J. Phyaiol. (1978), 278, pp. 403-423 With 13 text-figures Printed in Great Britain.
403
IONIC CURRENTS IN MAMMALIAN FAST SKELETAL MUSCLE
BY A. DUVAL AND C. L1OTY From the Laboratory of Animal Physiology and Cellular Physiology of Contractile Structures (CNRS associated team no. 111), University of Poitiers, Poitiers 86022, France
(Received 26 July 1977) SUMMARY
1. The double sucrose-gap technique has been applied to rat skeletal muscle fibres to study the ionic currents under voltage-clamp conditions. 2. The iliacus muscle was found to be of 'fast' type according to the characteristics of the twitch generated by an action potential. 3. Micro-electrode measurements have shown that the intracellular potential is under good control even when an inward current develops. 4. The components of an equivalent circuit with two time constants have been estimated from the records of the capacitive current. 5. In rat muscle, between 15 and 21 TC, inward and outward currents are similar to sodium and potassium currents found in frog muscle at lower temperature
(1-3 0C).
6. The inward current which depends on [Na]o and is abolished by tetrodotoxin is carried by sodium ions. Related to the mean value for the holding potential (- 90 5 mV) this current reaches its maximum amplitude at + 40 and + 50 mV, reverses between + 130 and + 150 mV and its half inactivation occurs between +14 and +22 mV. The effect of low doses of tetrodotoxin suggests that two components participate in the sodium current. 7. The delayed outward current which shows inactivation is divided in two components: (i) the fast has a linear instantaneous current-voltage relation and differs from the fast component of frog muscle in that its equilibrium potential is more negative than the resting potential; (ii) the slow has a linear instantaneous currentvoltage relation and the mean value for its equilibrium potential is 26 mV less negative than the resting potential. 8. Inward-going rectification is present in rat muscle. INTRODUCTION
The electrical event which occurs at the surface membrane of vertebrate skeletal muscle has been widely studied using the voltage-clamp technique. The great number of results reported so far are based largely on experiments with frog (Adrian, Chandler & Hodgkin, 1966, 1968, 1970a, b; Kao & Stanfield, 1967, 1968; Stanfield, 1969, 1970a, b; Ildefonse & Rougier, 1972), snake (Hunt & Heistracher, 1967) and fish muscles (Stanfield, 1972). Apart from the results obtained by Pappone (1977) and by Adrian & Marshall (1977) on rat muscle only a little is known about
A. DUVAL AND C. LLOTY mammalian skeletal muscles. So the aim of the present experiments was to investigate the changes in ionic conductances of rat skeletal muscle. The double sucrose-gap method was modified in such a way that it appeared to be satisfactory for voltage clamping isolated muscle cells even when early current develops (Leoty & Poindessault, 1974; Leoty & Alix, 1976). Mammalian muscles are obviously composed of different types of fibres but the diversity of definitions found in the literature made the classification not as clear as in the frog where only two types of fibres have been described (for reviews, see Peachey, 1961; Hess, 1970). Even so, according to their mechanical properties (Burke, Levine, Tsairis & Zajac, 1973) and their ultrastructural components (Eisenberg & Kuda, 1976) mammalian skeletal muscles can be divided into two major types: 'slow' and 'fast'. In the present work the characteristics of the contraction associated with an action potential were used to select fibres from iliacus muscle which were found to be of fast-type by comparison with typical slow fibres from soleus muscle. Some of the results have been described previously (Duval & Leoty, 1977). 404
METHODS
Electronics The voltage-clamp system used to obtain the present results was essentially identical to that already described by Poindessault, Duval & Leoty (1976). For current clamp experiments the circuit was modified by the addition of a constant generator calibrated by steps of 0-01 flA (maximum output 5 1tA/ 1 M.0). Under voltage-clamp conditions, the liquid surrounding the preparation in the central compartment is held at ground potential by the inverting input of the current to voltage converter. The output voltage is proportional to the total current flowing in the test node. An appropriate calibration allows an accurate measurement of the ionic current which was expressed in ,#A. The internal potential of the fibre follows the command voltage. The experimental chamber, built in Perspex according to the method reported by L6oty & Alix (1976) has a series resistance lower than 1 kQ, so the electronic circuit proposed by L6oty & Poindessault (1974) to compensate for the external series resistance was not used. However, such a low value induces some ringing in the current recorded at the rise and fall of the voltage step which can be reduced or fully damped by an adjustable resistance connected in series with the central electrode. In the majority of cases values no higher than 3 3 kM were used, but if these conditions are necessary for potential stability an analysis of the capacitive current cannot be properly done. In some experiments the intracellular potential or the holding potential of the muscular cell in the test compartment was measured with conventional 3 m-KCl-filled glass micro-electrodes
(10-20 Mn).
