ABSTRACT
The effects of GABA and picrotoxin on the input resistance of the muscle of the earthworm, Pheretima communissima, in Ringer solution and in solutions containing various foreign anions were observed.
Substitution of Cl− by I− and Br− reduced the input resistance and hyperpolarized the membrane. Although anions larger than chloride in hydrated size increased the input resistance, no change of the membrane potential was observed.
GABA reduced the input resistance of the membrane and picrotoxin increased it in Ringer solution. The dose-response curve for the changes of the input resistance under various concentrations of GABA shifted parallel-wise under treatment with picrotoxin.
In the presence of foreign anions which had larger hydration size than Br−, GABA reduced the input resistance. Picrotoxin did not, however, increase the input resistance when the solutions contained anions of smaller hydration size than C1O4−.
Reversal potential levels for the miniature inhibitory junction potential in various concentrations of chloride were measured. The change of the reversal potential levels produced by a tenfold change of chloride concentration was 25 mV.
INTRODUCTION
Hidaka et al. (1969b) have investigated neuromuscular transmission in the longitudinal muscle of the earthworm. They reported that electrical stimulation of diffusely innervating inhibitory nerves released GABA which increased the conductance only for chloride ion, thus hyperpolarizing the membrane and blocking the spontaneously generating spikes and the spikes evoked by electrical stimulation. The inhibitory actions of GABA were completely blocked by treatment with picrotoxin. They estimated the chloride equilibrium potential (EC1) to be approximately −55 mV. from the reversal potential levels for the miniature inhibitory junction potential (m.i.j.p.) and the inhibitory junction potential (i.j.p.). This level of the membrane potential was 20 mV. more negative than the resting membrane potential of −35 mV.
Hidaka et al. (1969 a) have also observed the effects of various ions on the membrane potential using the longitudinal muscle of earthworm. When the chloride in the medium was replaced with less permeant anions than chloride ion, the membrane potential level remained the same before and after treatments but the input resistance of the membrane was increased to twice the input resistance observed in the normal solution.
Del Castillo, De Mello & Morales (1964) have investigated the effect of anions on the somatic muscle of Ascaris lumbricoides. They observed that if Cl− was replaced by SO42− a depolarization was produced. If however, Cl− was replaced by SO3− and I− the membrane was hyperpolarized. Recently Del Castillo, De Mello & Morales (1967) reported the effect of nerve stimulation on the membrane activity of the somatic muscle of Ascaris-, they postulated that inhibitory nerves might release GABA from the nerve terminals. The somatic muscle of both Ascaris and the earthworm show oblique arrangement of the contractile protein but they show quite different morphological structures.
The primary aim of this experiment was to investigate the competitive action of GABA and picrotoxin on the anion permeability of the membrane in the presence of the various foreign anions.
METHOD
A longitudinal layer of the somatic muscle of the earthworm, Pheretimma communissima, 5-8 cm. in length, was used. The earthworm was pinned on a rubber plate and dissected lengthwise from the dorsal side. The alimentary canal was carefully dissected out and removed from the body wall. A 1–1·5 cm-section of the remaining tissue was pinned on the plate. The tissue was immersed in an organ bath through which various solutions at room temperature flowed continuously. The physiological solution (Ringer solution) used for this tissue was of the following composition: Na, 140 mM; K, 2 ·7 mM; Ca, 1 ·8 mM; Mg, 1·0 mM; Cl, 148·3 mM, and pH adjusted to 7·3–7·5.
Microelectrodes were used for making the electrical recording and for stimulating, by means of the Wheatstone bridge method. The range of the applied current was between 10−10 and 5 x 10−9 A. The microelectrodes were filled with 3 M-KCI for the measurement of the various properties of the membrane and with 3 M-K-citrate for the measurement of the reversal potential for the m.i.j.p. The resting potential of the membrane under the various foreign anions was measured with 3 M-KCI. TO reduce the junction potential produced between microelectrode and the perfusing solution containing foreign anions, another microelectrode was inserted in the bath as an indifferent electrode.
Chloride ion was substituted by the various foreign anions Br−, I−, NO3−, C1O4−, C2H5SO3− and D-glutamate, and pH was adjusted to 7·3–7·5. Concentrations of the drugs used in the experiment are described in the Results.
