bluuebunny/crossref_metadata_2025 huggingface dataset:
5‐Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin‐Sensitive Site Distinct from the 2‐[125I]Iodomelatonin Binding Site
Authors: Iuvone P. Michael , Gan Jiwei , Alonso‐Gómez Angel L.
Published: July 2010

Abstract: Melatonin and 5‐methoxytryptamine inhibited forskolin‐stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC50 values of 0.8 nM for melatonin and 7.2 nM for 5‐methoxytryptamine. Inhibition of cyclic AMP formation by 5‐methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5‐methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5‐methoxytryptamine but not that to 2‐iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2‐[125I]iodomelatonin. Melatonin displaced specific 2‐[125I]iodomelatonin with a Ki value (0.8 nM) similar to the EC50 for inhibition of cyclic AMP formation. In contrast, 5‐methoxytryptamine only inhibited 2‐[125I]iodomelatonin binding at very high concentrations (Ki = 650 nM). Pretreating cultured cells for 24 h with 2‐iodomelatonin or melatonin, but not with 5‐methoxytryptamine, reduced subsequent 2‐[125I]iodomelatonin binding. Thus, 5‐methoxytryptamine appears to inhibit forskolin‐stimulated cyclic AMP formation at a site distinct from the 2‐iodomelatonin binding site.
Eukaryotic Initiation Factor 4E Degradation During Brain Ischemia
Authors: Neumar Robert W. , DeGracia Donald J. , White Blaine C. , McDermott Paul J. , Evans David R. , Krause Gary S.
Published: July 2010

Abstract: Suppression of protein synthesis in the brain following an ischemic insult has been thought to occur because of inhibition of translation initiation. All eukaryotic mRNAs, with the exception of heat‐shock transcripts, require the activity of eukaryotic initiation factor (eIF) 4E for formation of the translation initiation complex, and eIF‐4E availability is rate‐limiting. The response of brain eIF‐4E concentration and phosphorylation following decapitation ischemia was studied in rat brain homogenates after electrophoresis and western blotting with antibodies against eIF‐4E and phosphoserine, respectively. There was no change in level of eIF‐4E after 5 min of ischemia (p = 0.82 vs. time 0), but it had decreased 32 (p = 0.01) and 57% (p = 0.006) after 10 and 20 min of ischemia, respectively. There was no loss of serine phosphorylation on eIF‐4E beyond signal loss observed due to degradation of the protein itself (p = 0.31). In vitro exposure of eIF‐4E to activated μ‐calpain resulted in a 50% loss in 10 min of eIF‐4E on western blots. If active eIF‐4E is required for translation of its own mRNA, degradation of this protein during ischemia, possibly by activated μ‐calpain, could be a direct mechanism of irreversible neuronal injury, and the rate of proteolysis of eIF‐4E could place an upper time limit on the maximal duration of global brain ischemia compatible with neurologic recovery.
Angiotensin II Type 2 Receptor‐Mediated Stimulation of Protein Phosphatase 2A in Rat Hypothalamic/Brainstem Neuronal Cocultures
Authors: Huang Xian‐Cheng , Richards Elaine M. , Sumners Colin
Published: July 2010

Abstract: Recent studies have suggested a role for an inhibitory guanine nucleotide binding (Gi) protein and protein (serine/threonine) phosphatase 2A (PP2A) in the angiotensin II type 2 (AT2) receptor‐mediated stimulation of neuronal K+ currents. In the present study we have directly analyzed the effects of angiotensin II on PP2A activity in neurons cultured from newborn rat hypothalamus and brainstem. Angiotensin II elicited time (30 min–24 h)‐ and concentration (10 nM‐1 µM)‐dependent increases in PP2A activity in these cells, an effect mimicked by the AT2 receptor ligand CGP‐42112A. These effects of angiotensin II and CGP‐42112A involve AT2 receptors, because they were inhibited by the AT2 receptor‐selective ligand PD 123,319 (1 µM) but not by the angiotensin II type 1 receptor antagonist losartan (1 µM). Furthermore, the stimulatory effects of angiotensin II and CGP‐42112A on PP2A activity were inhibited by pretreatment of cultures with pertussis toxin (200 ng/ml; 24 h), indicating the involvement of a Gi protein. These effects of angiotensin II and CGP‐42112A appear to be via activation of PP2A, and western blot analyses revealed no effects of either peptide on the protein levels of the catalytic subunit of PP2A in cultured neurons. In summary, these data suggest that PP2A is a cellular target modified following neuronal AT2 receptor activation.
Cholinomimetics Increase Glutamate Outflow via an Action on the Corticostriatal Pathway: Implications for Alzheimer's Disease
Authors: Dijk Sas N. , Francis Paul T. , Stratmann Gary C. , Bowen David M.
Published: July 2010

