bluuebunny/crossref_metadata_2025 huggingface dataset:
Accumulation of N‐Arachidonoylethanolamine (Anandamide) into Cerebellar Granule Cells Occurs via Facilitated Diffusion
Authors: Hillard Cecilia J. , Edgemond William S. , Jarrahian Abbas , Campbell William B.
Published: July 2010

Abstract: N‐Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [3H]AEA accumulation by cerebellar granule cells is dependent on incubation time (t1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent Km of 41 ± 15 µM and a Vmax of 0.61 ± 0.04 nmol/min/106 cells. [3H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µM phloretin (57.4 ± 4% of control) in a noncompetitive manner. [3H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [3H]AEA accumulation is fairly selective for AEA among other naturally occurring N‐acylethanolamines; only N‐oleoylethanolamine significantly inhibited [3H]AEA accumulation at a concentration of 10 µM. The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µM. N‐Arachidonoylbenzylamine and N‐arachidonoylpropylamine, but not arachidonic acid, 15‐hydroxy‐AEA, or 12‐hydroxy‐AEA, compete for AEA accumulation. When cells are preloaded with [3H]AEA, temperature‐dependent efflux occurs with a half‐life of 1.9 ± 1.0 min. Phloretin does not inhibit [3H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein‐mediated transport process that has the characteristics of facilitated diffusion.
Comparison of [3H]Phosphatidylinositol and [3H]Phosphatidylinositol 4,5‐Bisphosphate Hydrolysis in Postmortem Human Brain Membranes and Characterization of Stimulation by Dopamine D1 Receptors
Authors: Pacheco Mary A. , Jope Richard S.
Published: July 2010

Abstract: Assessing the function of the phosphoinositide signal transduction system in membranes prepared from postmortem human brain by measuring the hydrolysis of exogenous labeled phosphoinositides has been applied to studies of a variety of CNS disorders in recent years. Two issues concerning such studies were addressed in the current investigation: how do [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5‐bisphosphate compare as substrates, and how do dopamine D1 receptors influence phosphoinositide signaling? Comparisons of [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5‐bisphosphate hydrolysis stimulated by guanosine‐5′‐O‐(3‐thiotriphosphate)‐activated G proteins and by several receptor agonists demonstrated that in most cases each substrate gave similar relative results in membranes prepared from prefrontal cortices of six individuals. However, using optimal assay conditions, [3H]phosphatidylinositol produced a greater signal‐to‐noise ratio compared with [3H]phosphatidylinositol 4,5‐bisphosphate. Dopamine D1 receptors were demonstrated to be directly coupled to phosphoinositide hydrolysis in human brain membranes, and this response was shown to be mediated by the Gq/11 G protein subtype and by the β‐subtype of phospholipase C. Therefore, these results demonstrate that [3H]phosphatidylinositol is a suitable substrate to measure phosphoinositide hydrolysis in human brain membranes and that dopamine D1 receptors directly stimulate this signaling system.
Dopaminergic Properties of Cultured Rat Carotid Body Chemoreceptors Grown in Normoxic and Hypoxic Environments
Authors: Jackson Adele , Nurse Colin
Published: July 2010

Abstract: Using dissociated carotid body (CB) cultures prepared from neonatal (postnatal days 5–7; P7) or juvenile (postnatal day 19–20; P20) rats, we compared catecholaminergic properties and mechanisms of O2 sensing in glomus cells grown in normoxic (Nox; 20% O2) and chronically hypoxic (CHox; 6% O2) environments for up to 2 weeks. In Nox cultures, basal dopamine (DA) release, determined by HPLC and normalized to the number of tyrosine hydroxylase‐positive glomus cells present, was similar for P7 and P20 cultures (∼0.3 pmol/1,000 cells/15 min) and was unaffected by culture duration (2 vs. 12 days). Acute hypoxia (5 and 10% O2) caused a dose‐dependent stimulation (6× and 3× basal, respectively) in DA release, that was inhibited by nifedipine (10 µM). DA release was also stimulated by high extracellular K+ (30 mM) and iberiotoxin (200 nM), a selective blocker of Po2‐regulated, Ca‐dependent K+ channel in glomus cells. The stimulatory effect of iberiotoxin was similar to 5% O2 in P20 cultures, but substantially less (about one‐half) in P7 cultures. In contrast, in CHox cultures, basal DA release was substantially elevated, ∼8× Nox levels, although this did not correlate with significant differences in stores. Further, whereas acute hypoxia (5% O2) and high K+ also stimulated DA release in CHox cultures (∼2× and ∼3× basal), iberiotoxin (200 nM) did not. Thus, after chronic hypoxia in vitro, there is an enhanced basal catecholamine release and an apparent down‐regulation of functional Ca‐dependent K+ channels in CB chemoreceptors. These cellular adaptations may relate to changes in CB chemosensitivity during chronic hypoxemia.
Effects of Ketone Bodies on Astrocyte Amino Acid Metabolism
Authors: Yudkoff Marc , Daikhin Yevgeny , Nissim Ilana , Grunstein Renee , Nissim Itzhak
Published: July 2010

