NO decreases evoked quantal ACh release at a synapse of Aplysia by a mechanism independent of Ca2+ influx and protein kinase G.

1. The exogenous nitric oxide (NO) donor, SIN-1, decreased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic neuro-neuronal synapse in the buccal ganglion of Aplysia californica. 2. The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage-clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was decreased by SIN-1, pointing to a presynaptic action of the drug. 3. Vitamin E, a scavenger of free radicals, prevented the effects of SIN-1 on ACh release. SIN-1 still decreased ACh release in the presence of superoxide dismutase, whereas haemoglobin suppressed the effects of SIN-1. These results showed that NO is the active compound. 4. 8-Bromoguanosine 3', 5' cyclic monophosphate (8-Br-cGMP) mimicked the inhibitory effect of NO on ACh release suggesting the involvement of a NO-sensitive guanylate cyclase. This was reinforced by the reversibility of the effects of SIN-1 by inhibitors of guanylate cyclase, Methylene Blue, cystamine or LY83583. Methylene Blue partially reduced the inhibitory effect of NO. In addition, in the presence of superoxide dismutase, Methylene Blue blocked and cystamine significantly reduced the NO-induced inhibition of ACh release. 5. In the presence of KT5823 or R-p-8-pCPT-cGMPS, two inhibitors of protein kinase G, the reduction of ACh release by SIN-1 still took place indicating that the effects of NO most probably did not involve protein kinase G-dependent phosphorylation. 6. Presynaptic voltage-dependent Ca2+ (L-, N- and P-types) and K+ (IA and late outward rectifier) currents were unmodified by SIN-1. 7. The modulation of ACh release in opposite ways by L-arginine and N omega-nitro-L-arginine points to the involvement of an endogenous NO synthase-dependent regulation of transmitter release.