Extracellular ATP promotes protein synthesis in skeletal muscle through activation of the Akt-mTOR signaling pathway.

Objective
Extracellular nucleotides are released from different cells surrounding skeletal muscle, like motoneuron, endothelial cells, fibroblasts and bone cells during exercise. Even the muscle fibers themselves release ATP during membrane depolarization and contraction. Could this ATP-enriched environment modulate protein synthesys in skeletal muscle? In this study, we assessed if extracellular ATP leads to protein synthesis in adult muscle through activation of Akt-mTOR signaling pathway.

Methods
Flexor Digitorium Brevis (FDB) muscles from 6–7 weeks-old BalbC mice were dissected. Isolated muscles were stimulated with exogenous ATP in a time- and concentration-dependent manner (3–20 min, 0.1 – 100 μM). The relative protein levels of p-Akt(Ser473), p-mTOR( Ser2448), p-p70S6K(Thr389) and p-4E-BP1(Thr37/46) were evaluated by immunoblot. Normalization with the non-phosphorylated protein and GAPDH was performed. The protein synthesis was estimated by the nonradioactive SUrface SEnsing of Translation (SUnSET) technique.

Results
100 μM ATP increased p-Akt(Ser473) levels by 3-fold in 10–15 min. Akt phosphorylation was concentration-dependent, with a bell-shaped curve that peaked in 3 μM ATP. Phosphorylation of Akt signalling-related proteins (mTOR, p70S6K, and 4E-BP1) and protein synthesis levels were also increased after 10–20 min with 3 μM ATP and UTP. Preincubation for 30 min with a general P2Y/P2X receptors antagonist (100 μM suramin), PI3-kinase inhibitor (50 μM LY294002) or mTOR inhibitor (100 nM Rapamycin) abolished the effect of ATP on the protein synthesis.

Conclusion
These findings suggest that extracellular ATP promotes protein synthesis in skeletal muscle through the activation of P2YRs and the Akt-mTOR signaling pathway. Considering that effect on protein synthesis was mimicked by UTP (P2Y2R/P2Y4R agonist), and that P2Y2R is the mostly expressed nucleotide receptor in FDB muscle (as we previously described), a relevant role of P2Y2R in controlling protein synthesis in skeletal muscles is proposed. This proposed pathway could have a fine-tuned role in the regulation of muscle mass during the integrated musculoskeletal activity.