EFFECTS OF VARIOUS IONS ON THE RESTING AND ACTIVE MEMBRANE OF THE SOMATIC MUSCLE OF THE EARTHWORM

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1 J. Exp. BioL (1969), 50, s 405 With 8 text-figures Printed in Great Britain EFFECTS OF VARIOUS IONS ON THE RESTING AND ACTIVE MEMBRANE OF THE SOMATIC MUSCLE OF THE EARTHWORM BY T. HIDAKA, Y. ITO, H. KURIYAMA AND N. TASHIRO Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan {Received 10 July 1968) INTRODUCTION The membrane potential of the longitudinal muscle of the earthworm is only 35 mv. and this value is much lower than those of the Loligo giant fibres ( 60 mv.), frog striated muscle ( 90 mv.) and mammalian smooth muscle ( 50 mv.). The difference in the membrane potential might suggest a specificity in the ionic distribution or ionic permeability in the longitudinal muscle of the earthworm different from that of other excitable cells. The mechanism of spike generation in Loligo giant fibres and frog skeletal muscle was investigated in detail by Hodgkin & Katz (1948), Hodgkin, Huxley & Katz (1952) and Hodgkin & Huxley (1952a, b). Their results supported the conclusion that the upstroke of the spikes was due to the inward movement of sodium ions. In contrast, in the frog spinal ganglion (Koketsu, Cerf & Nishi, 1959; Nishi, Soeda & Koketsu, 1965), crustacean muscle (Fatt & Katz, 1953; Fatt & Ginsborg, 1958) and barnacle muscle (Hagiwara, Chichibu & Naka, 1964) spikes could be generated in sodium-free solution (sodium was substituted by hydrazine, barium strontium or tetra-ethyl ammonium). Furthermore, in barnacle muscle, calcium-dependent spikes were clearly demonstrated by a most skilful technique; and tetrodotoxin, known to block any increase in the sodium conductance in the frog nerve fibre and muscle during the active state, had no effect on spike generation in the barnacle muscle (Narahashi, Moore & Scott, 1964; Hagiwara & Nakajima, 1965). As described in the previous paper, tetrodotoxin did not influence either the spike amplitude or the numbers of train discharges in the longitudinal muscle of the earthworm, generated either spontaneously or in response to electrical stimulation (Hidaka, Ito & Kuriyama, 1969). The present experiments were intended to investigate the effects of various ions on the resting and active states of the membrane. The results led to the conclusion that the spikes are generated by the inward movement of calcium ion, and the low membrane potential is due to relatively high sodium permeability and low potassium permeability. METHODS The experimental methods and procedure were the same as described previously (Hidaka, Ito & Kuriyama, 1969). The concentrations of the various drugs used in the present experiment will be described in the results. 26-3

2 406 T. HffiAKA AND OTHERS RESULTS The resting membrane potential The relationship between the membrane potential and the external potassium concentration was observed in the cell membrane of the longitudinal muscle. High potassium concentrations, [K] o, were prepared by adding solid KC1 to the solution. Figure i shows the changes in the membrane potential against [K] o on a logarithmic scale, in normal concentration of sodium and in sodium-free (tris) solution. In solutions containing NaCl the curve relating the membrane potential to [K] o had two Or 10 r 20 S 30 S 40 Normal saline. Na-free saline K + concentration (mm) Fig. i Ionic concentrations (HIM) Fig. 2 Fig. i. Changes in the membrane potential against (K) o on a logarithmic scale, in a normal concentration of sodium and in sodium-free (tris) solution. Arrow indicates the normal potassium concentration. Individual points indicate the mean values of measurements. Fig. 2. Effects of external sodium, calcium and chloride ions on the membrane potential. Arrows indicate normal concentration in the physiological solution. Individual pointb in the figure indicate the mean values of measurements. 100 different forms, one above and one below a [K] o of 14 mm. The maximum slope of the membrane potential, i.e. the change in potential produced by a tenfold change in [KJ, was 27 mv. above 14 mm [K] o, was 7 mv. below 14 mm (the mean of five preparations). These values were much smaller than the values expected from the Nernst equation. Even when the solutions were kept isosmotic by reducing (Na) 0, the maximum slope was only 30 mv. (the mean of thefivespecimens). In sodium-free solution, above 14 mm, the maximum slope was 42 mv. and, below 14 mm, the slope was 30 mv. (n = 5 specimens). It is significant that at the normal [K] o, i.e. 2*8 mm., the membrane was hyperpolarized from 36 mv. (n = 55, S.D. = ±3*1) to 58 mv.

