Aims: 

In human skeletal muscle, ATP is released at the neuromuscular junction. We could recently show that ATP activates ERK1/2 via P2Y1 receptor under inhibitory conditions of extracellular nucleotide hydrolysis. Here we allowed hydrolysis of ATP to test the hypothesis that adenosine receptor might be activated.

Methods: 

In the absence of an ATP regenerating system ATP was applied to differentiated human skeletal muscle cells and the formation of cAMP and the phosphorylation of ERK1/2 was investigated.

Results: 

ATP is extensively hydrolysed when applied to differentiated human skeletal muscle cells in the absence of an ATP regenerating system. Basal and forskolin stimulated formation of cAMP was significantly enhanced in the presence of 100 mM ATP. This ATP induced increment in adenylyl cyclase activity was completely abrogated by the universal adenosine receptor antagonist xanthine amine congener (XAC). A cross-activation of the cAMP formation by ATP via P2Y receptors can be excluded because suramin failed to inhibit. Adenosine receptor mediated cAMP production was further corroborated by stimulation with the adenosine analogue 5'-N-ethyl-carboxamidoadenosine (NECA) which again was inhibited by XAC. Using the specific A2B receptor antagonist PSB-1115 and the potent inhibitor ZM241385 allowed for the pharmacological identification of an A2B receptor. This is further confirmed by the observation that already nanomolar concentrations of NECA are sufficient to activate ERK1/2.

Conclusion: 

These results demonstrate for the first time that nanomolar concentrations of NECA are sufficient to trigger ERK1/2 phosphorylation via an A2B receptor while adenylyl cyclase was not activated under these conditions.