bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysophosphatidic Acid‐Induced Neurite Retraction in PC12 Cells: Neurite‐Protective Effects of Cyclic AMP Signaling
Authors: Tigyi Gabor , Fischer David J. , Sebök Ágnes , Marshall Frakeetta , Dyer David L. , Miledi Ricardo
Published: July 2010

Abstract: Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric Gi proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl‐cyclic AMP prevented neurite retraction. The neurite‐protective effect of forskolin was blocked by Rp‐adenosine 3′,5′‐phosphorothioate. Forskolin and dibutyryl‐cyclic AMP both failed to protect neurites in A126‐1B2 and 123.7 cells, which lack cyclic AMP‐activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP‐activated protein kinase‐dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP‐ribosylation of Rho by the Clostridium botulinum C‐3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA‐induced neurite retraction. LPA activates Gq‐ and Gi‐linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor‐activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.
Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats
Authors: McKinzie Sanna L. , Hammond Donna L. , Grabau Curtis , Tyce Gertrude M.
Published: July 2010

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 mM K+. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10−5M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.
Synthetic Analogues of Conantokin‐G: NMDA Antagonists Acting Through a Novel Polyamine‐Coupled Site
Authors: Zhou Li‐Ming , Szendrei Gyorgyi I. , Fossom Linda H. , Maccecchini Maria‐Luisa , Skolnick Phil , Otvos Laszlo
Published: July 2010

Abstract: Conantokin‐G (con‐G) is a 17‐amino‐acid polypeptide that acts as an N‐methyl‐d‐aspartate (NMDA) antagonist. This action has been attributed to a specific but noncompetitive inhibition of the positive modulatory effects of polyamines at NMDA receptors. Con‐G possesses several unusual structural features, including five γ‐carboxyglutamate (Gla) residues and a high degree of helicity in aqueous media. Previous structure‐activity studies indicated that one or more Gla residues are necessary for NMDA antagonist activity. Con‐G analogues were synthesized with alanine (Ala), serine (Ser), and phosphoserine substituted for Gla to assess the contribution of individual Gla residues to biological activity and secondary structure. Replacement of Gla in positions 3 and 4 resulted in polypeptides with markedly reduced and no NMDA antagonist actions, respectively. In contrast, Gla residues in positions 7, 10, and 14 are not required for NMDA antagonist actions because the potencies of con‐G analogues containing Ser7, Ser10, Ala14, and Ser14 to inhibit spermine‐stimulated [3H]MK‐801 binding are similar to the parent peptide. Moreover, the Ala7 derivative of con‐G was about fourfold more potent than the parent peptide both as an inhibitor of spermine‐stimulated increases in [3H]MK‐801 binding (IC50 of ∼45 nM) and in reducing NMDA‐stimulated increases in cyclic GMP levels (IC50 of ∼77 nM) in cerebellar granule cell cultures. Although con‐G and its analogues assumed mixtures of 310 and α‐helices, no clear‐cut relationship was evinced between the NMDA antagonist properties of these peptides and the degree of helicity they assumed in aqueous solutions. Together with the inability of con‐G to affect 5,7‐dichloro[3H]kynurenic acid, [3H]CGP‐39653, and [3H]ifenprodil binding, these data are consistent with the hypothesis that this polypeptide acts at a unique, polyamine‐associated site on NMDA receptors.
Ceramide Induces Apoptosis in Cultured Mesencephalic Neurons
Authors: Brugg Bernard , Michel Patrick P. , Agid Yves , Ruberg Merle
Published: July 2010

Abstract: The death of dopaminergic and other neurons in primary cultures of the mesencephalon could be induced by treatment with ceramide, as in lymphocytes where it mediates activation by the cytokines tumor necrosis factor‐α and interleukin‐1β of a novel sphingomyelin‐dependent signaling pathway leading to apoptosis. The morphological hallmarks of this form of cell death—bleb formation, cell body shrinkage, nuclear chromatin condensation, and fragmentation—were observed in degenerating neurons. Internucleosomal DNA degradation could also be evidenced by gel electrophoresis. The C2 and C6 analogues as well as native ceramide, administered in a dodecane suspension, had a similar effect, whereas the closely related C2‐dihydroceramide, which lacks the 4–5 trans double bond in the sphingosine chain, failed to induce apoptosis. Neuronal death could be delayed by serum factors, dibutyryl cyclic AMP, and the protein synthesis inhibitor cycloheximide.
Mechanisms of Sodium Transport at the Blood‐Brain Barrier Studied with In Situ Perfusion of Rat Brain
Authors: Ennis Steven R. , Ren Xiao‐dan , Betz A. Lorris
Published: July 2010

Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital‐anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis‐Menten saturation kinetics with a Vmax of 50.1 nmol/g/min and a Km of 17.7 mM in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood‐brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µM), an inhibitor of Na+/H+ exchange, reduced sodium transport by 28%, whereas phenamil (25 µM), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µM) and hydrochlorothiazide (1.5 mM), inhibitors of Na+‐K+‐2Cl−/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 mM), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate Pco2 of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na+/H+ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl−/HCO3− exchange, lead to net NaCl secretion and obligate water transport into the brain.
Casein Kinase II Phosphorylates the Neural Cell Adhesion Molecule L1
Authors: Wong Eric V. , Schaefer Andrew W. , Landreth Gary , Lemmon Vance
Published: July 2010

Abstract: L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane‐spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144–1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173–1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177–1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser1,181. This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction.
Influence of C Terminus on Monoamine Oxidase A and B Catalytic Activity
Authors: Chen Kevin , Wu Hsiu‐Fei , Shih Jean Chen
Published: July 2010

Abstract: Monoamine oxidase (MAO) A and B play important roles in the metabolism of neurotransmitters and dietary amines. The domains important for enzyme specificities were studied by construction of chimeric MAOA/B enzymes. Exchange of the N‐terminal 45 amino acids of MAOA with the N‐terminal 36 residues of MAOB (chimeric enzymes B36A and A45B) resulted in the same substrate and inhibitor sensitivities as the wild‐type MAOA or B. Thus, the N terminus may not be responsible for MAOA or B enzyme specificities. When MAOB C‐terminal residues 393–520 were replaced with MAOA C‐terminal residues 402–527 (chimeric B393A) catalytic activity was not detectable. Chimeric B393A consists of eight residues with different charges, three less proline residues (458, 476, and 490), and one additional proline at 518 compared with wild‐type MAOB. These differences may have induced conformational changes and affected MAOB catalytic activity. Thus, the C terminus of MAOB is critical for maintaining MAOB in an active form. It is interesting that when the C terminus of MAOA was switched with MAOB (chimeric A402B), little effect was observed on MAOA catalytic activity. This new information is valuable for further studies of the structure and function relationship of this important enzyme.
Increased Neostriatal Tyrosine Hydroxylation During Stress: Role of Extracellular Dopamine and Excitatory Amino Acids
Authors: Castro Sandra L. , Sved Alan F. , Zigmond Michael J.
Published: July 2010

Abstract: We examined the regulation of neostriatal tyrosine hydroxylation during acute stress, testing the hypothesis that excitatory amino acids (EAAs) contribute to the stress‐evoked increase in dopamine (DA) synthesis. Dialysis probes implanted into neostriatum permitted delivery of drugs and sampling of extracellular fluid. Rats were exposed to 30 min of intermittent tail shock during infusion of an inhibitor of aromatic amino acid decarboxylase (AAAD), NSD‐1015 (100 µM), and DOPA was measured in the dialysate. Tail shock was applied beginning either 15 min after the onset of NSD‐1015 treatment (the initial rate of DOPA accumulation) or 75 min after the onset of treatment (when DOPA had approached steady state). Tail shock increased the steady‐state levels of extracellular DOPA in neostriatum (+40%). However, there was no change in the initial rate of DOPA accumulation unless animals also received the D2 receptor antagonist eticlopride (50 nM), in which case an increase was observed (+228%). The impact of tail shock on the steady‐state level of DOPA was attenuated by the D2 agonist quinpirole (100 µM), or by 2‐amino‐5‐phosphonovalerate (APV) (100 µM) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) (100 µM), EAA antagonists acting at NMDA or d,l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐4‐propionate (AMPA) receptors, respectively. These data suggest that acute stress normally has little effect on tyrosine hydroxylation in neostriatum due to the inhibitory influence of DA in the extracellular fluid. However, when that influence is absent (e.g., during extended inhibition of DOPA decarboxylation or blockade of DA receptors), stress increases tyrosine hydroxylation via EAAs acting on NMDA and AMPA receptors. Thus, EAAs released from corticostriatal projections may stimulate DA synthesis and thereby restore dopaminergic activity under conditions in which the availability of DA for release has been compromised.
Serotonin Metabolism and Release in Frontal Cortex of Rats on a Vitamin E‐Deficient Diet
Authors: Heslop K. E. , Goss‐Sampson M. A. , Muller D. P. R. , Curzon G.
Published: July 2010