The micro-electrode was connected to an electrometer-input negative capacitance amplifier. This set-up was also used for a study of the potential distribution inside the muscle.
Tension recording In the sucrose-gap arrangement the contraction of the fibre in the test channel was estimated using a force transducer (Endeveo 8107). The sensing probe end of the transducer was extended by a pin with a tip diameter of 20 jam. The tip was placed in contact with the preparation. The transducer was calibrated with a series of known weights and the Wheatstone bridge circuit was set for an output signal amplitude of 4 mV/10 Ilg.
Preparations The fibres were isolated from the iliacus or soleus muscle of the rat (rattus norvegicus 400-500 g). The rat was anaesthetized by ether then killed by a blow on the neck. The isolated muscle was placed in a dissecting dish containing oxygenated Ringer (95 % 02, 5 % C02) kept at 25 'C. The muscle was gently stretched and both tendons pinned in the dish; the physiological solution
IONIC CURRENTS IN MAMMALIAN MUSCLE
405
was periodically changed. Good preparations could be maintained in good conditions for more than 5 hr. Under microscope all connective tissue was removed with fine scissors and small bundles of ten to twenty fibres were isolated. From each bundle the largest cell was selected (50-70 ,Am in diameter) and isolated for a length of 1-2 cm, then transported to the voltageclamp dish on a cover slide. All the results reported in the present paper were from cells showing no damage along their whole length; however, all attempts to use cut fragments of fibres (0*51 cm long) were successful. The shape of the action potential in cut fibres was identical to that of intact fibres but the after potential was less pronounced (Fig. 3). Electrical activity and contraction could be maintained for up to an hour. Contrary to the report of Hille & Campbell (1976) on frog muscle, the amplitude and duration of action potentials in cut fibres were not significantly different from those recorded on isolated fibres. This could be due to the fact that both extremities of our cut fibres were placed in normal Ringer and showed some sealing. From the muscle, such fragments can be dissected fairly rapidly but this method offers some disadvantages in that the preparation cannot be stretched when placed in the experimental chamber. Under unstretched conditions, a slow shortening of the fragment was observed as contractions were elicited by depolarization. This shortening appeared to be responsible for some instability in the amplitude and shape of the currents recorded. In some experiments isolated fibres were subjected to glycerol treatment according to the method described by Eisenberg, Howell & Vauighan (1971). Solutions The normal chloride Ringer used as bathing and perfusing solution consisted of (mM) NaCl 140, KCl 6, MgCl2 1, NaHCO3 4, NaH2PO4 0 5, Na2HPO4 2, glucose 2 g/l. The calcium concentration was achieved by the addition of calcium chloride from a stock solution 1 M (B.D.H. volumetric Standard Analar grade). Different calcium concentrations were tried (1 8-3-2 mM) and as the highest appeared to maintain the fibres in the most stable condition, especially for the dissection, it was used for all the experiments presently reported. Tile low chloride solution was made by replacing all the sodium chloride with an equivalent amount of sodium methylsulphate (Merck-Schuchardt). The concentration of ionized calcium was measured by means of a calcium electrode (ORION ref. 93.20) and the results showed that the calcium concentration is not modified by methylsulphate. The depolarizing Ringer was made by replacing a given amount of sodium chloride by potassium chloride. The 30 % sodium solution was made by replacing 98 mm sodium chloride by an equivalent amount of choline chloride (atropine 10-4 M) or by 186 mm sucrose. For all solutions the pH was adjusted to 7 3-7 4. The sucrose solution was made isotonic with the Ringer. All solutions were passed through heat exchange coils and could be maintained at a stable temperature. A Thermistor located near the preparation in the central channel allowed a continuous control of the Ringer temperature.
