bluuebunny/crossref_metadata_2025 huggingface dataset:
Secretory Proteins from Adrenal Medullary Cells Are Carboxyl‐Methylated In Vivo and Released Under Their Methylated Form by Acetylcholine
Authors: Nguyen M. H. , Harbour D. , Gagnon C.
Published: October 2006

Abstract: The carboxyl methylation of secretory proteins in vivo was investigated in bovine adrenal medullary cells in culture. Chromogranin A., the major intragranular secretory protein in adrenal medullary cells, and other secretory proteins were found to be carboxyl‐methylated within secretory vesicles. The in vivo labeling pattern using [methyl‐3H]methionine and the in vitro labeling pattern using S‐adenosyl‐[methyl‐14C]methionine of intravesicular secretory proteins were similar. The detection of methylated chromogranin A in mature secretory vesicles required 3–6 h, a time consistent with the synthesis and storage of secretory proteins in this tissue. Carboxyl‐methylated chromogranin A was secreted from medullary cells by exocytosis via activation of nicotinic cholinergic receptor and recovered still under the methylated form in the incubation medium. Since protein‐carboxylmethylase is cytosolic, these results suggest that methylation of secretory proteins is a co‐translational phenomenon.
Lipid Composition and Temperature Adaptation of the Nervous System of the Leech Hirudo medicinalis L.
Authors: Spinedi Angelo , Rufini Stefano , Luly Paolo
Published: October 2006

Abstract: The lipid composition of the nervous system of the leech Hirudo medicinalis was investigated following acclimatization of animals at 25°C and 5°C. Choline, ethanolamine, and serine plus inositol phosphoglycerides are the major phospholipid classes of the leech ganglionic chain; minor amounts of lysophosphatidylcholine, phosphatidic acid, and sphingomyelin are also present. Neither the phospholipid pattern nor the cholesterol to total phospholipid molar ratio was dependent on the acclimatization temperature, whereas the fatty acid patterns of choline and serine plus inositol phosphoglycerides were significantly affected. Both for choline and serine plus inositol phosphoglycerides, a significant increase of the unsaturation index and a decrease of saturated to unsaturated fatty acid ratio was observed in animals acclimatized at 5°C in comparison with those acclimatized at 25°C. These observations, which point to increased lipid fluidity of the nervous system of cold‐adapted leeches, are strengthened by results obtained by the fluorescence polarization method using 1,6‐diphenyl‐1,3,5‐hexatriene as a probe: a decrease of the fluorescence polarization value was observed throughout the temperature range selected (5–40°C).
Pentobarbital Antagonizes the A1 Adenosine Receptor‐Mediated Inhibition of Hippocampal Neurotransmitter Release
Authors: Lohse Martini , Brenner Angelika S. , Jackisch Rolf
Published: October 2006

Abstract: Barbiturates have been shown to be competitive antagonists at A1 adenosine receptors in radioligand binding studies. The present study investigates the effects of pentobarbital on the A1 receptor‐mediated inhibition of neuro‐transmitter release from rabbit hippocampal slices. The inhibition of the electrically evoked release of [3H]noracrenaline by the AI receptor agonist (R)‐N6‐phenylisopro‐pyladenosine (R‐PIA) was antagonized by pentobarbital with an apparent pA2 value of 3.5. Low concentrations of pentobarbital alone altered neither basal nor evoked release of [3H]noradrenaline, whereas 1,000 μM pentobarbital enhanced the basal and reduced the evoked release. In the presence of 8‐phenyltheophylline, pentobarbital (200 μM and 1,000 μM) reduced the evoked noradrenaline release. Pentobarbital also antagonized the inhibition of [3H]acetyl‐choline release by R‐PIA. In contrast to the noradrenaline release model, the evoked release of acetylcholine was enhanced by the presence of pentobarbital (50–500 μM), an effect that was lost in the presence of 8‐phenyltheophylline. These results indicate that pentobarbital, in addition to a direct inhibitory action at higher concentrations, has a faci‐litatory effect on neurotransmitter release by blocking pre‐synaptic A1 adenosine receptors. The possible relevance of these findings for the excitatory effects of barbiturates is discussed.
Trifluoperazine Activates and Releases Latent ATP‐Generating Enzymes Associated with the Synaptic Plasma Membrane
Authors: Leung Thomas K. C. , Hall Christine , Monfries Clinton , Lim Louis
Published: October 2006

Abstract: Neurone‐specific enolase (NSE) and the brain form of creatine phosphokinase (CPK‐BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two‐dimensional gel (2‐D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be “cryptic” unless the SPM were treated with Triton X‐100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for “unmasking” the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK‐BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2‐D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70‐kilodalton heat‐shock cognate protein. Tubulin, synapsin I, and a 35‐kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase on the basis of 2‐D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.
Inefficient Transcription of the Myelin Basic Protein Gene Possibly Causes Hypomyelination in Myelin‐Deficient Mutant Mice
Authors: Okano Hideyuki , Miura Masayuki , Moriguchi Akira , Ikenaka Kazuhiro , Tsukada Yasuzo , Mikoshiba Katsuhiko
Published: October 2006

