bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic Activation of Muscarinic and Metabotropic Glutamate Receptors Down‐Regulates Type I Inositol 1,4,5‐Trisphosphate Receptor Expression in Cerebellar Granule Cells
Authors: Simpson Peter B. , Challiss R. A. John , Nahorski Stefan R.
Published: July 2010

Abstract: The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5‐trisphosphate (InsP3) over extended incubation periods, and consequently to affect the level of InsP3 receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 mM) evoked a biphasic accumulation of InsP3, a rapid three‐ to fourfold peak increase over control levels at ∼10 s, decreasing within 1 min to a long‐lasting plateau elevation. Using an antibody against the type I InsP3 receptor, it was demonstrated that >50% down‐regulation of type I InsP3 receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 mM CCh. Over 24 h, 1 mM CCh caused an ∼85% decrease in type I InsP3 receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP3 receptor expression using a radioligand binding method also detected down‐regulation, but to an apparently lesser extent. 1‐Aminocyclopentane‐1S,3R‐dicarboxylic acid (200 µM), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP3 receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP3 production in cerebellar granule cells is the down‐regulation of InsP3 receptor expression and that this down‐regulation may be a common mechanism of action of phosphoinositide‐linked receptors during prolonged stimulation.
β2‐Adrenoreceptors Stimulate c‐fos Transcription Through Multiple Cyclic AMP‐ and Ca2+‐Responsive Elements in Cerebellar Granular Neurons
Authors: Barthel F. , Loeffler J. Ph.
Published: July 2010

Abstract: Neurons from the granular layer of the cerebellum express functional β2‐adrenoreceptors (β2‐ARs). We show that stimulation of β2‐ARs with isoprenaline increases cyclic AMP (cAMP) production and stimulates transcription of genes containing the cAMP‐responsive element (CRE; TGACGTCA). This effect is mediated by cAMP‐dependent protein kinase and the trans‐acting factor CRE binding protein. Transcriptional regulation by the β2‐AR was investigated by using the c‐fos protooncogene as a model system. We show that β2‐ARs stimulate c‐fos mRNA accumulation, increase AP1 binding activity, and stimulate transcription through the phorbol ester‐responsive element (TGACTCA). The transcriptional regulation of c‐fos itself was studied with reporter constructs driven by c‐fos promoter sequences. Deletion studies revealed that β2‐ARs stimulate c‐fos transcription through at least three distinct regulatory sequences: (a) the CRE located at −60 bp 5′ to the initiation site, (b) the fos AP1‐like element (−291 to −297), and (c) the serum‐responsive element (−297 to −317). The regulation of these elements by the two putative second messengers of the β2‐AR, cAMP and Ca2+, was analyzed. We report that all three of these regulatory sequences are coregulated by both second messengers. These results indicate that β2‐ARs stimulate c‐fos transcription by multiple cAMP‐ and Ca2+‐dependent regulatory elements in neurons.
Ca2+ ‐ and Nitric Oxide‐Dependent Stimulation of Cyclic GMP Synthesis in Neuronal Cell Line Induced by P2‐Purinergic/Pyrimidinergic Receptor
Authors: Reiser Georg
Published: July 2010

Abstract: The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma × rat glioma hybrid cells [108CC15 (NG 108‐15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for ∼1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5′‐(γ‐thio)triphosphate > UTP = 2‐methylthio‐ATP > ADP ≫ CTP, AMP, α,β‐methylene‐ATP, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)ATP. The EC50 of 1 ± 0.2 µM for UTP was significantly lower than that for ATP (14 ± 8 µM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC50 = 40–60 µM) or the pyridoxal phosphate analogue pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (IC50 = 20–30 µM). Pretreatment of cells with the Ca2+ ionophore ionomycin or with 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone, an inhibitor of Ca2+‐ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca2+ stores, suppressed the ATP‐ or UTP‐induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP‐induced rise in cyclic GMP level by 60%. The cyclic GMP response caused by ATP was suppressed either by arginine analogues (IC50 for nitroarginine = 1 µM) or by hemoglobin (IC50 = 2 µM). This indicates that ATP/UTP via a P2‐receptor causes formation of nitric oxide, which activates guanylate cyclase. The synthesis of nitric oxide depends on a preceding rise in cytosolic Ca2+ level, mostly due to release of Ca2+ from internal stores. Bradykinin induces a rise in cyclic GMP level with an amplitude and time course comparable to that caused by ATP. Therefore, we studied cross‐desensitization between ATP and bradykinin receptors. Pretreatment with bradykinin completely suppressed a subsequent response to ATP. However, stimulation with ATP reduced a following response to bradykinin by ∼40% only. This indicates a heterologous cross‐desensitization predominantly in one direction (bradykinin ⇒ ATP).
5′‐(N‐Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells
Authors: Mateo Jesús , Castro Enrique , Zwiller Jean , Aunis Dominique , Miras‐Portugal M. T.
Published: July 2010