Recording Current and/or voltage were displayed on an oscilloscope (Tektronix 5115 N) and photographed.
Nomenclature and definition The holding potential is defined as 0 mV. All clamps were carried out from the holding potential; positive change of the potential (V) corresponds to depolarization and negative change to hyperpolarization. The membrane current of the central segment of the fibre (I) is positive for an outward movement of cations and negative for an inward movement of cations. Under voltage-clamp conditions the holding potential was set for the fast inward current induced by a given depolarization to be near its maximum (ho 1). In an attempt to compare our results to those obtained with other methods, this holding potential was measured by intracellular recording on eleven fibres and was found to range between -86 and -95 mV (mean - 90 5 mV ± 0 9; + S.E. of mean). In the same fibres the resting potential ranged between -70 and -85 mV (mean -78-3 mV + 1.4; +S.E. of mean). Several problems arise concerning the use of the double sucrose-gap method. One concerns the difficulty of measuring accurately the length and surface area of the fibre in the central node.
A. DUVAL AND C. LE.OTY
406
Even if subject to criticism, membrane characteristics and magnitudes of current were estimated in an attempt to compare the present results with those obtained on other preparations. The rate at which the stimulation is applied induces strong modification in the amplitude and time course of the current. A frequency of 1 or 2/min was used to avoid such effects.
Control of the method Previous experiments have shown that, in the presence of a series resistance, a variation in the potential of the solution surrounding the central node can occurduring current flow (Poindessault et al. 1976). In the present experiments it was also necessary to check that the potential inside the fibre was well controlled. Temporal and spacial uniformity were investigated by intracellular micro-electrode impalements of the muscle fibre within the central node. Fig. 1 B
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Temperature (C') Fig. 3. Action potentials recorded under current clamp condition in Ringer solution. A and B, from an intact fibre (19 'C) at two different time bases; C and D, from a cut fibre (23 'C). E and F illustrate the effect of temperature on the time to peak of the action potential and the time for 50 % repolarization. The triangles are from records A to D and the filled circles are from five other intact fibres.
The characteristics of the action potential are very dependent on the temperature. The time to peak and the time for 50 % of the repolarization (Fig. 3E, F) show a Q10 of respectively 1.6-1 and 1.9-1 (between 12 and 22 'C). After allowing
IONIC CURRENTS IN MAMMALIAN MUSCLE 409 for the effect of temperature, the action potentials recorded with the double sucrosegap are similar to those recorded with intracellular micro-electrodes on fast muscle from cat (Buller, Lewis & Ridge, 1966; Lewis, 1972) and from rat (Yonemura, 1967). In normal Ringer at 18 TC, depolarizing stimulations which last more than 100 msec and have an intensity lower than 0-2,uA produce unsustained repetitive firing in isolated iliacus fibres. These results resemble those reported by Adrian & Bryant (1974) on goat myotonic muscle fibres. The low temperature at which the present experiments were carried out could be, in part, responsible for such responses. However, this behaviour was not observed on soleus fibres investigated under identical experimental conditions.
