bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin Causes a Transient Tyrosine Phosphorylation of NR2A and NR2B NMDA Receptor Subunits in Rat Hippocampus
Authors: Christie J. M. , Wenthold R. J. , Monaghan D. T.
Published: March 2003

Abstract: NMDA receptors play a critical role in various aspects of CNS function. Hence, it is important to identify mechanisms that regulate NMDA receptor activity. We have shown previously that insulin rapidly potentiates NMDA receptor activity in both native and recombinant expression systems. Here we report that insulin causes a transient phosphorylation of NR2A and NR2B NMDA receptor subunits on tyrosine residues. Rat hippocampal slices were exposed to 1 μM insulin for 20 and 60 min and then solubilized. NR2A and NR2B subunits were immunoprecipitated and probed for tyrosine phosphorylation. Insulin incubation of hippocampal slices for 20 min elicited an increase in tyrosine phosphorylation to 176 ± 16% (NR2A) and 203 ± 15% (NR2B) of control levels. In contrast, 60 min of insulin incubation did not alter NR2 tyrosine phosphorylation levels (NR2A: 85 ± 13% of control; NR2B: 93 ± 10% of control). Although the consequence of insulin‐stimulated tyrosine phosphorylation is unknown, it is possible that this site(s) is responsible for insulin potentiation of NMDA receptor activity. This possibility is consistent with our earlier finding that insulin potentiates hippocampal NMDA receptor activity after 20 min, but not after 60 min, of insulin exposure.
Brain Neurotransmitter Deficits in Mice Transgenic for the Huntington’s Disease Mutation
Authors: Reynolds Gavin P. , Dalton Caroline F. , Tillery Claire L. , Mangiarini Laura , Davies Stephen W. , Bates Gillian P.
Published: March 2003

Abstract: Huntington’s disease (HD) is associated with an expansion in the CAG repeat sequence of a gene on chromosome 4, resulting in a neurodegenerative process particularly affecting the striatum and with profound but selective changes in content of various neurotransmitters. Recently, transgenic mice expressing a fragment of the human HD gene containing a large CAG expansion have been generated; these mice exhibit a progressive neurological phenotype that includes motor disturbances, as well as neuronal deficits. To investigate their underlying neurotransmitter pathology, we have determined concentrations of GABA, glutamate, and the monoamine neurotransmitters in several brain regions in these mice and control animals at times before and after the emergence of the behavioural phenotype. In contrast to the findings in HD, striatal GABA was unaffected, although a deficit was observed in the cerebellum, consistent with a dysfunction of Purkinje cells. Losses of the monoamine transmitters were observed, some of which are not seen in HD. Thus, 5‐hydroxytryptamine and, to a greater extent, 5‐hydroxyindoleacetic acid levels were diminished in all brain regions studied, and noradrenaline was particularly affected in the hippocampus. Dopamine was decreased in the striatum in older animals, parallelling evidence for diminished dopaminergic activity in HD.
Depletion of Intracellular Glutathione Increases Susceptibility to Nitric Oxide in Mesencephalic Dopaminergic Neurons
Authors: Ibi Masakazu , Sawada Hideyuki , Kume Toshiaki , Katsuki Hiroshi , Kaneko Shuji , Shimohama Shun , Akaike Akinori
Published: March 2003

Abstract : Using primary neuronal cultures, we investigated the

effects of GSH depletion on the cytotoxic effects of glutamate and NO in

dopaminergic neurons. Intracellular GSH was depleted by 24‐h exposure to

L‐buthionine‐[S,R]‐sulfoximine (BSO), an irreversible inhibitor of

GSH synthase. BSO exposure caused concentration‐dependent reduction of the

viability of both dopaminergic and nondopaminergic neurons. In contrast, 24‐h

exposure of cultures to glutamate or NOC18, an NO‐releasing agent,

significantly reduced the viability of non‐dopaminergic neurons without

affecting that of dopaminergic neurons. Pretreatment with

N‐acetyl‐L‐cysteine for 24 h ameliorated the NOC18‐induced toxicity in nondopaminergic neurons. In dopaminergic neurons, sublethal concentrations of BSO reduced intracellular GSH content and markedly potentiated glutamate‐ and NOC‐18‐induced toxicity. These results suggested that glutamate toxicity was enhanced in dopaminergic neurons by suppression of defense mechanisms against NO toxicity under conditions of GSH depletion. Under such conditions, free iron plays an important role because BSO‐enhanced NO toxicity was ameliorated by the iron‐chelating agent, deferoxamine. These results suggest that GSH plays an important role in the expression of NO‐mediated glutamate cytotoxicity in dopaminergic neurons. Free iron may be related to enhanced NO cytotoxicity under GSH depletion.
Role of the Conserved Lysine Residue in the Middle of the Predicted Extracellular Loop Between M2 and M3 in the GABAA Receptor
Authors: Sigel Erwin , Buhr Andreas , Baur Roland
Published: March 2003

Abstract : In α1, β2, and γ2 subunits of the

γ‐aminobutyric acid A (GABAA) receptor, a conserved lysine

residue occupies the position in the middle of the predicted extracellular

loop between the transmembrane M2 and M3 regions. In all three subunits, this

residue was mutated to alanine. Whereas the mutation in α1 and β2

subunits results each in about a sixfold shift of the concentration‐response

curve for GABA to higher concentrations, no significant effect by mutation in

the γ subunit was detected. The affinity for the competitive inhibitor

bicuculline methiodide was not affected by the mutations in either the

α1 subunit or the β2 subunit. Concentration‐response curves for

channel activation by pentobarbital were also shifted to higher concentrations

by the mutation in the α and β subunits. Binding of

[3H]Ro 15‐1788 was unaffected by the mutation in the α

subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the α1 subunit to E, Q, or R resulted in an about eight‐, 10‐, or fivefold shift, respectively, to higher concentrations of the concentration‐response curve for GABA. From these observations, it is concluded that the corresponding residues on the α1 and β2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.
Delayed and Sustained Activation of p42/p44 Mitogen‐Activated Protein Kinase Induced by Proteasome Inhibitors Through p21ras in PC12 Cells
Authors: Hashimoto Keiko , Guroff Gordon , Katagiri Yasuhiro
Published: March 2003