Abstract: We investigated the effect of the adenosine receptor agonist 5′‐(N‐ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) in a time‐dependent manner. Inhibition reached 25% after 30–40‐min exposure to NECA. This effect on DMPP‐evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca2+]i transients induced by DMPP that peaked at 30‐min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short‐term treatment with NECA failed both to modify [Ca2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP‐evoked Ca2+ influx pathway (e.g., L‐type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.
Inducible Nitric Oxide Synthase in a Human Glioblastoma Cell Line
Authors: Fujisawa Hironori , Ogura Tsutomu , Hokari Atsushi , Weisz Alessandro , Yamashita Junkoh , Esumi Hiroyasu
Published: July 2010

Abstract: Nitric oxide synthase (NOS) activity was induced in the cytosol of A‐172 cells by treatment with lipopolysaccharide, tumor necrosis factor‐α, and interferon‐γ. A 130‐kDa human inducible NOS (iNOS) protein was detected with anti‐rat iNOS antibody by western blot analysis. Northern blot analysis showed that the iNOS mRNA was ∼4.5 kb, using a cDNA fragment for human iNOS, isolated from stimulated A‐172 cells by reverse transcriptase‐PCR, as a probe. The mRNA was induced by interferon‐γ at a trace level, and its expression was synergistically enhanced by simultaneous addition of lipopolysaccharide, tumor necrosis factor‐α, and, to a larger extent, interleukin‐1β. The mRNA expression was blocked by coincubation with actinomycin D or cycloheximide. Furthermore, by transfecting the mouse iNOS gene promoter into A‐172 cells, transcriptional activation of the iNOS gene was detected in these cells upon stimulation with lipopolysaccharide and cytokines. The pattern of promoter activation correlated well with that of iNOS mRNA expression upon stimulation. These data indicate that expression of iNOS is transcriptionally regulated in A‐172 cells. This process requires de novo protein synthesis with a mechanism similar to that in place in mouse macrophages.
Melatonin Receptor‐Mediated Stimulation of Phosphoinositide Breakdown in Chick Brain Slices
Authors: Popova Juliana S. , Dubocovich Margarita L.
Published: July 2010

Abstract: The direct effect of melatonin and related agonists on Li+‐amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo‐[2‐3H]‐inositol. The melatonin receptor agonist 6‐chloromelatonin (10–100 µM) increased, in a concentration‐dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6‐chloromelatonin (10 µM) was rapid as transient increases in IP3/IP4 (maximal increase, 29% at 20 s) and IP2 levels (maximal increase, 36% at 1 min) were observed, followed by a slower but sustained increase in IP1 level (30% at 5 min), when the amount of IP3/IP4 and IP2 had already been decreased to the control level. The phosphoinositide response elicited by 6‐chloromelatonin (10 µM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6‐chloromelatonin (10 µM) in isolated myo‐[2‐3H]inositol‐prelabeled optic tectum membranes was dependent on the presence of guanosine‐5′‐O‐(3‐thio)triphosphate (1 µM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10–100 µM) inhibited in a concentration‐dependent manner the IP1 accumulation stimulated by 6‐chloromelatonin (10–100 µM); however, it did not affect the accumulation stimulated by 5‐hydroxytryptamine (10 µM). By contrast, methysergide (10 µM) completely inhibited 5‐hydroxytryptamine (10 µM)‐, but not 6‐chloromelatonin (10 µM)‐, induced IP1 accumulation. Melatonin receptor agonists increased IP1 accumulation in a concentration‐dependent manner reaching different maximal responses. N‐Acetyl‐5‐hydroxytryptamine was more potent than melatonin in increasing IP1 accumulation, suggesting activation of a melatonin receptor site other than the ML‐1 melatonin receptor (i.e., N‐acetyl‐5‐hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML‐1 subtype leads to activation of the phospholipase C‐mediated signal transduction pathway.
Evidence for Glutamatergic Projections from the Cochlear Nucleus to the Superior Olive and the Ventral Nucleus of the Lateral Lemniscus
Authors: Suneja S. K. , Benson C. G. , Gross J. , Potashner S. J.
Published: July 2010

