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Query: UNIPROT:P06889 (Mol)
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Y79 human retinoblastoma cells are known to contain receptors for both insulin and insulin-like growth factors (IGFs), to produce these cytokines and release them in the culture medium. Previously we have demonstrated that IGFs and insulin stimulate Y79 cell proliferation through the involvement of type I IGF receptor and Insulin Receptor Substrate 1 (IRS-1). This paper studies the effect of prolonged exposure to insulin on Y79 cells. Cells grown for 10 days in the presence of insulin were reseeded and incubated once more with insulin. In the reseeded cells proliferation lowered and morphological changes appeared. After 10 days of reseeding, cells stopped proliferating and showed long ramifying neurite processes and varicosities consistent with neuronal differentiation. Morphological differentiation was accompanied by a marked increase in the content of total protein and in that of tubulin, the major protein constituent of microtubules, a marked increase in the content of specialized protein markers of dopaminergic and cholinergic differentiation (dopamine beta-hydroxylase and choline acetyltransferase activities, respectively); a contemporaneous decrease in the content of glial fibrillary acidic protein (GFAP), a specific marker of glial cells, was also observed. Our results demonstrate that prolonged exposure to insulin induces Y79 cells to differentiate into a neuronal-like phenotype. At this moment it is not possible to establish the mechanism by which insulin induces this differentiative effect.
Mol Cell Biochem 1997 May
PMID:Differentiation of Y79 cells induced by prolonged exposure to insulin. 914 31

The P19 embryonal carcinoma cells differentiate into neurons, astrocytes, and fibroblast-like cells following induction with retinoic acid. The cells mature into functional neurons, as determined by their ability to release neurotransmitters in a Ca(2+)- and depolarization-dependent manner. P19 neurons in culture represent a mixed population in terms of their neurotransmitter phenotype. The cholinergic phenotype of these neurons is modulated by culture density. Cholinergic markers, such as the vesicular acetylcholine transporter, acetyl cholinesterase, and choline acetyltransferase, are expressed in about 85% of the cells in sparse cultures and are largely suppressed at high cell densities. In contrast, glutamate release is enhanced in dense P19 neuronal cultures. The factor mediating the density effect is concentrated exclusively on the cell membrane of P19 neurons and not on the nonneuronal cells, which also differentiate from P19 embryonal carcinoma cells. This membrane-associated component retains its functionality, even after membrane fixation. The downregulation of the cholinergic properties in dense cultures is paralleled by a downregulation of the alpha subunit of the ciliary neurotrophic factor (CNTF) receptor. Thus, it is suggested that the membrane-associated factor, which mediates the density effect, downregulates the cholinergic phenotype by inhibiting the responsiveness of these neurons to CNTF. We further suggest that the P19 cell line can serve as a model system for the study of neurotransmitter phenotype acquisition and plasticity throughout neuronal differentiation.
J Mol Neurosci 1997 Apr
PMID:Culture density regulates both the cholinergic phenotype and the expression of the CNTF receptor in P19 neurons. 918 41