Abstract: A hereditary dysmyelination mutation, named myelin deficient (mld), is considered to be allelic to shiverer, a deletion mutation of the myelin basic protein (MBP) gene. The present study showed that MBP expression is greatly reduced in mld, but that it is still detectable. Northern blot analysis revealed that the pronounced decrease in the MBP level in mld resulted from a reduced mRNA level and was not caused by deletion of a large portion of the MBP gene as in shiverer. Southern blot studies with BamHI‐digested chromosomal DNA suggested some part of the MBP gene, at least the 5′‐portion, was duplicated in mld. These results indicated that the mld and shiverer mutations were different from each other, even though genetic allelism between the two was reconfirmed. We also examined the developmental pattern of the gene expression of MBP and that of another protein, myelin proteolipid protein (PLP), specifically expressed in the oligodendrocyte, in mld by RNA dot blot study. The mRNA level of MBP in mld was greatly reduced during the active myelination stages, gradually increasing and remaining constant in the later stages. The PLP‐mRNA content in mld was almost normal (60–80% that of control) at any stage of development. All these findings imply that the primary defect in mld is due to reduced transcriptional activity of the MBP gene.
Opposite Effects of δ and μ Opioid Receptor Agonists on the In Vitro Release of Substance P‐Like Material from the Rat Spinal Cord
Authors: Mauborgne Annie , Lutz Olivier , Legrand Jean‐Claude , Hamon Michel , Cesselin Frangois
Published: October 2006

Abstract: Superfusion of slices from the dorsal half of the lumbar enlargement of rat spinal cord with Krebs‐Henseleit medium supplemented with 30 μM bacitracin allowed the collection of substance P‐like immunoreactive material (SPLI), which was released at a rate of ∼ 10 pg/4 min. Tissue depolarization by an excess of K+ (30–60 mM) or veratridine (50 μM) induced a marked increase in SPLI outflow, provided that Ca2+ was present in the superfusing fluid. K+‐or veratridine‐induced SPLI overflow could be modulated in opposite directions by μ and δ opioid receptor agonists. Thus, the two preferential μ agonists Tyr‐d‐Ala‐Gly‐MePhe‐Gly‐ol (DAGO; 10 μM) and Tyr‐d‐Ala‐Gly‐MePhe‐Met(O)5‐OH (FK‐33824; 0.1 μM) enhanced SPLI overflow from depolarized tissues, whereas the selective δ agonists Tyr‐d‐Thr‐Gly‐Phe‐Leu‐Thr (deltakephalin; 3 μM) and [2‐d‐penicillamine, 5‐d‐penicillamine]enkephalin (50 μM) reduced it. The effect of DAGO was antagonized by a low concentration (1 μM) of naloxone but not by the selective δ antagonist ICI‐154129 (50 μM). In contrast, the latter drug prevented the inhibitory influence of δ agonists on K+‐induced SPLI release. Complementary experiments with morphine (10 μM) and [2‐d‐alanine, 5‐d‐leucine]enkephalinamide (3 μM), in combination with 1 μM naloxone or 50 μM ICI‐154129 for the selective blockade of μ or δ receptors, respectively, confirmed that the stimulation of μ receptors increased, whereas the stimulation of δ receptors reduced, SPLI overflow. The results suggest that, at the spinal level, the antinociceptive action of δ but not μ agonists might involve a presynaptic inhibition of substance P‐containing primary afferent fibers.
A Simple and Rapid Method for the Purification of Ovine Pineal Arylalkylamine N‐Acetyltransferase
Authors: Namboodiri M. A. A. , Brownstein M. J. , Voisin Pierre , Weller Joan L. , Klein David C.
Published: October 2006

Abstract: A two‐step chromatographic procedure has been developed for the purification of ovine pineal arylalkylamine N‐acetyltransferase (EC 2.3.1.87), based on the principles of disulfide exchange and anion exchange. The enzyme from 20 ovine pineal glands can be purified about 500‐fold in a day; recovery is about 5%. Polyacrylamide gel electrophoretic analysis of the final preparation shows four major bands; one appears to be arylalkylamine N‐acetyl‐transferase.
Modulation of Dopamine Release from Neuron‐Enriched Tissue Cultures by Cholinergic Agents
Authors: Barochovsky O. , Bradford H. F.
Published: October 2006