Abstract: Physostigmine, the acetylcholinesterase inhibitor (0.3 mg/kg, i.m.), increased extracellular glutamate but not aspartate concentrations in the striatum of anaesthetised rats, determined using microdialysis and HPLC. The rise was both tetrodotoxin and calcium dependent. In contrast, neither physostigmine (10 µM) added to the perfusion fluid nor vehicle (injected intramuscularly) affected amino acid concentrations. To obtain evidence that the action of acetylcholine was to modulate positively cortical pyramidal neurone activity via the M1 receptor, the selective M1 agonist PD 142505‐0028 (10 µM) was topically applied to the frontal cortex. Like physostigmine, PD 142505‐0028 rapidly increased glutamate but not aspartate concentrations in the striatum. Moreover, the effect of intramuscular physostigmine was blocked by a topically applied M1 antagonist. These new data add to our hypothesis that cholinomimetics increase pyramidal neurone function.
Effect of Oxysterol Treatment on Cholesterol Biosynthesis and Reactive Astrocyte Proliferation in Injured Rat Brain Cortex
Authors: Bochelen D. , Mersel M. , Behr P. , Lutz P. , Kupferberg A.
Published: July 2010

Abstract: We have reported previously that oxysterols inhibit astrogliosis and intracranial glioblastoma growth. To elucidate the mechanism of action of these molecules in vivo, we have investigated their effect on the cholesterol biosynthesis in the injured brain. In a bilateral lesion model, injection of liposomes containing 7β‐hydroxycholesterol decreased [3H]acetate incorporation into neutral lipids and cholesterol by 30% and 40%, respectively. Structural analogues were tested using a unilateral lesion model. The injury did not significantly affect cholesterogenesis; injection of 7β‐hydroxycholesterol or 7β‐hydroxycholesteryl‐3‐oleate reduced acetate incorporation into cholesterol by 47% and 43%, respectively. Both 7‐ketocholesteryl‐3‐oleate and 7α‐hydroxycholesteryl‐3‐oleate inhibited cholesterogenesis by 32%. As cholesterol and by‐products of the cholesterol pathway play a key role in cell division, we have assessed the effect of oxysterols on reactive astrocyte proliferation. The incorporation of bromodeoxyuridine showed that up to 46% of astrocytes were proliferating 24 h after the injury. Injection of 12 nmol of 7β‐hydroxycholesterol or 7β‐hydroxycholesteryl‐3‐oleate reduced the labelling index to 26%, whereas the labelling index in the 7‐ketocholesteryl‐3‐oleate‐treated cortex was 37%. These findings demonstrate that oxysterols are potent inhibitors of the endogenous cholesterol biosynthesis in brain and show a correlation between cholesterogenesis and reactive astrocyte proliferation.
Affinity Purification of Proteoglycans that Bind to the Amyloid Protein Precursor of Alzheimer's Disease
Authors: Williamson Timothy G. , Nurcombe Victor , Beyreuther Konrad , Masters Colin L. , Small David H.
Published: July 2010

Abstract: The binding of the amyloid protein precursor (APP) to heparan sulfate proteoglycans has been shown to stimulate the neurite‐promoting activity of APP. In this study, proteoglycans that bind with high affinity to APP were characterized. Conditioned medium from cultures of postnatal day 3 mouse brain cells was applied to an affinity column containing a peptide homologous to a heparin‐binding domain of APP. A fraction 17‐fold enriched in proteoglycans was recovered by elution with a salt gradient. APP bound saturably and with high affinity to the affinity‐purified proteoglycan fraction. Scatchard analysis of the binding showed that APP bound to high‐ and low‐affinity sites with equilibrium dissociation constants of 1.4 × 10−11 and 6.5 × 10−10M, respectively. APP, in conjunction with the affinity‐purified proteoglycan fraction, promoted neurite outgrowth. The affinity‐purified proteoglycan fraction contained a heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan. Digestion of the affinity‐purified fraction with heparitinase I revealed a core protein of 63–69‐kDa molecular mass, whereas digestion with chondroitinase ABC revealed a core protein of 100–110 kDa. The results suggest that expression of specific APP‐binding proteoglycans may be an important step in the regulation of the neurite outgrowth‐promoting activity of APP.
Nerve Growth Factor Administration Attenuates Cognitive but Not Neurobehavioral Motor Dysfunction or Hippocampal Cell Loss Following Fluid‐Percussion Brain Injury in Rats
Authors: Sinson Grant , Voddi Madhu , McIntosh Tracy K.
Published: July 2010