Capacitive current and membrane resistance Under voltage-clamp conditions, when a small voltage step is applied to a fibre, the initial surge of current corresponds to the capacitive current (Fig. 4A, B). The current trace shows two phases of decay only when the series resistance '(R.) is
increased for a complete damping (see Methods) so the two components can be analysed. In semilogarithmic plot the faster phase has a time constant r1 between 0 012 and 0 03 msec (Table 1A) and the slower one has a time constant 12 between 0 37 and 0*9 msec. The presence of two exponential phases in the decay of the capacitive current has already been reported for frog skeletal muscle (Eisenberg & Gage, 1967; Adrian et al. 1970a; Ildefonse & Rougier, 1972), and according to Falk & Fatt (1964) could be related to the presence of two capacitive-resistance systems. Following glycerol treatment (Fig. 4B) the decay of the capacitive current shows only one component with a time constant r1 between 0-015 and 0-033 msec (Table 1 A) similar to that of the fast component found in normal fibre. As in frog muscle, the absence of the slow capacitive component on rat glycerol-treated preparations could be related to an alteration of the transverse tubular system. An equivalent circuit which gives a capacitive current with a time course similar to that recorded is shown in Fig. 4D. Although a detailed analysis of the equivalent circuit of the fibre has not been performed so far, a simplification was made so that a comparison with values from frog muscle could be made; in fact a simple analysis is possible by using the equation described by Ildefonse & Rougier (1972) for the value of the different elements of the membrane (Fig. 4C, E). The results obtained in glycerol-treated preparations suggest that R8 and C2 correspond to the transverse tubular system, as suggested for frog fibres (Falk & Fatt, 1964; Freygang, Rapoport & Peachey, 1967). R81, C1 could correspond to the surface membrane of the preparation. Membrane resistance Rm was calculated from the steady-state outward current following the transient. Table 1 A gives the mean values of fibre parameters obtained on eleven preparations. The membrane resistance found in rat iliacus is smaller than previously reported values for cat (Boyd & Martin, 1959) and rat (Kiyohara & Sato, 1967; Adrian & Marshall, 1967). This discrepancy could be due to the method of measurement and as a check, experiments were done on isolated skeletal fibres from frog (Rana esculenta) so that the results could be compared with previous frog results. Table 1 gives mean values for ten preparations. The value for C1 is similar to previous measurements
A. DUVAL AND C. LJOTY
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411 IONIC CURRENTS IN MAMMALIAN MUSCLE done on frog (Falk & Fatt, 1964; Eisenberg & Gage, 1967; Gage & Eisenberg, 1969; Valdosiera, Clausen & Eisenberg, 1974) and more than twice that estimated for surface membrane by Ildefonse & Rougier (1972). Although the value of R.9 is not far from the calculated value of 67 n cm2 for the radial resistance of the tubular fluid (Adrian. Costantin & Peachey, 1969), it is smaller than the value of 330 Q cm2 obtained A
403
IONIC CURRENTS IN MAMMALIAN FAST SKELETAL MUSCLE
BY A. DUVAL AND C. L1OTY From the Laboratory of Animal Physiology and Cellular Physiology of Contractile Structures (CNRS associated team no. 111), University of Poitiers, Poitiers 86022, France
(Received 26 July 1977) SUMMARY
1. The double sucrose-gap technique has been applied to rat skeletal muscle fibres to study the ionic currents under voltage-clamp conditions. 2. The iliacus muscle was found to be of 'fast' type according to the characteristics of the twitch generated by an action potential. 3. Micro-electrode measurements have shown that the intracellular potential is under good control even when an inward current develops. 4. The components of an equivalent circuit with two time constants have been estimated from the records of the capacitive current. 5. In rat muscle, between 15 and 21 TC, inward and outward currents are similar to sodium and potassium currents found in frog muscle at lower temperature
(1-3 0C).
