bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Stimulatory and Inhibitory Inputs to the Hypothalamic‐Pituitary‐Adrenal Axis During Hypoglycaemia or Transport In Ewes
Authors: Smith R. F. , French N. P. , Saphier P. W. , Lowry P. J. , Veldhuis J. D. , Dobson H.
Published: July 2010

This study used the novel approach of statistical modelling to investigate the control of hypothalamic‐pituitary‐adrenal (HPA) axis and quantify temporal relationships between hormones. Two experimental paradigms were chosen, insulin‐induced hypoglycaemia and 2 h transport, to assess differences in control between noncognitive and cognitive stimuli. Vasopressin and corticotropin‐releasing hormone (CRH) were measured in hypophysial portal plasma, and adrenocorticotropin hormone (ACTH) and cortisol in jugular plasma of conscious sheep, and deconvolution analysis was used to calculate secretory rates, before modelling. During hypoglycaemia, the relationship between plasma glucose and vasopressin or CRH was best described by log10 transforming variables (i.e. a positive power–curve relationship). A negative‐feedback relationship with log10 cortisol concentration 2 h previously was detected. Analysis of the ‘transport’ stimulus suggested that the strength of the perceived stimulus decreased over time after accounting for cortisol facilitation and negative‐feedback. The time course of vasopressin and CRH responses to each stimulus were different However, at the pituitary level, the data suggested that log10 ACTH secretion rate was related to log10 vasopressin and CRH concentrations with very similar regression coefficients and an identical ratio of actions (2.3 : 1) for both stimuli. Similar magnitude negative‐feedback effects of log10 cortisol at −110 min (hypoglycaemia) or −40 min (transport) were detected, and both models contained a stimulatory relationship with cortisol at 0 min (facilitation). At adrenal gland level, cortisol secretory rates were related to simultaneously measured untransformed ACTH concentration but the regression coefficient for the hypoglycaemia model was 2.5‐fold greater than for transport. No individual sustained maximum cortisol secretion for longer than 20 min during hypoglycaemia and 40 min during transport. These unique models demonstrate that corticosteroid negative‐feedback is a significant control mechanism at both the pituitary and hypothalamus. The amplitude of HPA response may be related to stimulus intensity and corticosteroid negative‐feedback, while duration depended on feedback alone.
Activation of Muscarinic Receptors in Rat Subfornical Organ Neurones
Authors: Honda E. , Ono K. , Toyono T. , Kawano H. , Masuko S. , Inenaga K.
Published: July 2010

Cholinergic muscarinic inputs to subfornical organ (SFO) neurones in rats were studied using histochemical, molecular–biological and electrophysiological techniques. Neurones in the medial septum and the diagonal band (MS‐DBB) were retrogradely labelled by a tracer wheat germ agglutinin‐conjugated horseradish peroxidase–colloidal gold complex injected into the SFO. Some in the MS‐DBB were double‐labelled by choline acetyltransferase (ChAT) antibody. Many ChAT‐immunoreactive fibres were observed in the SFO. M3 muscarinic receptor subtype‐like immunoreactivity, detected using a polyclonal antiserum, was observed in the SFO. In slice preparations, muscarine induced inward currents in a dose‐related manner. The inward currents were suppressed by the relatively M3 muscarinic receptor selective antagonist 4‐diphenylacetoxy‐N‐methylpiredine methiodide. In the whole‐cell current mode, muscarine depolarized the membrane with increased frequency of action potentials. Reverse transcriptase‐polymerase chain reaction showed the presence of M2–M5 receptor mRNA in the SFO tissues. These results suggest that the SFO receives cholinergic muscarinic synaptic inputs from the MS‐DBB. Acetylcholine postsynaptically activates and depolarizes neurones in the SFO partly through specific muscarinic receptors, including M3 receptor subtypes.
Oxytocin, But Not Oxytocin Receptor, is Regulated by Oestrogen Receptor β in the Female Mouse Hypothalamus
Authors: Patisaul H. B. , Scordalakes E. M. , Young L. J. , Rissman E. F.
Published: July 2010

In the female rat, oestrogen receptor (ER) β is colocalized with both oxytocin‐ and vasopressin‐producing neurones in the paraventricular nucleus of the hypothalamus (PVN). In this study, we demonstrate that the same pattern of colocalization between ERβ and oxytocin exists in the female mouse. Because this nucleus contains only a negligible quantity of ERα, it is likely that the oestrogen‐dependent regulation of oxytocin and vasopressin synthesis in the PVN is mediated by ERβ. Thus, we compared the effect of ovarian hormones on oxytocin and vasopressin mRNA expression in the PVN of wild‐type (WT) and ERβ knockout (βERKO) mice. We also compared the effects of ovarian hormones on oxytocin receptor (OTR) expression in the medial amygdala (MeA) and ventromedial nucleus of the hypothalamus (VMN) in female WT and βERKO mice. Ovariectomized mice underwent long‐term treatment with oestradiol or oil. Progesterone was given concurrently on the final 7 days of treatment, and all mice were killed 48 h after the final progesterone injection. In the PVN, hormone treatment increased oxytocin mRNA expression in WT but not βERKO females. These results suggest that ERβ is necessary for the regulation of the expression of oxytocin in the PVN. Hormone treatment had no effect on vasopressin mRNA expression in the PVN, but significantly increased OTR binding in both the VMN and the MeA in both genotypes. Collectively, our data show region and peptide specific regulation by ERα and ERβ in the mouse hypothalamus.
Alternative Splicing of a 51‐Nucleotide Exon that Encodes a Putative Protein Kinase C Phosphorylation Site Generates Two Forms of the Chicken γ‐Aminobutyric AcidAReceptor β2 Subunit
Authors: Harvey Robert J. , Chinchetru Miguel A. , Darlison Mark G.
Published: July 2010

