Antagonism of calcium currents and neurotransmitter release by barium ions at frog motor nerve endings

Abstract

1. The effects of Ba(2+) (0.1 - 2 mM) on the component of the perineural voltage change associated with nerve terminal calcium currents (prejunctional Ca(2+) currents) were compared with the effects of this ion to antagonize calcium-dependent acetylcholine (ACh) release. These experiments were made on isolated neuromuscular junctions of the frog. 2. In the presence of sufficient concentrations of K(+) channel blockers to eliminate measurable prejunctional K(+) currents, low concentrations of Ba(2+) selectively antagonized prejunctional Ca(2+) currents in normal Ca(2+) solutions. Higher concentrations of Ba(2+) also substantially reduced the Na(+) component of the perineural waveform. 3. Ba(2+) inhibited the prolonged prejunctional Ca(2+) currents that developed in the presence of higher concentrations of K(+) channel blockers. 4. Simultaneous measurements of the prejunctional Ca(2+) currents and the electrophysiological correlates of ACh release (i.e. end-plate potentials, EPPs) were made under conditions of modest K(+) channel blockade. Under these conditions, Ba(2+) generally produced simultaneous decreases in both Ca(2+) currents and EPP amplitudes. In some instances, a prolongation of prejunctional Ca(2+) currents and a transient increase in EPP amplitudes preceded the decreases in both electrophysiological events. 5. These results suggest that Ba(2+) ions can antagonize the entry of calcium into motor nerve endings and this effect is likely to be responsible for the inhibitory effects of Ba(2+) on evoked ACh release.

Figure 1

Prejunctional Ca²⁺ currents in normal Ca²⁺ current  Ringer and its antagonism by Ba²⁺ (0.1 mM). The averaged control response shown (a) is characterized by a downward (inward) tetrodotoxin-sensitive Na⁺ current (Na⁺) and an upward (outward) deflection that largely reflects the prejunctional calcium current (Ca²⁺) that mediates ACh release. For the control record, the peak of the Ca²⁺ component ranged from 1.9–2.2 mV (mean±1 s.e.mean=2.1±0.04 mV, n=5 stimuli). For details of the repetitive firing, see text. After 15 min of superfusion with ringer containing 0.1 mM Ba²⁺, the peak of the prejunctional Ca²⁺ current was decreased without a change in the Na⁺ component. The peak of the Ca²⁺ component in (b) ranged from 1.3–1.6 mV (mean±1 s.e.mean=1.5±0.06 mV, n=5 stimuli). The decrease was highly statistically significant (Mann Whitney rank sum test, P<<0.01). Note also the elimination of repetitive firing (also indicative of a reduction in Ca²⁺ entry). Each trace is the averaged response to five stimuli delivered at 0.3 Hz.

Figure 2

Ba²⁺ (2 mM) antagonizes prejunctional Ca²⁺ currents in normal Ca²⁺ current Ringer. (a) shows the control perineural trace. The mean Ca²⁺ component was 1.63 mV. (b) shows perineural records after 5 min in 2 mM Ba²⁺. Note the inhibition of the perineural Ca²⁺ current (mean amplitude=0.74 mV) without an accompanying effect on the Na⁺ component in this experiment. Each trace is the averaged response to 64 stimuli (0.3 Hz).

Figure 3

Ba²⁺ antagonizes Ca²⁺ currents in the presence of higher concentration of K⁺ channel blockers. (a) shows control response. (b) shows response 6 min after the beginning of superfusion with 2 mM Ba²⁺. This solution contained normal calcium with 10 mM TEA, 200 μM DAP, and procaine (100 μM, to prevent repetitive firing) and generated a longer duration Ca²⁺ current. This effect of Ba²⁺ was reversible (data not shown, but see Figure 6). Each trace is the average of two responses (frequency of stimulation=0.01 Hz).

