bluuebunny/crossref_metadata_2025 huggingface dataset:
A Monoclonal Antibody to Rabbit Brain GABA Transaminase
Authors: Edwardson J. Michael , Phillips Nicholas I. , Kirby Neil , Fowler Leslie J.
Published: October 2006

A monoclonal antibody of class IgG (subclass IgG1) has been prepared to rabbit brain GABA transaminase (GABA‐T). This antibody reveals a single band of molecular weight 52,000 on a nitrocellulose filter blotted with purified GABA‐T. On a filter blotted with unfractionated rabbit brain supernatant a major band of molecular weight 58,000 is revealed. An immunoaffinity column was prepared by coupling proteins from ascites fluid containing anti‐rabbit GABA‐T antibody to Bio‐Rad Affi‐Gel 15. This column bound purified GABA‐T and extracted from unfractionated rabbit brain supernatant a protein of molecular weight 58,000, which was almost homogeneous and which had GABA‐T enzyme activity. Using immunoaffinity chromatography, therefore, a high degree of purification of GABA‐T may be achieved in a single step. Further, this technique may preserve an authentic form of the enzyme that is lost during the conventional purification procedure. The antibody inhibits GABA‐T enzyme activity, up to a maximum of 35%.
Methylation of Chromosomal Proteins in Neuronal and Glial Nuclei Purified from Cerebral Hemispheres of Rat During Postnatal Development
Authors: Serra I. , Avola R. , Lombardo B. , Kamiyama M. , Hashim G. A. , Giuffrida A. M.
Published: October 2006

The process of methylation of chromosomal proteins [histones and nonhistone proteins (NHP)] in neuronal and glial cell nuclei obtained from cerebral hemispheres of rats at 1, 10, and 30 days of age was investigated. Purified neuronal and glial nuclei were incubated in the presence of S‐adensyl[methyl‐3H]methi‐onine. Histone and NHPs were extracted and fractionated by polyacrylamide gel electrophoresis. The results obtained indicate remarkable differences in the process of methylation of histones and NHPs between neuronal and glial nuclei, especially during the first period of postnatal development. In both nuclear populations the histone fraction H3 was labeled to a greater degree than the other fractions and showed the major changes during postnatal development. The densitometric and radioactive patterns of NHPs show considerable changes in the two nuclear populations at the various ages examined. The main difference between neuronal and glial nuclei consists in the intense methylation of proteins with a molecular weight of approximately 100,000, which are present in neuronal nuclei and virtually absent in glial ones. The results obtained may be correlated with the different chromatin structures of neuronal and glial nuclei and with the patterns of maturation and differentiation of neuronal and glial cells during postnatal development.
Uptake and Anterograde Axonal Transport of Aleuria Lectin in Retinal Ganglion Cells of the Rabbit
Authors: Ohlson C. , Nilsson E. , Karlsson J.‐O.
Published: October 2006

A fucose‐specific lectin from Aleuria aurantia was used to study the dynamics of neuronal membrane glycoproteins. Albino rabbits received vitreal injections of affinity‐purified 125I‐Aleuria lectin. The radioactive probe was internalized by adsorptive endocytosis into retinal ganglion cells, and transported intact down to the nerve terminals in the contralateral geniculate bodies and superior colliculi. We found that the radiolabeled lectin was transported with at least two distinct rates (I, $205 mm/day; II, $45 mm/day) corresponding to the two rapid phases of anterograde transport of endogenous polypeptides described earlier in this system. This is the first evidence that an exogenous macromolecule may be transported along the axon at more than one velocity.
Binding Sites for l‐[3H]Glutamate on Hippocampal Synaptic Membranes: Three Populations Differentially Affected by Chloride and Calcium Ions
Authors: Nadler J. Victor , Wang Andrew , Werling Linda L.
Published: October 2006

