Effect of Lanthanum Ions on Neuromuscular Transmission in Insects

Lanthanum produces blockage of neuromuscular transmission, perhaps due to a prevention of the influx of calcium ions into presynaptic nerve terminals (Miledi, 1966; Heuser & Miledi, 1971; Miledi, 1971). However, lanthanum also gives rise to a substantial increase in discharges of acetylcholine quanta (Blioch, Glagoleva, Liberman & Nenashev, 1968; Heuser & Miledi, 1971; DeBassio, Schnitzler & Parsons, 1972).

Recently, cobalt ions were found to have a dual effect on the spontaneous release of transmitter at insect motor nerve terminals, an inhibitory action being followed by an acceleratory action (Washio, 1982a). There was an antagonism by calcium of the inhibitory effect of cobalt. Since both cobalt (Weakly, 1973) and lanthanum (Blioch et al. 1968; Heuser & Miledi, 1971; DeBassio et al. 1972) are more effective than magnesium in suppressing evoked transmitter release, we have studied the presynaptic effect of lanthanum at insect neuromuscular junctions. In this paper evidence is also presented to suggest that lanthanum has a postsynaptic action on the insect neuromuscular synapse.

Experiments were performed on coxal depressor muscles of the cockroach, Periplaneta americana, muscle 178 from the metathoracic leg and 136 from the mesothoracic leg (Carbonell, 1947), and on the longitudinal ventral muscles of the larval mealworm, Tenebrio molitor. The standard bathing solution for cockroach muscles had the following composition (mm): NaCl, 158; KC1, 10·8; CaCl₂, 5; HEPES, 5; adjusted to pH 7·0 with NaOH. For mealworm muscles composition was NaCl, 70; KC1, 30; CaCl₂, 0·7; MgCh, 14·3; glucose, 445; HEPES, 5; adjusted to pH 7·2. Test solutions were prepared by substituting the appropriate cation chloride, for NaCl in the cockroach saline, and for glucose in the mealworm saline.

MEPSPs of cockroach muscle fibres were recorded intracellularly and extracellularly with micropipettes filled with 3 M-KCI and 2M-NaCl, respectively. The EPSPs in Tenebrio muscle fibres were evoked by stimulating the segmental nerve innervating the muscle with supramaximal square pulses (0·01 ms) using a pair of silver hook electrodes. The iontophoretic electrodes were filled with 1 M-Na L-glutamate at pH 8·0 and had resistances of about 80 MΩ. The preparation was constantly perfused at a rate of 5·0 ml min⁻¹. Experiments were all performed at 20—24 °C.

The experiments on spontaneous release of transmitter were performed on the cockroach muscle in order to compare with the effect of cobalt at the cockroach neuromuscular junction (Washio, 1982a), whereas the experiments on transmitter release by nerve impulses and on the response to L-glutamate applied iontophoreti-cally were performed at the Tenebrio neuromuscular junction, since these mechanisms have been well documented in those muscles (Yamamoto & Washio, 1980).

In the presence of 5mm-Ca²⁺, increasing the concentration of La³⁺ caused an increase in frequency of MEPSPs recorded intracellularly. The higher the concentration, the faster the rate of rise of the frequency and the shorter the time lag between the application of the test solution and increase in the frequency (Fig. 1). Although the effect of external La³⁺ was almost the same in both normal and high K⁺ (20·8 HIM) saline, it seemed that the time lag and time course were shorter in high K⁺ saline (Fig. 1). The high frequency caused by 5 mm-lanthanum lasted for less than 1 h at a K⁺ concentration of either 10·8 or 20·8 mm (Fig. 1). Raising Ca²⁺ concentration while keeping La³⁺ concentration constant reduced the potentiation, but not below a control level (Fig. 2B). For example, when 0·1 mm-La³⁺ was introduced (at 0·1 mm-Ca²⁺ and 20·8 mm-K⁺), it produced a sharp rise (more than one order of magnitude) in the frequency of occurrence of MEPSPs after a lag of several minutes (Fig. 2A). After reaching a peak, MEPSP frequency declined to a very low level along a roughly exponential time course with a time constant of about 14 min in this fibre.