Abstract: Proteolysis by the ubiquitin/proteasome pathway regulates the intracellular level of several proteins, some of which control cell proliferation and cell cycle progression. To determine what kinds of signaling cascades are activated or inhibited by proteasome inhibition, we treated PC12 cells with specific proteasome inhibitors and subsequently performed in‐gel kinase assays. N‐Acetyl‐Leu‐Leu‐norleucinal and lactacystin, which inhibit the activity of the proteasome, induced the activation of p42/p44 mitogen‐activated protein (MAP) kinases [extracellular signal‐regulated kinases (ERKs) 1 and 2]. In contrast, N‐acetyl‐Leu‐Leu‐methional, which inhibits the activity of calpains, but not of the proteasome, failed to induce ERK activation. Uniquely, the kinetics of MAP kinase activation induced by proteasome inhibitors are very slow compared with those resulting from activation by nerve growth factor; ERK activation is detectable only after a 5‐h treatment with the inhibitors, and its activity remained unchanged for at least until 27 h. Proteasome inhibitor‐initiated ERK activation is inhibited by pretreatment with the ERK kinase inhibitor PD 98059, as well as by overexpression of a dominant‐negative form of Ras. Thus, proteasome inhibitors induce sustained ERK activation in a Ras‐dependent manner. Proteasome inhibitor‐induced neurite outgrowth, however, is not inhibited by PD 98059, indicating that sustained activation of ERKs is not the factor responsible for proteasome inhibitor‐induced morphological differentiation. Our data suggest the presence of a novel mechanism for activation of the MAP kinase cascade that involves proteasome activity.
Increased Secretion of Adrenomedullin from Cultured Human Astrocytes by Cytokines
Authors: Takahashi Kazuhiro , Nakayama Masaharu , Totsune Kazuhito , Murakami Osamu , Sone Masahiko , Kitamuro Tomomi , Yoshinoya Ayako , Shibahara Shigeki
Published: March 2003

Abstract: Adrenomedullin, originally discovered from pheochromocytoma, is a member of the calcitonin gene‐related peptide family. The production and secretion of adrenomedullin by cultured human astrocytes were studied by northern blot analysis and radioimmunoassay. Northern blot analysis showed the expression of adrenomedullin mRNA in cultured human astrocytes. Immunoreactive adrenomedullin concentrations in the culture medium were 29.6 ± 1.2 fmol/105 cells/24 h (mean ± SEM, n = 4). Treatment with interferon‐γ (100 U/ml), tumor necrosis factor‐α (1 and 10 ng/ml), or interleukin‐1β (1 and 10 ng/ml) for 24 h caused >20‐fold increases in immunoreactive adrenomedullin levels in the culture medium of human astrocytes. On the other hand, northern blot analysis showed only small increases (∼40%) in the adrenomedullin mRNA expression of human astrocytes with either 100 U/ml interferon‐γ or 10 ng/ml interleukin‐1β and no noticeable change with tumor necrosis factor‐α. Reverse phase HPLC of the medium extracts of human astrocytes treated with interferon‐γ, tumor necrosis factor‐α, or interleukin‐1β showed that most of immunoreactive adrenomedullin was eluted in the position of adrenomedullin‐(1‐52). On the other hand, immunoreactive adrenomedullin in the medium of human astrocytes without cytokine treatment was eluted earlier than the adrenomedullin standard, suggesting that this immunoreactive adrenomedullin represents adrenomedullin with some modifications or fragments of the adrenomedullin precursor. The present study has shown the production and secretion of adrenomedullin by human astrocytes and increased secretion of adrenomedullin by cytokines.
PD98059 Prevents Neurite Degeneration Induced by Fibrillar β‐Amyloid in Mature Hippocampal Neurons
Authors: Rapoport Mark , Ferreira Adriana
Published: March 2003

Abstract: How senile plaques and neurofibrillary tangles are linked represents a major gap in our understanding of the pathophysiology of Alzheimer’s disease (AD). We have previously shown that the addition of fibrillar β‐amyloid (Aβ) to mature hippocampal neurons results in progressive neuritic degeneration accompanied by the enhanced phosphorylation of adult tau isoforms. In the present study, we sought to obtain more direct evidence of the signal transduction pathway(s) activated by fibrillar Aβ leading to tau phosphorylation and the generation of dystrophic neurites. Our results indicated that fibrillar Aβ induced the progressive and sustained activation of the mitogen‐activated protein kinase (MAPK) in mature hippocampal neurons. On the other hand, the specific inhibition of the MAPK signal transduction pathway by means of PD98059, a MAPK kinase (MEK) specific inhibitor, prevented the phosphorylation of tau (at Ser199/Ser202) induced by fibrillar Aβ. In addition, the inhibition of MAPK activation partially prevented neurite degeneration. Taken collectively, our results suggest that the sustained activation of the MAPK signal transduction pathway induced by fibrillar Aβ may lead to the abnormal phosphorylation of tau and the neuritic degeneration observed in AD.
Characterization of the AMPA‐Activated Receptors Present on Motoneurons
Authors: Greig Ann , Donevan Sean D. , Mujtaba Tahmina J. , Parks Thomas N. , Rao Mahendra S.
Published: March 2003

