal preparations showed a decreased susceptibility to digestion during aging, although the onset of this alteration varies. In addition, both neuronal populations showed dramatic increases in the nucleosome spacing of the chromatin. Cerebral neuronal chromatin has a repeat length (nucleosome core and linker region) of 164 base pairs at 22 days and 11 months, 186 base pairs at 24 months, and 199 base pairs at 30 months. Cerebellar neuronal chromatin has a repeat of 188 base pairs at both 22 days and 11 months, 208 base pairs at 24 months, and 243 base pairs at 30 months. Neuroglial chromatin, on the other hand, showed no change in either accessibility to nuclease or repeat length.
Muscarinic Acetylcholine Receptors in Torpedo Electric Organ: Effect of Guanine Nucleotides
Authors: Dowdall Michael J. , Strange Philip G. , Golds Peter R.
Published: October 2006

Abstract: The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, [3H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites [slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 mM GTP. The binding of the N‐methylated antagonists, N‐methylatropine and N‐methyl‐scopolamine was more complex (slope factors <1) but also insensitive (N‐methylatropine) to 0.1 mM GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high‐affinity sites varied with the nature of the agonist (15–80%). Agonist binding was depressed by 0.1 mM GTP, and the order of sensitivity was oxotremorine‐M > carbamoylcholine > muscarine > acetylcholine > arecoline > oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half‐maximal at 12 μM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two‐site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.
Biochemical Studies of Olfaction: Binding Specificity of Odorants to a Cilia Preparation from Rainbow Trout Olfactory Rosettes
Authors: Rhein Linda D. , Cagan Robert H.
Published: October 2006

Abstract: Cilia isolated from the olfactory epithelium (olfactory rosettes) of rainbow trout (Salmo gairdneri) bind amino acids, which are odor stimuli to this species. We demonstrate that L‐threonine, l‐serine, and l‐alanine bind to a common site, TSA, in the cilia preparation. All possible mixtures of two of the amino acids as competitors, with the third as the 3H‐labeled ligand, were studied. The effect of two combined (unlabeled) competitors was always substantially less than additive compared with their actions singly. Along with additional inhibition studies using mixtures of inhibitors, the data show that the three odorants must interact with at least one common binding site, TSA. Binding of l‐[3H]lysine to site L was unaffected by addition of l‐threonine, l‐serine, or l‐alanine, establishing its independence from site TSA. l‐Arginine inhibited binding of l‐[3H]lysine, showing that both of these basic amino acids interact with site L. The data establish the presence, in trout olfactory cilia, of at least two separate and noninteracting populations of odorant binding sites, TSA and L.
Metabolically Active Synaptosomes Can Be Prepared from Frozen Rat and Human Brain
Authors: Hardy John Anthony , Dodd Peter R. , Oakley Arthur Ernest , Perry Robert Henry , Edwardson James Alexander , Kidd Alison Maria
Published: October 2006

Abstract: Nerve ending particles (synaptosomes) were prepared from pieces of rat and human brain and from brain homogenate that had been frozen and thawed under a variety of conditions. Their purity, as judged by electron microscopy, and performance in terms of a number of metabolic and functional parameters [accumulation of tissue potassium, respiration, release of transmitter amino acids, and the responses on these indices to depolarisation by veratrine (VX)] were compared with those of fresh tissue‐derived synaptosomes. It was found that rapid freezing and/or slow thawing severely impaired the subsequent performance of incubated synaptosomes. In contrast, synaptosomes from tissue frozen slowly and thawed rapidly showed relatively good retention of morphology and metabolic performance. It was better to use whole (1‐5 g) pieces of tissue than tissue homogenate: the synaptosome fraction from frozen tissue pieces contained 80% of the proportion of identified synaptosomes found in the fresh tissue synaptosome fraction, its respiratory rate was 65%, and its tissue potassium content 70% of that of fresh controls. Moreover, it responded to VX or potassium stimulation by showing increased respiratory rate, decreased tissue potassium, and increased release of neurotransmitter amino acids, to an extent that was comparable to that of fresh tissue fractions. Thus, preparations from frozen rat and human brain were shown to be metabolically and functionally active, and can be used for a variety of neurotransmitter‐related studies.
The Release and Neosynthesis of Glutamic Acid Are Increased in Experimental Models of Hepatic Encephalopathy
Authors: Moroni F. , Lombardi G. , Moneti G. , Cortesini C.
Published: October 2006

