Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g., neostigmine). Nicotinic α7 receptors (α7nAChR) on perisynaptic Schwann cells (PSCs) can control ACh spillover by unknown mechanisms. We hypothesized that adenosine might be the gliotransmitter underlying PSCs‐nerve terminal communication. Rat isolated hemidiaphragm preparations were used to measure (1) the outflow of [3H]ACh, (2) real‐time transmitter exocytosis by video‐microscopy with the FM4‐64 fluorescent dye, and (3) skeletal muscle contractions during high‐frequency (50 Hz) nerve stimulation bursts in the presence of a selective α7nAChR agonist, PNU 282987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that α7nAChR‐mediated effects require direct activation of PSCs, we used fluorescence video‐microscopy in the real‐time mode to measure PNU 282987‐induced [Ca2+]i transients from Fluo‐4 NW loaded PSCs in non‐stimulated preparations. The α7nAChR agonist, PNU 282987, decreased nerve‐evoked diaphragm tetanic contractions. PNU 282987‐induced inhibition was mimicked by neostigmine and results from the reduction of ACh exocytosis measured as decreases in [3H]ACh release and FM4‐64 fluorescent dye unloading. Methyllycaconitine blockage of α7nAChR and the fluoroacetate gliotoxin both prevented inhibition of nerve‐evoked ACh release and PSCs [Ca2+]i transients triggered by PNU 282987 and neostigmine. Adenosine deamination, inhibition of the ENT1 nucleoside outflow, and blockage of A1 receptors prevented PNU 282987‐induced inhibition of transmitter release. Data suggest that α7nAChR controls tetanic‐induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A1 inhibitory receptors. image

Background/Aims: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A_2A and muscarinic M₁ receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. Methods: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500–750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. Results: Activation of A_2A and M₁ receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 μmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 μmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 μmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 μmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 μmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 μmol/L) was also prevented by HC-3 (4 μmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 μmol/L). Conclusion: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A_2A and M₁ receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.