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A cDNA encoding Xenopus P2Y(1) receptor was isolated by cross-hybridization with chicken homolog. The deduced amino acid sequence of Xenopus P2Y(1) receptor with 361 amino residues is 80-85\% identical to human, rodent and avian homologs. When the cDNA was expressed in mammalian cells, the activation of Xenopus P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphate, and adenosine 3',5'-bismonophosphate (A3P5P) or other antagonists blocked its action; these pharmacological properties showed resemblance of P2Y(1) receptor family in higher vertebrate. A transcript encoding Xenopus P2Y(1) receptor at similar to 3.2 kb was revealed in the brain, spinal cord and muscle of adult Xenopus, and it is strongly expressed in developing brain, spinal cord and myotomal muscles of the embryos by in situ hybridization. P2Y(1) receptor was shown to be restricted to the neuromuscular junctions and co-localized with AChRs in adult muscle. These results support the notion that ATP and its P2Y(1) receptor subtype are effectors in organizing the post-synaptic apparatus.

A cDNA encoding Xenopus P2Y(1) receptor was isolated by cross-hybridization with chicken homolog. The deduced amino acid sequence of Xenopus P2Y(1) receptor with 361 amino residues is 80-85\% identical to human, rodent and avian homologs. When the cDNA was expressed in mammalian cells, the activation of Xenopus P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphate, and adenosine 3',5'-bismonophosphate (A3P5P) or other antagonists blocked its action; these pharmacological properties showed resemblance of P2Y(1) receptor family in higher vertebrate. A transcript encoding Xenopus P2Y(1) receptor at similar to 3.2 kb was revealed in the brain, spinal cord and muscle of adult Xenopus, and it is strongly expressed in developing brain, spinal cord and myotomal muscles of the embryos by in situ hybridization. P2Y(1) receptor was shown to be restricted to the neuromuscular junctions and co-localized with AChRs in adult muscle. These results support the notion that ATP and its P2Y(1) receptor subtype are effectors in organizing the post-synaptic apparatus.

A cDNA encoding Xenopus P2Y(1) receptor was isolated by cross-hybridization with chicken homolog. The deduced amino acid sequence of Xenopus P2Y(1) receptor with 361 amino residues is 80-85\% identical to human, rodent and avian homologs. When the cDNA was expressed in mammalian cells, the activation of Xenopus P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphate, and adenosine 3',5'-bismonophosphate (A3P5P) or other antagonists blocked its action; these pharmacological properties showed resemblance of P2Y(1) receptor family in higher vertebrate. A transcript encoding Xenopus P2Y(1) receptor at similar to 3.2 kb was revealed in the brain, spinal cord and muscle of adult Xenopus, and it is strongly expressed in developing brain, spinal cord and myotomal muscles of the embryos by in situ hybridization. P2Y(1) receptor was shown to be restricted to the neuromuscular junctions and co-localized with AChRs in adult muscle. These results support the notion that ATP and its P2Y(1) receptor subtype are effectors in organizing the post-synaptic apparatus.

The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a similar to2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP-responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.

The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a similar to2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP-responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to similar to 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors, in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant G alpha(s) increased the expression of AChE mRNA and protein to similar to 2-fold, while the constitutively active mutant G alpha(i) cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at similar to 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway. NeuroReport 11:801-806 (C) 2000 Lippincott Williams & Wilkins.

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to similar to 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors, in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant G alpha(s) increased the expression of AChE mRNA and protein to similar to 2-fold, while the constitutively active mutant G alpha(i) cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at similar to 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway. NeuroReport 11:801-806 (C) 2000 Lippincott Williams & Wilkins.

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to similar to 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors, in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant G alpha(s) increased the expression of AChE mRNA and protein to similar to 2-fold, while the constitutively active mutant G alpha(i) cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at similar to 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway. NeuroReport 11:801-806 (C) 2000 Lippincott Williams & Wilkins.