Abstract: This study attempts to determine if projections ascending from the guinea pig cochlear nucleus (CN) could be glutamatergic and/or aspartatergic. Multiple radio frequency lesions were made to ablate the right CN. The ablation was verified histologically. To identify the principal targets of CN efferents, silver impregnation methods were used to localize the preterminal degeneration of fibers in transverse sections of the brainstem 5 and 7 days after CN ablation. CN efferents projected heavily to the lateral superior olive (LSO) ipsilaterally, the medial superior olive (MSO) bilaterally, and contralaterally to the medial (MNTB) and ventral (VNTB) nuclei of the trapezoid body, the ventral (VNLL) and intermediate nuclei of the lateral lemniscus and the central nucleus of the inferior colliculus (ICc). There were smaller projections to the lateral nucleus of the trapezoid body ipsilaterally, the dorsal and dorsomedial periolivary nuclei bilaterally, and the dorsal nucleus of the lateral lemniscus contralaterally. There were sparse projections to the VNLL and ICc ipsilaterally and the CN contralaterally, and a very sparse projection to the contralateral LSO. To determine if CN efferents were glutamatergic and/or aspartatergic, the fresh brainstem was sectioned transversely and samples of the LSO, MSO, MNTB, VNLL, and ICc were taken to measure the electrically evoked release and the uptake of d‐[3H]Asp and [14C]Gly or [14C]GABA 3–5 days after the CN ablation. The release studies suggest that only certain of the histologically identified projections ascending from the CN may be glutamatergic and/or aspartatergic. CN ablation depressed d‐[3H]Asp release in the MSO bilaterally and in the contralateral MNTB and VNLL, suggesting that the CN efferents to these nuclei may use glutamate or aspartate as a transmitter. It was unclear whether a marginal depression of d‐[3H]Asp release in the ipsilateral LSO reflected the presence of glutamatergic CN projections to this nucleus. d‐[3H]Asp release in the ICc was unaffected, suggesting that CN efferents to this nucleus may not be glutamatergic. There were no deficits in d‐[3H]Asp uptake. [14C]Gly release from the LSO and MSO was unchanged. [14C]Gly uptake was unchanged in the MSO and depressed only in the contralateral LSO, possibly reflecting subnormal uptake activity in endings contributed by contralateral MNTB cells that had lost their CN efferents. [14C]GABA uptake in the MNTB, VNLL, and ICc was unchanged. [14C]GABA release was unchanged in the VNLL and ICc. [14C]GABA release was depressed only in the contralateral MNTB, possibly reflecting the loss of a small complement of GABAergic CN efferents and the reaction of GABAergic projections from the contralateral VNTB to their loss of CN efferents.
Serotonin 5‐HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus
Authors: Kaufman Marc J. , Hartig Paul R. , Hoffman Beth J.
Published: July 2010

Abstract: The serotonin 5‐HT2C receptor (formerly designated the 5‐HT1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5‐HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five‐fold basal level, with an EC50 of 11 nM. This response was not inhibited by muscarinic or β‐adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent Ki values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin‐stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin‐stimulated cGMP formation was calcium, phospholipase A2, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A2 or lipoxygenase inhibitors p‐bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5‐HT2C receptors trigger cGMP formation in a calcium‐sensitive manner.
Glucocorticoids Enhance Histamine‐Evoked Catecholamine Secretion from Bovine Chromaffin Cells
Authors: Choi Alice Y. , Fukui Hiroyuki , Perlman Robert L.
Published: July 2010

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine‐evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine‐rich) and noradrenergic (norepinephrine‐rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine‐evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC50 for dexamethasone was ∼1 nM. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine‐evoked catecholamine secretion in both dexamethasone‐treated and untreated cells was blocked by the H1 histamine receptor antagonist mepyramine but was not affected by the H2 antagonist cimetidine; thus, dexamethasone appeared to enhance an H1 receptor‐mediated process. In the absence of glucocorticoids, H1 receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H1 receptor mRNA levels in both cell types. The increased expression of H1 receptors presumably contributes to the enhancement of histamine‐evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.
Allosteric Regulation of the N‐Methyl‐d‐Aspartate Receptor‐Linked Ion Channel Complex and Effects of Ethanol in Ethanol‐Withdrawal Seizure‐Prone and ‐Resistant Mice
Authors: Carter Leslie A. , Belknap John K. , Crabbe John C. , Janowsky Aaron
Published: July 2010

Abstract: The effects of ethanol, glycine, and spermidine on the specific binding of [3H]MK‐801 were characterized in Triton‐treated membranes prepared from the hippocampus and cortex of ethanol‐withdrawal seizure‐prone (WSP) and ‐resistant (WSR) mice. Glycine, an allosteric agonist at the NMDA receptor‐linked ion channel complex, caused an increase in specific [3H]MK‐801 binding to hippocampal membrane preparations. There were no significant differences in EC50 values between the selected lines for the effect of glycine (WSP, 391.7 ± 48.4 nM; WSR, 313.4 ± 77 nM) in the presence of 10 µM NMDA or in the maximal response to the agonist (WSP, 1.75 ± 0.26 pmol/mg of protein; WSR, 1.67 ± 0.22 pmol/mg of protein). The EC50 values for the spermidine‐induced increase in [3H]MK‐801 binding in membranes from hippocampus in the absence (WSP, 11.7 ± 0.83 µM; WSR, 9.98 ± 1.29 µM) or in the presence of 10 µM glycine and 10 µM NMDA (WSP, 2.1 ± 0.35 µM; WSR, 2.37 ± 0.42 µM) also did not differ. Similar results were obtained in cortical membranes. Saturation isotherms indicated that there was no difference in the density of [3H]MK‐801 binding sites, or in their affinity for the radioligand, between the mouse lines. In addition, administration of ethanol by inhalation (24 h) to WSP and WSR mice did not cause an increase in the density of [3H]MK‐801 binding sites, and there was no difference in the density or affinity of binding sites between the mouse lines. Withdrawal from ethanol (6 h), which causes an increase in the severity of handling‐induced convulsions in WSP mice, also did not alter the binding site density or affinity for radioligand. The results suggest that the characteristics of the NMDA receptor‐linked ion channel complex in the tissue preparations described here do not differ in WSP and WSR mice. Thus, genetic differences in seizure susceptibility during ethanol withdrawal can be dissociated from the total density of hippocampal or cortex NMDA receptors under activating conditions.
Heat‐Shock 70 Messenger RNA Levels in Human Brain: Correlation with Agonal Fever
Authors: Morrison‐Bogorad Marcelle , Zimmerman Anton L. , Pardue Sibile
Published: July 2010