Abstract: The effects of ammonium ions on the release of glutamic acid from the rat cerebral cortex were measured in vivo using cortical cups and a multiple ion detection technique. The neosynthesis of this amino acid from glucose was also studied in two experimental models of hepatic encephalopathy: (1) rats receiving large amounts of ammonium acetate (i.p.) and (2) rats with a surgically constructed portocaval anastomosis. Intraperitoneal administration of 8 mmol/kg of ammonium acetate increased the cortical release of glutamic acid from 9.1.0.8 to 19.2 (nmol. cm−2. min−1). Moreover, 20 min after ammonium acetate administration the rate of incorporation of 13C2, originating from [13C]glucose, into glutamic acid increased by 65%. In several brain areas of rats bearing a portocaval anastomosis and fed ad libitum for 4 weeks, the content of glutamic acid slightly increased and the rate of formation of [13C2]glutamate from [13C]glucose approximately doubled. These results indicate that ammonium ions increase the release and the formation of glutamic acid in the brain. The resulting increased concentration of this amino acid in the extracellular spaces may be one of the mechanisms of ammonia toxicity in vivo.
Characterization of Separated Cell Types from the Developing Rat Cerebellum: Transport of Glutamate and Aspartate by Preparations Enriched in Purkinje Cells, Granule Neurones, and Astrocytes
Authors: Gordon R. D. , Balàzs R.
Published: October 2006

Preparations of structurally preserved cerebellar perikarya (cells) were found to express high‐affinity transport systems for glutamate but not for certain putative transmitter substances (including monoamines, glycine and taurine) and non‐transmitter amino acids. The characteristics of the high‐affinity glutamate transport system were similar to those of other preparations of brain tissue: [3H]glutamate uptake by the cells was Na+‐dependent and was inhibited competetively by other acidic amino acids. The rank order of apparent affinities of the carrier for acidic amino acids was L‐aspartate > L‐glutamate > D‐aspartate ≫ D‐glutamate (the affinity for D‐glutamate being over two orders of magnitude lower than for the other three amino acids). Comparison of high‐affinity [3H]glutamate uptake in preparations enriched in different cell types showed that although the affinities are similar (2‐4 fiM), the rate is outstandingly high in astrocytes (Vmax 18 nmol/min per mg protein). Significantly, uptake into the putatively glutamatergic granule cells was very low. These observations were supported by autoradiographic findings which showed that the predominant sites of [3H]glutamate uptake in cerebellar cultures enriched in interneurones are the astrocytes. Furthermore, the Vmax in cultures enriched in astrocytes was as high as that in separated astrocytes. Thus, it seems that the principal cell type involved in acidic amino acid uptake in the cerebellum is the astrocyte, and this must be taken into consideration when high‐affinity uptake is used as a marker for glutamatergic transmitter systems. Furthermore, the selective cellular distribution of glutamate transport sites, together with the uneven distribution of enzymes related to glutamate metabolism observed previously, indicates that a metabolic interaction takes place between the different cell types, supporting the current hypothesis on metabolic compartmentation in the brain.
Kainate‐Enhanced Release of d‐[3H]Aspartate from Cerebral Cortex and Striatum: Reversal by Baclofen and Pentobarbital
Authors: Potashner S. J. , Gerard D.
Published: October 2006

