bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Expression of Phospholipase C Specific for Inositol Phospholipids at the Cell Surface of Rat Glial Cells and REF52 Rat Embryo Fibroblasts
Authors: Birrell G. Bruce , Hedberg Karen K. , Volwerk Johannes J. , Griffith O. Hayes
Published: October 2006

Abstract: Phosphatidylinositol(PI)‐specific phospholipase C activity was detected on the surface of rat astrocytes, rat C6 glioma cells, and rat embryo (REF52) fibroblasts. The cell surface phospholipase C (ecto‐PLC) activity was calcium‐dependent, did not result from secreted phopholipase C, and was not released from the cell surface by bacterial PI‐specific phospholipase C. Agents known to stimulate intracellular PI turnover, including carbachol, L‐glutamic acid, acetylcholine, and orthovanadate, did not induce measurable alterations in the activity of the ecto‐PLC. The expression of ecto‐PLC activity by REF52 fibroblasts was density‐dependent: subconfluent cultures of REF52 exhibited low levels of activity (less than 80 pmol of inositol phosphate formed/min/106 cells), whereas in confluent cultures ecto‐PLC activity increased to approximately 300 pmol/min/106 cells. In contrast to this behavior and that exhibited by previously reported ecto‐PLC‐positive cell types, the ecto‐PLC activity exhibited by astrocytes (approximately 1,000 pmol/min/106 cells) and by C6 glioma cells (approximately 100 pmol/min/106 cells) was independent of cell culture density up to confluence. The constitutive expression of ecto‐PLC activity of astroglial cells may be related to their function as accessory cells in close association with neurons.
Cell Specificity of Molecular Changes During Memory Storage
Authors: McPhie D. L. , Matzel L. D. , Olds J. L. , Lester D. S. , Kuzirian A. M. , Alkon D. L.
Published: October 2006

Abstract: The aeolid nudibranch, Hermissenda crassicornis, exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C (PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B‐cell photoreceptor. The present study was designed to further examine learning‐specific PKC involvement in identified cellular areas, particularly those in the visual‐vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [3H]phorbol‐12,13‐dibutyrate ([3H]‐PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [3H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [3H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.
Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor
Authors: Pawlowska Zofia , Hogan Michael V. , Kornecki Elizabeth , Ehrlich Yigal H.
Published: October 2006

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ‐32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32Pi. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled Pi. As also expected from ecto‐protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho‐α‐casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto‐protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto‐protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca2+‐dependent, high‐affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K‐252a inhibited both intra‐ and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K‐252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto‐protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto‐protein kinase in the reported inhibition of NGF‐induced neurite extension by K‐252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto‐protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto‐protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF‐induced neuronal differentiation. In the concentration range (10–100 μM) at which 6‐thioguanine blocked NGF‐promoted neurite outgrowth in PC12 cells, 6‐thioguanine effectively inhibited the phosphorylation of specific proteins by ecto‐protein kinase. This study provides the basis for continued investigation of the involvement of ecto‐protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.
Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes
Authors: Kitanaka Jun‐ichi , Ishibashi Tadashi , Baba Akemichi
Published: October 2006

Abstract: The effect of phloretin on prostaglandin (PG) F2α‐induced phosphoinositide hydrolysis and elevation of intracellular Ca2+ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF2α (1 μM)‐induced phosphoinositide hydrolysis in a concentration‐dependent manner with an IC50 value of 16 μM. The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose‐response curves of PGF2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μM) inhibited the stimulation induced by PGF2α (1 μM) plus GTPγS (50 μM) without affecting that induced by GTPγS alone. PGF2α at 1 μM transiently increased astrocytic intracellular Ca2+ concentration in 39% of the cells tested. The response was completely blocked by 100 μM phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF2α receptor linked to phospholipase C in astrocytes.
Pharmacological and Regional Characterization of [3H]LY278584 Binding Sites in Human Brain
Authors: Abi‐Dargham Anissa , Laruelle Marc , Wong David T. , Robertson David W. , Weinberger Daniel R. , Kleinman Joel E.
Published: October 2006

