bluuebunny/crossref_metadata_2025 huggingface dataset:
Catecholamine Secretion by Chemically Skinned Cultured Chromaffm Cells
Authors: Brooks Jack C. , Treml Suzanne
Published: October 2006

Abstract: The secretory system of intact chromaffin cells is not accessible to direct chemical manipulation because of the selective permeability of the plasmalemma. We have devised a simple procedure for chemically “skinning” (permeabilizing) cultured adrenal medullary chromaffin cells by brief exposure to the detergent saponin. This procedure disrupts the continuity of the plasmalemma, thus allowing us to bypass those aspects of the secretory process controlled by the cell membrane and giving direct access of exogenous substances to the cellular secretory machinery. We report here that the skinned cells retain a fully competent secretory mechanism dependent only on exogenous calcium and MgATP. Saponin treatment had no significant effect on the total catecholamine content of the cells. Secretion could be initiated by either MgATP or calcium as long as the other was present in the medium. Catecholamine and dopamine‐β‐hydroxylase release by the skinned cells was dependent on the calcium concentration of the medium. The ratio of secreted catecholamine and enzyme was similar to that of the cells, indicating that secretion occurred by an exocytotic mechanism. About half the total cellular content of the cytoplasmic enzyme lactic dehydrogenase was released during the permeabilization process and subsequent incubations, indicating plasmalemma permeability to molecules as large as protein. Calcium‐induced secretion was unaffected by several drugs known to affect catecholamines and granule function. Saponin treatment of chromaffin cells in culture appears to be a simple means for allowing access of exogenous substances to the cell's secretory machinery. Therefore, it offers the opportunity to use chemical treatments and perhaps specific antibodies to cellular components, to determine the role of these elements in the secretory process. These techniques should also be applicable to other cells known to secrete by an exocytotic mechanism.
Receptor‐Mediated Increases in Phosphatidylinositol Turnover in Neuron‐Like Cell Lines
Authors: Cohen Neri M. , Schmidt David M. , McGlennen Ronald C. , Klein William L.
Published: October 2006

Abstract: Muscarinic receptors found in the N IE‐115 mouse neuroblastoma cell line were tested for their ability to mediate stimulation of phosphatidylinositol (PI) turnover. This study was facilitated by the development of a new solvent system (acetone: butanol: acetic acid: water, 5: 5: 1: 1) for the rapid and consistent separation of PI by one‐dimensional thin‐layer chromatography. Cholinergic stimulation caused as much as a 680% increase in the incorporation of 32P into PI. Enhanced incorporation of 32P into PI could be measured as early as 4 min after stimulation began. By 20 min, the rate of incorporation by stimulated cells had decreased to that of unstimulated cells, indicating desensitization. The magnitude of the response was dependent on the extent of receptor occupancy and the response elicited by a saturating dose of carbamylcholine was blocked completely by 10−7 M at‐ropine, a specific muscarinic antagonist. Chronic stimulation, known to cause a loss of receptor binding sites, led to a 90% decrease in the maximum response even after a 40‐min withdrawal period. Replacement of Na+ ions in the medium with choline or K+ severely impaired the ability of the cells to incorporate added 32P into PI (90 and 50%, respectively). Removal of the putative second messenger Ca2+ for short periods of time by the addition of excess EGTA did not alter either basal or muscarinic‐stimulated PI turnover.
The Effects of Apomorphine upon Local Cerebral Glucose Utilization in Conscious Rats and in Rats Anesthetized with Chloral Hydrate
Authors: Grome John J. , McCulloch James
Published: October 2006

Abstract: The effects of the dopaminergic agonist apomorphine (1 mg‐kg−1i.v.) upon local cerebral glucose utilization in 43 anatomically discrete regions of the CNS were examined in conscious, lightly restrained rats and in rats anesthetized with chloral hydrate by means of the quantitative autoradiographic [14C]2‐deoxyglucose technique. In animals anesthetized with chloral hydrate, glucose utilization was reduced throughout all regions of the CNS from the levels observed in conscious animals, although the magnitude of the reductions in glucose use displayed considerable regional heterogeneity. With chloral hydrate anesthesia, the proportionately most marked reductions in glucose use (by 40–60% from conscious levels) were noted in primary auditory nuclei, thalmaic relay nuclei and neocortex and the least pronounced reductions in glucose use (by 15–25% from conscious levels) were observed in limbic areas, some motor relay nuclei and white matter. In conscious, lightly restrained rats, the administration of apomorphine (1 mg‐kg−1) effected significant increases in glucose utilization in 15 regions of the CNS (e.g., subthalamic nucleus, ventral thalamic nucleus, rostral neocortex, substantia nigra, pars reticulata) and significant reductions in glucose utilization in two regions of the CNS (lateral habenular nucleus and anterior cingulate cortex). In rats anesthetized with chloral hydrate, the effects of apomorphine upon local glucose utilization were less widespread and less marked than in conscious animals. In only two of the regions (the globus pallidus and septal nucleus), which displayed increased glucose use following apomorphine in conscious rats, were significant increases in local glucose utilization observed with this agent in chloral hydrate‐anesthetized rats. In the pars compacta of the substantia nigra, in which apomorphine increased glucose utilization in conscious animals, significant reductions in glucose utilization were observed following apomorphine in rats anesthetized with chloral hydrate. The profound effects of chloral hydrate anesthesia upon local cerebral glucose use and the modification by this anesthetic regime of the local metabolic responses to apomorphine, emphasize the difficulties which exist in the extrapolation of data from anesthetized animals to the conditions which prevail in the conscious animal.
Calelectrin, a Calcium‐Dependent Membrane‐Binding Protein Associated with Secretory Granules in Torpedo Cholinergic Electromotor Nerve Endings and Rat Adrenal Medulla
Authors: Walker J. H. , Obrocki J. , Südhof T. C.
Published: October 2006

