Nitric oxide-cGMP pathway facilitates acetylcholine release and bradycardia during vagal nerve stimulation in the guinea-pig in vitro

1. We tested the hypothesis that nitric oxide (NO) augments vagal neurotran smission and bradycardia via phosphorylation of presynaptic calcium channel s to increase vesicular release of acetylcholine. 2. The effects of enzyme inhibitors and calcium channel blockers on the act ions of the NO donor sodium nitroprusside (SNP) were evaluated in isolated guinea-pig atrial-right vagal nerve preparations. 3. SNP (10 muM) augmented the heart rate response to vagal nerve stimulatio n but not to the acetylcholine analogue carbamylcholine (100 nm). SNP also increased the release of [H-3]acetylcholine in response to field stimulatio n. No effect of SNP was observed on either the release of [H-3] acetylcholi ne or the HR response to vagal nerve stimulation in the presence of the gua nylyl cyclase inhibitor 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ , 10 muM). 4. The phosphodiesterase 3 (PDE 3) inhibitor milrinone (1 muM) increased th e release of [H-3] acetylcholine and the vagal bradycardia and prevented an y further increase by SNP. SNP was still able to augment the vagal bradycar dia in the presence of the protein kinase G inhibitor KT5823 (1 muM) but no t after protein kinase A (PKA) inhibition with H-89 (0.5 muM) or KT5720 (1, um) had reduced the HR response to vagal nerve stimulation. Neither milrino ne nor H-89 changed the HR response to carbamylcholine. 5. SNP had no effect on the magnitude of the vagal bradycardia after inhibi tion of N-type calcium channels with omega -conotoxin GVIA (100 nM). 6. These results suggests that NO acts presynaptically to facilitate vagal neurotransmission via a cGMP-PDE 3-dependent pathway leading to an increase in cAMP-PKA-dependent phosphorylation of presynaptic N-type calcium channe ls. This pathway may augment the HR response to vagal nerve stimulation by increasing presynaptic calcium influx and vesicular release of acetylcholin e.