Abstract: Motoneurons have been shown to be particularly sensitive to Ca2+‐dependent glutamate excitotoxicity, mediated via AMPA receptors (AMPARs). To determine the molecular basis for this susceptibility we have used immunocytochemistry, RT‐PCR, and electrophysiology to profile AMPARs on embryonic day 14.5 rat motoneurons. Motoneurons show detectable AMPAR‐mediated calcium permeability in vitro and in vivo as determined by cobalt uptake and electrophysiology. Motoneurons express all four AMPAR subunit mRNAs, with glutamate receptor (GluR) 2 being the most abundant (63.9 ± 4.8%). GluR2 is present almost exclusively in the edited form, and electrophysiology confirms that most AMPARs present are calcium‐impermeant. However, the kainate current in motoneurons was blocked an average of 32.0% by Joro spider toxin, indicating that a subset of the AMPARs is Ca2+‐permeable. Therefore, heterogeneity of AMPARs, rather than the absence of GluR2 or the presence of unedited GluR2, explains AMPAR‐mediated Ca2+ permeability. The relative levels of flip/flop isoforms of each subunit were also examined by semiquantitative PCR. Both isoforms were present, but the relative proportion varied for each subunit, and the flip isoform predominated. Thus, our data show that despite high levels of edited GluR2 mRNA, some AMPARs are Ca2+‐permeable, and this subset of AMPARs can account for the AMPAR‐mediated Ca2+ inflow inferred from cobalt uptake and electrophysiology studies.
Brainstem Activation of Platelet‐Derived Growth Factor‐β Receptor Modulates the Late Phase of the Hypoxic Ventilatory Response
Authors: Gozal David , Simakajornboon Narong , Czapla Marc A. , Xue Ying‐Dan , Gozal Evelyne , Vlasic Vukmir , Lasky Joseph A. , Liu Jing‐Yao
Published: March 2003

Abstract: The early phase of the biphasic ventilatory response to hypoxia in mammals is critically dependent on NMDA glutamate receptor activation within the nucleus of the solitary tract. However, the mechanisms underlying the subsequent development of the typical ventilatory roll‐off are unclear and could underlie important roles in the functional and molecular adaptation to oxygen deprivation. Because the growth factor platelet‐derived growth factor (PDGF)‐BB can modulate the open channel probability of NMDA receptors by activating PDGF‐β receptors, its contribution to hypoxic ventilatory roll‐off was examined. Administration of PDGF‐BB, but not PDGF‐AA, in the nucleus of the solitary tract was associated with significant attenuations of the early hypoxic ventilatory response in conscious rats. Furthermore, marked reductions in the magnitude of hypoxic ventilatory roll‐off occurred in mice heterozygous for a mutation in the PDGF‐β receptor. Administration of a PDGF‐β receptor antagonist to wild‐type littermates elicited similar declines in hypoxic ventilatory roll‐off. The relative abundance of PDGF‐β receptors was confirmed in the nucleus of the solitary tract and other nuclei implicated in the hypoxic ventilatory response. In nucleus of the solitary tract lysates, PDGF‐β receptor tyrosine phosphorylation was temporally correlated with hypoxic ventilatory roll‐off formation. Increased PDGF‐B chain mRNA expression was induced by hypoxia in the nucleus of the solitary tract, and PDGF‐B chain immunoreactivity colocalized with ≅40% of nucleus of the solitary tract neurons, demonstrating hypoxia‐induced c‐Fos enhancements. Thus, PDGF‐BB release and PDGF‐β receptor activation in the nucleus of the solitary tract are critical components of hypoxic ventilatory roll‐off and may have important functional implications in processes underlying survival and acclimatization to hypoxic environments.
Changes in Histamine H3 Receptor Responsiveness in Mouse Brain
Authors: Morisset S. , Traiffort E. , Arrang J. M. , Schwartz J.‐C.
Published: March 2003

Abstract: Changes in various histamine (HA) H3 receptor‐mediated responses and H3 receptor binding in brain were investigated in mice receiving single or repeated administration of ciproxifan, a potent brain‐penetrating and selective H3 receptor antagonist. Blockade of the H3 autoreceptor was nearly as effective in enhancing levels of tele‐methylhistamine (t‐MeHA), a major HA metabolite, in brain areas when ciproxifan was administered once either at 7 a.m. or 8 p.m., in spite of the large differences of basal levels at these two phases of the circadian cycle. Blockade after a single ciproxifan administration was, however, followed by a transient decrease in striatal t‐MeHA levels, possibly reflecting rapid development of autoreceptor hypersensitivity. Following a 5‐day administration of ciproxifan and a 2‐day drug‐free period, basal t‐MeHA levels were significantly decreased (approximately ‐20%) in three brain areas, and the ED50 values of the drug to enhance t‐MeHA levels were increased by 5‐15 times without significant change in maximal response, indicating that H3 autoreceptor hypersensitivity had developed. However, in synaptosomes from the cerebral cortex of these animals, the H3 receptor‐mediated inhibition of K+‐induced [3H]HA release was not significantly modified. Subchronic administration of ciproxifan for 10 days also resulted in an increased binding of [125I]iodoproxyfan to the H3 receptor of striatal and hypothalamic membranes by 40‐54%. Hypersensitivity at H3 somatodendritic autoreceptors and at heteroreceptors attributable to an increased number of HA binding sites could account for the various changes observed in this study.
Changes in the Kinetics of Dopamine Release and Uptake Have Differential Effects on the Spatial Distribution of Extracellular Dopamine Concentration in Rat Striatum
Authors: Peters Jennifer L. , Michael Adrian C.
Published: March 2003