Abstract: Calelectrin, a calcium‐dependent membrane‐binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine β‐hydroxylase, although the two antisera did not cross‐react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vescicles and also self aggregates in a calcium‐dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30–80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.
Differential Effects of Protein Malnutrition and Ascorbic Acid Deficiency on Histidine Metabolism in the Brains of Infant Nonhuman Primates
Authors: Enwonwu Cyril O. , Okolie Eugene E.
Published: October 2006

Abstract:Male infant nonhuman primates (M. nemes‐trina) born in captivity were used in the study. They were divided into three groups. The first group of three animals was fed a 20% casein diet and the second group of six monkeys received a 2.0% casein diet. The third group of four monkeys received a 20% casein diet totally devoid of ascorbic acid for 3.5 weeks before the diet was supplemented with ascorbic acid (20 mg/kg diet). All the diets were given to the animals in two daily rations of 100 g/animal. The monkeys fed a 2% casein diet failed to grow, and after about 3.5 months showed variable degrees of edema, hypoalbuminemia, evidence of psychomotor disturbance, depressed plasma levels of many essential amino acids, and other features consistent with the diagnosis of protein‐energy malnutrition. Examination of the brains revealed significant alterations in the levels of histidine (+ 172%) and homocarnosine (+ 146%) in comparison with the control well‐fed monkeys. Associated with the increase in brain histidine was a marked elevation of brain histamine level. Protein deficiency also led to poor brain retention of ascorbic acid but not to the same degree observed in the ascorbic acid‐deficient animals. The latter group of animals, after receiving their diet for about 8 months, demonstrated a modest elevation in the plasma levels of most amino acids in comparison with controls. Ascorbic acid deficiency elicited a significant reduction (p < 0.01) in brain level of histidine, with hardly any change in homocarnosine level. In addition, vitamin C deficiency produced elevation of brain histamine level comparable to findings in the protein‐energy‐deficient monkeys. The results suggested that protein deficiency raised brain histamine level mainly through increased availability of the precursor amino acid histidine, while defective degradation might account for the increased brain level of this amine in ascorbic acid‐deficient monkeys. Histamine has been proposed to have a predominantly depressant action on relevant neurons, and has also been shown to participate with other neuro‐transmitters in influencing the function of the pituitary gland by regulating release of the hypothalamic hormones into the portal vessels. The relevance of the findings of marked increases in brain histamine in experimental protein and ascorbic acid deficiencies to the behavioral and extensive endocrinological alterations seen in human malnutrition deserves some intensive investigation.
Cerebrovascular Endothelial Cell Culture: Metabolism and Synthesis of 5‐Hydroxytryptamine
Authors: Maruki C. , Spatz M. , Ueki Y. , Nagatsu I. , Bembry J.
Published: October 2006

Abstract: The presence of 5‐hydroxytryptamine was investigated in cultured and propagated Cerebrovascular endothelium using immunohistochemistry and high pressure liquid chromatography. These studies demonstrate that the endothelium has the ability to take up and metabolize 5‐hydroxytryptamine as well as to synthesize this amine from its precursor l‐tryptophan, thus providing evidence for extraneural synthesis of 5‐hydroxtryptamine in the central nervous system.
Hypomyelination in the Cerebrum of the Congenitally Hypothyroid Mouse (hyt)
Authors: Noguchi Tetsuya , Sugisaki Tetsuro
Published: October 2006

Abstract: 2′,3′‐Cyclic nucleotide 3′‐phosphohydrolase activity in the cerebrum of the inherited primary hypothyroid mouse (hyt/hyt) is reduced in comparison with the normal heterozygate (hyt/+). No differences were observed with regard to DNA and RNA content and the RNA/DNA ratio. The results of this study indicate that hypomyelination in the hypothyroid mouse is restricted to the cerebrum, and is not related to arrested glial proliferation.
Flunitrazepam Binding to Intact and Homogenized Astrocytes and Neurons in Primary Cultures
Authors: Bender Alexander S. , Hertz Leif
Published: October 2006