Abstract: Modulation of [3H]dopamine release by cholinergic agents (acetylcholine, atropine, d‐tubocurarine, oxo‐tremorine, and nicotine) was studied in primary cell cultures derived from whole brains of foetal rats (17 days of gestation). Monolayer and aggregated neuron‐enriched cultures were maintained for 17 days in vitro. [3H]Dopamine basal outflow was enhanced by acetylcholine, nicotine, and atropine and was unaffected by oxotremorine, hexametho‐nium, and d‐tubocurarine. The action of nicotine was antagonized by d‐tubocurarine, and that of atropine was partially blocked by oxotremorine. A similar picture was seen when the influence of cholinergic agents was studied under depolarizing conditions. The action of oxotremorine was dependent on nerve activity. The presence of both musca‐rinic and nicotinic antagonists was necessary for abolishing the effect of acetylcholine on the dopamine outflow. These results show that dopamine release in both types of neuron‐enriched cultures can be influenced by cholinergic agents and that both muscarinic and nicotinic receptors are involved in regulation of the amine's outflow.
Identification and Characterization of a Polypeptide from a Lobster Neurosecretory Gland that Induces Cyclic GMP Accumulation in Lobster Neuromuscular Preparations
Authors: Goy Michael F. , Mandelbrot Didier A. , York Carey M.
Published: October 2006

Abstract: Several observations suggest that cyclic GMP might regulate some aspect of neuromuscular physiology or metabolism in the lobster, Homarus americanus: (1) lobster muscle is one of the richest known sources of cyclic GMP‐dependent protein kinase, (2) the preparation contains several phosphoproteins whose state of phosphorylation is affected by cyclic GMP more effectively than by cyclic AMP, and (3) guanylate cyclase and phosphodiesterase are active in this tissue. However, no factor has yet been identified that alters lobster muscle cyclic GMP levels. We have screened extracts of neural and neurosecretory structures for the capacity to promote cyclic GMP accumulation in isolated exoskeletal muscles. Extracts of the sinus gland (a neurohemal organ found in the eyestalk) contain a factor that induces up to 100‐fold increases in muscle cyclic GMP content, whereas extracts of other tissues are ineffective. This factor can also act on targets other than muscle, with hepatopancreas, testis, and neuronal tissue showing the largest responses. The sinus gland factor does not appear to affect cyclic GMP metabolism by depolarizing the preparation or by mobilizing extracellular Ca2+. The effect on cyclic GMP levels is dose‐dependent and linear with time. Biological activity is destroyed by boiling and by 90% ethanol. It is also destroyed by trypsin, chymotrypsin, or pronase, which suggests that the factor is a protein or peptide. Both gel filtration chromatography and experiments using dialysis tubing with different molecular weight exclusion limits indicate that the factor has an apparent molecular weight of 5,000–12,000 daltons. A preliminary fractionation scheme, based on gel filtration, ion‐exchange, and reverse‐phase chromatography, gives >1,300‐fold purification. Our long‐range goal is to purify this factor to homogeneity, compare it to other peptide hormones, and use it as a probe to evaluate the role of cyclic GMP at the neuromuscular junction.
Cytoplasmic Poly(ADP‐Ribose) Polymerase Associated with Free Messenger Ribonucleoprotein Particles in Rat Brain
Authors: Chypre Camille , Maniez Christine , Mandel Paul
Published: October 2006

Abstract: Poly(ADP‐ribose) polymerase associated with free cytoplasmic messenger ribonucleoprotein particles (free mRNP particles) carrying messenger RNA has been characterized in rat brain. There were first‐order kinetics for NAD with an apparent Km for NAD of 90.5 ± 0.70 μM and Vmax of 19.7 ± 2.8 pmol ADP‐ribose incorporated min‐1 mg protein‐1. Five poly(ADP‐ribose) protein acceptors were identified in the Mr 37,000–120,000 range. It is hypothesized that ADP‐ribosylation of specific free mRNP proteins might play a role in the derepression and translation of the silent mRNAs of free mRNP particles.
Synaptic Protein Tyrosine Kinase: Partial Characterization and Identification of Endogenous Substrates
Authors: Ellis Paul D. , Bissoon N. , Gurd James W.
Published: October 2006

Abstract: The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]‐angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2‐ and 2.5‐fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 ± 1.6 and 142 ± 4 pmol/min/ mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33–37% of the angiotensin and 43–70% of the pGT‐phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 ± 5 and 23 ± 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent‐insoluble residue obtained following the extraction of SMs with Triton X‐100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X‐100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre‐ and postsynaptic compartments and suggest a role for this enzyme in synaptic function.
Prenatal Diazepam Exposure Affects β‐Adrenergic Receptors in Brain Regions of Adult Rat Offspring
Authors: Rothe Thomas , Langer Matthias
Published: October 2006