Abstract: Systematic review of antemortem clinical information on randomly selected Alzheimer disease (AD) patients revealed that ∼40% of the patients had a recorded fever of ≥39.2°C at or near death. Using isolation and quantitation techniques appropriate for analysis of human brain mRNAs, we found that low levels of inducible heat‐shock protein 70 (hsp70) mRNAs were present in cerebellum of afebrile AD patients and that mRNA levels were usually lower in two brain regions affected in AD, i.e., hippocampus and temporal cortex. Levels of hsp70 mRNAs were increased three‐ to 33‐fold in cerebellum of febrile patients compared with levels in patients whose recorded temperatures were ≤37.5°C. Levels of hsp70 mRNAs were also increased in hippocampus and cortex of these febrile patients, but to a lesser extent than cerebellum. Heat‐shock cognate 70 (hsc70) mRNAs were present at highest levels in afebrile cerebellum and were also present in the other brain regions. In cerebellum of patients with the highest temperatures, hsc70 mRNAs were induced severalfold over basal levels. Although there was a low and variable induction of hsc70 mRNAs in temporal cortex of these patients, there was no evidence for any induction in hippocampus. Increased heat‐shock 70 mRNA levels did not correlate with hypoxia, coma, hypertension, hypoglycemia, seizures, or medication. These results indicate that a specific agonal stress, namely fever, can increase the levels of heat shock 70 mRNAs in AD brain; however, there is no evidence to suggest that affected regions of AD brain have higher overall levels of these mRNAs. Failure to obtain adequate agonal state information could result in inaccurately identifying short‐term stress‐related changes in postmortem brain as neuropathology characteristic of a chronic disease state.
Loss of Protein Kinase C‐αβ in Brain of Heroin Addicts and Morphine‐Dependent Rats
Authors: Busquets Xavier , Escriba Pablo V. , Sastre Magdalena , García‐Sevilla Jesús A.
Published: July 2010

Abstract: The biochemical status of human brain protein kinase C (PKC)‐αβ during opiate dependence was studied by means of immunoblotting techniques in postmortem brain of heroin addicts who had died by opiate overdose. In the frontal cortex, a marked decrease (53%, p < 0.05) in the immunoreactivity of PKC‐αβ was found in heroin addicts compared with matched controls. The loss of PKC‐αβ in the brain of human addicts paralleled that observed in the frontal cortex of rats after chronic treatment with morphine (10–100 mg/kg i.p. for 5 days) (PKC‐αβ decreased by 34%, p < 0.05). Chronic treatment with naloxone (1 mg/kg i.p. every 12 h for 5 days) did not alter PKC‐αβ immunoreactivity in the rat brain. However, in morphine‐dependent rats, naloxone‐precipitated withdrawal induced a rapid and strong behavioral reaction with a concomitant up‐regulation of PKC‐αβ immunoreactivity to control values. These results indicated that the decrease of brain PKC‐αβ induced by heroin/morphine is a μ‐opioid receptor‐mediated effect. The chronic administration of opiates has been associated with a marked sensitization of the adenylyl cyclase/cyclic AMP system, although this phenomenon is not exclusive of the opioid system but the general cellular adaptation to chronic inhibition of adenylyl cyclase. In this context, chronic treatment of rats with other inhibitory agonists (e.g., clonidine, 1 mg/kg i.p. every 12 h for 14 days) acting through receptors (e.g., α2‐adrenoceptors) also coupled to adenylyl cyclase did not alter brain PKC‐αβ immunoreactivity. Together these findings suggest that the brain PKC system might play a major role in opiate addiction.
Structure‐Activity Analyses of β‐Amyloid Peptides: Contributions of the β25–35 Region to Aggregation and Neurotoxicity
Authors: Pike Christian J. , Walencewicz‐Wasserman Andrea J. , Kosmoski Joseph , Cribbs David H. , Glabe Charles G. , Cotman Carl W.
Published: July 2010

Abstract: The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of β‐amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β‐amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure‐activity analyses of a series of peptides derived from both the proposed active fragment of β‐amyloid, β25–35, and the full‐length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β‐amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β‐amyloid‐induced neurotoxicity. Further, both N‐ and C‐terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β‐sheet secondary structure in aggregating, toxic β‐amyloid peptides but not in nonaggregating, nontoxic β‐amyloid peptides. Together, these data further define the primary and secondary structures of β‐amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.
Production of Polyclonal Antisera that Recognize and Distinguish Between the Extracellular Domains of Neuronal Nicotinic Acetylcholine Receptor Subunits
Authors: Neff Shawn , Dineley‐Miller Kelly , Char David , Quik Maryka , Patrick Jim
Published: July 2010