Abstract: Lateral fluid‐percussion brain injury in rats results in cognitive deficits, motor dysfunction, and selective hippocampal cell loss. Neurotrophic factors have been shown to have potential therapeutic applications in neurodegenerative diseases, and nerve growth factor (NGF) has been shown to be neuroprotective in models of excitotoxicity. This study evaluated the neuroprotective efficacy of intracerebral NGF infusion after traumatic brain injury. Male Sprague‐Dawley rats received lateral fluid‐percussion brain injury of moderate severity (2.1–2.3 atm). A miniosmotic pump was implanted 24 h after injury to infuse NGF (n = 34) or vehicle (n = 16) directly into the region of maximal cortical injury. Infusions of NGF continued until the animal was killed at 72 h, 1 week, or 2 weeks after injury. Animals were evaluated for cognitive dysfunction (Morris Water Maze) and regional neuronal cell loss (Nissl staining) at each of the three time points. Animals surviving for 1 or 2 weeks were also evaluated for neurobehavioral motor function. Although an improvement in memory scores was not observed at 72 h after injury, animals receiving NGF infusions showed significantly improved memory scores when tested at 1 or 2 weeks after injury compared with injured animals receiving vehicle infusions (p < 0.05). Motor scores and CA3 hippocampal cell loss were not significantly different in any group of NGF‐treated animals when compared with controls. These data suggest that NGF administration, in the acute, posttraumatic period following fluid‐percussion brain injury, may have potential in improving post‐traumatic cognitive deficits.
Metabolism of l‐Tryptophan to Kynurenate and Quinolinate in the Central Nervous System: Effects of 6‐Chlorotryptophan and 4‐Chloro‐3‐Hydroxyanthranilate
Authors: Naritsin Dmitry B. , Saito Kuniaki , Markey S. P. , Chen Cai Y. , Heyes Melvyn P.
Published: July 2010

Abstract: The metabolism of l‐tryptophan to the neuroactive kynurenine pathway metabolites, l‐kynurenine, kynurenate and quinolinate, and the effects of two inhibitors of quinolinate synthesis (6‐chlorotryptophan and 4‐chloro‐3‐hydroxyanthranilate) were investigated by mass spectrometric assays in cultured cells and in vivo. Cell lines obtained from astrocytoma, neuroblastoma, macrophage/monocytes, lung, and liver metabolized l‐[13C6]‐tryptophan to l‐[13C6]kynurenine and [13C6]kynurenate, particularly after indoleamine‐2,3‐dioxygenase induction by interferon‐γ. Kynurenine aminotransferase activity was measurable in all cell types examined but was unaffected by interferon‐γ. These results suggest that many cell types can be sources of kynurenate following immune activation. In vivo synthesis of l‐[13C6]kynurenine and [13C6]kynurenate from l‐[13C6]tryptophan was studied in the CSF of macaques infected with poliovirus, as a model of inflammatory neurologic disease. The effects of 6‐chlorotryptophan and 4‐chloro‐3‐hydroxyanthranilate on the synthesis of kynurenate were different. 6‐Chlorotryptophan attenuated formation of l‐[13C6]kynurenine and [13C6]kynurenate and was converted to 4‐chlorokynurenine and 7‐chlorokynurenate. It may be an effective prodrug for the delivery of 7‐chlorokynurenate, which is a potent antagonist of NMDA receptors. In contrast, 4‐chloro‐3‐hydroxyanthranilate did not reduce accumulation of l‐[13C6]kynurenine and [13C6]kynurenate. 6‐Chlorotryptophan and 4‐chloro‐3‐hydroxyanthranilate are useful tools to manipulate concentrations of quinolinate and kynurenate in the animal models of neurologic disease to evaluate physiological roles of these neuroactive metabolites.
Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?
Authors: Hassel Bjørnar , Sonnewald Ursula
Published: July 2010

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2‐13C]acetate and [1‐13C]glucose as shown by 13C NMR spectroscopy. In fasted mice that had received [2‐13C]acetate intravenously, brain lactate C‐2 and C‐3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1‐13C]glucose strongly labeled lactate C‐3, reflecting glycolysis, whereas lactate C‐2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate‐metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2‐13C]acetate reached ∼1% in both the C‐2 and the C‐3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C‐2 and C‐3 from [2‐13C]acetate, whereas in fed mice, lactate C‐3 was more highly labeled than the C‐2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.
Functional and Binding Properties of σ Receptors in Rat Cerebellum
Authors: Gonzalez‐Alvear Grace M. , Thompson‐Montgomery DeDeene , Deben Sophia E. , Werling Linda L.
Published: July 2010

Abstract: Autoradiographic studies have shown that σ receptors are enriched in the locus coeruleus, the origin of noradrenergic projections to the cerebellum, as well as in the Purkinje, molecular, and granular layers and the interpositus cerebellar nucleus of the cerebellum itself. In contrast, the cerebellum is relatively poor in phencyclidine (PCP) binding sites, which have been historically confused with σ sites. The high ratio of σ to PCP receptors in cerebellum is advantageous for discriminating σ‐mediated physiological effects. σ agonists and antagonists have been shown to regulate N‐methyl‐d‐aspartate (NMDA)‐stimulated norepinephrine release in hippocampus, which is innervated by locus coeruleus projections. We now report that σ drugs also regulate norepinephrine release from cerebellum. In contrast to findings in the hippocampus, where regulation is via σ1 and σ2 receptors, σ‐mediated regulation in cerebellum seems to be primarily via σ1 receptors. In radioligand binding studies, we find that σ receptors primarily of the σ1 type are present in the cerebellum. We further report that binding to σ receptors in cerebellum is not affected by the addition of NMDA or glycine or by the presence of NMDA antagonists, suggesting that σ receptors are not located within the NMDA‐operated cation channel in this brain region.
Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells
Authors: Graham Margaret E. , Burgoyne Robert D.
Published: July 2010