Abstract: [3H]Flunitrazepam binds to intact and homogenized mouse astrocytes and neurons in primary cultures. In intact cells, the binding is to a single, high‐affinity, saturable population of benzodiazepine binding sites with a KD of 7 nM and Bmax of 6,033 fmol/mg protein in astrocytic cells and a KD of 5 nM and Bmax of 924 fmol/mg protein in neurons. After homogenization, the Bmax values decrease drastically in both cell types, but most in astrocytes. The temperature and time dependency are different for the two cell types, with a faster association and dissociation in astrocytes than in neurons and a greater temperature sensitivity in the astrocytes. Moreover, flunitrazepam binding sites on neuronal and astrocytic cells have different pharmacological profiles. In intact astrocytic cells, Ro 5–4864 (K1= 4 nM) is the most potent displacing compound, followed by diazepam (K1= 6 nM) and clonazepam (K1= 600 nM). In intact neurons, the relative order of potency of these three compounds is different: diazepam (K1= 7 nM) is the most potent, followed by clonazepam (K1= 26 nM) and Ro 5–4864, which has little effect. After homogenization the potency of diazepam decreases. We conclude that both neuronal and astrocytic cells possess high‐affinity [3H]flunitrazepam binding sites. The pharmacological profile and kinetic characteristics differ between the two cell types and are further altered by homogenization.
Effect of Lipid Structure on the Capacity of Myelin Basic Protein to Alter Vesicle Properties: Potent Effects of Aliphatic Aldehydes in Promoting Basic Protein‐Induced Vesicle Aggregation
Authors: Epand Richard M. , Dell Karen , Surewicz Witold K. , Moscarello Mario A.
Published: October 2006

The capacity of myelin basic protein or of poly‐l‐lysine to promote leakage of carboxyfluorescein from vesicles or the aggregation of vesicles was studied. The vesicles were composed of phosphatidylcholine as the sole or major lipid component. Addition of 10% sphingomyelin, 10% phosphatidylglycerol, 10% egg or bovine brain phosphatidylethanolamine, or 30% dodecanal had relatively little effect on the extent of carboxyfluorescein release in the presence of either myelin basic protein or poly‐l‐lysine. In contrast with these results, the extent of vesicle aggregation was very sensitive to lipid composition. Addition of 10% phosphatidylglycerol induced more aggregation than the other phospholipids tested. Admixing 10% of a partially degraded sample of bovine brain phosphatidylethanolamine also led to a large amount of aggregation induced by the myelin basic protein. This latter aggregation appeared more specific for the basic protein, as it occurred to a much smaller extent with poly‐l‐lysine. In general, the effects of the myelin basic protein on either carboxyfluorescein release or vesicle aggregation were similar to, although somewhat greater than, that of poly‐l‐lysine. The aggregation of vesicles containing degradation products of phosphatidylethanolamine can be ascribed largely to the presence of aliphatic aldehydes. The effect of aliphatic aldehydes was specific in that the aliphatic alcohol, hexadecanol, or the short‐chain aldehydes, acetaldehyde or butyraldehyde, did not promote myelin basic protein‐induced vesicle aggregation. In addition, poly‐l‐lysine was less effective than the basic protein in aggregating vesicles containing aliphatic aldehydes. Other proteins such as bovine serum albumin and ribonuclease had no effect. Aliphatic aldehydes are produced as a result of the degradation of plasmalogens, a class of phospholipids abundant in myelin. Plasmalogenase activity is elevated in multiple sclerosis. The interaction between myelin basic protein and membranes containing aldehydes may thus be of particular biological importance.
Serotonin Receptor Changes in Dementia of the Alzheimer Type
Authors: Cross A. J. , Crow T. J. , Ferrier I. N. , Johnson J. A. , Bloom S. R. , Corsellis J. A. N.
Published: October 2006

Serotonin receptors were assessed in postmortem brains of control and Alzheimer‐type dementia (ATD) patients using ligand binding techniques. Differential losses of serotonin S1 and S2 receptors were present in neocortex, hippocampus, and amygdala of ATD patients, whereas no significant changes were observed in basal forebrain and basal ganglia. Losses of S1 receptors were significantly age‐related in the ATD group, suggesting they occurred at a later stage of the disease process. Losses of S2 receptors were considerably greater (with a reduction to 35% of control in temporal cortex) and were not age‐related in ATD. Significant correlations were observed within the ATD group between S2 receptor binding and somatostatin immuno‐reactivity in temporal and frontal cortices. Thus the loss of S2 receptors in ATD may be a relatively early change in the disease process, and may precede the changes in ascending serotonergic neurones.
Benzodiazepine Receptor Binding Following Amygdala‐Kindled Convulsions: Differing Results in Washed and Unwashed Brain Membranes
Authors: Niznik H. B. , Burnham W. M. , Kish S. J.
Published: October 2006