The effects of Cl− and Ca2+ were studied on the specific binding of l‐[3H]glutamate to multiple sites on rat hippocampal synaptic membranes. Quisqualate (5 μM) or dl‐2‐amino‐4‐phosphonobutyrate (2‐APB) (300 μM) was used to discriminate two previously identified classes of binding sites. Saturation isotherms and displacement curves constructed under different ionic conditions suggested that the effects of Cl− and Ca2+ could best be explained by postulating the existence of three major binding site populations in this preparation rather than two. The binding of l‐glutamate to Glu A sites exhibits an absolute dependence on Cl−, and Ca2+ markedly increases the maximum density of these sites. Glu A sites bind quisqualate and 2‐APB with relatively high affinity. Cl− (47 μM) more than doubles the maximum density of Glu B sites, but Ca2+ appears to have no effect. Glu B sites can be discriminated from the other classes by their relatively low affinity for quisqualate and 2‐APB. There is reason to think that the Glu B population is heterogeneous. The novel Glu C population can be virtually selectively labeled by exposing 2‐APB‐sensitive binding sites to radioligand in Tris‐HOAc buffer with Ca2+. Binding of l‐[3H]glutamate to these sites is enhanced by both Cl− and Ca2+, but requires neither ion. Ca2+ appears to increase both the affinity of Glu C sites for l‐glutamate and their maximum binding site density. In the presence of Ca2+ and Cl−, Glu C sites bind the radioligand with micromolar affinity (KD > 2 μM) and high capacity (Bmax > 160 pmol/mg protein). The structural specificity of these binding sites closely resembles that of Glu A sites, but these sites bind excitants with about an order of magnitude lower affinity. The Glu C binding sites exhibit characteristics compatible with a synaptic receptor function in the hippocampal formation.
Subcellular Distribution and Developmental Expression of Cholesterol Ester Hydrolases in Fetal Rat Brain Cultures
Authors: Bhat Shama , Pfeiffer S. E.
Published: October 2006

Abstract: Cholesterol ester hydrolase activities previously have been identified in brain and linked to the production of myelin, which has very low levels of esterified cholesterol. We have studied two cholesterol ester hydrolase activities (termed the pH 6.0 and pH 7.2 activities) in cultures derived from 19‐ to 21‐day‐old dissociated fetal rat brains and in developing rat brain. In vivo the levels of both the pH 6.0 and pH 7.2 activities began to increase by about 10 postnatal days, reached maximal levels at 20 days (20 and 1.5 nmol/h/mg protein, respectively), and thereafter remained nearly constant (pH 6.0) or decreased somewhat before becoming constant (pH 7.2). In contrast, in the cultures the pH 6.0 cholesterol ester hydrolase activity was low until 21 days in culture (DIC; 20 nmol/h/mg protein), increased to a peak activity at 31 DIC (60 nmol/h/mg protein), remained high for 24 days, and finally decreased (18 nmol/h/mg protein at 63 DIC); the pH 7.2 cholesterol ester hydrolase activity was very low until 20 DIC, increased to a peak activity at 31 days (3 nmol/h/mg protein), and thereafter decreased to a lower level (2 nmol/h/mg protein) that was maintained for about 24 days before decreasing (0.7 nmol/h/mg protein at 63 DIC). Therefore, (a) the time courses of appearance of both cholesterol ester hydrolase activities were delayed by 10–14 days relative to that seen in vivo, and (b) the specific activities observed in the cultures were transiently two‐ to three‐fold higher than in rat brain, but then declined to levels characteristic of whole brain homogenates. Subcellular fractionation of the cultures demonstrated that the pH 7.2 cholesterol ester hydrolase activity, along with myelin basic protein and 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase activity, was enriched in a membrane fraction collected at an interface between 0.32 M and 0.9 M sucrose; the pH 6.0 cholesterol ester hydrolase activity, in contrast, was enriched in the microsomal fraction.
Palmitoyl‐CoA Elongation in Brain Microsomes: Dependence on Cytochrome b5 and NADH‐Cytochrome b5 Reductase
Authors: Takeshita Masazumi , Tamura Minoru , Yoshida Satoshi , Yubisui Toshitsugu
Published: October 2006

Abstract: Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p‐chloromercuriphenylsulfonate, inhibitors on NADH‐cytochrome b5 reductase, at 32 μM inhibited NADH‐supported palmitoyl‐CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH‐supported palmitoyl‐CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH‐cytochrome b5 reductase inhibited brain microsomal NADH‐cytochrome b5 reductase activity and NADH‐dependent palmitoyl‐CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH‐ and NADPH‐cytochrome c reductase activities were significantly decreased, but the decrease in NADH‐cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl‐CoA into palmitoyl‐CoA was observed with trypsintreated microsomes, addition of detergent‐solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH‐NADH‐cytochrome b5 reductase–cytochrome b5‐fatty acid elongation, in brain microsomes.
Partial Restoration of Cerebral Myelination of the Congenitally Hypothyroid Mouse by Parenteral or Breast Milk Administration of Thyroxine
Authors: Noguchi Tetsuya , Sugisaki Tetsuro , Satoh Isamu , Kudo Motoshige
Published: October 2006