Abstract: The effects of acetoacetate and 3‐hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with 15N as a metabolic probe and gas chromatography‐mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3‐hydroxybutyrate (5 mM) to the incubation medium did not alter the initial rate of appearance of [15N]glutamate in the glia, but it did inhibit transamination of glutamate to [15N]aspartate. Addition of acetoacetate also inhibited formation of [2‐15N]glutamine, but 3‐hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 mM) was also associated with diminished production of [15N]aspartate and [2‐15N]glutamine with [15N]glutamate as precursor. Studies with [2‐15N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [15N]GABA to [2‐15N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3‐hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.
Oleic Acid Inhibits Gap Junction Permeability and Increases Glucose Uptake in Cultured Rat Astrocytes
Authors: Lavado Eva , Sánchez‐Abarca Luis I. , Tabernero Arantxa , Bolaños Juan P. , Medina José M.
Published: July 2010

Abstract: The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape‐loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose‐dependent inhibition of gap junction permeability by 79.5% at 50 µM, and no further inhibition was observed by increasing the oleic acid concentration to 100 µM. The oleic acid‐mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid‐related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.
Inhibition of [3H]MK‐801 Binding by Ferrous (II) but Not Ferric (III) Ions in a Manner Different from That by Sodium Nitroprusside (II) in Rat Brain Synaptic Membranes
Authors: Shuto Makoto , Ogita Kiyokazu , Minami Takao , Maeda Hiroko , Yoneda Yukio
Published: July 2010

Abstract: The addition of sodium nitroprusside (SNP) significantly inhibited binding of (+)‐5‐[3H]methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine ([3H]MK‐801) to an ion channel associated with the N‐methyl‐d‐aspartate (NMDA) receptor in a concentration‐dependent manner at concentrations of >1 µM in rat brain synaptic membranes not extensively washed. However, neither S‐nitroso‐N‐acetylpenicillamine nor S‐nitroso‐l‐glutathione inhibited binding even at 100 µM. Of the two compounds structurally related to SNP (II), similarly potent inhibition was induced by potassium ferrocyanide (II) but not by potassium ferricyanide (III). In addition, ferrous chloride (II) induced much more potent inhibition of binding than ferric chloride (III), at a similar concentration range. In contrast, iron chelators prevented the inhibition by ferrous chloride (II) without markedly affecting that by SNP (II) and potassium ferrocyanide (II). Pretreatment with ferrous chloride (II) also led to potent inhibition of [3H]MK‐801 binding in a manner insensitive to subsequent addition of the iron chelators. Pretreatment with Triton X‐100 resulted in significant potentiation of the ability of ferrous chloride (II) to inhibit [3H]MK‐801 binding irrespective of the addition of agonists, moreover, although binding of other radioligands to the non‐NMDA receptors was unaltered after pretreatment first with Triton X‐100 and then with ferrous chloride (II). These results suggest that ferrous ions (II) may interfere selectively with opening processes of the NMDA channel through mechanisms entirely different from those underlying the inhibition by both SNP (II) and potassium ferrocyanide (II) in rat brain.
Mechanisms of GABA‐ and Glycine‐Induced Increases of Cytosolic Ca2+ Concentrations in Chick Embryo Ciliary Ganglion Cells
Authors: Sorimachi M. , Rhee J. S. , Shimura M. , Akaike N.
Published: July 2010

