bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of Myristoylated Alanine‐Rich C Kinase Substrate (MARCKS) by Proline‐Directed Protein Kinases and Its Dephosphorylation
Authors: Yamamoto Hideyuki , Arakane Futosi , Ono Tsunehiko , Tashima Kazuhiro , Okumura Eiichi , Yamada Keisuke , Hisanaga Shin‐ichi , Fukunaga Kohji , Kishimoto Takeo , Miyamoto Eishichi
Published: July 2010

Abstract: We identified two major substrates for the proline‐directed protein kinases—cdc2 kinase and tau protein kinase II (TPKII)—in the cytosol fraction from rat brains. The molecular masses of the proteins were 80 and 46 kDa. Because the 80‐kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine‐rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80‐kDa protein and purified MARCKS, we concluded that the 80‐kDa protein is indeed MARCKS. The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two‐dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline‐directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it. These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.
Effect of Nerve Growth Factor in Ethylcholine Mustard Aziridinium (AF64A)‐Treated Rats: Sensitization of Cholinergic Enzyme Activity in the Septohippocampal Pathway
Authors: Willson Christopher A. , Hanin Israel
Published: July 2010

Abstract: In this study, we examined the effects of nerve growth factor (NGF) administration on cholinergic enzyme activity in both normal and ethylcholine mustard aziridinium (AF64A)‐treated rats. Choline acetyltransferase (ChAT) and acetylcholinesterase activity were measured in the hippocampus and septum of rats chronically administered NGF (0.36–2.85 µg/day) into the lateral ventricle for 14 days. In both normal and AF64A‐treated rats, NGF increased cholinergic enzyme activity in a dose‐dependent manner. Furthermore, although NGF increased ChAT activity in normal rats by 147%, it had a greater effect in AF64A‐treated rats, increasing ChAT activity as much as 273%. NGF increased acetylcholinesterase activity in normal rats by only 125% but produced a 221% increase in this activity in AF64A‐treated rats. These data indicate that AF64A produces an increased sensitivity to NGF in cholinergic neurons.
[3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes
Authors: Fallarino F. , Genazzani A. A. , Silla S. , L'Episcopo M. R. , Camici O. , Corazzi L. , Nicoletti F. , Fioretti M. C.
Published: July 2010

Abstract: [3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4°C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of ∼70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone‐containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37°C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA‐sensitive glutamate receptors, specifically bound [3H]aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.
3‐Nitropropionic Acid Neurotoxicity Is Attenuated in Copper/Zinc Superoxide Dismutase Transgenic Mice
Authors: Beal M. Flint , Ferrante Robert J. , Henshaw Ross , Matthews Russell T. , Chan Pak H. , Kowall Neil W. , Epstein Charles J. , Schulz Jörg B.
Published: July 2010

Abstract: The mitochondrial toxin 3‐nitropropionic acid (3‐NP) produces selective striatal lesions in both experimental animals and humans. The pathogenesis of the lesions involves secondary excitotoxicity that may then lead to free radical generation. To test this further we examined the effects of 3‐NP in both transgenic (Tg) mice that carry the complete sequence for the human copper/zinc superoxide dismutase (SOD) gene as well as non‐Tg littermate controls. The Tg‐SOD mice showed a pronounced attenuation of Nissl‐stained striatal lesions compared with non‐Tg mice. Systemic administration of 3‐NP resulted in production of hydroxyl free radicals as assessed by the conversion of salicylate to 2,3‐ and 2,5‐dihydroxybenzoic acid. This production was attenuated significantly in Tg‐SOD mice. In a similar way, 3‐NP produced significant increases in 3‐nitrotyrosine/tyrosine, a marker for peroxynitrite‐mediated damage, which were significantly attenuated in Tg‐SOD mice. These results support that oxygen free radicals and peroxynitrite play an important role in the pathogenesis of 3‐NP neurotoxicity.
Adrenergic Control of Rat Pineal NO Synthase
Authors: Schaad Nicolas C. , Vanecek Jiri , Kosar Evzen , Aubry Jean‐Michel , Schulz Pierre E.
Published: July 2010

