Question: 

In the absence of nerve impulses, the release of ACh from the motor nerve ending mostly occurs non-quantally.The effect of ATP, which is released together with acetylcholine (ACh), on the non-quantal ACh release (NQR) in rat diaphragm endplates, was studied. NQR release can perform a important neurotrophic function in endplate development and in maintaining the morphological and functional properties of skeletal muscles and probably also of the heart and airways. We therefore checked what kind of purine receptors are involved.

Method: 

Intracellular microelectrodes were used. NQR was estimated by the amplitude of endplate hyperpolarization (the H-effect) following the blockade of postsynaptic nicotinic receptors and cholinesterase.

Results: 

We found that 100 mM 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. Howewer, the effect of ATP disappeared after the application of the broad spectrum P2 receptor antagonist suramin, metabotrophic P2Y receptor blocker reactive blue 2 and U73122, an inhibitor of phospholipase C. P2Y-mediated regulation is not coupled to presynaptic voltage-dependent Ca²⁺ 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.

Conclusions: 

Unlike the effects of purines on the spontaneous quantal secretion of ACh, its NQR is regulated via P2Y receptors coupled to G_q/11 and and PLC-PKC route. ATP thus regulates the neuromuscular synapse by two different pathways. Mechanism and three main regulations of NQR (purinergic, NO and glutamatergic) in neuromuscular junction are also presented (for details see Vysko[ccaron]il, Malomouzh, Nikolsky, Physiol. Res. 2010, 58:763–784 and Malomouzh, Nikolsky, Vysko[ccaron]il, Neurosci Res. 2011, 71:219–225).

Figure: Mechanism and regulation of non-quantal release (NQR) of acetylcholine (ACh) in neuromuscular junction.

Cytosolic ACh is thought to transit the nerve terminal membrane through vesicle-associated ACh transporters (VAChT) (1), which retain their orientation following the incorporation of the vesicle membrane into the axon membrane (2), mostly during full collapse exocytosis (kiss-and-stay). VAChTs move ACh from the axoplasm into the extracellular space and the synaptic cleft until they are retracted from the membrane during endocytosis (3). Dotted arrows- ATP pathway (ATP) from axoplasm to vesicles, from vesicles to synaptic cleft, GQ/11-coupled P2Y receptors and PCL and PKC, which negatively (-) influence NQR. Another pathway is glutamatergic (Glu). Glutamate exerts a strong inhibitory effect on the NQR of ACh, mediated by the activation of NMDA receptors and the entry of Ca²⁺ due to a subsequent increase in the activity of a Ca²⁺-dependent NO-synthase. NO is formed in the muscle fibers, it then diffuses through the synaptic cleft and acts retrogradely at the motor nerve terminals, where it activates NO-sensitive guanylyl cyclase. This ultimately leads to a reduction in NQR. Glutamate, which decreases NQR, can be released not only from the motor nerves, but can also be formed from the very common neuropeptide N-acetylaspartylglutamate in the synaptic gap. Last but not least, there is cholinergic regulation (ACh). Classical muscarinic agonists reduce NQR. via M1 receptors and requires extracellular Ca².This points to the possibility that in both cases there is a Ca²⁺-dependent synthesis of NO which inhibits the NQR. The inhibitory effect of ACh and Glu is completely absent in the presence of NO-cascade inhibitors, namely L-NAME, (NO-synthase inhibitor), hemoglobin (a scavenger of extracellular NO molecules), ODQ (an inhibitor of NO-sensitive guanylyl cyclase) and the calmodulin (CaM) antagonist calmidazolium.

Figure 1