Abstract: Binding of [3H]LY278584, which has been previously shown to label 5‐hydroxytryptamine3 (5‐HT3) receptors in rat cortex, was studied in human brain. Saturation experiments revealed a homogeneous population of saturable binding sites in amygdala (KD= 3.08 ± 0.67 nM, Bmax= 11.86 ± 1.87 fmol/mg of protein) as well as in hippocampus, caudate, and putamen. Specific binding was also high in nucleus accumbens and entorhinal cortex. Specific binding was negligible in neocortical areas. Kinetic studies conducted in human hippocampus revealed a Kon of 0.025 ± 0.009 nM−1 min−1 and a Koff of 0.010 ± 0.002 min−1. The kinetics of [3H]LY278584 binding were similar in the caudate. Pharmacological characterization of [3H]LY278584 specific binding in caudate and amygdala indicated the compound was binding to 5‐HT3 receptors. We conclude that 5‐HT3 receptors labeled by [3H]LY278584 are present in both limbic and striatal areas in human brain, suggesting that 5‐HT3 receptor antagonists may be able to influence the dopamine system in humans, similarly to their effects in rodent studies.
p‐Chlorophenylalanine Increases Tryptophan‐5‐Hydroxylase mRNA Levels in the Rat Dorsal Raphe: A Time Course Study Using In Situ Hybridization
Authors: Cortés Roser , Mengod Guadalupe , Celada Pau , Artigas Francesc
Published: October 2006

Abstract: The effects of a single dose of p‐chlorophenylalanine on the mRNA encoding tryptophan‐5‐hydroxylase (EC 1.14.16.4) in the rat dorsal raphe nucleus were analyzed using in situ hybridization. The levels of tryptophan‐5‐hydroxylase mRNA were markedly increased in cell bodies located in the ventromedial part of the dorsal raphe 1–2 days after p‐chlorophenylalanine (300 mg/kg, i.p.) administration. This was followed by a decrease in the amount of tryptophan‐5‐hydroxylase mRNA, which returned to basal values by 5 days after treatment. An almost symmetric time course was observed for the midbrain serotonin concentration. Our results on the temporal pattern of changes in tryptophan‐5‐hydroxylase mRNA levels in the ventromedial part of the dorsal raphe are opposite to those reported for the enzyme activity and serotonin concentration after p‐chlorophenylalanine treatment. These changes may result from modifications in enzyme mRNA expression, suggesting that tryptophan‐5‐hydroxylase gene transcription is involved in feedback mechanisms regulating serotonin synthesis.
β‐Endorphin Is a Potent Inhibitor of Thymidine Incorporation into DNA via μ and κ‐Opioid Receptors in Fetal Rat Brain Cell Aggregates in Culture
Authors: Barg Jacob , Belcheva Mariana , McHale Robert , Levy Rivka , Vogel Zvi , Coscia Carmine J.
Published: October 2006

Abstract: Thymidine incorporation into DNA was inhibited dose‐dependently by β‐endorphin in rat fetal brain cell aggregate cultures. The inhibition was reversed partially by μ (cyclic D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr amide) or k (norbinaltorphimine) antagonists. Complete blockade of the β‐endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Eadie–Hofstee analysis revealed that β‐endorphin inhibited thymidine incorporation noncompetitively. In the presence of protease inhibitors, β‐endorphin decreased thymidine incorporation with an IC50 of 0.7 nM. Truncated and N‐acetylated β‐endorphin derivatives, which bind with low affinity to opioid receptors, did not affect thymidine incorporation. These findings indicate that β‐endorphin at physiological concentrations can regulate thymidine incorporation in cultured brain cells.
Serotonin 5‐HT1A Autoreceptor Blockade Potentiates the Ability of the 5‐HT Reuptake Inhibitor Citalopram to Increase Nerve Terminal Output of 5‐HT In Vivo: A Microdialysis Study
Authors: Hjorth Stephan
Published: October 2006

Abstract: The present study addressed the possibility that disinhibition of serotonin (5‐HT) autoreceptor‐mediated negative feedback might potentiate the elevation of nerve terminal 5‐HT output induced by selective 5‐HT reuptake blockade. To this end, rats were given citalopram and the 5‐HT autoreceptor‐blocking agents (S)‐UH‐301 (5‐HT1A) and (−)‐penbutolol (5‐HT1A/1B), and the effect on extracellular 5‐HT in the ventral hippocampus was monitored by means of in vivo microdialysis. Citalopram (5 mg/kg, s.c.) approximately doubled the 5‐HT output, a response that was markedly augmented by (S)‐UH‐301 (3 mg/kg, s.c.) and (−)‐penbutolol (8 mg/kg, s.c.) and by combined treatment with (S)‐UH‐301 (3 mg/kg, s.c.) plus (−)‐penbutolol (1 μM; via the dialysis perfusion medium), but not by (−)‐penbutolol (1 μM) alone. These findings provide evidence that 5‐HT, in particular 5‐HT1A, autoreceptor‐mediated negative feedback mechanisms are pivotal in determining the nerve terminal 5‐HT output level after 5‐HT reuptake inhibition. These findings have important implications for the interplay between different processes controlling 5‐HT transmission in vivo and might possibly offer a lead toward novel, therapeutically exploitable principles.
Endogenous d‐Serine in Rat Brain: N‐Methyl‐d‐Aspartate Receptor‐Related Distribution and Aging
Authors: Hashimoto Atsushi , Nishikawa Toru , Oka Takae , Takahashi Kiyohisa
Published: October 2006

