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  • Nuclear choline acetyltransferase activates transcription of a high-affinity choline transporter. 21163949

    Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter (VACHT) as candidate genes, which function together with ChAT in acetylcholine production. Using SH-SY5Y human neuroblastoma cells stably expressing wild-type human ChAT, we found that overexpressed ChAT enhanced transcription of the CHT1 gene but not the VACHT gene. In contrast, nuclear localization signal disrupted, and catalytically inactive mutant ChATs could not induce, CHT1 expression. Additionally, ChAT did not alter CHT1 expression in non-neuronal HEK293 cells. Our results suggest that ChAT activates the transcription of selected target genes in neuronal cells. Both enzymatic activity and nuclear translocation of ChAT are required for its transcriptional enhancement.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation. 20132834

    Previous work by our group demonstrated that homomeric alpha7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca(2+) increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific alpha7 agonist PNU 282987 with IC(50) values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human alpha7 but not with alpha4beta2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and alpha-bungarotoxin but not by dihydro-beta-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on alpha7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca(2+) release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca(2+) levels and induced an increase in alpha-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and alpha7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca(2+)-dependent enzymes such as protein kinase C and nitric oxide synthase, which are involved in the generation of ROS and the blockade of the dopamine transporter. This, together with caspase 3 activation, must play a role in MDMA-induced cytotoxicity.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1622
    Nombre del producto:
    Anti-Spectrin alpha chain (nonerythroid) Antibody, clone AA6

  • Preconditioning-mimetics bradykinin and DADLE activate PI3-kinase through divergent pathways. 17292392

    We previously reported that pharmacological preconditioning of rabbit hearts with acetylcholine involves activation of phosphatidylinositol 3-kinase (PI3-K) through transactivation of the epidermal growth factor receptor (EGFR). Transactivation is thought to be initiated by cleavage of membrane-bound pro-heparin-binding EGF-like growth factor (HB-EGF) by a membrane metalloproteinase thus releasing HB-EGF which binds to the EGFR. This pathway leads to redox signaling with the generation of reactive oxygen species (ROS) by mitochondria. We tested whether preconditioning's physiological triggers, bradykinin and opioid, also signal through the EGFR. Both bradykinin and the synthetic delta-opioid agonist DADLE increased ROS production in isolated cardiomyocytes by approximately 50%. DADLE's effect was abrogated by either metalloproteinase inhibitor III (MPI) or the diphtheria toxin mutant CRM-197 which blocks heparin-binding EGF shedding indicating that DADLE signals through EGFR transactivation. MPI also blocked DADLE's infarct-sparing effect in whole hearts. Additionally, blocking Src kinase (a component of the EGFR's signaling complex) with PP2 or PI3-K with wortmannin blocked DADLE's effect on cardiomyocyte ROS production and PP2 blocked DADLE's salvage of ischemic myocardium. Finally, DADLE increased phosphorylation of Akt and extracellular signal-regulated protein kinases (ERK) 1/2 in left ventricular myocardium, and this increase was blocked by the EGFR antagonist AG1478. On the other hand, neither MPI nor CRM-197 prevented bradykinin from increasing ROS production, and MPI did not affect bradykinin's infarct-sparing effect in intact hearts. Conversely, both PP2 and wortmannin blocked bradykinin's effect on ROS generation and also aborted bradykinin's cardioprotective effect in intact hearts. While bradykinin also increased phosphorylation of Akt and ERK in myocardium, that increase was not affected by AG1478. Hence bradykinin, unlike acetylcholine or opioid, does not transactivate EGFR, although all 3 agonists do signal through Src and PI3-K.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-481
    Nombre del producto:
    Anti-phospho-MAP Kinase1/2 Antibody

  • Choline transporter 1 maintains cholinergic function in choline acetyltransferase haploinsufficiency. 15201317

    Choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine (ACh), is thought to be present in kinetic excess in cholinergic neurons. The rate-limiting factor in ACh production is the provision of choline to ChAT. Cholinergic neurons are relatively unique in their expression of the choline transporter 1 (CHT1), which exhibits high-affinity for choline and catalyzes its uptake from the extracellular space to the neuron. Multiple lines of evidence indicate that the activity of CHT1 is a key determinant of choline supply for ACh synthesis. We examined the interaction of ChAT and ChT activity using mice heterozygous for a null mutation in the Chat gene (Chat+/-). In these mice, brain ChAT activity was reduced by 40-50% relative to the wild type, but brain ACh levels as well as ACh content and depolarization-evoked ACh release in hippocampal slices were normal. However, the amount of choline taken up by CHT1 and ACh synthesized de novo from choline transported by CHT1 in hippocampal slices, as well as levels of CHT1 mRNA in the septum and CHT1 protein in several regions of the CNS, were 50-100% higher in Chat+/- than in Chat+/+ mice. Thus, haploinsufficiency of ChAT leads to an increased expression of CHT1. Increased ChT activity may compensate for the reduced ChAT activity in Chat+/- mice, contributing to the maintenance of apparently normal cholinergic function as reflected by normal performance of these mice in several behavioral assays.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5966

  • Localization of muscarinic receptors M1R, M2R and M3R in the human colon. 20146726

    Muscarinic acetylcholine receptors (MR) are involved in multiple intestinal reflexes. The cellular localization of subtypes of MRs within enteric circuits mediating muscle and mucosal reflexes remains to be demonstrated. This study aimed to localize the three functionally significant subtypes of MRs in human colon.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1529
    Nombre del producto:
    Anti-Nitric Oxide Synthase I Antibody

  • Activated cholinergic signaling provides a target in squamous cell lung carcinoma. 18559515

    The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non-small cell lung carcinomas. Understanding how cholinergic signaling is up-regulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of alpha5 and beta3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogen-activated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth in vitro and also blocked H520 growth in nude mice in vivo. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB305
    Nombre del producto:
    Anti-Choline Acetyltransferase Antibody, clone 1E6

  • Nicotinic acetylcholine receptor subunit mRNA expression and channel function in medial habenula neurons. 11044729

    Relationships between nicotinic acetylcholine receptor (nAChR) channel function and nAChR subunit mRNA expression were explored in acutely isolated rat medial habenula (MHb) neurons using a combination of whole-cell recording and single cell RT-PCR techniques. Following amplification using subunit-specific primers, subunits could be categorized in one of three ways: (i) present in 95-100% cells: alpha3, alpha4, alpha5, beta2 and beta4; (ii) never present: alpha2; and (iii) sometimes present ( approximately 40% cells): alpha6, alpha7 and beta3. These data imply that alpha2 subunits do not participate in nAChRs on MHb cells, that alpha6, alpha7 and beta3 subunits are not necessary for functional channels but may contribute in some cells, and that nAChRs may require combinations of all or subsets of alpha3, alpha4, alpha5, beta2 and beta4 subunits. Little difference in the patterns of subunit expression between nicotine-sensitive and insensitive cells were revealed based on this qualitative analysis, implying that gene transcription per se may be an insufficient determinant of nAChR channel function. Normalization of nAChR subunit levels to the amount of actin mRNA, however, revealed that cells with functional channels were associated with high levels (>0.78 relative to actin; 11/12 cells) of all of the category (i) subunits: alpha3, alpha4, alpha5, beta2 and beta4. Conversely, one or more of these subunits was always low (0.40 relative to actin) in all cells with no detectable response to nicotine. Thus the formation of functional nAChR channels on MHb cells may require critical levels of several subunit mRNAs.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB305
    Nombre del producto:
    Anti-Choline Acetyltransferase Antibody, clone 1E6

  • Tonic Modulation of GABA Release by Nicotinic Acetylcholine Receptors in Layer V of the Murine Prefrontal Cortex. 19812239

    By regulating the neocortical excitability, nicotinic acetylcholine receptors (nAChRs) control vigilance and cognition and are implicated in epileptogenesis. Modulation of gamma-aminobutyric acid (GABA) release often accompanies these processes. We studied how nAChRs regulate GABAergic transmission in the murine neocortex with immunocytochemical and patch-clamp methods. The cholinergic fibers densely innervated the somatosensory, visual, motor, and prefrontal cortices (PFC). Laminar distribution was broadly homogeneous, especially in the PFC. The cholinergic terminals were often adjacent to the soma and dendrites of GABAergic interneurons, but well-differentiated synapses were rare. Tonically applied nicotine (1-100 muM) increased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) on pyramidal neurons in PFC layer V. The contribution of nAChR types was assessed by using 1 muM dihydro-beta-erythroidine (DHbetaE), to block heteromeric nAChRs, and 10 nM methyllycaconitine (MLA), to block homomeric nAChRs. Both inhibitors antagonized the effect of nicotine on IPSCs, suggesting that mixed nAChR types control pyramidal neuron inhibition in layer V. To determine whether nAChRs are expressed on basket cells' terminals, we studied miniature IPSCs (mIPSCs). These were revealed using 0.5 muM tetrodotoxin and 50 muM Cd(2+) to isolate the GABAergic terminals from the action potential drive. The nicotinic stimulation of mIPSCs was antagonized by DHbetaE, but not MLA, indicating that heteromeric nAChRs prevail in GABAergic terminals. Immunocytochemistry confirmed the expression of nAChRs on basket cells' somata and terminals. Finally, when the ionotropic glutamatergic transmission was blocked, nicotine partially inhibited the IPSCs, an effect counteracted by both DHbetaE and MLA. Therefore, a fraction of nAChRs are capable of activating GABAergic interneurons that in turn inhibit other GABAergic interneurons, thereby reducing the IPSCs. We conclude that heteromeric nAChRs control GABA release presynaptically, whereas mixed nAChRs regulate both excitation and inhibition of interneurons, the balance depending on the overall glutamatergic drive.,
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5590

