Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

ALEJANDRO CINALLI; JUAN GUARRACINO; VERÓNICA FERNANDEZ; LILIANA ROQUEL; ADRIANA LOSAVIO

BRITISH JOURNAL OF PHARMACOLOGY

WILEY-BLACKWELL PUBLISHING, INC

Lugar: Londres; Año: 2013 vol. 169 p. 1810 - 1823

0007-1188

BACKGROUND AND PURPOSE The role of inosine at mammalian neuromuscular junction (NMJ) was not clearly defined. Moreover, the nucleoside was classically considered inactive metabolite of adenosine. We investigated the effect of inosine upon spontaneous and evoked ACh release, the receptor type the nucleoside binds to, the mechanism underlying its modulatory action, and the signal transduction pathway involved. EXPERIMENTAL APPROACH End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from mouse phrenic-diaphragm preparations using conventional intracellular electrophysiological techniques. KEY RESULTS Inosine (100 μM) reduced MEPP frequency as well as the amplitude and quantal content of EPPs. This effect was inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd2+ or the absence of extracellular Ca2+ prevented inosine action. The same happened with the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively. Additionally, inosine decreased the hypertonic response in the absence of endogenous adenosine. Inosine modulation was prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine, and calmodulin antagonist W-7, while PKA antagonists, H-89 or KT-5720, and the inhibitor of CaMKII KN-62 failed to do so. CONCLUSION AND IMPLICATIONS Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release when activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in the action of the nucleoside.