bluuebunny/crossref_metadata_2025 huggingface dataset:
Lithium Chloride Inhibits the Phosphorylation of Newly Synthesized Neurofilament Protein, NF‐M, in Cultured Chick Sensory Neurons
Authors: Bennett Gudrun S. , Laskowska Danuta , DiLullo Camille
Published: October 2006

Abstract: The middle and high molecular weight members of the neurofilament triplet, NF‐M and NF‐H, undergo extensive posttranslational polyphosphorylation, a process requiring 24 h or more for completion. We have investigated ways of perturbing this process in intact cells and have found that phosphorylation of newly synthesized NF‐M in cultured chick sensory neurons is inhibited by Li+. [35S]Methionine pulse‐chase experiments were carried out with pure neuronal cultures, and the phosphorylation of newly synthesized NF‐M was monitored by following the accompanying change, with chase time, in apparent size and charge of the polypeptide. Addition of LiCl to the medium inhibited this mobility shift in a dose‐dependent manner over concentrations between 2 and 25 mM. Incorporation of 32P into NF‐M, as well as NF‐H, was also inhibited, whereas incorporation into the low molecular weight neurofilament protein, β‐tubulin, and total protein was unaffected. Protein synthesis was not altered.

Exposure to 25 mM LiCl for up to 72 h was not toxic, and the inhibition of NF‐M phosphorylation was completely reversible. When 25 mM Li+ was added after NF‐M had become partially phosphorylated, further progression was blocked, but there was no net dephosphorylation or degradation of NF‐M. Additional experiments suggest that this action of Li+ is probably not due to effects on second messenger levels or to effects on tubulin metabolism and assembly state presented in our accompanying article, but rather to interference by Li+ itself, with the phosphorylation of NF‐M and NF‐H by specific neurofilament kinase(s).
Potassium Channel Blockers Inhibit D2 Dopamine, but Not A1Adenosine, Receptor‐Mediated Inhibition of Striatal Dopamine Release
Authors: Cass Wayne A. , Zahniser Nancy R.
Published: October 2006

Abstract: D2 dopamine autoreceptors and A1 adenosine heteroreceptors inhibit the evoked release of dopamine from rat striatum. We examined the role of potassium channels in this modulation by determining the effects of two potassium channel blockers, 4‐aminopyridine and tetraethylammonium, on the modulation of electrically stimulated release of endogenous dopamine from rat striatal slices. Maximally effective concentrations of the D2 dopamine receptor agonist N‐0437 (10 nM) and of adenosine (50 μM) caused a 30% inhibition of evoked dopamine overflow, and their effects were additive. When coperfused with N‐0437, both 4‐aminopyridine and tetraethylammonium blocked the inhibition caused by N‐0437 in a dose‐dependent manner. 4‐Aminopyridine was approximately three orders of magnitude more potent than tetraethylammonium, with complete blockade occurring at 3μM and 1 mM, respectively. Binding experiments confirmed that neither 4‐aminopyridine nor tetraethylammonium was a direct‐acting D2 dopamine receptor antagonist at the concentration necessary to block the release‐modulatory effect of D2 receptor activation. In contrast, the inhibitory modulation produced by adenosine was not affected by 4‐aminopyridine (30 μM) or tetraethylammonium (1 mM). These results suggest that D2 dopamine and A1 adenosine receptors inhibit dopamine release in the striatum by different mechanisms. D2 dopamine autoreceptor action appears to involve potassium channels, whereas A1 adenosine receptor action does not.
Regulation and Glucocorticoid‐Independent Induction of Lipocortin I in Cultured Astrocytes
Authors: Gebicke‐Haerter Peter J. , Schobert Angelika , Dieter Peter , Honegger Paul , Hertting Georg
Published: October 2006

Abstract: Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP‐, calcium ionophore A23187‐, or tetradecanoyl phorbol acetate (TPA)‐stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC‐mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2(PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2–3‐week‐old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum‐and GC‐free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed. The data raise the hypothesis that enriched astrocyte cultures synthesize steroid‐like compounds (neurosteroids).
Immunocytochemical Demonstration of Na+,K+‐ATPase in Internodal Axolemma of Myelinated Fibers of Rat Sciatic and Optic Nerves
Authors: Mata Marina , Fink David J. , Ernst Stephen A. , Siegel George J.
Published: October 2006

Abstract: We used postembedding electron microscopic immunocytochemistry with colloidal gold to determine the ultrastructural distribution of Na+,K+‐ATPase in the sciatic and optic nerves of the rat. Using a polyclonal antiserum raised against the denatured catalytic subunit of brain Na+,K+‐ATPase, we found immunoreactivity along the internodal axolemma of myelinated fibers in both nerves. This antiserum did not produce labeling of nodal axolemma. These results suggest that an important site of energy‐dependent sodium‐potassium exchange is along the internodal axolemma of myelinated fibers in the mammalian CNS and PNS and that there may be differences between the internodal and nodal forms of the enzyme.
Fructose 2, 6‐Bisphosphate in Hypoglycemic Rat Brain
Authors: Ambrosio Santiago , Ventura Francesc , Rosa Jose Luis , Bartrons Ramon
Published: October 2006

Abstract: Fructose 2,6‐bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6‐bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6‐bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6‐bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6‐phosphofructo‐2‐kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6‐phosphate). These results suggest that fructose 2,6‐bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6‐phosphofructo‐l‐kinase and glycolysis.
Differential Effects of Acidic and Basic Fibroblast Growth Factors on Spinal Cord Cholinergic, GABAergic, and Glutamatergic Neurons
Authors: Sweetnam Paul M. , Sanon Henry R. , White Linda A. , Brass Barry J. , Jaye Michael , Whittemore Scott R.
Published: October 2006

Abstract: When spinal cord cultures from embryonic day 12 rats were cultured at low density, both acidic and basic fibroblast growth factors significantly increased neuronal survival and neurite outgrowth in a dose‐dependent manner. The effects of acidic fibroblast growth factor were independent of heparin, in contrast to its mitogenic effects on both NIH3T3 cells and cerebral cortical astrocytes. In high‐density cultures, acidic fibroblast growth factor increased choline acetyltransferase activity by 57%, glutamic acid decarboxylase activity by 58%, and aspartate aminotransferase activity by 65%. Basic fibroblast growth factor increased choline acetyltransferase activity by 73% and glutamic acid decarboxylase activity by 200% but decreased aspartate aminotransferase activity by 40%. Growing these cultures in the presence of a mitotic inhibitor did not significantly alter the effect of acidic or basic fibroblast growth factor on these enzyme activities. These results demonstrate that acidic and basic fibroblast growth factors differentially affect neurotransmitter enzyme levels of multiple classes of neurons, rather man having effects on a single neuronal population.
Receptor Binding, Endocytosis, and Mitogenesis of Insulin‐Like Growth Factors I and II in Fetal Rat Brain Neurons
Authors: Nielsen Finn C. , Wang Enmei , Gammeltoft Steen
Published: October 2006

Abstract: Cell surface binding, internalization, and biological effects of insulin‐like growth factors (IGFs) I and II have been studied in primary neuronal cultures from developing rat brain (embryonic day 15). Two types of IGF binding sites are present on the cell surface. The IGF‐I receptor α‐subunit (Mr 125,000) binds IGF‐I with a KD of 1 nM and IGF‐II with 10 times lower affinity. The mannose‐6‐phosphate (Man‐6‐P)/IGF‐II receptor (Mr 250,000) binds IGF‐II with a KD of 0.5 nM and IGF‐I with 100 times lower affinity. Surface‐bound IGF‐I and IGF‐II are internalized by their respective receptors and degraded to amino acids. Man‐6‐P increases the receptor binding and internalization of IGF‐II but not those of IGF‐I. Neuronal synthesis of RNA and DNA is increased twofold by IGF‐I with 10 times higher potency than IGF‐II. Antibody 3637, which blocks receptor binding of IGF‐II, has no effect on the DNA response to IGF‐I or IGF‐II. Double immunocytochemical staining with antibodies to bromodeoxyuridine and neurofilament shows that >80% of the bromodeoxyuridine‐positive cells become neurofilament positive. It is concluded that IGF‐I and IGF‐II bind to two receptors on the surface of neuronal precursor cells that mediate endocytosis and degradation of IGF‐I and IGF‐II. Proliferation of neuronal precursor cells is stimulated by IGF‐I and IGF‐II via activation of the IGF‐I receptor.
Ca2+o‐Independent Veratridine‐Evoked Acetylcholine Release from Striatal Slices Is Not Inhibited by Vesamicol (AH5183): Mobilization of Distinct Transmitter Pools
Authors: Adam‐Vizi Vera , Deri Z. , Vizi E. S. , Sershen H. , Lajtha A.
Published: October 2006

Abstract: The effect of 2‐(4‐phenylpiperidino)cyclohexanol (AH5183 or vesamicol), a compound known to block the uptake of acetylcholine (ACh) into cholinergic synaptic vesicles, on the release of endogenous and [14C]ACh from slices of rat striatum was investigated. ACh release was evoked either by electrical stimulation or by veratridine. The effect of electrical stimulation was entirely dependent on external Ca2+. By contrast, veratridine (40 μM) also enhanced ACh release in the absence of Ca2+. Indeed, with veratridine two components were clearly distinguished: one dependent on external Ca2+ and the other not. Vesamicol inhibited [14C]ACh release evoked by both veratridine and electrical stimulation in the presence of external Ca2+, provided it was added to the tissue prior to loading with [14C]choline. With the same treatment vesamicol only slightly affected the release of endogenous ACh. Under the same conditions the Ca2+‐independent [14C]ACh release evoked by veratridine was not prevented by vesamicol. The differential responsiveness to vesamicol suggests that ACh pools involved in Ca2+o‐dependent ACh release are different from those mobilized during Ca2+o‐independent ACh release.
Metabolism and Release of Glutamate in Cerebellar Granule Cells Cocultured with Astrocytes from Cerebellum or Cerebral Cortex
Authors: Westergaard Niels , Fosmark Hanne , Schousboe Arne
Published: October 2006

Abstract: Cerebellar granule cells were cocultured with astrocytes from either cerebral cortex or cerebellum in two different systems. In one system the cells were plated next to each other only sharing the culture medium (separated cocultures) and in the other system the granule cells were plated on top of a preformed layer of astrocytes (sandwich cocultures). Using astrocytes from cerebellum, granule cells developed morphologically and functionally showing a characteristic high activity of the glutamate synthesizing enzyme aspartate aminotransferase (AAT) as well as a high stimuluscoupled transmitter release regardless of the culture system, i.e., granule cells could grow on top of cerebellar astrocytes as well as next to these cells. In the case of cerebral cortex astrocytes it was found that cerebellar granule cells did not develop (11% survival) when seeded on top of these astrocytes. This was indicated by the morphological appearance of the cultures as well as by a negligible difference between the AAT activity in sandwich cocultures and astrocytes cultured alone. On the other hand, granule cells in separated cocultures with cerebral cortex astrocytes exhibited a normal morphology and a high activity of AAT as well as a large stimulus‐coupled transmitter release. Cerebellar and cortical astrocytes expressed the astrocyte specific enzyme glutamine synthetase in a glucocorticoid‐inducible form regardless of the culture system. The results show that under conditions of direct contact between granule cells and astrocytes, regional specificity exists with regard to neuron‐glia contacts. This specificity does not seem to involve soluble factors present in the culture medium because in separated cocultures the cerebellar granule cells developed normally regardless of the regional origin of the astrocytes.
Soluble 5′‐Nucleotidase Activities in Rat Brain
Authors: Orford Michael , Mazurkiewicz Dorota , Saggerson David
Published: October 2006

Abstract: 5′‐Nucleotidase activity was assayed in 105,000‐g supernatants from rat brain by following conversion of [3H]AMP into adenosine. The effect of ATP on this process was complex and suggested the presence of at least two soluble 5′‐nucleotidase activities: one inhibited by ATP and another activated by ATP. The relative proportions of these activities differed considerably among brain regions. Activity changes induced by hypothyroidism also suggested that these activities may be regulated independently. These findings may have consequences for the regional regulation of adenosine formation in the brain.
Caffeine‐Sensitive Calcium Stores in Bovine Adrenal Chromaffin Cells
Authors: Liu Pei‐Shan , Lin Yi‐Jen , Kao Lung‐Sen
Published: October 2006

Abstract: Caffeine was used to study the intracellular Ca2+ pools of bovine chromaffin cells. Its effects on cytosolic Ca2+ concentration ([Ca2+]i) were examined using fura‐2. Caffeine caused a transient increase in [Ca2+]i in the presence or absence of extracellular Ca2+. In the former case, the caffeineinduced [Ca2+]i increase was higher and stayed above the basal value for several minutes. In the latter case, the [Ca2+]i rise was lower and fell to the basal level within 1 min. These results suggest that caffeine increases [Ca2+]i by causing both Ca2+ influx and Ca2+ release from intracellular pools. In the absence of extracellular Ca2+, ionomycin but not caffeine caused a further increase in [Ca2+]i in cells that had been treated with caffeine. Apparently there are at least two intracellular Ca2+ pools, only one of which is sensitive to caffeine. The caffeine‐induced [Ca2+]i rise became smaller when the cells were pretreated with the inositol trisphosphate‐generating agonists, methacholine and bradykinin. In addition, metha‐choline was unable to initiate a [Ca2+]i transient after the cells had been treated with caffeine. The results indicate that the caffeine‐sensitive Ca2+ pools overlap with the inositol trisphosphate‐sensitive pool and that the size, of the latter pool is smaller than that of the former. The caffeine‐sensitive Ca2+ pools were refilled after high K+ treatment, which suggests that the caffeine‐sensitive Ca2+ pools may be important in buffering the cytosolic Ca2+. The effect of caffeine on [Ca2+]i is not due to inhibition of phosphodiesterase. Our results support a Ca2+ entry model in which depletion of intracellular Ca2+ pools controls the rate of Ca2+ entry across the plasma membrane.
Regulation of Intracellular Calcium in Cerebellar Granule Neurons: Effects of Depolarization and of Glutamatergic and Cholinergic Stimulation
Authors: Ciardo Alberto , Meldolesi Jacopo
Published: October 2006

