Relative contribution of ecto-ATPase and ecto-ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons.
OBJECTIVE
The relative contribution of distinct ecto-nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.


METHODS
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.


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
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.
Duarte-Araújo
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Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Portugal.
Nascimento
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C
Timóteo
M A
MA
Magalhães-Cardoso
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MT
Correia-de-Sá
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D015720:5'-Nucleotidase / Q000378:metabolism*; D000109:Acetylcholine / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001081:Apyrase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007082:Ileum / Q000294:innervation; D066298:In Vitro Techniques; D008297:Male; D009046:Motor Neurons / Q000187:drug effects* / Q000201:enzymology; D009197:Myenteric Plexus / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058476:Receptors, Purinergic P2X2
D015703:Antigens, CD; C546403:P2rx2 protein, rat; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen; D000109:Acetylcholine