Stimulation-dependent release, breakdown, and action of endogenous ATP in mouse hemidiaphragm preparation: the possible role of ATP in neuromuscular transmission

In this study the in vitro mouse phrenic nerve- hemidiaphragm preparation w as utilized to study the release and extracellular catabolism of endogenous ATP and its action on the postsynaptic site, i.e. on the contraction force evoked by nerve stimulation. ATP, measured by the luciferin-luciferase ass ay, was released stimulation-dependently from the mouse hemidiaphragm in re sponse to electrical field stimulation at 10 Hz. Blockade of the Na+ channe l activity by tetrodotoxin inhibited the majority of the release of ATP in response to stimulation, showing that it is related to neuronal activity. T he nicotinic receptor antagonists d-tubocurarine, and alpha-bungarotoxin an d cooling the bath temperature to 7 degrees C also reduced stimulation-indu ced ATP outflow, suggesting that nicotinic receptors are responsible for th e part of the release of ATP that is released from postsynaptic sites in a carrier-mediated manner. Exogenous ATP (20-500 mu M) added to the bath was degraded to ADP and AMP by the action of ectoATPase and ectoATPdiphosphohyd rolase; the K-m and nu(max) values of these enzymes were 185.8 mu M and 55. 16 nmol/min.g respectively. However, the total amount of nucleotides ([ATP + ADP + AMP]) was increased after the addition of ATP. indicating that ATP itself promoted further adenine nucleotide release. Twitch contractions of the rat hemidiaphragm preparation evoked by low frequency electrical stimul ation was blocked concentration-dependently by the nondepolarizing muscle r elaxants d-tubocurarine and pancuronium. Suramin (100 mu M-1 mM) reversed n euromuscular blockade by d-tubocurarine and pancuronium; i.e., it shifted t heir concentration-response curves to the right Taken together our data, th at endogenous ATP is released by stimulation and subsequently catabolized i n the hemidiaphragm preparation and that suramin inhibits ecto-ATPase activ ity could be interpreted as meaning that suramin prolongs the action of end ogenous ATP to elicit twitch contraction, which points to a new, undefined role of ATP in neuromuscular transmission. The source of ATP is partly post synaptic, released from the muscle in response to activation of nicotinic A Ch receptors expressed on the muscle. (C) 2000 Elsevier Science B.V. All ri ghts reserved.