Study was made of the influence of external sodium, calcium and magnesium concentrations on the directly elicited response of the segmental muscle fibres of the larva of Galleria mellonella, as recorded with intracellular microelectrodes. The graded response caused by an outward current pulse was converted into an all-or-none spike in the presence of 10 mM tetraethylammonium. The amplitude of these spikes increased with increasing external Ca concentration but not with increasing Na or Mg concentration. The spikes were not influenced by tetrodotoxin (3 × 10−5 M) but were suppressed by Mn ions (20 mM). After replacement of TEA-containing saline with an isotonic CaCl2 solution, spikes were still produced in the latter. It appears that only calcium ions act as charge carriers of inward current at the non-synaptic membrane.
Crustacean muscle fibres do not produce action potentials under normal conditions, and a strong depolarizing current pulse elicits oscillatory and graded responses only. These responses can be converted into all-or-none spikes by treatment with tetraalkylammonium ions externally (Fatt & Ginsborg, 1958; Werman & Grundfest, 1961) or internally (Hagiwara, Naka & Chichibu, 1964), or by elimination of internal calcium ions with chelating agents such as EDTA and EGTA (Hagiwara & Naka, 1964). Whereas sodium ions play a major role in generating action potentials in many excitable membranes (Hodgkin & Huxley, 1952; Katz, 1966), these crustacean muscle fibres produce action potentials involving an inflow of calcium ions instead of sodium ions (Hagiwara, Hayashi & Takahashi, 1969). Barium and strontium ions are also capable of generating all-or-none spikes.
Many insect muscle fibres seem to have similar properties to those of crustacean fibres. Graded responses elicited by outward current pulses in normal saline solution can be converted into all-or-none spikes by treatment with tetraethylammonium (TEA) (Belton & Grundfest, 1961 ; Washio, 1972) or ryanodine (Usherwood, 1962). Such action potentials are independent of external sodium concentration in the skeletal muscle fibres of a locust (Washio, 1972) and a fly (Patlak, 1976). Ba and Sr ions can also generate spikes. On the other hand, Na-dependent electrogenesis has been reported by many investigators in a variety of insect muscle fibres ; such as the skeletal muscle fibres of a grasshopper (Werman, McCann & Grundfest, 1961), Tenebrio (Belton & Grundfest, 1962) and a moth (Piek, 1975), the proctodeal muscle fibres of a cockroach (Nagai, 1972), and the heart muscle fibres of Orthoptera and Mantodea (S-Rózsa & V-Szöke, 1972).
In this paper, study was made of the ionic requirements for the generation of directly elicited responses in the muscle fibres of Galleria mellonella. This lepidopterous insect has a haemolymph which is characterized by a low concentration of sodium ions and high concentrations of potassium and magnesium ions (Duchâteau, Florkin & Leclercq, 1953; Florkin & Jeuniaux, 1974).
MATERIALS AND METHODS
The segmental muscle fibres of the larva of the waxmoth Galleria mellonella were used. The electrophysiological techniques were similar to those described in a previous paper (Yamamoto & Fukami, 1976). The muscle fibre membrane was depolarised intracellularly by square current pulses. These currents were injected through glass microelectrodes filled with 2 M potassium citrate having a resistance of 1-10Ω when tested in normal saline. Membrane potentials were recorded intracellularly with a second microelectrode filled with 2 M potassium citrate. The resistance of the recording electrode ranged from 5 to 20 MΩ. The recording and current electrodes were inserted near one another into the same fibre. The normal saline (Belton, 1969) had the following composition (mm): NaCl, 35; KC1, 36; CaCl2, 12; MgCl2, 16; glucose, 269; Tris(hydroxymethyl)aminomethane, 5. The pH of the solution was adjusted to 7.4 with HC1. Test solutions were prepared by replacing glucose with an osmotically equivalent amount of test ions. To maintain the isoosmolarity of the TEA-containing 100 mM Ca-saline, CaCl2 replaced part of the NaCl also. Salines containing lower concentrations of Na, K, Ca and/or Mg ions were prepared by substitution of an osmotically equivalent amount of glucose for the ions.
Experiments were conducted at a room temperature of 25 °C.
Graded responses were converted into spikes by TEA in concentrations above 10 mM (Fig. 1). No further effect was observed up to 50 mM. This contrasts with the situation in Romalea, in which the conversion is achieved at 1 × 10−3 M, and a block is caused at higher concentrations (Belton & Grundfest, 1961).