Abstract: The regulation of the cytosolic free Ca2+ concentration ([Ca2+]i]) was investigated by microfluorimetry in single cerebellar granule neurons exposed to various treatments (high K+, glutamate, or acetylcholine) and drugs. The responses to the treatments developed asynchronously during cell culture, with high K+ and glutamate reaching their maxima at 6 and 7 days in vitro and acetylcholine at 9 days in vitro. The biphasic [Ca2+]i] transients induced by high K+ (an initial peak, followed by a plateau 30–40% of the peak, both sustained by dihydropyridine‐sensitive voltage‐gated Ca2+ channels) were dissipated by washing with fresh medium or, more rapidly, by addition of excess EGTA (t1/2= 11 ± 2 and 3 ± 0.6 s, respectively). Compared to those induced by high K+, the [Ca2+]i] transients induced by glutamate administered in Mg2+‐free medium were much more variable. An initial peak, sustained by voltage‐gated Ca2+ channels, was visible in only ∼50% of the cells and disappeared when multiple glutamate pulses were administered. In the rest of the population, the transients were monophasic, with persistent plateaus sustained only in part (30–40%) by voltage‐gated Ca2+] channels. These plateaus were either stable or (in 10% of the cells) slowly growing to high [Ca2+]i] values. Addition of excess EGTA caused (Ca2+]i] to recover almost as fast as in the cells depolarized with K+ (t1/2 = 5 ± 1.8s), whereas after washing or N‐methyl‐D‐aspartate (NMDA) receptor blockade the recovery occurred, but was slow. These results indicate that, in glutamate‐stimulated cells, Ca2+ extrusion is affected only slightly, whereas Ca2+ influx is stimulated persistently via multiple pathways [voltage‐gated channels, NMDA receptors, and possibly other(s) to be characterized]. A third type of stimulatory response, a rapid Ca2+]i] spike (resembling spontaneous fluctuations observed in a few cells only), was elicited by acetylcholine administered in Mg2+‐free medium. Because this response was prevented by the blockade of not only muscarinic, but also NMDA receptors, as well as Na+ and Ca2+ channels, it appears to require the activation of action potentials and synaptic connections between the cells of the culture. An additional effect of acetylcholine was inhibitory and consisted in a depression of the glutamate‐ and K+‐induced plateaus, possibly occurring via a negative modulation of voltage‐gated Ca2+ channels.
Release of Endogenous and Newly Synthesized Glutamate and of Other Amino Acids Induced by Non‐N‐Methyl‐D‐Aspartate Receptor Activation in Cerebellar Granule Cell Cultures
Authors: Levi Giulio , Patrizio Mario , Gallo Vittorio
Published: October 2006

Abstract: Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures. Kainic acid (KA; 50 μM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine. Quisqualic acid (QA) and, to a lesser degree, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) (both 50 μM) enhanced the release of the following amino acids in the order glutamate > aspartate ≥ taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way. The release of glutamate induced by KA was partially (43%) Ca2+ dependent. The other release‐inducing effects of KA and QA were not Ca2+ dependent. In all cases, the evoked release could be prevented by the non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor antagonist 6‐cyano‐2,3‐hydroxy‐7‐nitroquinoxaline, and thus appeared to be receptor mediated. NMDA (5 and 50 μM) had no releaseinducing activity. The KA‐, QA‐, and AMPA‐evoked release of newly synthesized [3H]glutamate and [3H]aspartate (formed in the cells exposed to [3H]glutamine) was very similar to the evoked release of endogenous glutamate and aspartate. On the other hand, the release of preloaded D‐[3H]aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 μM KA was twice as high as that of endogenous glutamate. In the case of high [K+] depolarization, in contrast, the release of preloaded D‐[3H]aspartate was ∼30% lower than that of endogenous glutamate. The reasons for the above differences in the susceptibility of the various glutamate pools to being released may be the following: Non‐NMDA receptor agonists cause essentially a carrier‐mediated efflux of glutamate from a cytoplasmic pool that is preferentially labeled by exogenous D‐[3H]aspartate, whereas depolarization with high [K+] causes mainly an exocytotic‐like release from a vesicular pool to which exogenous D‐[3H]aspartate has a more limited access.
Inhibitors of Urokinase and Thrombin in Cultured Neural Cells
Authors: Wagner Steven L. , Lau Alice L. , Nguyen Ann , Mimuro Jun , Loskutoff David J. , Isackson Paul J. , Cunningham Dennis D.
Published: October 2006

Abstract: Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum‐free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate‐stable complexes with 125I‐urokinase or 125I‐thrombin. Rinsed glioblastoma cells possessed two components that complexed 125I‐urokinase. One was type 1 plasminogen activator inhibitor (PAI‐1), because the 125I‐urokinase‐containing complexes were immunoprecipitated with anti‐PAI‐1 antibodies. The other component formed complexes with 125I‐urokinase that were not recognized by antibodies to PAI‐1 or protease nexin‐1 (PN‐1). Its identity is unknown. In addition to these cell‐bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I‐urokinase; one was PAI‐1, and the other was PN‐1. The secreted PN‐1 also formed complexes with 125I‐thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I‐urokinase or 125I‐thrombin. However, human neuroblastoma cells did contain very low levels of PN‐1 mRNA and PN‐1 protein. Added PN‐1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN‐1 and blocked the ability of PN‐1 to form complexes with 125I‐urokinase. Thus, cell‐bound PN‐1 was a specific thrombin inhibitor. Together, these studies show that urokinase and thrombin inhibition is mediated primarily by nonneuronal cells of the CNS, although neuroblastoma cells can regulate both the activity and target protease specificity of PN‐1.
Substance P‐Induced Release of Prostaglandins from Astrocytes: Regional Specialisation and Correlation with Phosphoinositol Metabolism
Authors: Marriott Derek R. , Wilkin Graham P. , Wood John N.
Published: October 2006

