Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm

► We studied the action of ATP on non-quantal release of ACh in rat endplates. ► ATP inhibits non-quantal release of ACh via P2Y but not P2X receptors. ► Action of ATP occurs through the activation of phospholipase C. ► Action of ATP is independent of the activity of NO-synthase and Ca 2+ channels. ► Mechanisms of ATP and glutamate inhibitory actions on ACh release are independent. We established the effect of ATP, which is released together with acetylcholine (ACh), on the non-quantal ACh release (NQR) in rat diaphragm endplates and checked what kind of purine receptors are involved. NQR was estimated by the amplitude of endplate hyperpolarization (the H-effect) following the blockade of postsynaptic nicotinic receptors and cholinesterase. 100 μM ATP reduced the H-effect to 66% of the control. The action of ATP remained unchanged after the inhibition of ionotropic P2X receptors by Evans blue and PPADS, but disappeared after the application of the broad spectrum P2 receptor antagonist suramin, metabotropic P2Y receptor blocker reactive blue 2 and U73122, an inhibitor of phospholipase C. P2Y-mediated regulation is not coupled to presynaptic voltage-dependent Ca 2+ channels. During the simultaneous application of ATP and glutamate (which is another ACh cotransmitter reducing non-quantal release), the additive depressant effect led to a disappearance of the H-effect. This can be explained by the independence of the action of ATP and glutamate. Unlike the effects of purines on the spontaneous quantal secretion of ACh, its non-quantal release is regulated via P2Y receptors coupled to G q/11 and PLC. ATP thus regulates the neuromuscular synapse by two different pathways.