During the first trimester of pregnancy, the human placental trophoblast contains very low levels of choline acetyltransferase (ChA), a specific enzyme for synthesis of acetylcholine (ACh) and a reliable cholinergic marker. There is a relationship between ChA levels and development and maturation of syncytiotrophoblast during the first six months of pregnancy, when ChA levels reach maximum. These observations suggest that ChA and its product, ACh, have significant roles during differentiation of cytotrophoblast into syncytium. Therefore, we have measured ChA levels in malignant trophoblast cultures before and after differentiation. Two pure trophoblast cell lines (BeWo and JAr) are used in these studies. ChA in these cells was determined by a standard radiometric method in which 14C-acetyl groups were transferred from 14C-acetylcoenzyme A to choline. Methotrexate (1 microM) was used to differentiate the cells. The following results were obtained: 1) Undifferentiated BeWo cells contained ChA level of 311.0 +/- 8.3 pmoles ACh formed (M +/- S.E., N = 6)/mg protein/10 min. Differentiation decreased ChA level to 213.0 +/- 9.3 pmoles ACh/mg protein/10 min.; b) Undifferentiated JAr cells contained ChA levels of 279.0 +/- 15.0 ACh pmoles formed/mg protein/10 min. This decreased to 166.8 +/- 10.0 upon differentiation; c) Upon differentiation, ChA levels decreased by 31.5-40.2% in BeWo and JAr cell lines. In term human placenta, the ChA levels falls by about 46.0-73% after syncytiotrophoblast is fully developed; d) Chorionic gonadotropin (hCG), which is produced in cytotrophoblast, is a standard tumor marker for chorionic cancer. As the syncytiotrophoblast is formed, production of hCG decreases. Similarly, production of hCG by BeWo and JAr cells decreases upon their differentiation, and e) In hydatidiform mole, which can undergo malignant transformation into choriocarcinoma, there were no significant levels of ChA. These observations suggest that ChA and ACh are necessary for development and maturation of syncytiotrophoblast.
Cell Mol Biol (Noisy-le-grand) 1997 Jun
PMID:Cholinergic markers in transformed trophoblast cells: BeWo and JAr cells. 922 Jan 49

Ginsenoside Rb1 (Rb1), a saponin of North American ginseng (Panax quinquefolium L.), has been found to exert beneficial effects on memory and learning, putatively through its actions on the cholinergic system. In situ hybridization studies show that Rb1 increases the expression of choline acetyltransferase and trkA mRNAs in the basal forebrain and nerve growth factor mRNA in the hippocampus. Other neurotrophins (brain-derived neurotrophic factor, neurotrophin-3), genes encoding neuropeptides (preproenkephalin, preprotachykinin) and amyloid protein precursor were also studied, but no significant change was observed. These findings support the specificity of the effects of Rb1 on certain aspects of the cholinergic and neurotrophic systems.
Brain Res Mol Brain Res 1997 Jul
PMID:Ginsenoside Rb1 regulates ChAT, NGF and trkA mRNA expression in the rat brain. 922 15

Metabotropic glutamate receptors (mGluRs) can be divided into three groups based on sequence homology and pharmacology. We studied expression of group I mGluRs (mGluR1 and mGluR5) in identified neurons of the rat neostriatum, neocortex, and hippocampus using in situ hybridization. Tissue sections were hybridized with radiolabeled RNA probes for mGluR1 or mGluR5 and digoxygenin labeled RNA probes detecting somatostatin (SOM), preproenkephalin (ENK), preprotachykinin (SP), glutamic acid decarboxylase 67 (GAD67), parvalbumin (PARV), or choline acetyltransferase (ChAT) mRNA. In the striatum, mGluR1 hybridization signal was observed in all six neuronal populations. The strongest signal was found in SP-positive neurons, with a lower signal in ENK-positive neurons. All striatal interneurons were labeled less intensely than ENK- and SP-positive projection neurons. For striatal mGluR5 mRNA, both SP- and ENK-positive projection neurons were intensely labeled, but only GAD67-positive interneurons exhibited a significant signal. In the neocortex and hippocampus, mGluR1 and mGluR5 hybridization signals were studied in SOM-, GAD67-, and PARV-positive neurons. Hybridization signal for mGluR1 mRNA was intense in SOM-positive neurons of the cortex, CA1, CA3, and dentate gyrus, and weaker in GAD67-positive neurons of CA3 and dentate gyrus. MGluR5 signals were intensely labeled in SOM-, GAD67- and PARV-positive neuronal populations of the cortex and hippocampus. SOM-positive neurons were more intensely labeled in the hippocampus than cortex.
Brain Res Mol Brain Res 1997 Sep
PMID:Expression of group one metabotropic glutamate receptor subunit mRNAs in neurochemically identified neurons in the rat neostriatum, neocortex, and hippocampus. 933 23