RESULTS
Effects of various anions on the membrane potential and input resistance of the muscle membrane
Figure 1 shows the effects of rapid replacements of Cl− by Br− and glutamate on the membrane potential. Individual spots in the figure indicated the mean values of the 3-5 measurements within every minute. The external chloride concentration, [Cl]0, remained at 5·6 mM in the solution as CaCL2 and MgCl2. When Cl− was rapidly replaced with other anions which had a larger hydration size than chloride ion (Robinson & Stokes, 1959), the membrane was transiently depolarized. For example in an experiment, glutamate depolarized membrane from −37 mV. to −22 mV. In another experiment, C2H6SO3− solution depolarized the membrane from −35 mV. to −23 mV. Their depolarization, however, did not last more than a few minutes. Replacement of glutamate solution by Ringer solution transiently hyperpolarized the membrane from −35 mV. to −56 mV. at peak (Fig. 1). In the case of C2H5SO3−, it was hyperpolarized to −55 mV.
When Cl− was substituted by Br− the membrane was transiently hyperpolarized, gradually depolarizing within a few minutes, but did not return to the level before the treatment. When Br− solution was washed out with Ringer solution, the membrane was transiently depolarized within a few minutes.
Figure 2 shows the effects of the various anions on the membrane potential. The mean membrane potential was calculated from the measurements of 50-100 fibres. When Cl− was substituted by anions which had a larger hydration size than Cl−, no change of the membrane potential could be observed. The anions of hydration size smaller than that of Cl− hyperpolarized the membrane. For example, the membrane potential ( –35 mV., S.D. = ± 1 ·0, n = 50) was hyperpolarized to-37·5 mV. (S.D. = ±0 ·8, n = 50) in 18 ·2 mM Br−, to −38 ·9 mV. (S.D. = ±o-8, n = 50) in 72 ·8 mM Br− and to-39·4 mV. (S.D. = ± 1 ·0, n = 50) in 142 ·7 mM Br−.
The input resistance of the membrane was measured from electrotonic potentials evoked by weak inward currents. Current-voltage relation observed in the solution containing either chloride or foreign anions showed a linear relation with the ranges of displacement of the membrane potential from −100 mV. to −20 mV. The input resistance was measured after 15 min. perfusion with the substituted solutions. Figure 3 shows the effects of intracellular polarizing currents on the membrane applied by Wheatstone bridge method. When outward current was applied to the fibre the membrane was depolarized and finally triggered the spike. The critical membrane potential for spike generation was –20 mV. The elicited spike showed an overshoot potential which was followed by after-hyperpolarization, as observed from the spontaneous discharges. The spikes were always recorded in the solutions containing the foreign anions used in the present experiments. The input resistance of the membrane varied in the individual fibres and also during the season. For example, the mean input resistance of the membrane measured during the summer season (May-October) was 37 MΩ (22-56 MΩ, S.D. = ± 2 ·4, n = 42). During the winter season (November-March) it was 67 MΩ (42-102 MΩ, S.D. = + 3·1, n = 55). Therefore values of the input resistance in Ringer solution was recorded as 100% (see also Table 1). Figure 4 shows the effects of various anions on the input resistance of the membrane. The input resistances were measured on 3-15 different fibres in a series of experiments. When Cl− was replaced with or glutamate, the input resistance was increased up to 150%. In the input resistance was increased to 118%, although the membrane potentials measured in solutions containing this anion remained the same as before the substitution. When, however, Cl− was replaced with I− or Br−, both the membrane conductance and the membrane potential were increased.
The results showed that the changes of the input resistance did not closely correlate with the changes of the membrane potential.