Abstract: The objective of this study was to examine whether the
limited diffusion distance of dopamine in rat striatum produces spatial
heterogeneity in the extracellular dopamine concentration on a dimensional
scale of a few micrometers. Such heterogeneity would be significant because it
would imply that the concentration of dopamine at a given receptor depends on
the receptor's ultrastructural location. Spatially resolved measurements of
extracellular dopamine were performed in the striatum of chloral
hydrate‐anesthetized rats with carbon fiber microdisk electrodes. Dopamine was
monitored during electrical stimulation of the nigrostriatal pathway before
and after administration of drugs that selectively affect the kinetics of
evoked dopamine release and dopamine uptake. The effects of nomifensine (20
mg/kg), L‐DOPA (250 mg/kg), and α‐methyl‐p‐tyrosine (250 mg/kg) on the amplitude of the stimulation responses were examined. The outcome of these experiments was compared with predictions derived from a mathematical model that combines diffusion with the kinetics of release and uptake. The results demonstrate that the extracellular dopamine concentration is spatially heterogeneous on a micrometer scale and that changing the kinetics of dopamine release and uptake has different effects on this spatial distribution. The impact of these results on brain neurochemistry is considered.
Elevated Cortical Extracellular Fluid Glutamate in Transgenic Mice Expressing Human Mutant (G93A) Cu/Zn Superoxide Dismutase
Authors: Alexander Guillermo M. , Deitch Jeffrey S. , Seeburger Jeffrey L. , Valle Luis Del , Heiman‐Patterson Terry D.
Published: March 2003

Abstract: Transgenic mice expressing a mutated (G93A) human Cu/Zn
superoxide dismutase (SOD1) develop motor neuron pathology and clinical
symptoms similar to those seen in patients with amyotrophic lateral sclerosis.
Loss of motor neurons is most prominent in lumbar, followed by cervical cord
and then brainstem. No significant cell death has been reported in motor
cortex. The integrity of the cortical glutamate reuptake systems was evaluated
using intracerebral microdialysis and western immunoblot assays for the
glutamate transporters GLT‐1, GLAST, and EAAC1. The basal extracellular fluid
levels of aspartate, glutamate, glutamine, 3,4‐dihydroxyphenylacetic acid, and
5‐hydroxyindole‐3‐acetic acid were evaluated by HPLC. The extraction fraction
of L‐[3H]glutamate, corrected with [14C]mannitol, was
also evaluated. GLT‐1, EAAC1, and GLAST protein levels were determined by
semiquantitative chemiluminescence immunoblot of proteins from
membrane‐enriched fractions. The relative optical density of film was
translated into relative protein level by comparison with a standard control
mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05)
increase in basal levels of extracellular aspartate and glutamate. In
addition, when the glutamate extraction fraction was challenged with exogenous
unlabeled glutamate (500 μM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space.
Glutamate‐Stimulated Activation of DNA Synthesis via Mitogen‐Activated Protein Kinase in Primary Astrocytes
Authors: Schinkmann Karin A. , Kim Tae‐Aug , Avraham Shalom
Published: March 2003

Abstract: Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time‐ and dose‐dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca2+ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen‐activated protein kinases [extracellular signal‐related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca2+ and by PKC inhibition, suggesting that RAFTK might mediate the glutamate‐dependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate‐stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation.
Inflammatory Cytokines Enhance Muscarinic‐Mediated Arachidonic Acid Release Through p38 Mitogen‐Activated Protein Kinase in A2058 Cells
Authors: Wood Michael W. , Segal Jeff A. , Mark Robert J. , Ogden Ann Marie , Felder Christian C.
Published: March 2003

Abstract: The human melanoma cell line A2058 expresses the Gq‐coupled M5 subtype of muscarinic receptor. Stimulation with the cholinergic agonist, carbachol, induces a dose‐dependent increase in arachidonic acid release. The carbachol‐induced arachidonate release is potentiated two‐ to threefold by pretreatment of A2058 cells with either of the inflammatory cytokines, tumor necrosis factor‐α or interleukin‐1β. Cytokine‐induced enhancement of muscarinic‐mediated arachidonic acid release peaks near 1 h. Western analysis suggests that both cytokines are capable of activating the nuclear factor‐κB (NF‐κB) and p38 mitogen‐activated protein kinase (MAPK) pathways. Anisomycin (1 μM) treatment mimics the cytokine‐induced enhancement of arachidonic acid production and activates the p38 MAPK pathway, but does not activate the NF‐κB pathway. Furthermore, pretreatment of A2058 cells with the putative p38 MAPK inhibitor, SB202190, ablates the cytokine‐dependent augmentation without interfering with the muscarinic‐mediated arachidonic acid release in untreated cells. Moreover, cytokine treatment does not affect other M5‐coupled pathways (e.g., phospholipase C activity or intracellular Ca2+ mobilization), suggesting that p38 MAPK activation principally modulates muscarinic‐mediated phospholipase A2 activity. Finally, in primary cultures of cells taken from rat cerebellum, key aspects of this finding are repeated in cultures enriched for glia, but not in cultures enriched for granule neurons.
Calpain Inhibitors Confer Biochemical, but Not Electrophysiological, Protection Against Anoxia in Rat Optic Nerves
Authors: Jiang Qiubo , Stys Peter K.
Published: March 2003

