ABSTRACT
Electrical and mechanical properties of the red muscle (M. levator pinnae pectoralis) and white muscle (M. levator pinnae lateralis abdominis) in the silver carp (Carasstus auratus Linné) were investigated by using caffeine and thymol.
A complete tetanus could be produced in the red muscle. But in the white muscle no tetanus was produced and there was a gradual decrease in tension during continuous stimulation, even at a frequency of 1 c/s or less.
Caffeine (0·5–1 mM) and thymol (0·25–0·5 mM) potentiated the twitch tension in both muscles without an increase in the resting tension; they produced a contracture in both muscles when the concentration was increased further.
The falling phase of the active state of contraction was nearly the same in both muscles and was prolonged by caffeine (0·5 mM) and by thymol (0·25 mM).
The resting membrane potential of the red muscle was scarcely affected by caffeine (0·5–5 HIM), whereas in the white muscles a depolarization of 10 mV was observed with caffeine of more than 2 mM. The resting potential of both muscles was little changed by 0·25 mM thymol. However, at a concentration of more than 0·5 mM thymol depolarized the membrane in both muscles to the same extent.
In caffeine (2–3 mM) solution the mean specific membrane resistance was reduced from 8-8Ω cm2 to 6·0 kΩ cm2 in the red muscle, and from 5·0 kΩ cm2 to 2·7 kΩ cm2 in the white muscle. In thymol (0·5–1 mM) solution it was reduced from 11·2 kΩ cm2 to 6·5 kΩcm2 in the red muscle, and from 5·4 kΩ cm2 to 3·1 kΩ cm2 in the white muscle. The specific membrane capacitance, calculated from the time constant and the membrane resistance, remained more or less the same in both muscles after a treatment with these agents.
Electrical properties of the muscles and the effects of caffeine and thymol on mechanical responses suggest that there are no fundamental differences between red and white muscles except for the excitation-contraction coupling. A lack of summation of twitch, a successive decline of twitch, and inability to produce potassium contracture in the white muscle may be due to the fact that the Ca-releasing mechanism is easily inactivated by depolarization of the membrane.
INTRODUCTION
In the silver carp there are distinct differences in tension development between the red muscle (M. levator pinnae pectoralis) and the white muscle (M. levator pinnae lateralis abdominis) (Hidaka & Toida, 1969a, b). In red muscle the twitch tension has a slower time course than in white muscle, and stimulation at high frequencies produce summation and a complete tetanus at 30 c/s or more. However, in the white muscle no summation is observed, and the twitch tension gradually declines with successive stimuli when the frequency is higher than 1 c/s. Furthermore, when the external potassium concentration is increased, a sustained contracture is observed in the red muscle but not in the white muscle.
These differences cannot be explained in terms of the fine structure of the muscles, since no differences between the red and white muscle fibres are observed with respect to the tubular system, the sarcoplasmic reticulum, the triads or the myofilaments (Nishihara, 1967). Furthermore, there are no fundamental differences in their innervations. Both muscles receive multiple innervation. The aim of the present experiments was to further investigate the electrical and mechanical properties of muscle fibres in the red and white muscles. For this purpose, effects of caffeine and thymol on electrical and mechanical properties were studied, and the active state of contraction was measured. Caffeine and thymol are known to act directly on the sarcoplasmic reticulum and to cause the contracture.
It was concluded from the experiments that the red and white muscles have similar properties as to membrane excitation and contractile mechanism, and that the main difference between the red and white muscle lies in the mechanism of Ca release from the sarcoplasmic reticulum triggered by membrane depolarization.
METHODS
Red muscle (M. levator pinnae pectoralis) and white muscle (M. levator pinnae lateralis abdominis) were dissected from the silver carp, Carassius auratus (Linné). Muscles used were 1·5–2 cm in length and 1 mm in width.
One end of the preparation was connected with a thread to the hook of the tension recorder, and the other end was fixed. The tension development was measured isometrically with a mechano-electric transducer (RCA 5734 tube). The active state was measured using the quick-release method described by Ritchie (1954). Electrical stimulation was applied with multigrid stimulating electrodes placed in parallel with the long axis of the muscle fibres.