Effects of GABA and picrotoxin on the input resistance of the membrane
In the present experiment it was difficult to distinguish between the receptor and non-receptor regions of the membrane, since the fibre diameter was only 5-10 pm. and the length of the fibre was about 1 mm. Wherever microelectrodes were inserted, the outward current elicited the spike and m.i.j.p.’s could also be recorded. Moreover, the space constant calculated in this fibre from the equation introduced for a limited cable by Fatt & Ginsborg (1958) was only 140 /μm. Concentrations of more than 10−7 g./ml. of GABA reduced, and more than 10−8 g./ml. of picrotoxin increased, the input resistance of the membrane. In one series of experiments the mean input resistance of the membrane in Ringer solution was 51·2 MΩ (S.D. = ±3 ·8, n = 10). GABA (10−8 g./ml.) reduced the input resistance to 37·8 MΩ (S.D. = ±2 ·8, n = 17). On the other hand, picrotoxin (10−5 g./ml.) increased the input resistance from 32-2 MΩ (S.D. = ±3 ·1, n = 11) to 47 ·9 MΩ (S.D. = ±3 ·8, n = 15).
When picrotoxin (10−5 g./ml.) and GABA (10−5 g./ml.) were applied simultaneously to the tissue, the input resistance remained the same as that measured before the treatment (control; 38-9 MΩ, S.D. = ±4·1, n = 6, test solution; 40·7 MΩ, S.D. = ±2·8, n = 13). Strychnine had no effect on the input resistance of the membrane (control; 35-4 MΩ, S.D. = +6·0, n = 6, strychnine 10−8 g./ml.; 34-2 MΩ, S.D. = ± 5·5, n = 5). These results might indicate competitive action of GABA and picrotoxin on the input resistance of the membrane. Figure 5 shows the effects of various concentrations of GABA (10_8-10−5 g./ml.) on the input resistance of the membrane in the presence or absence of picrotoxin. 10−8 g./ml. of GABA had no effect on the input resistance of the membrane. Increased concentrations of GABA, however, reduced the input resistance from 39 MΩ (S.D. = + 4·2, n = 7) to 30 MΩ (S.D. = ±4·6, n = 5) in 10−7 g./ml. and to 28 MΩ (S.D. = ±4·8, n = 4) in 10−6 g./ml. of GABA. Further increased concentrations of GABA only slightly reduced the input resistance. On the other hand, picrotoxin increased the input resistance of the membrane; that is, 31-6 MΩ (S.D. = ±4·8, n = 4) in the normal solution increased to 35·8 MΩ (S.D. = ±2·3, n = 6) in 10−7 g./ml. to 52·8 MΩ (S.D. = ±7·6, n = 4) in 10−6 g./ml. and to 53·8 MΩ (S.D. = ±5·2, n = 9) in 10−5 g./ml. of picrotoxin.
Picrotoxin (10−6 g./ml.) increased the input resistance even in the presence of GABA (10−7-10−6 g./ml.). The dose-response curve observed under the various concentrations of GABA shifted, in the presence of picrotoxin (10−6 g./ml.), to the side of the higher concentrations of GABA. These results might indicate an antagonistic action of GABA and picrotoxin on the input resistance of the membrane, particularly affecting the anion channel.
Effects of GABA and picrotoxin on the input resistance of the membrane in the presence of various foreign anions
Effects of GABA (10−6 g./ml.) and picrotoxin (10−6 g./ml.) on the input resistance of the membrane in solutions containing Br−, I−, NO3−, C1O4−, C2H5SO3− and glutamate were investigated. Table 1 shows the measured values of the input resistance of the membrane in solutions containing the above anions, in the presence and in the absence of GABA and picrotoxin. As described previously, the input resistance even in the physiological solution varied remarkably. However, the effects of picrotoxin and GABA on the input resistance of the membrane were consistent. For example, in the case of Cl− and of anions whose hydration size is larger than that of chloride, GABA reduced the input resistance. Conversely, when the hydration size of the anion is smaller than that of Cl−, e.g. Br−, no reduction of the input resistance was observed. When, however, the hydration size is larger than C1O4−, no effect of picrotoxin was observed. With anions smaller than C104−;, picrotoxin increased the input resistance of the membranes.
Figure 6 shows the effects of GABA (10−6 g./ml.) on the input resistance of the membrane in the presence of various foreign anions; Figure 7 shows the effects of picrotoxin. In the figures the input resistances of the membrane in the physiological solution are shown as 100%. The relative changes of input resistance under the various conditions were also compared. The mean value of the input resistance observed under treatment with picrotoxin was 150%, When the input resistance was increased to 150% by foreign anions no further increase of the input resistance was observed by treatment with picrotoxin. In contrast, under treatment with GABA anions of hydration size larger than Cl− reduced the input resistance nearly to the value obtained in the physiological solution.
