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

Abstract

In this study the in vitro mouse phrenic nerve- hemidiaphragm preparation was 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 assay, was released stimulation-dependently from the mouse hemidiaphragm in response to electrical field stimulation at 10 Hz. Blockade of the Na⁺ channel activity by tetrodotoxin inhibited the majority of the release of ATP in response to stimulation, showing that it is related to neuronal activity. The nicotinic receptor antagonists d-tubocurarine, and α-bungarotoxin and cooling the bath temperature to 7°C also reduced stimulation-induced ATP outflow, suggesting that nicotinic receptors are responsible for the part of the release of ATP that is released from postsynaptic sites in a carrier-mediated manner. Exogenous ATP (20–500 μM) added to the bath was degraded to ADP and AMP by the action of ectoATPase and ectoATPdiphosphohydrolase; the K_m and v_max values of these enzymes were 185.8 μM 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 stimulation was blocked concentration–dependently by the non-depolarizing muscle relaxants d-tubocurarine and pancuronium. Suramin (100 μM–1 mM) reversed neuromuscular blockade by d-tubocurarine and pancuronium; i.e., it shifted their concentration–response curves to the right Taken together our data, that endogenous ATP is released by stimulation and subsequently catabolized in the hemidiaphragm preparation and that suramin inhibits ecto-ATPase activity could be interpreted as meaning that suramin prolongs the action of endogenous ATP to elicit twitch contraction, which points to a new, undefined role of ATP in neuromuscular transmission. The source of ATP is partly postsynaptic, released from the muscle in response to activation of nicotinic ACh receptors expressed on the muscle.