Abstract: We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We present here the evidence that chronic (5 days) norepinephrine (NE) or isoproterenol treatment prevents the effect of LL and enhances pineal NOS activity in LL animals. This effect of NE appears to be mediated by β‐adrenoceptors, because it was not mimicked by the α‐agonist phenylephrine. Pineal NOS activity was reduced in 16‐h light/8‐h dark animals treated for 4 days with the β‐adrenergic antagonist propranolol but not with the α1‐antagonist prazosin, indicating again an involvement of β‐adrenergic receptor in the control of NOS. Treatment with adrenergic antagonists did not affect cortical NOS activity, suggesting that the control of NOS is different in these two tissues or that the pineal expresses a specific isoform of the enzyme. Taken together, these data suggest that NE controls NOS in the pineal gland through β‐adrenergic receptors. To our knowledge, this represent the first demonstration of a regulation of NOS by a neurotransmitter in the CNS, as assayed under Vmax conditions.
Selective Alterations of Extracellular Brain Amino Acids in Relation to Function in Experimental Portal‐Systemic Encephalopathy: Results of an In Vivo Microdialysis Study
Authors: Raghavendra Rao V. L. , Audet R. M. , Butterworth R. F.
Published: July 2010

Abstract: Portal‐systemic encephalopathy (PSE) is characterized by neuropsychiatric symptoms progressing through stupor and coma. Previous studies in human autopsy tissue and in experimental animal models of PSE suggest that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. To assess this possibility, levels of amino acids were measured using in vivo cerebral microdialysis in frontal cortex of portacaval‐shunted rats administered ammonium acetate (3.85 mmol/kg, i.p.) to precipitate severe PSE. Sham‐operated rats served as controls. Portacaval shunting resulted in significant increases of levels of extracellular glutamine (threefold, p < 0.001), alanine (38%, p < 0.01), aspartate (44%, p < 0.05), phenylalanine (170%, p < 0.001), tyrosine (140%, p < 0.001), tryptophan (63%, p < 0.001), leucine (75%, p < 0.001), and serine (60%, p < 0.001). Administration of ammonium acetate to sham‐operated animals led to a significant increase in extracellular glutamine and taurine content, but this response was absent in shunted rats. The lack of taurine release into extracellular fluid following ammonium acetate administration in portacaval‐shunted rats could relate to the phenomenon of brain edema in these animals. Ammonium acetate administration resulted in significant increases in the extracellular concentrations of phenylalanine and tyrosine in both sham‐operated and portacaval‐shunted rats. Severe PSE was not accompanied by significant increases in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, or serine, suggesting that increased spontaneous release of these amino acids in cerebral cortex is not implicated in the pathogenesis of hepatic coma.
Detection of Free Radical Activity During Transient Global Ischemia and Recirculation: Effects of Intraischemic Brain Temperature Modulation
Authors: Globus Mordecai Y.‐T. , Busto Raul , Lin Baowan , Schnippering Holger , Ginsberg Myron D.
Published: July 2010

Abstract: To obtain direct evidence of oxygen radical activity in the course of cerebral ischemia under different intraischemic temperatures, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA) following salicylate administration. Wistar rats were subjected to 20 min of global forebrain ischemia by two‐vessel occlusion plus systemic hypotension (50 mm Hg). Intraischemic striatal temperature was maintained as normothermic (37°C), hypothermic (30°C), or hyperthermic (39°C) but was held at 37°C before and following ischemia. Salicylate was administered either systemically (200 mg/kg, i.p.) or by continuous infusion (5 mM) through a microdialysis probe implanted in the striatum. Striatal extracellular fluid was sampled at regular intervals before, during, and after ischemia, and levels of 2,3‐ and 2,5‐DHBA were assayed by HPLC with electrochemical detection. Following systemic administration of salicylate, stable baseline levels of 2,3‐ and 2,5‐DHBA were observed before ischemia. During 20 min of normothermic ischemia, a 50% reduction in mean levels of both DHBAs was documented, suggesting a baseline level of hydroxyl radical that was diminished during ischemia, presumably owing to oxygen restriction to tissue at that time. During recirculation, 2,3‐ and 2,5‐DHBA levels increased by 2.5‐ and 2.8‐fold, respectively. Levels of 2,3‐DHBA remained elevated during 1 h of reperfusion, whereas the increase in 2,5‐DHBA levels persisted for 2 h. The increases in 2,3‐ and 2,5‐DHBA levels observed following hyperthermic ischemia were significantly higher (3.8‐ and fivefold, respectively). In contrast, no significant changes in DHBA levels were observed following hypothermic ischemia. The postischemic changes in DHBA content observed following local administration of salicylate were comparable to the results obtained with systemic administration, thus confirming that the hydroxyl radicals arose within brain parenchyma itself. These results provide evidence that hydroxyl radical levels are increased during postischemic recirculation, and this process is modulated by intraischemic brain temperature. Hence, these data suggest a possible mechanism for the effects of temperature on ischemic outcome and support a key role for free radical‐induced injury in the development of ischemic damage.
Characterization of the Binding of [3H]NS 257, a Novel Competitive AMPA Receptor Antagonist, to Rat Brain Membranes and Brain Sections
Authors: Nielsen Elsebet Ø. , Johansen Tina H. , Wätjen Frank , Drejer Jørgen
Published: July 2010

