Nicotinic alpha 7 receptor-induced adenosine release from perisynaptic Schwann cells controls acetylcholine spillover from motor endplates

Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g., neostigmine). Nicotinic alpha 7 receptors (alpha 7nAChR) 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 [H-3]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 alpha 7nAChR agonist, PNU 282987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that alpha 7nAChR-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 alpha 7nAChR 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 [H-3]ACh release and FM4-64 fluorescent dye unloading. Methyllycaconitine blockage of alpha 7nAChR 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 A(1) receptors prevented PNU 282987-induced inhibition of transmitter release. Data suggest that alpha 7nAChR controls tetanic-induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A(1) inhibitory receptors.