Adenosine decreases both presynaptic calcium currents and neurotransmitter release at the mouse neuromuscular junction

Abstract

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.

Figure 1. General observations on the development (A) and antagonism (B) of perineural Ca²⁺ currents in mouse phrenic nerve hemidiaphragm

A shows the Na⁺ currents and K⁺ currents as downward deflections prior to application of K ⁺ channel blockers (5 superimposed sweeps, 0.1 Hz). Superfusion with solution containing potassium channel blockers (standard Ca²⁺ current solution; see Methods) produces the blockade of K⁺ currents and the progressive development of Ca²⁺ currents (1–4) during stimulation of the phrenic nerve (0.016 Hz). For details of current polarities and description of the perineural currents, which reflect voltage changes in the extracellular space and are thus calibrated in mV, see Methods. A shows the typical result from n = 10 preparations. B shows averaged perineural Ca²⁺ currents (Ca²⁺) and EPPs recorded simultaneously in standard Ca²⁺ current solution. In the absence of agatoxin, both the Ca²⁺ currents and EPPs in B were stable for a period of approximately 23 min. The fast and slow phases of the Ca²⁺ current are shown (for discussion see Methods and Xu & Atchison, 1996). The lower two traces in B show the progressive decreases in both the averaged Ca²⁺ currents and EPPs (average of 5–15 stimuli, 0.012 Hz) in the presence of the P/Q-type Ca²⁺ channel blocker ω-agatoxin-IVA (100 nm). Intervals of approximately 14 min elapsed between the traces in B (B shows the typical result from n = 4 preparations).

Figure 2. Antagonism of calcium currents by adenosine in wild-type and Rab3A^−/− mutant mice

Traces in A show the effects of 500 μm adenosine (averaged data from a wild-type mouse). B shows the effect of 1 mm adenosine in another experiment made on a wild-type mouse. The raw data in B were obtained using the protocol for rapid solution changes as described in the Methods. C shows averaged traces before and after application of 50 μm adenosine to a preparation from a Rab3A^−/− mutant mouse. The phrenic nerve was stimulated at 0.016 Hz.

Figure 3. Simultaneous reduction in ACh release (EPPs) and Ca²⁺ currents produced by adenosine

A shows the reversible inhibition of ACh release and perineural Ca²⁺ currents in low Ca²⁺ solutions (0.7 mm Ca²⁺, 2 mm Mg²⁺ and reduced concentrations of K⁺ channel blockers; see Methods). B and C show experiments in the standard perineural solution (Xu & Atchison, 1996). B shows raw data from another experiment that illustrate the onset of inhibition by adenosine. The phrenic nerve was stimulated at 0.015 Hz. C shows averaged data (n = 4–5 stimuli) from a Rab3A^−/− mouse depicting the inhibitory effect adenosine (50 μm) on both the EPPs and perineural currents.

Figure 4. Absence of effects of adenosine on nerve terminal Na⁺ and K⁺ currents

In A, currents (lower traces) were measured from the perineurium at the junction of the myelinated and non-myelinated axon (the heminode). Upper traces show averaged EPPs. Note the absence of effects of 50 μm adenosine in this experiment on a Rab3A^−/− mouse on Na⁺ or K⁺ currents under conditions in which the averaged EPPs are decreased to approximately 58% of the control level by this concentration of adenosine. The average responses to 5 stimuli (0.1 Hz) are depicted. B shows experiments made using a loose patch electrode at the nerve terminal from a wild-type mouse. Spontaneous focal mEPCs, were recorded first, the preparation curarized, the motor nerve stimulated (arrow marks the stimulus artifact) and the nerve terminal currents (NTCs) and end-plate currents (EPCs) measured. Note the absence of effects of adenosine (5 mm) on NTCs under conditions in which it produces a reversible inhibition of end-plate currents (EPCs) to approximately 60% of the control level. Each trace is the averaged response to 256 stimuli (0.25 Hz). C shows recording of Na⁺ and K⁺ currents from the nerve terminal proper using the perineural recording method. The currents are mirror-images of those recorded from the heminode, and represent a higher resolution image of the NTCs recorded with a patch electrode in B. Again, no effects of adenosine were observed on the Na⁺ or K⁺ currents in this wild-type mouse. Traces in C are the average response to 20 stimuli (0.25 Hz).

Figure 5. Absence of effects of adenosine on the residual ionic currents after treatment with Cd²⁺ (B and C) or ω-agatoxin-IVA (D and E)

A shows progressive antagonism of the perineural Ca²⁺ current by Cd²⁺ (0.017 Hz). B shows the averaged perineural waveform after 1 mm Cd²⁺ treatment (1.60 mV, n = 6 stimuli). C shows that the residual current in the presence of Cd²⁺ is not affected by the application of 1 mm adenosine (1.56 mV, n = 6 stimuli). D shows the averaged perineural waveform after ω-agatoxin-IVA (100 nm) in another experiment (1.65 mV, n = 5 stimuli). E shows that the residual current in the presence of ω-agatoxin-IVA is not affected by the application of 10 mm adenosine (1.60 mV, n = 6 stimuli). Prior to treatment with ω-agatoxin-IVA the control Ca²⁺ currents for traces D and E resembled that of A but with an average peak of 2.74 mV (n = 6 stimuli).

Figure 6. Dose–response curves for the inhibition of Ca²⁺ currents (A) and EPPs (B) by adenosine in wild-type (○) and Rab3A^−/− mutant mice (•)

Note the differences in potency for adenosine in the two species of mouse for both Ca²⁺ currents (A) and EPPs (B) under the same conditions. Each data point represents the mean of 4–9 experiments. Error bars are ± 1 s.e.m. 0.016 Hz stimulation rate.

Figure 7. Comparison of the relationship between the simultaneous inhibition of EPPs and perineural Ca²⁺ currents by adenosine and by equi-effective concentrations of the Ca²⁺ channel blocker Cd²⁺

For each of the 5 experiments that contributed to the averaged data in Fig. 7, simultaneous measurements of EPPs and perineural Ca²⁺ currents were made from the same site for the duration of the experiments. It should be noted that these results with Cd²⁺ on both EPPs and perineural Ca²⁺ currents are submaximal as higher concentrations of Cd²⁺ (≥100 μm) produce complete inhibition of the EPPs and further reductions in the Ca²⁺ current (see Figs 1 and 5). Due to cost and the experimental impracticality of the long incubation times required for after ω-agatoxin-IVA (see, e.g. Fig. 1B), Cd²⁺ was used as the Ca²⁺ channel blocker in these experiments. Error bars are +1 s.e.m. 0.017 Hz, frequency of stimulation.