3 Effect of ions on membrane of muscle of earthworm 407 (n = 55, s.d. = + 3-8) in sodium-free solution, indicating a very dominant influence of sodium permeability in normal saline in determining the membrane potential. Figure 2 shows the effects of external sodium, calcium and chloride ions on the membrane potential. When the sodium was replaced with tris in stages, the membrane was hyperpolarized in proportion to [Na] 0. The maximum slope in relation to [Na] 0 was 21 mv. (n = 5 specimens). Below [Na] 0 of 7 mm the membrane potential remained at nearly the same level of -557 mv. (n = 25, S.D. = ±4-39). The actual hyperpolarization of the membrane caused by a reduction in [Na] 0 to one-twentieth of normal in this experiment was 21 mv. The changes in the membrane resistance (i? e a) were measured by applying a weak electrical current. The membrane conductance was reduced in sodium-free solution to 77% of its value in the normal solution ( Cl 5-6 mw K27IHM Cl mm» l l I I I I l Minutes Fig. 3. Effect of sudden change of external chloride concentration on the membrane potential. Each point in the figure indicates the mean value of measurements. External potassium concentration remains the same. Calcium-deficient solutions depolarized the membrane. When [Ca] 0 was reduced to a tenth of the normal concentration, the membrane was depolarized from 32-5 mv. (n = 25, s.d. = ±3-6) to -i6-omv. (n = 25, s.d. = ±2-1). The maximum slope in relation to [Ca] 0 was 17 mv. This value was very low compared with the value expected from the Nernst equation, which is 28 mv. Increased [Ca] 0 hyperpolarized the membrane but only by a few mv., i.e. 2-5 times the normal concentration hyperpolarized the membrane from 35 to -39 mv. Further increase in [Ca] 0 did not increase the membrane potential, and ten times the normal concentration hyperpolarized the membrane by only a further 2-5 mv. beyond the 39 mv. induced by a 2-5-fold increase. The effect of varying the chloride concentration in the external solution was also observed. The chloride was replaced by D-glutamate in the form of sodium glutamate and potassium glutamate at ph 7-6, but the solution still contained 5-6 mm chloride from the CaClj, and MgCl 2. Figure 3 shows the effect of sudden change of external chloride concentration on the membrane potential. Each point in the figure indicates the mean value of 12-20