Abstract: Recently, a substantial amount of free d‐serine has been demonstrated in rat brain, although it has long been presumed that d‐amino acids are uncommon in mammals. The anatomical distribution and age‐related changes in endogenous d‐serine have been examined here to obtain insight into its physiological functions. Free d‐serine exclusively occurs in brains, with a persistent high content from birth to at least 86 postnatal weeks. The patterns of the regional variations and the postnatal changes in brain d‐serine are closely correlated with those of the N‐methyl‐d‐aspartate (NMDA)‐type excitatory amino acid receptor. Because d‐serine potentiates NMDA receptor‐mediated transmission by selective stimulation of the strychnine‐insensitive glycine site of the NMDA receptor, it is proposed that d‐serine is a novel candidate as an intrinsic ligand for the glycine site in mammalian brain.
Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes
Authors: Fontana Giovanni , Blaustein Mordecai P.
Published: October 2006

Abstract: The effects of K+ depolarization and of stimulation by veratridine on apparent cytosolic free Ca2+ ([Ca2+]cyt) and net Ca2+ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca2+]cyt was determined with fura‐2, and Ca2+ accumulation was measured with tracer 45Ca. [Ca2+]cyt was ∼ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K+]0, was increased from the normal 5 mM to 30 or 50 mM, 45Ca uptake and [Ca2+]cyt both increased within 1 s. Both increases were directly related to [Ca2+]0 for [Ca2+]0= 0.02–1.2 mM; however, the increase in 45Ca uptake greatly exceeded the increase in [Ca2+]cyt. With small Ca2+ loads ≤100 μmol/L of cell water, equivalent to the Ca2+ entry during a train of ≤60 impulses), the 45Ca uptake exceeded the increase in [Ca2+]cyt by a factor of nearly 1,000. This indicates that ∼99.9% of the entering Ca2+ was buffered and/or sequestered within ∼ 1 s. With larger Ca2+ loads, a larger fraction of the entering Ca2+ was buffered; ∼99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca2+ entry and the increase in [Ca2+]cyt, the “calcium buffer ratio”β, was therefore ∼ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca2+]cyt, which ranged between ∼7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca2+]0= 0.2–0.6 mM), 45Ca influx and [Ca2+]cyt increased progressively for ∼10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in 45Ca influx and [Ca2+]cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca2+]cyt and the veratridine‐stimulated 45Ca within the terminals to decline, thereby demonstrating that the Ca2+ loading is reversible in the presence of extracellular Ca2+. These data show that synaptosomes are capable of buffering and metabolizing Ca2+ in a manner expected for intact neurons.
The Synaptic Vesicle‐Associated G Protein o‐rab3 Is Expressed in Subpopulations of Neurons
Authors: Volknandt Walter , Hausinger Angela , Wittich Beate , Zimmermann Herbert
Published: October 2006

Abstract: The distribution of o‐rab3—a synaptic vesicle‐associated low‐molecular‐weight GTP‐binding protein—was studied in various neural tissues of the electric ray Torpedo marmorata. o‐rab3 was shown to be associated selectively with isolated cholinergic synaptic vesicles derived from the electric organ. Gel filtration of cholinergic synaptic vesicles using Sephacryl S‐1000 column chromatography demonstrated a copurification of o‐rab3 with the synaptic vesicle content marker ATP and with SV2—a synaptic vesicle transmembrane glycoprotein. Indirect immunofluorescence using antibodies against o‐rab3 and SV2 and a double labeling protocol revealed an identical distribution of both antigens in the cholinergic nerve terminals within the electric organ and at neuromuscular junctions. An immunoelectron microscopic analysis demonstrated the presence of o‐rab3 at the surface of the synaptic vesicle membrane. In the CNS immunofluorescence of o‐rab3 and SV2 overlap only in small and distinct areas. Whereas SV2 has an overall distribution in nerve terminals of the entire CNS, o‐rab3 is restricted to a subpopulation of nerve terminals in the dorsolateral neuropile of the rhombencephalon and in the dorsal horn of the spinal cord. Our results demonstrate that the synaptic vesicle‐associated G protein o‐rab3 is specifically expressed only in subpopulations of neurons in the Torpedo CNS.
Expression of Glia Maturation Factor β mRNA and Protein in Rat Organs and Cells
Authors: Zaheer Asgar , Fink Brian D. , Lim Ramon
Published: October 2006