Abstract: Complementary DNAs that encode two forms of the chicken ‐γ‐aminobutyric acid type A (GABAA) receptor β2 subunit have been isolated. These polypeptides differ by the presence (β2L) or absence (β2S) of 17 amino acids, which contain a possible target for phosphorylation by protein kinase C, in the large intracellular loop between the third and fourth membrane‐spanning domains. The extra sequence in the chicken β2L subunit is not found in previously published GABAA receptor β2‐subunit sequences. Analysis of genomic DNA has revealed that the two β2‐subunit mRNAs arise by alternative splicing of a novel 51‐nucleotide exon. Although the two β2‐subunit transcripts appear to be present in 1 ‐day‐old chick brain at similar steady‐state levels, we have been unable to detect an mRNA for the long form of the β2 subunit in either the bovine or the rat. Because the various GABAA receptor genes are thought to have arisen by duplication of a common ancestor, our data, taken together with that on the γ2 subunit, which occurs in two forms that arise by alternative splicing of a 24‐nucleotide exon, suggest that the coding region of the primordial gene or one of its very early descendants contained 10 exons, not nine as previously thought.
Purification and Characterization of B2Bradykinin Receptor from Rat Uterus
Authors: Yaqoob Mohammed , Snell Christopher R.
Published: July 2010

Abstract:A B2bradykinin (BK) receptor was solubilised and partially purified from rat uterine membranes by a combination of ammonium sulphate precipitation, desalting on Sephadex G‐50, and hydroxyapatite and wheat germ agglutinin affinity chromatography. The partially purified BK receptor, enriched 1,500‐fold, was then cross‐linked to125l‐Tyr0‐BK using disuccinimidyl suberate and purified to homogeneity as a single protein species on two‐dimensional gel electrophoresis with a molecular mass of 81 kDa. This molecular size was in agreement with the value of 80–120 kDa estimated from Sephacryl 300 size exclusion column chromatography of the B2receptor. The partially purified and the crude solubilised B2BK receptor from rat uterus showed similar affinities for BK and the BK analogues iodo‐Tyr0‐BK, D‐Phe7‐BK, and des‐Arg9‐BK, indicating that the ligand binding specificity of the receptor had been retained during the purification procedures. The biochemical properties of the solubilised B2BK receptor correspond to those of a hydrophobic acidic glycoprotein (isoelectric focusing gave a value of 4.5–4.7) that binds specifically to wheat germ agglutinin but has no affinity for either concanavalin A or lentil lectin, suggesting the absence of terminal mannose or glucose residues.
Hydroxyl Radicals Generated In Vivo Kill Neurons in the Rat Spinal Cord: Electrophysiological, Histological, and Neurochemical Results
Authors: Liu Danxia , Yang Ruoming , Yan Xiaoxia , McAdoo David J.
Published: July 2010

Abstract:We have used microdialysis to establish an experimental model to characterize mechanisms whereby released substances cause secondary damage in spinal cord injury. We use this model here to characterize damaging effects of the hydroxyl radical (OH') in vivo in the spinal cord. OH'was generatad in vivo by pumping H2O2and FeCI2/EDTA through parallel microdialysis fibers inserted into the spinal cord. These agents mixed in the tissue to produce OH'by Fenton's reaction. Two types of control experiments were also conducted, one administering only 5mM H2O2and the other only 0.5 mMFeCI2/0.82mM EDTA. During administration of these chemicals, electrical conduction was recorded as one test for deterioration. OH'blocked conduction completely in 2.5‐5 h and Fe2+/EDTA partly blocked conduction, but H2O2alone did not cause detectable blockage. Histological examination supported the hypothesis that neurons were killed by OH', as Fe2+/EDTA and H2O2alone did not destroy significant numbers of neurons. OH', H2O2, and Fe2+all caused gradual increases in extracellular amino acid levels. These results are consistent with Fe2+‐catalyzed free radical generation playing a role in tissue damage upon spinal cord injury.
Vitamin E, Ascorbate, Glutathione, Glutathicne Disulfide, and Enzymes of Glutathione Metabolism in Cultures of Chick Astrocytes and Neurons: Evidence that Astrocytes Play an Important Role in Antioxidative Processes in the Brain
Authors: Makar Tapas K. , Nedergaard Maiken , Preuss Axel , Gelbard Alan S. , Perumal Arthur S. , Cooper Arthur J. L.
Published: July 2010