ChAT (choline acetyltransferase) is the enzyme responsible for acetylcholine synthesis and is specifically expressed in cholinergic neurons. To further characterize the transcriptional regulation of the hCHAT (human ChAT) gene by NGF, we examined the effects upon ChAT promoter activity of a family of transcription factors which are activated by NGF and several extracellular stimuli and encoded by immediate-early genes. These include NGFI-A (Egr1, zif268), NGFI-C (Egr2), Krox-20 and NGFI-B (Nurr77). Two fragments of the hChAT gene were used for functional analysis carrying 944 bp (P1) and 4000 bp (P1 + P2) of the 5' flanking region in front of the chloramphenicol acetyltransferase (CAT) reporter gene. They were transiently co-transfected with NGFI-A, NGFI-C, Krox-20 and NGFI-B expression vectors in NG108-15, SN6 and COS-1 cells. CAT activity after transfection of the p4000 ChAT-CAT reporter into both neuronal cell lines (NG108-15 and SN6 cells) was increased up to 5-fold in the presence of co-transfected NGFI-A and up to 5- and 12-fold after co-transfection of NGFI-C expression vector in NG108-15 and SN6 cells, respectively. In NG108-15 cells, dbcAMP excerted a strong enhancing activity on the transactivation properties of NGFI-C while this was not observed when cells were transfected with NGFI-A. These trans-activation effects were specific for neuronal cells. When NG108-15 cells were treated with dbcAMP in the presence of H89, a specific PKA inhibitor, the increase of transcriptional activity of NGFI-C was abolished, indicating that a signalling transduction mechanism through PKA plays a role in NGFI-C-induced trans-activation. Electrophoretic mobility-shift assays showed that the sequence GCCCGGGGAG (NGFRE) located 1205 bp upstream of the first coding ATG (E1) can bind NGFI-A but not NGFI-C. Several possibilities explaining the observed results are discussed. Finally, transfections of ChAT-CAT reporters including the P1 + P2 region or a minimal ChAT enhancer present in the P2 region in front of a heterologous promoter indicated the presence of a regulatory element which conferred AP2-dependent trans-activation with homologous as well as with heterologous promoter constructs.
Brain Res Mol Brain Res 1997 Oct 03
PMID:Transcriptional activation of human choline acetyltransferase by AP2- and NGF-induced factors. 938 76

A number of pharmacological, anatomical, and immunological studies have previously addressed the subtype identity of the hippocampal muscarinic pre-synaptic autoreceptor. A preponderance of findings indicate that it is of the M2 pharmacological type. Both the m2 and m4 molecular subtypes exhibit M2 pharmacology and there are few drugs that differentiate between these receptors. Pharmacological attempts at defining the hippocampal autoreceptor have yielded conflicting results. The basal forebrain is relatively enriched in m2 muscarinic receptor mRNA and protein, and lesions that denervate the hippocampus of its basal forebrain cholinergic input have shown a decrement in m2, but not m4, receptor protein in the hippocampus. Thus, the anatomical data obtained to date tend to support the view that the m2 subtype is expressed as the hippocampal autoreceptor. We have combined in situ hybridization histochemistry (ISHH) with immunocytochemistry to choline acetyltransferase to examine whether mRNA for the m4 subtype of muscarinic receptor is expressed in central cholinergic neurons. The m4 muscarinic mRNA was found at moderate levels in all subdivisions of the cholinergic basal forebrain, including the medial septum/diagonal band complex (MS/DB). The m4 mRNA was also found in striatal cholinergic interneurons, in the cholinergic reticular core of the upper brainstem, and in brainstem cholinergic motor neurons. Muscarinic m4 receptor mRNA was also found in many non-cholinergic cells in the brain. For example, the hippocampal pyramidal neurons, dentate gyrus granule cells, and entorhinal cortical pyramidal neurons express relatively high levels of m4 mRNA, while in the brainstem the dorsal raphe and pontine reticular nuclei express relatively high levels of this mRNA. The finding of m4 mRNA in the MS/DB cholinergic neurons suggests that this receptor protein might be expressed as an autoreceptor in hippocampal cholinergic terminals.
Brain Res Mol Brain Res 1997 Oct 15
PMID:mRNA for the m4 muscarinic receptor subtype is expressed in adult rat brain cholinergic neurons. 940 47