Abstract: Rats were fed a control or vitamin E (all‐rac‐α‐tocopheryl acetate)‐deficient diet for 3 or 12 weeks. Serotonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA), tryptophan, and α‐tocopherol concentrations were determined in the frontal cortex using HPLC. α‐Tocopherol concentrations fell significantly to 27% of control values at 12 weeks. Tissue 5‐HT, 5‐HIAA, and tryptophan concentrations were not significantly altered by the vitamin E‐deficient diet at either time point. In vivo microdialysis revealed normal basal and K+‐stimulated concentrations of 5‐HT and 5‐HIAA, but extracellular concentrations of tryptophan were significantly decreased after 3 weeks on the vitamin E‐deficient diet, which resulted in an increase in the tissue/extracellular ratio and suggested a change in compartmentation. However, after 12 weeks on the deficient diet these values had returned to normal. Results in general indicate that a prolonged and substantial depletion of brain vitamin E can occur without major disturbance of serotonergic function.
Presynaptic NMDA Receptors Display Physiological Characteristics of Homomeric Complexes of NR1 Subunits that Contain the Exon 5 Insert in the N‐Terminal Domain
Authors: Wang James K. T. , Thukral Vijay
Published: July 2010

Abstract: The subunit composition of the N‐methyl‐d‐aspartate (NMDA) glutamate receptor affects both its channel activity and its sensitivity to modulation by a wide variety of substances. Expression studies in oocytes and physiological studies in neurons indicate that endogenous postsynaptic NMDA receptors are heterooligomeric complexes of NR1 and NR2 subunits. To deduce the subunit composition of the presynaptic NMDA receptor on noradrenergic nerve terminals, we examined the modulation of NMDA‐evoked norepinephrine (NE) release from hippocampal synaptosomes. At high glycine concentrations, the NMDA‐evoked release was not potentiated by reducing reagents, low micromolar Zn2+ or Ni2+, polyamines, or 100 µM histamine. It was also not inhibited by oxidizing agents or physiological concentrations of protons but was inhibited by high micromolar Co2+, Zn2+, and Ni2+, but not Fe3+, by high micromolar ifenprodil, and by 1 mM histamine. At low glycine concentrations, it was potentiated by spermine. These characteristics are similar to those displayed by homooligomeric complexes of NR1 subunits that contain in the N‐terminal domain the 21‐amino‐acid insert encoded by exon 5. These data provide physiological evidence that some endogenous NMDA receptor complexes may contain only the NR1 (+ exon 5) subunits.
Role of PC2 in Proenkephalin Processing: Antisense and Overexpression Studies
Authors: Johanning Karla , Mathis John P. , Lindberg Iris
Published: July 2010

Abstract: The contribution of the prohormone‐processing enzyme PC2 to the proteolytic maturation of proenkephalin was examined in three sets of studies. In the first, the processing of this precursor was compared in PC2‐rich (Rin5f) and PC2‐lacking (AtT‐20) cell lines expressing proenkephalin by virtue of stable transfection. These studies showed that the time frame for processing of this precursor is cell line specific, with AtT‐20 cells processing proenkephalin to peptide B much more rapidly than Rin cells. However, the latter cell line processed proenkephalin much more extensively, i.e., produced a greater proportion of the penta‐ to octapeptide enkephalins. The involvement of PC2 in these later processing events was analyzed by examining the processing of proenkephalin in PC2‐overexpressing AtT‐20 cell lines. These experiments yielded a processing profile similar to that observed for Rin cells, although the time frame of initial processing was similar to that found in AtT‐20 cells. To confirm the physiological involvement of proenkephalin in the production of the small opioid peptides, we generated a Rin cell line in which the production of PC2 was impaired due to stable expression of antisense mRNA to this enzyme. These experiments provided conclusive evidence that the generation of Met‐enkephalin‐Arg‐Phe and Met‐enkephalin‐Arg‐Gly‐Leu, but not the larger enkephalin‐containing peptides, is mediated by PC2. Taken together, our data support the idea that PC2 is physiologically capable of mediating only the later processing steps of neuropeptide precursors. PC2 thus appears to be the primary enzyme responsible for the generation of bioactive opioid peptide species from proenkephalin.
Identification of Gln313 and Pro327 as Residues Critical for Substrate Inhibition in Tyrosine Hydroxylase
Authors: Quinsey Noelene S. , Lenaghan Catherine M. , Dickson Phillip W.
Published: July 2010