Abstract: Addition of substance P (SP) to astrocytes cultured from rat neonatal spinal cord evoked a time‐and concentration‐dependent increase in the accumulation of phosphoinositol and the release of prostaglandin (PG) D2 and PGE2. Both basal and stimulated releases were reduced to similar levels by indomethacin. In contrast, astrocytes cultured from cerebral cortex and cerebellum showed no SP‐stimulated increase in phosphoinositol accumulation or release of PGs. Release of PGD2 and PGE2 was, however, stimulated by the calcium ionophore A23187, and both phosphoinositol accumulation and PG release were stimulated from cortical astrocytes incubated in the presence of serum. The results from this study suggest that SP‐stimulated phosphoinositol accumulation and release of PGs from cultured rat neonatal astrocytes are regionally specialised in favour of cells derived from spinal cord.
Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence
Authors: Tansey Francine A. , Farooq Muhammad , Cammer Wendy
Published: October 2006

Abstract: The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white‐matter regions of rat brain, and in Vibratome sections as well as paraffin sections.
t‐[35S]Butylbicyclophosphorothionate Binding under Equilibrium and Nonequilibrium Conditions: Differential Effects of Barbiturates and γ‐Aminobutyric Acid in the Long‐Sleep and Short‐Sleep Selected Mouse Lines
Authors: McIntyre Todd , Skolnick Phil
Published: October 2006

Abstract: Significant differences were demonstrated between the long‐sleep (LS) and short‐sleep (SS) selected mouse lines in the abilities of barbiturates and γ‐aminobutyric acid (GABA) to inhibit t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to well‐washed cerebral cortical membranes. Thus, using phenobarbital to initiate the dissociation of [35S]TBPS, the extent of inhibition was significantly greater in LS mice (but not SS mice) than would be predicted using equilibrium conditions. Pentobarbital had the opposite effect, causing [35S]TBPS to dissociate to a greater extent in SS than LS membranes. [35S]TBPS binding was dissociated from LS and SS membranes by GABA to a greater and lesser extent, respectively, than would be predicted from equilibrium studies. Because no line differences in the potencies of these drugs to inhibit [35S]TBPS binding were found using equilibrium conditions, these results indicate that the association rates of barbiturates and GABA may be different between these lines. These findings are consistent with neurochemical studies indicating differences in the benzodiazepine/GABA receptor‐chloride channel complex in these selected lines and may explain their differential sensitivities to certain agents acting through this supramolecular complex.
Characterisation of the Glycine Modulatory Site of the N‐Methyl‐d‐Aspartate Receptor‐Ionophore Complex in Human Brain
Authors: Procter A. W. , Stratmann G. C. , Francis P. T. , Lowe S. L. , Bertolucci P. H. F. , Bowen D. M.
Published: October 2006

Abstract: [3H]Glycine binding and glycine modulation of [3H]MK‐801 binding have been used to study the glycine allosteric site associated with the N‐methyl‐D‐aspartate receptor complex in postmortem human brain. The effect of glycine on [3H]MK‐801 binding appeared sensitive to duration of terminal coma, and possibly postmortem delay. Thirty percent of the binding occurred in a subfraction of brain tissue and did not show enhancement by glycine and glutamic acid. [3H]Glycine binding to a subfraction free from this component was studied and showed high specific binding. KD and Bmax values showed considerable intersubject variability which did not appear to be due to demographic features or to tissue content of amino acids with an affinity for this site. The pharmacological characteristics of binding in this subfraction and a correlation between Bmax values and the maximal enhancement of [3H]MK‐801 binding by glycine are consistent with [3H]glycine binding occurring to an N‐methyl‐D‐aspartate receptor complex associated site. Further support for this is provided by a significantly lower Bmax value for [3H]glycine binding in subjects with Alzheimer's disease and reduced glycine enhancement of [3H]MK‐801 binding. However, the effect of perimortem factors makes it difficult to confidently attribute this solely to a disease‐related change in the receptor. The possible role of the glycine allosteric site in the treatment of neuropsychiatric disorders is discussed.
The Turnover of Rat Cortical α1‐Adrenoceptors Is Not Modified by Repeated Electroconvulsive Treatment
Authors: Nowak Gabriel , Zak Joanna
Published: October 2006