The time constant of the decay of the potentials recorded extracellularly depended on La³⁺ concentration. For example, in Fig. 3B, the time constant of the decay of the largest MEPSP was increased by 5 mm-La³⁺ from 0·61 ±0·05 ms (mean ± s.d., N= 12) to 0·97 ±0·18 ms (N = 10), (P<0·05, Student’s t-test). The frequency of MEPSPs recorded extracellularly was much lower than that recorded intracellularly in insect muscle fibres (Washio & Inouye, 1980).

As in frog muscle (Miledi, 1966; Heuser & Miledi, 1971), La³⁺ could not replace Ca²⁺ as a requirement for transmitter release in Tenebrio muscle. Release was not restored by 0·5 mm-La³⁺ after blockage in Ca-deficient medium.

Fig. 4A illustrates the reduction of the excitatory postsynaptic potential (EPSP) of the Tenebrio muscle fibre during the application of 0·5 mm-La³⁺. The amplitude of the EPSP elicited by repetitive stimulation at 0·5 Hz was suppressed by 60% with perfusion for 2 min. The suppression of EPSP amplitude was almost irreversible (Fig External MEPSP amplitude (%) 4A3). The relationship between the amplitude of EPSP and extracellular La³⁺ concentration, and the effect of La³⁺ on the time course of the EPSP are shown in Figs 5 and 6A, respectively. The reduction in the amplitude of EPSP produced by La³⁺ could be antagonized to some degree by elevation of the external Ca²⁺ concentration (Fig. 7).

The quanta! content was calculated from the number of failures of EPSP (del Castillo & Katz, 1954) in the absence and presence of La³⁺ at low Ca²⁺ concentration (0·2mm-Ca²⁺ and 14·8mm-Mg²⁺) during repetitive nerve stimulation at 0·5 Hz (Table 1). The quantal content was reduced by 80% in the presence of 0·1 mm-La³⁺ Thus, the reduction in the EPSP amplitude by La³⁺ may be due to a decrease in the amount of transmitter released by a nerve impulse.

Since glutamate is a putative transmitter in the Tenebrio muscle (Yamamoto & Washio, 1980), we studied the effect of La³⁺ on sensitivity to L-glutamate applied iontophoretically.

During the application of 0-5 mm-La³⁺, the amplitude of the glutamate potential decreased by 50% in 2 min after the application and recovered completely 10 min after washing with a La³⁺-free saline (Fig. 4B). The time course of the recovery after washing was quite different from that of the neurally evoked response (cf. Fig. 4A and B). Also the threshold concentration of La³⁺ on the glutamate potential was found to be lower than that for the EPSP (Fig. 5). The difference of the threshold concentration and time course of the recovery between the response to L-glutamate applied iontophoretically and the EPSP may be attributed to the fact that the former is produced at a restricted junctional site while the latter is the integrated potential change of the junctions distributed over the whole muscle fibre.

Lanthanum produced a reduction in the apparent maximum of the dose-response curve for glutamate-induced depolarization (Fig. 8). Double logarithmic plots of the amplitude of the depolarizing response to L-glutamate against coulomb strength of the ejection currents showed that the limiting slope values were 2·6 and 1 ·8 in the absence and presence of 0·1mm-La³⁺ respectively (Fig. 8B). Thus, it seems unlikely that glutamate and lanthanum are competing for a common binding site on the postsynaptic membrane.

The present study in insect muscle fibres confirms the finding previously reported at frog neuromuscular junctions (Heuser & Miledi, 1971; DeBassio et al. 1972) that La³⁺ produces a substantial increase in spontaneous release of transmitter and blockage of neuromuscular transmission. Furthermore, our results indicate that La³⁺ brings about a reduction in the response to L-glutamate applied iontophoretically. The depression of the EPSP amplitude by La³⁺ could be accounted for by a decrease in the quantal content released by nerve impulses.