  • Polysialic acid limits choline acetyltransferase activity induced by brain-derived neurotrophic factor. 16903870

    Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine, is known to be activated by brain derived neurotrophic factor (BDNF). We found that the specific removal of the carbohydrate polysialic acid (PSA) significantly increased BDNF-induced ChAT-activity in embryonic septal neurons. Using a p75 neurotrophin receptor (p75(NTR)) function-blocking antibody and K252a, a-pan tropomyosin related kinase (Trk) inhibitor, we demonstrate that BDNF-induced ChAT activity requires the stimulation of p75(NTR) and TrkB. PSA removal drastically increased radioactive iodinated ([(125)I])BDNF's maximal binding capacity (Bmax), derived from concentrations of [(125)I]BDNF ranging from 1 pM to 3.2 nM. In the presence of unlabeled nerve growth factor to prevent the binding of [(125)I]BDNF to p75(NTR) sites, the impact of PSA removal on the binding capacity of [(125)I]BDNF was greatly reduced. In conclusion, PSA limits BDNF-induced ChAT activity and BDNF-receptor interactions. BDNF-induced ChAT activity is TrkB and p75(NTR) dependent, and upon PSA removal the additional binding of BDNF to its receptors, especially p75(NTR), likely contributes to the maximal ChAT activity observed. In vivo, the ontogenetic loss of PSA in the postnatal period may allow more interactions between BDNF and its receptors to increase ChAT activity and assure the proper development of the cholinergic septal neurons.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5312
    Nombre del producto:
    Anti-Growth Associated Protein 43 Antibody

  • An immunocytochemical study of choline acetyltransferase-containing neurons and axon terminals in normal and partially deafferented hippocampal formation. 3548884

    Monoclonal antibodies to the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), have been used to study putative cholinergic structures in immunocytochemical preparations of normal rat hippocampal formation and of hippocampal formation deprived of its septal innervation. Small numbers of ChAT-positive (ChAT+) neuronal somata were observed scattered throughout the septotemporal extent of the normal hippocampal formation. They were most common in stratum lacunosum-moleculare of regio superior, but were also found in various layers of the dentate gyrus and occasionally in the remaining hippocampal laminae. In addition, light microscopy demonstrated that ChAT+ terminal fields in normal hippocampal formation were organized in discrete bands and laminae. Pronounced dense bands were observed: immediately superficial to stratum granulosum; deep to stratum pyramidale; and at the border between stratum radiatum and stratum lacunosum-moleculare. In the dentate gyrus, ChAT+ staining was pronounced in the hilus at temporal levels, but only moderate staining occurred in the anterior hilus and throughout the molecular layer. A close correspondence was observed in the density and distribution of ChAT+ immunoreactivity and acetylcholinesterase staining. Electrolytic lesions of the medial septal nucleus/diagonal band complex had no effect on the occurrence of ChAT+ somata, but virtually abolished the ChAT+ laminar staining pattern and eliminated all but occasional small patches of ChAT+ terminals. These results confirm that the vast majority of hippocampal cholinergic terminals originate either from neurons of the medial septum/diagonal band complex or from fibers of passage. The newly observed intrinsic hippocampal neurons can account for at least some of the ChAT activity remaining after septal lesions, and they apparently contribute to the cholinergic innervation of the hippocampal formation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB305
    Nombre del producto:
    Anti-Choline Acetyltransferase Antibody, clone 1E6