Abstract: The effect of repeated treatment with electroconvulsive shock (ECS) on the turnover of cortical α1‐adrenoceptors in rats was measured using the N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ)‐induced irreversible receptor inactivation method. Repeated treatment with ECS did not affect parameters (the synthesis rate constant r, the degradation rate constant k) of α1‐adrenoceptor turnover. Because increase in the density of α1‐adrenoceptors in the ECS‐treated group disappears later during measurement of turnover, several calculation possibilities were discussed. The present data confirm that repeated treatment with ECS produces a short‐lasting up‐regulation of cortical α1‐adrenoceptors, but does not affect the turnover of this receptor type.
Evidence Against Impaired Brain Microtubule Protein Polymerization at High Glucose Concentrations or During Diabetes Mellitus
Authors: Eaker Ervin Y. , Angelastro James M. , Purich Daniel L. , Sninsky Charles A.
Published: October 2006

Abstract: Previous studies suggest that brain microtubule protein exposed to high glucose levels or isolated from diabetic rats can become glucosylated and that this impairs GTP‐induced microtubule polymerization. We set out to extend that investigation to define the mechanistic basis for inhibition of microtubule assembly during diabetes or on incubation at high glucose levels. Rat and bovine brain microtubule protein was purified by cycles of polymerization/depolymerization. When microtubules were incubated for 1 h in either buffer or buffer containing glucose (up to 165 mM), there was no difference in polymerization, a finding contrary to the earlier study. Other rats were injected with vehicle or streptozotocin (90 mg/kg) to induce diabetes as evidenced by serum glucose in excess of 300 mg%, and at 4 weeks, brain microtubule protein was isolated by the polymerization cycling method. Again, there was no difference in the amount or purity of isolated microtubule protein between control or diabetic rats. We also observed no increase in microtubule glucosylation, and GTP‐induced polymerization in vitro was indistinguishable for protein derived from brains of normal rats and rats with diabetes as measured by turbidity or electron microscopy. Our results suggest that in vitro incubation with glucose or in vivo elevation of glucose during diabetes fails to impair microtubule polymerization, pointing to other mechanisms for the neuropathy associated with diabetes.
Isolation of a Novel cDNA Corresponding to a Transcript Expressed in the Choroid Plexus and Leptomeninges
Authors: Lecain Eric , Zelenika Diana , Laine Marie‐Christine , Rhyner Thomas , Pessac Bernard
Published: October 2006

Abstract: The CNS is composed of neurons and glial cells (i.e., astrocytes, oligodendrocytes, and microglia). The brain communicates with the blood circulation through choroid plexus and meninges as well as through the blood‐brain barrier. To identify transcripts specifically expressed in a distinct brain cell type, we have previously constructed a subtracted cDNA library from the poly(A)+ RNAs of a velate protoplasmic‐like astrocytic cell line, designated D19. This library was screened in order to isolate transcripts over‐expressed in this astroglial cell line versus another astroglial cell line. Of the six recombinants which have been isolated, three sequences have not been described. Their sizes ranged from 100 to 200 bp and they hybridized to mRNAs expressed in vivo inside and outside the CNS. We have constructed a sizeselected D19 cDNA library in order to obtain the full length cDNAs corresponding to the three undescribed sequences. We report here the isolation of a 1.6 kb cDNA corresponding to one of these recombinants, named p14. Its sequence has not yet been described and its deduced amino acid sequence codes for a 43 kDa protein with a putative signal peptide. In situ hybridization shows that this transcript is expressed at a high level in choroid plexus and leptomeninges and also in perivascular cells in the adult mouse brain. It is also expressed in cell subsets of kidney and gonads as well as in perivascular cells in skeletal and cardiac muscles. These localizations suggest that the encoded protein might be involved in transport processes and hormonally controlled.
Characterization of Two Chromaffin Cell Populations Isolated from Bovine Adrenal Medulla
Authors: Moro M. A. , Garcia A. G. , Langley O. K.
Published: October 2006

Cultures of chromaffin cells isolated from the bovine adrenal medulla have been extremely useful for investigating secretory mechanisms, but such cultures used up to the present time represent mixed populations of adrenergic and nor‐adrenergic cells. This report describes how, with slight modifications to standard procedures, two separate chromaffin cell populations may be separated from bovine adrenal medullae. These two cell fractions have been characterized by biochemical, immunocytochemical, and morphological techniques as enriched populations of adrenergic or noradrenergic cells, respectively. The adrenergic cell‐enriched fraction consists of >90% adrenergic cells, whereas the noradrenergic cell‐enriched fraction contains >60% noradrenergic cells. We also demonstrate that these cells may be cultured with their secretory machinery intact: analysis of secreted catecholamines from nicotine‐ or high K+ concentration‐stimulated cells cultured from each fraction confirms that adrenaline is the major catecholamine secreted by one fraction, whereas noradrenaline is mainly secreted by the other.
A Novel Synaptic Vesicle‐Associated Phosphoprotein: SVAPP‐120
Authors: Bahler Martin , Klein Ronald L. , Wang James K. T. , Benfenati Fabio , Greengard Paul
Published: October 2006