6. The inward current which depends on [Na]o and is abolished by tetrodotoxin is carried by sodium ions. Related to the mean value for the holding potential (- 90 5 mV) this current reaches its maximum amplitude at + 40 and + 50 mV, reverses between + 130 and + 150 mV and its half inactivation occurs between +14 and +22 mV. The effect of low doses of tetrodotoxin suggests that two components participate in the sodium current. 7. The delayed outward current which shows inactivation is divided in two components: (i) the fast has a linear instantaneous current-voltage relation and differs from the fast component of frog muscle in that its equilibrium potential is more negative than the resting potential; (ii) the slow has a linear instantaneous currentvoltage relation and the mean value for its equilibrium potential is 26 mV less negative than the resting potential. 8. Inward-going rectification is present in rat muscle. INTRODUCTION
The electrical event which occurs at the surface membrane of vertebrate skeletal muscle has been widely studied using the voltage-clamp technique. The great number of results reported so far are based largely on experiments with frog (Adrian, Chandler & Hodgkin, 1966, 1968, 1970a, b; Kao & Stanfield, 1967, 1968; Stanfield, 1969, 1970a, b; Ildefonse & Rougier, 1972), snake (Hunt & Heistracher, 1967) and fish muscles (Stanfield, 1972). Apart from the results obtained by Pappone (1977) and by Adrian & Marshall (1977) on rat muscle only a little is known about
A. DUVAL AND C. LLOTY mammalian skeletal muscles. So the aim of the present experiments was to investigate the changes in ionic conductances of rat skeletal muscle. The double sucrose-gap method was modified in such a way that it appeared to be satisfactory for voltage clamping isolated muscle cells even when early current develops (Leoty & Poindessault, 1974; Leoty & Alix, 1976). Mammalian muscles are obviously composed of different types of fibres but the diversity of definitions found in the literature made the classification not as clear as in the frog where only two types of fibres have been described (for reviews, see Peachey, 1961; Hess, 1970). Even so, according to their mechanical properties (Burke, Levine, Tsairis & Zajac, 1973) and their ultrastructural components (Eisenberg & Kuda, 1976) mammalian skeletal muscles can be divided into two major types: 'slow' and 'fast'. In the present work the characteristics of the contraction associated with an action potential were used to select fibres from iliacus muscle which were found to be of fast-type by comparison with typical slow fibres from soleus muscle. Some of the results have been described previously (Duval & Leoty, 1977). 404
METHODS
Electronics The voltage-clamp system used to obtain the present results was essentially identical to that already described by Poindessault, Duval & Leoty (1976). For current clamp experiments the circuit was modified by the addition of a constant generator calibrated by steps of 0-01 flA (maximum output 5 1tA/ 1 M.0). Under voltage-clamp conditions, the liquid surrounding the preparation in the central compartment is held at ground potential by the inverting input of the current to voltage converter. The output voltage is proportional to the total current flowing in the test node. An appropriate calibration allows an accurate measurement of the ionic current which was expressed in ,#A. The internal potential of the fibre follows the command voltage. The experimental chamber, built in Perspex according to the method reported by L6oty & Alix (1976) has a series resistance lower than 1 kQ, so the electronic circuit proposed by L6oty & Poindessault (1974) to compensate for the external series resistance was not used. However, such a low value induces some ringing in the current recorded at the rise and fall of the voltage step which can be reduced or fully damped by an adjustable resistance connected in series with the central electrode. In the majority of cases values no higher than 3 3 kM were used, but if these conditions are necessary for potential stability an analysis of the capacitive current cannot be properly done. In some experiments the intracellular potential or the holding potential of the muscular cell in the test compartment was measured with conventional 3 m-KCl-filled glass micro-electrodes
(10-20 Mn).
The micro-electrode was connected to an electrometer-input negative capacitance amplifier. This set-up was also used for a study of the potential distribution inside the muscle.
Tension recording In the sucrose-gap arrangement the contraction of the fibre in the test channel was estimated using a force transducer (Endeveo 8107). The sensing probe end of the transducer was extended by a pin with a tip diameter of 20 jam. The tip was placed in contact with the preparation. The transducer was calibrated with a series of known weights and the Wheatstone bridge circuit was set for an output signal amplitude of 4 mV/10 Ilg.