Abstract: The binding of [3H]NS 257 {1,2,3,6,7,8‐hexahydro‐3‐(hydroxyimino)‐N,N‐[3H]dimethyl‐7‐methyl‐2‐oxobenzo[2,1‐b:3,4‐c′] dipyrrole‐5‐sulfonamide} to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [3H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 mM. In the presence of thiocyanate [3H]NS 257 bound to a single population of binding sites with an affinity of 225 ± 8 nM and a binding site density of 0.61 ± 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [3H]NS 257 for both α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) and l‐glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)‐quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > l‐glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [3H]NS 257 binding sites in rat brain was similar to that of [3H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.
Calcium and Photoentrainment in Chick Pineal Cells Revisited: Effects of Caffeine, Thapsigargin, EGTA, and Light on the Melatonin Rhythm
Authors: Zatz Martin , Heath James R.
Published: July 2010

Abstract: Chick pineal cells in dispersed cell culture display a persistent, photosensitive, circadian rhythm of melatonin production and release. Light pulses have at least two distinguishable effects on these cells, i.e., acute suppression of melatonin output and phase shifts (entrainment) of the underlying circadian pacemaker. Previous results linked calcium influx through voltage‐sensitive calcium channels in the plasma membrane to acute regulation of melatonin synthesis but denied a role for such influx in entrainment. Those experiments did not, however, address the role of intracellular calcium metabolism. Here we describe the effects of pulses of caffeine, thapsigargin, and EGTA on the melatonin rhythm, and their interactions with the effects of light pulses. Caffeine had two distinguishable effects on these cells, acute enhancement of melatonin output (attributable to phosphodiesterase inhibition) and phase shifts of the circadian pacemaker with a light‐like pattern (attributable to effects on intracellular calcium). Phase shifts induced by light and caffeine were not additive. Thapsigargin (which specifically blocks the pump that replenishes intracellular calcium stores, thereby increasing cytoplasmic calcium and depleting intracellular stores) had no phase‐shifting effects by itself but reduced the size of the phase advances induced by caffeine or light. Low calcium solution acutely suppressed melatonin output without inducing phase shifts or affecting those induced by caffeine or light. However, addition of EGTA (which specifically chelates calcium, thereby lowering cytoplasmic calcium and depleting intracellular stores) did reduce the size of phase advances induced by caffeine or light, in normal medium or in low calcium solution, without inducing a phase shift by itself at that phase. Taken together, these results point toward a role for intracellular calcium fluxes in entrainment of the circadian pacemaker.
Reconstitution of High‐Affinity Binding of a β‐Scorpion Toxin to Neurotoxin Receptor Site 4 on Purified Sodium Channels
Authors: Thomsen William , Martin‐Eauclaire M. F. , Rochat Hervé , Catterall William A.
Published: July 2010

Abstract: Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high‐affinity neurotoxin binding and action at neurotoxin receptor sites 1–3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a β‐scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). 125I‐Css IV, purified by reversed‐phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half‐times of 0.22 min−1 and 0.015 min−1. At equilibrium, the toxin bound to two classes of specific high‐affinity sites, a variable minor class with KD of ∼0.1 nM and a major class with a KD of ∼5 nM. Approximately 0.8 mol 125I‐Css IV was bound per mole of reconstituted, right‐side‐out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1–3 or 5, consistent with the characteristics of binding of β‐scorpion toxins to sodium channels in cells and membrane preparations. Our results show that specific, high‐affinity binding at neurotoxin receptor site 4 on purified sodium channels can be restored by reconstitution into phospholipid vesicles and provide an experimental approach to analysis of the peptide components of the toxin receptor site.
The Expression of Two Splice Variants of Metabotropic Glutamate Receptor Subtype 5 in the Rat Brain and Neuronal Cells During Development
Authors: Minakami Reiko , Iida Ken‐ichiro , Hirakawa Noriko , Sugiyama Hiroyuki
Published: July 2010