A study was made of the actions of the excitant neurotoxin, kainic acid, on the uptake and the release of d‐[2,3‐3H]aspartate (d‐ASP) in slices of guinea pig cerebral neocortex and striatum. The slices took up d‐ASP, reaching concentrations of the amino acid in the tissue which were 14–23 times that in the medium. Subsequently, electrical stimulation of the slices evoked a Ca2+‐dependent release of a portion of the d‐ASP. Kainic acid (10‐5‐10‐3M) produced a dose‐dependent inhibition of d‐ASP uptake. The electrically evoked release of d‐ASP was increased 1.6–2.0 fold by 10‐5 and 10‐4M kainic acid. The kainate‐enlarged release was Ca2+‐dependent. Dihydrokainic acid, an analogue of kainic acid with little excitatory or toxic action, did not increase d‐ASP release but depressed d‐ASP uptake. Attempts were made to block the action of kainic acid with baclofen and pentobarbital, compounds which depress the electrically evoked release of l‐glutamate (l‐GLU) and l‐aspartate (l‐ASP). Baclofen (4 × 10‐6M), an antispastic drug, and pentobarbital (10‐4M), an anesthetic agent, each inhibited the electrically evoked release of d‐ASP and prevented the enhancement of the release above control levels usually produced by 10‐4M kainic acid. It is proposed that 10‐5 and 10‐4M kainic acid may enhance the synaptic release of l‐GLU and l‐ASP from neurons which use these amino acids as transmitters. This action is prevented by baclofen and pentobarbital. In view of the possibility that cell death in Huntington's disease could involve excessive depolarization of striatal and other cells by glutamate, baclofen might be effective in delaying the loss of neurons associated with this condition.
Isoguvacine Binding, Uptake, and Release: Relation to the GABA System
Authors: White W. F. , Snodgrass S. R.
Published: October 2006

Isoguvacine (1,2,3,6‐tetrahydropyridine‐4‐car‐boxylic acid) is a GABA (γ‐aminobutyric acid) agonist with limited conformational flexibility. In these studies we investigated the binding, uptake, and release of [3H] isoguvacine by use of tissue preparations of rat CNS, comparing the results with similar studies of [3H]GABA. The results from these investigations indicate that isoguvacine binds to membrane preparations of rat forebrain with pharmacological characteristics similar to the post‐synaptic GABA recognition site; that it is transported into synaptosomal preparations by an uptake system similar to the high‐affinity GABA uptake system; and that recently accumulated isoguvacine is released in a Ca2+‐dependent manner and by heteroexchange with external GABA. The ability of isoguvacine and γ‐hydroxybutyric acid to decrease the K+‐stimulated Ca2+‐dependent release process was also investigated. The results indicate that isoguvacine interactions have many of the biochemical features of GABA synaptic function, isoguvacine being, however, less potent than GABA.
Glucocorticoids Increase Catecholamine Synthesis and Storage in PC 12 Pheochromocytoma Cell Cultures
Authors: Tischler Arthur S. , Perlman Robert L. , Morse Gardiner M. , Sheard Beth E.
Published: October 2006

Abstract: Glucocorticoids, cholera toxin and high plating density all increase the activity of tyrosine 3‐monooxygenase (TH) in cultured PC12 pheochromocytoma cells. Glucocorticoids increase enzyme activity in cells treated with cholera toxin and in cells grown at high plating density. Glucocorticoids also increase the content of stored catecholamines in the cells. In cells cultured under routine conditions, glucocorticoids primarily increase the stores of dopamine. The addition of ascorbate to the culture medium increases the storage of norepinephrine in both steroid‐treated and untreated cells. Incubation of the cells in media containing 56 nM K+ causes the release of the same percentage of stored dopamine from steroid‐treated as from untreated cells. Steroid‐treated cells contain more dopamine than do untreated cells and therefore, in response to high K+, the steroid‐treated cells secrete more dopamine than do untreated cells. We conclude that the activity of tyrosine 3‐monooxygenase in PC12 cells can be regulated by several distinct mechanisms; that glucocorticoids cause a coordinate increase in TH activity and in catecholamine storage; that steroids increase the storage of catecholamines in a releasable pool; and that the steroid‐induced increase in catecholamine storage may result in increased secretion of catecholamines from steroid‐treated cells.
Presynaptic Cholinergic Dysfunction in Patients with Dementia
Authors: Sims N. R. , Bowen D. M. , Allen S. J. , Smith C. C. T. , Neary D. , Thomas D. J. , Davison A. N.
Published: October 2006

Abstract: Indices of presynaptic cholinergic nerve endings were assayed in neocortical biopsy samples from patients with presenile dementia. For those patients in whom Alzheimer's disease was histologically confirmed, [14C]acetylcholine synthesis, choline acetyltransferase activity and choline uptake were all found to be markedly reduced (at least 40%) below mean control values. The changes occurred in samples from both the frontal and temporal lobes and for [14C]acetylcholine synthesis the decrease was similar under conditions of high and low neuronal activity (as assessed by incubations in 31 mM and 5 mM K+ respectively). Samples from other demented patients, in whom the histological features of Alzheimer's disease were not detected, produced values for all three biochemical parameters which were similar to controls. For the total group of patients with presenile dementia there were correlations between values for the three markers of presynaptic cholinergic nerve endings suggestive of a loss of functional activity at these sites in Alzheimer's disease.
The Suppression of Stimulus‐Evoked Release of Amino Acid Neurotransmitters from Synaptosomes by Verapamil
Authors: Norris P. J. , Dhaliwal D. K. , Druce D. P. , Bradford H. F.
Published: October 2006