Abstract: The postnatal development of [3H]dihydro‐alprenolol binding to β‐adrenergic receptors has been studied in frontal cortex, cerebellum, striatum, and hypothalamus of the rat after prenatal and perinatal exposure to diazepam. Dams were injected subcutaneously with single daily doses of 1 mg of diazepam/kg from day 7 to 20 of gestation or from day 15 of gestation to day 6 after birth. Prenatal exposure had no effect on litter size or length of gestation or on the postnatal development of body and brain weights of the progeny. However, a reduced mortality of the pups was observed in relation to vehicle‐treated controls until postnatal day 10. Prenatal diazepam administration decreased [3H]dihydroalprenolol binding in frontal cortex, striatum, and hypothalamus but not in cerebellum. This decrease in β‐adrenergic receptor binding was due to a decrease in receptor density rather than in receptor affinity. In contrast, perinatal diazepam exposure led to a transient decrease in [3H]dihydroalprenolol binding limited to the frontal cortex. The permanent reduction in number of β‐adrenergic receptors, which depends on the scaling and duration of the drug application period, points to the necessity of a prolonged evaluation of effects of exposure to psychotropic drugs in early stages of brain development.
Effect of Thyroid Hormones on Benzodiazepine Receptors in Neuron‐Enriched Primary Cultures
Authors: Go Tohshin , Ito Masatoshi , Okuno Takehiko , Mikawa Haruki
Published: October 2006

Abstract:The effect of administration of thyroid hormones on central benzodiazepine receptors was investigated using neuron‐enriched primary cultures obtained from the neo‐pallium of 16‐day‐old embryonic rats. Addition of l‐triio‐dothyronine for 3 days decreased the maximal number of benzodiazepine receptor binding sites without any change in affinity at 10−5and 10−6M. l‐Thyroxine administered for 3 days had the same effect at 10−5M. No significant change was observed over periods of <3 days, a finding indicating that this inhibition was not a direct in vitro effect. This down‐regulation seems to be a direct modulatory effect of thyroid hormones on cerebral cortical neurons. Addition for 3 days ofd‐thyroxine and d‐triiodothyronine, which are physiologically inactive isomers of the thyroid hormones, did not induce any significant alterations in benzodiazepine receptors. The decrease in number of cerebral cortical neuronal benzodiazepine receptors due to l‐isomers of thyroid hormones may be related to the convulsions and anxiety observed in thyrotoxicosis in humans.
An Immunochemical Analysis of the Distribution of a Brain‐Specific Polypeptide, PEP‐19
Authors: Ziai M. R. , Sangameswaran L. , Hempstead J. L. , Danho W. , Morgan J. I.
Published: October 2006

Abstract: Immunochemical and immunohistochemical techniques were used to map the tissue distribution and cellular localization of a rat brain‐specific polypeptide, termed PEP‐19. PEP‐19 was found to be abundant in the cerebellum and olfactory bulbs but was present at much lower levels in other gross brain regions. It was undetectable in all nonneural tissues examined but was present in the cerebellum of several vertebrates, including rat, mouse, guinea pig, monkey, and human. Immunohistochemical analysis revealed that PEP‐19 was localized to the soma, axon, and dendritic processes of rat cerebellar Purkinje cells with no demonstrable immunoreactivity in nonneuronal cell types. Furthermore, mutant mice showing degeneration of Purkinje cells exhibit markedly decreased levels of PEP‐19. Because PEP‐19 appears during the final stages of maturation of Purkinje cells, it may be utilized as a probe to monitor the development of these neurons in vivo.
Affinity Labelling and Identification of the High‐Affinity Choline Carrier from Synaptic Membranes of Torpedo Electromotor Nerve Terminals with [3H]Choline Mustard
Authors: Rylett R. Jane
Published: October 2006

Abstract: The physiological mechanisms regulating activity of the sodium‐dependent, high‐affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion ([3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium‐sensitive, and sodium‐, time‐, and temperature‐dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30°C. Separation of the 3H‐labelled membrane proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of ∼42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC‐3. Two other 3H‐labelled polypeptides were detected, with apparent molecular masses of ∼58,000 and 90,000 daltons; radiolabelling of the former was also HC‐3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.
Functional Properties of the Subtype of Insulin Receptor Found on Neurons
Authors: Heidenreich Kim A. , de Vellis Genevieve , Gilmore Patricia R.
Published: October 2006

Abstract: In this report, we have examined the structure, regulation, and function of insulin receptors in cultured neurons from fetal chicken brain. The apparent molecular weight of the α‐subunit of neuronal insulin receptors, analyzed by photoaffinity labeling and sodium dodecyl sulfate gel electrophoresis under reducing conditions, was 115,000. The number of insulin receptors in the cultures increased from day 2 to day 4 during a period of extensive process formation. After 5 days in culture, there were approximately 40,000 high‐affinity insulin receptors per neuron. When neurons were photoaffinity labeled at 16°C and then warmed to 37°C for 30 min, approximately 40% of the cell‐surface receptors were recovered in the intracellular, trypsin‐insensitive pool. Chronic exposure of neurons to insulin (100 ng/ml) resulted in a time‐dependent loss of neuronal insulin receptors with a maximal decrease of 50% after 24 h. Insulin had no effect on glucose transport, glucose oxidation, or glycogen synthase activity in neurons. On the other hand, insulin supported the growth and differentiation of a fraction of neurons isolated from chick forebrain. We conclude that (1) cultured neurons from fetal chicken brain express the same subtype of insulin receptor previously identified in adult rat and human brain, (2) the neuronal subtype of insulin receptor undergoes internalization and down‐regulation in response to insulin, and (3) neuronal insulin receptors do not acutely regulate glucose metabolism but mediate growth in neurons.
Age‐Associated Changes in Pineal Adrenergic Receptors and Melatonin Synthesizing Enzymes in the Wistar Rat
Authors: Dax Elizabeth M. , Sugden David
Published: October 2006