Abstract: GSH, GSSG, vitamin E, and ascorbate were measured in 14‐day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included ‐γ‐glutamylcysteine synthetase, GSH synthetase, γ‐glutamyl cyclotransferase, γ‐glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid‐soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. γ‐Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain. Because enzymes of GSH metabolism are generally well represented in cultured astrocytes and neurons these cells may be ideally suited as probes for manipulating GSH levels in neural tissues in vitro. Cultured astrocytes and neurons should be amenable to the study of the effects of various metabolic insults on the GSH system. Such studies may provide insights into the design of therapeutic strategies to combat oxidative and xenobiotic stresses.
GABAergic Modulation of Striatal Cholinergic Interneurons: An In Vivo Microdialysis Study
Authors: DeBoer P. , Westerink B. H. C.
Published: July 2010

Abstract:Striatal cholinergic interneurons have been shown to receive input from Striatal γ‐aminobutyric acid (GABA)‐containing cell elements. GABA is known to act on two different types of receptors, the GABAAand the GABA6receptor. Using in vivo microdialysis, we have studied the effect of intrastriatal application of the GABAA‐selective compounds muscimol and bicuculline and the GA‐ BAB‐selective compounds baclofen and 2‐hydroxysaclofen, agonists and antagonists, respectively, at GABA receptors, on the output of Striatal acetylcholine (ACh). Intrastriatal infusion of 1 and 10 μmol/L concentrations of the GABAAantagonist bicuculline resulted in a significant increase in Striatal ACh output, whereas infusion of 1 and 10 /μmol/L concentrations of the GABAAagonist muscimol significantly decreased the output of Striatal ACh. Both compounds were ineffective in changing the output of Striatal ACh at lower concentrations. Infusion of concentrations up to 100 μmol/L of the GABAB‐selective antagonist 2‐hydroxy‐saclofen failed to affect Striatal ACh output, whereas infusion of 10 and 100 μmol/L baclofen, but not 0.1 and 1 μmol/L baclofen, significantly decreased the output of Striatal ACh. Thus, agonist‐stimulation of GABAA and GABABreceptors decreases the output of striatal ACh in a dose‐dependent fashion, whereas the GABAergic system appears to inhibit tonically the output of striatal ACh via GABAAreceptors, but not via GABABreceptors. We hypothesize that although GABAAmediated regulation of striatal ACh occurs via GABA receptors on the cholinergic neuron, the GABABmediated effects may be explained by presynaptic inhibition of the glutamatergic input of the striatal cholinergic neuron.
Modulatory Effects of Testosterone on 1‐Methyl‐4‐Phenyl‐ 1, 2, 3, 6‐Tetrahydropyridine‐Induced Neurotoxicity
Authors: Dluzen Dean , Jain Rupesh , Liu Binjun
Published: July 2010

Abstract:In this experiment, we examined the modulatory effects of testosterone on the parkinsonism‐inducing drug 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) in two strains of mice. Orchidectomized male CD‐1 and C57/BI mice were implanted with either empty Silastic capsules or capsules containing testosterone and subsequently treated with MPTP. A small area of the corpus striatum was removed for determination of dopamine (DA) content, whereas the remainder was superfused and used to measure L‐DOPA (5 μM)‐evoked DA release. In animals treated with MPTP, L‐DOPA‐evoked DA release was reduced significantly in CD‐1 mice, but not in C57/BI mice, treated with testosterone. No differences in L‐DOPA‐stimulated DA release between MPTP‐ versus vehicle‐treated mice was observed in either the CD‐1 or C57/BI mice receiving empty Silastic capsules. Corpus striatum DA contents were more severely depleted in the MPTP‐sensitive C57/BI versus the CD‐1 mouse strain irrespective of hormone treatment. These results confirm previous results demonstrating differences in these two mouse strains in response to the neurotoxic effects of MPTP upon corpus striatum DA content. More interestingly, they show an important differential modulatory effect of testosterone upon L‐DOPA‐evoked DA release as a function of MPTP treatment and indicate that testosterone significantly alters the neurotoxic effects of MPTP in the CD‐1 mouse.
Formation, Metabolism, and Action of Hepoxilin A3in the Rat Pineal Gland
Authors: Reynaud Denis , Delton Isabelle , Gharib Abdallah , Sarda Nicole , Lagarde Michel , Pace‐Asciak Cecil R.
Published: July 2010

Abstract: The present study was undertaken to investigate the possible formation of hepoxilin A3 in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 μM) led to the appearance of hepoxilin A3 (HxA3) analyzed as its stable trihydroxy derivative, trioxilin A3 by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA3 is estimated to be 1.43 ± 0.66 ng//μg of protein. This amount is not modified when the tissue is boiled (2.07 ± 0.66 ng/μg of protein). However, the formation of this compound was stimulated to 21.26 ±5.82 ng/μg of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 /μg) resulted in a partial blockade of hepoxilin formation. Using [1‐14C] H×A3, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA3 into trioxilin A3. This hydrolysis was inhibited by 1 μmol/L of 3, 3, 3‐trichloropropene‐1, 2‐oxide. In a separate study, HxA3 in the presence of 3, 3, 3‐trichloropropene‐1, 2‐oxide to block the hydrolysis of HxA3 decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5′‐N‐ethylcarboxamidoadenosine, an A1/A2 adenosine receptor agonist. This effect is stereospecific because the (8S)‐enantiomer is more active in decreasing cyclic AMP production (−88.7%) than the (8R)‐enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA3 in the rat pineal gland.
Preservation of Redox, Polyamine, and Glycine Domains of theN‐Methyl‐D‐Aspartate in Alzheimer's Disease
Authors: Palmer Alan M. , Burns Mark A.
Published: July 2010