Abstract: Rat tyrosine hydroxylase was expressed in Escherichia coli. High‐level expression was obtained after incubation at 27°C for 18 h. The smallest fragment of tyrosine hydroxylase that gave a soluble active molecule was from Leu188 to Phe456. This fragment corresponds directly to the section of phenylalanine hydroxylase that had previously been shown to be this enzyme's catalytic core region. It has been shown that Glu286 plays a critical role in pterin function in phenylalanine hydroxylase. The corresponding residue in tyrosine hydroxylase (Glu332) has no significant role in pterin function. Substitution of a leucine for a proline at position 327 in tyrosine hydroxylase produces a molecule with a Km for tetrahydrobiopterin 20‐fold higher than that of the wild‐type molecule, whereas the same substitution at the corresponding residue in phenylalanine hydroxylase (Pro281) has no effect on the kinetic constant for the cofactor. This suggests that corresponding residues in phenylalanine hydroxylase and tyrosine hydroxylase can have different roles in pterin function. Substitution of a leucine for a proline at position 281 in phenylalanine hydroxylase increases the Km for phenylalanine >20‐fold over that of the wild‐type. Substitution of leucine or alanine for Pro327 or a glutamic acid for Gln313 in tyrosine hydroxylase eliminates the substrate inhibition shown by wild‐type tyrosine hydroxylase.
Low Levels of mRNA for Dopamine D4 Receptor in Human Cerebral Cortex and Striatum
Authors: Matsumoto Mitsuyuki , Hidaka Kazuyuki , Tada Shoko , Tasaki Yoshikazu , Yamaguchi Tokio
Published: July 2010

Abstract: Significant densities of mRNA for the dopamine D4 receptor in cerebral cortex, particularly in frontal lobe, have been reported in rats and monkeys, supporting the D4 hypothesis in the pathology of schizophrenia. Using northern blot analysis and the competitive reverse transcription‐polymerase chain reaction (RT‐PCR) method, we determined the relative levels of D4 mRNA in human brain regions to clarify whether the cortical level is also higher in humans. Northern blot analysis revealed an unexpected profile of D4 mRNA in the brain. The detected mRNA size, 1.5 kb, was quite different from the 5.3 kb reported in human neuroblastoma SK‐N‐MC cells. Higher levels of D4 mRNA were detected not only in the mesolimbic system but also in the corpus callosum, spinal cord, medulla, and subthalamic nucleus. It was surprising that in the cerebral cortex regions as well as the striatum, D4 mRNA was hardly detected. The competitive RT‐PCR revealed these relative densities to be at least three orders of magnitude lower than that of the striatal D2 receptor. Our results demonstrate a remarkable difference in cortical D4 mRNA density in humans compared with that in rats and monkeys. Furthermore, the mRNA distribution suggests that the higher density of D4‐like binding sites reported recently in normal human striatum is not due to the D4 receptor.
Regulation of the Kynurenine Metabolic Pathway by Interferon‐γ in Murine Cloned Macrophages and Microglial Cells
Authors: Alberati‐Giani Daniela , Ricciardi‐Castagnoli Paola , Köhler C. , Cesura Andrea M.
Published: July 2010

Abstract: Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon‐γ (IFN‐γ) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN‐γ induced the expression of indoleamine 2,3‐dioxygenase (IDO) activity. Whereas tumor necrosis factor‐α did not affect enzyme induction by IFN‐γ, lipopolysaccharide modulated IDO activity differently in the two IFN‐γ‐activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3‐hydroxylase, and 3‐hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3‐hydroxylase activity was stimulated by IFN‐γ. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN‐γ markedly stimulated the activity of this enzyme only in MT2 cells. IFN‐γ‐treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3‐hydroxyanthranilic acid quinolinic acids from l‐[5‐3H]tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.
Differentiation of NG108‐15 Neuroblastoma Cells by Serum Starvation or Dimethyl Sulfoxide Results in Marked Differences in Angiotensin II Receptor Subtype Expression
Authors: Seidman K. J. N. , Barsuk J. H. , Johnson R. F. , Weyhenmeyer J. A.
Published: July 2010

