Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction

DE LORENZO S, VEGGETTI M, MUCHNIK S, LOSAVIO A

BRITISH JOURNAL OF PHARMACOLOGY

WILEY-BLACKWELL PUBLISHING, INC

Lugar: Londres; Año: 2004 vol. 142 p. 113 - 124

0007-1188

1- At the mouse neuromuscular junction, adenosine (AD) and the A1 agonist 2-chloro-N6- cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A1 AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L- and N-type VDCCs), we measured the miniature endplate potential (mepp) frequency in mouse diaphragm muscles. 2- Blockade of VDCCs by Cd2+ prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not o-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3- As A1 receptors are coupled to a Gi/o protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4- The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1- naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(β-aminoethyl ether)- N,N,N?,N?-tetraacetic acid-acetoxymethyl ester e6TD-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca2+-calmodulin. 5- To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K+ concentrations. The effect of CCPA on ACh secretion evoked by 10mM K+ was prevented by the P/Q-type VDCC antagonist o-agatoxin IVA. 6- CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20mM K+. We demonstrated that, at high K+ concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20mM K+ in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K+ concentrations, the activation of A1 receptors by endogenous AD prevents excessive neurotransmitter release.