Abstract:This study used [3H] dizocilpine ([3H] MK‐801) binding to theN‐methyl‐D‐aspartate (NMDA) receptor to examine redox, polyamine, and glycine modulatory sites in membranes derived from the superior frontal and the superior temporal cortex of patients with Alzheimer's disease. In control subjects the competitive polyamine site antagonist arcaine inhibited [3H] dizocilpine binding in a dose‐dependent fashion and this curve was shifted to the right by the addition of 50 μMspermidine. Arcaine inhibition of binding was more potent in the temporal cortex than in the frontal cortex, in both the absence and presence of 50 μMspermidine. In Alzheimer's disease, arcaine inhibition of [3H] dizocilpine binding (in both the absence and the presence of spermidine) was not different from control in either of the two brain areas examined. The sulfhydryl redox site of the NMDA receptor was assessed using the oxidizing agent 5, 5′‐dithio‐bis(2‐nitrobenzoic acid), which inhibited binding in a dose‐dependent fashion. This inhibition was similar in patients with Alzheimer's disease and control subjects. Glycine‐stimulated [3H] dizocilpine binding was also unaffected in patients with Alzheimer's disease. However, in the temporal cortex there was a significant age‐associated decline in [3H] dizocilpine binding in the presence of 100 μMglutamate (R8=‐0.71) and 100 μMglutamate plus 30 μMglycine (R8=−0.90). There was also an age‐related increase in arcaine IC50(which reflects an age‐related decrease in arcaine affinity) in the frontal cortex, determined both in the absence (R8= 0.83) and the presence (R8= 0.79) of spermidine. These data indicate that the NMDA receptor and its modulatory redox, polyamine, and glycine subsites are intact in patients with Alzheimer's disease and that the modulatory activity of polyamine and glycine sites decline with aging.
Brain Ion and Amino Acid Contents During Edema Development in Hepatic Encephalopathy
Authors: Hilgier Wojciech , Olson James E.
Published: July 2010

Abstract: Brain edema in hepatic encephalopathy has been associated with circulating ammonia that is metabolized to glutamine. We measured alterations in blood chemistry and brain regional specific gravity and ion and amino acid contents in models of simple hyperammonemia and liver failure induced by daily administrations of ammonium acetate (AAc) or thioacetamide (TAA), respectively. Serum and brain ammonia increased to similar levels (200 and 170% of control, respectively) in both experimental groups. Serum transaminase activities increased 10‐fold in animals injected with TAA but were unchanged in animals given AAc injections. In both experimental groups glutamine was elevated in cerebral white matter, cerebral gray matter, and basal ganglia, whereas brain tissue specific gravity decreased in all brain regions, indicating edema formation. In the AAc group, we observed a decrease in glutamate and taurine contents concomitant with the development of brain edema. In these animals, cerebral gray matter specific gravity and taurine contents returned to control levels 24 h after the third AAc injection. TAA‐injected animals demonstrated similar decreases in brain tissue specific gravity, whereas glutamine, glutamate, and taurine contents were all elevated. During hepatic encephalopathy, ammonia‐induced changes in brain amino acid content may contribute to brain edema development.
Affinity Purification of Angiotensin Type 2 Receptors from N1E‐115 cells: Evidence for Agonist‐Induced Formation of Multimeric Complexes
Authors: Siemens Ivo R. , Yee Daniel K. , Reagan Lawrence P. , Fluharty Stever J.
Published: July 2010

Abstract:The murine neuroblastoma N1E‐115 cell line possesses type 1 and type 2 angiotensin II (Angll) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT2‐receptor subtype, whereas the density of the AT1receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐ 1‐propanesulfonate (CHAPS) selectively solubilized AT2receptors from N1E‐115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (Angll) during affinity purification of AT2receptors resulted in the elution of high (110‐kDa) and low (66‐kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar1, lle8‐Angll was used during purification, only the lower 66‐kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2‐receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66‐kDa protein, but unlike that in the presence of Sar1, lle8‐Angll, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1‐receptor antagonist, was completely ineffective in eluting any Angll receptors from the affinity column, further confirming their AT2identity. After agonist elution, the 110‐kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate‐gel electrophoresis was run under reducing conditions. The 110‐kDa and 66‐kDa proteins, but not smaller, affinity‐purified proteins, specifically bound125l‐Angll as determined by covalent cross‐linking of125l‐Angll to the receptors with the homobifunctional cross‐linker disuccinimidyl suberate, or by size exclusion chromatography on a TSK 3000 SW column. Lastly, immunoblot analysis of affinity‐ purified material with antibodies selective for AT2receptors revealed major immunoreactive proteins of 110 kDa and 66 kDa in the presence of an agonist, whereas the same 66‐kDa protein, as well as a smaller (54‐kDa) immunoreactive protein, was detected under reducing conditions. Collectively, these data suggest that CHAPS‐solubilized AT2receptors from N1E‐115 cells may consist of a binding protein of approximately 66 kDa, which in the presence of an agonist readily associates with other smaller proteins to form larger multimeric complexes.
The Regional Distribution ofN‐Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System
Authors: Fuhrman Stefanie , Palkovits Miklos , Cassidy Martha , Neale Joseph H.
Published: July 2010