Preparations The fibres were isolated from the iliacus or soleus muscle of the rat (rattus norvegicus 400-500 g). The rat was anaesthetized by ether then killed by a blow on the neck. The isolated muscle was placed in a dissecting dish containing oxygenated Ringer (95 % 02, 5 % C02) kept at 25 'C. The muscle was gently stretched and both tendons pinned in the dish; the physiological solution
IONIC CURRENTS IN MAMMALIAN MUSCLE
405
was periodically changed. Good preparations could be maintained in good conditions for more than 5 hr. Under microscope all connective tissue was removed with fine scissors and small bundles of ten to twenty fibres were isolated. From each bundle the largest cell was selected (50-70 ,Am in diameter) and isolated for a length of 1-2 cm, then transported to the voltageclamp dish on a cover slide. All the results reported in the present paper were from cells showing no damage along their whole length; however, all attempts to use cut fragments of fibres (0*51 cm long) were successful. The shape of the action potential in cut fibres was identical to that of intact fibres but the after potential was less pronounced (Fig. 3). Electrical activity and contraction could be maintained for up to an hour. Contrary to the report of Hille & Campbell (1976) on frog muscle, the amplitude and duration of action potentials in cut fibres were not significantly different from those recorded on isolated fibres. This could be due to the fact that both extremities of our cut fibres were placed in normal Ringer and showed some sealing. From the muscle, such fragments can be dissected fairly rapidly but this method offers some disadvantages in that the preparation cannot be stretched when placed in the experimental chamber. Under unstretched conditions, a slow shortening of the fragment was observed as contractions were elicited by depolarization. This shortening appeared to be responsible for some instability in the amplitude and shape of the currents recorded. In some experiments isolated fibres were subjected to glycerol treatment according to the method described by Eisenberg, Howell & Vauighan (1971). Solutions The normal chloride Ringer used as bathing and perfusing solution consisted of (mM) NaCl 140, KCl 6, MgCl2 1, NaHCO3 4, NaH2PO4 0 5, Na2HPO4 2, glucose 2 g/l. The calcium concentration was achieved by the addition of calcium chloride from a stock solution 1 M (B.D.H. volumetric Standard Analar grade). Different calcium concentrations were tried (1 8-3-2 mM) and as the highest appeared to maintain the fibres in the most stable condition, especially for the dissection, it was used for all the experiments presently reported. Tile low chloride solution was made by replacing all the sodium chloride with an equivalent amount of sodium methylsulphate (Merck-Schuchardt). The concentration of ionized calcium was measured by means of a calcium electrode (ORION ref. 93.20) and the results showed that the calcium concentration is not modified by methylsulphate. The depolarizing Ringer was made by replacing a given amount of sodium chloride by potassium chloride. The 30 % sodium solution was made by replacing 98 mm sodium chloride by an equivalent amount of choline chloride (atropine 10-4 M) or by 186 mm sucrose. For all solutions the pH was adjusted to 7 3-7 4. The sucrose solution was made isotonic with the Ringer. All solutions were passed through heat exchange coils and could be maintained at a stable temperature. A Thermistor located near the preparation in the central channel allowed a continuous control of the Ringer temperature.
Recording Current and/or voltage were displayed on an oscilloscope (Tektronix 5115 N) and photographed.
Nomenclature and definition The holding potential is defined as 0 mV. All clamps were carried out from the holding potential; positive change of the potential (V) corresponds to depolarization and negative change to hyperpolarization. The membrane current of the central segment of the fibre (I) is positive for an outward movement of cations and negative for an inward movement of cations. Under voltage-clamp conditions the holding potential was set for the fast inward current induced by a given depolarization to be near its maximum (ho 1). In an attempt to compare our results to those obtained with other methods, this holding potential was measured by intracellular recording on eleven fibres and was found to range between -86 and -95 mV (mean - 90 5 mV ± 0 9; + S.E. of mean). In the same fibres the resting potential ranged between -70 and -85 mV (mean -78-3 mV + 1.4; +S.E. of mean). Several problems arise concerning the use of the double sucrose-gap method. One concerns the difficulty of measuring accurately the length and surface area of the fibre in the central node.
A. DUVAL AND C. LE.OTY
406
Even if subject to criticism, membrane characteristics and magnitudes of current were estimated in an attempt to compare the present results with those obtained on other preparations. The rate at which the stimulation is applied induces strong modification in the amplitude and time course of the current. A frequency of 1 or 2/min was used to avoid such effects.
Control of the method Previous experiments have shown that, in the presence of a series resistance, a variation in the potential of the solution surrounding the central node can occurduring current flow (Poindessault et al. 1976). In the present experiments it was also necessary to check that the potential inside the fibre was well controlled. Temporal and spacial uniformity were investigated by intracellular micro-electrode impalements of the muscle fibre within the central node. Fig. 1 B
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Temperature (C') Fig. 3. Action potentials recorded under current clamp condition in Ringer solution. A and B, from an intact fibre (19 'C) at two different time bases; C and D, from a cut fibre (23 'C). E and F illustrate the effect of temperature on the time to peak of the action potential and the time for 50 % repolarization. The triangles are from records A to D and the filled circles are from five other intact fibres.