The binding of [3H]flunitrazepam and [3H]RO5–4864 was measured in unwashed brain homogenates and in extensively washed brain membranes from amygdala‐kindled and “yoked” control rats sacrificed 2 weeks following the sixth stage 5 convulsion. In unwashed homogenates, [3H]flunitrazepam binding was reduced in both the hypothalamus and ipsilateral right cortex of kindled rats (unchanged in other areas). In washed brain membranes, [3H]flunitrazepam binding was unaltered in these regions; it was bilaterally elevated, however, in both the amygdala and hippocampus (unchanged in other areas). In washed membranes, the in vitro addition of ‐γ‐aminobutyric acid enhanced [3H]flunitrazepam binding to a similar extent in kindled and control membranes. These data indicate that the type of benzodiazepine binding abnormality observed after kindling depends on the type of tissue preparation employed in the assay procedure.
Regulation of Cyclic AMP Accumulation by Peptide Hormone Receptors in Immunocytochemically: Defined Astroglial Cells
Authors: Evans Tony , McCarthy Ken D. , Harden T. Kendall
Published: October 2006

Abstract: Primary cultures of neonatal murine brain have been reported to express multiple receptors that regulate adenylate cyclase activity. Since for the most part these results were obtained with mixed cell cultures, it has been difficult to define receptor profiles for specific cell types. With this concern in mind a series of studies has been initiated designed to identify specific receptors present on highly purified, immunocytochemically defined astroglia derived from the cerebral cortices of neonatal rats. In this study the capacity of a variety of peptide hormones to regulate cyclic AMP metabolism in these cells was examined. Fibroblasts derived from the meninges represent a predictable source of contamination in primary CNS culture. Thus, to assign more clearly specific receptors to the astroglial cell population, receptor‐mediated regulation of cyclic AMP accumulation was also examined in fibroblasts. Cyclic AMP accumulation in astroglia was stimulated by catecholamines (acting at β1,‐ adrenergic receptors), prostaglandin E1, vasoactive intestinal polypeptide, α‐melanocyte‐stimulating hormone, and adrenocorticotropin. Bombesin, luteinizing hormonereleasing hormone, neurotensin, thyrotropin‐releasing hormone, somatostatin, secretin, and vasopressin did not significantly increase cyclic AMP levels in these cultures. Catecholamines, acting at α2‐adrenergic receptors, and somatostatin inhibited agonist‐stimulated cyclic AMP ac cumulation. In meningeal cell cultures catecholamines (acting at β2‐ and α2‐adrenergic receptors) and prostaglandin E1, regulated cyclic AMP levels. However, vasoactive intestinal peptide did not stimulate and somatostatin did not inhibit cyclic AMP accumulation in these cells.
In Vivo Brain Dialysis of Amino Acids and Simultaneous EEG Measurements Following Intrahippocampal Quinolinic Acid Injection: Evidence for a Dissociation Between Neurochemical Changes and Seizures
Authors: Vezzani Annamaria , Ungerstedt Urban , French Edward D. , Schwarcz Robert
Published: October 2006

Abstract: The extracellular content of taurine, glutamate, and glycine was measured by the novel method of brain dialysis in the acute phases following an intrahippocampal injection of the excitotoxic convulsant brain metabolite quinolinic acid (QUIN). Using bilaterally depth electrodes physically combined with hollow fibers for dialysis, it was possible to collect continuously brain perfusates while simultaneously monitoring brain activity in the unanesthetized rat. In separate animals, hippocampal amino acid tissue levels were measured 2 h after an intracerebral injection of a convulsant dose (156 nmol) of QUIN. When compared with those in animal receiving the nonconvulsant decarboxylation product of QUIN, nicotinic acid, no differences in tissue levels were detected. In contrast, the same dose of QUIN caused a selective increase (2.24‐fold) in taurine levels in perfusates from the injected hippocampus. These changes were apparent prior to the onset of electrographic seizures and did not occur in the contralateral hippocampus where seizure activity was equally severe. Thus, increases in extracellular taurine, triggered by the presence of QUIN in the hippocampus, may reflect a selective tissue response to the neurotoxic (rather than the convulsant) effects of this excitotoxin.
Expression of Myelin Basic Protein Genes in Several Dysmyelinating Mouse Mutants During Early Postnatal Brain Development
Authors: Roth H. John , Hunkeler Markus J. , Campagnoni Anthony T.
Published: October 2006