4 408 T. HlDAKA AND OTHERS measurements. [Cl] 0 was reduced from to 5-8 mm. without any change in [KJ. The membrane was transiently depolarized for 3-5 min., and then repolarized to the value before [Cl] 0 was reduced. When the solution was restored to normal, the membrane was transiently hyperpolarized (14 mv.) and returned to the normal level after being rinsed for 3-5 min. Figure 4 shows the changes in the electrotonic potentials after treatment with chloride-deficient solution. The membrane resistance and capacitance were measured from the electrotonic potentials. The membrane resistance (i?en) was increased in the chloride-deficient solution and the time constant of the membrane was slightly prolonged. Control (Cl=144 mho CI-<Jefldency (CI=5-6 mi^ 50 mv. 400 msec 400 msec " ^ ^ ^ ^ ^ ^ ^ 1 1 2x 10-* A. Fig. 4. Effect of chloride-deficient solution on the electrotonic potentials produced by intracellular polarizing currents, (a) Control, (6) after treatment with chloride-deficient medium. (Cl = 5-6 min. D-glutamate = mm.) The values, before treatment, of the membrane resistance (R et t), the time constant (T), the specific membrane resistance (R^, the capacitance (C m ) and the total conductance of the membrane (G^) were compared with those obtained after treatment with chloride-deficient solution. The specific resistance and capacitance were calculated from the equation for a limited cable (Hidaka, Ito & Kuriyama, 1969); the controli? eff was44 MQ.(n = 12, s.d. = ±3-1), T was 72 msec, (n = 12, S.D. = ±5-8), R m was 14 KQ cm 2, (n = 12, S.D. = ±0-9) and C m was 5-2/iF./cm a. After the treatment, R^ was 79 Mli (n = 10, s.d. = ±8-i), r was 85 msec, (n = 10, S.D. = ±8-3), Rn was 24-5 Kli cm 2, (n = 10, s.d. = ±2*1) and C m was 3-5/iF./cm. 2 (n = 10, s.d. = ± 3*4) respectively. The conductance (G m ) was reduced to 56% of the control value. Figure 2 also shows the effect of the various external chloride concentrations on the membrane potential. Reduction of [Cl] 0 transiently depolarized the membrane, but after mm - *^e membrane potential returned to the normal level and [Cl] 0 did not influence the membrane potential. The active membrane potential Figure 5 shows the effect of excess potassium on spontaneous spikes discharges. Excess [K] o depolarized the membrane as described previously. The spike height and after hyperpolarization were decreased and the spike duration was prolonged. When [K] o was increased to 27 mm., a spike could be triggered but no after-hyperpolarization

5 Ejfect of ions on membrane of muscle of earthworm 409 Control KCI 2-7 m SxKCI (c) nox KCI 50 mv. 200 msec- Fig. 5. Effect of excess potassiuin on spontaneous spike discharges. The spikes are recorded after 30 min. of replacement, (a) Control, (b) 13-5 rnm-k + (5 times of normal (K) o ), (c) 27 mm-k + (10 times). 50 mv miec 200miec Fig. 6. Effects of sodium-free (tris) solution on the spikes elicited by intracellular polarization, (a) Control, (6) sodium-free solution after 30 min.

6 T. HlDAKA AND OTHERS was observed. In shape the spike exactly resembled the spike of the Purkinje fibres of the mammalian heart (Weidmann, 1956). The fact that increase in the external potassium ions reduced the after-hyperpolarization indicates that the after-hyperpolarization was due to increased potassium conductance. Figure 6 shows the effect of sodium-free (tris) solution on the membrane activity. Intracellular depolarizing currents triggered spikes with overshoot and afterhyperpolarization (a). When the tissue was bathed for 3 hr. in continuously flowing sodium-free (tris) solution, the membrane was hyperpolarized to about 60 mv. An intracellular depolarizing current could trigger a spike with an overshoot potential but no after-hyperpolarization was observed (b). The increased amplitude of the spike was due to hyperpolarization of the membrane. The effective membrane resistance was increased from 30 MQ {n = 6, S.D. = + 2-8) to 39 MQ (n = 6, S.D. = ± 4-1). (a) Ca 0-18 mm Na-free (Trl5) "100miec 100 msec Fig. 7. Effects of various external calcium concentrations on spike generation in sodium-free solution, (a) Ca; o-i8 mm. (6) Ca; i-8 mm (control), (c) Ca; 18 DIM. To investigate the nature of the inward movement of ions the effect of calcium ions on membrane activity in sodium-free (tris) solution was observed. Figure 7 shows the effects of various calcium concentrations on the membrane activity. The spikes were triggered by extracellular stimulation with pulses 1 msec, in duration. As shown in Fig. 7, in a tenth the normal concentration of [Ca] 0 small graded responses to the external stimulation could be generated. At normal [Ca] 0 (i-8 mm), electrical stimulation could generate spikes with an overshoot potential. Increasing [Ca] 0 further increased the amplitude of the spikes. The relationship between the membrane potential and the maximum rate of rise and the amplitude of the spikes, under various conditions of [Ca] 0, are illustrated in Fig. 8. As described previously, the membrane was significantly depolarized in calcium-deficient solution; however, when the tissue was immersed in calcium-deficient, sodium-free solution the membrane was not depolarized. Therefore, the depolarization of the membrane in calcium-deficient solution might be due to increased sodium conductance, controlled by calcium but not due to the direct action of calcium itself. The amplitude of the spikes elicited by extracellular stimulation increased in proportion to the increased [Ca] 0 up to 2-5 times the normal concentration; further increase in [Ca] 0 did not increase the spike amplitude, as expected from the extrapolated relation observed from the concentration changes between 015 and 36 mm. The maximum slope of the changes in amplitude of the spikes per tenfold change of [Ca] 0 was 39 mv. (n = 3 specimens). The change in the amplitude of the overshoot potential produced by a tenfold change in the [Ca] 0 was