The characteristics of the action potential are very dependent on the temperature. The time to peak and the time for 50 % of the repolarization (Fig. 3E, F) show a Q10 of respectively 1.6-1 and 1.9-1 (between 12 and 22 'C). After allowing
IONIC CURRENTS IN MAMMALIAN MUSCLE 409 for the effect of temperature, the action potentials recorded with the double sucrosegap are similar to those recorded with intracellular micro-electrodes on fast muscle from cat (Buller, Lewis & Ridge, 1966; Lewis, 1972) and from rat (Yonemura, 1967). In normal Ringer at 18 TC, depolarizing stimulations which last more than 100 msec and have an intensity lower than 0-2,uA produce unsustained repetitive firing in isolated iliacus fibres. These results resemble those reported by Adrian & Bryant (1974) on goat myotonic muscle fibres. The low temperature at which the present experiments were carried out could be, in part, responsible for such responses. However, this behaviour was not observed on soleus fibres investigated under identical experimental conditions.
Capacitive current and membrane resistance Under voltage-clamp conditions, when a small voltage step is applied to a fibre, the initial surge of current corresponds to the capacitive current (Fig. 4A, B). The current trace shows two phases of decay only when the series resistance '(R.) is
increased for a complete damping (see Methods) so the two components can be analysed. In semilogarithmic plot the faster phase has a time constant r1 between 0 012 and 0 03 msec (Table 1A) and the slower one has a time constant 12 between 0 37 and 0*9 msec. The presence of two exponential phases in the decay of the capacitive current has already been reported for frog skeletal muscle (Eisenberg & Gage, 1967; Adrian et al. 1970a; Ildefonse & Rougier, 1972), and according to Falk & Fatt (1964) could be related to the presence of two capacitive-resistance systems. Following glycerol treatment (Fig. 4B) the decay of the capacitive current shows only one component with a time constant r1 between 0-015 and 0-033 msec (Table 1 A) similar to that of the fast component found in normal fibre. As in frog muscle, the absence of the slow capacitive component on rat glycerol-treated preparations could be related to an alteration of the transverse tubular system. An equivalent circuit which gives a capacitive current with a time course similar to that recorded is shown in Fig. 4D. Although a detailed analysis of the equivalent circuit of the fibre has not been performed so far, a simplification was made so that a comparison with values from frog muscle could be made; in fact a simple analysis is possible by using the equation described by Ildefonse & Rougier (1972) for the value of the different elements of the membrane (Fig. 4C, E). The results obtained in glycerol-treated preparations suggest that R8 and C2 correspond to the transverse tubular system, as suggested for frog fibres (Falk & Fatt, 1964; Freygang, Rapoport & Peachey, 1967). R81, C1 could correspond to the surface membrane of the preparation. Membrane resistance Rm was calculated from the steady-state outward current following the transient. Table 1 A gives the mean values of fibre parameters obtained on eleven preparations. The membrane resistance found in rat iliacus is smaller than previously reported values for cat (Boyd & Martin, 1959) and rat (Kiyohara & Sato, 1967; Adrian & Marshall, 1967). This discrepancy could be due to the method of measurement and as a check, experiments were done on isolated skeletal fibres from frog (Rana esculenta) so that the results could be compared with previous frog results. Table 1 gives mean values for ten preparations. The value for C1 is similar to previous measurements
A. DUVAL AND C. LJOTY
410
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411 IONIC CURRENTS IN MAMMALIAN MUSCLE done on frog (Falk & Fatt, 1964; Eisenberg & Gage, 1967; Gage & Eisenberg, 1969; Valdosiera, Clausen & Eisenberg, 1974) and more than twice that estimated for surface membrane by Ildefonse & Rougier (1972). Although the value of R.9 is not far from the calculated value of 67 n cm2 for the radial resistance of the tubular fluid (Adrian. Costantin & Peachey, 1969), it is smaller than the value of 330 Q cm2 obtained A
