ATP potentiates the formation of AChR aggregate in the co‐culture of NG108‐15 cells with C2C12 myotubes

Ling, Kky; Siow, Nina L.; Choi, Roy Chi Yan; Tsim, Karl Wah Keung

doi:10.1016/j.febslet.2005.03.054

Cited by 12 publications

(9 citation statements)

References 32 publications

(56 reference statements)

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“…The correlation between extracellular degradation of cAMP and formation of adenosine observed in rat primary muscle cultures is in agreement with the expression of ecto-NTs described in chicken (Delgado et al, 1997) and mouse skeletal muscle (Martínez-Martínez et al, 1998) and C2C12 cultured cells (Ling et al, 2005). However, in the previous studies, this cell-surface enzyme was essentially associated with the extracellular metabolism of ATP, considered to be the only relevant source of adenosine outside the cell (Cunha et al, 1996;Delgado et al, 1997;Martínez-Martínez et al, 1998).…”

Section: Discussionsupporting

confidence: 88%

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

Chiavegatti¹,

Costa²,

Araújo³

et al. 2008

British J Pharmacology

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Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5 0 -AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5 0 -AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications:Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine.

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Section: Discussionsupporting

confidence: 88%

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

Chiavegatti¹,

Costa²,

Araújo³

et al. 2008

British J Pharmacology

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“…The concentration of ambient ATP in the bulk phase above the monolayer of cortical neurons in our conditions was measured and found to be low, being limited by the high surface ectonucleotidase activity, but it was increased ϳ6-fold when the cultures contained freely growing glial cells (Supplemental Data S3). In agreement, we have found a similar endogenous release of glial ATP from the NG108-15 neuroblastoma/glioma cells cultured alone (Ling et al, 2005). For comparison, activation of P2YRs on cultured cortical astrocytes strongly mobilizes their intracellular Ca 2ϩ to release glutamate, which, when occurring in situ, would activate adjacent neuronal NMDA receptors (Lee et al, 2007).…”

Section: Resultssupporting

confidence: 70%

ATP Induces Synaptic Gene Expressions in Cortical Neurons: Transduction and Transcription Control via P2Y₁ Receptors

Siow¹,

Choi²,

Xie³

et al. 2010

Mol Pharmacol

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Studies in vertebrate neuromuscular synapses have revealed previously that ATP, via P2Y receptors, plays a critical role in regulating postsynaptic gene expressions. An equivalent regulatory role of ATP and its P2Y receptors would not necessarily be expected for the very different situation of the brain synapses, but we provide evidence here for a brain version of that role. In cultured cortical neurons, the expression of P2Y 1 receptors increased sharply during neuronal differentiation. Those receptors were found mainly colocalized with the postsynaptic scaffold postsynaptic density protein 95 . This arises through a direct interaction of a PDZ domain of PSD-95 with the C-terminal PDZ-binding motif, D-T-S-L of the P2Y 1 receptor, confirmed by the full suppression of the colocalization upon mutation of two amino acids therein. This interaction is effective in recruiting PSD-95 to the membrane.Specific activation of P2Y 1 (G-protein-coupled) receptors induced the elevation of intracellular Ca 2ϩ and activation of a mitogen-activated protein kinase/Raf-1 signaling cascade. This led to distinct up-regulation of the genes encoding acetylcholinesterase (AChE T variant), choline acetyltransferase, and the N-methyl-D-aspartate receptor subunit NR2A. This was confirmed, in the example of AChE, to arise from P2Y 1 -dependent stimulation of a human ACHE gene promoter. That involved activation of the transcription factor Elk-1; mutagenesis of the ACHE promoter revealed that Elk-1 binding at its specific responsive elements in that promoter was induced by P2Y 1 receptor activation. The combined findings reveal that ATP, via its P2Y 1 receptor, can act trophically in brain neurons to regulate the gene expression of direct effectors of synaptic transmission.

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“…Nerve-muscle co-culture procedure was performed as it was first described by Chen et al (2005) and Ling et al (2005). Briefly, approximately 2.5 × 10 5 cells were grown as myoblasts on 35-mm culture dishes in culture medium: DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic for three days.…”

Section: Methodsmentioning

confidence: 99%

“…A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma × neuroblastoma hybrid cells (NG108-15) were used to study nerve-muscle interactions in vitro . Because of their similar characteristics to their counterparts in vivo , the cell lines used in this study have been widely used by researchers in physiological and pharmacological studies (Ling et al 2005). …”

Section: Introductionmentioning

confidence: 99%

Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture

Vianney

Spitsbergen

2011

Brain Research

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Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent survival factor for both central and peripheral neurons. GDNF has been shown to be a potent survival factor for motor neurons during programmed cell death and continuous treatment with GDNF maintains hyperinnervation of skeletal muscle in adulthood. However, little is known about factors regulating normal production of endogenous GDNF in skeletal muscle. This study aimed to examine the role that motor neurons play in regulating GDNF secretion by skeletal muscle. A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma × neuroblastoma hybrid cells (NG108-15) were used to create nerve muscle interactions in vitro. Acetylcholine receptors (AChRs) on nerve-myotube co-cultures were blocked with alpha bungarotoxin (α-BTX). GDNF protein content in cells and in culture medium was analyzed by enzyme-linked immunosorbant assay (ELISA) and western blotting. GDNF localization was examined by immunocytochemistry. The nerve-muscle co-culture study indicated that the addition of motor neurons to skeletal muscle cells reduced the secretion of GDNF by skeletal muscle. The results also showed that blocking AChRs with α-BTX reversed the action of neural cells on GDNF secretion by skeletal muscle. Although ELISA results showed no GDNF in differentiated NG108-15 cells grown alone, immunocytochemical analysis showed that GDNF was localized in NG108-15 cells co-cultured with C2C12 myotubes. These results suggest that motor neurons may be regulating their own supply of GDNF secreted by skeletal muscle and that activation of AChRs may be involved in this process.

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ATP potentiates the formation of AChR aggregate in the co‐culture of NG108‐15 cells with C2C12 myotubes

Cited by 12 publications

References 32 publications

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

ATP Induces Synaptic Gene Expressions in Cortical Neurons: Transduction and Transcription Control via P2Y₁ Receptors

Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture

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ATP potentiates the formation of AChR aggregate in the co‐culture of NG108‐15 cells with C2C12 myotubes

Cited by 12 publications

References 32 publications

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

ATP Induces Synaptic Gene Expressions in Cortical Neurons: Transduction and Transcription Control via P2Y1 Receptors

Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture

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ATP Induces Synaptic Gene Expressions in Cortical Neurons: Transduction and Transcription Control via P2Y₁ Receptors