Abstract: We previously reported that a variant with extra amino acid residues exists in the metabotropic glutamate receptor subtype 5 (mGluR5). Either of the two isoforms, named mGluR5b and mGluR5a for the isoforms with and without the inserted sequence, respectively, generated Ca2+‐activated Cl− current when expressed in Xenopus oocytes. We herein report that these two isoforms are produced by the alternative splicing of the exon skipping type. When examined during the course of postnatal development, the major mGluR5 isotype mRNA was observed to switch from mGluR5a to mGluR5b in the rat hippocampus and the cerebral cortex. We also investigated two cell lines that could be differentiated into neuron‐like cells in vitro. Whereas the mGluR5b mRNA was hardly detectable in either undifferentiated or differentiated NG108‐15 cells, the relative amounts of the two variant mRNAs changed after the induction of differentiation in the P19 cells. An extracellular application of trans‐d,l‐1‐amino‐1,3‐cyclopentanedicarboxylate on the neuron‐like P19 cells induced intracellular Ca2+ mobilization, thus suggesting that the cells could express functional mGluR(s) coupled to phospholipase C and other components that could mediate the signal transduction pathway. This cell line may thus provide a model system for studying both mGluR5 expression and other mGluR‐induced phenomena at the molecular level.
Evidence that the Modulation of Membrane‐Associated Protein Kinase C Activity by an Endogenous Inhibitor Plays a Role in N1E‐115 Murine Neuroblastoma Cell Differentiation
Authors: Chakravarthy Balu R. , Wong Josée , Durkin Jon P.
Published: July 2010

Abstract: Murine neuroblastoma cells, N1E‐115, were induced to differentiate into neuron‐like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro “direct” assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC‐selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane‐associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E‐115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane‐associated PKC activity in cells and thereby modulate PKC‐related neuronal functions.
Endothelin 1 Stimulates Na+,K+‐ATPase and Na+‐K+‐Cl− Cotransport Through ETA Receptors and Protein Kinase C‐Dependent Pathway in Cerebral Capillary Endothelium
Authors: Kawai Nobutoshi , Yamamoto Toshifumi , Yamamoto Hideko , McCarron Richard M. , Spatz Maria
Published: July 2010

Abstract: The effect of endothelins (ET‐1 and ET‐3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET‐1 or ET‐3 dose‐dependently enhanced K+ uptake (EC50 = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo‐d‐Trp‐d‐Asp‐Pro‐d‐Val‐Leu). Neither the selective ETB agonists IRL 1620 [N‐succinyl‐(Glu9,‐Ala11,15)‐ET‐1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)‐ET‐1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K+‐ATPase) and bumetanide (inhibitor of Na+‐K+‐Cl− cotransport) reduced (up to 40% and up to 70%, respectively) the ET‐1‐stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12‐myristate 13‐acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K+‐ATPase and Na+‐K+‐Cl− cotransport. ET‐1‐but not PMA‐stimulated K+ uptake was inhibited by 5‐(N‐ethyl‐N‐isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET‐1‐stimulated Na+,K+‐ATPase and Na+‐K+‐Cl− cotransport activity that is not mediated by PKC.
Comparison of In Vitro Ischemia‐Induced Disturbances in Energy Metabolism and Protein Synthesis in the Hippocampus of Rats and Gerbils
Authors: Paschen Wulf , Djuricic Bogdan
Published: July 2010