Abstract: Ligand‐gated ion channels are oligomeric transmembrane proteins that usually contain more than one kind of monomer. The variety of monomers available to participate in oligomer formation and the apparent latitude in acceptable monomer combinations allows considerable diversity. Mechanisms for identifying the monomers comprising specific receptors are needed. We have generated affinity‐purified polyclonal antisera that recognize the extracellular domain of nine neuronal nicotinic acetylcholine receptor (nAChR) subunits and distinguish between them. We prepared these antisera by immunizing rabbits with bacterially expressed recombinant protein representing the N‐terminal extracellular domain of each neuronal nAChR subunit followed by affinity purification of antibodies against synthetic peptides corresponding to residues 68–81 of the α1 subunit. We demonstrate subunit specificity of each affinity‐purified antisera by western blots of the bacterially expressed protein and immunoblot against peptide. We further used these antibodies to demonstrate expression of neuronal nAChR subunits on the surface of transiently transfected simian kidney (COS‐7) cells.
Fatty Acyl‐CoA Inhibits 1‐Alkyl‐sn‐Glycero‐3‐Phosphate Acetyltransferase in Microsomes of Immature Rabbit Cerebral Cortex: Control of the First Committed Step in the De Novo Pathway of Platelet‐Activating Factor Synthesis
Authors: Baker R. Roy , Chang Huu‐Yi
Published: July 2010

Abstract: Microsomal fractions of cerebral cortices of 15‐day‐old rabbits were used to study the 1‐alkyl‐sn‐glycero‐3‐phosphate (AGP) acetyltransferase that generates 1‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphate in the de novo path of platelet‐activating factor synthesis. The AGP acetyltransferase activity was inhibited by small concentrations of medium‐long chain fatty acyl‐CoA thioesters. In contrast, the AGP acyltransferase used oleoyl‐CoA as substrate and was not inhibited by the presence of acetyl‐CoA in high molar excess. The inhibition of AGP acetyltransferase was seen at concentrations of oleoyl‐CoA as low as 0.5 µM using 12.5 µM AGP and 200 µM acetyl‐CoA. The inhibition by oleoyl‐CoA was noncompetitive for the acetyl‐CoA substrate. However, there was evidence that the oleoyl‐CoA was competing with AGP in the acetyltransferase reaction, as the inhibition was lessened by increasing the AGP substrate concentration. Several acyl‐CoA thioesters were effective as inhibitors of the AGP acetyltransferase, including oleoyl‐, palmitoyl‐, lauroyl‐, and octanoyl‐CoA. Propionyl‐ and butyryl‐CoA were less effective as inhibitors, and propionyl‐CoA was found to be a competitive inhibitor for acetyl‐CoA. We have noted earlier that MgATP is an effective inhibitor of the AGP acetyltransferase and here we show that the inhibition by oleoyl‐CoA can be increased by the presence of 0.1 mM MgATP. In brain ischemia, a decline in ATP levels would likely lead to a corresponding fall in acyl‐CoA concentrations, thereby relieving the inhibition of AGP acetyltransferase and permitting the flow of AGP into the de novo pathway of platelet‐activating factor synthesis.
Human Y‐79 Retinoblastoma Cells Exhibit Specific Corticotropin‐Releasing Hormone Binding Sites
Authors: Olianas Maria C. , Lampis Giorgio , Onali Pierluigi
Published: July 2010

Abstract: In this study we have identified specific binding sites for corticotropin‐releasing hormone (CRH) in human Y‐79 retinoblastoma cell membranes by using 125I‐Tyrovine CRH (125I‐oCRH) as radioligand. Binding at 19°C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The 125I‐oCRH binding was enhanced by Mg2+ and inhibited by the GTP analogue guanosine 5′‐O‐(3′‐thiotriphosphate). Y‐79 cell membranes exhibited two populations of binding sites, a high‐affinity site with an apparent dissociation constant (KD) of 1 nM and a low‐affinity site with an apparent KD of 500 nM. 125I‐oCRH binding was completely antagonized by human/rat CRH, [Met(O)21]oCRH, α‐helical CRH9–41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH‐binding sites in retinal cells. The Y‐79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.
Characterization of the Excitotoxic Potential of the Reversible Succinate Dehydrogenase Inhibitor Malonate
Authors: Greene James G. , Greenamyre J. Timothy
Published: July 2010