Abstract: We used fura‐2 microfluorometry and the gramicidin‐perforated patch clamp technique in an attempt to clarify the mechanisms underlying the GABA‐and glycine‐induced increases in the cytosolic Ca2+ concentration ([Ca]in) in acutely isolated chick embryo ciliary ganglion neurons. GABA, glycine, and isoguvacine, but not baclofen, increased [Ca]in in a dose‐ and a Ca2+‐dependent manner. The GABA‐induced [Ca]in increase was inhibited by bicuculline and picrotoxin, and potentiated by pentobarbital, flunitrazepam, and alphaxalone, whereas the glycine‐induced [Ca]in increase was inhibited by strychnine but not by bicuculline or picrotoxin. L‐and N‐type Ca2+ channel blockers inhibited the GABA‐and glycine‐induced [Ca]in increases, whereas Bay K‐8644 potentiated these responses. These responses were also substantially potentiated by blockers of various K+ channels and by lowering the external Cl− concentrations. The high KCI‐ and nicotine‐induced [Ca]in increases were substantially reduced during continuous stimulation with either 2 µM GABA or 1 mM glycine. Electrophysiological studies indicated that the reversal potential of the GABA‐induced current exhibited a more depolarized value than the resting membrane potential in 17 of the 25 cells examined. Taken together, these results suggest that both GABA and glycine depolarize the membrane potentials by increasing Cl− conductance via respective receptors and thus increase the Ca2+ influxes through L‐ and N‐type voltage‐dependent Ca2+ channels.
Interferon‐β Is a Potent Promoter of Nerve Growth Factor Production by Astrocytes
Authors: Boutros Tarek , Croze Ed , Yong Voon Wee
Published: July 2010

Abstract: Recent clinical evidence has suggested that interferon‐β is efficacious in the treatment of the demyelinating disease, multiple sclerosis. The mechanism of its efficacy remains unclear, and suggested modes of action have focused on immune modulation. Nonimmune effects of interferon‐β may also contribute to its efficacy. Given that astrocytes produce a range of neurotrophic factors, we examined the possibility that interferon‐β could increase the astrocytic production of nerve growth factor (NGF), which has been reported to cause oligodendrocytes to proliferate and to extend their processes; these phenotypes can impact favorably on remyelination. When the recombinant form of mouse interferon‐β was added to mouse astrocyte cultures, a dose‐dependent increase in NGF mRNA was obtained. The 40‐fold increase in NGF mRNA elicited by 1,000 U/ml interferon‐β was far more potent than that produced by other NGF‐elevating agents in this study. In concordance, the protein for NGF was elevated by interferon‐β. The production of NGF by interferon‐β may be relevant to its clinical efficacy in multiple sclerosis. Furthermore, we suggest the potential utility of interferon‐β in Alzheimer's disease.
Inhibition of Glial Cell Line‐Derived Neurotrophic Factor Induced Intracellular Activity by K‐252b on Dopaminergic Neurons
Authors: Pong Kevin , Xu Ren Y. , Beck Klaus D. , Zhang T. J. , Louis Jean‐Claude
Published: July 2010

Abstract: The c‐ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line‐derived neurotrophic factor (GDNF). K‐252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain‐derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin‐3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K‐252b would modulate GDNF‐induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 µM K‐252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K‐252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase‐immunoreactive neurons. K‐252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K‐252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K‐252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.
A Novel Gelsolin Isoform Expressed by Oligodendrocytes in the Central Nervous System
Authors: Vouyiouklis Demetrius A. , Brophy Peter J.
Published: July 2010

Abstract: Two isoforms of the Ca2+‐sensitive, actin‐binding protein gelsolin have been identified thus far; one is an intracellular protein, cytoplasmic gelsolin, and the other is a secretory protein called plasma gelsolin. Gelsolin expression in the mammalian CNS appears to be localized mainly to oligodendrocytes where it is presumed that the cytoplasmic isoform predominates. Here, we show that oligodendrocytes not only contain cytoplasmic gelsolin, but they also express a novel gelsolin isoform that we have named gelsolin‐3. Cytoplasmic gelsolin, plasma gelsolin, and gelsolin‐3 arise by alternative splicing from the same gene. The N‐terminal amino acid sequence unique to gelsolin‐3 is shown to be encoded by a single exon in a region previously thought to be an intron in the human gelsolin gene. In situ hybridization analysis confirmed that gelsolin‐3 mRNA is localized primarily to oligodendrocytes in rat brain. In other tissues, gelsolin‐3 shows a more restricted pattern of expression than cytoplasmic gelsolin. These data support the view that the gelsolin isoforms have differential roles in the regulation of the actin cytoskeleton.
Stimulation of the Sympathetic Perimesenteric Arterial Nerves Releases Neuropeptide Y Potentiating the Vasomotor Activity of Noradrenaline: Involvement of Neuropeptide Y‐Y1 Receptors
Authors: Donoso M. V. , Brown N. , Carrasco C. , Cortes V. , Fournier A. , Huidobro‐Toro J. P.
Published: July 2010

