Adenosine Promotes Endplate nAChR Channel Activity in Adult Mouse Skeletal Muscle Fibers via Low Affinity P1 Receptors

Abstract

Adenosine is a powerful modulator of skeletal neuromuscular transmission, operating via inhibitory or facilitatory purinergic-type P1 receptors. To date, studies have been focused mainly on the effect of adenosine on presynaptic P1 receptors controlling transmitter release. In this study, using two-microelectrode voltage-clamp and single-channel patch-clamp recording techniques, we have explored potential postsynaptic targets of adenosine and their modulatory effect on nicotinic acetylcholine receptor (nAChR)-mediated synaptic responses in adult mouse skeletal muscle fibers in vitro. In the whole-mount neuromuscular junction (NMJ) preparation, adenosine (100 μM) significantly reduced the frequency of the miniature endplate currents (MEPCs) and slowed their rising and decay time. Consistent with a postsynaptic site of action, adenosine and the potent P1 receptor agonist NECA significantly increased the open probability, the frequency and the open time of single nAChR channels, recorded at the endplate region. Using specific ligands for the P1 receptor subtypes, we found that the low-affinity P1 receptor subtype A was responsible for mediating the effects of adenosine on the nAChR channel openings. Our data suggest that at the adult mammalian NMJ, adenosine acts not only presynaptically to modulate acetylcholine transmitter release, but also at the postsynaptic level, to enhance the activity of nAChRs. Our findings open a new scenario in understanding of purinergic regulation of nAChR activity at the mammalian endplate region.

“Time window” effect of Yoda1‐evoked Piezo1 channel activity during mouse skeletal muscle differentiation

“…Postsynaptic EPCs and MEPCs recordings were performed using intracellular glass microelectrodes (tip diameter ~1 µm, resistance 3‐5 MΩ, filled with 2.5 M KCl) and the two‐electrode voltage‐clamp technique, as previously described 48‐50 . To maintain the physiological level of quantal release and avoid contractions 51 during the EPC recordings, the muscle fibres were transversely cut (“cut muscles”).…”

“…48 Postsynaptic EPCs and MEPCs recordings were performed using intracellular glass microelectrodes (tip diameter ~1 µm, resistance 3-5 MΩ, filled with 2.5 M KCl) and the two-electrode voltage-clamp technique, as previously described. [48][49][50] To maintain the physiological level of quantal release and avoid contractions 51 during the EPC recordings, the muscle fibres were transversely cut ("cut muscles"). In this case, the V h (holding potential) was kept at −40 mV and the recording started after the stabilization of the cell membrane potential (in about 40 minutes), as described elsewhere.…”

Preliminary Observations on Skeletal Muscle Adaptation and Plasticity in Homer 2-/- Mice

“…ACh (200 nM) was used in the recording pipette solution. The activity of single nAChR channels was measured in membrane patches at the level of the endplate region ( Figure 6 A), identifiable by phase-contrast microscopy as a roughness of the cell surface [ 30 ]. The unitary current amplitude distribution was best fitted by a single Gaussian curve and the open-time distribution by a single exponential, revealing the presence of a single type of nAChR channel ( Figure 6 B).…”

“…Signals were sampled at 50 kHz, filtered at 2 kHz with a low pass Bessel filter, and analysed by the pCLAMP 8.0 software package (Molecular Devices, Union City, CA, USA), using a threshold crossing criterion. All records were performed in the presence of 200 nM of ACh (Sigma-Aldrich), and for each stable patch, more than 500 events were analysed [ 30 ]. The open time distribution was best fitted with one or more exponentials if appropriate by maximum likelihood method.…”

Modulatory Roles of ATP and Adenosine in Cholinergic Neuromuscular Transmission

“…It is known that the potentiating effect of ATP is manifested in embryonic and developing muscles [89]. Adenosine increases ε-nAChR-channel activity at the end-plate in adult skeletal muscle fibers where the interplay between adenosine and cholinergic systems is mainly mediated by A 2B receptors and partially by A 3 receptors [90]. ATP increases the time of the open state and the frequency of opening of acetylcholine-activated ion channels in embryonic muscles [19,91].…”