Abstract: We attempted to define whether thyroid hormone can ameliorate the cerebral hypomyelination present in the congenitally hypothyroid (hyt) neonatal mouse, and to define the critical time period during early postnatal life when thyroxine (T4) is essential for myelin formation. We administered T4) to the hyt mouse by breast milk during the first 20 days of postnatal life, and through the diet during the second 20 days of postnatal life. Positive results were obtained only when hormone was given during the first 20 days of postnatal life. A distinct increase in cerebral 2′,3′‐cyclic nucleotide 3′‐phosphohy drolase activity was noted, and brain sections stained for myelin basic protein correlated with the biochemical findings. The later administration of hormone through diet was ineffective.
Transglutaminase Activity in Rat Brain: Characterization, Distribution, and Changes with Age
Authors: Gilad Gad M. , Varon Larry E.
Published: October 2006

Abstract: The activity of transglutaminase was characterized in the rat brain. In adults, comparable levels of transglutaminase activity are present in all brain regions examined. The activity is present in all subcellular fractions, as studied by differential centrifugation, but the soluble fraction contains the highest specific activity. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate casein) is very low in all subcellular fractions, except in the synaptosomal fraction where its highest levels are about 40–60% of the activity assayed in the presence of casein. Furthermore, enzyme activity is present on the external surface of synaptosomes. In the soluble fraction, maximal activity can be detected between pH values of 9 and 10 when assayed in the presence of 5 mM CaCl2 (with half‐maximal activity requiring 0.75 mM CaCl2) and 0.4 mM putrescine (with an apparent Km for putrescine of 0.1 mM). The activity can be partially inhibited by ZnCl2 (with an IC50 of 4.5 mM) and by AlCl3 (with an IC50 of 5.1 mM). In the cerebellum, where the full span of neuronal development can be studied after birth, the highest specific activity is observed just after birth, thereafter the activity starts to decline and by 14 days, after a reduction of about 65%, it reaches levels observed throughout life.
Characterization of an Insulin Receptor in Human Y79 Retinoblastoma Cells
Authors: Yorek Mark A. , Spector Arthur A. , Ginsberg Barry H.
Published: October 2006

Abstract: Cultured human Y79 retinoblastoma cells bind [125I]iodoinsulin in a manner similar to that of other CNS and peripheral tissues. The only difference noted between the insulin binding properties of the Y79 cells and other CNS preparations is that insulin binding to Y79 cells is down‐regulated by prolonged exposure of the cells to insulin. By contrast, studies with the various brain preparations indicate that the brain insulin receptor is not down‐regulated by circulating levels of insulin. Insulin binding to Y79 cells exhibits negative cooperativity, has a pH optimum of 7.8, is responsive to cations, and gives a curvilinear Scatchard plot. Y79 cell insulin binding capacity is 26 fmol/100 μg of cell protein, corresponding to about 125,000 binding sites per cell. These findings are the first to report insulin binding in a human cell line of retinal origin. The characterization of the insulin binding in this cell line may facilitate an understanding of the relationship between insulin and its specific functions in the human retina.
Neonatal Steroid Treatment Reduces Catecholamine‐Induced Increases in Pineal Serotonin N‐Acetyltransferase Activity
Authors: Yuwiler A.
Published: October 2006

Abstract: Hydrocortisone acetate given to the neonatal rat diminishes subsequent elevations in pineal serotonin N‐acetyltransferase (acetyl‐coenzyme A:arylamine N‐acetyltransferase; EC 2.3.1.5; NAT) activity produced by administration of catecholamines to the intact animal or to pineals in organ culture. The time required for development of this decrease in sensitivity varies inversely with age at treatment. A minimal dose of 200 μg of hydrocortisone acetate/rat is required to elicit this decreased response to agonist. Other glucocorticoids have qualitative effects similar to hydrocortisone acetate, but cholesterol and the gonadal steroids testosterone, estradiol, and progesterone are without effect. In addition to showing a smaller rise in NAT activity on stimulation, pineals from steroid‐treated neonates also synthesize less N‐acetylserotonin and melatonin from tryptophan. The decrease in NAT response to stimulation after steroid treatment appears due to actions beyond cyclic AMP generation and may involve inhibition of protein synthesis.
Effects of Repeated Administration of Estradiol Benzoate on Tubero‐Infundibular GABAergic Activity in Male Rats
Authors: Nicoletti Ferdinando , Grandison Lindsey , Meek James L.
Published: October 2006

Abstract: Repeated (once a day for 8 days) but not single administration of estradiol benzoate (10 μg/kg, s.c.) induced a sevenfold increase in anterior pituitary γ‐aminobutyric acid (GABA) concentration in male rats. GABA concentration also increased in the median eminence whereas no changes or decreases were observed in other brain regions including hypothalamic arcuate nucleus, lateral septum, hippocampus, caudate nucleus, and substantia nigra. Eight‐day estradiol benzoate injection also enhanced the Vmax of median eminence glutamate decarboxylase activity without affecting the Km of the enzyme for glutamic acid. Taken together, these results suggest that repeated administration of estradiol benzoate increases the activity of the tubero‐infundibular GABAergic system in male rats.
Metabolism of [3H]Nipecotic Acid in the Rabbit Retina
Authors: Jr. Paul Madtes , Redburn Dianna
Published: October 2006