Abstract: Using a range of Ca2+ channel blockers we have investigated the Ca2+ channel subtypes that mediate the depolarisation‐induced elevation of the intracellular free Ca2+ concentration ([Ca2+]i) and glutamate release from cultured rat cerebellar granule cells. ω‐Conotoxin‐GVIA had little effect on either the transient or plateau phase of the depolarisation‐induced [Ca2+]i rise or on glutamate release, ruling out a significant role for N‐type Ca2+ channels. Nifedipine substantially inhibited the initial transient rise in [Ca2+]i and the plateau phase of the [Ca2+]i rise and glutamate release, suggesting the involvement of L‐type Ca2+ channels. Both ω‐agatoxin and ω‐conotoxin‐MVIIC also inhibited the transient rise in [Ca2+]i and glutamate release but not the plateau phase of the [Ca2+]i rise. The inhibitions by nifedipine were not increased by coaddition of ω‐conotoxin‐MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca2+ currents that are sensitive to nifedipine, ω‐agatoxin‐IVA, and also ω‐conotoxin‐MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca2+]i rise or glutamate release to distinct P‐, Q‐, or O‐type Ca2+ currents.
Ethanol Increases Cytochrome P4502E1 and Induces Oxidative Stress in Astrocytes
Authors: Montoliu C. , Sancho‐Tello M. , Azorin I. , Burgal M. , Vallés S. , Renau‐Piqueras J. , Guerri C.
Published: July 2010

Abstract: We demonstrate the presence of cytochrome P4502E1 (CYP2E1) in astrocytes in primary culture, its induction by ethanol, and the concomitant generation of free radical species. Double immunofluorescence using anti‐CYP2E1 and anti‐glial fibrillary acidic protein showed that CYP2E1 was distributed over the cytoplasm and processes, although labeling was more pronounced over the nuclear membrane. Immunogold labeling confirmed this pattern of distribution. Addition of 25 mM ethanol to the astrocyte culture medium for 14 days resulted in an increase in the CYP2E1 content, as determined by confocal microscopy and dot blot. In addition, ethanol induced a dose‐dependent increase in the formation of reactive oxygen species that was partially prevented by incubating the astrocytes with anti‐CYP2E1. Alcohol also induced a dose‐dependent increase in malonaldehyde and hydroxynonenal formation and a depletion of the glutathione (GSH) content. These results suggest that ethanol induces oxidative damage in astrocytes, which could explain some of the toxic effects of ethanol on these cells, such as cytoskeletal alterations. This assumption is supported here by the fact that an increase in GSH content prevents the deleterious effects of alcohol on the cytoskeleton of astrocytes. These results suggest that importance of oxidative stress as a mechanism involved in alcohol‐induced neural and brain damage.
The Distribution and Biochemical Properties of a Cdc2‐Related Kinase, KKIALRE, in Normal and Alzheimer Brains
Authors: Yen Shu‐Hui , Kenessey Agnes , Lee Sunhee C. , Dickson Dennis W.
Published: July 2010

Abstract: The biochemical properties and distribution of a Cdc2‐related kinase, KKIALRE, were studied in brain tissues and cultured cells with antibodies to a subregion of KKIALRE protein deduced from cDNA. In adult human brain, the KKIALRE‐immunoreactive protein consisted of four or five isoforms having a molecular size of 40–52 kDa, whereas in fetal brain, there was one protein of ∼48 kDa. Cultured astrocytes, neuroblastoma cells, and mouse brains contained the fetal form of KKIALRE protein. KKIALRE‐immunoreactive proteins were capable of phosphorylating histone and synthetic peptides with the X‐Ser‐Pro‐X motif, indicating that these proteins belong to the proline‐directed Ser/Thr protein kinase family. The KKIALRE immunoreactivity was detected primarily in fibrous astrocytes in white matter and perivascular and subpial spaces, as well as in Bergmann glia in the cerebellum. In fetal brains radial glia were weakly immunoreactive. Reactive astrocytes were more intensely labeled than other glia. Neurons in normal brains and brains with Alzheimer's disease (AD) displayed no KKIALRE immunoreactivity. KKIALRE immunoreactivity was similar in neurons with and without neurofibrillary tangles. The results indicate that in CNS, the KKIALRE protein is mainly a glial protein that is up‐regulated in gliosis and that it probably plays no role in the hyperphosphorylation of τ in AD brains.
Metabolism of [5‐3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway
Authors: Guidetti Paolo , Eastman Clifford L. , Schwarcz Robert
Published: July 2010

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN‐lesioned rat striata after a focal injection of [5‐3H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5‐3H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µM [5‐3H]KYN. Labeled QUIN, KYNA, 3‐hydroxykynurenine (3‐HK), 3‐hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µM [5‐3H]KYN, a lesion‐induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [3H]KYNA (5.0 vs. 1.8%), [3H]3‐HK (20.9 vs. 4.5%), [3H]XA (1.5 vs. 0.4%), and [3H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion‐induced increases of the activities of kynurenine‐3‐hydroxylase (3.6‐fold), kynureninase (7.6‐fold), kynurenine aminotransferase (1.8‐fold), and 3‐hydroxyanthranilic acid oxygenase (4.2‐fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.
1‐Benzyl‐1,2,3,4‐Tetrahydroisoquinoline as a Parkinsonism‐Inducing Agent: A Novel Endogenous Amine in Mouse Brain and Parkinsonian CSF
Authors: Kotake Yaichiro , Tasaki Yoshikazu , Makino Yukiko , Ohta Shigeru , Hirobe Masaaki
Published: July 2010