Abstract: Rat glia maturation factor β (GMF‐β) cDNA was obtained by reverse transcription of rat brain mRNA followed by polymerase chain reaction amplification, using primers from the human sequence. The deduced amino acid sequence of rat GMF‐β differed from the human counterpart in only three places: His27 in place of Asn, Val51 in place of Ile, and Leu93 in place of Val. The high degree of evolutionary conservation suggests that GMF‐β plays an essential role in animal cell physiology. The expression of GMF‐β mRNA in the rat was studied by the northern blot technique, using a rat cRNA probe corresponding to the entire coding region. GMF‐β mRNA was predominantly expressed in the brain and spinal cord, although trace levels were found in other organs, including testis and ovary. In the brain GMF‐β mRNA was detectable at as early as embryonic day 10, and persisted through as late as postnatal month 14, with minor variations in between. On the other hand, GMF‐β protein exhibited more obvious developmental changes, with its level increasing slowly prenatally and plateauing at 1 week after birth. GMF‐β mRNA and protein were also observed in several cultured cells. Some cells of neural origin contained higher levels of GMF‐β protein compared with cells derived from other sources. Through demonstration of mRNA and confirmation by immunoblotting, we conclude that GMF‐β is synthesized by rat organs and that GMF‐β is predominantly a brain protein.
Amino Acid Levels and γ‐Aminobutyric AcidA Receptors in Rat Neostriatum, Cortex, and Thalamus After Neonatal 6‐Hydroxydopamine Lesion
Authors: Molina‐Holgado Eduardo , Dewar Karen M. , Grondin Louise , van Gelder Nico M. , Reader Tomás A.
Published: October 2006

Abstract: The amino acid γ‐aminobutyric acid (GABA) is the main inhibitory neurotransmitter in brain, and GABAergic neurons have been proposed to play a major role in basal ganglia physiology. In the neostriatum (caudate putamen), medium‐sized aspiny interneurons, as well as neostriatal output neurons that project to several brain regions, use GABA as their neurotransmitter. Dopamine fibers arising from the substantia nigra represent a major input to the neostriatum where, besides their classic neurotransmitter role, they are seemingly involved in the regulation of amino acid neurotransmitter release. To further characterize the nature of some of the amino acid/dopamine interactions, selective dopaminergic deafferentations were produced in neonatal rats (3 days postnatal) by intraventricular administration of the neurotoxin 6‐hydroxydopamine (6‐OHDA); the noradrenergic neurons were protected by prior administration of desmethylimipramine. After a 3‐month survival, levels of catecholamines, indoleamines, and amino acids were determined in cingulate cortex, thalamus, and neostriatum. In addition, GABAA receptors were measured in membrane preparations from these three regions, using the specific agonist [3H]muscimol. In the 6‐hydroxydopamine‐lesioned rats, levels of dopamine and its metabolites homovanillic acid, 3,4‐dihydroxyphenylacetic acid, and 3‐methoxytyramine were decreased, as expected, in cortex and neostriatum, but remained unmodified in thalamus. In all three regions, serotonin content was increased; its metabolite, 5‐hydroxyindole‐3‐acetic acid, was also elevated, but only in cortex and neostriatum. The levels of GABA were increased in neostriatum and thalamus, but remained unmodified in cortex. Glycine was increased in all three regions examined. There were also increases of phosphatylethanolamine and serine in thalamus, and of aspartic acid and alanine in neostriatum. The density of GABAA binding sites was increased in neostriatum, but remained unchanged in cortex and thalamus. The changes in amino acid levels and [3H]muscimol binding sites induced by a neonatal 6‐hydroxydopamine treatment differ from those found after similar lesions in adult animals, possibly because of the plastic and synaptic rearrangements that can still occur during early postnatal development. The present results also demonstrate that adaptations occur in response to a dopaminergic deafferentation at an early age and that these exhibit a regional specificity.
Transcriptional Organization of a Drosophila Glutamic Acid Decarboxylase Gene
Authors: Newby Laurel M. , Kulkarni Shankar J. , Jackson F. Rob
Published: October 2006