Abstract: Calpains are ubiquitous Ca2+‐activated neutral proteases that have been implicated in ischemic and traumatic CNS injury. Ischemia and trauma of central white matter are dependent on Ca2+ accumulation, and calpain overactivation likely plays a significant role in the pathogenesis. Adult rat optic nerves, representative central white matter tracts, were studied in an in vitro anoxic model. Functional recovery following 60 min of anoxia and reoxygenation was measured electrophysiologically. Calpain activation was assessed using western blots with antibodies against calpain‐cleaved spectrin breakdown products. Sixty minutes of in vitro anoxia increased the amount of spectrin breakdown ≈20‐fold over control, with a further increase after reoxygenation to >70 times control, almost as much as 2 h of continuous anoxia. Blocking voltage‐gated Na+ channels with tetrodotoxin or removing bath Ca2+ was highly neuroprotective electrophysiologically and resulted in a marked reduction of spectrin degradation. The membrane‐permeable calpain inhibitors MDL 28,170 and calpain inhibitor‐l (10‐100 μM) were effective at reducing spectrin breakdown in anoxic and reoxygenated optic nerves, but no electrophysiological improvement was observed. We conclude that calpain activation is an important step in anoxic white matter injury, but inhibition of this Ca2+‐dependent process in isolation does not improve functional outcome, probably because other deleterious Ca2+‐activated pathways proceed unchecked.
Mice Homozygous for the L250T Mutation in the α7 Nicotinic Acetylcholine Receptor Show Increased Neuronal Apoptosis and Die Within 1 Day of Birth
Authors: Orr‐Urtreger Avi , Broide Ron S. , Kasten Michael R. , Dang Hong , Dani John A. , Beaudet Arthur L. , Patrick James W.
Published: March 2003

Abstract: The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in modulating neurotransmitter release and may play a role in the regulation of neuronal growth and differentiation. A threonine for leucine 247 substitution in the channel domain of the chick α7 nAChR increases agonist affinity and decreases the rate of desensitization, creating a “gain of function” model for this receptor. We have generated mice that express the analogous mutation (L250T) in the α7 nAChR using the techniques of homologous recombination and here report their characteristics. Mice heterozygous (+/T) for the L250T mutation are viable, fertile, and anatomically normal compared with wild‐type littermates. In contrast, homozygous (T/T) L250T mice die within 2‐24 h of birth. Brains of T/T mouse pups exhibit a marked reduction in α7 nAChR protein levels and show extensive apoptotic cell death throughout the somatosensory cortex. Furthermore, α7 L250T nAChRs are functionally expressed on neurons within the brains of T/T neonatal mice and have properties that are consistent with those observed for the rat α7 L250T and the chick α7 L247T mutant nAChRs expressed in oocytes. These findings indicate that neurons in the developing brain expressing only α7 L250T mutant nAChRs are susceptible to abnormal apoptosis, possibly due to increased Ca2+ influx.
Molecular Mechanisms Underlying the Activity‐Linked Alterations in Acetylcholinesterase mRNAs in Developing Versus Adult Rat Skeletal Muscles
Authors: Boudreau‐Larivière Céline , Chan Roxanne Y. Y. , Wu Jun , Jasmin Bernard J.
Published: March 2003

Abstract: The molecular mechanisms underlying the activity‐linked plasticity of acetylcholinesterase (AChE) mRNA levels in mammalian skeletal muscle have yet to be established. Here, we demonstrate that denervation of adult muscle induces a dramatic (up to 90%) and rapid (within 24 h) decrease in the abundance of AChE mRNAs. By contrast, denervation of 14‐day‐old rats leads to a significantly less pronounced reduction (50% of control) in the expression of AChE mRNAs. Assessment of the transcriptional activity of the AChE gene reveals that it remains essentially unchanged in adult denervated muscles, whereas it displays an approximately two‐ to three‐fold increase (p < 0.05) in denervated muscles from 2‐ to 14‐day‐old rats. In addition, we observed a higher rate of degradation of in vitro transcribed AChE mRNAs upon incubation with protein extracts from denervated muscles. Finally, UV‐crosslinking experiments reveal that denervation increases the abundance of RNA‐protein interactions in the 3′ untranslated region of AChE transcripts. Taken together, these data suggest that the abundance of AChE transcripts in mature muscles is controlled primarily via posttranscriptional regulatory mechanisms, whereas in neo‐ and postnatal muscles, both transcriptional and posttranscriptional regulation appears critical in dictating AChE mRNA levels. Accordingly, the activity‐linked transcriptional regulation of the AChE gene appears to demonstrate a high level of plasticity during muscle development when maturation of the neuromuscular junctions is still occurring.
Preferential Resistance of Dopaminergic Neurons to the Toxicity of Glutathione Depletion Is Independent of Cellular Glutathione Peroxidase and Is Mediated by Tetrahydrobiopterin
Authors: Nakamura Ken , Wright David A. , Wiatr Ted , Kowlessur Devanand , Milstien Sheldon , Lei Xin Gen , Kang Un Jung
Published: March 2003

