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Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.
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