title: Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques.
  
  
  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 Cd(2+) , the removal of extracellular Ca(2+) 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 Gi/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.
  
  
  CONCLUSIONS
  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 Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.
authors.affiliation: Laboratorio de Neurofisiología, Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
authors.forename: A R
authors.initials: AR
authors.lastname: Cinalli
authors.forename: J F
authors.initials: JF
authors.lastname: Guarracino
authors.forename: V
authors.initials: V
authors.lastname: Fernandez
authors.forename: L I
authors.initials: LI
authors.lastname: Roquel
authors.forename: A S
authors.initials: AS
authors.lastname: Losavio
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D003964:Diaphragm / Q000294:innervation; D004095:Dihydropyridines / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D007288:Inosine / Q000494:pharmacology*; D008297:Male; D051379:Mice; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000378:metabolism; D010791:Phrenic Nerve; D011758:Pyrroles / Q000494:pharmacology; D043684:Receptor, Adenosine A3 / Q000187:drug effects* / Q000378:metabolism; D018047:Receptors, Purinergic P1
chemical_list: D002121:Calcium Channel Blockers; D002227:Carbazoles; D004095:Dihydropyridines; C107728:MRS 1191; D011758:Pyrroles; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; C057416:KT 5720; D007288:Inosine; D000109:Acetylcholine; D005033:Ethylmaleimide
keywords: A3 adenosine receptor; Ca2+- independent mechanism; PKC; calmodulin; inosine; mammalian neuromuscular junction; presynaptic inhibition; voltage-gated calcium channels