Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca2+ content of nerve terminals. The Ca2+ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca2+ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 μM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, ω-conotoxin GVIA (ω-CgTx, 5 μM). On the other hand, nifedipine and ω-CgTx had no effect on K+-induced transmitter secretion. ω-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K+ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca2+ enters nerve terminals through at least three different channels, two of them (L- and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca2+-binding molecule-related spontaneous release apparently binds Ca2+ very rapidly and would probably be located very close to Ca2+ channels, since the fast Ca2+ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca2+concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca2+ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by “bulk” intracellular Ca2+concentration increases.

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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.

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