The nocturnal stimulation of pineal melatonin synthesis and elevation of serum melatonin is known to be reduced in old age in several species. In Wistar rats the capacity of the β‐adrenoceptor to develop supersensitivity (increase in Bmax) during the light period of the diurnal light/dark cycle is lost during maturation (3–6 months) rather than old age. Further, the present study shows that neither the α1‐ nor β‐adrenoceptor density of the pineal declines as rats age. Pineal hydroxyindole‐O‐methyltransferase activity does fall (17–55%) in rats after 18 months of age, but nocturnal pineal arylalkylamine N‐acetyltransferase activity is not significantly altered. Thus, from examination of these parameters across the life span of the rat, it seems likely that the reported reduction in serum melatonin in old animals is related to a reduced capacity of the pineal to synthesize melatonin, rather than an altered responsiveness of the gland to neural stimulation.
Developmental Pattern for Deoxyhypusine Hydroxylase in Rat Brain
Authors: Abbruzzese Alberto
Published: October 2006

Deoxyhypusine hydroxylase catalyzes the formation of hypusine from deoxyhypusine in a precursor form of eukaryotic initiation factor 4D (eIF‐4D). The enzymatic activity was examined in mammalian brain homogenates and the results were consistent with the existence of deoxyhypusine hydroxylase levels comparable to those occurring in other mammalian tissues. Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in cow brain (1.82 units/ mg of protein). In the rat the enzyme was found to be unevenly distributed among various brain regions. The parietal cortex contained the highest specific activity (2.1 units/mg of protein). Rat brain deoxyhypusine hydroxylase was mainly present in the postmicrosomal supernatant (81% of the total activity). The highest specific activity (3 units/mg of protein) was observed in the rat brain during the first few days of life. Thereafter the activity started to decline, and continued to do so for 15 days, remaining throughout the rest of life at levels of less than one‐half that of newborn.
Choline Transport and Metabolism in Soman‐or Sarin‐Intoxicated Brain
Authors: Drewes Lester R. , Singh Ashok K.
Published: October 2006

The metabolism and blood‐brain transport of choline (Ch) were investigated in perfused canine brain under control conditions and for 60 min after inhibition of brain cholinesterases by the organophosphorus (OP) compounds soman (pinacolylmethylphosphonofluoridate) and sarin (isopropylmethylphosphonofluoridate). Ch and acetylcholine (ACh) in blood and brain samples were analyzed using gas chromatography‐mass spectrometry methods. Net transport of Ch was determined by Ch analysis in arterial and venous samples. Unidirectional transport of [3H]Ch was determined using the indicator dilution method. During control perfusion periods of 90 min, net efflux of brain Ch occurred at a rate of 1.6 ± 0.4 nmol/g/ min, and the Ch content of the recirculated perfusate increased 10‐fold to ˜8 μM. Brain Ch content increased in proportion to the increase in perfusate Ch level, but brain ACh was unaltered. Rapid administration of soman (100 Hg) or sarin (400 μg) into the arterial perfusate after a 40‐min control period resulted in a > 10‐fold increase in ACh content in cerebral cortex, brainstem, and hippocampus.

The ACh content of cerebellum increased only slightly. The Ch level in all four brain regions studied also increased two‐to fourfold above control levels. Ch efflux from brain, however, decreased to 0.2 ± 0.1 nmol/g/min during the 60 min after OP exposure. Unidirectional influx of [3H)Ch was 0.49 ± 0.07 nmol/g/min before and did not change significantly 10 or 40 min after OP exposure, thus indicating that the Ch transporter of the brain endothelial cell is not directly inhibited. Based on these results, it is proposed that (a) efflux of brain Ch occurs from the extracellular compartment, which becomes depleted when ACh breakdown is inhibited; (b) brain Ch is sequestered intracellularly; and (c) inhibition of cholinesterases by OP compounds stimulates synthesis of Ch, the source of which is not yet clear but which may involve hydrolysis of Ch‐containing brain lipids.
Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training
Authors: Ali Shahid M. , Bullock Sarah , Rose Steven P. R.
Published: October 2006