Abstract: Differentiation of NG108‐15 neuroblastoma cells following exposure to either 1.5% dimethyl sulfoxide (DMSO)/0.5% fetal bovine serum (FBS) or serum starvation resulted in significant differences in angiotensin (AT) receptor levels and the AT1/AT2 receptor ratio. When NG108 cells were differentiated for 4 days with DMSO/low serum, the number of AT binding sites increased 30‐fold compared with the binding levels on undifferentiated (blast) cells. However, cells differentiated by serum starvation for 4 or 14 days resulted in only a modest 2.5‐ and fivefold increase in AT receptor levels, respectively, over the levels seen with the undifferentiated cells. KD values for all treatment conditions were not significantly different (0.71 ± 0.11 nM, p = 0.06). Using the AT1 and AT2 isoform‐specific receptor antagonists losartan and PD123319, the relative numbers of AT receptor subtypes on undifferentiated and differentiated cells were determined by competitive inhibition against 125I‐[Sar1,Ile8]‐angiotensin II (sarile). A majority of the AT receptors on undifferentiated NG108 cells were the AT1 subtype (AT1/AT2 receptor ratio of 8:3). Differentiation by serum starvation and DMSO/low serum treatment resulted in fivefold and 30‐fold increases in AT receptor levels, respectively, compared with the levels seen with the undifferentiated cells. Although serum starvation increased the total number of AT1 and AT2 receptors, it did not significantly alter the AT1/AT2 receptor ratio. In contrast, differentiation with DMSO/low serum both increased the total number of AT1 and AT2 receptors and reversed the AT1/AT2 receptor ratio (1:3). The increase in AT receptors following differentiation with DMSO/low serum for 4 days was largely accounted for by an 80‐fold increase in the AT2 receptor level. Previous studies by Tallant et al. (1991) and Bryson et al. (1992) reported increased AT2 receptor expression following neuroblastoma differentiation with dibutyryl cyclic AMP and DMSO/low serum, respectively, and suggested a role for the AT2 receptor in neuronal differentiation. In the present study, we have extended these earlier observations by demonstrating that the method of differentiation significantly affects both the AT receptor level and the ratio of AT1 to AT2 receptor expression. Finally, our findings indicate that the AT2 receptor is expressed as a consequence of neuronal maturation and does not mediate morphological differentiation.
Cholera Toxin Induces Cyclic AMP‐Independent Down‐Regulation of Gsα and Sensitization of α2‐Autoreceptors in Chick Sympathetic Neurons
Authors: Boehm Stefan , Huck Sigismund , Motejlek Andrea , Drobny Helmut , Singer Ernst A. , Freissmuth Michael
Published: July 2010

Abstract: The role of the stimulatory GTP‐binding protein (GS) in the α2‐autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The α2‐adrenoceptor agonist UK 14,304 caused a concentration‐dependent reduction of electrically evoked [3H]noradrenaline release with half‐maximal effects at 14.0 ± 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half‐maximal concentration was lowered to 3.2 ± 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 µM forskolin or 100 µM 8‐bromo‐cyclic AMP, neither the half‐maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8‐bromo‐cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8‐bromo‐cyclic AMP, induced a translocation of α‐subunits of Gs (Gsα) from particulate to soluble fractions and led ultimately to a complete loss of Gsα from the neurons. In contrast, no effect was seen on the distribution of either α‐subunits of Gi‐ or Go‐type G proteins or of β‐subunits. These results indicate that cholera toxin causes a selective, cyclic AMP‐independent down‐regulation of Gsα. This down‐regulation of Gsα is associated with the sensitization of α2‐autoreceptors.
MAP2a, an Alternatively Spliced Variant of Microtubule‐Associated Protein 2
Authors: Chung Wook Joon , Kindler Stefan , Seidenbecher Constanze , Garner Craig C.
Published: July 2010

Abstract: MAP2, a dendritically localized microtubule‐associated protein (MAP), consists of a pair of high molecular mass (280 kDa) polypeptides, MAP2a and MAP2b, and several low molecular mass (70 kDa) proteins called MAP2c. Although MAP2b and MAP2c have been shown to arise via alternative splicing, it was not clear whether MAP2a is also created by alternative splicing or by posttranslational modification. Using epitope peptide mapping, we have demonstrated that an element specific to MAP2a is situated at its N‐terminal end. A cDNA clone from an adult rat brain library was found to contain an additional 246 nucleotides situated at the 5′ end of the 9‐kb MAP2 mRNA. Antibodies generated against the encoded protein sequence recognize specifically MAP2a in rat brain homogenates. Moreover, although MAP2a, like MAP2b, is found in dendrites and cell bodies, its temporal appearance and cell type‐specific distribution in rat brain differs from MAP2b.
The Cerebellum‐Enriched Form of Nuclear Factor I Is Functionally Different from Ubiquitous Nuclear Factor I in Glial‐Specific Promoter Regulation
Authors: Krebs Christopher J. , Dey Barna , Kumar Gyanendra
Published: July 2010