Abstract: Although the mechanism of neuronal death in neurodegenerative diseases remains unknown, it has been hypothesized that relatively minor metabolic defects may predispose neurons to N‐methyl‐d‐aspartate (NMDA) receptor‐mediated excitotoxic damage in these disorders. To further investigate this possibility, we have characterized the excitotoxic potential of the reversible succinate dehydrogenase (SDH) inhibitor malonate. After its intrastriatal stereotaxic injection into male Sprague‐Dawley rats, malonate produced a dose‐dependent lesion when assessed 3 days after surgery using cytochrome oxidase histochemistry. This lesion was attenuated by coadministration of excess succinate, indicating that it was caused by specific inhibition of SDH. The lesion was also prevented by administration of the noncompetitive NMDA antagonist MK‐801. MK‐801 did not induce hypothermia, and hypothermia itself was not neuroprotective, suggesting that the neuroprotective effect of MK‐801 was due to blockade of the NMDA receptor ion channel and not to any nonspecific effect. The competitive NMDA antagonist LY274614 and the glycine site antagonist 7‐chlorokynurenate also profoundly attenuated malonate neurotoxicity, further indicating an NMDA receptor‐mediated event. Finally, the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) antagonist NBQX (2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)‐quinoxaline) was ineffective at preventing malonate toxicity at a dose that effectively reduced S‐AMPA toxicity, indicating that non‐NMDA receptors are involved minimally, if at all, in the production of the malonate lesion. We conclude that inhibition of SDH by malonate results in NMDA receptor‐mediated excitotoxic neuronal death. If this mechanism of “secondary” or “weak” excitotoxicity plays a role in neurodegenerative disease, NMDA antagonists and other “antiexcitotoxic” strategies may have therapeutic potential for these diseases.
The Degradative Pathway of Gangliosides GM1 and GM2 in Neuro2a Cells by Sialidase
Authors: Riboni Laura , Caminiti Antonella , Bassi Rosaria , Tettamanti Guido
Published: July 2010

Abstract: Gangliosides GM1 [3H‐labeled at the sphingosine (Sph) moiety] and GM2 [3H‐labeled at the Sph or N‐acetylgalactosamine (GalNAc) moiety] were administered to cultured Neuro2a cells for varying pulse (1–4 h) and chase (up to 4 h) periods, and their metabolic processing was followed. The main and earliest formed 3H‐metabolites of [Sph‐3H]GM1 were GM2, asialo‐GM1, asialo‐GM2, and lactose‐ceramide, and those of [Sph‐3H]GM2 were asialo‐GM2 and lactose‐ceramide. The asialo‐GM1 and asialo‐GM2 formed were isolated and chemically characterized. [3H]Asialo‐GM2 was produced in identical amounts after treatment with equimolar [Sph‐3H]GM2 and [GalNAc‐3H]GM2. At low temperature or in the presence of chloroquine, the formation of all 3H‐metabolites, including asialo‐GM2 and asialo‐GM1, was undetectable, indicating that ganglioside metabolic processing was an endocytosis‐ and lysosome‐dependent process. These results demonstrate that in Neuro2a cells exogenous GM1 (and GM2) is mainly degraded through the pathway GM1 → GM2 → asialo‐GM2 →→ Sph, with a minor fraction of GM1 undergoing degradation with the sequence GM1 → asialo‐GM1 → asialo‐GM2 →→ Sph. These findings are consistent with the hypothesis that Neuro2a cells contain a sialidase (likely of lysosomal nature) affecting ganglioside GM1 and GM2. The sialidase‐mediated degradative pathway of GM1 and GM2 in Neuro2a cells might be related to the tumoral nature of these cells.
Proadrenomedullin N‐Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla
Authors: Katoh Fumi , Kitamura Kazuo , Niina Hiromi , Yamamoto Ryuichi , Washimine Hisanori , Kangawa Kenji , Yamamoto Yoshitaka , Kobayashi Hideyuki , Eto Tanenao , Wada Akihiko
Published: July 2010

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca2+‐dependent exocytotic cosecretion of proadrenomedullin N‐terminal 20 peptide (PAMP) (EC50 = 50.1 µM) and catecholamines (EC50 = 63.0 µM), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH2 inhibited carbachol‐induced 22Na+ influx via nicotinic receptors (IC50 = 2.5 µM) in a noncompetitive manner and thereby reduced carbachol‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels (IC50 = 1.0 µM) and catecholamine secretion (IC50 = 1.6 µM). It did not alter high K+‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels or veratridine‐induced 22Na+ influx via voltage‐dependent Na+ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca2+‐dependent exocytosis in response to nicotinic receptor stimulation.
Recombinant Human NMDA Homomeric NR1 Receptors Expressed in Mammalian Cells Form a High‐Affinity Glycine Antagonist Binding Site
Authors: Grimwood Sarah , Le Bourdellès Béatrice , Whiting Paul J.
Published: July 2010

Abstract: The cDNA NMDAR1 (NR1) encodes a single polypeptide that forms a receptor‐channel complex with electrophysiological and pharmacological properties characteristic of the N‐methyl‐d‐aspartate receptor. Homomeric NR1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C‐terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (±)‐4‐(trans)‐2‐carboxy‐5,7‐dichloro‐4‐[3H]phenylaminocarbonylamino‐1,2,3,4‐tetrahydroquinoline ([3H]L‐689,560) bound to NR1a‐ and NR1e‐transfected cells with high affinity (KD = 3.29 and 1.61 nM, respectively). Bmax values for NR1a‐ and NR1e‐transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity. Ki values for glycine site antagonists inhibiting [3H]L‐689,560 binding to NR1e‐transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four‐ to 16‐fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist‐preferring state. Ki values obtained with NR1a‐transfected cells were approximately twofold lower than those obtained with NR1e‐transfected cells. High‐affinity binding to NR1‐transfected cells was not observed with the transmitter recognition site radioligands l‐[3H]glutamate and d,l‐(ε)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentanoic acid ([3H]CGP‐39653) or the ion‐channel radioligand [3H]dizocilpine ([3H]MK‐801). These results indicate that although transfection of mammalian cells with homomeric NR1 recombinant receptors does not appear to result in functional receptors, a glycine binding site is formed that may have a physiological role if present in vivo.
Differential Effects of 4′‐Chlorodiazepam on Expressed Human GABAA Receptors
Authors: Lan Nancy C. , Chen Jie‐Sheng , Johnson Deborah , Gee Kelvin W.
Published: July 2010