Abstract: Northern blot and “dot” blot analyses using a myelin basic protein (MBP) specific cDNA probe and in vitro translation techniques were utilized to estimate the relative levels of myelin basic protein messenger RNA (mRNA) in the brains of C57BL/6J control mice, three dysmyelinating mutants (qk/qk, jp/Y, and shi/shi), and three heterozygote controls (qkl+, jpl+, and shi+) during early postnatal development. In general, the MBP mRNA levels measured directly by Northern blot and “dot” blot analyses correlated well with the indirect in vitro translation measurements. The Northern blots indicated that the size of MBP mRNAs in quaking and jimpy brain polysomes appeared to be similar to controls. Very low levels of MBP mRNAs were observed in shi/shi brain polyribosomes throughout early postnatal development. Compared to C57BL/6J controls, accumulation of MBP mRNAs in qk/qk and qk/+ brain polyribosomes was delayed by several days. That is, whereas MBP mRNA levels were below normal between 12 and 18 days, normal levels of message had accumulated in both qk/qk and qk/+ brain polyribosomes by 21 days. Furthermore, normal levels of MBP mRNAs were observed to be maintained until at least 27 days. MBP mRNA levels remained well below control levels in jp/Y brain polyribosomes throughout early postnatal development. The levels did, however, fluctuate slightly and peaked at 15 days in both jp/Y and jp/+ brains, 3 days earlier than in normal mice. Thus, it appears that jimpy and quaking mice exhibit developmental patterns of MBP expression different from each other and from C57BL/6J control mice.
Effect of Acute Hypoxia on Ascorbate Content of Plasma, Cerebral Cortex, and Adrenal Gland
Authors: Arad I. , Sidi A. , Shohami E.
Published: October 2006

Abstract: Levels of ascorbic acid (AA) in the plasma, brain, and adrenal gland of rats were determined after 15 min of hypoxia (PaO2 < 25 mm Hg) and following asphyxia. In rabbits, AA plasma levels were followed up to 75 min of reoxygenation following 15 min of hypoxia of the same severity. A significant increase (∼70%) in AA levels was found in plasma of rats and rabbits after hypoxia and asphyxia. This increase was found to be transient, with a return to normal levels within 1 h after resumption of normal oxygenation. Pretreatment with dexamethasone reduced the increase in AA level in both rabbits and rats. Adrenalectomy in rats, performed 24 h before the experiment, abolished the response to hypoxia. Ascorbate levels in the cerebral cortex, hypothalamus, and adrenal gland of awake rats subjected to hypoxia or asphyxia were found to be the same as in normoxic rats. Our results suggest that the observed changes in plasma AA levels are probably mediated through adrenocorticotropic hormone and that the adrenal gland is the major source of ascorbate efflux into the circulation during oxygen deprivation.
Rapid Nocturnal Increase in Ovine Pineal N‐Acetyltransferase Activity and Melatonin Synthesis: Effects of Cycloheximide
Authors: Aryan Namboodiri M. A. , Sugden D. , Klein D. C. , Grady R. , Mefford I. N.
Published: October 2006

Abstract: Thirty minutes after the onset of darkness, ovine pineal arylalkylamine N‐acetyltransferase, N‐acetylserotonin, and melatonin increase 5‐ to 10‐fold. No significant changes in hydroxyindole‐O‐methyltransferase, 5‐hydroxytryptamine, 5‐hydroxyindoleacetic acid, 5‐hydroxytryptophol, 5‐methoxyindoleacetic acid, and 5‐methoxytryptophol are detected at this time. Administration of cycloheximide inhibits the rise in N‐acetyltransferase and N‐acetylserotonin, but not melatonin. Unexpectedly, 5‐methoxytryptophol increases after cycloheximide treatment. Taken together, these results, although consistent in part with a role for serotonin N‐acetylation in the regulation of melatonin synthesis in sheep, indicate that an N‐acetyltransferase‐independent mechanism may also be involved.
D2 Dopamine Receptors in Calf Globus Pallidus: Agonist High‐ and Low‐Affinity Sites Not Regulated by Guanine Nucleotide
Authors: De Keyser Jacques , De Backer Jean‐Paul , Convents André , Ebinger Guy , Vauquelin Georges
Published: October 2006

Abstract: By use of the radioligand [3H]spiroperidol, D2 3,4‐dihydroxyphenylethylamine (dopamine) receptor binding characteristics were studied in calf globus pallidus and compared with those of neostriatum. Antagonist competition curves were monophasic and revealed similar affinities for neostriatum and globus pallidus, suggesting a uniform receptor population with one affinity state for antagonists. In both regions, competition curves with the agonist dopamine were biphasic, distinguishing a high‐ and low‐agonist‐affinity state. In neostriatum and globus pallidus, respectively, 45% and 19% of [3H]spiroperidol binding was displaced with high affinity and the remainder with low affinity. In neostriatum, the addition of 0.4 mM GTP resulted in a partial conversion from high‐ to low‐affinity state with a remaining highaffinity component of 15%. In globus pallidus, dopamine binding was not altered by GTP. The capability of GTP to modulate agonist binding to D2 receptors appears to be dependent on their neuroanatomical localization.
In the Beginning: To Celebrate 20 Years of the International Society for Neurochemistry (ISN)
Authors: McIlwain Henry
Published: October 2006