Abstract: Nipecotic acid has been demonstrated to block the γ‐aminobutyric acid transport systems. Previous studies have shown that the uptake system is the first transmitter‐specific parameter to appear during the development of the rabbit retina. Use of these observations has been made to study the influence on the development of γ‐aminobutyric acid receptors of altering the uptake mechanism by treating newborn pups with nipecotic acid to block GABA transport. The present study of the in vivo metabolism of [3H]nipecotic acid in the CNS measured the changes in the levels of [3H]nipecotic acid in both adult and newborn rabbit retinas after injection of the label into the vitreal chamber. It was found that the effective half‐life of [3H]nipecotic acid in the vitreous is about 5 h for adult tissue and 3 h for newborn. In contrast, all retinal fractions retained the label longer, the effective half‐lives being about 60 h (adult) and 45 h (newborn). Further, no labeled metabolites of nipecotic acid were detected in either adult or newborn tissue. This study gives evidence that the degradation of nipecotic acid in nervous tissue is minimal and suggests that, although the rate of clearance is faster in neonates, the fate of nipecotic acid in vivo may be similar in both adult and newborn tissues.
Proteolytic Digestion Patterns of “Soluble”and “Detergent‐Soluble”Bovine Caudate Nucleus Acetylcholinesterases
Authors: Marsh David J. , Massoulié Jean
Published: October 2006

Abstract: The structures of purified “soluble”and “detergent‐soluble”bovine caudate nucleus acetylcholinesterases were compared by peptide mapping on polyacrylamide gels. The digestion products generated from the two acetylcholinesterases on proteolysis by a given protease (Staphylococcus aureus V8 protease, α‐chymotrypsin, or papain) are remarkably similar as judged from the electrophoretic band patterns. We conclude that the “soluble”and “detergent‐soluble”acetylcholinesterases from bovine caudate nucleus share a common evolutionary origin.
Regional Distribution of Calmodulin Activity in Rat Brain
Authors: Zhou L‐W. , Moyer J. A. , Muth E. A. , Clark B. , Palkovits M. , Weiss B.
Published: October 2006

Abstract: Calmodulin activity in 68 discrete areas of rat brain, obtained by micropunch technique, was assessed by its capacity to activate a calmodulin‐sensitive form of phosphodiesterase. In general, the activity of calmodulin was higher in the telencephalon, limbic system, and hypothalamus than in the mesencephalon, pons, cerebellum, and medulla. However, there were substantial differences in calmodulin activity in discrete nuclei of each region. The regional distribution of calmodulin activity in rat brain does not appear to correlate with that of any of the known putative neurotransmitters or peptides.
Leptinotoxin‐h Action in Synaptosomes, Neurosecretory Cells, and Artificial Membranes: Stimulation of Ion Fluxes
Authors: Madeddu Luisa , Pozzan Tullio , Robello Mauro , Rolandi Ranieri , Hsiao Ting H. , Meldolesi Jacopo
Published: October 2006

Abstract: Leptinotoxin‐h (LPTx), a neurotoxin (otherwise designated β‐leptinotarsin‐h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry23, 734–741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+‐containing Ringer medium, at concentrations in the 10−11‐10−10M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage‐dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx: and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+‐free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono‐ and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of α‐latrotoxin (α‐LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit α‐LTx binding.
Maternal Undernutrition During Lactation: Effect on Amino Acids in Brain Regions of Offspring
Authors: Rathbun Wayne E. , Druse Mary J.
Published: October 2006

