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The relative contribution of distinct ecto-nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.Extracellular ATP catabolism (assessed by high-performance liquid chromatography) and its influence on [3H]acetylcholine ([3H]ACh) release were investigated in the myenteric plexus of rat ileum in vitro.ATP was primarily metabolized via ecto-ATPDase (adenosine 5′-triphosphate diphosphohydrolase) into AMP, which was then dephosphorylated into adenosine by ecto-5′-nucleotidase. Alternative conversion of ATP into ADP by ecto-ATPase (adenosine 5′-triphosphatase) was more relevant at high ATP concentrations. ATP transiently increased basal [3H]ACh outflow in a 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP)-dependent, tetrodotoxin-independent manner. ATP and ATPγS (adenosine 5′-[γ-thio]triphosphate), but not α,β-methyleneATP, decreased [3H]ACh release induced by electrical stimulation. ADP and ADPβS (adenosine 5′[β-thio]diphosphate) only decreased evoked [3H]ACh release. Inhibition by ADPβS was prevented by MRS 2179 (2′-deoxy-N6-methyl adenosine 3′,5′-diphosphate diammonium salt, a selective P2Y1 antagonist); blockade of ADP inhibition required co-application of MRS 2179 plus adenosine deaminase (which inactivates endogenous adenosine). Blockade of adenosine A1 receptors with 1,3-dipropyl-8-cyclopentyl xanthine enhanced ADPβS inhibition, indicating that P2Y1 stimulation is cut short by tonic adenosine A1 receptor activation. MRS 2179 facilitated evoked [3H]ACh release, an effect reversed by the ecto-ATPase inhibitor, ARL67156, which delayed ATP conversion into ADP without affecting adenosine levels.ATP transiently facilitated [3H]ACh release from non-stimulated nerve terminals via prejunctional P2X (probably P2X2) receptors. Hydrolysis of ATP directly into AMP by ecto-ATPDase and subsequent formation of adenosine by ecto-5′-nucleotidase reduced [3H]ACh release via inhibitory adenosine A1 receptors. Stimulation of inhibitory P2Y1 receptors by ADP generated alternatively via ecto-ATPase might be relevant in restraining ACh exocytosis when ATP saturates ecto-ATPDase activity.