Abstract: Nuclear factor I (NFI) binding sites are present in a wide range of brain‐specific gene enhancer and promoter sequences and appear to play a role in establishing cell type‐specific expression within the CNS. The precise mechanisms used by various members of the NFI family of proteins to confer brain‐specific expression are unclear. We have addressed this issue by comparing the transactivating capabilities of two forms of NFI in directing gliotropic expression from two different JC virus (JCV) promoter configurations. The JCV is an opportunistic pathogen of humans that causes lytic destruction of the oligodendrocytes and thus demyelination in immunocompromised patients. Our results show that the cerebellum‐enriched form of NFI (NFI‐A1) transactivates two gliotropic JCV early promoters to a greater extent than the ubiquitous form of NFI (NFI‐C1). Activation by NFI‐A1 was dramatically greater in glial than in nonglial cells. These results suggest that NFI proteins direct brain‐specific expression through combinatorial interactions with cell specific coactivators and/or transcription factors that recognize adjacent sites within brain specific promoters.
Thrombin Attenuates Neuronal Cell Death and Modulates Astrocyte Reactivity Induced by β‐Amyloid In Vitro
Authors: Pike Christian J. , Vaughan Patrick J. , Cunningham Dennis D. , Cotman Carl W.
Published: July 2010

Abstract: β‐Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that β‐amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of β‐amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of β1–42 and β25–35 peptides. In cultured astrocytes, the stellate morphology induced by β1–42 and β25–35 aggregates can be prevented and reversed by thrombin exposures between 10 pM and 1 µM. In contrast, thrombin potentiates rather than attenuates the β‐amyloid‐induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of β‐amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor‐activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin‐1. These data provide both new insight into the signaling pathways underlying the cellular effects of β‐amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.
Adenosine A2b Receptors Mediate an Increase in Interleukin (IL)‐6 mRNA and IL‐6 Protein Synthesis in Human Astroglioma Cells
Authors: Fiebich Bernd L. , Biber Knut , Gyufko Kacin , Berger Mathias , Bauer Joachim , Van Calker Dietrich
Published: July 2010

Abstract: The cytokine interleukin (IL)‐6 has recently been demonstrated to play a role in the pathology of Alzheimer's disease (AD). The mechanisms leading to increased IL‐6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the level of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL‐6 gene expression in the brain. Here we show that the mixed A1 and A2 agonist 5′‐(N‐ethylcarboxamido)adenosine (NECA) induces an increase in IL‐6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A1‐specific agonists R‐phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A2a‐specific agonist CGS‐21680 shows only marginal effects. Increased levels of mRNA are already found within 30 min after NECA treatment. The A2a‐selective antagonists 8‐(3‐chlorostyryl)caffeine and KF17837 [(E)‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐7‐methylxanthine], which have also some antagonistic properties at A2b receptors, and the nonspecific adenosine antagonist 8‐phenyltheophylline were equipotent at inhibiting the NECA‐induced increase in IL‐6 protein synthesis, whereas the specific A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine is much less potent. The results indicate that adenosine A2b receptors participate in the regulation of the IL‐6 gene in astrocytoma cells.
Inhibition of Na+,K+‐ATPase by Ouabain Opens Calcium Channels Coupled to Acetylcholine Release in Guinea Pig Myenteric Plexus
Authors: Gomez Renato S. , Gomez Marcus Vinicius , Prado Marco Antônio M.
Published: July 2010

Abstract: Ouabain, an Na+,K+‐ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca2+‐independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain‐evoked release of [3H]ACh. Release of [3H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N‐type calcium channel blockage by the ω‐conotoxins GVIA (selective N‐type calcium channel blocker) and MVIIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [3H]ACh. L‐type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [3H]ACh. The release of transmitter was also not affected significantly by nickel, a T‐type calcium channel blocker. In addition, ω‐agatoxin‐IVA, at concentrations that block P‐ and Q‐type calcium channels, did not affect significantly the release of [3H]ACh. Thus, extracellular Ca2+ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N‐type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na+,K+‐ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain.
Characterization of Excitatory Amino Acid Modulation of Dopamine Release in the Prefrontal Cortex of Conscious Rats
Authors: Jedema Hank Peter , Moghaddam Bita
Published: July 2010

Abstract: The effect of various classes of excitatory amino acid agonists on the release of dopamine in the medial prefrontal cortex (PFC) of awake rats was examined using intracerebral microdialysis. Local infusion of 20 µMα‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA), through the microdialysis probe, produced a significant increase of more than twofold in extracellular levels of dopamine. Application of 100 µM AMPA increased these levels nearly 15 fold. The AMPA/kainate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) (50 µM) blocked the increase in dopamine release produced by 20 µM AMPA. Local infusion of kainate at concentrations of 5 and 20 µM increased dopamine release by nearly 150 and 500%, respectively. Local application of CNQX (50 µM) before 20 µM kainate significantly attenuated the stimulatory effect of kainate on dopamine levels. In contrast to AMPA and kainate, infusion of N‐methyl‐d‐aspartate (NMDA) at 20 or 100 µM did not increase dopamine release. In fact, a trend toward a decrease in dopamine release was evident after 100 µM NMDA. The present study indicates that the in vivo release of dopamine in the PFC is facilitated by AMPA and kainate receptors. This modulation is more profound than that previously reported in the basal ganglia. The lack of an excitatory effect of NMDA is in agreement with recent reports that the NMDA receptor may inhibit indirectly dopaminergic neurotransmission in the PFC.
Quantitative Changes of Amino Acid Distributions in the Rat Vestibular Nuclear Complex After Unilateral Vestibular Ganglionectomy
Authors: Li Hongyan , Godfrey Timothy G. , Godfrey Donald A. , Rubin Allan M.
Published: July 2010

Abstract: Changes of amino acid concentrations in the vestibular nuclear complex (VNC) during lesion‐induced vestibular compensation were studied in rats after unilateral vestibular ganglionectomy. Distributions of 12 amino acids within the VNC were measured at 2, 4, 7, and 30 days after surgery, using microdissection of freeze‐dried brain sections and HPLC. Glutamate decreased on the lesioned side in nearly all VNC regions. Changes were fully developed 2 days after lesion and persisted through 30 days. In some regions, glutamate decreased also on the unlesioned side, especially at longer survival times, so that bilateral asymmetries became reduced. Aspartate changes were similar to those of glutamate on either side. Lesion‐induced glutamine asymmetry was usually opposite to that of glutamate. Although GABA concentration decreased at early survival times, it recovered at later times and sometimes increased in dorsal parts of lateral and medial nuclei. Taurine changes were similar to those of GABA in most regions. Glycine change was primarily limited to a bilateral decrease in the dorsal part of the lateral vestibular nucleus. Concentrations of other amino acids were much lower, but some showed postlesion changes.
Presynaptic Serotonergic Markers in Community‐Acquired Cases of Alzheimer's Disease: Correlations with Depression and Neuroleptic Medication
Authors: Chen Christopher P. L.‐H. , Alder J. Tracy , Bowen David M. , Esiri Margaret M. , McDonald Brendan , Hope Tony , Jobst Kim A. , Francis Paul T.
Published: July 2010

Abstract: Presynaptic serotonergic markers, serotonin uptake sites, and concentrations of serotonin and 5‐hydroxyindoleacetic acid were studied in the frontal and temporal cortex of 20 community‐acquired cases of Alzheimer's disease and 16 controls matched for age, sex, postmortem delay, and storage. Clinical assessments, including behavioural symptoms, of the Alzheimer patients were made at 4‐month intervals during life. There was a significant reduction in the number of serotonin uptake sites in Alzheimer cases in temporal but not frontal cortex. There was no significant alteration in the concentrations of serotonin or 5‐hydroxyindoleacetic acid in either region. Alzheimer patients who had persistent depressive symptoms during life had significantly fewer serotonin uptake sites in both cortical areas compared with Alzheimer patients without these symptoms. In addition, Alzheimer patients who were receiving chronic neuroleptic medication had significantly lower concentrations of serotonin in frontal cortex and 5‐hydroxyindoleacetic acid in temporal cortex than those patients not receiving such treatment. These data suggest previous studies that reported uniform serotonergic dysfunction may have been subject to unintentional selection of behaviourally disturbed Alzheimer cases or those receiving chronic neuroleptic medication. This study also provides a basis for the treatment of behaviourally disturbed Alzheimer patients with serotonomimetics.
CCK‐4‐Induced Calcium Mobilization in T Cells Is Enhanced in Panic Disorder
Authors: Akiyoshi Jotaro , Moriyama Tamie , Isogawa Koichi , Miyamoto Masafumi , Sasaki Ichiro , Kuga Kiyomi , Yamamoto Hisao , Yamada Kenji , Fujii Isao
Published: July 2010