Abstract: Neuropeptide Y (NPY) appears to be involved in the sympathetic regulation of vascular tone. To assess the putative role of NPY in mesenteric circulation, the release and biological effect of NPY were examined after electrical stimulation of perimesenteric arterial nerves. Nerve stimulation with trains of 2–30 Hz increased the perfusion pressure of the arterially perfused rat mesenteric bed in a frequency‐ and time‐dependent fashion. Trains of 15–30 Hz significantly displaced to the left, approximately threefold, the noradrenaline (NA)‐induced pressor concentration‐response curve, in addition to increasing significantly its efficacy. Perfusion with 10 nM exogenous NPY mimicked the electrical stimulation effect, causing a threefold leftward shift of the NA concentration‐response curve and increasing the maximal NA response. These effects were antagonized by 100 nM BIBP 3226, indicating the activity of NPY‐Y1 receptors. Electrical stimulation of the perimesenteric nerves released immunoreactive NPY (ir‐NPY) in a frequency‐dependent fashion; the ir‐NPY coelutes with synthetic NPY as confirmed by HPLC. Both the electrically induced pressor response and the calcium‐dependent release of NPY were obliterated in preparations perfused with 1 µM guanethidine or in rats pretreated intravenously for 48 h with 6‐hydroxydopamine, thus revealing the sympathetic origin of these phenomena. Only a small proportion of the total NPY content in the perimesenteric arterial nerves is released after electrical stimulation. Chromatographic studies of the physiological sources of the ir‐NPY support that NPY fragments are generated via peptidase degradation. The present findings demonstrate that NPY is released from the perimesenteric arterial sympathetic nerves and acts, via the activation of NPY‐Y1 receptors, as the mediator responsible for the potentiation of NA's effect on perfusion pressure in the isolated rat mesenteric bed.
M3 Muscarinic Receptor‐Mediated Enhancement of NMDA‐Evoked Adenosine Release in Rat Cortical Slices In Vitro
Authors: Semba Kazue , White Thomas D.
Published: July 2010

Abstract: Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep‐promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA‐evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 µM) alone did not affect the basal release of adenosine. However, carbachol (100 µM) induced a 253% increase in NMDA (20 µM)‐evoked adenosine release in the presence of Mg2+. In the absence of Mg2+, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 µM) blocked the facilitatory effect of carbachol on NMDA‐evoked adenosine release, and this was mimicked by the M3‐selective antagonist 4‐diphenylacetoxy‐N‐methylpiperidine (1 µM). Neither an M1‐selective dose of pirenzepine (50 nM) nor the M2‐selective antagonist methoctramine (1 µM) affected carbachol's action on NMDA‐evoked adenosine release. Carbachol had no effect on adenosine release evoked by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor‐mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long‐term basis.
Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion
Authors: Wu Qun , Coffey Lori L. , Reith Maarten E. A.
Published: July 2010

Abstract: The present study addresses the possibility that there are different cocaine‐related and mazindol‐related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn2+ (30–100 µM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1–100 µM) had no effect; Hg2+ at ∼3 µM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0°C, and at 30–100 µM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30–100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21°C and Hg2+ at 0°C.
Roles for Protein Kinase C and Mitogen‐Activated Protein Kinase in Nicotine‐Induced Secretion from Bovine Adrenal Chromaffin Cells
Authors: Cox Michael E. , Parsons Sarah J.
Published: July 2010

Abstract: Both the Ca2+/phospholipid‐dependent protein kinases (protein kinases C, PKCs) and mitogen‐activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal‐regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long‐term phorbol 12‐myristate 13‐acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine‐induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl‐induced events, and little, if any, effect on Ca2+ ionophore‐elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca2+ ionophore. Treatment of cells with the MEK‐activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC50 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC50∼10 µM) than that induced by nicotine (IC50∼30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short‐term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca2+ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.
The Lipid Peroxidation Product, 4‐Hydroxy‐2‐trans‐Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins
Authors: Subramaniam Ram , Roediger Fred , Jordan Brad , Mattson Mark P. , Keller Jeffrey N. , Waeg Georg , Butterfield D. Allan
Published: July 2010