Abstract: Verapamil, at 200 μM, prevented the respiratory stimulation, K+ loss, transmitter release and 45Ca2+ entry into incubated synaptosomes evoked by veratrine (25 to 75 μM) or by high K+ (56 mM). Verapamil (100 μM) also blocked γ‐aminobutyric acid homoexchange, whilst tetrodotoxin was ineffective. Much lower concentrations of verapamil (< 1 μM) blocked the 45Ca2+ entry caused by veratrine, but not its action in releasing neurotransmitter or K+. It is concluded that verapamil, at 30 to 200 μM, blocks active Na+ channels, thereby preventing depolarization. At >1 μM, verapamil blocks Ca+ channels selectively.
Multiple Molecular Forms of Acetylcholinesterase in the Nematode Caenorhabditis elegans
Authors: Johnson Carl D. , Russell Richard L.
Published: October 2006

Abstract: Extracts of the nematode Caenorhabditis elegans contain five molecular forms of acetylcholinesterase (AChE) activity that can be separated by a combination of selective solubilization, velocity sedimentation, and ion‐exchange chromatography. These are called form IA (5.2s), form IB (4.9.s), form II (6.7s), form III (11.3s), and form IV (13.0s). All except form III are present in significant amounts in rapidly prepared extracts and are probably native; form III is probably derived autolytically from form IV. Most of forms IA and IB can be solubilized by repeated extractions without detergent, whereas forms II, III, and IV require detergent for effective solubilization and may therefore be membrane‐bound. High salt concentrations are not required for, and do not aid in, the solubilization of these forms. For all forms, molecular weights and frictional ratios have been estimated by a combination of gel permeation chromatography and velocity sedimentations in both H2O and D2O. The molecular weight estimates range from 83,000 to 357,000 and only form II shows extensive asymmetry. The separated forms have been characterized with respect to substrate affinity, substrate specificity, inhibitor sensitivity, thermal inactivation, and detergent sensitivity. Judging by these properties, C. elegans is like other invertebrates in that none of its cholinesterase forms resembles either the “true” or the “pseudo” cholinesterase of vertebrates. However, internal comparison of the C. elegans forms clearly distinguishes forms IA, III, and IV as a group from forms IB and II; the former are therefore designated “class A” forms, the latter “class B” forms. Genetic evidence indicates that separate genes control class A and class B forms, and that these two classes overlap functionally. Several factors, including kinetic properties, molecular asymmetry, molecular size, and solubility, all suggest that a molecular model of the multiple cholinesterase forms observed in vertebrate electric organs probably does not apply in C. elegans. Potential functional roles and subunit structures of the multiple AChE forms within each C. elegans class are discussed.
Vasoactive Intestinal Peptide Stimulation of Pineal Serotonin‐N‐Acetyltransferase Activity: General Characteristics
Authors: Yuwiler Arthur
Published: October 2006

Abstract: Vasoactive intestinal polypeptide (VIP) stimulates basal serotonin‐N‐acetyltransferase (NAT) activity in pineals in organ culture and enhances the effects of catecholamines in inducing the enzyme. VIP appears to act postsynaptically; its action is independent of the beta receptor and is dependent upon protein synthesis. Its effects may be mediated by a receptor. The magnitude of the pineal response to VIP varies with age, is greater in pineals maintained in 48‐h organ culture than in those in acute culture, and can be detected in pineals from newborns after 48‐h organ culture. Intravenous administration of VIP can increase pineal NAT activity in vivo.
Retina Maturation Following Administration of Thyroxine in Developing Rats: Effects on Polyamine Metabolism and Glutamate Decarboxylase
Authors: Macaione Salvatore , Di Giorgio Rosa M. , Nicotina Piero A. , Ientile Riccardo
Published: October 2006

Abstract: The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and γ‐aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9–12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S‐Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15‐fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.