Abstract: We previously described the sequence and expression pattern of a Drosophila mRNA (Gad) that encodes the major soluble form of glutamic acid decarboxylase (GAD). We now report the transcriptional organization of the Drosophila Gad gene. Based on a combination of DNA sequence, RNase protection, primer extension, and polymerase chain reaction analyses, we conclude that the transcription unit for a 3.1‐kb Gad mRNA is composed of eight exons that span an ∼17‐kb genomic interval. By this analysis, the site of Gad transcript initiation overlaps with a recognition sequence that confers binding of the zeste transcription factor to other promoter elements. We emphasize that our analysis of the Gad transcription unit provides no evidence for alternative RNA splicing as a mechanism for the generation of GAD isoforms. Thus, the several GAD‐immunoreactive proteins (putative GAD isoforms) that can be detected in Drosophila extracts are probably encoded by distinct genes.
Structure of the Rat Aromatic L‐Amino Acid Decarboxylase Gene: Evidence for an Alternative Promoter Usage
Authors: Hahn Soonjung L. , Hahn Mounou , Kang Un Jung , Joh Tong H.
Published: October 2006

Abstract: Aromatic L‐amino acid decarboxylase catalyzes the biosynthesis of the neurotransmitters dopamine and serotonin. This enzyme is also expressed in nonneuronal tissues. Two reported cDNA sequences show that the pheochromocytoma message differs from the liver message only at the 5’untranslated region. We present the complete exonal organization and promoter sequences of the rat gene encoding this enzyme. The rat aromatic L‐amino acid decarboxylase gene is composed of two promoters and 16 exons spanning more than 80 kb in the genome. The first exon carries the majority of the 5’untranslated sequence of the liver cDNA, and the second exon carries that of the pheochromocytoma cDNA. In the third exon, there are two alternatively utilized splicing acceptors specific to the first and second exons. Therefore, both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. The sequence of each promoter region shows putative binding sites for octamer factors and AP‐2.
Effect of α‐Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+‐Dependent and Na+‐Independent Components
Authors: Deri Z. , Bors P. , Adam‐Vizi Vera
Published: October 2006

Abstract: We studied the effect of α‐latrotoxin (αLTX) on [14C]acetylcholine ([14C]ACh) release, intracellular Ca2+ concentration ([Ca2+]i), plasma membrane potential, and high‐affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10−10‐10−8M) caused an elevation of the [Ca2+]i as detected by Fura 2 fluorescence and evoked [14C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na+ in the external medium and another that did not. Displacement of Na+ by sucrose or N‐methylglucamine in the medium considerably decreased the elevation of [Ca2+]i and [14C]ACh release by αLTX. The Na+‐dependent component of the αLTX action was obvious in the inhibition of the high‐affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca2+]i and [14C]ACh release remained in the absence of Na+. Both the Na+‐dependent and the Na+‐independent components of the αLTX‐evoked [14C]ACh release partly required the presence of either Mg2+ or Ca2+. The nonneurotransmitter [14C]choline was released along with [14C]ACh, but this release did not depend on the presence of either Na+ or Ca2+, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation‐dependent ACh release, which is related to (a) Na+‐dependent divalent cation entry and (b) Na+‐independent divalent cation entry, and (2) nonspecific Na+‐ and divalent cation‐independent leakage.
Neurosecretory Vesicles Contain Soluble and Membrane‐Associated Monofunctional and Bifunctional Peptidylglycine α‐Amidating Monooxygenase Proteins
Authors: Oyarce Ana Maria , Eipper Betty A.
Published: October 2006

Abstract: Peptidylglycine α‐Amidating monooxygenase (PAM) catalyzes the COOH‐terminal amidation of neuro‐peptides in a reaction requiring the sequential action of two enzymes contained within this bifunctional protein. The CNS contains primarily transcripts encoding rPAM‐1 and rPAM‐2, integral membrane proteins differing by the presence or absence of a noncatalytic domain separating the two enzymes. Subcellular fractionation of adult rat hypothala‐mus and hippocampus demonstrated the localization of both enzymatic activities to fractions enriched in neurose‐cretory vesicles. Upon separation of the soluble contents from the membranes of neurosecretory vesicles, 30–40° of both enzymatic activities was recovered in the soluble fraction. Over 40° of both enzymatic activities remained membrane‐associated following removal of peripheral membrane proteins. Antisera specific to different regions of PAM were used to identify intact rPAM‐1 and rPAM‐2, a monofunctional integral membrane peptidyl‐α‐hydroxyglycine α‐Amidating lyase protein generated from rPAM‐1, and a noncatalytic COOH‐terminal fragment as the major PAM proteins in carbonate‐washed membranes, Endopro‐teolytic processing generated large amounts of soluble, monofunctional forms of both enzymes from rPAM‐1 and smaller amounts of a soluble, bifunctional PAM protein from rPAM‐2. A significant amount of both monofunctional enzymes lacking the transmembrane domain was tightly associated with membranes. Whereas soluble mono‐and bifunctional enzymes may be released upon exocytosis of neurosecretory vesicles, membrane‐associated PAM proteins may remain on the cell surface or be internalized.
Signal Transduction Pathways Coupled to a P2U Receptor in Neuroblastoma × Glioma (NG108‐15) Cells
Authors: Lin Tai‐An , Lustig Kevin D. , Sportiello Michael G. , Weisman Gary A. , Sun Grace Y.
Published: October 2006