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β‐peptide (Aβ). Aβ‐associated free radicals cause lipid peroxidation, a major product of which is 4‐hydroxy‐2‐trans‐nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein‐specific spin label, MAL‐6(2,2,6,6‐tetramethyl‐4‐maleimidopiperidin‐1‐oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid‐specific stearic acid label, 5‐nitroxide stearate, was used to probe for HNE‐induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration‐ and time‐dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque‐ and Aβ‐rich AD hippocampal and inferior parietal brain regions. HNE‐induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE‐caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µM). The results obtained are discussed with relevance to the hypothesis of Aβ‐induced free radical‐mediated lipid peroxidation, leading to subsequent HNE‐induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.
The Amyloid β‐Protein of Alzheimer's Disease Increases Acetylcholinesterase Expression by Increasing Intracellular Calcium in Embryonal Carcinoma P19 Cells
Authors: Sberna Gian , Sáez‐Valero Javier , Beyreuther Konrad , Masters Colin L. , Small David H.
Published: July 2010

Abstract: One of the characteristic changes that occurs in Alzheimer's disease is the loss of acetylcholinesterase (AChE) from both cholinergic and noncholinergic neurons of the brain. However, AChE activity is increased around amyloid plaques. This increase in AChE may be of significance for therapeutic strategies using AChE inhibitors. The aim of this study was to examine the effect of amyloid β‐protein (Aβ), the major component of amyloid plaques, on AChE expression. Aβ peptides spanning residues 1–40 or 25–35 increased AChE activity in P19 embryonal carcinoma cells. A peptide containing a scrambled Aβ25–35 sequence did not stimulate AChE expression. To examine the possibility that the increase in AChE expression was mediated by an influx of calcium through voltage‐dependent calcium channels (VDCCs), drugs acting on VDCCs were tested for their effects. Inhibitors of L‐type VDCCs (diltiazem, nifedipine, and verapamil), but not N‐ or P‐ or Q‐type VDCCs, resulted in a decrease in AChE expression. Agonists of L‐type VDCCs (maitotoxin and S(−)‐Bay K 8644) increased AChE expression. As L‐type VDCCs are known to be modulated by cyclic AMP‐dependent protein kinase, the effect of the adenylate cyclase activator forskolin was also examined. Forskolin stimulated AChE expression, an action that was blocked by the L‐type VDCC antagonist nifedipine. The Aβ25–35‐induced increase in AChE expression was mediated by an L‐type VDCC, as the effect was also blocked by nifedipine. The results suggest that the increase in AChE expression around amyloid plaques could be due to a disturbance in calcium homeostasis involving the opening of L‐type VDCCs.
Oxidative DNA Damage in the Parkinsonian Brain: An Apparent Selective Increase in 8‐Hydroxyguanine Levels in Substantia Nigra
Authors: Alam Z. I. , Jenner A. , Daniel S. E. , Lees A. J. , Cairns N. , Marsden C. D. , Jenner P. , Halliwell B.
Published: July 2010

Abstract: Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8‐hydroxy‐2′‐deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8‐hydroxyguanine (8‐OHG) tended to be elevated and levels of 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8‐OHG in PD substantia nigra (p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8‐OHG is unlikely to be due to peroxynitrite (ONOO−) or hydroxyl radicals (OH•), or to be a prooxidant effect of treatment with l‐Dopa. However, some or all of the rise in 8‐OHG could be due to a change in 8‐OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.
Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet‐Activating Factor Antagonist BN 50739
Authors: Kintner Douglas B. , Fitzpatrick James H. , Gilboe David D.
Published: July 2010

Abstract: The Pulsinelli‐Brierley four‐vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo‐ or hyperglycemic ischemia or 30 min of normo‐ or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet‐activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid‐reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet‐activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.
An Extensive Repeat Structure Down‐Regulates Human Monoamine Oxidase A Promoter Activity Independent of an Initiator‐Like Sequence
Authors: Zhu Qin‐shi , Shih Jean Chen
Published: July 2010