Abstract: Sprague‐Dawley dams were fed either a protein‐calorie deficient or control diet from day 5 to day 21 after parturition. The concentrations of seven amino acids (aspartate, glutamate, γ‐aminobutyric acid, glycine, glutamine, serine, and taurine) were determined in brain regions from 17‐day‐old undernourished offspring and from 35‐day‐old rehabilitated rats. The brain regions examined were the cortex, cerebellum, corpus striatum, hippocampus, hypothalamus, brainstem, and midbrain. At 17 days of age, taurine was the amino acid with the highest concentration, whereas at 35 days glutamate had the highest concentration. This change was due to the fact that the concentration of taurine decreased significantly in all brain regions between 17 and 35 days, whereas the concentration of glutamate remained high or increased somewhat in all brain regions except the hypothalamus and brainstem. When the age‐matched offspring of control and undernourished rats were compared, several interesting and significant differences were found. The concentrations of glutamate and aspartate were significantly lower (decreased 16–34%) in the cerebellum, brainstem, cortex, and midbrain in 17‐day‐old undernourished rats. The aspartate level was also significantly decreased in the corpus striatum and hypothalamus in 17‐day‐old offspring. However, the deficiencies of aspartate and glutamate were transient and reversible. In contrast, the concentration of taurine was increased in the hypothalamus (31%) and hippocampus (12–33%) at both 17 and 35 days of age and in the midbrain (17%) at 17 days. Other transient abnormalities in amino acid levels were found in undernourished offspring. The results of these experiments suggest that undernutrition during lactation causes delayed CNS development, which is manifested in altered concentrations of the neurotransmitters aspartate, glutamate, and taurine.
Down‐Regulation of [3H]5‐Hydroxytryptamine Binding Sites in Chick Embryo Brain by Monoamine Oxidase Inhibitors or Fenfluramine and Potentiation by d,l‐5‐Hydroxytryptophan
Authors: Soblosky Joseph S. , DuMontier Gary , Jeng Ingming
Published: October 2006

Abstract: Three monoamine oxidase (MAO) inhibitors—pargyline, clorgyline and deprenyl—as well as the serotonin (5‐HT, agent hydroxytryptamine) releasing agent fenfluramine were administered to developing chick embryos and the effects on [3H]5‐HT binding parameters and endogenous 5‐HT levels were assessed. Multiple, but not acute, pretreatments with any of the three MAO inhibitors significantly increased 5‐HT levels (p < 0.01) and decreased receptor number (Bmax) to a maximum of 20% (p < 0.01) without affecting the affinity (KD). When d,l‐5‐hydroxytryptophan (d,l‐5‐HTP) was similarly administered there were large increases in 5‐HT levels (p < 0.01), but no significant effects on either Bmax or KD. However, if d,l‐5‐HTP was co‐administered with any of the MAO inhibitors there was a significant (p < 0.01) enhancement of the MAO inhibitor‐induced down‐regulation to a maximum of about 40%. Multiple pretreatments with fenfluramine resulted in dose‐related decreases in 5‐HT levels (p < 0.01) and Bmax (p < 0.01) without affecting KD. The largest decrease in [3H]5‐HT binding sites inducible by fenfluramine treatment alone was also about 40%. When given in combination with d,l‐5‐HTP, there was a potentiation of the down‐regulation capabilities of fenfluramine at several different dosage levels; however, maximal down‐regulation was also limited to 40%. Evidence was presented suggesting that these effects were not due to endogenous 5‐HT or drugs remaining in the tissue preparation. The overall evidence implies that merely increasing endogenous 5‐HT levels, as by precursor administration, does not necessarily induce down‐regulation unless the 5‐HT is also made available as functional 5‐HT.
Identification of Dopamine “D3′’ and “D4′’ Binding Sites, Labelled with [3H]2‐Amino‐6,7‐Dihydroxy‐ 1,2,3,4‐ Tetrahydronaphthalene, as High Agonist Affinity States of the D1 and D2 Dopamine Receptors, Respectively
Authors: Urwyler Stephan , Markstein Rudolf
Published: October 2006

Abstract: On the basis of affinity differences for spiperone, two binding sites for [3H](±)‐2‐amino‐6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene ([3H]ADTN) in the rat brain could be distinguished: “D3′’ with a low and “D4′’ with a high affinity for spiperone. Evidence is provided that D3 and D4 sites are related to high agonist affinity states of the D1 and D2 dopamine receptors, respectively. Various well‐known selective D1 and D2 agonists and antagonists showed potencies at these sites in agreement with this hypothesis. A comparison of the Bmax values for [3H]ADTN binding to D3 and D4 sites with the numbers of D1 receptors (labelled by [3H]SCH 23390) and of D2 receptors (labelled by [3H]spiperone), both in the striatum and in the mesolimbic system, indicated that under the conditions used for 3H‐agonist binding experiments, both populations of D1 and D2 receptors were converted to their high agonist affinity states to a considerable, although different extent. In fact, when competition experiments with [3H]spiperone were performed under the conditions otherwise used for [3H]ADTN binding experiments (instead of the conditions usually used for antagonist binding), substantial shifts of the displacement curves of 3,4‐dihydroxyphenylethylamine (dopamine) and ADTN toward higher affinities were observed. A comparison of the effects of various agonists and antagonists in the [3H]ADTN binding experiments and in functional tests revealed a significant correlation between their potencies at D4 binding sites and at D2 receptors modulating the release of [3H]acetylcholine from striatal slices. However, in the situation of the D1/D3 pair, when the measurement of adenylate cyclase activity was taken as a functional test for D1 receptors, agonists were more active in the binding than in the functional test, whereas for many antagonists the opposite was found. The results are discussed with regard to the classification and functional aspects of brain dopamine receptors.
A Study of the Cerebral Cortex Cholecystokinin Receptor Using Two Radiolabelled Probes: Evidence for a Common CCK 8 and CCK 4 Cholecystokinin Receptor Binding Site
Authors: Clark C. R. , Daum P. , Hughes J.
Published: October 2006