Abstract: Depletion of glutathione in the substantia nigra is one of the earliest changes observed in Parkinson’s disease (PD) and could initiate dopaminergic neuronal degeneration. Nevertheless, experimental glutathione depletion does not result in preferential toxicity to dopaminergic neurons either in vivo or in vitro. Moreover, dopaminergic neurons in culture are preferentially resistant to the toxicity of glutathione depletion, possibly owing to differences in cellular glutathione peroxidase (GPx1) function. However, mesencephalic cultures from GPx1‐knockout and wild‐type mice were equally susceptible to the toxicity of glutathione depletion, indicating that glutathione also has GPx1‐independent functions in neuronal survival. In addition, dopaminergic neurons were more resistant to the toxicity of both glutathione depletion and treatment with peroxides than nondopaminergic neurons regardless of their GPx1 status. To explain this enhanced antioxidant capacity, we hypothesized that tetrahydrobiopterin (BH4) may function as an antioxidant in dopaminergic neurons. In agreement, inhibition of BH4 synthesis increased the susceptibility of dopaminergic neurons to the toxicity of glutathione depletion, whereas increasing BH4 levels completely protected nondopaminergic neurons against it. Our results suggest that BH4 functions as a complementary antioxidant to the glutathione/glutathione peroxidase system and that changes in BH4 levels may contribute to the pathogenesis of PD.
Lithium Ions Enhance Cysteine String Protein Gene Expression In Vivo and In Vitro
Authors: Cordeiro Mara L. , Umbach Joy A. , Gundersen Cameron B.
Published: March 2003

Abstract: Lithium is a well established pharmacotherapy for the treatment of recurrent manic‐depressive illness. However, the mechanism by which lithium exerts its therapeutic action remains elusive. Here we report that lithium at 1 mM significantly increased the expression of cysteine string proteins (CSPs) in a pheochromocytoma cell line (PC12 cells) differentiated by nerve growth factor. These cells concomitantly exhibited increased expression of CSPs in their cell bodies and boutons. Enhanced CSP expression was also observed in the brain of rats fed a lithium‐containing diet, which elevated serum lithium to a therapeutically relevant concentration of ∼1.0 mM. However, both in vitro and in vivo, the expression of another synaptic vesicle protein, synaptophysin, and the t‐SNARE, synaptosomal‐associated protein of 25 kDa (SNAP‐25), was not significantly altered by lithium. These observations indicate that lithium‐induced changes of CSP gene expression may contribute to the therapeutic efficacy of this monovalent cation.
Nongenomic Inhibition of Catecholamine Secretion by 17β‐Estradiol in PC12 Cells
Authors: Kim Youn‐Jae , Hur Eun‐Mi , Park Tae‐Ju , Kim Kyong‐Tai
Published: March 2003

Abstract: We investigated the effects of 17β‐estradiol, an estrogen, on [3H]norepinephrine ([3H]NE) secretion in PC12 cells. Pretreatment with 17β‐estradiol reduced 70 mM K+‐induced [3H]NE secretion in a concentration‐dependent manner with a half‐maximal inhibitory concentration (IC50) of 2 ± 1 μM. The 70 mM K+‐induced cytosolic free Ca2+ concentration ([Ca2+]i) rise was also reduced when the cells were treated with 17β‐estradiol (IC50 = 15 ± 2 μM). Studies with voltage‐sensitive calcium channel (VSCC) antagonists such as nifedipine and ω‐conotoxin GVIA revealed that both L‐ and N‐type VSCCs were affected by 17β‐estradiol treatment. The 17β‐estradiol effect was not changed by pretreatment of the cells with actinomycin D and cycloheximide for 5 h. In addition, treatment with pertussis or cholera toxin did not affect the inhibitory effect of 17β‐estradiol. 17β‐Estradiol also inhibited the ATP‐induced [3H]NE secretion and [Ca2+]i rise. In PC12 cells, the ATP‐induced [Ca2+]i rise is known to occur through P2X2 receptors, the P2Y2‐mediated phospholipase C (PLC) pathway, and VSCCs. 17β‐Estradiol pretreatment during complete inhibition of the PLC pathway and VSCCs inhibited the ATP‐induced [Ca2+]i rise. Our results suggest that 17β‐estradiol inhibits catecholamine secretion by inhibiting L‐ and N‐type Ca2+ channels and P2X2 receptors in a nongenomic manner.
Contribution of Anion Transporters to the Acidosis‐Induced Swelling and Intracellular Acidification of Glial Cells
Authors: Ringel Florian , Chang Raymond C. C. , Staub Frank , Baethmann Alexander , Plesnila Nikolaus
Published: March 2003

Abstract : This study examines the contribution of anion

transporters to the swelling and intracellular acidification of glial cells

from an extracellular lactacidosis, a condition well‐known to accompany

cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were

exposed to lactacidosis in physiological or anion‐depleted media, and

different anion transport inhibitors were applied. Changes in cell volume and

intracellular pH (pHi) were simultaneously quantified by flow

cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell

volume to 125.1 ± 2.5% of baseline within 60 min, whereas the

pHi dropped from the physiological value of 7.13 ± 0.05 to

6.32 ± 0.03. Suspension in Cl‐‐free or

HCO3‐/CO2‐free media or application of anion

transport inhibitors [0.1 mM bumetanide or 0.5 mM

4,4′‐diisothio‐cyanatostilbene‐2,2′‐disulfonic acid (DIDS)] did

not affect cell volume during baseline conditions but significantly reduced

cell swelling from lactacidosis. In addition, the Cl‐‐free or

HCO3‐/CO2‐free media and DIDS attenuated

intracellular acidosis on extracellular acidification. From these findings it

is concluded that besides the known activation of the

Na+/H+ exchanger, activation of the

Na+‐independent Cl‐/HCO3‐

exchanger and the Na+‐K+‐Cl‐ cotransporter contributes to acidosis‐induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.
Stoichiometry of Tyrosine Hydroxylase Phosphorylation in the Nigrostriatal and Mesolimbic Systems In Vivo
Authors: Salvatore Michael F. , Garcia‐Espana Antonio , Goldstein Menek , Deutch Ariel Y. , Haycock John W.
Published: March 2003