Two microelectrodes were inserted, 50–100 μm apart, in the same fibre. One electrode was used for recording the potential change and the other for polarizing the membrane by passing current pulses. The microelectrodes were micropipettes filled with 3 M-KCI. Their resistance varied from 10 to 30 MΩ.
Physiological saline had the following composition: NaCl 129·6 mM; KC1 2·7 mM; CaCl2 1·8 mM ; NaHCO3 2·5 mM. All measurements were carried out at room temperature (20-24 °C), except for measurements of the active state which were made at 10 °C.
RESULTS
Effect of caffeine and thymol on mechanical properties
Twitch tensions elicited by maximal stimulation with 10 msec pulses at 1 min intervals were reduced to about 70% by d-tubocurarine (10−6 g/ml) in both the red and white muscles. The chemical transmitter in the neuromuscular transmission of the red muscle in the silver carp is acetylcholine (Hidaka & Toida, 1969b). An increase in the concentration of d-tubocurarine had little further effect, but an addition of tetrodotoxin (1 × 10−7g/ml) suppressed the twitch tension to about 10% of control levels, as shown in Fig. 1. Effects of d-tubocurarine and tetrodotoxin were similar in both muscles. It is therefore likely that the twitch evoked by a 10 msec pulse is mainly mediated by the excitation of the muscle fibre in response to direct stimulation in the red muscle as well as in the white muscle. Since the presence or the absence of d-tubocurarine made no essential difference to the results, most of the present experiments were carried out without d-tubocurarine.
There were clear differences between the red and white muscles in their mechanical responses, as previously reported (Hidaka & Toida, 1969a). In the red muscle a tetanus was produced by a stimulation frequency of more than 5 c/s at 20 °C, as observed in the frog skeletal muscle. However, in the white muscle there was a gradual decrease in tension during continuous stimulation at 1 c/s or more, as shown in Fig. 2. In order to maintain a constant amplitude of twitch tension in the white muscle, stimuli had to be applied at intervals of 10–30 sec. When the white muscle was stimulated at 20 c/s it took about 5 min for twitch tension to recover completely to control levels.
A contracture was produced in response to excess potassium in the red muscle, whereas no potassium contracture was observed in the white muscle (Hidaka & Toida, 1969a). However, a contracture was produced both in the red and white muscles by the addition of caffeine or thymol to Ringer solution.
Figs. 3 and 4 show the effects of caffeine (1–5 mw) on the tension development of the red and white muscles. The muscles were stimulated at a frequency of 0·5 c/s, except for the first 2 min after caffeine application. The final concentration of caffeine in the external solution was reached within 2–3 sec, and kept constant for 4 min. At a low concentration (1 HIM), only twitch potentiation was produced without any change in resting tension. When the concentration was increased to more than 3 mM, not only potentiation but also a contracture developed in both muscles.
In another series of experiments caffeine was applied while the muscles were continuously stimulated without interruption. The potentiation was clearly observed with 0·5 mM caffeine. The effects appeared gradually taking about 2 min for complete development.
Experiments similar to those of Figs. 3 and 4 were repeated using thymol. The effects were essentially similar to those of caffeine. However, thymol was more effective than caffeine, as shown in Figs. 5 and 6. In a low concentration (0·5 mM) the twitch was potentiated in both muscles. At higher concentrations (1–3 mM) contractures were observed.
Although the potentiation was stronger with higher concentrations of caffeine or thymol until the contracture developed, the twitch tension had a tendency to be reduced following development of a large contracture.
Effects of caffeine and thymol on electrical properties
The resting potential of red muscles was about io mV lower than that of white muscles (Hidaka & Toida, 1969a). In the red muscles the membrane potential was little affected by caffeine, whereas in the white muscles depolarization of about 10 mV was observed with a treatment of caffeine of more than 2 mM (Fig. 7a).
The membranes in red and white muscles were not depolarized by 0·25 mM thymol, but large depolarization was observed when concentration of thymol was increased to more than 0·5 mM, as shown in Fig. 7(b). The degree of depolarization was roughly the same in both muscles.