Generation of antibodies and direct protein sequencing were used to identify and characterize proteins associated with highly purified synaptic vesicles from rat brain. A protein doublet of low abundance of 119 and 124 kDa apparent molecular mass [synaptic vesicle‐associated phosphoprotein with a molecular mass of 120 kDa (SVAPP‐120)] was identified using polyclonal antibodies. SVAPP‐120 was found to copurify with synaptic vesicles and to be enriched in the purified synaptic vesicle fraction to the same extent as synapsin I. Like synapsin I, SVAPP‐120 is not an integral membrane protein because it was released from synaptic vesicles by high salt concentrations. This protein was demonstrated to be brain specific, and its distribution in various brain regions paralleled the distribution of synapsin I and synaptophysin. During the postnatal development of the rat cortex and cerebellum, its expression correlated with synaptogenesis. SVAPP‐120 was demonstrated to be a phosphoprotein both in vivo and in vitro. It was shown to be phosphorylated on serine and to a lesser extent on threonine residues. These results provide evidence that SVAPP‐120 represents a novel synaptic vesicle‐associated phosphoprotein. In addition, aldolase, a glycolytic enzyme, and αc‐adaptin, a clathrin assembly‐promoting protein, were identified on purified synaptic vesicles by direct protein sequencing.
Protein Kinase C (αand β) Immunoreactivity in Rabbit and Rat Retina: Effect of Phorbol Esters and Transmitter Agonists on Immunoreactivity and the Translocation of the Enzyme from Cytosolic to Membrane Compartments
Authors: Osborne N. N. , Broyden N. J. , Barnett N. L. , Morris N. J.
Published: October 2006

Using a monoclonal antibody against protein kinase C (PKC) that recognises the isoenzymes α, βI, and βII, positive immunoreactivity was observed throughout the cytoplasm of bipolar cells in both rat and rabbit retinas. PKC immunoreactivity was also associated with the outer segment of photoreceptors in the rabbit retina and presumed amacrine cells in the rat retina. The PKC immunoreactivity in the retina was unaffected in content or localisation in rats kept in continuous dark or light conditions over a period of 6 days. The localisation of PKC immunoreactivity in retinas was similar in 6‐day‐old, 16 day‐old, or adult rabbits. However, the content of PKC was lowest at the youngest stage and highest in the adult rabbit retinas. Of the two active phorbol esters studied, only phorbol 12, 13‐dibutyrate (PDbut) at a concentration of 1 μM caused the PKC immunoreactivity in rabbit retina bipolar cells to be „transported” from the perikarya towards the axonal terminal processes. Biochemical analyses showed that most of the cytosolic PKC was translocated to the membrane compartment following such treatment. The other phorbol ester, phorbol 12‐myristate 13‐acetate, even at a concentration of 10 μM did not cause a similar transport of PKC immunoreactivity in the bipolar cells, although a partial translocation of the enzyme could be followed biochemically. Both the translocation and transport of PKC by PDbut could be reversed by simply incubating the retinas in physiological solution for 60 min. The „transport” and translocation processes were not obviously affected by the transport inhibitor colchicine or by known PKC inhibitors such as staurosporine, H‐7, sphingosine, or polymyxin B. In addition, agonists known to stimulate inositol phosphates in the retina, viz., carbachol, noradrenaline, and quisqualate, or 4‐aminopyridine did not cause a translocation or „transport” of PKC as observed for the phorbol esters.
Effect of Zinc on Calmodulin‐Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex
Authors: Weinberger R. P. , Rostas J. A. P.
Published: October 2006

Abstract: The effect of increasing concentrations of Zn2+ (1 μM‐5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2‐M) fractions from rat cerebral cortex and purified calmodulinstimulated protein kinase II (CMKII). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2‐M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin‐stimulated conditions showed a dose‐dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 μM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin‐stimulated phosphoproteins in P2‐M and S3 showed biphasic behavior inhibition at <100 μM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the a and β subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin‐stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn2+‐stimulated, Ca2+/ calmodulin‐independent activity at concentrations of > 100 μM that produces a redistribution of activity biased toward autophosphorylation and an subunit with an altered mobility on sodium dodecyl sulfate‐containing gels.
Effects of Inhibitors of Oligosaccharide Processing on P0Protein Synthesis and Incorporation into PNS Myelin
Authors: Smith Marion E.
Published: October 2006

Four inhibitors of Oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the Oligosaccharide chains of the Po and 19‐kDa glycoproteins. Several different inhibitors of Oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21–24 days of age) and the uptake of 14C‐amino acid and [3H] fucose or [3H] mannose was measured in Po and the 19‐kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H] Mannose was not found as suitable as [3H] fucose as an Oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H] mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated Oligosaccharide chains. Endoglycosidase H cleaved ∼50% of the Po labeled with [3H] fucose and 14C‐amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one‐half the [3H] fucose label on the protein, whereas one‐half remained on the Oligosaccharide chain of the undegraded Po, indicating that at least one‐half the Pohas a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H] fucose into the Oligosaccharide chains of Po and the 19‐kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. Po synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19‐kDa glycoprotein. Both the incompletely processed Po and the 19‐kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.
Interstitial 3‐Methoxytyramine Reflects Striatal Dopamine Release: An In Vivo Microdialysis Study
Authors: Brown Erin E. , Damsma Geert , Gumming Paul , Fibiger Hans C.
Published: October 2006