7 Effect of ions on membrane of muscle of earthworm mv. (n = 3 specimens); this value was slightly lower than the value of 28 mv. expected from the Nernst equation. The maximum rate of rise of the spikes was increased from 10-5 V./sec. to 32 V./sec. (n = 10) in ten times normal [Ca] 0. Thespike duration at 50% height was reduced by increasing [Ca] 0. These results strongly support the hypothesis that the ions moving inward during the active state of the membrane are calcium ions. +20 T20 i i i -20 A' Max. rate of rise N»-fre«10-60 M.p.Na-fre«- / / mm-ca f+ Fig. 8. Effects of various external calcium concentrations on the membrane potential, maximum rate of rise and amplitude of spike. Maximum rate of rise of spike must be read by V./sec. (scale of right side). The membrane potentials were measured in sodium-free solutions and in solutions containing sodium. Individual points in the figure indicate the mean values of measurements. It has been clearly demonstrated in barnacle muscle that spike generation caused by the inward movement of calcium ions is competitively blocked by the presence of Mn*+ (Hagiwara & Nakajima, 1965). Effect of manganate ions on the membrane activity of the longitudinal muscle of the earthworm was observed. 3 mm Mn^ in the normal solution gradually prolonged the spike duration, mainly due to prolongation of the second component of the falling phase. The plateau phase sometimes exceeded 2 sec, with sustained depolarization between the two membrane potential levels. Higher Mn 2 " 1 " concentrations (> 5 mm) depolarized the membrane and blocked the spontaneous discharges completely. In this condition, electrical stimulation could elicit spikes with a very prolonged plateau phase. However, spike generation in re-

