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

The role of adenosine 5'-triphosphate (ATP) and P2Y(1) nucleotide receptor in potentiating agrin-induced acetylcholine receptor (AChR) aggregation is being demonstrated in a co-culture system of NG108-15 cell, a mouse neuroblastoma X rat glioma hybrid cell line that resembles spinal motor neuron, with C2Cl2 myotube. In the co-cultures, antagonized P2Y1 receptors showed a reduction in NG108-15 cell-induced AChR aggregation. Parallel to this observation, cultured NG108-15 cell secreted ATP into the conditioned medium in a time-dependent manner. Enhancement of ATP release from the cultured NG108-15 cells by overexpression of active mutants of small GTPases increased the aggregation of AChRs in co-culturing with C2C12 myotubes. In addition, ecto-nucleotidase was revealed in the co-culture, which rapidly degraded the applied ATP. These results support the notion that ATP has a role in directing the formation of post-synaptic apparatus in vertebrate neuromuscular junctions. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

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ATP induces post-synaptic gene expressions in vertebrate skeletal neuromuscular junctions

In vertebrate neuromuscular junctions (nmjs), adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Several lines of evidence suggest that the synaptic ATP can act as a synapse-organizing factor at the nmjs, mediated by metabotropic P2Y(1) receptors. P2Y(1) receptor mRNAs in chicken and rat muscles are low in embryo but increases markedly in the adult, and decreased after denervation. The P2Y(1) receptor protein is restricted to the nmjs and co-localized with AChRs in adult muscles. The activation of P2Y(1) receptor by adenine nucleotides in cultured chick myotubes stimulated the accumulation of inositol phosphates, intracellular Ca2+ mobilization, protein kinase C activity and phosphorylation of extracellular signal-regulated kinases. The receptor activation led to an increase in the expression of transcripts encoding AChE catalytic subunit and AChR subunits. The ATP-induced post-synaptic gene expression is possibly mediated by the activation of signaling cascades of mitogen-activated protein kinase. Therefore, a model is being proposed here that the synaptic ATP has a role of synergy with other regulatory signals, such as neuregulin, which act via their post-synaptic receptors to activate second signaling molecules locally to enhance the transcription of AChR/AChE genes specifically in the adjacent sub-synaptic nuclei during the formation and, especially, the maintenance of post-synaptic specializations at the nmjs