Extracellular ATP activates ERK1/ERK2 via a metabotropic P2Y 1 receptor in a Ca 2+ independent manner in differentiated human skeletal muscle cells

Abstract

ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca 2+ transients in these cells via P2Y receptors which were not attributable to Ca 2+ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC 50 of 21.3 μM. The Ca 2+ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca 2+ pool was not involved in ATP triggered Ca 2+ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y 1, P2Y 2, P2Y 4 and P2Y 6 receptors. Substantially, ATP activated IP formation via a P2Y 1 receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y 1 receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca 2+ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP 3 receptor mediated Ca 2+ transients and Ca 2+ insensitive phosphorylation of ERK1/2.