Abstract: The 5′ flanking sequence of the human monoamine oxidase A (MAO A) gene consists of an extensive repeat structure. Two 90‐bp repeats (I and II) were found in the core promoter (the 0.24‐kb PvuII/DraII fragment), each containing two Sp1 binding sites. An additional six repeats were found, five of which (III–VII) were upstream and one (‐I) downstream of the core promoter. Using transient transfection assay with a human growth hormone reporter gene, we found that the upstream repeating units III–VII (in a 0.78‐kb BamHI/DraII fragment) down‐regulate core promoter activity to 13 ± 10% in a human glioma cell line (1242 MG) and 2 ± 1% in a cervical carcinoma cell line (HeLa), respectively. The 0.24‐kb core promoter activity was taken as 100%. Addition of the initiator (Inr)‐like sequence to this 0.78‐kb fragment (0.82‐kb BamHI/−17 fragment) still showed decreased promoter activity (10 ± 9% in 1242 MG cells and 8 ± 1% in HeLa cells). Thus, the upstream sequence down‐regulates promoter activity with or without the Inr‐like sequence. When the Inr‐like sequence was added to the core promoter (0.28‐kb PvuII/−17 fragment), the promoter activity decreases significantly in both 1242 MG (55 ± 6%) and HeLa (60 ± 10%) cells. These results suggest that although the Inr‐like sequence is present in the human MAO A promoter, it acts as a negative cis element instead of a transcription initiator.
P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε
Authors: Chen Ching‐Chow , Chen Wei‐Chyuan
Published: July 2010

Abstract: Incubation of Neuro 2A mouse neuroblastoma cells with UTP and UDP results in a concentration‐dependent increase in the accumulation of inositol phosphates with equal potency and maximal effect; ATP, ADP, and 2‐methylthioadenosine 5′‐triphosphate were much less potent, indicating the expression of P2Y receptor in these cells. The effects of UTP and ATP were not affected by pretreatment of cells with pertussis toxin, indicating that the P2Y receptor in Neuro 2A cells is coupled to pertussis toxin‐insensitive Gq protein. Short‐term (10 min) treatment of cells with 1 µM 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) resulted in the inhibition of the UTP and ATP effects; this inhibitory effect was gradually attenuated with increased length of TPA treatment (1.5–6 h) and was not seen after long‐term (24 h) treatment. Western blot analysis showed the expression of protein kinase C (PKC) α, ε, θ, and ζ in Neuro 2A cells. Translocation of PKCα, ε, and θ from the cytosol to the membrane was seen after 10 min or 1.5 h of treatment with TPA. However, partial and complete down‐regulation of both membrane PKCα and θ were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA‐induced translocation of PKCε was maintained after 3–6 h of treatment, and almost complete down‐regulation occurred only after a 24‐h treatment. The observed TPA‐induced inhibition of UTP‐ or ATP‐stimulated phosphoinositide hydrolysis, therefore, correlated well with the extent of translocation of PKCε. Phosphoinositide hydrolysis induced by AlF4−, but not Ca2+ ionophores, was inhibited by a 10‐min treatment with TPA. This was not seen after a 24‐h treatment, indicating that the site of action of PKCε in the P2Y receptor/Gq protein/phospholipase Cβ pathway might be the Gq protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal PKCε in the regulation of the receptor‐mediated phosphoinositide turnover.
Rescue of Mesencephalic Dopamine Neurons by Anticancer Drug Cytosine Arabinoside
Authors: Michel Patrick P. , Ruberg Merle , Agid Yves
Published: July 2010

Abstract: Nanomolar concentrations of cytosine arabinoside (ara‐C), a structural analogue of 2′‐deoxycytidine (2′dC) used in the chemotherapy of cancer, proved to be highly effective in preventing the death of postmitotic dopaminergic neurons that occurs spontaneously by apoptosis in mesencephalic cultures. The rescued cells were totally functional and highly differentiated. The trophic/neuroprotective effects of ara‐C were (1) specific for dopaminergic neurons; (2) long‐lived, remaining detectable several days after withdrawal of the nucleoside analogue from the culture medium; (3) still observed when the treatment was delayed after plating; (4) abolished by an excess of 2′dC or dCTP, or by exposure to the neurotoxin 1‐methyl‐4‐phenylpyridinium; and (5) mimicked by ara‐CTP, 5‐fluoro‐2′‐deoxyuridine, and aphidicolin. Autoradiographic studies revealed that ara‐C was incorporated exclusively into astrocyte nuclei, suggesting that the dopaminotrophic activity was indirect and resulted from the antiproliferative action of the modified nucleoside on glial cells at concentrations that were not neurotoxic. No evidence was found for putative deleterious or trophic molecules secreted by proliferating or ara‐C‐treated astrocytes, respectively, suggesting that neuroglial contact may play a role. Our results suggest a possible mechanism underlying neurodegeneration in Parkinson's disease, where selective loss of dopaminergic neurons in the mesencephalon is accompanied by astrogliosis.
The Dietary Excitotoxins β‐N‐Methylamino‐l‐Alanine and β‐N‐Oxalylamino‐l‐Alanine Induce Necrotic‐ and Apoptotic‐Like Death of Rat Cerebellar Granule Cells
Authors: Staton Penelope C. , Bristow David R.
Published: July 2010