Abstract: Extracellular ATP has neurotransmitter‐like properties in the CNS and PNS that are mediated by a cell‐surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108‐15 cells). The addition of ≥1 μM ATP to NG108‐15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis off [32P]‐phosphatidylinositol 4,5‐bisphosphates and an increase in the level of inositol 1,4,5‐trisphosphate. ATP also caused a time‐ and dose‐dependent increase in levels of [3H]inositol monophosphates in lithium‐treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4‐monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4‐) in the medium and not with the concentration of magnesium‐ATP (MgATP2‐). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP‐γS, 2‐methylthio ATP, β,γ‐imidoATP or 3′‐O‐(4‐benzoyl)benzoylATP, but not CTP, AMP, β,γ‐methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]Pi or [14C]‐arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid‐labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108‐15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D.
Elevated Phosphatidyl‐CMP Is Not the Source of Diacylglycerol Accumulation Induced by Lithium in NG108‐15 Cells
Authors: Brami Brigitte A. , Leli Ubaldo , Hauser George
Published: October 2006

Abstract: Previous studies have shown that in the neuroblastoma X glioma hybrid cell line NG108‐15 lithium is able to induce an increase in diacylglycerol levels. This effect was shown to be enhanced by the presence of bradykinin. Another striking effect of lithium was a marked gain in the level of the liponucleotide phosphatidyl‐CMP. Increased phosphatidyl‐CMP levels were detected in the presence of lithium alone but were considerably more pronounced in the presence of both lithium and bradykinin. These results are consistent with the inhibitory action of lithium on key enzymes of the degradation pathway of inositol phosphates, resulting in a decrease in cellular inositol content and in an elevation in levels of phosphorylated inositols. Comparison of the mass of the inositol phosphates and diacylglycerol showed that the lithium‐induced diacylglycerol levels were substantially greater than would be expected from phosphoinositide hydrolysis alone. One possible reason for the increase in the level of diacylglycerol through the action of lithium is the reversal of the reaction for the formation of phosphatidyl‐CMP. The resulting phosphatidic acid would then need to be further dephosphorylated to diacylglycerol. The lithium‐induced elevation of phosphatidyl‐CMP was prevented by addition of myo‐inositol (10–30 mM), suggesting that the increase in liponucleotide level was due to depletion of cellular inositol. Under the same conditions the elevated diacylglycerol concentration remained unchanged. Consequently, phosphatidyl‐CMP is not its source, and diacylglycerol may arise through an effect of lithium on the degradation of phospholipids other than phosphoinositides. The action of phospholipase C or D on phosphatidylcholine is the most likely mechanism.
Characterization of Two Cytosolic Diacylglycerol Kinase Forms
Authors: Chen Qing , Klemm Nancy , Jeng Ingming
Published: October 2006

Abstract: Two forms of rat brain cytosolic diacylglycerol kinase (EC 2.7.1.107) were separated by heparin‐agarose column chromatography. These forms, designated DGK‐I and DGK‐II, were not interconvertible as determined by rechromatography. DGK‐I and DGK‐II had respective molecular masses of 88 and 180 kDa, as measured by Sepharose 6B chromatography. Both forms preferred diacylglycerol over monoacylglycerol and were insensitive to R59022. DGK‐II, but not DGK‐I, was activated by an activator substance prepared from chicken egg yolk. DGK‐II was activated by a rat brain cytosolic activator and was exclusively sensitive to 5′‐AMP‐mediated inactivation. Further studies revealed that these two forms had the following distinct characteristics: (a) substrate specificity, (b) inhibition by heparin, (c) sensitivity to lysine‐containing polyamino acids, and (d) responses to different phospholipids. In general, DGK‐II was more responsive to various inhibitors and activators, making it a prime candidate for a regulatable enzyme.
The Effect of Neurocatin on Protein Phosphorylation in Striatal Synaptosomes from Rat Brain
Authors: Yee Daniel K. , Pastuszko Anna , Wilson David F.
Published: October 2006