Abstract:N‐Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate andN‐acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two‐stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate andN‐acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.
Postmortem Stability of Monoamines, Their Metabolites, and Receptor Binding in Rat Brain Regibns
Authors: Kontur P. J. , Al‐Tikriti M. , Innis R. B. , Roth R. H.
Published: July 2010

Abstract:The effects of postmortem delay, time of storage, and freezing, thawing, and refreezing tissue samples were studied in postmortem rat brain using conditions that reflect the handling of postmortem human brain before neurochemical analysis. The levels of monoamines and metabolites in the striatum and cingulate and occipital cortex were measured using alumina extraction and HPLC methods. Binding of raclopride to dopamine D2, SCH‐ 23390 to dopamine D1, ketanserin to serotonin 5‐HT2, 8‐ hydroxy‐2‐(di‐n‐propylamino)tetralin to serotonin 5‐HT1A, and cholecystokinin (CCK)‐8 to CCK‐B sites was measured in tissue homogenates from the striatum or frontoparietal cortex. An 18‐h postmortem delay before dissection and storage resulted in region‐specific changes in monoamine and metabolite levels. Binding to striatal D1and frontoparietal cortex CCK‐B sites was reduced over the course of a 27‐h postmortem delay. Binding to D2and 5‐HT sites was relatively stable. Storage of tissue for up to 8 months also resulted in region‐specific changes in monoamine and metabolite levels. No changes in receptor binding were seen after long‐term storage. Freezing, thawing, and refreezing tissue samples resulted in increased levels of striatal 3, 4‐dihydroxyphenylacetic acid and decreased binding to striatal D2sites. These results demonstrate time‐, temperature‐, and storage‐dependent regional differences in.the stability of monoamines and their metabolites and in binding to various receptor sites. These differences in stability and binding should be accounted for to interpret accurately the effects of neurological disorders on neurotransmitter dynamics in postmortem human brain tissue.
Effects of Gangliosides GM1 and GD1 a on GAP‐43 Phosphorylation and Dephosphorylation in Isolated Growth Cones
Authors: Meiri Karina F. , Beverly Marcia
Published: July 2010

Abstract: Phosphorylation of the nervous system‐specific protein GAP‐43 in growth cones in vivo increases as the growth cones near their targets, at a time when the gangliosides GM1 and GD1a are being accumulated in the growth cone membrane, thus raising the possibility that the gangliosides could modulate GAP‐43 behavior. We used a subcellular fraction of intact isolated growth cones to show that both GM1 and GD1a affected the calcium‐ dependent posttranslational regulation of GAP‐43 in several similar ways. Both gangliosides induced rapid incorporation of phosphate into GAP‐43; however, the induction was undetectable with our antibody 2G12 that is specific for kinase C‐phosphorylated GAP‐43. Furthermore, neither ganglioside stimulated kinase C activity in isolated growth cones, suggesting that the rapid Phosphorylation may not be on Ser41, the kinase C site. However, both gangliosides did induce a slower accumulation of GAP‐43 phosphorylated on Ser41, apparently by inhibiting a phosphatase. Finally, calcium‐dependent proteolysis of GAP‐43 was also stimulated by both GM1 and GD1a. In contrast, GD1a, but not GM1, caused the redistribution of GAP‐43 into the isolated growth cone cytoskeleton. The results demonstrate that both gangliosides can modulate the calcium‐dependent regulation of GAP‐43.
Evidence that the Major Membrane Lipids, Except Cholesterol, Are Made in Axons of Cultured Rat Sympathetic Neurons
Authors: Vance Jean E. , Pan Debao , Campenot Robert B. , Bussiere Miguel , Vance Dennis E.
Published: July 2010