Abstract: This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [125I] Bolton‐Hunter (BH) CCK 8 and [3H]pentagastrin (Boc‐β‐Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (KD) of 1.9 nM and 1.4 nM for [3H]pentagastrin and [125I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one‐site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C‐terminal fragment CCK 4 bind to a single class of high‐affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity.
Developmental Pattern for Phosphatidylserine Decarboxylase in Rat Brain
Authors: White Frances V. , Toews Arrel D. , Morell Pierre
Published: October 2006

Abstract: In adult rats, a significant portion of brain ethanolamine glycerophospholipids are synthesized by a pathway involving phosphatidylserine decarboxylase, a mitochondrial enzyme. We have now examined whether this enzyme plays a particularly prominent role during development. Activities for both phosphatidylserine decarboxylase and succinate dehydrogenase (another mitochondrial enzyme) were determined in brain homogenates from rats 5 days of age to adulthood. Succinate dehydrogenase activity, expressed on a per unit brain protein basis, increased markedly during development. This pattern has been reported previously and is as expected from the postnatal increase in oxidative metabolism. In contrast, phosphatidylserine decarboxylase activity decreased 40% from 5 to 30 days of age. The apparent Km for brain phosphatidylserine decarboxylase was 85 μM in both young (8‐ and 20‐day‐old) and adult animals. Parallel studies in vivo were carried out to determine the contribution of the phosphatidylserine decarboxylase pathway, relative to pathways utilizing ethanolamine directly, to the synthesis of brain ethanolamine glycerophospholipids. Animals were injected intracranially with a mixture of L‐[G‐3H]serine and [2‐14C]ethanolamine and incorporation into the base moieties of the phospholipids determined. The 3H/14C ratio of ethanolamine glycerophospholipids decreased about 50% during development. Our studies in vitro and in vivo both suggest that phosphatidylserine decarboxylase plays a significant role in the synthesis of brain ethanolamine glycerophospholipids at all ages, although it is relatively more prominent early in development.
Effects of Hypothermia on the In Vivo Measurement of Rapid Axonal Transport in the Rat: A Cautionary Note
Authors: Padilla Stephanie , Lyerly Donald
Published: October 2006

Abstract: Rapid axonal transport of glycoproteins was examined in the retinofugal projections of hypothermic and normothermic adult male Long‐Evans hooded rats previously receiving intraocular injections of [3H]fucose. The amount of retinal fucosylation appeared normal in the hypothermic animals 3.5 h after isotope injection, but glycoprotein transport was reduced relative to normothermic controls. This reduction was especially pronounced in the most distal structure of the retinofugal tract (superior colliculus). We conclude that rapid axonal transport decreases with reductions in mammalian body temperature. This finding emphasizes the importance of controlling body temperature in in vivo studies of mammalian axonal transport.
Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures
Authors: Langan Thomas J. , Volpe Joseph J.
Published: October 2006

Abstract: Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3‐hydroxy‐3‐methylglutaryl‐CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24‐h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.
Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum
Authors: Cross Alan , Skan Wendy , Slater Paul
Published: October 2006

Abstract The binding of [3H]aspartate and [3H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [3H]glutamate binding was to sites similar to those labelled by [3H]aspartate. An additional component of [3H]gluta‐mate binding (about 50%) was displaced by quisqualate and aL‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid, and may represent a “quisqualate‐preferring” receptor. Neither N‐methyl‐d‐aspartic acid‐sensitive nor dl‐2‐amino‐4‐phosphonobutyric acid‐sensitive [3H]glutamate binding was detected.
Cyclic GMP Formation and Inositol Phosphate Accumulation Do Not Share Common Origins in Rat Brain Slices
Authors: Kendall David A.
Published: October 2006