Abstract: 1‐Benzyl‐1,2,3,4‐tetrahydroisoquinoline (1BnTIQ) was detected as a novel endogenous amine in mouse brain and parkinsonian CSF by using the gas chromatography‐selected ion‐monitoring method. The level of 1BnTIQ was very high in CSF of some parkinsonian patients compared with that of controls with other neurological diseases, the mean value being three times higher (parkinsonians: 1.17 ± 0.35 ng/ml of CSF, n = 18; vs. controls: 0.40 ± 0.10 ng/ml of CSF, n = 11; mean ± SEM, not significantly different). The pole test, a toxicological examination to evaluate behavior abnormalities related to Parkinson's disease, was used to examine the pharmacological effect of 1BnTIQ in mice. Repeated administration of 1BnTIQ induced behavior abnormalities, which pretreatment with 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline could prevent. We suggest that 1BnTIQ may be related to the idiopathic Parkinson's disease.
Induction of Prostanoid Biosynthesis by Bacterial Lipopolysaccharide and Isoproterenol in Rat Microglial Cultures
Authors: Minghetti Luisa , Levi Giulio
Published: July 2010

Abstract: We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E2, prostaglandin D2, and thromboxane A2 was low in basal conditions and increased in a dose‐ and time‐dependent way upon lipopolysaccharide treatment (1–100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100‐fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B4; although lipopolysaccharide failed to stimulate leukotriene B4 release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [3H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of β‐adrenergic receptor activation. The β‐agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide. The drug appeared to act mainly through the inducible cyclooxygenase; because it did not stimulate arachidonic acid release, it enhanced the lipopolysaccharide‐evoked prostanoid production observed after aspirin pretreatment and induced de novo synthesis of cyclooxygenase detectable by western blot analysis. We suggest that during cerebral inflammatory processes microglia can contribute to the establishment of high prostanoid levels, which can be further elevated by β‐adrenergic activation.
Oligomerization of Chicken Acetylcholinesterase Does Not Require Intersubunit Disulfide Bonds
Authors: Gough Nancy R. , Randall William R.
Published: July 2010

Abstract: Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C‐terminus is thought to play an important role in these interactions. We generated mutants in the C‐terminus of the catalytic T‐subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild‐type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C‐terminal Cys764, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C‐terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild‐type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys764 is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C‐terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.
Differential Biological Effects of K252 Kinase Inhibitors Are Related to Membrane Solubility but Not to Permeability
Authors: Ross Alonzo H. , McKinnon Christine A. , Daou Marie‐Claire , Ratliff Kristin , Wolf David E.
Published: July 2010

Abstract: K252a and K252b are related protein kinase inhibitors that, dependent on conditions, can either inhibit or potentiate the effects of neurotrophic factors. K252a, an ester, is more potent and more cytotoxic on intact cells than K252b, a carboxylic acid. To understand better why these drugs elicit different degrees of biological responses, we analyzed their hydrophobicity, cell permeability, and subcellular distribution. As judged by partitioning between organic and aqueous phases, both compounds are hydrophobic. The partition coefficients were 15.6:1 (organic/aqueous phases) for K252a and 4.4:1 for K252b. The ratio of fluorescence excitation at 352 nm to that at 340 nm for the K252 compounds in the organic alcohol 1‐decanol versus water provides a simple assay of binding of these compounds to phospholipid membranes. This ratio shifted for K252a, but not K252b, in the presence of phospholipid vesicles, indicating that K252a dissolved in the hydrophobic interior of the membrane. Using quantitative video fluorescence microscopy, we found that K252a strongly labeled both Sf9 insect cells and PC12 rat pheochromocytoma cells, probably staining intracellular membranes. The uptake of K252a was rapid and apparently irreversible. K252b also quickly entered Sf9 and PC12 cells, but staining was much weaker. Hence, K252a and K252b are similar in that they both rapidly enter cells but greatly differ in their membrane solubility.
In Vivo Microdialysis Evidence for Transient Dopamine Release by Benzazepines in Rat Striatum
Authors: Tomiyama Katsunori , Koshikawa Noriaki , Funada Kunie , Oka Koichi , Kobayashi Masafumi
Published: July 2010