Abstract:Membrane lipids and proteins required for axonal growth and regeneration are generally believed to be synthesized in the cell bodies of neurons and transported into the axons. However, we have demonstrated recently that, in cultured rat sympathetic neurons, axons themselves have the capacity to synthesize phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine. In these experiments, we employed a compartment model of neuron culture in which pure axons grow in a fluid environment separate from that containing the cell bodies. In the present study, we again used compartmented cultures to confirm and extend the previous results. We have shown that three enzymes of phosphatidylcholine biosynthesis via the CDP‐choline pathway are present in axons. We have also shown that the rate‐limiting step in the biosynthesis of phosphatidylcholine by this route in neurons, and locally in axons, is catalyzed by the enzyme CTP:phosphocholine cytidylyltransferase. The biosynthesis of other membrane lipids, such as phosphatidylserine, phosphatidylethanolamine derived by decarboxylation of phosphatidylserine, phosphatidylinositol, and fatty acids, also occurs in axons. However, the methylation pathway for the conversion of phosphatidylethanolamine into phosphatidylcholine appears to be a quantitatively insignificant route for phosphatidylcholine synthesis in neurons. Moreover, our data provided no evidence for the biosynthesis of another important membrane lipid, cholesterol, in axons.
NMR Identification of the Formic Acid‐Modified Residue in Alzheimer's Amyloid Protein
Authors: Klunk William E. , Xu Chong‐Jun , Pettegrew Jay W.
Published: July 2010

Abstract: The β/A4‐amyloid protein (β/A4) and many synthetic fragments of this protein have proved to be very difficult to solubilize, leading to the use of relatively harsh chemical methods, most notably, formic acid. This treatment has previously been shown to cause a covalent modification of this peptide. In this study, one‐ and two‐dimensional NMR techniques are used to show that the nature of this covalent modification is formation of a formate ester to a serine residue. This finding is consistent with our previously reported kinetic studies of formic acid‐induced modification ofβ/A4 and further illustrates the potential danger of solubilizing fragments of β/A4 in formic acid. Alternative methods of solubilization are discussed.
Role of Mg‐ATP in Norepinephrine Biosynthesis in Intact Chromaffin Granules
Authors: Dhariwal Kuldeep R. , Shirvan Mitchell H. , Levine Mark
Published: July 2010

Abstract: Dopamine β‐mdriooxygenase converts dopamine to norepinephrine in intact chromaffin granules using intragranular ascorbic acid as a cosubstrate. Mg‐ATP with external ascorbic acid is required for maximal norepinephrine biosynthesis. Mechanisms to explain these requirements were investigated specifically using intact granules. The effect of Mg‐ATP was independent of membrane potential (ΔΨ) because norepinephrine biosynthesis was unchanged whether ΔΨ was positive or collapsed. Furthermore, the effect of Mg‐ATP was independent of absolute intragranular and extragranular pH as well as the pH difference across the chromaffin granule membrane (ΔpH). Nevertheless, norepinephrine biosynthesis was inhibited by N‐ethylmaleimide, 4‐chloro‐7‐nitrobenzofurazane, and N, N‐dicyclohexylcarbodiimide, specific inhibitors of the secretory vesicle ATPase that may directly affect proton pumping. Biosynthesis occurred normally with other ATPase inhibitors that do not inhibit the ATPase in secretory vesicles. The data indicate that the effect of Mg‐ATP with ascorbic acid is mediated by the granule membrane ATPase but independent of maintaining ΔΨ and ApH. An explanation of these findings is that Mg‐ATP, via the granule ATPase, may change the rate at which protons or dopamine are made available to dopamine β‐monooxygenase.
Compared Binding Properties of125I‐Labeled Analogues of Neurotensin and Neuromedin N in Rat and Mouse Brain
Authors: Gaudriault Georges , Zsürger Nicole , Vincent Jean‐Pierre
Published: July 2010

Abstract:Neurotensin and neuromedin N are two structurally related peptides that are synthesized by a common precursor. The purpose of the present work was to characterize neuromedin N receptors in rat and mouse brain and to compare these receptors with those of neurotensin. A radiolabeled analogue of neuromedin N has been prepared by acylation of the N‐terminal amino group of the peptide with the125I‐labeled Bolton‐Hunter reagent. This125I‐labeled derivative of neuromedin N bound to newborn mouse brain homogenate with high affinity (Kd= 0.5 nM). Cross‐competition experiments between radiolabeled and unlabeled neurotensin and neuromedin N indicated that each peptide was able to displace completely and specifically the other peptide from its interaction with its receptor. Independently of the radioligand used, the affinity of neurotensin was always better than that of neuromedin N. Quantitative radioautographic studies demonstrated that the ratio of labeling intensities obtained with125I‐labeled analogues of neurotensin and neuromedin N remained constant in all the brain areas. Our results do not support the existence of a specific neuromedin N receptor in rat and mouse brain and can be explained by the presence of a common receptor for both peptides.
Rapid Communication: Oxidative Stress Induces Apoptosis in Embryonic Cortical Neurons
Authors: Ratan Rajiv R. , Murphy Timothy H. , Baraban Jay M.
Published: July 2010

Abstract: Glutamate‐induced glutathione depletion in immature embryonic cortical neurons has been shown to lead to oxidative stress and cell death. We have used this in vitro model to investigate the mechanism(s) by which free radicals induce neuronal degeneration. We find that glutathione depletion leads to hypercondensation and fragmentation of chromatin into spherical or irregular shapes, a morphologic signature of apoptosis. These morphologic changes are accompanied by laddering of DNA into multiple oligonucleosomal fragments and can be prevented by the antioxidants idebenone and butylated hydroxyanisole. Cell death induced by glutathione depletion can also be prevented by inhibitors of macromolecular synthesis. Taken together, these observations suggest that oxidative stress can induce apoptosis in neurons.
Rapid Communication: Attenuation of Methamphetamine‐Induced Neurotoxicity in Copper/Zinc Superoxide Dismutase Transgenic Mice
Authors: Cadet Jean Lud , Sheng Peilin , All Syed , Rothman Richard , Carlson Elaine , Epstein Charles
Published: July 2010