Abstract: The antecedents of the International Society for Neurochemistry are described, especially the parts played by the International Neurochemical Symposia held between 1954 and 1962; the Journal of Neurochemistry between 1956 and 1965; and by other organizations concerned with neural systems though not primarily neurochemical. Description is also given of the foundation of the Society in 1965 and of its first International Meeting in 1967.
Ca2+ and Phospholipid‐Dependent Protein Kinase Activity and Phosphorylation of Endogenous Proteins in Bovine Adrenal Medulla
Authors: Wise Bradley C. , Costa Erminio
Published: October 2006

Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca2+/ phospholipid‐dependent and cyclic AMP‐dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo‐daltons) showed an absolute requirement for the presence of both Ca2+ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca2+/phospholipid‐dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca2+/phospholipid‐dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP‐dependent enzyme was predominantly membrane‐bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca2+/phos‐pholipid‐dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca2+/phospholipid‐dependent protein kinase in chromaffin cell function.
Age‐Dependent Changes in Brain GABA Turnover Rates in Two Inbred Strains of Mice
Authors: Ciesielski L. , Simler S. , Clement J. , Mandel P.
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) steady‐state levels and turnover rates have been determined in 15 brain areas of 21‐day‐ and 3‐month‐old DBA/2J (DBA) and C57B1/6J (C57) mice. These two inbred strains differ by their susceptiblity to audiogenic seizures; moreover, the involvement of GABAergic neurotransmission has been suggested in the control of this behavior. Turnover rates are generally higher at 21 days than at 3 months of age. There are few significant differences in the GABA steady‐state levels between 21‐day‐old seizure‐prone DBA mice when compared with seizure‐resistant C57 mice. In the DBA mice, the steady‐state level is higher in the olfactory bulbs and lower in the posterior colliculus and the olfactory tubercles than in the C57 mice. Although there are some significant differences in GABA turnover rates and steady‐state levels, intra or inter strains, it is difficult to correlate directly these differences with seizure susceptibility.
Neurochemical Characterization of Canine α‐L‐Iduronidase Deficiency Disease (Model of Human Mucopolysaccharidosis I)
Authors: Constantopoulos George , Shull Robert M. , Hastings Nancy , Neufeld Elizabeth F.
Published: October 2006

Abstract: This report presents the neurochemical findings on the first dog to die with deficiency of α‐L‐iduronidase (mucopolysaccharide α‐L‐iduronohydrolase; EC 3.2.1.76). The principal findings were (a) markedly increased glycosaminoglycan content in all neural tissues examined (from threefold in sciatic nerve to 15‐fold in brainstem), (b) a modest increase in levels of gangliosides GM2, GM3, and GD3, particularly in gray matter, (c) excessive accumulation of glycosaminoglycans in the CSF, (d) the increased glycosaminoglycans were dermatan sulfate and heparan sulfate, and (e) the molecular weights of the liver glycosaminoglycans were shifted toward smaller sizes, indicating partial degradation. The canine disorder thus resembles mucopolysaccharidosis I in all aspects.
Acetylcholine Synthesis by Adult Bovine Adrenal Chromaffin Cell Cultures
Authors: Boksa P.
Published: October 2006

Abstract: Adrenal chromaffin cells normally synthesize and release catecholamines. In the present study, [3H]acetylcholine synthesis and another characteristic of cholinergic neurons, [3H]choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells. Chromaffin cell cultures took up [3H]choline from the medium and acetylated the [3H]choline to form [3H]acetylcholine. The rate of [3H]acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture. [3H]Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+. The ability for K+ to stimulate synthesis of [3H]acetylcholine developed only after 28 days in culture. [3H]Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline. [3H]Choline uptake was enhanced by Na+ omission in day‐14 cultures, but was at least partially Na+‐dependent in day‐29 cultures. Hemicholinium‐3 (IC50< 10 μM) inhibited [3H]choline uptake into chromaffin cell cultures. It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.
Photoaffinity Labelling of Benzodiazepine Receptors: Lack of Effect on Ligand Binding to the Nucleoside Transport System
Authors: Hammond James R.
Published: October 2006

Abstract: This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl‐β‐carboline‐3‐carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (∼ 16‐fold) and dipyridamole (∼sevenfold) for the [3H]Ro 15–1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.
Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme
Authors: Laitinen Paivi H. , Huhtinen Riitta‐Liisa , Hietala Oili A. , Pajunen Antti E. I.
Published: October 2006