Abstract Cyclic GMP formation and inositol phospholipid hydrolysis were studied in rat brain slices to determine if the two processes have common origins. Muscarinic choliner‐gic stimulation enhanced [3H]inositol phosphate ([3H]IP) accumulation from slices prelabelled with [3H]inositol but did not affect cyclic GMP formation in the cortex, striatum, or cerebellum. An elevated level of extracellular K+ stimulated accumulation of both cyclic GMP and [3H]IP in cortex slices. The former, but not the latter, was reduced by lipoxy‐genase and phospholipase A2 inhibition. Calcium channel activation enhanced and blockade reduced K+‐stimulated [3H]IP formation without affecting the cyclic GMP level, and there were differences in the Ca2+ requirements for the two responses. Thus, there is no support for the concept that guanylate cyclase activation inevitably accompanies inositol phospholipid breakdown, and the evidence presented demonstrates that K+ stimulation promotes cyclic GMP and [3H]IP accumulation by different transducing pathways.
Characterization of High‐Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate‐Solubilized Bovine Striatal Preparations
Authors: Kazmi Syed M. I. , Ramwani Jai , Srivastava Lalit K. , Rajakumar G. , Ross Gregory M. , Cullen Marjorie , Mishra Ram K.
Published: October 2006

Abstract 3,4‐Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid‐NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane‐bound receptors. Computer‐assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high‐ and low‐affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high‐affinity sites to low‐affinity sites for the agonists. The binding of the high‐affinity agonist [3H]N‐n‐propylnor‐apomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of gua‐nylyl‐imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl‐Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six‐ to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high‐affinity binding sites than the second peak. The solubilized preparation also showed high‐affinity binding of [35S]guanosine‐5′‐(γ‐thio)triphos‐phate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor‐guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor‐effector interactions.
Age‐Related Changes of Myelin Proteins in the Rat Peripheral Nervous System
Authors: Uchida Yoko , Tomonaga Masanori , Nomura Kohji
Published: October 2006

Abstract: Age‐related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS‐PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS‐PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.
Stimulation of Amino Acid Uptake and Na+, K+‐ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats
Authors: Ando Masato , Nagata Yutaka
Published: October 2006

Abstract: Active uptake of a labelled nonmetabolizable amino acid, α‐aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 μM) or 3,4‐dihydroxy‐phenylethylamine (dopamine, DA, 100 μM) to the medium during aerobic incubation for 2 h at 37°C. The NE‐induced increase in AIB uptake was significantly antagonized by the addition of an α1‐adrenoceptor antagonist (prazosin, 10 μM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE‐evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+,K+‐ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE‐induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+,K+‐ATPase.
Cell Cycle‐Dependent Modulation of Biosynthesis and Stimulus‐Evoked Release of Catecholamines in PC12 Pheochromocytoma Cells
Authors: Koike Tatsuro , Takashima Akihiko
Published: October 2006

Abstract: Catecholamine biosynthesis and its stimulusevoked release in PC12 pheochromocytoma cells were studied as a function of cell cycle by means of HPLC with electrochemical detection. We found that 3,4‐dihydroxyphenylethylamine (dopamine) levels in PC12 cells remained constant throughout the period of cell cycle. In contrast, the noradrenaline content was dependent on the cell cycle: it increased during the S + G2 phase followed by a decrease in the M phase. These results were confirmed further by measuring the activities catalyzing the catecholamine biosynthesis. Thus, activities of tyrosine 3‐monooxygenase and 3,4‐dihydroxyphenylalanine decarboxylase were independent of the cell cycle, whereas both soluble and membrane‐bound dopamine β‐monooxygenase activities were modulated during the cell cycle. On the other hand, release of the catecholamines stimulated with 50 mM KCI increased in the G1 phase, reached a maximum in the late G1, and then gradually decreased in later periods. We also found that carbamylcholine‐induced release of the catecholamines occurred maximally in the early S + G2 phase followed by a decrease during the M phase. Cell cycle dependence of the catecholamine release was in good agreement with that of 45Ca2+ uptake. Thus, this study provides evidence that the catecholamine biosynthesis and its release in PC12 cells are modulated during the period of cell cycle.
Peptide Uptake by Astroglia‐Rich Brain Cultures
Authors: Schulz Michael , Hamprecht Bernd , Kleinkauf Horst , Bauer Karl
Published: October 2006