Abstract:Administration of methamphetamine (METH) to rats and nonhuman primates causes loss of terminals in the nigrostriatal dopaminergic system. The mechanism by which METH causes its neurotoxicity is not known. To evaluate further the role of oxyradicals in METH‐induced neurotoxicity, we have tested its effects in CuZn superoxide dismutase (SOD) transgenic (Tg) mice, which express the human CuZnSOD gene. In non‐Tg mice, acute METH administration causes significant decreases in levels of dopamine (DA) and 3, 4‐dihydroxyphenylacetic acid (DOPAC) in the striata and cortices of non‐Tg mice. In contrast, there were no significant decreases in cortical or striatal DA in the SOD‐Tg mice. The effects of METH on DOPAC were also attenuated in both structures of these SOD‐Tg mice. Chronic METH administration caused decreases in levels of striatal DA and DOPAC in the non‐ Tg mice, whereas the SOD‐Tg mice were not affected. These results suggest that METH‐induced dopaminergic toxicity in mice may be secondary to increased production of reactive oxygen species such as the superoxide radical.
Rapid Communication: A cis‐Acting Element, Tα‐1, in the Upstream Region of Rod α‐Transducin Gene that Binds a Developmentally Regulated Retina‐Specific Nuclear Factor
Authors: Ahmad Iqbal , Yu Xiu , Barnstable Colin J.
Published: July 2010

Abstract: The G protein transducin (T) is an integral component of the signal transduction pathway in photoreceptors. We have identified a cis‐acting element, Ta‐1, in the upstream region of the mouse rod a‐T (Trα) gene that may be important for tissue‐specific expression. Tα‐1 binds a retina‐specific nuclear factor of apparent molecular mass of 90 kDa. Binding to the Tα‐1 site is developmentally regulated and peaks between postnatal days 6 and 9. This corresponds to the time of rod photoreceptor maturation and the rise in Trα gene expression. The sequence of Tα‐1 shows homology with RET‐1, a cis‐acting element in the proximal promoter of opsin gene that binds a distinct retina‐specific factor. Tα‐1 and RET‐1 sequences may have been derived from a prototype Tα‐1/RET‐1 sequence, evolved to confer photoreceptor specificity on retina‐specific genes.
Rapid Communication: The Role of P‐Type Calcium Channels in the Depolarization‐Induced Activation of Nitric Oxide Synthase in Frontal Cortex
Authors: Alagarsamy Sudarkodi , Lonart Gyorgy , Johnson Kenneth M.
Published: July 2010

Abstract:In this study we demonstrate that 50 mRS K+stimulates the conversion of L‐[3H] arginine to L‐[3H] citrulline and that this effect is blocked by 10 μMAT‐nitro‐l‐arginine, a nitric oxide synthase inhibitor, and Ca2+‐free conditions. Amiloride (1 mM) and low Na+conditions were used to test the possible involvement of the Na+‐Ca2+exchanger. These treatments were without effect. The calcium channel blockers 10 mRS Mg2+, 100 μMCd2+, and 10 mRS Co2+also blocked the K+response, suggesting the involvement of voltage‐dependent calcium channels (VDCCs). The specific VDCC involved seems to be the P type, as funnel‐web spider toxin blocked the response whereas 200 μMNi2+, 10 μMnifedipine, and 100 nMω‐conotoxin did not.
Rapid Communication: Neuropeptide Expression and Processing as Revealed by Direct Matrix‐Assisted Laser Desorption Ionization Mass Spectrometry of Single Neurons
Authors: Jimenez C. R. , Veelen P. A. van , Li K. W. , Wildering W. C. , Geraerts W. P. M. , Tjaden U. R. , Greef J. van der
Published: July 2010

Abstract: Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI‐MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI‐ MS study of direct peptide fingerprinting in the single neuron these experients demonstrate that MALDI‐MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single‐cell studies in vertebrates, including humans.
Production and Characterization of Recombinant Rat Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 from Insect Cells
Authors: Negro Alessandro , Tavella Adriana , Grandi Claudio , Skaper Stephen D.
Published: July 2010