Abstract ; Electrical stimulation of the medial forebrain bundle

increases 32P incorporation into striatal tyrosine hydroxylase (TH)

at Ser 19, Ser31, and Ser40. In the present

studies, the effects of acute haloperidol and related drugs on sitespecific TH

phosphorylation stoichiometry (PS) in the nigrostriatal and mesolimbic systems

were determined by quantitative blot immunolabeling using phosphorylation

statespecific antibodies. The striatum (Str), substantia nigra (SN), nucleus

accumbens (NAc), and ventral tegmental area (VTA) from Sprague‐Dawley rats

were harvested 30‐40 min after a single injection of either vehicle,

haloperidol (2 mg/kg), raclopride (2 mg/kg), clozapine (30 mg/kg), or SCH23390

(0.5 mg/kg). In vehicle‐injected control rats, Ser19 PS was 1.5‐ to

2.5‐fold lower in Str and NAc than in SN and VTA, Ser31 PS was

two‐to fourfold higher in Str and NAc than in SN and VTA, and Ser40

PS was similar between the terminal field and cell body regions. After

haloperidol, Ser40 PS increased twofold in Str and NAc, whereas a

smaller increase in SN and VTA was observed. The effects of haloperidol on

Ser19 PS were similar to those on Ser40 in each region ;

however, haloperidol treatment increased Ser31 PS at least 1.6‐fold in all regions. The effects of raclopride on TH PS were comparable to those of haloperidol, whereas clozapine treatment increased TH PS at all sites in all regions. By contrast, the effects of SCH23390 on TH PS were relatively small and restricted to the NAc. The stoichiometries of site‐specific TH phosphorylation in vivo are presented for the first time. The nigrostriatal and mesolimbic systems have common features of TH PS, distinguished by differences in TH PS between the terminal field and cell body regions and by dissimilar increases in TH PS in the terminal field and cell body regions after acute haloperidol.
Light‐Mediated Activation of Diacylglycerol Kinase in Rat and Bovine Rod Outer Segments
Authors: Huang Zhong , Ghalayini Abboud , Guo Xia Xiao , Alvarez Kathleen M. , Anderson Robert E.
Published: March 2003

The hydrolysis of phosphatidylinositol 4,5‐bisphosphate is regulated by light in retinal rod outer segment (ROS) membranes. We recently reported that the activities of phosphatidylinositol synthetase and phosphatidylinositol 3‐kinase are also higher in bleached (light‐exposed) ROS (B‐ROS). In this study, we investigated the effect of bleaching on diacylglycerol (DAG) kinase (DAG‐kinase) activity in bovine and rat ROS membranes prepared from dark‐adapted (D‐ROS) or bleached (B‐ROS) retinas. In bovine ROS, DAG‐kinase activity toward endogenous DAG substrate was higher in B‐ROS than in D‐ROS. Quantification of DAG in both sets of membranes showed that the levels were the same, eliminating the possibility that the greater DAG‐kinase activity was due to higher levels of endogenous substrate in B‐ROS. DAG‐kinase activity was also higher in B‐ROS against an exogenous, water‐soluable substrate (1,2‐didecanoyl‐rac‐glycerol), which competed with endogenous DAG

substrate and saturated at ~2 mM. Immunoblot analysis with an anti‐DAG‐kinase γ polyclonal antibody demonstrated that the γ isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal sections confirmed the presence of DAG‐kinase γ immunoreactivity in ROS, as well as other retinal cells. Quantification of the immunoreactive products on western blots showed that more DAG‐kinase γ was present in B‐ROS than in D‐ROS. In an in vivo experiment, ROS prepared from rats exposed to 30 min of room light had greater DAG‐kinase activity than ROS prepared from dark‐adapted animals. Taken together, these data suggest that light exposure leads to the translocation of DAG‐kinase from the cytosol to ROS membranes and that the greater DAG‐kinase activity in B‐ROS is due to the presence of more protein associated with ROS membranes.
Assessment of Stereoselectivity of Trimethylphenylalanine Analogues of δ‐Opioid [D‐Pen2,D‐Pen5]‐Enkephalin
Authors: Witt Ken A. , Slate Cheryl A. , Egleton Richard D. , Huber Jason D. , Yamamura Henry I. , Hruby Victor J. , Davis Thomas P.
Published: March 2003

Abstract : [D‐Pen2,D‐Pen5]‐Enkephalin (DPDPE)

is an enzymatically stable δ‐opioid receptor‐selective peptide, which

was modified by the trimethylation of the Phe4 residue to give

β‐methyl‐2′,6′‐dimethylphenylalanine (TMP), resulting in four

conformations : (2R,3S)‐β‐Phe‐DPDPE,

(2R,3R)‐β‐Phe‐DPDPE,

(2R,3S)‐β‐Phe‐DPDPE, and

(2S,3R)‐β‐Phe‐DPDPE. Synthesis was by solid‐phase

techniques using enantiomerically pure amino acids to give the four optically

pure diastereoisomer peptides. The potency and selectivity (δ‐ versus

μ‐opioid receptor) were evaluated by radioreceptor binding in rat brain,

with a μ/δ ratio decrease for all TMP conformations, compared with

the parent compound (DPDPE). Octanol/buffer distribution analysis showed

enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated

with (2S,3S)‐TMP. In situ vascular perfusion in anesthetized

rats showed a 1.6‐fold (p < 0.01) increase in the ratio of brain

uptake for (2S,3S)‐TMP and a 1.5‐fold (p < 0.01)