Abstract: Uptake of carnosine has been investigated in as‐troglia‐rich primary cultures derived from brains of newborn mice. It could be demonstrated that carnosine is not degraded by these cells but rapidly taken up in an energy and sodium‐dependent process. Uptake and release of carnosine by these cells were found to be mediated by a saturable, high‐affinity transport system with apparent kinetic constants of Km=50 μMand Vmax= 22.7 nmol h1 mg protein1. Uptake of carnosine is strongly inhibited by other dipeptides as well as by various oligopeptides, e.g., Leu‐en‐kephalin. However, uptake of the radiolabeled tripeptide D Ala‐L‐Ala‐L‐Ala was not observed. Radiolabeled Leu‐en‐kephalin also did not accumulate intracellularly, even if degradation of the peptide was prevented by use of peptidase inhibitors. These results suggest that uptake of carnosine is catalyzed by a dipeptide‐specific transport system with broad substrate specificity. With neuronal cells in primary culture, uptake of carnosine or other peptides was not observed.
Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices
Authors: Routledge C. , Marsden C. A.
Published: October 2006

Abstract: The uptake of [3H]adrenaline and [3H]‐ noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high‐affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose‐dependent manner, but the selective dopamine and 5‐hydroxytryptamine up‐ take inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]‐ noradrenaline and by unlabelled noradrenaline with [3H] adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline‐containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high‐affinity sites on noradrenergic nerve terminals.
Angiotensinogen in Cerebrospinal Fluid Corresponds Chromatographically to the γ‐Form of Plasma Angiotensinogen
Authors: Moffett R. Bruce
Published: October 2006

Abstract: Angiotensinogen (Aogen) (CA 11002–13–14), the prohormone of the neuro‐ and vasoactive peptide angioten‐ sin II (Ang II) (CA 11128–99–7), is found in dog brain as well as in dog plasma. At 2–4 μg/ml CSF, Aogen comprises 1‐ 2% of the total protein in dog CSF. Immunopurified CSF and plasma Aogen were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and anion‐exchange HFLC. Two major (α‐ and β‐) forms and one minor (γ‐) form of Aogen were observed in dog plasma. The majority of Aogen in dog CSF was Chromatographically identical to the γ‐form of plasma Aogen; α‐ and β‐Aogen forms comprised <5% of the total CSF Aogen. The N‐terminal amino acid sequences of α‐, β‐, and γ‐Aogen identified these proteins as members of the Aogen family. The N‐terminal amino acid sequence of CSF γ‐Aogen was Asp‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐Phe‐His‐Leu‐Leu‐Val‐Tyr‐Ser‐Lys‐Ser‐ Ser‐(X)‐Glu‐. More basic than either a‐ or β‐Aogen, γ‐ Aogen was shown to be a glycoprotein with an apparent molecular weight (Mr) of 58,000. CSF [des Ang I]‐Aogen exhibited a greater anion‐exchange HPLC retention. CSF, however, contained only minor amounts of [des Ang I]‐ Aogen. These analyses have demonstrated that brain overwhelmingly releases one particular Aogen into the CSF; however, very little of this brain Aogen is utilized for the production of Ang I.
Interactions Among Lithium, Calcium, Diacylglycerides, and Phorbol Esters in the Regulation of Adrenocorticotropin Hormone Release from AtT‐20 Cells
Authors: Reisine Terry , Zatz Martin
Published: October 2006

Abstract: Interactions among lithium, calcium, and phorbolesters in the regulation of adrenocorticotropin hormone (ACTH) release were examined in a tumor cell line (AtT‐ 20) of the anterior pituitary. Lithium, which blocks the phosphatase that converts inositol phosphates (IPs) to inositol, increases the levels of IPs in these cells and stimulates ACTH release. This ion potentiates the ability of calcium, an activator of phospholipase C, to raise levels of IPs in these cells and to stimulate ACTH secretion. Pretreatment of AtT‐20 cells with calcium specifically abolishes the ACTH release response to lithium or calcium, a result suggesting that these secretagogues may act through a common mechanism to induce hormone secretion. Prior exposure of AtT‐ 20 cells to either lithium or calcium also attenuates the ACTH release induced by phorbol ester, an activator of protein kinase C. To examine the link among lithium, calcium, phosphatidylinositol (PI) turnover, and phorbol esterevoked ACTH secretion, AtT‐20 cells were treated with 1‐oleoyl‐2‐acetoyl‐ns‐3‐glycerol (OAG), an analogue of the diacylgylcerols that are formed by phospholipase C during PI metabolism and that also activate protein kinase C. OAG itself does not alter ACTH release or the levels of IPs in AtT‐ 20 cells. Pretreatment of AtT‐20 cells with OAG, however, selectively blocks the ACTH release response to lithium, calcium, or phorbol ester. Furthermore, such pretreatment reduced the ability of lithium to increase levels of IPs. The results suggest that one mechanism of action of lithium is to potentiate selectively an action of calcium, possibly the stimulation of phospholipase C activity. In addition, diacylglycerol, a product of phospholipase C activity, acts to feedback inhibit the secretory response to lithium, calcium, and phorbol esters.