Mouse brain ornithine decarboxylase activity is about 70‐fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immuno‐reactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme‐mediated mechanism in adult brain.
Subcellular Fractionation of Rat Sciatic Nerve and Specific Localization of Ganglioside LM1 in Rat Nerve Myelin
Authors: Chou K. H. , Nolan C. E. , Jungalwala F. B.
Published: October 2006

Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane‐like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl‐lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl‐lactoneonorhexaosylceramide and sialosyl‐lactoneooctasylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (< 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane‐like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were –70–75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane‐like fractions were GM3 (–40%) and GM1 (–20%). LM1 in these fractions was minimal (less than –5%). Significant amounts of GM3 with N‐glycolylneuraminic acid (–10%) and GM1b (4–14%) were also identified in the microsomal and plasma membrane‐like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.
Limited Blood‐Brain Barrier Transport of Polyamines
Authors: Shin W.‐W. , Fong W.‐F. , Pang S.‐F. , Wong Patrick C.‐L.
Published: October 2006

Abstract: Transport of polyamines across the blood‐brain barrier of adult rats was examined by measuring the amount of radioactivity that reached the forebrain 5 s after a “bolus’ intracarotid injection. The values were expressed by the brain uptake index (BUI), which is the percentage of material transported in relation to freely diffusible water in a single passage through the brain. Transport was restricted as indicated by the respective BUI values, presented as means ± SD (number of animals): putrescine, 5.3 ± 0.8 (11); spermidine, 6.1 ± 1.3 (7); and spermine, 5.8 ± 0.5 (4). A kinetic study of the transport of [14C]putrescine showed that transport due to passive diffusion accounted for the majority of the observed influx (66% at 1 mM putrescine). However, a small saturable component exists with a Km value of 4–5 mM and a Vmax of 30 nmol ± min−1± g−1. This Km value is considerably higher than the circulating levels of the polyamine in the normal mature animal, and thus is unlikely to be of physiological significance. Competition studies indicated that putrescine does not interact with carriers for adenosine, arginine, choline, or leucine.
A Histochemical Study of the Distribution of β‐Hydroxybutyrate Dehydrogenase in Developing Rat Cerebellum
Authors: Gesink Dirk S. , Wilson John E.
Published: October 2006

Abstract: The distribution of β‐hydroxybutyrate dehydrogenase (3‐hydroxybutyrate dehydrogenase, EC 1.1.1.30) in the developing rat cerebellum has been determined using a histochemical method. Staining of Purkinje cells, particularly the soma, was seen at all ages examined. Intense staining of the proximal portions of Purkinje dendrites was noted at 8–11 days postnatally, with less prominent staining of Purkinje dendrites and surrounding structures of the molecular layer seen at later times. Development of glomeruli in the granule cell layer could also be observed due to the intense staining of these structures. (Although noncerebellar structures were not the focus of this study, intense staining of the choroid plexus of the fourth ventricle was also noted.) The transient external germinal layer of the cerebellum did not show appreciable staining. Since β‐hydroxybutyrate dehydrogenase is required for ketone body metabolism, the apparent low level of this enzyme in the external germinal layer suggests that the cells of this layer are not particularly well adapted for utilization of ketone bodies. Thus these results do not provide support for the suggestion that ketone bodies may serve as major substrates for energy metabolism in the external germinal layer of the developing cerebellum. Indeed, the rather restricted distribution of this enzyme in both developing and mature cerebellum (and presumably elsewhere in brain) suggests that ketone body metabolism may be largely confined to relatively few specific cellular compartments.
Benzodiazepine Receptor and Thyroid Hormones: In Vivo and In Vitro Modulation
Authors: Medina Rjorge H. , De Robertis Eduardo
Published: October 2006

Abstract: In rats rendered hyperthyroid by chronic treatment with L‐triiodothyronine (T3) hormone there was a 21 and 27% decrease, respectively, in the number of binding sites for [3H]flunitrazepam ([3H]FNZ) and [3H]ethyl‐β‐carboline‐3‐carboxylate ([3H]β‐CCE) without changes in affinity for the two ligands. Two weeks after thyroidectomy there was a 44% increase in [3H]FNZ sites and a 17% increase in [3H]β‐CCE binding sites. In vitro we found that T3 produces a decrease in Bmax and an increase in KD, both changes being characteristic of a mixed type of inhibition. Thyroid status dramatically affected the Ki of T3 in displacing [3H]FNZ from sites on isolated membranes of the cerebral cortex: in hypothyroid rats the Ki value was 0.9 μM, whereas in hyperthyroid rats it was 83 μM, a 92‐fold difference. In control rats, the Ki was 11 μM These findings are discussed in relation to a possible modulation of benzodiazepine receptors by thyroid hormones.
Glycemia, Ketonemia, and Brain Enzymes of Ketone Body Utilization in Suckling and Adult Rats Undernourished from Intrauterine Life
Authors: Escrivá F. , Rodriguez C. , Pascual‐Leone A. M.
Published: October 2006