Abstract: The neurotoxic properties of the dietary excitotoxins β‐N‐methylamino‐l‐alanine and β‐N‐oxalylamino‐l‐alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose‐dependent death of cerebellar granule cells after a 30‐min exposure when viability was assessed 24 h later. β‐N‐Methylamino‐l‐alanine and β‐N‐oxalylamino‐l‐alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of β‐N‐methylamino‐l‐alanine were blocked by d(−)‐2‐amino‐5‐phosphonopentanoic acid, and those of β‐N‐oxalylamino‐l‐alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K+ (30 mM)‐ to a low K+ (10 mM)‐containing medium. In cerebellar granule cells exposed to β‐N‐methylamino‐l‐alanine or β‐N‐oxalylamino‐l‐alanine (3 mM), hallmarks of necrotic‐ and apoptotic‐like death were observed at various time points over a 72‐h period. Therefore, in cerebellar granule cells, β‐N‐methylamino‐l‐alanine and β‐N‐oxalylamino‐l‐alanine induce death over 12–72 h of exposure via a mechanism that involves both necrotic‐ and apoptotic‐like cell death.
l‐DOPA Is a Substrate for Tyrosine Hydroxylase
Authors: Haavik Jan
Published: July 2010

Abstract: In the presence of thiols, tyrosine hydroxylase (TH) oxidizes l‐dihydroxyphenylalanine (l‐DOPA) with a specific activity of up to 140 nmol min−1 mg−1 at 37°C and pH 7.0, which is ∼12–50% of its TH activity under similar experimental conditions. Using assay conditions that are optimal for measuring TH activity, the specific DOPA oxidase activity of human TH is similar to that of mushroom tyrosinase, but the two enzymes are clearly different in terms of substrate specificities, cofactor dependencies, and selectivity with respect to the effects of metal chelators and other inhibitors. In the presence of an excess of dithiothreitol, 2‐mercaptoethanol, cysteine, or reduced glutathione, the reaction products of the two enzymes are identical and have been identified tentatively as thioether derivatives of DOPA. Theoretically, the oxidation of l‐DOPA by TH may contribute to the formation of neuromelanin (pheomelanin) in catecholaminergic neurons and in the metabolism of DOPA to reactive intermediates that can react with free thiol groups in cellular proteins. The DOPA oxidase activity of TH can lead to errors in the estimation of in vivo or in vitro TH activity, and currently used assay protocols may have to be modified to avoid interference from this activity.
Lipid Binding to Amyloid β‐Peptide Aggregates: Preferential Binding of Cholesterol as Compared with Phosphatidylcholine and Fatty Acids
Authors: Avdulov Nicolai A. , Chochina Svetlana V. , Igbavboa Urule , Warden Christopher S. , Vassiliev Alexei V. , Wood W. Gibson
Published: July 2010

Abstract: Amyloid β‐peptide (Aβ) aggregates are one of the key neuropathological characteristics of Alzheimer's disease. Aβ belongs to a group of proteins that aggregate and form β‐sheets, and some of these proteins bind cholesterol and other lipids. The purpose of the experiments reported here was to determine if cholesterol, fatty acids, and phosphatidylcholine (PC) would bind to Aβ1–40 and if such binding would be dependent on aggregation of Aβ1–40. Lipid binding was determined using fluorescent‐labeled lipids. Incubation of Aβ1–40 for 0, 1, 3, 6, 21, and 24 h resulted in aggregation of the peptide with formation of dimers, trimers (1–24 h), and polymers (6–24 h) as determined by sodium dodecyl sulfate‐gel electrophoresis. No change in the fluorescence of the lipids was observed when lipids were added to Aβ1–40 that had been incubated for 0, 1, or 3 h. However, the fluorescence intensities of cholesterol, saturated fatty acids, and PC were significantly increased (p < 0.0001) when added to Aβ1–40 that had been incubated for 6, 21, and 24 h in which Aβ1–40 polymers were detected. The binding affinity of cholesterol to Aβ1–40 polymers (KD of 3.24 ± 0.315 × 10−9M) was markedly higher as compared with the other lipids (stearic acid, 9.42 ± 0.41 × 10−8M; PC, 7.07 ± 0.12 × 10−7M). The results of this study indicate that Aβ1–40 polymers bind lipids and have a higher affinity for cholesterol than PC or saturated fatty acids. Aggregated Aβ1–40 may affect lipid transport between cells or remove specific lipids from membranes, and such effects could contribute to neuronal dysfunction.
Site Mutation in the Rat μ‐Opioid Receptor Demonstrates the Involvement of Calcium/Calmodulin‐Dependent Protein Kinase II in Agonist‐Mediated Desensitization
Authors: Koch Thomas , Kroslak Thomas , Mayer Peter , Raulf Evelyn , Höllt Volker
Published: July 2010