Abstract: The interactions of the atypical benzodiazepine 4′‐chlorodiazepam (Ro 5‐4864) with functionally expressed human GABAA receptor cDNAs were determined. Cotransfection of human α2, β1, and γ2 subunits was capable of reconstituting a 4′‐chlorodiazepam recognition site as revealed by a dose‐dependent potentiation of t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to the GABA‐activated chloride channel. This site is found on GABAA receptor complexes containing sites for GABA agonist‐like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α1 or α3 for the α2 subunit did not reconstitute a 4′‐chlorodiazepam recognition site that was capable of modulating [35S]TBPS binding under the same experimental conditions. The 4′‐chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4′‐chlorodiazepam, consistent with the previous analysis of the 4′‐chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α2 and β1 alone reconstituted a 4′‐chlorodiazepam recognition site. It is interesting, however, that the 4′‐chlorodiazepam site was found to inhibit [35S]TBPS binding to the GABA‐activated chloride channel. Thus, the 4′‐chlorodiazepam site may be reconstituted with only the α and β polypeptides.
Dynamic Storage of Dopamine in Rat Brain Synaptic Vesicles In Vitro
Authors: Floor Erik , Leventhal Phillip S. , Wang Yalin , Meng Lihao , Chen WeiQing
Published: July 2010

Abstract: The dynamics of catecholamine storage were studied in highly purified, small synaptic vesicles from rat brain both during active uptake or after inhibiting uptake with reserpine, tetrabenazine, or removal of external dopamine. To assess turnover during active uptake, synaptic vesicles were allowed to accumulate [3H]dopamine ([3H]DA) for ∼10 min and then diluted 20‐fold into a solution containing unlabeled DA under conditions such that active uptake could continue. After dilution, [3H]DA was lost with single exponential kinetics at a half‐time of ∼4 min at 30°C in 8 mM Cl− medium, in which both voltage and H+ gradients are present in the vesicles. In 90 mM Cl− medium, in which high H+ and Cl− gradients but no voltage gradient are present, [3H]DA escaped at a half‐time of ∼7 min. In both high and low Cl− media, ∼40% of [3H]DA efflux was blocked by reserpine or tetrabenazine. The residual efflux also followed first‐order kinetics. These results indicate that two efflux pathways were present, one dependent on DA uptake (and thus on the presence of external DA) and the other independent of uptake, and that both pathways function regardless of the type of electrochemical H+ gradient in the vesicles. The presence of both uptake‐dependent and ‐independent efflux was observed in experiments using DA‐free medium, instead of uptake inhibitors, to prevent uptake. Uptake‐independent efflux showed molecular selectivity for catecholamines; [14C]DA was lost about three times faster than [3H]norepinephrine after adding tetrabenazine directly (without dilution) to vesicles that had taken up comparable amounts of each amine. In addition, the first‐order rate constant for uptake‐independent efflux showed little change over a 60‐fold range of internal DA concentrations, which suggests that this pathway had a high transport capacity. All efflux was blocked at 0°C, suggesting that efflux did not occur through a large pore. There was little or no change in the proton gradient in synaptic vesicles, monitored by [14C]methylamine equilibration, during the experimental manipulations used here. Thus, the driving force for catecholamine uptake remained approximately constant. The physiological role of uptake‐independent efflux could be to allow the monoamine content of synaptic vesicles to be regulated over a time range of minutes and, thereby, control the amount released by exocytosis. These results imply that catecholamines turn over with a half‐time of minutes during active uptake by brain synaptic vesicles in vitro.
High Resolution Proton NMR Spectroscopy of Multiple Sclerosis Lesions
Authors: Davies Siân E. C. , Newcombe Jia , Williams Stephen R. , McDonald W. Ian , Clark John B.
Published: July 2010