Abstract: Rat brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) were engineered for expression in a baculovirus‐infected Spodoptera frugiperda insect cell system. The BDNF and NT‐3 from the culture supernatants were purified by ion‐exchange and reverse‐phase chromatography to apparent homogeneity. The purification procedure yielded ∼2 mg of pure rat BDNF or NT‐3 per liter of culture supernatant. A single N‐terminus only was found for either secreted molecule and was analogous to that predicted from the corresponding cDNA sequence. The recombinant neurotrophins obtained were also homogeneous with regard to molecular weight and amino acid sequence. In their native conformation, the insect cell‐produced rat BDNF and NT‐3 molecules were homodimers consisting of 119 amino acid polypeptide chains. Thus, although the genes transfected into the S. frugiperda cells coded for proBDNF or proNT‐3, the BDNF and NT‐3 recovered after purification were >95% fully processed, mature protein. Mature recombinant rat BDNF and NT‐3 were found not to be significantly glycosylated. Pure, recombinant rat BDNF and NT‐3 promoted the survival of embryonic dorsal root ganglion neurons in the low picomolar range. Because recombinant rat BDNF and NT‐3 can be obtained in large quantities, purified to near homogeneity, and are identical in amino acid sequence to the corresponding human proteins, they are suitable for evaluation in animal models.
Astroglial Gap Junction Communication Is Increased by Treatment with Either Glutamate or High K+ Concentration
Authors: Kristian Enkvist M. O. , McCarthy Ken D.
Published: July 2010

Abstract: Astroglia are extensively coupled by gap junctions and form a functional syncytium. Astroglial gap junctions are thought to be involved in the spatial buffering of K+ in vivo and in the Ca2+ waves seen on glutamate receptor activation. The conductivity of gap junctions is regulated by several second messengers, with up‐regulation by cyclic AMP and down‐regulation through activation of protein kinase C, decreases in intracellular pH, or increases in the free cytosolic Ca2+ concentration. The results presented here indicate that dye coupling of astroglia is significantly up‐regulated by membrane depolarization, both by increases in the extracellular K+ concentration and directly by ionophores. Furthermore, glutamate, kainate, and quisqualate, which depolarize astroglial cells through activation of ionotropic receptors, also increase dye coupling in astroglia. The effect of kainate and quisqualate was reversed by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, an inhibitor of the ionotropic glutamate receptor. A dose‐dependent decrease in dye coupling was seen when the cells were injected with increasing concentrations of Ca2+. However, if the cells were simultaneously depolarized, the inhibitory effect of Ca2+ on gap junctional conductance was reversed. Significant increases over basal coupling was attained when the cells were injected with Ca2+ if they were treated with kainate or K+. These data suggest that ligands that depolarize astroglia enhance gap junction communication between astroglia and that this enhancement may be important in maintaining communication between astroglia in the face of elevated Ca2+ levels.
Guinea Pig Histamine H1 Receptor. II. Stable Expression in Chinese Hamster Ovary Cells Reveals the Interaction with Three Major Signal Transduction Pathways
Authors: Leurs R. , Traiffort E. , Arrang J. M. , Tardivel‐Lacombe J. , Ruat M. , Schwartz J.‐C.
Published: July 2010

Abstract: A cDNA encoding a guinea pig histamine H1 receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H1), the H1 receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a Gi/Go protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca2+ and of protein kinase C activation by phorbol 12‐myristate 13‐acetate (PMA). Histamine induced, in a PTX‐ and PMA‐insensitive manner, a biphasic increase in the intracellular Ca2+ level of which only the second sustained phase was dependent on the extracellular Ca2+ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX‐insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca2+. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca2+‐ and PMA‐sensitive manner, probably through the activation of a cytosolic phospholipase A2, which partly involves coupling to a PTX‐sensitive G protein. In comparison, in HeLa cells endowed with a native H1 receptor, the histamine‐induced arachidonic acid release was also Ca2+‐ and PMA‐sensitive, but totally PTX‐insensitive. Finally, in CHO(H1) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca2+, and PMA. These various observations show that stimulation of a single receptor subtype, the guinea pig H1 receptor, can trigger four major intracellular signals through coupling to several G proteins that are variously modulated by extracellular Ca2+ and protein kinase C activation.
Characterization of Serotonin Transporter in Goldfish Retina by the Binding of [3H]Paroxetine and the Uptake of [3H]Serotonin: Modulation by Light
Authors: Lima L. , Schmeer C.
Published: July 2010

Abstract: The serotonin (5‐HT) uptake system of goldfish retina was evaluated by the binding of [3H]paroxetine to membrane preparations and the uptake of [3H]5‐HT into isolated cells from goldfish retina. The order of potency of inhibitors of [3H]paroxetine binding was imipramine > 5‐methoxy‐N,N‐dimethyltryptamine > desipramine > fluoxetine > citalopram > 5‐HT. The saturation experiments indicated a high‐affinity binding site, and positive cooperativity with Hill coefficient higher than unity. The association reached equilibrium at about one hour of incubation and was efficiently displaced by imipramine. The equilibrium dissociation constants calculated by the antilog of the log concentration of ligand giving 50% of occupation, and by the ratio of dissociation/association constants, were similar: 5.84 and 2.34 nM, respectively. The binding was not significantly reduced by decreasing the temperature of incubation and was sodium dependent. The lesion with 5,7‐dihydroxytryptamine reduced the binding to 60%. The uptake of [3H]5‐HT into isolated cells also showed positive cooperativity. The order of potency of inhibitors was similar to the one obtained for the binding of [3H]paroxetine. Darkness increased the uptake of 5‐HT. The allosteric regulation of the 5‐HT transporter and the modulation by light could be related to the physiological role of the monoamine, as a neurotransmitter and as a precursor of melatonin synthesis in the retina.