Abstract: To elucidate whether the high sensitivity of gerbil compared with rat hippocampus to metabolic stress results from tissue‐specific or hemodynamic factors, ischemia‐induced metabolic disturbances [energy metabolism and protein synthesis rate (PSR)] were studied using the in vitro model of the hippocampal slice preparation. At the end of in vitro ischemia, ATP content was measured in individual slices with HPLC. In other groups of slices, PSR was measured after 120 min of recovery after in vitro ischemia. ATP breakdown was almost identical in rat and gerbil slices at all temperatures (37°C, 34°C, or 31°C) and periods of ischemia (5, 10, or 15 min) studied. In contrast to the identical rate of ATP depletion during ischemia, however, postischemic disturbances in PSR were significantly increased in gerbil slices compared with rat slices and this relationship was stable after different periods of ischemia and at different incubation temperatures. The results illustrate that the pattern of ischemia‐induced disturbances observed in vivo can also be reproduced using the in vitro model of hippocampal slice preparation, as evidenced by the postischemic disturbance in PSR. It is concluded that comparison of the extent of metabolic disturbances in gerbil and rat hippocampal slices after transient in vitro ischemia may help to elucidate the mechanisms of ischemic cell damage.
Phosphorylation of VAMP/Synaptobrevin in Synaptic Vesicles by Endogenous Protein Kinases
Authors: Nielander Henk B. , Onofri Franco , Valtorta Flavia , Schiavo Giampietro , Montecucco Cesare , Greengard Paul , Benfenati Fabio
Published: July 2010

Abstract: VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G and is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca2+/calmodulin‐dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.
Differential Targeting to the Plasma Membrane of the Torpedo 15‐kDa Proteolipid Expressed in Oocytes
Authors: Leroy Christine , Meunier François‐Marie
Published: July 2010

Abstract: Xenopus laevis oocytes were injected with poly(A)+ RNAs extracted from the electric lobes of Torpedo marmorata, which contain a homogeneous population of cholinergic neurons. These primed oocytes were able to synthesize acetylcholine and to release the neurotransmitter in a calcium‐dependent manner. Fractionation of oocyte membranes as well as immunofluorescence experiments showed that the 15‐kDa proteolipid, a common subunit of the vacuolar H+‐ATPase and of a presynaptic membrane protein capable of calcium‐dependent acetylcholine translocation called the mediatophore, was located at the oocyte plasma membrane. In contrast, oocytes injected with separate transcripts encoding the 15‐kDa proteolipid and choline acetyltransferase were unable to release acetylcholine in spite of an equivalent acetylcholine content and a higher level of 15‐kDa proteolipid expression. We observed by immunofluorescence that under these conditions, the 15‐kDa proteolipid was expressed in granular cytoplasmic membranes, which were then identified as being Golgi vesicles by cell fractionation. The striking difference in the distribution of the 15‐kDa proteolipid expressed in oocytes primed with Torpedo electric lobe mRNA as compared with that seen in oocytes injected with the cRNA alone suggests that another protein endogenous to the electric lobe may be implicated in the localization of the 15‐kDa proteolipid at the plasma membrane. Moreover, such a targeting mechanism could contribute to the capacity of electric lobe mRNA‐injected oocytes to release acetylcholine.
Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo
Authors: Schmid Lorenz , Bottlaender Michel , Fuseau Chantal , Fournier Denis , Brouillet Emmanuel , Mazière Mariannick
Published: July 2010

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [11C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [11C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half‐maximal inhibitory doses for the high‐affinity binding site were similar to those found in cerebellum and neocortex and ∼100‐fold higher for the low‐affinity binding site. The low‐affinity binding site accounted for <32% of the specific [11C]‐flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.
Schwannoma‐Derived Growth Factor Interacts with the Epidermal Growth Factor Receptor
Authors: Maher Pamela A. , Schubert David
Published: July 2010

Abstract: Schwannoma‐derived growth factor (SDGF) is a potent mitogen and neuronal differentiation factor. Because of its relationship to epidermal growth factor (EGF) and the heregulins, it was asked if SDGF interacts with the EGF receptor or HER2/neu. SDGF binds to and causes the phosphorylation on tyrosine of the EGF receptor but not HER2/neu.
Differentiation Alters the Expression of the Two Splice Variants of the Serotonin 5‐HT3 Receptor‐A mRNA in NG108‐15 Cells
Authors: Emerit M. B. , Martres M. P. , Miquel M. C. , El Mestikawy S. , Hamon M.
Published: July 2010

