The activation of P Rs modulates

The activation of P1Rs modulates the activity of many ion channels and receptors (Sebastião and Ribeiro, 2009) and the recent detection of P1Rs at the postsynaptic level of the adult NMJ (Garcia et al., 2014) makes potentially possible an adenosine-mediated modulation of the nicotinic Imiloxan hydrochloride receptor (nAChR) channels located on the motor endplate under physiological conditions. After innervation and the release of trophic factors, the embryonic nAChR (α2βγδ) expressed in immature skeletal muscle is replaced by the adult isoform by the substitution of the γ subunit with the ε (Steinbach, 1989). The developmental switch of the subunits confers to the channel a higher conductance and a reduction in the mean open time with respect to the embryonic isoform. Moreover, after innervation, the nAChRs become mainly clustered at the endplate region and not spread along the cell surface as in immature muscle (Kummer et al., 2006). The channel activity of the embryonic and the adult nAChR isoforms is differentially modulated by protein kinases and by second messenger signaling pathways (Huganir and Miles, 1989). In a previous study, we demonstrated that endogenous adenosine released by contracting myotubes in culture, increased the open probability and mean open time of the embryonic isoform of the nAChR-ion channel by acting via A2B type receptors (Bernareggi et al., 2015). Therefore, adenosine, as well as ACh (Bandi et al., 2005), could play an important role to sustain mechanical activity and muscle trophism during the early stages of myogenesis, when nerve contact is not yet established. In the present work, the possible interplay between postsynaptic P1Rs and the adult isoform of the nAChRs was investigated, because a possible adenosinergic regulation of the cholinergic receptors at the mature endplate region of skeletal muscle fibers could result in a significant change of muscle activity. To address this issue, we recorded nAChR-mediated miniature endplate currents (MEPCs) at the mouse NMJ and nAChR-activated single-channel currents at the motor end plate region of isolated mouse muscle fibers.
Experimental procedures
Results
Discussion Recent reports have clearly demonstrated the presence of postsynaptic P1Rs at the adult mammalian NMJ (Garcia et al., 2013, Garcia et al., 2014). However, their functional role (if any) has so far remained unknown. In this study, we provide the first evidence in favor of a distinct postsynaptic modulatory effect of adenosine mediated via P1Rs. Our data also suggest the A2B subtype as the major contributor in the adenosine-mediated modulation of nAChR channel activity in mammalian skeletal muscle fibers. There is a general consensus in considering adenosine as an important neuromodulator of synaptic transmission in the central nervous system, where it elicits its effects by binding to P1Rs expressed both at the presynaptic and postsynaptic level as well as on glial cells (Burnstock, 2015). At the level of the NMJ, pre- and post-synaptic actions of adenosine might also occur. The presynaptic effects are well established in many species, included rodents, where adenosine acts on high-affinity P1Rs at the cholinergic nerve endings (Ginsborg and Hirst, 1972, Ribeiro and Sebastião, 1987, Ribeiro and Sebastião, 1988, Sebastião et al., 1990, Silinsky, 2004, De Lorenzo et al., 2004, Garcia et al., 2013). The postsynaptic effects of adenosine however, are only now starting to be identified (Garcia et al., 2013, Garcia et al., 2014). The main result of the present work is the observation that, at the adult NMJ, adenosine significantly modulates the activity of nAChRs also at the postsynaptic level. We provide two pieces of experimental evidence in favor of this novel mechanism. The first evidence is at the mouse diaphragm NMJ where a change of MEPC kinetics was observed in the presence of adenosine. This is in addition to the modulation of MEPC frequency due to the known presynaptic effect, well described at the rat (Ginsborg and Hirst, 1972, Correia-de-Sá et al., 1991, Pousinha et al., 2010), frog (Giniatullin and Sokolova, 1998) and mouse (Silinsky, 2004, De Lorenzo et al., 2004, Garcia et al., 2013) NMJs.