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Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.
Choi RC
,
Man ML
,
Ling KK
,
Ip NY
,
Simon J
,
Barnard EA
,
Tsim KW
.
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In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.
Fig. 7.
The localizations of P2Y1 receptor in muscles and motor neurons. A, Chick, rat, orXenopus muscle section (20 μm) was used. For each, the same field is shown stained by the anti-P2Y1-receptor antibody (green) or for AChR (red) by TMR-BuTX (10 nM) or superimposed (yellow). Controls are noted in Results. Scale bar, 20 μm. B, Spinal cord from adult chicken. Peroxidase-conjugated secondary antibody was used here to reveal (brown) the P2Y1 receptor sites in the ventral horn. In the control, the antibody was pretreated with an excess of the recombinant P2Y1 receptor antigen. A high-power magnification is shown for two adjacent positive cells seen in the stained low-power field. Scale bars, 500 μm (low power); 100 μm (high power).
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