Abstract: We investigated the effects of brain cholecystokinin (CCK) receptors on the intracellular calcium concentration and protein kinase C in human T cells. CCK‐4 produced a transient increase in calcium in the absence of extracellular calcium. CCK‐B agonists stimulated calcium mobilization in a dose‐dependent manner in T cells. CCK‐B antagonists suppressed CCK‐4‐induced calcium mobilization more potently than CCK‐A antagonist. The recovery of desensitization of the CCK‐4‐induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A. The responsiveness to CCK‐4 was also reduced by phorbol 12,13‐dibutyrate (PDBu), and this effect of PDBu was abolished completely by preincubation with staurosporine. CCK‐4‐induced calcium mobilization was too small to attribute the desensitization to the protein kinase C transduction pathway. T cells from patients with untreated panic disorder exhibited significantly higher cholecystokinin‐4‐induced calcium mobilization than those from healthy controls or patients with treated panic disorder. These results suggest that cholecystokinin‐B receptor function in T cells of patients with panic disorder is enhanced. Cholecystokinin‐4‐induced calcium mobilization in T cells may be state dependent and useful as a biological marker of panic disorder.
Modulation of Serine Proteinases and Metalloproteinases During Morphogenic Glial‐Endothelial Interactions
Authors: Rao Jasti S. , Sawaya Raymond , Gokaslan Ziya L. , Yung W. K. Alfred , Goldstein Gary W. , Laterra John
Published: July 2010

Abstract: The regulation of microvessel formation and the expression of CNS‐specific endothelial properties are attributed to perivascular astroglia. Specific proteolytic pathways mediate processes such as tissue remodeling, differentiation, invasion, and metastasis. We used a coculture system in which C6 glial cells induce CNS microvascular endothelial cells to form capillary‐like structures to examine the role of plasminogen activators and collagenases in CNS microvessel morphogenesis. Fibrin zymography revealed the presence of high‐molecular weight urokinase‐type plasminogen activator (uPA), low‐molecular weight uPA, and uPA/inhibitor complexes within endothelial cultures and cocultures. Gelatin zymography revealed the presence of 92‐, 72‐, and 62‐kDa type IV collagenases within endothelial cultures and cocultures. uPA activity was confirmed by incubating the extracts with amiloride, an inhibitor of uPA. Collagenase activity was confirmed by incubating the gels with EDTA, an inhibitor of metalloproteinases. Quantitative densitometry showed a six‐ to eightfold decrease in coculture uPA during capillary‐like structure formation. Substantially less change in type IV 72‐kDa procollagenase activity was seen in cocultures during capillary‐like structure formation, but active type IV 62‐kDa collagenase activity was significantly increased during capillary‐like structure formation. These findings establish that uPA and activated type IV collagenase activity specifically regulates morphogenic endothelial responses to glial interactions and suggest mechanisms by which microvessels respond within the CNS.
Glutamine Transport in Mouse Cerebral Astrocytes
Authors: Nagaraja Tavarekere N. , Brookes Neville
Published: July 2010

Abstract: We measured initial influx and exchange of [14C]glutamine in primary astrocyte cultures in the presence and absence of Na+. Kinetic analysis of transport in Na+‐free solution indicated two saturable Na+‐independent components, one of which was identifiable functionally as system L1 transport. In the presence of Na+, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na+‐dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by α‐(methylamino)isobutyric acid. However, vigorous system A‐mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans‐acceleration of Na+‐dependent uptake, preferential inhibition of an Li+‐intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and α‐(methylamino)isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na+, low‐affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 µM glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li+ in place of Na+.
O‐Linked Oligosaccharide on the 75‐kDa Neurotrophin Receptor
Authors: Chapman Barbara S. , Eckart Michael R. , Kaufman Susan E. , Lapointe Gena R.
Published: July 2010

Abstract: Four neurotrophic factors, important for survival and function of neurons, bind a common receptor, the 75‐kDa neurotrophin receptor (NTR). An O‐glycosylated peptide connects the ligand‐binding domain of NTR to its transmembrane helix. This peptide, the transmembrane helix, and intracellular sequences are highly conserved in vertebrate evolution. To investigate the structure and function of O‐glycosylation on NTR, we produced the extracellular domains by expression in mammalian cells. Addition during biosynthesis of O‐linked glycans was evaluated, and structures were characterized by lectin blotting and glycosidase digestion. Effects of desialylation, deglycosylation, and lectin attachment on the equilibrium binding constant were measured. Addition of O‐linked glycans during biosynthesis was found to have a large effect on NTR structure assessed by mobility in polyacrylamide gels. NTR O‐linked glycans synthesized by cultured cells had the structure (NeuNAc)1–2‐Galβ1‐3GalNAc. Modification of the O‐linked oligosaccharide produced small, possibly significant effects on the binding constant of NTR for nerve growth factor. The results are discussed in reference to a potential role for the stalk region in ligand binding and signaling.