Abstract: The effect of undernutrition from the 16th day of pregnancy up to 70th day of life on blood glucose and ketone bodies and on several brain mitochondrial enzymes related to energy metabolism or biosynthetic function was investigated. Undernutrition in perinatal period was established by means of a food restriction to pregnant rats and, later, to the lactating mother; undernourished postweaned rats received half the diet consumed by the controls. Body and brain weight from undernourished rats was less than controls throughout the entire period studied. Glycemia and ketonemia were also always lower than controls. Cytochrome c oxidase, citrate synthase, 3‐hydroxybutyrate dehydrogenase, 3‐oxoacid coenzyme A transferase, and acetoacetyl‐coenzyme A thiolase activities during the suckling period were in most stages lower than controls; subsequently, activities in undernourished rats reached or surpassed the control values. These results could explain the “catch up”phenomenon in several ultrastructural parameters found by other authors in undernourished postweaned rats.
Reduced Proteins in Temporal Cortex in Alzheimer's Disease: An Electrophoretic Study
Authors: Borthwick Neil M. , Yates Celia M. , Gordon Alexander
Published: October 2006

Abstract: Cytoplasmic and pellet fractions from postmortem temporal cortex from eight cases of neuropathologically confirmed Alzheimer's disease, one case of cerebrovascular dementia, and five controls were examined by sodium dodecylsulphate‐polyacrylamide gel electrophoresis. No differences were observed in the cytoplasmic proteins from the five controls and the case of cerebrovascular dementia. In five cases of Alzheimer's disease with neurone loss, there was a major loss of a cytoplasmic 55,000‐dalton protein identified as tubulin and variable reductions in cytoplasmic proteins of molecular weights of 28,000, 30,000, 92,000, and 200,000 daltons. Three cases of Alzheimer's disease had no detectable neurone loss; two of these cases had protein patterns indistinguishable from the controls and one showed some reduction in soluble tubulin only. These results indicate that decreases of particular proteins in the temporal cortex in Alzheimer's disease may be associated with neurone loss.
Mg‐ATPase and Torpedo Cholinergic Synaptic Vesicles
Authors: Diebler M. F. , Lazereg S.
Published: October 2006

Abstract: The reported presence of Mg‐ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that a ouabain‐insensitive Mg‐ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg‐ATPase associated with the synaptic vesicles to oligomycin, N,N′‐dicyclohexylcarbodiimide, and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg‐ATPase, which suggests the involvement of a phosphorylated intermediate. On the basis of both the difference in inhibitor sensitivity between untreated and detergent‐treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.
Subsynaptosomal Calcium Distribution During Hypoxia and 3,4‐Diaminopyridine Treatment
Authors: Peterson Christine , Nicholls David G. , Gibson Gary E.
Published: October 2006

Abstract: Previous results demonstrate that hypoxia (low oxygen) diminishes calcium uptake by synaptosomes. The present studies examined the effects of low oxygen on calcium homeostasis in the digitonin‐resistant (mitochondrial) and the digitonin‐labile (nonmitochondrial) compartments of intact synaptosomes and their relation to altered membrane potentials. A 10‐min hypoxic incubation in low‐potassium media reduced total (‐38.3%), mitochondrial (‐43.3%), and nonmitochondrial (‐27.8%) calcium uptake. In high‐potassium media, low oxygen reduced mitochondrial (‐41.2%) and total (‐34.4%) uptake whereas nonmitochondrial (+6%) calcium uptake was essentially unaffected. A temporal analysis of nonmitochondrial calcium uptake revealed an initial depression (0–5 min) followed by a stimulation (5–10 min). Hypoxic‐induced alterations in the subsynaptosomal distribution of calcium resembled those produced by uncouplers [FCCP (carbonylcyanide‐p‐trifluoro‐methoxyphenylhydrazone) or rotenone plus oligomycin]. 3,4‐Diaminopyridine partially ameliorated the hypoxic‐and FCCP‐induced decreases in synaptosomal calcium uptake. Low oxygen reduced the total synaptosomal membrane potential (i.e., plasma plus mitochondrial membrane potential) as measured by an increased efflux of tetraphenylphosphonium ion. This hypoxic‐induced efflux of tetraphenylphosphonium was slowed by pretreatment with 3,4‐diaminopyridine. Thus, both drug and membrane potential studies suggest that hypoxic‐induced alterations in the subcellular distribution of calcium may be due to an uncoupling mechanism and a collapse of the synaptosomal mitochondrial membrane potential.