Abstract: We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1‐day‐old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n‐octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n‐octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1‐day‐old, 14‐day‐old, and 21‐day‐old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphor ylation and in the components phosphorylated. A time‐course study was carried out in which phosphorylation of SPMs and PSDs from 1‐day‐old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water‐coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (‐21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water‐trained and untrained chicks. After 30 min, this decrease was greater (‐34%, p < 0.05) and was significant. There was a concomitant but nonsignificant increase (+22%) in phosphorylation of a 45 kDa component in the SPM fraction. No significant changes in phosphorylation patterns were seen in PSD fractions or in SPMs and PSDs isolated from cerebella of chicks with passive avoidance training at any time.
Synaptic Vesicles from Mammalian Brain: Large‐Scale Purification and Physical and Immunochemical Characterization
Authors: Floor E. , Schaeffer S. F. , Feist B. E. , Leeman S. E.
Published: October 2006

Abstract: Purification of synaptic vesicles directly from homogenates of mammalian brain is compared with a classical method based on osmotic lysis of brain synaptosomes. The direct method affords increased yield and purity of synaptic vesicles prepared under isoosmotic conditions. Antigen SV2 and the antigens (primarily synaptophysin) recognized by rabbit antiserum R10, raised to purified rat brain synaptic vesicles, are localized specifically on ∼40‐nm‐diameter microsomal vesicles from rat brain. Rat brain synaptic vesicles have equilibrium densities of ∼1.11 g/ml on Nycodenz density gradients, 1.12 g/ml on glycerol/Nycodenz, and 1.07 g/ml on Ficoll gradients. Both SV2 and the R10 antigens are enriched ∼50‐fold in purified rat brain synaptic vesicles. Synaptic vesicles purified from rat or cow brain show active uptake of [3H]norepinephrine that is reserpine sensitive and dependent on ATP and Mg2+. Synaptic vesicles exhibiting [3H]norepinephrine uptake comigrate with ∼40‐nm‐diameter synaptic vesicles earning SV2 or R10 antigens during permeation chromatography. After the Sephacryl S‐1000 chromatography step, [3H]‐norepinephrine uptake activity is purified ∼90‐fold. Highly purified brain synaptic vesicles should facilitate studies at the molecular level of the roles of these organelles in neurotransmission at mammalian synapses.
Oxidation of γ‐Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide‐Independent Enzyme
Authors: Kaufman Elaine E. , Nelson Thomas , Miller David , Stadlan Noam
Published: October 2006

Abstract: An antibody that inhibits over 95% of the cytosolic NADP+‐dependent 7‐hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1–14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1–14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADPT and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+‐depen‐dent cytosolic oxidoreductase which catalyzes this reaction.
l‐Glutamate Binding Site on N18‐RE‐105 Neuroblastoma Hybrid Cells Is Not Coupled to an Ion Channel
Authors: Berry Bill W. , Boland Linda M. , Hoch Daniel B. , Dingledine Raymond
Published: October 2006

Abstract: We studied the properties of the N18‐RE‐105 neuronal cell line to determine if its glutamate binding site represents a neurotransmitter receptor. In immunocytochemical experiments, these cells stained strongly for neurofilament, but not for glial fibrillary acidic protein. In whole‐cell patch clamp experiments, cells exhibited voltage‐dependent Na+, Ca2+, and K+ currents characteristic of neurons. However, perfusion with l‐glutamate or other excitatory amino acids did not evoke the inward current expected of a receptor/ channel complex. In binding studies, the maximum accumulation of l‐[3H]glutamate by washed membrane vesicles at 37°C was 69 pmol/mg protein, and half‐maximal accumulation occurred at 0.64 μM. This accumulation was blocked completely by quisqualate, partially by dl‐2‐amino‐4‐phosphonobutyric acid and l‐cystine, but not at all by 1 mM kainate or N‐methylaspartate. l‐[3H]Glutamate accumulation was stimulated by Cl−, but reduced by Na+, 0.01 % digitonin, or hyperosmotic (400 mM glucose) assay medium. The release of l‐[3H]glutamate from vesicles was much faster in the presence of 100 μM unlabelled glutamate than 100 μM unlabelled quisqualate or dl‐2‐amino‐4‐phosphonobutyric acid. Thus, although N18‐RE‐105 cells possess many neuronal properties, the results obtained are not those expected from reversible binding of l‐glutamate to a receptor/ channel complex, but are consistent with a Cl−‐stimulated sequestration or exchange process.
Different Subcellular Localization of Neurotensin‐Receptor and Neurotensin‐Acceptor Sites in the Rat Brain Dopaminergic System
Authors: Schotte Alain , Rostène William , Laduron Pierre M.
Published: October 2006

Abstract: The subcellular localization of neurotensin‐receptor sites (NT2 sites) and neurotensin‐acceptor sites (NT1 sites) was studied in rat caudate‐putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT, sites occurred as a single large peak at an intermediate density. 6‐Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate‐putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate‐putamen led to a partial decrease of NT, sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate‐putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.
Neuromodulator‐Mediated Phosphorylation of Specific Proteins in a Neurotumor Hybrid Cell Line (NCB‐20)
Authors: Berry‐Kravis Elizabeth , Kazmierczak Barbara I. , Derechin Vivianna , Dawson Glyn
Published: October 2006