Abstract: Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of ∼80 and ∼60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60‐kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to ∼7.5 ng/100 μl of suspension), incorporation of 32P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin >7.5 ng/100 μl caused progressive decrease in incorporation of 32P into many synaptosomal proteins; by a concentration of neurocatin of ∼45 ng/100 μ/l, the level of 32P incorporation into many proteins was ≤70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of ∼7.5 ng/100 μl of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times >2.0 min, showed progressive decrease in 32P incorporation. Removing extrasynaptosomal Ca2+ with EGTA attenuated the increased 32P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin‐induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of 32P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane.
Calcium Permeability of Non‐N‐Methyl‐D‐Aspartate Receptor Channels in Immature Cerebellar Purkinje Cells: Studies Using Fura‐2 Microfluorometry
Authors: Sorimachi Masaru
Published: October 2006

Abstract: Using fura‐2 microfluorometry, I investigated the mechanism by which non‐N‐methyl‐d‐aspartate (NMDA) receptor agonists increase the cytosolic free calcium concentration ([Ca]in) in single cerebellar Purkinje cells isolated from 3–10‐day‐old rats. Kainate and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate dose‐dependently increased the cytosolic free Na+ concentration, which was measured using sodium‐binding benzofuran isophthalate microfluorometry, confirming the Na+ influx through ion channels linked to non‐NMDA receptors. The [Ca2+] increases induced by relatively lower concentrations of agonists were entirely dependent on external Ca2+ and were reduced by removal of external Na+ or by addition of a Ca2+ channel blocker, D600. The results indicate that the non‐NMDA agonist–induced [Ca]in increase was due mainly to Ca2+ influx through voltage‐dependent Ca2+ channels, which were activated by a massive Na+ influx. On the other hand, higher concentrations of agonists dose‐dependently increased [Ca]in under conditions in which activation of voltage‐dependent Ca2+ channels were blocked by a combination of Na+ removal with D600. These [Ca]in increases were Ca2+ dependent and little affected by adding a competitive NMDA antagonist. Non‐NMDA agonists also stimulated influxes of Mn2+ and Co2+, both of which can be monitored by quenching fura‐2 fluorescence under the same conditions. These results suggest that ion channels linked to non‐NMDA receptors on immature Purkinje cells are permeable to Ca2+, Mn2+, and Co2+.
Induction of Ornithine Decarboxylase by N‐Methyl‐D‐Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons
Authors: Lombardi G. , Szekely A. M. , Bristol L. A. , Guidotti A. , Manev H.
Published: October 2006

Abstract: Polyamines positively modulate the activity of the N‐methyl‐d‐aspartate (NMDA)‐sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate‐induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate‐triggered neuronal death, but the glutamate‐induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.
Regionally Distinct N‐Methyl‐D‐Aspartate Receptors Distinguished by Quantitative Autoradiography of [3H]MK‐801 Binding in Rat Brain
Authors: Sakurai Sharin Y. , Penney John B. , Young Anne B.
Published: October 2006

Abstract: Quantitative autoradiography of [3H]MK‐801 binding was used to characterize regional differences in N‐methyl‐d‐aspartate (NMDA) receptor pharmacology in rat CNS. Regionally distinct populations of NMDA receptors were distinguished on the basis of regulation of [3H]MK‐801 binding by the NMDA antagonist 3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP). CPP inhibited [3H]MK‐801 binding in outer cortex (OC) and medial cortex (MC) with apparent Ki values of 0.32‐0.48 μM, whereas in the medial striatum (MS), lateral striatum (LS), CA1, and dentate gyrus (DG) of hippocampus, apparent Ki values were 1.1‐1.6 μM. In medial thalamus (MT) and lateral thalamus (LT) the apparent Ki values were 0.78 μM. In the presence of added glutamate (3 μM), the relative differences in apparent Ki values between regions maintained a similar relationship with the exception of the OC. Inhibition of [3H]MK‐801 binding by the glycine site antagonist 7‐chlorokynurenic acid (7‐ClKyn) distinguished at least two populations of NMDA receptors that differed from populations defined by CPP displacement. 7‐ClKyn inhibited [3H]MK‐801 binding in OC, MC, MS, and LS with apparent Ki values of 6.3‐8.6 μM, whereas in CA1, DG, LT, and MT, Ki values were 11.4‐13.6 μM. In the presence of added glycine (1 μM), the relative differences in apparent Ki values were maintained. Under conditions of differential receptor activation, regional differences in NMDA receptor pharmacology can be detected using [3H]MK‐801 binding.
Activity of Tyrosine Hydroxylase in the Striatum of Newborn Piglets in Response to Hypocapnic Hypoxia
Authors: Tammela Outi , Pastuszko Anna , Lajevardi Nasser S. , Delivoria‐Papadopoulos Maria , Wilson David F.
Published: October 2006

