Noronha-Matos_2024_Biochem.Pharmacol__116649

Nicotinic alpha7 receptors (alpha7 nAChRs) present in perisynaptic Schwann cells (PSCs) control acetylcholine (ACh) spillover from the neuromuscular synapse by transiently increasing intracellular Ca(2+), which fosters adenosine release via type 1 equilibrative nucleoside transporters (ENT1) and retrograde activation of presynaptic A(1) inhibitory receptors. The putative Ca(2+)-dependent pathways downstream alpha7 nAChRs involved in the sensing inhibitory drive operated by PSCs is unknown. Herein, we used phrenic nerve-hemidiaphragm preparations from Wistar rats. Time-lapse video-microscopy was instrumental to assess nerve-evoked (50-Hz bursts) transmitter exocytosis and intracellular NO oscillations in nerve terminals and PSCs loaded with FM4-64 and DAF-FM diacetate fluorescent dyes, respectively. Selective activation of alpha7 nAChRs with PNU 282987 reduced transmitter exocytosis (FM4-64 dye unloading) during 50-Hz bursts. Inhibition of calmodulin activity (with W-7), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII; with KN-62) and Rho-kinase (ROCK; with H1152) all prevented the release inhibitory effect of PNU 282987. The alpha7 nAChR agonist transiently increased NO inside PSCs; the same occurred during phrenic nerve stimulation with 50-Hz bursts in the presence of the cholinesterase inhibitor, neostigmine. The nitric oxide synthase (NOS) inhibitor, L-NOARG, but not with the guanylylcyclase (GC) inhibitor, ODQ, prevented inhibition of transmitter exocytosis by PNU 282987. Inhibition of adenosine kinase with ABT 702 favors the intracellular accumulation and translocation of the nucleoside to the synaptic cleft, thus overcoming prevention of the PNU 282987 effect caused by H1152, but not by L-NOARG. In conclusion, the alpha7nAChR-mediated cholinergic inhibitory drive operated by PSCs involves two distinct Ca(2+)-dependent intracellular pathways: a CaMKII/ROCK cascade along with a GC-independent NO pathway with divergent end-effects concerning ADK inhibition.