The effects of Na, Ca and Mg ions on the action potential produced by an outward current pulse were examined in the presence of 10 mM TEA. Changing the external Na or Mg concentration had only negligible effects on the overshoot of the action potential (Fig. 2). In contrast, the overshoot of the action potential and the rate of rise of the spike were markedly increased as the external concentration of Ca ions was raised (Fig. 3). The relationship between the peak potential of the spike and the logarithm of the external calcium concentration was almost linear, with a slope of 30 mV for tenfold change in the concentration (Fig. 4). The threshold membrane potential was not significantly changed in the Ca concentration range between 10 and 100 mM (Fig. 4).
Fig. 5 illustrates the effects of TTX (3 × 10−5M) and manganous ions (20 mM) on the action potentials elicited by outward current in the presence of TEA. TTX had no effect on the action potential (record B), whereas Mn ions markedly suppressed both the amplitude and the rate of rise of the action potential (record D).
Once treated with TEA Galleria muscle fibres were capable of producing action potentials in a saline solution containing 100 mM-CaCl2, 195 mM glucose and 5 mM Tris-HCl, but devoid of Na, Mg, K and TEA ions. Fig. 6 illustrates an example, in which the spike was first recorded in TEA-containing 100 mM Ca-saline (see Materials and Methods) (record A), and then in a solution devoid of Na, K, Mg and TEA ions (record B). The spikes could be recorded even 1 h after the change of saline, although the critical firing level was shifted in the positive direction and the falling phase of the action potential became faster. It should be pointed out that withdrawal of Ca ions from the bathing medium effectively suppressed the action potential in the presence of a high concentration of sodium or magnesium ions.
The experiments indicated that calcium influx was responsible for the rising phase of the TEA-induced spikes in the non-synaptic membrane of muscle fibre of Galleria. TEA was shown not to be involved by the observation that the spikes persisted after its withdrawal (Fig. 6). A similar observation has been made for tetraalkylammonium salts in crustacean muscle fibres (Fatt & Ginsborg, 1958). Evidence for the role of calcium was as follows. Firstly, the overshoot of the spikes increased with external Ca concentration, but was unaffected by change in Na and Mg concentrations (Figs. 2-4). Secondly, in fibres pretreated with TEA, spikes were generated in a solution which contained only calcium as a cation (Fig. 6). Finally, in the presence of TEA, the spikes were not affected by high concentration of TTX (3 × 10−5 M) but were suppressed by 20 mM manganous ions (Fig. 5). In many excitable membranes, the Na channel is readily blocked by 10−7 to 10−8 M TTX (Narahashi, Moore & Scott, 1964; Narahashi, 1974), and the Ca channel is selectively suppressed by Mn ions (Hagiwara & Nakajima, 1965; 1966; Narahashi, 1974). Since it has been established that TTX and TEA act independently upon Na and K channels, respectively (Hille, 1967), the possibility that TEA prevents the TTX action appears unlikely.
Among insects, calcium-dependent action potentials have been reported in skeletal muscle of a locust (Washio, 1972), Drosophila (Ikeda, Ozawa & Hagiwara, 1976), Sarcophaga (Patlak, 1976) and Ephestia (Deitmer & Rathmayer, 1976); the heart muscle of Hyalophora (McCann, 1971); and a motoneurone cell-body (Pitman, 1976). Na-independent and probably Ca dependent action potentials have been reported in the myocardia of Locusta migratoria, Phaneroptera nana and Mantis religiosa (S-Rózsa & V-Szöoke, 1971, cited by Miller, 1975), and the hind-gut muscle of Leucophaea maderae (Cook & Reinecke, 1973).
One observation in this study of Galleria muscle was not in keeping with previous results; there is no effect of Ca concentration on threshold potential (Fig. 4), whereas the stabilizing action of Ca ions has been clearly demonstrated in locust muscle fibres (Washio, 1972). This may be due, at least in part, to the relatively high concentrations of Mg ions in the bathing media (Hagiwara & Takahashi, 1967).
The authors wish to express their indebtedness to Dr Y. Obara for his constant guidance. Their cordial thanks are due to Prof. T. Narahashi for his reading and commenting on the manuscript. Thanks are also due to Messrs H. Wago and H. Yoshimoto for many valuable references, and to Prof. H. Sugi and Dr T. Ichinose for their kind advice.
TTX was kindly supplied by Dr S. Tsuda.