Although transmitter release by nerve impulses was decreased by extracellular La³⁺, spontaneous release of transmitter was substantially increased. The potentiation of MEPSP frequency by La³⁺ was suppressed in a high Ca²⁺ saline and enhanced in the absence of Ca²⁺. Thus, there might be an antagonism by Ca²⁺ of the La³⁺ acceleration of spontaneous release. DeBassio et al. (1972) have reported an antagonism between them and suggested that the competition might occur at anionic binding sites on the external surface of the nerve terminal. An inhibitory action on the spontaneous release of transmitter which has been found in the presence of Ca²⁺ (Washio, 1982a) has not been clear in the presence of La³⁺. The increase in the frequency was recorded in the absence of external Ca²⁺ or the presence of a very low concentration. Therefore, it seems more likely that La³⁺ acts on an intracellular site (DeBassio et al. 1972; Washio, 1982b). For example, La³⁺ has been reported to be an extremely potent inhibitor of the energy-linked accumulation of Ca²⁺ by mitochondria (Mela, 1968, 1969) and thereby increases the intracellular calcium concentration (Rahamimoff, 1978). However, MEPSP frequency was found to be lowered slightly in the presence of 5 mm-Ca²⁺ just after the application of 0·5 mm-La³⁺ in some cockroach muscle fibres partially depolarized with high K⁺ (Fig. IB). It might be possible that an inhibitory effect of La³⁺ on the external surface of the nerve terminal was followed by the acceleratory effect which masked the former.

Our results also showed that lanthanum ions decreased the amplitude of the transient depolarization (glutamate potential) produced by iontophoretically applied L-glutamate. Double logarithmic plots of the amplitude of the depolarizing response to L-glutamate against coulomb strength of the ejection currents showed that La³⁺ reduced the value for the slope of the junctional receptors. Therefore, it seems unlikely that L-glutamate and La³⁺ are competing for a common binding site on the post-synaptic membrane. Alternatively, La³⁺ might affect the kinetics of the channels activated by L-glutamate, since the trivalent ion can modify the time course of the MEPSP recorded extracellularly. It has been shown that the kinetics of L-glutamate-induced channels may be altered by the species of permeable cations (Cull-Candy & Miledi, 1982) as well as the kinetics of acetylcholine-induced ones (Gage & Van Helden, 1979; Bregestovski, Miledi & Parker, 1979; Magleby & Weinstock, 1980). On the other hand, it has been proposed that the change in the decay time of the endplate current can be explained by screening fixed negative charges on the end-plate membrane (Mallart & Molgó, 1978; Cohen & Van der Kloot, 1978). The kinetics of the channels depend upon the voltage gradient through the postsynaptic membrane. In insect muscles, hyperpolarization shortens the decay time of MEPSCs and depolarization lengthens the decay time (Cull-Candy & Miledi, 1982) which is in the opposite direction to the kinetics in vertebrate muscles (Anderson, Cull-Candy & Miledi, 1978) and to the behaviour expected from screening fixed negative charges (Cohen & Van der Kloot, 1978). Thus, the results obtained in insect muscles are incompatible with the surface charge interpretation. Recently, (+)-tubocurarine-induced block of the glutamate synapse resulting from plugging the open channels was reported in Tenebrio muscle fibres (Yamamoto & Washio, 1983). Thus, it might be possible that La³⁺ can alter the kinetics of glutamate channels by acting on channel properties as a non-competitive antagonist, like a local anaesthetic, or by changing the rates with which L-glutamate interacts with receptors, like acetylcholine and strontium (Miledi & Parker, 1980).

The authors wish to thank Professor David M. J. Quastel, Department of Pharmacology, University of British Columbia, for his helpful comments on the manuscript. Thanks are also due to Miss Junko Kaneko for her technical assistance.