Reversal potential levels for the m.i.j.p.’s in various concentrations of chlorideion
When Cl− was replaced in steps with D-glutamate, the membrane potential remained the same but the m.i.j.p. was gradually reduced in amplitude. Further reduction of [Cl]0 reversed the polarity of m.i.j.p.s. In all the experiments D-tubocurarine (10−6 g./ml.) was added to prevent the generation of m.e.j.p.s.
Figure 8 shows the effects of variations in [Cl]0 on the m.i.j.p.’s. When the [Cl]0 was reduced to 17-7 mM, the m.i.j.p.’s were nearly abolished and in 5’6 mM the polarity of the m.i.j.p.’s was reversed.
Figure 9 shows a typical example of the change of polarity of the m.i.j.p.’s in a chloride-deficient solution ([Cl]0 = 5·6 mM) at various membrane potential levels. D-tubocurarine (10−6 g./ml.) was continuously perfused through the tissue during the experiments. In the chloride-deficient solution (substituted by glutamate) m.i.j.p.’s could be recorded with a polarity in the depolarizing direction. Outward current applied to the muscle fibre by the Wheatstone bridge method elicited spikes with overshoot and after hyperpolarization (see Fig. 9). To measure the reversal potential level, intracellular polarizing currents were applied to the muscle fibre with the minimum gradient of the increased current for the prevention of the spike generation. In this particular muscle fibre the reversal potential level for the m.i.j.p.’s in 5·6 mM [Cl]0 was –26 mV. In the physiological solution, the reversal potential level in this experiment was −60 mV. (S.E. = ± 4·1, n = 3) and −55 mV. (S.D. = + 4·1, n = 3) in 75·4 mM [Cl]0,-44 mV. (S.D. = ±5·1, n = 3) in 33·6 mM [Cl]0,-35 mV. (S.D. = ±4-3, n = 4) in 14·5 mM [Cl]0 and −24 mV. (S.D. = ±2·8, n = 5) in 5·6 mM [Cl]0. Reversal potential level plotted against [Cl]0 on a logarithmic scale was linear, with a slope of 25 mV. per tenfold change of [Cl]0. Assuming that the internal concentration of chloride ion remains the same, the value was much smaller than that expected from the Nernst equation, namely 56 mV. The simple explanation of the above results is that reduction of the external chloride concentration may be followed by reduction of the internal chloride concentration of the muscle fibre.
DISCUSSION
Del Castillo et al. (1964) observed the effects of various anions on the membrane potential of the somatic muscle of Ascaris. They reported that the membrane potential decreased only when chloride was substituted by SO42−, but increased in the presence of Br−, I−, and N0a−. They thought that these three anions passed through the muscle membrane more readily than chloride. Their observations were roughly in agreement with what is reported for the visceral smooth muscle of mammals (Holman, 1958; Kuriyama, 1963; Bûlbring & Kuriyama, 1963a, b).
The observations made on the above tissues were quite different from those observed in the frog skeletal and cardiac muscles (Hutter & Noble, 1961 ; Hutter & Padsha, 1959; Hodgkin & Horowicz, 1959, 1960; Adrian, 1961; Harris, 1963). In frog skeletal muscle two thirds of the resting membrane conductance is due to chloride. Anion like NO3−, Br−, I− and thiocynate−, which slow down the chloride efflux, appear to do so by decreasing the permeability of the sarcolemma to chloride, thus reflecting in the increase in the resistance of the sarcolemma caused by these anions (see review of Caldwell, 1968).
The membrane potential of the somatic muscle in the earthworm, in respect of changes in the external medium, behaved differently when compared with the skeletal muscle of the frog and the somatic muscle of Ascaris’, that is, changes in the input resistance of the membrane did not cause changes in the membrane potential. Furthermore, the chloride equilibrium potential (EC1) estimated from the reversal potential level for the m.i.j.p. was about −60 mV., assuming that chloride pump was inactive during the determination of the chloride equilibrium potential. This value was 25 mV., which was more negative than the membrane potential of −35 mV. Therefore, the role of the chloride ion in determining the membrane potential cannot be explained by the Boyle-Conway type of Donnan equilibrium as invoked for frog muscle by Hodgkin & Horowicz (1959).