Abstract: The effects of benzazepine derivatives on extracellular levels of dopamine (DA) and l‐3,4‐dihydroxyphenylacetic acid (DOPAC) in the dorsal striatum of freely moving rats were studied using in vivo microdialysis. Direct injection of SKF‐38393 (0.5 or 1.5 µg/0.5 µl), a selective D1 receptor agonist, into the striatum through a cannula secured alongside a microdialysis probe produced a rapid dose‐dependent transient increase in striatal DA efflux and a more gradual reduction in efflux of DOPAC. The rapid increase in DA efflux was not affected by infusion of tetrodotoxin (TTX; 2 µM) or Ca2+‐free Ringer's solution and occurred after either enantiomer of SKF‐38393. A TTX‐insensitive increase in DA level similar to that induced by SKF‐38393 was also seen after other benzazepines acting as agonists (SKF‐75670 and SKF‐82958, each 1.5 µg in 0.5 µl) and antagonists (SCH‐23390, 1.5 µg in 0.5 µl) at the D1 receptor and after (+)‐amphetamine. These effects were inhibited by infusion of nomifensine (100 µM). It is concluded that the transient increases in striatal DA efflux seen after intrastriatal injection of SKF‐38393 and other benzazepines are not mediated by presynaptic D1 receptors but by an amphetamine‐like action on the dopamine transporter.
Memory Consolidation Induces a Transient and Time‐Dependent Increase in the Frequency of Neural Cell Adhesion Molecule Polysialylated Cells in the Adult Rat Hippocampus
Authors: Fox Gerard B. , O'Connell Alan W. , Murphy Keith J. , Regan Ciaran M.
Published: July 2010

Abstract: Animals trained in a passive avoidance task exhibit a transient time‐dependent increase in hippocampal neural cell adhesion molecule (NCAM) polysialylation at 12–24 h following the initial learning trial. Using immunocytochemical techniques with a monoclonal antibody that specifically recognises NCAM‐polysialic acid homopolymers, a distinct population of granule‐like cells, at the border of the granule cell layer and the hilus in the dentate gyrus of the adult rat hippocampus, has been demonstrated to exhibit time‐dependent change in frequency at 10–12 h following the initial learning of a one‐trial, step‐through, passive avoidance response. These changes were paradigm specific as they failed to occur in those animals rendered amnesic with scopolamine. These polysialylated dentate neurons are not de novo granule cell precursors as administration of 5‐bromo‐2′‐deoxyuridine every 2 h from the point of learning to the 12‐h posttraining time showed no significant difference between trained and passive animals in the small number of heterogeneously distributed, labelled cells. These findings directly identify a morphological substrate of memory, implied by previous correlative and interventive studies on NCAM function.
Cyclic AMP‐Independent Inhibition of Voltage‐Sensitive Calcium Channels by Forskolin in PC12 Cells
Authors: Park Tae‐Ju , Kim Kyong‐Tai
Published: July 2010

Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage‐sensitive Ca2+ channels (VSCCs) in a cyclic AMP (cAMP)‐independent manner in PC12 cells. Ca2+ influx induced by membrane depolarization with 70 mM K+ was inhibited when cells were preincubated with 10 µM forskolin. Almost maximum inhibitory effect on Ca2+ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca2+ influx was not affected by the presence of 2′,5′‐dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin‐induced cAMP production. 1,9‐Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca2+ influx induced by 70 mM K+ without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.
Comparison of the Effects of Retinoic Acid and Nerve Growth Factor on PC12 Cell Proliferation, Differentiation, and Gene Expression
Authors: Cosgaya Jose Miguel , Garcia‐Villalba Pilar , Perona Rosario , Aranda Ana
Published: July 2010

Abstract: Retinoic acid (RA) produced a dose‐dependent inhibition of PC12 cell growth and the appearance of cell clusters without neurite extension. RA‐induced cell clumping was similar to that caused by dexamethasone (Dx). Nerve growth factor (NGF) induced neurite extension, and the combination of RA plus NGF produced a maximal decrease in cell proliferation with a mixed morphology in which part of the cell population had neurites and part formed clumps. Transcriptional effects of RA were demonstrated by the increase in the activity of reporter constructs that contain an RA response element. RA also regulated expression of endogenous genes in PC12 cells. The retinoid produced a two‐ to threefold increase in level of p75LNGFR mRNA (the low‐affinity NGF receptor), without altering expression of the trk protooncogene (the high‐affinity NGF receptor carrying tyrosine kinase activity). RA also caused a transient increase in level of tyrosine hydroxylase (TH) mRNA (twofold after 16 h), which returned to basal levels and then decreased relative to basal levels at 48 h. The effect of NGF on the expression of these genes was identical to that produced by RA. However, incubation with Dx did not induce p75LNGFR mRNA and produced a strong and sustained increase of TH mRNA level (three‐ to fivefold after 48 h). These results show that, despite the common morphological changes produced by RA and glucocorticoids in PC12 cells, the biochemical changes caused by RA are similar to those produced by NGF. Therefore, RA could initiate a biochemical program of neuronal differentiation in PC12 cells, although a fully differentiated phenotype with neurite extension is not obtained.
Evidence that Nitric Oxide Causes Calcium‐Independent Release of [3H]Dopamine from Rat Striatum In Vitro
Authors: Stewart Tracy L. , Michel Anton D. , Black Mark D. , Humphrey Patrick P. A.
Published: July 2010

