Purinergic signalling during myogenesis: a role for adenosine and its receptors

During embryogenesis, before functional activation of the motor nerve, the nicotinic acetylcholine receptor (nAChR; 2a1b1cd) is regulated mainly by autocrine processes that control the maturation of skeletal muscle. A lack of, or alteration to, this regulation can lead to a series of potentially serious modifications of the differentiation processes of muscle fibres. In the early phases of the myogenic process, the application of exogenous ACh and/or cholinergic agonists promotes the fusion of myoblasts into myotubes through the activation of nAChR channels. The release of ACh or similar substances from immature skeletal muscle fibres was proposed almost 40 years ago by Miledi et al. (1977) and subsequently confirmed by Hamann et al. (1995). Moreover, considering that the opening of the embryonic muscle nAChR channel contributes to spontaneous transients of increased intracellular Ca concentration ([Ca]i) and to twitches that are characteristic of developing myotubes before innervation, it has also been proposed that autocrine actions of ACh-like substances that can increase the opening probability of the nAChR channels might be associated with the release of Ca from the sarcoplasmic reticulum. In mature muscle, several substances are believed to have roles as endogenous ACh-releasing agents. Among these, special attention needs to be given to the endogenous purine nucleosides, as these can modulate many physiological processes through their links with at least four known subtypes of the P1 G-protein-coupled adenosine receptors: A1, A2A, A2B and A3 (Li et al. 2014). Until the late 1970s, experimental evidence in mature muscle indicated that ATP and adenosine functions inhibit the release of ACh from nerve terminals (Ribeiro & Dominguez 1978, Silinsky 1984). However, not only do the sites of ATP and adenosine actions appear to be different, but also their influences on ACh release from nerve endings appear specific. Indeed, ATP has different actions according to the level of maturation of the neuromuscular junction. During the maturation of neuromuscular junctions, the main site of ATP action is at the level of the postsynaptic P2X receptor ion channels, where it acts as a co-transmitter, to facilitate the actions of ACh. In contrast, in the mature neuromuscular system, ATP acts as a modulator at both pre-junctional and post-junctional sites (Burnstock 2007). It has also been shown that ACh and ATP (the deamination of which produces adenosine outside the cell) are both released by the exocytosis of synaptic vesicles and that there is a mechanism for pre-synaptic adenosine re-uptake for the reconstitution of stored ATP (Burnstock et al. 2013). This adenosine that is formed extracellularly can act on the A1 adenosine receptors, which results in negative effects on the pre-synaptic release of ACh (Barroso et al. 2007). Also, it has been demonstrated that pre-synaptically, there are both A1 inhibitory and A2A facilitatory adenosine receptors, although the A1 receptors are more abundant in adults and the A2A receptors are more abundant in the newborn (Garcia et al. 2013). Therefore, there is an apparent discrepancy between the assumed inhibitory action of adenosine when there are nerve endings present (in the adult) and the stimulatory effects that are induced by adenosine during skeletal muscle development when there is an absence of active nerve endings. The first consideration here is to determine whether there are significant levels of adenosine or ATP, its precursor, in the interstitial space of immature muscle cells (i.e. myoblasts) and during their progressive differentiation and fusion to form myotubes. The second important issue is to determine the distribution of the P1 adenosine receptors to which adenosine can bind with high specificity on the outer membranes of muscle fibres during their differentiation. However, it appears that both A1 and A2A adenosine receptors are required at the motor endings if they are to be directly involved in the modulation of ACh release. In mature muscle, there are relatively high levels of ATP in the interstitium between the muscle fibres, which can also be released directly from damaged muscle fibres as a consequence of muscle exercise, as well as from necrotic muscle cells and vascular endothelial cells of the epithelium (Mortensen et al. 2009, Bustamante et al. 2014, Le et al. 2014). To generate extracellular adenosine from such released adenine nucleotides, the dephosphorylation of AMP by ecto-50nucleotidase is the rate-limiting step. Indeed, adenosine derived from the breakdown of ATP is known to