Relative contribution of ecto-ATPase and ecto-ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons.

BACKGROUND AND
PURPOSE: The relative contribution of distinct ecto-nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference. EXPERIMENTAL APPROACH: Extracellular ATP catabolism (assessed by high-performance liquid chromatography) and its influence on [(3)H]acetylcholine ([(3)H]ACh) release were investigated in the myenteric plexus of rat ileum in vitro. KEY
RESULTS: 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 [(3)H]ACh outflow in a 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP)-dependent, tetrodotoxin-independent manner. ATP and ATPgammaS (adenosine 5'-[gamma-thio]triphosphate), but not alpha,beta-methyleneATP, decreased [(3)H]ACh release induced by electrical stimulation. ADP and ADPbetaS (adenosine 5'[beta-thio]diphosphate) only decreased evoked [(3)H]ACh release. Inhibition by ADPbetaS was prevented by MRS 2179 (2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate diammonium salt, a selective P2Y(1) antagonist); blockade of ADP inhibition required co-application of MRS 2179 plus adenosine deaminase (which inactivates endogenous adenosine). Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine enhanced ADPbetaS inhibition, indicating that P2Y(1) stimulation is cut short by tonic adenosine A(1) receptor activation. MRS 2179 facilitated evoked [(3)H]ACh release, an effect reversed by the ecto-ATPase inhibitor, ARL67156, which delayed ATP conversion into ADP without affecting adenosine levels. CONCLUSIONS AND IMPLICATIONS: ATP transiently facilitated [(3)H]ACh release from non-stimulated nerve terminals via prejunctional P2X (probably P2X(2)) receptors. Hydrolysis of ATP directly into AMP by ecto-ATPDase and subsequent formation of adenosine by ecto-5'-nucleotidase reduced [(3)H]ACh release via inhibitory adenosine A(1) receptors. Stimulation of inhibitory P2Y(1) receptors by ADP generated alternatively via ecto-ATPase might be relevant in restraining ACh exocytosis when ATP saturates ecto-ATPDase activity.