8 412 T. HlDAKA AND OTHERS sponse to repetitive stimulation failed when the frequency of stimulation exceeded o-2 c./sec. Prolonged perfusion of the tissue in this Mn^ solution finally caused a depolarization block of the membrane activity. DISCUSSION The membrane potential The membrane potential in the longitudinal muscle fibre of the earthworm is mainly governed by the potassium and sodium concentration gradients across the cell membrane and the permeability to potassium and sodium. In this muscle the ratio of sodium permeability to potassium permeability seems very high compared with other excitable tissues. Sodium-free solution hyperpolarized the membrane from 37 to 60 mv, and the membrane conductance was reduced to 77 % of the normal value. It is considered that chloride permeation in this tissue takes place in a passive manner since reduction of the external chloride concentration did not influence the membrane potential in the steady state. When the normal solution was replaced with chloridedeficient solution, the membrane was transiently depolarized and reached a steady potential after 3-5 min. When the tissue was rinsed with normal solution, the membrane was transiently hyperpolarized as observed in skeletal muscle by Hodgkin & Horowic2 (1959). From measurements of the membrane resistance the chloride resistance accounted for nearly 50% of the total membrane resistance; this high ratio was also observed in skeletal muscle (Hutter & Padsha, 1959; Hutter & Noble, 1961). However, it is unlikely that the chloride ions are distributed passively, since, as will be described in the following paper, chloride-deficient solution reverse the polarity of the inhibitory potential almost without any change in the membrane potential. Furthermore, the chloride equilibrium potential predicted from the reversal potential level of the miniature inhibitory junction potentials and inhibitory junction potentials was nearly 60 mv. and this value was 25 mv. higher than the measured membrane potential (Hidaka, Ito, Kuriyama & Tashiro, 1969). Alternative explanations of the chloride permeability of the membrane might be that if the membrane potential is kept at the same level in chloride-deficient solution as the control, then either the chloride permeability should be reduced, or an increase of potassium permeability and a decrease of sodium permeability should appear in proportion of the changes in [Cl] 0, if the internal ionic concentrations are not altered by treatment. Or again, the chloride flux across the membrane might be coupled with other ions as anion pairs (Shanes, 1958), or active transport (i.e. an electrogenic chloride pump) might be involved. del Castillo, de Mello & Morales (1964) have studied extensively the anion permeabilities of the somatic muscle of Ascaris. They concluded from their experiments that the chloride battery with a low internal resistance seems to be largely responsible for the maintenance of the membrane potential, whereas the contribution of potassium appears to be limited by a low membrane conductance to those ions. For instance, an increase of [KJ from 3-45 mm in the presence of the normal [Cl] 0 reduces the average membrane potential by only 5 mv. None the less, when [Cl] 0 is low, a similar increment in [K] o causes an almost complete depolarization of the membrane. Furthermore, both K and Cl batteries are shunted by a relatively large conductance to sodium

9 Effect of ions on membrane of muscle of earthworm 413 ions. These properties of the Ascaris muscle membrane resembled those obtained in mammalian smooth muscle (Kuriyama, 1963; Bulbring & Kuriyama, 1963). Effects of various foreign anions on the membrane potential and membrane conductance of the earthworm somatic muscle are now studied. Changes in [Ca] 0 also modified the membrane potential level. In normal solution reduction of [Ca] 0 depolarized the membrane significantly. Yet in sodium-free solution reduction of [Ca] 0 changed the level of the membrane potential only slightly. The importance of calcium ions in the regulation of membrane permeability as well as of the membrane potential appears to be elucidated by ability of bound to the membrane. The present results might suggest that changes in [Ca] 0 modified the sodium permeability and produced changes in the level of the membrane potential. The action potential It has been shown that the electrical activities of some excitable tissues are maintained in sodium-free solution containing alkali-earth cations. The longitudinal muscle of earthworm produced spikes in both normal and sodium-free solution. This fact indicates in the active state that the membrane becomes selectively permeable to calcium ions, which move across the membrane as charge carriers for the inward positive current. Thus calcium ions would be substituted for sodium ions in the production of the action potential, or calcium ions themselves could produce spikes, even in the presence of sodium ions. In the present experiments, however, theobserved facts did not make it possible to decide between the above two possibilities, although tetrodotoxin, a selective inhibitor of the sodium-carrying system in skeletal muscle and nerve fibres (Narahashi, et al, 1964) did not influence spike generation, whether sodium was present or not, indicating that calcium spikes might occur in physiological solution. The relationship between the peak level of the action potential and [Ca] 0 in sodiumfree solution in the present experiments indicated that the spike amplitude was roughly linearly proportional to the logarithm of [Ca] 0 and also that a tenfold change in [Ca] 0 produced a change of 39 mv. in the spike amplitude. This potential gradient might be explained according to the theory put forward by Kimizuka & Koketsu (1964) and Kimizuka (1966). According to this theory, the spike amplitude could be expressed as follows: 2 RT Va = --=-ln (C) o +constant 3 * (cf. Kimizuka (1966, equation (63c)) and Nishi, et al. (1965, equation (5))), where Va is the spike amplitude, F is the Faraday constant, R is the gas constant, T is the absolute temperature, and [C] o is the external divalent cation concentration. Thus, the spike amplitude would be proportional to the logarithm of [Ca] 0 and would be changed approximately 40 mv. by a tenfold change in [Ca] 0. However, the above equation is quite different from Goldman's constant-field theory, and Fatt & Ginsborg (1958) reported that the relationship between the spike amplitude and the external strontium concentration in crustacean muscle was in reasonable agreement with the relation predicted by Goldman's equation. Hagiwara & Takahashi (1967) reported that the overshoot potential of the barnacle muscle increased with a slope of approximately 29 mv. for a tenfold increase in [Ca] 0

