Regulatory role of ATP at developing neuromuscular junctions

Abstract

Neuronal factors co-released with neurotransmitters may play an important role in synaptic development and function. Extracellular application of adenosine 5'-triphosphate (ATP), a substance co-stored and co-released with acetylcholine (ACh) in peripheral nervous systems, potentiated the spontaneous secretion of ACh at developing neuromuscular synapses in Xenopus 1-day-old cell cultures, as shown by a marked increase in the frequency of spontaneous synaptic currents recorded in the post-synaptic muscle cell. ATP also increased the frequency of miniature endplate potentials in the isolated tails of 2-week-old Xenopus tadpoles, with much smaller effect than that observed in cell cultures. The potentiation effect of ATP on ACh release in Xenopus cell cultures was inhibited by L-type Ca2+ channel blockers, suggesting that the L-type Ca2+ channel is responsible for the positive regulation of spontaneous ACh secretion by ATP at the developing neuromuscular synapses. The frequency of spontaneous synaptic events was found to vary greatly from cell to cell in the culture, over two orders of magnitude. Synapses with high frequency events are probably under the influence of endogenously released ATP. In addition, ATP was shown to potentiate the responses of isolated myocytes to iontophoretically-applied ACh. Local application of ATP to one region of the elongated myocyte surface resulted in potentiated ACh responses only at the ATP-treated region. Single channel recording showed that ATP specifically increased the open time and opening frequency of embryonic-type, low conductance ACh channels. Pharmacological experiments suggest that ATP exerted both its pre- and post-synaptic effects by binding to P2-purinoceptors and activating protein kinase C. Moreover, the potentiation effects of ATP were restricted to the early stages of embryos. Taken together, these results suggest that ATP co-released with ACh or released from stimulated myocytes may promote synaptic development by potentiating pre-synaptic ACh release and post-synaptic ACh channel activity during the early phase of synaptogenesis.