Abstract: Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB‐20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB‐20 cells were labeled with [32P]orthophosphate under conditions where 2‐chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP‐differentiated PC12 (pheochromocytoma) cells. When NCB‐20 cells were exposed to activators [5‐hydroxytryptamine (5‐HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP‐dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5‐HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three‐ to fourfold increase, and forskolin a four‐ to sevenfold increase in 32P incorporation into the 90‐kDa protein. [D‐Ala2,d‐Leu5]‐enkephalin, which decreased cyclic AMP levels and reversed the 2‐chloroadenosine‐stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90‐kDa protein in NCB‐20 cells. Pretreatment of NCB‐20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90– and 130‐kDa proteins, but phorbol esters such as 12‐O‐tetradecanoylphorbol 13‐acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80‐kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid‐dependent kinase with tumor promoting agent. Neither the 90‐kDa nor the 80‐kDa protein showed any immu‐nological cross‐reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP‐dependent protein kinase and calcium/calmodulin‐dependent protein kinase, but not calcium/phospholipid‐dependent protein kinase. This suggests that in NCB‐20 cells, several unique proteins can be phosphorylated by cyclic AMP‐dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80‐kDa protein is the primary substrate for calcium/phospholipid‐dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP‐dependent protein kinase.
Glutamate Neurotoxicity in Culture Depends on the Presence of Glutamine: Implications for the Role of Glial Cells in Normal and Pathological Brain Development
Authors: Simantov Rabi
Published: October 2006

Abstract: Glutamate toxicity was studied in neuronal (SC9), glial (WC5), and neuroblastoma‐glioma hybrid cell lines. In all three cell types, glutamate had a dual effect, depending on the concentration of glutamine in the culture medium. An expected dose‐dependent cytotoxicity of the amino acid was observed when cells were cultured in medium containing the standard glutamine concentration (1‐4 mM), but when the culture's glutamine content was decreased to 0.15–0.5 mM, glutamate had an apparent opposite, growth‐promoting effect. The specificity of glutamate effect was indicated by the following: (a) it was stereospecific, with the L and not the D isomer being active; (b) monosodium aspartate was inactive in the presence of either high or low glutamine; and (c) monosodium glutamate and monopotassium glutamate had a similar dual effect. Furthermore, the glutamate receptor antagonist γ‐glutamylglycine blocked the amino acid cytotoxicity in a dose‐dependent fashion. As glial cells are a major source of glutamine in the brain, neuronal‐glial co‐cultures were used to analyze the possible role of glial cells in glutamate neurotoxicity. It was found that SC9 cells were more sensitive to glutamate when co‐cultured with WC5 cells. Continuous depolarization of the SC9 cells with KCl decreased cell number, but glutamate had no additive neurotoxic effect when added with KCl. We suggest that glutamine, glial cells, and neuronal activation play roles in modulating glutamate neurotoxicity, in developing as well as aged brains. It is tempting to speculate also that alterations in the glutamate/glutamine ratio under pathological conditions may take part in the etiology of some neurodegenerative diseases.
Presynaptic Mechanisms Underlying the γ‐Aminobutyric Acid‐Evoked Receptor‐Independent Release of [3H]Norepinephrine in Rat Hippocampus
Authors: Bonanno Giambattista , Fontana Giovanni , Fedele Ernesto , Robino Grazia , Raiteri Maurizio
Published: October 2006

Abstract: The effects of γ‐aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA‐induced [3H]NE release was counteracted by the GABA uptake inhibitor N‐(4,4‐diphenyl‐3‐butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA‐induced release of [3H]NE was Ca2+‐dependent and tetrodotoxin‐sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.
Proteolytic Cleavage of Tetanus Toxin Increases Activity
Authors: Bergey Gregory K. , Habig William H. , Bennett Jeffrey I. , Lin Clara S.
Published: October 2006

Abstract: Tetanus toxin is initially synthesized in the form of a single polypeptide chain and then proteolytically “nicked” by the bacteria to produce a two‐chain structure joined by a disulfide bond. This two‐chain form of the toxin is the form known to be biologically active. Whether such nicking is necessary for activity, as it is for certain other bacterial toxins, has not been demonstrated previously. Single‐chain toxin preparations produced by salt extraction from the bacteria are characterized and compared with pure two‐chain toxin obtained from extracellular filtrates. The ability of these various toxin preparations to produce paroxysmal activity in mouse spinal cord neurons grown in dissociated cell culture is described. The pure two‐chain toxin is demonstrated to have greater activity than the single‐chain toxin preparations. Indeed the activity of the single‐chain toxin preparations can be explained by the small amounts of residual two‐chain toxin present in these extracts. Using a protease from a toxin‐minus strain of Clostridium tetani to convert a single‐chain toxin preparation to two‐chain toxin increases toxin activity. In vivo the single‐chain toxin preparation is also less toxic. These findings indicate that proteolytic nicking of tetanus toxin increases activity. The unnicked, single‐chain form of tetanus toxin may be a relatively nontoxic protoxin form of the toxin; this is a structure‐function relationship similar to that of other bacterial protein toxins.
Changes in Cholinergic but Not in GABAergic Markers in Amygdala, Piriform Cortex, and Nucleus Basalis of the Rat Brain Following Systemic Administration of Kainic Acid
Authors: Schliebs Reinhard , Zivin Marko , Steinbach Jörg , Rothe Thomas
Published: October 2006

