A controversy currently exists as to the mechanism of action by which adenosine, an endogenous mediator of neurotransmitter depression, reduces the evoked release of the neurotransmitter acetylcholine (ACh) at the skeletal neuromuscular junction. Specifically, it is uncertain whether adenosine inhibits ACh release from mammalian motor nerve endings by reducing Ca(2+) calcium entry through voltage-gated calcium channels or, as is the case at amphibian motor nerve endings, by an effect downstream of Ca(2+) entry. In an attempt to address this controversy, the effects of adenosine on membrane ionic currents and neurotransmitter release were studied at neuromuscular junctions in adult mouse phrenic nerve hemidiaphragm preparations. In wild-type mice, adenosine (500 microm-1 mm) reduced prejunctional Ca(2+) currents simultaneously with a reduction in evoked ACh release. In Rab3A knockout mice, which have been shown to have an increased sensitivity to adenosine, the simultaneous reduction in Ca(2+) currents and ACh secretion occurred at significantly lower adenosine concentrations (< or = 50 microM). Measurements of nerve terminal Na(+) and K(+) currents made simultaneously with evoked ACh release demonstrated that the decreases in Ca(2+) currents were not attributable to changes in cation entry through voltage-gated Na(+) or K(+) channels. Furthermore, no effects of adenosine on residual ionic currents were observed when P/Q-type calcium channels were blocked by Cd(2+) or omega-agatoxin-IVA. The results demonstrate that inhibition of evoked neurotransmitter secretion by adenosine is associated with a reduction in Ca(2+) calcium entry through voltage-gated P/Q Ca(2+) channels at the mouse neuromuscular junction. Whilst it may be that adenosine inhibits ACh release by different mechanisms at amphibia and mammalian neuromuscular junctions, it is also possible that the secretory apparatus is more intimately coupled to the Ca(2+) channels in the mouse such that an effect on the secretory machinery is reflected as changes in Ca(2+) currents.