The Flinders sensitive line of rats (FSL rats) have an altered REM sleep pattern which includes a shorter REM sleep latency and an increased percentage of REM sleep [R.M. Benca, D.H. Overstreet, M.A. Gilliland, D. Russell, B.M. Bergmann, W.H. Obermeyer, Increased basal REM sleep but no difference in dark induction or light suppression of REM sleep in Flinders rats with cholinergic supersensitivity, Neuropsychopharmacology 15 (1996) 45-51; P.J. Shiromani, D.H. Overstreet, D. Levy, C.A. Goodrich, S.A. Campbell, J. C. Gillin, Increased REM sleep in rats selectively bred for cholinergic hyperactivity, Neuropsychopharmacology 1 (1988) 127-133]. Cholinergic mechanisms have been implicated in REM sleep generation [reviewed in P.J. Shiromani, J.C. Gillin, S.J. Henriksen, Acetylcholine and the regulation of REM sleep: basic mechanisms and clinical implication for affective illness and narcolepsy, Annu. Rev. Pharmacol. Toxicol. 27 (1987) 137-156]. In the present study, specific aspects of the cholinergic system were examined in the pontine region of the FSL rats. The number of cholinergic neurons in the LDT and PPT were not different in FSL and control rats. Analysis of steady state levels of mRNAs encoding the acetylcholine synthesizing protein, choline acetyltransferase (ChAT) or the m2, m3 and m5 muscarinic receptor subtypes were also comparable in FSL and control rats. These data raise the possibility that the cellular events underlying the altered REM sleep pattern in FSL rats may include mechanisms that effect the muscarinic or nicotinic receptor in the pons.
Brain Res Mol Brain Res 1998 Apr
PMID:Expression of cholinergic markers in the pons of Flinders rats. 958 25

We previously described that the major promoter (M) of human choline acetyltransferase (ChAT) gene is activated by three inhibitors of histone deacetylase, butyrate, trichostatin and trapoxin, in transfected CHP126 neuroepithelioma cells. We now show that trapoxin and butyrate triggered a rapid and transient phosphorylation of ERK1/2 kinases, that was suppressed by PD98059, a highly specific inhibitor of MAP kinase kinase MEK1. The stimulation of ChAT promoter activity by trapoxin or butyrate did not require ongoing protein synthesis, and was suppressed by PD98059. The overexpression of dominant negative mutants of H-ras or ERK2 proteins depressed ChAT promoter activation by trapoxin in transient transfection assays. Conversely, the overexpression of constitutively active mutants of H-ras or MEK1 proteins had little or no effect on ChAT promoter activity, but strongly synergized with trapoxin. These data thus suggest that the activation of the MEK/ERK kinase cascade plays a necessary, but not sufficient, role in the regulation of ChAT promoter by inhibitors of histone deacetylase.
Brain Res Mol Brain Res 1998 May
PMID:Activation of the MAP kinase cascade by histone deacetylase inhibitors is required for the stimulation of choline acetyltransferase gene promoter. 960 89

The objective of this study was to examine the role of nerve growth factor (NGF) in regulation of expression of the cholinergic phenotype. NGF was administered to PC12 cells or primary cultures of embryonic (E17) rat basal forebrain for 2 days, then steady-state levels of choline acetyltransferase (ChAT) mRNA was monitored. Expression of ChAT mRNA isoforms was investigated using reverse transcription-polymerase chain reaction (RT-PCR) to amplify different upstream regions of the ChAT transcripts, and Southern blot analysis was used to verify identity of the PCR products. An NGF-induced increase of 1.8- and 1.5-fold in steady-state level of the ChAT transcript containing the M-exon (M-ChAT) was observed in PC12 cells and embryonic rat basal forebrain neurons, respectively. Also, a 2-fold increase in ChAT protein as determined by western blot analysis was associated with an NGF-mediated increase of 1.7-fold in ChAT activity in rat basal forebrain neurons within the same cultures following 4 days of NGF treatment.
Brain Res Mol Brain Res 1998 Nov 12
PMID:NGF-induction of the expression of ChAT mRNA in PC12 cells and primary cultures of embryonic rat basal forebrain. 979 14


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