Abstract: The rat μ‐opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the μ agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin‐dependent protein kinase II (CaM kinase II) on μ‐opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist‐induced desensitization of the μ‐opioid receptor. The increase of desensitization could not be observed with a μ‐opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser261 and Ser266 by CaM kinase II is involved in the desensitization of the μ‐opioid receptor.
Inhibition of Monoamine Oxidase Contributes to the Protective Effect of 7‐Nitroindazole Against MPTP Neurotoxicity
Authors: Di Monte Donato A. , Royland Joyce E. , Anderson Andrea , Castagnoli Kay , Castagnoli Neal , Langston J. William
Published: July 2010

Abstract: The ability of 7‐nitroindazole (7‐NI) to protect against MPTP‐induced neurotoxicity has been attributed to its inhibition of neuronal nitric oxide synthase. In the present study, 7‐NI was found to counteract almost completely striatal dopamine depletion caused by a single subcutaneus injection of 20 mg/kg MPTP in mice. This effect, however, was accompanied by a significant reduction in the striatal levels of MPP+, the toxic metabolite generated via monoamine oxidase B‐catalyzed MPTP oxidation. In the presence of 7‐NI, a dose of 40 mg/kg MPTP produced MPP+ concentrations similar to those measured after treatment with 20 mg/kg MPTP alone. A comparison of neurotoxicity in these two experimental conditions (i.e., mice treated with 20 mg/kg alone versus 40 mg/kg MPTP plus 7‐NI) revealed only a slight (20%), but statistically significant, protection of dopamine depletion with 7‐NI. These data indicate that the mechanism by which 7‐NI counteracts MPTP neurotoxicity in mice is not due solely to inhibition of neuronal nitric oxide synthase, but involves a reduction in MPP+ formation.
Molecular Cloning and Characterization of a Lobster Gαs Protein Expressed in Neurons of Olfactory Organ and Brain
Authors: Xu Fuqiang , Hollins Bettye , Gress Anne M. , Landers Teresa M. , McClintock Timothy S.
Published: July 2010

Abstract: We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, lobGαs, with >70% identity to mammalian and arthropod Gαs sequences. In genomic Southern blots, a fragment of lobGαs detected only one band, suggesting the lobsters have a single Gαs gene. In brain and olfactory organ, lobGαs mRNA was expressed predominantly in neurons, including many of the neuronal cell body clusters of the brain. Gαs protein was also expressed broadly, appearing on western blots as a band of 51.8 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that lobGαs plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, and the expression of lobGαs mRNA in the olfactory receptor neurons of the olfactory organ indicate that lobGαs may mediate olfactory transduction. That virtually all ORNs express lobGαs mRNA equally predicts that hyperpolarizing odor responses mediated by cyclic AMP are a property of all lobster olfactory receptor neurons.
Cloning and Expression of a Human Serotonin 5‐HT4 Receptor cDNA
Authors: Van den Wyngaert Ilse , Gommeren Walter , Verhasselt Peter , Jurzak Mirek , Leysen Josée , Luyten Walter , Bender Eckhard
Published: July 2010

Abstract: Using a combination of library screening and nested PCR based on a partial human serotonin 5‐HT4 receptor sequence, we have cloned the complete coding region for a human 5‐HT4 receptor. The sequence shows extensive similarity to the published porcine 5‐HT4A and rat 5‐HT4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5‐HT4 sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5‐HT4 receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5‐HT4 receptor, transiently expressed in COS‐7 cells, showed radioligand binding properties similar to 5‐HT4 receptors in guinea pig striatal tissue. [3H]GR 113808 revealed KD values of 0.15 ± 0.01 nM for the human receptor and 0.3 ± 0.1 nM in the guinea pig tissue. Binding constants were determined for four investigated 5‐HT4 antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS‐7 cells with 5‐HT4‐specific agonists caused an increase in cyclic AMP levels.