Previous ex vivo studies have provided indirect evidence that the dopamine (DA) metabolite 3‐methoxytyramine (3‐MT) may be a useful index of DA release in vivo. In the present study, in vivo microdialysis was utilized to assess directly the relationship between extracellular DA and 3‐MT in the striatum of rats following a variety of pharmacological manipulations. Apomorphine, a DA receptor agonist, produced a rapid, transient decrease in both DA and 3‐MT. Conversely, the DA receptor antagonist haloperidol produced a concomitant increase in extracellular DA and 3‐MT. Increases in DA and 3‐MT were also noted following the administration of the DA uptake inhibitor, bupropion. Local application of tetrodotoxin resulted in the complete elimination of measurable amounts of DA and 3‐MT in the dialysate. γ‐Butyrolactone also greatly decreased DA and 3‐ MT. Finally, d‐amphetamine produced a large increase in DA and 3‐MT in animals that had been treated previously with γ‐butyrolactone. The Pearson correlation coefficients for DA and 3‐MT following these manipulations ranged from 0.87 to 0.97. These data indicate that interstitial 3‐MT is an accurate index of DA release. However, when compared with previous ex vivo findings, the present results also suggest that changes in tissue concentrations of 3‐MT may not reliably reflect DA release following certain pharmacological manipulations.
A Specific Trarisduction Mechanism for the Glutamate Action on Phosphoinositide Metabolism via the Quisqualate Metabotropic Receptor in Rat Brain Synaptoneurosomes: I. External Na+ Requirement
Authors: Guiramand Janique , Vignes Michel , Mayat Ebrahim , Lebrun Fabienne , Sassetti Isabella , Récasens Max
Published: October 2006

: The characteristics of the transduction mechanism(s) activated by glutamate (Glu) via the quisqualate metabotropic receptor, as well as by depolarizing agents, to trigger formation of inositol phosphates (IPs) were investigated in 8‐day‐old rat forebrain Synaptoneurosomes. The replacement of external Na+ by various compounds (Li+, Tris+, N‐methyl‐D‐glucamine+, and sucrose) induces an increase in basal accumulation of IPs and depolarizes synaptoneurosome membranes. Under these conditions, Gluand K+‐induced accumulations of IPs are inhibited, whereas the carbachol (Carb)‐elicited response of IPs parallels the basal one. Agents increasing Na+ influx, such as veratridine and monensin, depolarize Synaptoneurosomes and stimulate formation of IPs. These stimulations are not additive with responses of IPs elicited by Glu or K+. These data suggest that (a) Glu activates phosphoinositide metabolism via a specific mechanism (distinct from that of cholinergic agonists), (b) depolarizing agents and Glu share at least one common intermediate step in their mechanisms of activation of the metabolism of IPs, and (c) the depolarization may correspond to this common step. ID addition, Na+ seems to be required for Glu stimulation of metabolism of IPs. The depolarization associated with the action of Glu on formation of IPs results neither from an influx via tetrodotoxin‐sensitive voltage‐dependent Na+ channels nor from an entry via the classically characterized Na+/Ca2+ or Na+/H+ exchangers. In fact, tetrodotoxin (2 μM) has no effect on the Glu‐or K+‐elicited response of IPs. Amiloride (>50 μM) and some of its derivatives similarly inhibit not only Glu‐and K+‐but also Carb‐evoked formation of IPs.
Neuronal Nicotinic Acetylcholine Receptors in Drosophila: Antibodies Against an α‐Like and a Non‐α‐Subunit Recognize the Same High‐Affinity α‐Bungarotoxin Binding Complex
Authors: Schloss Patrick , Betz Heinrich , Schroder Christiane , Gundelfinger Eckart D.
Published: October 2006

: ALS and ARD proteins are thought to represent a ligand binding and a structural subunit, respectively, of Drosophila nicotinic acetylcholine receptors (nAChRs). Here, antibodies raised against fusion constructs encompassing specific regions of the ALS and ARD proteins were used to investigate a potential association of these two polypeptides. Both ALS and ARD antisera removed 20‐30% of the high‐affinity binding sites for the nicotinic antagonist 125I‐α‐bungarotoxin (125I‐α‐Btx) from detergent extracts of fly head membranes. Combinations of both types of antisera also precipitated the same fraction of α‐Btx binding sites, a result suggesting that both polypeptides are components of the previously defined class I 125I‐α‐Btx binding sites in the Drosophila CNS. 125I‐α‐Btx binding to a MS2 polymerase‐ALS fusion protein containing the predicted antagonist binding region showed that the ALS protein indeed constitutes the ligand binding subunit of a nicotinic receptor complex. These data are consistent with neuronal nAChRs in Drosophila containing at least two types of subunits, ligand binding and structural ones.