Breakdown of phospholipids and the elevated nitric oxide are involved in M3 muscarinic regulation of acetylcholine secretion in the frog motor synapse.

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. The scheme of ACh secretion regulation by signaling pathways triggered by M3 choline receptor activation (M3AChR - M3 muscarinic receptor, NOS - nitric oxide synthase, EC - endocannabinoids, CB1R–CB1 cannabinoid receptor, IP3 - inositol-3-phosphate, PLC - phospolipase C, PI3K - phosphoinositide-3-kinase). Image 1 • Muscarine attenuates ACh release from frog motor terminals via activation of PLC. • PI3K activity is crucial for ACh secretion recovery after muscarine removal. • Muscarine-induced decrease of ACh release is linked to upregulation of NO signaling. • CB1 receptors do not contribute significantly to in the depressant effect of muscarine. • M3 receptors may modulate ACh secretion via PLC, PI3K and NO-dependent manner. [ABSTRACT FROM AUTHOR]