Figure 4

Ba²⁺ decreases perineural Ca²⁺ currents (upper traces) and evoked ACh release (EPPs) measured simultaneously (lower traces). Each trace is the averaged response to 21 stimuli in Ca²⁺ current ACh release Ringer (frequency of stimulation=0.05 Hz). Upper traces show perineural currents, lower traces show EPPs. (a) shows control data. (b) shows effects of 0.5 mM Ba²⁺. The average perineural Ca²⁺ waveform was reduced from a mean control level of 3.2 mV to 2.0 mV after 21 min in 0.5 mM Ba²⁺. The average EPP measured simultaneously was reduced from 3.7–1 mV. It should be noted that in this solution, repetitive firing often develops in low Ca²⁺ solutions (see b, lower trace) and, in contrast to the experiments of Figures 1 and 2, is not indicative of the degree of Ca²⁺ entry (see Redman & Silinsky, 1995, Figure 4).

Figure 5

Increases in Ca²⁺ current duration and ACh release produced by Ba²⁺ when K⁺ channels are not fully blocked. Ringer solution was Ca²⁺ current-ACh release Ringer. Prior to superfusion with Ba²⁺, nine control perineural currents (lower trace, a) were recorded. The mean amplitude of the perineural Ca²⁺ voltage change=0.64±0.03 mV (mean±1 s.d., n=9, range 0.59–0.69 mV). Simultaneously, nine control EPPs were recorded (a, upper trace). The mean EPP amplitude=4.5±0.2 mV, mean±1 s.d., n=9, range 4.1–4.9 mV). The coefficient of variation (s.d.mean⁻¹) was thus 0.5% for the perineural Ca²⁺ component and 0.6% for the EPPs, justifying (i) the use of the Rahamimoff (1967) statistical method to quantify the perineural recordings (see Methods) and (ii) the stability of both types of electrophysiological measurements in this solution. At (b), superfusion was begun with Ba²⁺ containing solution. Note the progressive prolongation of the perineural Ca²⁺ current (lower traces) and the increase in EPP amplitudes in Ba²⁺ to 7.2 mV (±1.0 mV) (mean±1 s.e.mean, n=6). Shortly after these records, EPPs were eliminated completely and both the Na²⁺ and Ca²⁺ components of the perineural traces were decreased (data not shown). Frequency of stimulation=0.05 Hz.

Figure 6

Reversible effects of Ba²⁺ on Ca²⁺ currents in Ca²⁺ current-ACh release Ringer. (a) shows control response, (b) shows response after 18 min in 1 mM Ba²⁺ ringer. Note reduction in Ca²⁺ current without an effect on Na⁺ current. In (c) and Ca²⁺ current is restored after 16 min in Ba²⁺ free solution. The increase in Ca²⁺ response after Ba²⁺ treatment is likely to be due to an increase in the Na⁺ current, possibly produced by a small shift in the position of the recording electrode. EPPs were eliminated during this part of the experiment (see text). Each trace is the average response to 64 stimuli (0.3 Hz).

Figure 7

Reversible decreases in EPPs produced by brief exposure to Ba²⁺ (0.2 mM) in Ca²⁺ current-ACh release Ringer. Traces in (a) are averaged EPPs in control solution, after 2.5 min in 0.2 mM Ba²⁺, and after washing in control solution for 3 min. Each trace is the averaged response to 5 stimuli (frequency of stimulation=0.05 Hz). An analysis of variance followed by multiple comparisons revealed highly statistically-significant differences between Ba²⁺-containing solutions and control conditions. No statistically significant difference was observed between control and wash after Ba²⁺. Traces in (b) illustrates another experiment in which both perineural Ca²⁺ currents (upper traces) and EPPs (lower traces) were recorded. Note the concomitant decrease in the average Ca²⁺ component of the control perineural current and the EPP (n=11 stimuli). The effect on the EPPs was partly reversible (post Ba²⁺ wash). Recovery of the Ca²⁺ current also occurred (data not shown but see Figure 6).