Therefore, potentiation of twitches by caffeine (0·5 mM) or thymol (o·1 mM) can be seen without a change in membrane potential, although during the strong contracture the membrane is depolarized to some extent.
Fig. 8 shows electrotonic potentials produced by intracellular polarization and effects of caffeine. No spike could be evoked in the red muscles, and it was very rare to trigger the spike in the white muscle. The same results have been reported by Hidaka & Toida (1969a).
The mean time constant (τ) of the membrane, the time to reach 84% of the final steady value of hyperpolarization, was 29·5 msec for the red muscles and 52·4 msec for the white muscles. When caffeine (2–3 MM) was added, the time constant was reduced on average to 22·8 msec for the red muscles and ·8 msec for the white muscles.
In Figs. 9 and 10 examples of the current-voltage (I-V) relationships obtained in the red and white muscles are shown. The I-V relationship was linear in both muscles before and after treatments with caffeine and thymol. Caffeine (Fig. 9) and thymol (Fig. 10) reduced the membrane resistance. The effects of caffeine and thymol on the electrical membrane parameters are summarized in Tables 1 and 2 respectively. The specific membrane resistance (Rm) was calculated from the cable equation Rm= πd3 Re2 /Ri where d is the fibre diameter, and Rt is the effective membrane resistance. In this calculation Riwas taken to be 389 Ω cm for the red muscle and 294 Ω cm for the white muscle from the data by Hidaka & Toida (1969a). The specific membrane capacitance was obtained by dividing τ by the Re. In caffeine (2-3 mM) solution the mean specific membrane resistance was reduced from 8·8 kΩ cm2 to 6-o kΩ cm2 in the red muscle, and from 5·0 kΩcm2 to 2·7 kΩ cm2 in the white muscle. Similarly, in thymol (0·5–1 MM) solution it was reduced from 11·2 kΩcm2 to 6·5 kΩ cm2 in the red muscle, and from 5·4 kΩ cm2 to 3·1 kΩ cm2 in the white muscle. The specific membrane capacitance was little affected in both muscles.
Effects of caffeine and thymol on the active state of contraction
Since the measurement of the active state was easier when the contraction was prolonged by lowering the temperature, the experiments were carried out at 10 °C. The time to reach the peak tension (peak time), the time to decline to a half of the maximum tension (half time) and the total duration of twitch at 20 and 10 °C are Usted in Table 3 for the red and white muscles.
Fig. 11 shows the decay of the active state (broken lines) in both muscles in normal solution at 10 °C. Solid lines indicate twitch tensions. It is noted that fall in tension lagged considerably behind decay of the active state; for example, at a time when the tension had declined to 80 % of maximum the active state in the red and white muscles fell approximately to 13 % and 18 % of peak value respectively. At a time when active state had virtually disappeared, tensions were still approximately 50% maximum
Except for the final segment of the curves the time course of the declining phase of the active state was very similar in the two types of muscles.
The effect of caffeine (0·25 mM) on the active state is shown in Fig. 12. At a low temperature (10 °C) caffeine was more effective in potentiating the twitch than at a higher temperature (20 °C). Caffeine produced a pronounced potentiation of the twitch at a concentration of 0·25 mM in both muscles at 10 °C. The active states were measured after 5 and 15 min application of caffeine in the red muscle, and after 15 min in the white muscle. Rates of tension development were increased and the falling phases of active state were prolonged in both muscles.
In Fig. 13 effects of thymol (0· 25 mM) on the mechanical responses at 10 °C are shown. In thymol solution rates of tension development remained the same although peak tensions were dramatically increased. The active state was clearly prolonged.
