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 end-plate potential (mepp) frequency in mouse diaphragm muscles. 2. Blockade of VDCCs by Cd(2+) prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not omega-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(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester epsilon6TDelta-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca(2+)-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 10 mm K(+) was prevented by the P/Q-type VDCC antagonist omega-agatoxin IVA. 6. CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20 mm 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 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(+) 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 A(1) receptors by endogenous AD prevents excessive neurotransmitter release.

Figure 1

AD and CCPA inhibit spontaneous ACh release in mouse skeletal muscle. (a) Mepps recorded from diaphragm muscle fibers bathed with control Ringer solution (Vm: −74.4 mV), with 500 nM CCPA (Vm: −73.5 mV), and with 500 nM CCPA in the presence of the A₁-selective AD receptor antagonist DPCPX (0.1 μM, Vm: −73.9 mV). Calibration: 3 mV, 2.5 s. Mepp amplitude was not modified by CCPA and/or DPCPX. (b) Summary graph bar of the presynaptic inhibition induced by AD and CCPA. The effect of CCPA on mepp frequency (s⁻¹) as a function of concentration is shown in the inset. Control: 1.03±0.03 (n=8); CCPA: 1 nM: 1.07±0.03 (n=4); 10 nM: 0.88±0.04 (n=4), 100 nM: 0.68±0.03 (n=4), 250 nM: 0.63±0.01 (n=3), 500 nM: 0.55±0.03 (n=8), 700 nM: 0.55±0.04 (n=4). EC₅₀: 19.4 nM. (c) DPCPX prevented the modulatory effect of CCPA. In (b, c), data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.

Figure 2

CCPA failed to induce presynaptic inhibition in the presence of Cd²⁺ and ω-CgTx+nitrendipine. (a) The universal VDCC blocker Cd²⁺ (100 μM) decreased mepp frequency and blocked the effect of AD. (b) The combined application of 5 μM ω-CgTx (an N-type VDCC antagonist) and 5 μM nitrendipine (Nit; an L-type VDCC antagonist) also diminished mepp frequency and prevented the inhibition induced by CCPA. Data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.

Figure 3

CCPA reduced the nitrendipine (Nit)- but not the ω-CgTx-sensitive component of mepp frequency. (a) Lack of effect of CCPA on spontaneous ACh secretion in the presence of 5 μM Nit. Mepp frequency recorded in CCPA and Nit was not significantly different from that obtained in Nit alone. (b) Lack of effect of Nit on spontaneous ACh secretion in the presence of CCPA. The effect of Nit was not observed when preparations were preincubated with CCPA. (c) Effect of CCPA on spontaneous ACh secretion in the presence of 5 μM ω-CgTx. CCPA induced a further reduction in mepp frequency when applied after ω-CgTx. (d) Effect of ω-CgTx on spontaneous ACh secretion in the presence of CCPA. ω-CgTx further reduced mepp frequency induced by CCPA. (e) Effect of CCPA on spontaneous ACh secretion in the presence of 100 nM ω-Aga. The P/Q-type VDCC blocker did not exert any change in spontaneous secretion and did not block the action of CCPA. Data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.

Figure 4

cAMP-PKA and phospholipase C-PKC pathways are not involved directly in CCPA modulation. (a) The specific PKA inhibitor H-89 (1 μM) neither mimicked nor prevented the effects of CCPA. CCPA inhibited spontaneous ACh release in the presence of H-89. (b) PKC blocker H-7 (10 μM) did not reverse the presynaptic inhibition induced by CCPA. H-7 added to control saline did not alter mepp frequency (see Table 1). (c) The PKC activator PMA (200 nM) did not block the action of CCPA. CCPA reduced mepp frequency when applied after incubation with PMA. Data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.

Figure 5

Ca²⁺-calmodulin is involved in the presynaptic inhibition induced by CCPA. (a) W-7 (50 μM), an antagonist of calmodulin, did not alter mepp frequency, and prevented the effect of CCPA on spontaneous ACh release. (b) EGTA-AM decreased mepp frequency and blocked the action of CCPA. Data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.

Figure 6

CCPA reduced the 10 mM K⁺-evoked increase in mepp frequency, acting on P/Q-type VDCCs. (a) CCPA reduced the asynchronous ACh release induced by 10 mM K⁺. (b) DPCPX prevented the modulatory effect of CCPA at 10 mM K⁺. (c) ω-Aga (100 nM) reduced the mepp frequency evoked by 10 mM K⁺-and prevented the presynaptic inhibition induced by CCPA. Data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.

Figure 7

CCPA failed to reduce asynchronous ACh release induced by 15 and 20 mM K⁺. (a, b) Lack of effect of CCPA on the 15 and 20 mM K⁺-evoked increase in mepp frequency. CCPA did not affect mepp frequency when applied after 15 and 20 mM K⁺. (c, d) Effect of DPCPX on the mepp frequency induced by 15 and 20 mM K⁺. The blockade of A₁ AD receptors by DPCPX caused a greater increase in mepp frequency compared to that obtained before the application of DPCPX in the same muscles. Data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.

Figure 8

Effect of exogenous and endogenous AD at different external K⁺ concentrations. (a) Effect of CCPA on mepp frequency at 5, 10, 15 and 20 mM K⁺, expressed as percentage change compared to control solutions. (b) Effect of DPCPX on mepp frequency at 5, 10, 15 and 20 mM K⁺, expressed as percentage change with respect to mepp frequency obtained in 5, 10, 15 and 20 mM K⁺ without DPCPX. ^*P<0.05.

Figure 9

Binding of endogenous AD to A₁ AD receptors prevented the effect of CCPA at 15 and 20 mM K⁺. (a, b) Incubation of preparations with ADA (0.5 U ml⁻¹), which degraded AD into the inactive metabolite inosine, increased mepp frequency compared to that obtained in 15 and 20 mM K⁺ before the application of ADA in the same muscles, and allowed the action of CCPA. Data are expressed as percentages of control values (black bars). Error bars indicate s.e.m. ^*P<0.05.