Abstract: Three days after systemic administration of kainic acid (15 mg/kg, s.c), selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, and high‐affinity choline uptake) and GABAergic parameters [benzodiazepine and γ‐aminobutyric acid (GABA) receptors] were studied in the frontal and piriform cortex, dorsal hippocampus, amygdaloid complex, and nucleus basalis. Kainic acid treatment resulted in a significant reduction of choline acetyltransferase activity in the piriform cortex (by 20%), amygdala (by 19%), and nucleus basalis (by 31%) in comparison with vehicle‐injected control rats. A lower activity of acetylcholinesterase was also determined in the piriform cortex following parenteral kainic acid administration. [3H]Quinuclidinyl benzilate binding to muscarinic acetylcholine receptors was significantly decreased in the piriform cortex (by 33%), amygdala (by 39%), and nucleus basalis (by 33%) in the group treated with kainic acid, whereas such binding in the hippocampus and frontal cortex was not affected by kainic acid. Sodium‐dependent high‐affinity choline uptake into cholinergic nerve terminals was decreased in the piriform cortex (by 25%) and amygdala (by 24%) after kainic acid treatment. In contrast, [3H]flunitrazepam binding to benzodiazepine receptors and [3H]muscimol binding to GABA receptors were not affected 3 days after parenteral kainic acid application in any of the brain regions studied. The data indicate that kainic acid‐induced limbic seizures result in a loss of cholinergic cells in the nucleus basalis that is paralleled by degeneration of cholinergic fibers and choli‐noceptive structures in the piriform cortex and amygdala, a finding emphasizing the important role of cholinergic mechanisms in generating and/or maintaining seizure activity.
Calcium Is Released by Exocytosis Together with Catecholamines from Bovine Adrenal Medullary Cells
Authors: von Grafenstein Hermann R. K. , Powis David A.
Published: October 2006

Abstract: We have tested the hypothesis that exocytosis is a possible export route for calcium from bovine adrenal medullary cells. After prelabelling cells in primary tissue culture with 45Ca, evoked 45Ca export and catecholamine secretion show the same time course, a similar fraction of the total pool of 45Ca and catecholamine is released), and the same concentrations of carbamylcholine or KCl are required for half‐maximal triggered release. Increasing the osmolality of the extracellular medium or treating the cells with botulinum toxin type D inhibits both evoked catecholamine secretion and 45Ca export to the same extent without inhibiting 45Ca influx. Incorporation of 45Ca into chromaffin granules is very slow, however, and incorporated 45Ca is not immediately releasable. 45Ca entering the cell during short‐term stimulation is not found in the releasable pool during a second period of triggered secretion. Our data suggest that chromaffin granules are the largest pool of intracellular calcium in bovine adrenal medullary cells and that most of the calcium in chromaffin granules does not rapidly exchange with cytoplasmic Ca, but can be released directly by exocytosis. Exocytosis does not appear to play a major role in exporting Ca that enters the cell during short‐term stimulation.
Modulation of Histamine Release and Synthesis in the Brain Mediated by α2‐Adrenoceptors
Authors: Gulat‐Marnay Christiane , Lafitte Andrée , Arrang Jean‐Michel , Schwartz Jean‐Charles
Published: October 2006

Abstract: The adrenergic regulation of histamine release was studied in rat brain slices labeled with L‐[3H]histidine. Noradrenaline in increasing concentrations progressively inhibited K+‐evoked [3H]histamine release from cortical slices, whereas phenylephrine and isoprenaline were ineffective. Yohimbine, a preferential α2‐adrenoceptor antagonist, reversed the noradrenaline effect in an apparently competitive manner and with a mean Ki value of 30 nM. Phentolamine reversed the noradrenaline effect with a similar potency, whereas propranolol was ineffective. The imidazolines clo‐nidine and oxymetazoline acted as partial agonists, oxymeta‐zoline even behaving as an apparent antagonist. In vivo clo‐nidine also inhibited [3H]histamine formation in cerebral cortex, an effect reversed by the administration of yohimbine. However, yohimbine failed to increase significantly [3H]histamine release in vitro and [3H]histamine formation in vivo, suggesting that adrenergic receptors are not activated by endogenous noradrenaline released under basal conditions. It is concluded that adrenergic α2‐adrenoceptors presumably located on histaminergic axons control release and synthesis of histamine in the brain.