DISCUSSION
Since it is difficult in the red muscle of the silver carp to evoke a spike by nerve stimulation or by field stimulation of the tissue, and since the junction potentials are large enough to cause the twitch, it has been assumed that tension development in this muscle is usually mediated by the junction potential, whereas in the white muscle it is triggered by the spike (Hidaka & Toida, 1969a, b). However, when twitches were evoked by using 10 msec pulses, in both types of muscles d-tubocararine had only a weak suppressing effect; and tetrodotoxin, which probably blocks electrical excitation of the muscle fibre, reduced twitch tensions to 10% of the control. Therefore, as far as membrane excitation is concerned, properties of the red and white muscles are probably similar. Further investigations are necessary to explain inability (or difficulty) to trigger spikes by intracellular stimulation.
The red muscle can produce a complete tetanus under repetitive stimulation and a contracture under excess potassium. On the other hand, the white muscle produces neither the tetanus nor the contracture. These differences in mechanical responses between the red and white muscles are most probably attributable to differences in excitation-contraction coupling or in the contractile mechanism.
Since the degree of membrane depolarization by excess potassium is roughly the same in the red and white muscle, and since suppression of twitch by excess potassium occurs at about the same speed in both the muscles, neither electrical properties of the membrane nor a slow diffusion time is responsible for a lack of potassium contracture in the white muscle (Hidaka & Toida, 1969a).
It is believed that contraction and relaxation of muscle are controlled by the intra-cellular free Ca concentration (Ebashi & Endo, 1968; Sandow, 1970). It is suggested that contraction is triggered by the release of Ca from the terminal cisternae of the sarcoplasmic reticulum, and relaxation is initiated when Ca is reabsorbed into the tubular portion of the reticulum (Winegrad, 1968, 1970; Connolly, Gough & Wine-grad, 1971). Therefore, it is likely that the lack of summation of twitch and of potassium contracture in the white muscle are due to inability to raise the intracellular Ca concentration.
The effects of caffeine on other skeletal muscles have been investigated by many authors (Axelsson & Thesleff, 1958; Sandow, 1965 ; Sandow & Brust, 1966; Lüttgau & Oetliker, 1968; Weber & Herz, 1968; Weber 1968). According to these studies, caffeine causes the release of Ca from the sarcoplasmic reticulum, probably from the terminal cisternae, and prevents uptake of Ca by the reticulum. Thymol, which has similar effects to caffeine, induces a contracture in frog skeletal muscle in normal Ringer solution and also in excess-potassium solution (Sakai, Fujii & Takemoto, 1967; Sakai, Fujii & Shimizu, 1968). Furthermore, the thymol contracture can be produced in naked myofibrils (Endo, 1967). Therefore, it may be that thymol acts directly on the sarcoplasmic reticulum as does caffeine, although the membrane is depolarized when the concentration is at more than 0·5 mM.
In both the red and white muscles of the silver carp caffeine increased the rate of rise of twitch tension and prolonged the active state, while thymol prolonged the active state without changing the rate of rise of twitch tension. These suggest that caffeine increases Ca release and reduces Ca uptake, but that the main action of thymol at low concentration (0·25–0·5 mM) is probably to suppress Ca uptake rather than to release Ca.
In a solution containing caffeine at a concentration above 3 mM or thymol at a concentration above 1 mM a contracture is developed in the white muscle as well as the red muscle. Thus it is considered that there is no qualitative difference between the red and white muscles in the mechanisms of contraction after Ca release from the sarcoplasmic reticulum or in mechanisms of reabsorption of Ca.
The differences between the red and white muscles in the mechanical responses mediated by membrane depolarization may be explained by assuming that a fraction of the stored Ca is available for release and that in the white muscle the process by which Ca is transferred from the stored form to the readily available form is very slow, as is also postulated for the crayfish muscle (Chiarandini et al. 1970); and that this process is easily inactivated by membrane depolarization. For the Ca release the rate of depolarization may be a critical factor. Depolarization of the membrane by excess potassium is apparently too slow to cause Ca release. Caffeine and thymol probably have a fadlitatory action of the process increasing the readily available form. This tentative model would explain why tension in the white muscle declines successively with repetitive stimulation, and why no potassium contracture is observed.
In the semitendinosus of the frog an increase in the threshold depolarization for mechanical activation has been observed when the rate of linearly rising current is slowed in low-Ca (0·1–0·25 mM) solution (Sugi, 1968). This accommodation process in the excitation-contraction coupling mechanism is eliminated by caffeine (1 mM). The white muscle of the fish may have properties similar to the fast muscle in low-Ca solution.
