Endogenous purines modulate K+-evoked ACh secretion at the mouse neuromuscular junction

VEGGETTI, MARIELA I.; GUARRACINO, JUAN F.; LOSAVIO, ADRIANA S.; CINALLI, ALEJANDRO R.

JOURNAL OF NEUROSCIENCE RESEARCH

WILEY-LISS, DIV JOHN WILEY & SONS INC

Año: 2018 vol. 96 p. 1066 - 1079

0360-4012

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y13 and A1, A2A, and A3 receptors, respectively. To elucidate the action of endogenous purines on K+-dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K+, the P2Y13 antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5′-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A1, A3, and A2A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K+-evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K+, endogenous ATP/ADP and adenosine bind to inhibitory P2Y13 and A1 and A3 receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5′-nucleotidase inhibitor α,β-methyleneadenosine 5′-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K+, suggesting that more adenosine is needed to activate excitatory A2A receptors. At high K+ concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K+ concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors.