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

Background and Purpose The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. Experimental Approach End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Key Results Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects 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⁺, the removal of extracellular Ca²⁺ and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the G_i/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Conclusion and Implications Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by 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 G_i/o protein. PKC and calmodulin may be involved in these effects of inosine.