There are also some differences between the red and white muscles of the silver carp in their electrical properties. The membrane resistance is higher and the membrane capacitance is smaller in the red muscle than in the white muscle. In these respects the muscle fibres in the red and white muscles of the fish have electrical properties similar to those of the slow and fast muscle fibres respectively of the frog, as pointed out by Hidaka & Toida (1969a).
In the iliofibularis of the frog the specific membrane resistance of the slow fibres is nearly 10 times that of the fast fibres, and the specific membrane capacitance is three times larger in the fast fibres (Adrian & Peachey, 1965). In the frog these electrical differences are attributed to the fact that the transverse tubular system is well developed in the fast fibres compared with the slow fibres. However, as described in the introduction, no clear differences in the tubular system or arrangement of the myofibrils are found between the red and white muscles of the fish (Nishihara, 1967). The internal structure of muscle fibres in both muscles resembles that of the frog fast fibres. It is possible that muscle fibres of the fish red muscle have high electrical resistance in series with the capacity of the walls of the tubular system.
In the frog muscle the membrane resistance of the slow fibres and of the fast fibres falls with increasing hyperpolarization (Adrian & Peachey, 1965). On the other hand, in the fish the resistance remains constant with polarization of the membrane in both types of muscles. This may suggest a possibility that, in the fish, electrical properties of the triads or of the terminal cisternae of the longitudinal reticulum are different from those in the frog. However, at the present stage, no correlation between electrical and mechanical properties can be made in the different types of muscles of the silver carp.
SUMMARY
Electrical and mechanical properties of the red muscle (M. levator pinnae pectoralis) and white muscle (M. levator pinnae lateralis abdominis) in the silver carp (Carasstus auratus Linné) were investigated by using caffeine and thymol.
A complete tetanus could be produced in the red muscle. But in the white muscle no tetanus was produced and there was a gradual decrease in tension during continuous stimulation, even at a frequency of 1 c/s or less.
Caffeine (0·5–1 mM) and thymol (0·25–0·5 mM) potentiated the twitch tension in both muscles without an increase in the resting tension; they produced a contracture in both muscles when the concentration was increased further.
The falling phase of the active state of contraction was nearly the same in both muscles and was prolonged by caffeine (0·5 mM) and by thymol (0·25 mM).
The resting membrane potential of the red muscle was scarcely affected by caffeine (0·5–5 HIM), whereas in the white muscles a depolarization of 10 mV was observed with caffeine of more than 2 mM. The resting potential of both muscles was little changed by 0·25 mM thymol. However, at a concentration of more than 0·5 mM thymol depolarized the membrane in both muscles to the same extent.
In caffeine (2–3 mM) solution the mean specific membrane resistance was reduced from 8-8Ω cm2 to 6·0 kΩ cm2 in the red muscle, and from 5·0 kΩ cm2 to 2·7 kΩ cm2 in the white muscle. In thymol (0·5–1 mM) solution it was reduced from 11·2 kΩ cm2 to 6·5 kΩcm2 in the red muscle, and from 5·4 kΩ cm2 to 3·1 kΩ cm2 in the white muscle. The specific membrane capacitance, calculated from the time constant and the membrane resistance, remained more or less the same in both muscles after a treatment with these agents.
Electrical properties of the muscles and the effects of caffeine and thymol on mechanical responses suggest that there are no fundamental differences between red and white muscles except for the excitation-contraction coupling. A lack of summation of twitch, a successive decline of twitch, and inability to produce potassium contracture in the white muscle may be due to the fact that the Ca-releasing mechanism is easily inactivated by depolarization of the membrane.
ACKNOWLEDGEMENTS
The author expresses his appreciation to Professor H. Kuriyama and Professor T. Tomita for suggestions concerning the research and to Professor N. Toida for encouragement during the course of the work.
He also wishes to thank Dr R. F. Cobum for his help in preparing the manuscript.