Abstract: The serotonin 5‐HT3‐A receptor (5‐HT3R‐A) mRNA has been shown recently to be expressed as two forms (5‐HT3R‐AL and 5‐HT3R‐AS) varying by the presence or the absence of a sequence of 18 bases in the region corresponding to the second cytoplasmic domain of the receptor, and generated by alternative splicing at the level of the 3′ acceptor site of exon 9. As the long form of the receptor exhibits a potential phosphorylation site that is disrupted by the alternative splicing, the hypothesis of functional identity and stochastic expression of these two variants was questioned. In the present study, we used quantitative reverse transcriptase‐polymerase chain reaction to examine the possible influence of culture conditions on the expression and the alternative splicing of 5‐HT3R‐A mRNA in NG108‐15 clonal cells. Cell differentiation induced by dibutyryl cyclic AMP or theophyllin plus prostaglandin E1 in the presence of 10% serum reduced by threefold the expression of total 5‐HT3R‐A mRNA, and favored the short form of the message as the ratio S/L (5‐HT3R‐AS mRNA/5‐HT3R‐AL mRNA) shifted from 2.23 to 7.33 after 9 days of treatment. Culture with 0.3% serum (instead of 10%) lowered by 10‐fold the level of expression of total 5‐HT3R‐A mRNA, but only slightly reduced the S/L ratio. However, this ratio fell to 0.06 in the presence of 0.3% serum plus 10 ng/ml basic fibroblast growth factor. These results demonstrate that external factors can influence the differential expression of the two variants of the 5‐HT3R‐A in NG108‐15 cells. Appropriate culture conditions for the almost exclusive expression of 5‐HT3R‐AS mRNA or 5‐HT3R‐AL mRNA in NG108‐15 cells should allow the identification of possible differences in the respective functional properties of each of these two forms of the native 5‐HT3 receptor.
Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain
Authors: Bureau Michel , Laschet Jacques , Bureau‐Heeren Mercédès , Hennuy Benoît , Minet Arlette , Wins Pierre , Grisar Thierry
Published: July 2010

Abstract: GABAA receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high‐affinity binding sites for [3H]flunitrazepam and other “central‐type” benzodiazepine ligands. The amount of specific [3H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [3H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABAA receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin‐like convulsants were characterized. Allosteric interactions between the different sites were typical of central‐type GABAA receptors. The presence of α‐subunit(s), as revealed by [3H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABAA receptors are extrasynaptic and expressed in astroglia.
Surface Phosphorylation by Ecto‐Protein Kinase C in Brain Neurons: A Target for Alzheimer's β‐Amyloid Peptides
Authors: Hogan Michael V. , Pawlowska Zofia , Yang Hui‐Ai , Kornecki Elizabeth , Ehrlich Yigal H.
Published: July 2010

Abstract: The powerful regulatory machinery of protein phosphorylation operates in the extracellular environment of the brain. Enzymatic activity with the catalytic specificity of protein kinase C (PKC) was detected on the surface of brain neurons, where it can serve as a direct target for neurotrophic and neurotoxic substances that control neuronal development and cause neurodegeneration. This activity fulfilled all the criteria required of an ectoprotein kinase (ecto‐PK). Detailed analysis of surface protein phosphorylation in cultured brain neurons using specific exogenous substrates (casein, histones, and myelin basic protein), inhibitors (PKC‐pseudosubstrate 19–36; K252b) and antibodies (anti‐PKC catalytic region M.Ab.1.9, antibodies to the carboxy‐terminus of eight PKC isozymes) revealed several types of ecto‐PK activity, among them ecto‐PKs with catalytic specificity of the PKC isozymes ζ and δ. The activity of the neuronal ecto‐PKC is constitutive and not stimulated by phorbol esters. The phosphorylation of a 12K/13K surface protein duplex by ecto‐PKC‐δ was found to be developmentally regulated, with peak activity occurring during the onset of neuritogenesis. Alzheimer's amyloid peptides β1–40 and β25–35 applied at neurotrophic concentrations stimulated the phosphorylation of endogenous substrates of ecto‐PKC activity in brain neurons but inhibited specifically this surface phosphorylation activity with the same dose‐response relationships that cause neurodegeneration. As may be expected from a relevant pathophysiological activity, β‐amyloid peptide 1–28 did not inhibit this surface phosphorylation. The discovery that ecto‐PKC‐mediated protein phosphorylation serves as a target for β‐amyloid peptides at the very site they operate, i.e., at the neuronal cell surface, opens a new research direction in the investigation of molecular events that play a role in the etiology of developmental disabilities and neurodegenerative disorders.
Structural Requirements for Cocaine‐Sensitive and ‐Insensitive Uptake of Phenethylamines into the Adrenal Chromaffin Cell
Authors: Powers Jennifer L. , Plaskon R. Richard , Olsen Greta A. , May Sheldon W.
Published: July 2010

Abstract: The adrenal medullary chromaffin cell is a commonly used model for the adrenergic neuron. Although much work has been done to study the transport system in the adrenal chromaffin vesicles, relatively little is known about cellular transport, especially with regard to structural features of phenethylamines required for intracellular accumulation. We have now investigated the structural requirements of phenethylamine‐related compounds for their accumulation into cultured adrenal chromaffin cells. We find that two types of cellular uptake, previously described only for dopamine, norepinephrine, and epinephrine, are also present for [3H]tyramine. Although two types of accumulation occur, tyramine accumulation occurs mainly via a cocaine‐insensitive process, whereas dopamine accumulation occurs predominantly via a cocaine‐sensitive process. The accumulation of [14C]‐phenethylamine and p‐methoxyphenethylamine is not affected by cocaine, suggesting that a ring hydroxyl substituent is necessary for cocaine‐sensitive accumulation. The compounds p‐hydroxyphenylpropylamine and p‐hydroxyphenyl‐2‐aminoethyl sulfide accumulate in the cell only via a cocaine‐insensitive process, indicating that lengthening of the aminoalkyl side chain prevents cocaine‐sensitive accumulation. We have performed conformational analyses of this series of compounds to determine whether the conformation of these compounds can be related to the kinetic data. For dopamine, tyramine, phenethylamine, and p‐methoxyphenethylamine, two groups of energy‐minimized conformers were found. We find that there is an approximately linear relationship between the Km values for these phenethylamines and the differences in minimized energies between the low‐ and highest energy conformer groups of each compound. A similar correlation was found for p‐hydroxyphenyl‐2‐aminoethyl sulfide. These results are consistent with the hypothesis that these compounds undergo a conformational change from the low‐energy conformer to the highest energy conformer before their cocaine‐insensitive accumulation.
Nitric Oxide Regulates Substance P Release from Rat Spinal Cord Synaptosomes
Authors: Kamisaki Yoshinori , Nakamoto Kentaro , Wada Kouichirou , Itoh Tadao
Published: July 2010

Abstract: In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate the synaptic transmission at the level of spinal cord, effects of NO‐donors on release of substance P (SP) and glutamic acid (Glu) were investigated by superfusion of synaptosomes prepared from the rat spinal cord. Basal levels of endogenous SP and Glu release were 5.99 ± 2.50 fmol/min/mg of protein and 26.2 ± 4.8 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCI evoked 2.7‐ and 3.8‐fold increases in SP and Glu release in a calcium‐dependent manner, respectively. Sodium nitroprusside (NP) caused a reduction in the depolarization‐evoked overflow of SP in a concentration‐dependent manner without affecting its basal release, although it failed to affect either basal or evoked release of Glu. The reduction in SP overflow was also observed by the perfusion with S‐nitroso‐N‐acetyl‐penicillamine or membrane‐permeable cyclic GMP, but not with cyclic AMP. NP caused the concentration‐dependent increases in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP‐containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels.
GABAB Receptor‐Mediated Effects in Synaptosomes of Lethargic (lh/lh) Mice
Authors: Lin Fu‐hsiung , Wang Ying , Lin Susie , Cao Zhen , Hosford David A.
Published: July 2010

Abstract: Previously, we have shown a significant increase in number of GABAB receptor binding sites in neocortex and thalamus of lethargic (lh/lh) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABAB receptors would inhibit [3H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age‐matched male lh/lh and +/+ mice were similar in uptake of [3H]GABA. In the neocortical preparation, baclofen dose‐dependently inhibited [3H]GABA release evoked by 12 mM KCl, an effect mediated by GABAB receptors. The maximal inhibition (Imax) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC50 (3 µM) was unchanged. In the thalamic preparation, the effect of baclofen (50 µM) was 58% less robust in lh/lh mice. Other effects mediated by GABAB receptors (inhibitions in Ca2+ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABAB receptor‐mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABAB receptors on GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.
β‐Amyloid1–40 Increases Expression of β‐Amyloid Precursor Protein in Neuronal Hybrid Cells
Authors: Le Weidong , Xie Wen Jie , Nyormoi Okot , Ho Bao Kuang , Smith R. Glenn , Appel Stanley H.
Published: July 2010

Abstract: Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β‐amyloid peptide (β‐AP), a 40–43‐amino‐acid peptide derived from a larger membrane glycoprotein, β‐amyloid precursor protein (β‐APP). Previous experiments have demonstrated that β‐AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β‐APP mRNA content is increased 3.5‐fold in 24 h after treatment with β‐AP1–40. Accompanying β‐AP1–40‐induced cell injury, levels of cell‐associated β‐APP and a C‐terminal intermediate fragment are increased up to 15‐fold, and levels of secreted forms of β‐APP and 12‐ and 4‐kDa fragments are also increased. Application of β‐APP antisense oligodeoxynucleotide reduces both cytotoxicity and β‐APP expression. 6‐Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β‐APP expression. These results suggest a selective positive feedback mechanism whereby β‐AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β‐APP with the net consequence of further neuronal damage.
Effects of Methylprednisolone on Extracellular Lactic Acidosis and Amino Acids After Severe Compression Injury of Rat Spinal Cord
Authors: Farooque Mohammad , Hillered Lars , Holtz Anders , Olsson Yngve
Published: July 2010

Abstract: We evaluated in rats with severe spinal cord compression at T8–9 the influence of methylprednisolone (MP) on lactic acidosis and extracellular amino acids, which may cause secondary, perifocal injuries of the cord. MP (30 mg/kg) was given intravenously 30 min before compression and hourly thereafter (15 mg/kg). Other rats with compression, given saline, served as controls. Samples from the extracellular fluid of one dorsal horn were collected by microdialysis and analyzed by HPLC. Microdialysis was performed for 1.5 h to establish basal levels. Samples were collected for 3 h after compression. MP‐treated rats showed a reduction of dialysate lactic acid and arginine levels during the first 1–2 h after trauma. The mean dialysate levels of glutamate in MP‐treated rats were lower than those of the controls, but the difference was not statistically significant. MP treatment did not influence dialysate levels of aspartate, glutamine, histidine, glycine, threonine, taurine, alanine, GABA, and tyrosine. Our study shows that MP has several effects, including reduced lactic acid formation, reduced levels of arginine (the substrate for nitric oxide production), and a trend toward decreased extracellular accumulation of the excitotoxic amino acid glutamate. We conclude that MP has the capacity to change the composition of the extracellular edema fluid after trauma to the spinal cord. These changes may counteract free radical formation and may be important mechanisms by which MP exerts its beneficial actions.
Stimulus‐Evoked Increase of Glutamate in the Mediorostral Neostriatum/Hyperstriatum Ventrale of Domestic Chick After Auditory Filial Imprinting: An In Vivo Microdialysis Study
Authors: Gruss Michael , Braun Katharina
Published: July 2010

Abstract: Imprinting in chicks is a form of juvenile learning that has been used to study the basic cellular mechanisms of learning and memory. The forebrain area mediorostral neostriatum/hyperstriatum ventrale (MNH) is a center for acoustic imprinting. Electrophysiological and pharmacological behavioral studies in the MNH have demonstrated that the glutamatergic system and the associated receptors are critically involved in auditory filial imprinting. Accordingly, we investigated the hypothesis that stimulus‐evoked glutamate release may be altered after this learning process. Using an in vivo microdialysis technique, we observed a significantly higher increase of extracellular glutamate level in tone‐imprinted chicks during exposure to the previously imprinted tone than in socially imprinted control chicks. In a further series of experiments, where we exposed animals from both experimental groups to handling distress, glutamate levels in MNH showed only a slight increase, whereas we observed a pronounced increase of extracellular glutamate in the lobus parolfactorius (LPO), the avian analogue of the basal ganglia. No difference of distress‐evoked glutamate release was found in MNH and LPO between tone‐imprinted and socially imprinted chicks. The tone‐evoked enhanced glutamate response in tone‐imprinted chicks suggests that during auditory imprinting glutamatergic synapses develop the potential to increase transmitter release in response to the imprinting stimulus.
Tyrosine Phosphorylation of Extracellular Signal‐Regulated Protein Kinase 4 in Response to Growth Factors
Authors: Peng Xing , Angelastro James M. , Greene Lloyd A.
Published: July 2010

Abstract: Extracellular signal‐regulated protein kinases (ERKs) are members of the mitogen‐activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross‐reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti‐ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine‐phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras‐dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two‐dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.