Abstract: Nitric oxide (NO), liberated from the photoactive donor Roussin's black salt (RBS), was investigated for its ability to release tritium from [3H]dopamine‐loaded rat striatal slices. Our results show that illumination of RBS‐pretreated striatal slices caused an increase in basal dopamine release, which was reduced by ∼73% in the presence of oxyhaemoglobin (10 µM), indicating that it was mediated by liberation of NO. The release was insensitive to removal of extracellular calcium yet was not due to gross cellular damage of the tissue, as there was no detectable increase in lactate dehydrogenase release. Chelation of intracellular calcium with 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetra(acetoxymethyl) ester (BAPTA‐AM; 10 µM) had no effect on the dopamine release stimulated by illumination of RBS‐pretreated slices. The concentration of BAPTA‐AM was adequate to chelate intracellular calcium because it inhibited release evoked by the calcium ionophore ionomycin (10 µM). Superfusion with zaprinast (10 µM) had no effect on RBS‐induced dopamine release, suggesting that a mechanism independent of cyclic GMP is involved. This study indicates that NO has a stimulatory effect on striatal dopamine release in vitro that is independent of calcium.
Arachidonate 5‐Lipoxygenase and Its Activating Protein: Prominent Hippocampal Expression and Role in Somatostatin Signaling
Authors: Lammers C.‐H. , Schweitzer P. , Facchinetti P. , Arrang J.‐M. , Madamba S. G. , Siggins G. R. , Piomelli D.
Published: July 2010

Abstract: 5‐Lipoxygenase‐activating protein (FLAP) is an 18‐kDa integral membrane protein required, in peripheral cells, for the activation of 5‐lipoxygenase (5‐LO) and for the resulting synthesis of leukotrienes from arachidonic acid. In the brain, the leukotrienes have been implicated in several pathophysiological events and in the electrophysiological effect of somatostatin, yet the cellular origin and role of these messenger molecules are still poorly understood. In the present study, we used reverse transcriptase‐polymerase chain reaction, in situ hybridization, and immunohistochemistry to demonstrate that 5‐LO and FLAP are expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus, and brainstem. Highest levels of expression were observed in cerebellum and hippocampus. In the latter we demonstrate the colocalization of 5‐LO and FLAP in CA1 pyramidal neurons. Moreover, electrophysiological experiments show that selective inhibition of FLAP with the compound MK‐886 (0.25–1 µM) prevents the somatostatin‐induced augmentation of the hippocampal K+ M‐current. Our results provide necessary evidence for the presence and signaling role of 5‐LO and FLAP in central neurons and strongly support their proposed participation in somatostatin‐receptor transmembrane signaling.
Arachidonic Acid and Oleoylacetylglycerol Induce a Synergistic Facilitation of Ca2+‐Dependent Glutamate Release from Hippocampal Mossy Fiber Nerve Endings
Authors: Zhang Lian , Ruehr Mary Louise , Dorman Robert V.
Published: July 2010

Abstract: Arachidonic acid and oleoylacetylglycerol enhance depolarization‐evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca2+‐dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca2+ ionophore ionomycin. The Ca2+ dependence of this effect was established, as removal of Ca2+ eliminated evoked release and the lipid‐dependent potentiation. Also, Ca2+ channel blockers attenuated ionomycin‐ and KCI‐evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca2+, it may not involve an enhancement of evoked Ca2+ accumulation, because ionomycin‐dependent glutamate release was potentiated under conditions that did not increase ionomycin‐induced Ca2+ accumulation. Also, the facilitation may not depend on inhibition of K+ efflux, because enhanced release was observed in the presence of increasing concentrations of 4‐aminopyridine and diazoxide did not reduce the lipid‐dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid‐dependent facilitations of both KCI‐ and ionomycin‐evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid‐dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca2+ and involves multiple presynaptic effects, some of which depend on protein kinase C.
Dopaminergic Inhibition of Catecholamine Secretion from Chromaffin Cells: Evidence that Inhibition Is Mediated by D4 and D5 Dopamine Receptors
Authors: Dahmer Mary K. , Senogles Susan E.
Published: July 2010

Abstract: Previous studies have suggested that activation of D2‐like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1‐like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2‐like dopamine receptors. Both D1‐ and D2‐selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K+ levels. The D1‐selective agonists 6‐chloro‐7,8‐dihydroxy‐3‐allyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine (Cl‐APB) and SKF‐38393 inhibited DMPP‐stimulated catecholamine secretion in a concentration‐dependent manner; 50% inhibition was obtained with ∼10 µM Cl‐APB and ∼100 µM SKF‐38393. Of the D2‐selective agonists, bromocriptine was a more potent inhibitor of DMPP‐stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by Cl‐APB or SKF‐38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2‐selective, but not D1‐selective, dopamine agonists. In addition, forskolin‐stimulated adenylyl cyclase activity was inhibited by D2‐selective, but not D1‐selective, agonists. Neither D1‐ nor D2‐selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP‐stimulated Ca2+ uptake was inhibited by both D1‐ and D2‐selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1‐like and D2‐like subfamilies was responsible for the observed inhibition. PCR analysis indicated that mRNA for only D4 and D5 dopamine receptor subtypes was present in chromaffin cells. These combined data suggest that D1‐ and D2‐selective agonists inhibit Ca2+ uptake and catecholamine secretion by activating D4 and D5 dopamine receptors on chromaffin cells.
Carrier‐Mediated Release of Serotonin by 3,4‐Methylenedioxymethamphetamine: Implications for Serotonin‐Dopamine Interactions
Authors: Gudelsky Gary A. , Nash J. Frank
Published: July 2010