Abstract: The effect of graded hypoxia induced by hyperventilation on the activity of tyrosine hydroxylase was measured in vivo by microdialysis. Microdialysis probes were inserted into the striatum of newborn piglets and perfused with medium containing 3‐hydroxybenzylhydrazine, an inhibitor of L‐aromatic amino acid decarboxylase. The level of 3,4‐dihydroxyphenylalanine (DOPA) measured in the effluent dialysate was then an index of tyrosine hydroxylase activity. The oxygen pressure in the veins and capillaries of the cortex was measured, through a cranial window placed over the parietal cortex, by the phosphorescence lifetime of palladium‐meso‐tetra(4‐carboxyphenyl)porphine added to the blood. After baseline measurements, PaCO2 was decreased from 38 torr (control value) to 19, 13, and 11 torr resulting in decreases in the cortical oxygen pressure from 40 ± 6 torr to 26 ± 3, 23 ± 4, and 20 ± 4 torr, respectively. Decrease in the oxygen pressure to 26 ± 3 torr caused a statistically significant increase of 25–30% in the level of DOPA in the effluent perfusate. During the next step of increase in ventilator rate, when oxygen decreased only slightly, the level of DOPA remained at the higher level. Ventilation rates that lowered the oxygen pressure to below 20 torr, however, caused a progressive decrease in the level of DOPA. During recovery, the level of DOPA steadily increased, attaining 160% of control value after 1.5 h. When the oxygen pressure was decreased to 16 ± 2 torr by a single increase in ventilator rate, the DOPA level decreased in the effluent to 15–20% below control. With return of the ventilator rate to control values, the DOPA levels again increased to well above control and stayed higher even after 1.5 h. The slow return of tyrosine hydroxylase activity to control indicates relatively long‐term modification of the enzyme activity. Activation of tyrosine hydroxylase occurs when the oxygen pressure is decreased, but at <16 torr the reaction rate becomes limited by the availability of oxygen and decreases with further decrease in oxygen pressure. Our results showed that even small changes in cortical oxygen pressure modulate the activity of tyrosine hydroxylase. This alteration in the metabolism of catecholamines in newborn brain may have significant impact on later development of the organism.
Dissociation of Phosphorylation and Translocation of a Myristoylated Protein Kinase C Substrate (MARCKS Protein) in C6 Glioma and N1E‐115 Neuroblastoma Cells
Authors: Byers David M. , Palmer Frederick B. St. C. , Spence Matthew W. , Cook Harold W.
Published: October 2006

Abstract: An 80‐kDa protein labeled with [3H]myristic acid in C6 glioma and N1E‐115 neuroblastoma cells has been identified as the myristoylated alanine‐rich C kinase substrate (MARCKS protein) on the basis of its calmodulin‐binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (β‐TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X‐100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β‐TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β‐TPA–stimulated phosphorylation of MARCKS in N1E‐115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α‐TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell‐specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.
Protein Methylation in Cerebellar Synaptosomes
Authors: Wright Lynda S. , Siegel Frank L.
Published: October 2006

Abstract: Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21–26‐kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21–26‐kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17‐ and 35‐kDa methyl acceptor proteins to the synaptoplasm, the 21–26‐kDa family to the synaptic membranes, and the 52‐kDa to synaptic vesicles. The 21–26‐kDa family was identified as GTP‐binding proteins by [α‐32P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52‐kDa methylated protein also contained volatile methyl esters, but did not bind [α‐32P]GTP. When synaptosomes were screened for putative methyltransferases by S‐adenosyl‐L‐[methyl‐3H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.
Role of Histidine Residues in Agonist and Antagonist Binding Sites of A1 Adenosine Receptor
Authors: Allende Gonzalo , Casadó Vicent , Mallol Josefa , Franco Rafael , Lluis Carme , Canela Enric I.
Published: October 2006

Abstract: The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L‐N6‐[adenine‐2,8‐3H, ethyl‐2‐3H]phenylisopropyladenosine ([3H]R‐PIA) and antagonist 8‐cyclopentyl‐1,3‐[3H]‐dipropylxanthine ([3H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [3H]R‐PIA associations with high‐ and low‐affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low‐affinity state. No ionizable group is involved in the dissociation process for the high‐affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low‐affinity state. For [3H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A1 adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (KD) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R‐PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand. The pattern of protective action of R‐PIA, DPCPX, adenosine, and guanylylimidodiphosphate in DEP treatment and the displacement curves of radiolabeled agonist and antagonist by both unlabeled ligands indicated that the interaction site for agonist and antagonist binding is the same, although the complete mechanisms for recognition and binding differ.