Abstract: Tissue from postmortem multiple sclerosis and normal control brains was extracted with perchloric acid and analysed using proton NMR spectroscopy. The content of N‐acetyl‐derived groups (the sum of N‐acetylaspartate, acetate, and N‐acetylaspartylglutamate) was decreased in multiple sclerosis plaques compared with normal control white matter (mean, 4.36 vs. 6.64 µmol/g wet weight). In normal appearing white matter adjacent to plaques a corresponding decrease was seen, with no change in white matter distant from plaques. A decrease in the content of total creatine was observed in multiple sclerosis plaques in comparison with normal control white matter (mean, 4.64 vs. 6.56 µmol/g wet weight), which correlated strongly with the decrease in N‐acetyl‐derived groups. No changes in other metabolites such as total choline or myo‐inositol were seen. The decreases in content of N‐acetyl‐derived groups are in agreement with observations from in vivo proton NMR spectroscopy in multiple sclerosis patients. The decrease in total creatine is in contrast to most of the observations made in vivo where total creatine is assumed to be unchanged and metabolite levels are often expressed as a total creatine ratio. The use of a total creatine ratio in vivo could lead to an underestimation of reductions in N‐acetylaspartate and an apparent increase in other metabolites in the multiple sclerosis lesion.
Effects of the Serotonin1A Agonist, 8‐Hydroxy‐2‐(Di‐n‐Propylamino)tetralin on Neurochemical Responses to Stress
Authors: Saphier David , Welch Jon E.
Published: July 2010

Abstract: The effects of intracerebroventricular administration of the 5‐hydroxytryptamine (5‐HT)1A agonist, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT; 0.1 pmol) on adrenocortical and neurochemical responses to stress were examined in conscious male rats. The following stress paradigms were used: acoustic stimulation (105 dB for 2 min); footshock (0.2 mA, five shocks over 5 min); conditioned fear (animals placed in a footshock chamber for 5 min, 24 h after footshock); restraint (5 min); intraperitoneal (i.p.) injection of recombinant human interleukin‐1α (rHu‐IL‐1α, 20 µg/kg); and injection of cocaine hydrochloride (20 mg/kg, i.p.). As previously shown, 8‐OH‐DPAT was able to attenuate the adrenocortical response to acoustic stress, conditioned fear, rHu‐IL‐1α, and cocaine administration. Cocaine decreased 5‐hydroxyindoleacetic acid (5‐HIAA)/5‐HT and dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratios and norepinephrine (NE) concentration in the prefrontal cortex, hypothalamus, and brainstem in all experiments, and 8‐OH‐DPAT reversed the changes in DOPAC/DA ratio without affecting 5‐HIAA/5‐HT ratios or NE content. 8‐OH‐DPAT alone had no effect on these parameters, although it decreased NE content in the prefrontal cortex in several experiments, and in the brainstem in one experiment. Significant decreases in NE content were observed in some brain regions following some of the stressors, but these changes were not generally affected by 8‐OH‐DPAT. Increases in the 5‐HIAA/5‐HT and DOPAC/DA ratios were also observed in some brain sites following some stressors, but these changes were not affected by 8‐OH‐DPAT except in the case of the increased 5‐HIAA/5‐HT ratio in the prefrontal cortex following the conditioned fear response. These results indicate that although 8‐OH‐DPAT is able to decrease plasma corticosterone responses following acoustic stress, conditioned fear, rHu‐IL‐1α, and cocaine administration, these effects do not appear to be related to an action of the 5‐HT1A agonist on biogenic amine metabolism. This observation indicates that the predominant effect of 8‐OH‐DPAT on adrenocortical responses is mediated at postsynaptic sites not involved in the regulation of cerebral biogenic amine metabolism.
Guanosine Triphosphate Binding to β‐Subunit of Tubulin in Alzheimer's Disease Brain: Role of Microtubule‐Associated Protein τ
Authors: Khatoon Sabiha , Grundke‐Iqbal Inge , Iqbal Khalid
Published: July 2010

Abstract: In Alzheimer's disease, paired helical filaments composed mainly of abnormally phosphorylated τ accumulate in certain selected neurons of the brain, and microtubules are rarely seen in the affected cells. In the present study, the binding of 32P‐labeled 8‐azidoguanosine triphosphate ([γ‐32P]8N3GTP), the photoaffinity analogue of GTP, to the β‐subunit of tubulin in brain homogenates was found to be markedly lower in patients with Alzheimer's disease than in aged control human cases. No significant differences were observed in the levels of the α‐ and β‐subunits of tubulin between Alzheimer's disease and control brains obtained 2–7 h postmortem. In nine of 19 Alzheimer's disease and 11 of 12 control autopsied brains (2–7 h postmortem and stored at −75°C) tubulin was isolated successfully from brain cytosol by in vitro polymerization induced with DEAE‐dextran. The GTP binding was observed in the two cycled assembled microtubule preparations from all the normal control, and in eight of nine Alzheimer's disease cases. Alzheimer's disease microtubule preparations contained varying amounts of abnormally phosphorylated τ, whereas no abnormal τ was detected in the control brain preparations. Addition of bovine τ to bovine, normal human, and Alzheimer's disease brain tubulin preparations markedly increased GTP binding to the β‐subunit. An alkaline phosphatase‐treated paired helical filament‐enriched preparation increased by approximately twofold the GTP binding to bovine brain tubulin. GTP binding to tubulin prepared by phosphocellulose chromatography of two cycled microtubules from three Alzheimer's disease and three normal control brains, revealed insignificant differences between the two groups. These findings have suggested that (1) τ protein promotes the GTP binding to the β‐subunit of tubulin, and (2) the breakdown of the microtubule system in brains of patients with Alzheimer's disease might in part be due to the abnormal phosphorylation of τ which depresses the GTP binding.