GABA, thought to be an inhibitory chemical transmitter, selectively increased the chloride conductance in the somatic muscle of the earthworm. The m.i.j.p.’s and j.p’s elicited by externally applied electrical stimulation had a reversal potential level similar to that observed for the GABA potential evoked by iontophoretically applied GABA. The m.i.j.p.’s and i.j.p.’s were blocked by treatment with picrotoxin and their polarity was reversed in the chloride deficient solution. Furthermore, the cross-over point of the current-voltage relation observed in five times the normal [K]o and five times the normal [K]o with GABA (10−6 g./ml.) was −56 mV. In the K-free solution and the K-free solution with GABA (10−s g./ml.) the cross-over point was –55 mV., thus indicating that GABA had no effect on the potassium permeability. The reversal potential level for the m.i.j.p. in the Na-free solution was similar to that observed in the normal solution (Hidaka et al. 1969 b). These effects of GABA on the neuromuscular transmission in the somatic muscle fibres of the earthworm confirmed the observations made on the neuromuscular junction in many invertebrates (see Florey, 1961; Tauc, 1967; Gershenfeld, 1966; Grundfest, 1966).
The dose-response curve for the molluscan neurone was obtained by Gershenfeld & Stefani (1965) by plotting the effect of iontophoretically applied 5-hydroxytryptamine against the logarithm of the injecting current with or without the presence of bromolysergic acid (BOL 148). They found that the curves corresponded to a family of curves of similar slope and saturation level. From these findings it might be inferred that there was competition for the same receptor site between the 5-hydroxytryptamine and antagonist bromolysergic acid. In the present experiment changes of the input resistance under various concentrations of GABA were taken as an indicator of the response. The dose-response curves obtained in the presence of different concentrations of picrotoxin showed parallel shifts, perhaps indicating competitive actions of GABA and picrotoxin on the same chloride channel. The picrotoxin-sensitive anion channels were distributed among the receptive and non receptive regions in crustacean muscle (Grundfest, 1966). On the other hand, van der Kloot (1960) observed that in the crayfish stretch-receptor neurone the synaptic region is 1000 times more sensitive to GABA than the axonal region. Furthermore, locations of GABA-sensitive spots in crustacean muscle were confirmed by the iontophoretic application of GABA by Takeuchi & Takeuchi (1965, 1966). In this tissue it was difficult to distinguish receptive and non-receptive regions owing to the small diameter and the short length of the muscle fibres.
It was difficult to explain the reduction of the input resistance of the membrane under treatment with GABA, and the failure of the input resistance to increase under picrotoxin in the solutions containing larger foreign anions.
Coombs, Eccles & Fatt (1955), Araki, Ito & Oscarson (1961) and Ito, Kostyuk & Oshima (1962) have studied the anion channel of the neurone soma related to the action of inhibitory chemical transmitter. They concluded that despite the apparent exceptions to the main sequence the inhibitory transmitter converts the inhibitory subsynaptic membrane into a sieve-like structure that allows small hydrated ions to pass and blocks all larger ones. For all other ion species the critical pore diameter must be less than that of the PF6 anion. Two species of ions, HS− and HCO2− lies out of the main sequence in which permeability of the activated inhibitory membrane may be correlated with ionic size in the hydrated state. These ions, however, had special action on the membrane (Eccles, 1964).
GABA might increase the size of anion channel although ethansulphonate and glutamate are thought to be too large to penetrate the anion channel in the somatic muscle of the earthworm.
Finally it is not yet known whether sodium permeability and potassium permeability remain constant when [Cl]0 is reduced to th of the normal concentration. If the membrane properties were modified in the chloride-deficient solution, the changes of the input resistance of the membrane might not be explained solely by the changed anion permeability.
Further detailed studies on the properties of the muscle membrane of the earthworm will be required to explain the ionic mechanism involved in the drug actions.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the kindness of Professor N. Toida in making arrangements for these experiments. This work was supported by a grant from the Educational Ministry of Japan.