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

Abstract

1 At the mouse neuromuscular junction, adenosine (AD) and the A 1 agonist 2-chloro-N 6 -cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A 1 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 Cd 2 þ 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 A 1 receptors are coupled to a G i/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(b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid-acetoxymethyl ester e6TD-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca 2 þ -calmodulin. 5 To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K þ concentrations. The effect of CCPA on ACh secretion evoked by 10 mM K þ 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 20 mM K þ . We demonstrated that, at high K þ concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A 1 receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mM K þ 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 þ concentrations, the activation of A 1 receptors by endogenous AD prevents excessive neurotransmitter release.

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

“…As we have observed in our previous papers (Cinalli et al, ; De Lorenzo et al, ; Guarracino et al, ; Palma et al, ; Veggetti et al, ), our experiments do not reveal any involvement of endogenous purines at basal conditions (K + 5 mM), since incubation of the diaphragms with the antagonists for the P2Y 13 , A 1 , A 3 , or A 2A receptors (AR‐C69931MX, DPCPX, MRS‐1191, or SCH‐58261, respectively) did not affect spontaneous neurotransmitter secretion. On the contrary, the application of exogenous agonists for P2Y 13 , A 1 , or A 3 receptors (2‐MeSADP, CCPA, or inosine, respectively) decreases MEPP frequency, whereas the A 2A receptor agonist CGS‐21680 increases it.…”

“…It was demonstrated that both purines, ATP and adenosine, modulate neurotransmitter release operating via presynaptic P2 and P1 receptors, respectively (De Lorenzo, Veggeti, Muchnik, & Losavio, ; Giniatullin & Sokolova, ; Sebastião & Ribeiro, ; Sokolova, Grishin, Shakirzyanova, Talantova, & Giniatullin, ). In previous reports performed in mouse neuromuscular junctions, we have found that at basal conditions (K + 5 mM), ATP and adenosine regulate ACh secretion by activating presynaptic P2Y receptors (De Lorenzo et al, ; Guarracino, Cinalli, Fernández, Roquel, & Losavio, ; Veggetti, Muchnik, & Losavio, ) and A 1 and A 2A receptors (De Lorenzo et al, ; Palma, Muchnik, & Losavio, ), respectively. Moreover, we have recently demonstrated, by pharmacological and immunohistochemical assays, that A 3 receptors are also present at the motor nerve terminals and that these receptors may be activated by adenosine and its metabolite inosine (Cinalli, Guarracino, Fernandez, Roquel, & Losavio, ).…”

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

“…For example, acting on A 1 and A 2A receptors at the vascular smooth muscle, adenosine may increase muscle blood flow and thus nutrient availability (Poucher, 1996;Ray et al, 2002). Besides, adenosine has presynaptic effects, either facilitating or inhibiting the release of acetylcholine from the motor nerve through A 1 (Correia-de-Sá et al, 1991;De Lorenzo et al, 2004) and A 2A (Correia-de-Sá et al, 2000;Baxter et al, 2005) receptors, respectively . Figure 10 summarizes the sequential steps of extracellular cAMP-adenosine pathway in the skeletal muscle, triggered by activation of cell-surface receptors coupled to G s protein, indicating the possible paracrine sites of adenosine action.…”

Perisynaptic Glia Discriminate Patterns of Motor Nerve Activity and Influence Plasticity at the Neuromuscular Junction

“…Furthermore, direct glial activation would allow us to tease apart the underlying mechanisms, a task that would be difficult with endogenous activity alone since both the presynaptic terminal and PSCs bear many of the same types of receptors (e.g., A 1 and muscarinic ACh receptors) (Hamilton and Smith, 1991;Correia-de-Sá et al, 1996;Grafe et al, 1999;Galkin et al, 2001;Rochon et al, 2001;Oliveira et al, 2002Oliveira et al, , 2004De Lorenzo et al, 2004;Silinsky, 2004;Baxter et al, 2005;Moores et al, 2005;Todd and Robitaille, 2006;Dudel, 2007).…”

“…Glial regulation occurs through a balanced A 1 /A 2A receptor activation Activation of A 1 and A 2A receptors results in inhibition and activation of different types of presynaptic calcium channels to regulate transmitter release at synapses in general and at the mammalian NMJ in particular (Correia-de-Sá et al, 1996;De Lorenzo et al, 2004;Oliveira et al, 2004;Silinsky, 2005;Cunha, 2008). Moreover, an adenosine-dependent long-term depression has been reported (Redman and Silinsky, 1994) that involves an inhibition of presynaptic calcium currents (Silinsky, 2004(Silinsky, , 2005.…”