Abstract: In vivo microdialysis was used to determine whether the 3,4‐methylenedioxymethamphetamine (MDMA)‐induced release of serotonin (5‐HT) in vivo involves a carrier‐mediated process and to investigate further the state‐dependent interaction between 5‐HT and dopamine. MDMA produced a dose‐dependent increase in the extracellular concentration of 5‐HT in the striatum and prefrontal cortex that was attenuated by treatment with fluoxetine but not by tetrodotoxin. Suppression by fluoxetine of the MDMA‐induced release of 5‐HT was accompanied by a suppression of the MDMA‐induced release of dopamine. Administration of MDMA to rats treated with carbidopa and l‐5‐hydroxytryptophan resulted in a synergistic elevation of the extracellular concentration of 5‐HT that was much greater than that produced by either treatment alone. The MDMA‐induced release of dopamine by MDMA also was potentiated in 5‐hydroxytryptophan‐treated rats. These data are consistent with the view that MDMA increases the extracellular concentration of 5‐HT by facilitating carrier‐mediated 5‐HT release, which can be enhanced greatly under conditions in which 5‐HT synthesis is stimulated. Moreover, these data are supportive of a state‐dependent, stimulatory role of 5‐HT in the regulation of dopamine release.
Quinolinic Acid Levels in a Murine Retrovirus‐Induced Immunodeficiency Syndrome
Authors: Sei Yoshitatsu , Paul Ian A. , Saito Kuniaki , Layar Richard , Hartley Janet W. , Morse Herbert C. , Skolnick Phil , Heyes Melvyn P.
Published: July 2010

Abstract: Mice infected with the retrovirus mixture designated LP‐BM5 murine leukemia virus (MuLV) develop an immunosuppressive disease. Quinolinic acid (QUIN) is an endogenous neurotoxic N‐methyl‐d‐aspartate agonist that may contribute to the pathogenesis of HIV‐associated neurologic disease. In the present study, the levels of QUIN in brain and blood were measured in mice infected with LP‐BM5 MuLV and compared with those in uninfected mice and mice infected with the nonpathogenic strain of ecotropic MuLV (helper component of LP‐BM5 MuLV). Infection with LP‐BM5 MuLV resulted in progressive increases in blood QUIN levels beginning 2 weeks after inoculation that peaked by 16 weeks postinfection. QUIN levels were also increased in cerebral cortex, hippocampus, and striatum. In systemic tissues, QUIN levels were increased in lung, liver, and spleen. In contrast, infection with the ecotropic viral component of the LP‐BM5 MuLV mixture was not associated with any changes in brain, blood, or systemic tissue QUIN levels, even though helper virus burdens were comparable to those in mice infected with LP‐BM5 MuLV. Treatment of LP‐BM5 MuLV‐infected mice with the antiretroviral agent zidovudine (azidothymidine) significantly reduced blood and brain QUIN levels in association with reductions in viral load in brain and spleen. These observations suggest that elevated QUIN production is not attributable to productive infection with retrovirus per se but occurs in response to an agent or agents, such as cytokines, that are produced by the host in response to virus infection.
Various Isoforms of Myomodulin Identified from the Male Copulatory Organ of Lymnaea Show Overlapping yet Distinct Modulatory Effects on the Penis Muscle
Authors: Van Golen Floral A. , Li Ka Wan , Chen Su , Jiménez Connie R. , Geraerts Wijnand P. M.
Published: July 2010

Abstract: Male copulatory behavior in the snail Lymnaea stagnalis is controlled by several types of peptidergic neurons, including a cluster of neurons in the ventral lobe of the right cerebral ganglion that show immunoreactivity to myomodulin‐A of Aplysia and innervate the penis complex. We identified structurally myomodulin‐A and three related peptides from Lymnaea and showed that they are present in a characteristic ratio in both the penis nerve and penis complex, suggesting that they are processed from a single precursor and transported from the ventral lobe to the penis complex. All four peptides decreased the relaxation time of electrically evoked contractions of the penis retractor muscle. However, their effects on the amplitude of contraction were different, ranging from no effect to an increase or a decrease in the amplitude. A mixture of the peptides in a ratio as determined by direct mass spectrometry of the penis nerve decreased the contraction time, the relaxation time, and the amplitude. These effects resemble those of one particular peptide in the mixture. The direct mass spectrometry determinations of the peptide profile in the penis nerve suggest that many more, as yet unidentified, neuropeptides are involved in modulation of muscle activities of the penis complex.