10 414 T. HlDAKA AND OTHERS in the low range. However, when the external [Ca] 0 is further increased this value is lower than 29 mv. because the overshoot potential tends to approach a saturation level. The present result also showed that the value obtained by a tenfold change in the relatively high [Ca] 0, the slope of the overshoot potential, was lowered from 39 to 24 mv. Manganese ions blocked spike generation in this muscle both in normal and in sodium-free solution, indicating that Mn^ blocked the calcium-carrying system in a competitive manner. This observation agreed well with the observations made on the calcium spike recorded from barnacle muscle (Hagiwara & Nakajima, 1965) and from the smooth muscle cells of the taenia coli (Nonomura, Hotta & Ohashi, 1965). The mechanism of the plateau formation during active state of the membrane in the presence of Mn^ should be clarified. SUMMARY 1. The properties of the membrane, in both the resting and the active state, of the longitudinal muscle of the earthworm were studied under various ionic environments. 2. The maximum slope of the membrane potential change against a tenfold change in the external potassium concentration was 27 mv. in the presence of external sodium and 42 mv. in the absence of external sodium. 3. In the normal external potassium concentration the removal of sodium hyperpolarized the membrane from a normal resting potential of 36 to 58 mv. 4. Reduction of the external calcium concentration to a tenth of its normal value depolarized the membrane by about 16 mv. 5. In excess of external potassium the spike height and the after-hyperpolarization were decreased and the duration of the spike was prolonged. 6. In sodium-free solution spikes with an overshoot potential were generated both spontaneously and under the stimulus of an intracellularly depolarizing current. 7. The amplitude and the maximum rate of rise of the spike were dependent on the external calcium concentration, whether or not sodium was present externally. 8. Manganese modified the membrane activity by competition with calcium. REFERENCES BOLBRING, E. & KUWYAMA, H. (1963). Effects of changes in external sodium and calcium concentration, on spontaneous electrical activity in smooth muscle of guinea pig coli taenia. J. Phytiol., Lond DEL CASTILLO, J., DE MELLO, W. C. & MORALES, T. (1064). Influence of some ions on the membrane potential of Atcarit muscle. J. gen. Phytiol. 48, FATT, P. & GINSBORQ, B. L. (1958). The ionic requirements for the production of action potentials in crustacean muscle fibres. J. Physiol., Lond. 143, FATT, P. & KATZ, B. (1951). An analysis of the endplate potential recorded with an in tracellular electrode J. Physiol, Lond. 115, FATT, P. & KATZ.B. (1953). The electrical properties of crustaceanfibres.j. Phytiol. Lond.,120, HAGIWARA, S., CHICHIBU, S. & NAKA, K. (1964). The effects of various ions on resting and spike potentials of barnacle muscle fibres. J. gen. Phytiol. 48, HAOIWARA, S. & NAKAJIMA, S. (1965). Tetrodotoxin and manganese ion: Effects on action potential of the frog heart. Science, N.Y. 149, HAGIWARA, S. & TAKAHASHI, K. (1967). Resting and spike potential of skeletal muscle fibres of saltwater elasmoblanch and teleost fish. J. Phytiol., Lond. 190, HIDAKA, T., ITO, Y. & KURIYAMA, H. (1969). Membrane properties of the somatic muscle (obliquely striated muscle) of the earthworm. J. exp. Biol. 50,

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