decrease in uptake for (2R,3R)‐TMP. Saturability of

(2S,3S)‐TMP was shown (p < 0.01) against 100

μM unlabeled DPDPE, showing a shared nondiffusionary transport

system. P‐glycoprotein affinity was shown in situ for the parent and

(2S,3S)‐TMP (p < 0.01). Protein binding capacity

of the TMP compounds in rat plasma and in situ mammalian bovine serum

albumin‐Ringer showed (2R,3S)‐TMP and

(2S,3R)‐TMP with the lowest degree of protein binding

(p < 0.01), and (2S,3S)‐TMP and

(2R,3R)‐TMP with comparable affinities to DPDPE. Analgesia,

via intravenous administration, showed significantly reduced (p <

0.01) end effect and time course for (2R,3R)‐TMP,

(2R,3S)‐TMP, and (2S,3R)‐TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P‐glycoprotein affinity, and blood‐brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.
Apoptosis Induced by Doxorubicin in Neurotumor Cells Is Divorced from Drug Effects on Ceramide Accumulation and May Involve Cell Cycle‐Dependent Caspase Activation
Authors: Di Bartolomeo Sabrina , Di Sano Federica , Piacentini Mauro , Spinedi Angelo
Published: March 2003

Doxorubicin (0.5 μg/ml) induced caspase‐dependent apoptosis in SH‐SY5Y neuroblastoma and CHP‐100 neuroepithelioma cells. The apoptotic response started to be evident ~15 h after drug administration and, as monitored over a 48‐h period, was more pronounced in CHP‐100 than in SH‐SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B1 (25 μM) ; this compound, however, did not

prevent drug‐induced apoptosis. Untreated cells from both lines expressed

negligible p53 levels ; on the other hand, whereas p53 and

p21Cip1/Waf1 were rapidly up‐regulated in doxorubicin‐treated

SH‐SY5Y cells, such a response was not observed in CHP‐100 cells. Doxorubicin

induced a G2/M phase block in both cell lines, but whereas the

G1 phase was markedly depleted in CHP‐100 cells, it was

substantially retained in SH‐SY5Y cells. In the latter system, double

G1 and G2/M block largely preceded cell death ; however,

as apoptosis underwent completion, it selectively targeted late S and

G2/M cells. Moreover, apoptosis suppression by caspase inhibition

did not result in a recovery of the G1 cell population. These

results support the notion that doxorubicin‐induced apoptosis and ceramide

elevation are divorced events in neuroectodermal tumors and that p53 function

is at least dispensable for apoptosis completion. Indeed, as G1

cells appear to be refractory to doxorubicin‐induced apoptosis, p53

up‐regulation and p21Cip1/Waf1 expression may provide an unfavorable setting for the apoptotic action of the drug.
Identification of Multiple Forms of Membrane‐Associated Neutral Sphingomyelinase in Bovine Brain
Authors: Jung Sung Yun , Suh Jang Hyuk , Park Hong Jun , Jung Kwang Mook , Kim Mie Young , Na Doe Sun , Kim Dae Kyong
Published: March 2003

Abstract: Many different stimuli such as bioactive agents and

environmental stresses are known to cause the activation of sphingomyelinase

(SMase), which hydrolyzes sphingomyelin to generate ceramide as a second

messenger playing a key role in differentiation and apoptosis in various cell

types. Here we identified multiple forms of the membrane‐associated neutral

SMase (N‐mSMase) activity in bovine brain. They could be classified into two

groups according to extracting agents: group T‐mSMase, extracted with 0.2%

Triton X‐100, and group S‐mSMase, extracted with 0.5 M

(NH4)2SO4. Group T‐mSMase: α, β,

γ, and δ, which were extensively purified from 40,000‐g

pellets of bovine brain homogenates by 3,150‐, 5,275‐, 1,665‐, and 2,556‐fold

over the membrane extracts, respectively, by sequential use of several column

chromatographies. On the other hand, S‐mSMase was eluted as two active peaks

of S‐mSMase ε and ζ in a phenyl‐5PW hydrophobic HPLC column and

further purified by 1,119‐ and 976‐fold over 40,000‐g pellets of the

homogenates, respectively. These highly purified N‐mSMase enzyme preparations

migrated as several bands on sodium dodecyl sulfate‐polyacrylamide gel

electrophoresis and showed many different features in biochemical properties

such as pH dependence, Mg2+ requirements, and effects of detergents. Taken together, our data strongly suggest that mammalian brain N‐mSMase may exist as multiple forms different in both its chromatographic profiles and biochemical properties.
Presence of Dynamin—Syntaxin Complexes Associated with Secretory Granules in Adrenal Chromaffin Cells
Authors: Galas Marie‐Christine , Chasserot‐Golaz Sylvette , Dirrig‐Grosch Sylvie , Bader Marie‐France
Published: March 2003

Abstract: Dynamin proteins have been implicated in many aspects of endocytosis, including clathrin‐mediated endocytosis, internalization of caveolae, synaptic vesicle recycling, and, more recently, vesicular trafficking to and from the Golgi complex. To provide further insight into the function(s) of dynamin in neuroendocrine cells, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation, immunoreplica analysis, and confocal immunofluorescence. We found that dynamin, presumably the dynamin‐2 isoform, is associated specifically with the membrane of purified secretory chromaffin granules. Oligomerization state analysis by sucrose density velocity gradients indicated that the granule‐associated dynamin is in a monomeric form. Immunoprecipitation experiments coupled to double‐labeling immunofluorescence cytochemistry revealed that the granular dynamin is associated with a syntaxin component that is not involved in the granule‐bound SNARE complex. The possibility that dynamin participates in the coupling of the exocytotic and endocytotic reaction through the building of a granular membrane subset of proteins is discussed.