Hiroshi HASUO and Kyozo KoKETSU. Department of Physiology, Kurume University School of Medicine, Kurume, 830 Japan

Transcription

1 Japanese Journal of Physiology, 35, , 1985 Potential Dependency of the Electrogenic Na+ in Bullfrog Atrial Muscles -pump Current Hiroshi HASUO and Kyozo KoKETSU Department of Physiology, Kurume University School of Medicine, Kurume, 830 Japan Abstract Experiments were designed to evaluate the concept that the activity of the electrogenic Na+-pump is dependent on the transmembrane potential. Cardiac muscle preparations were used because the electrogenic Nay -pump current can be recorded at different potentials with the voltage clamp method in this preparation. Electrogenic Nat-pump current was identified as the membrane current which was abolished by ouabain (5 µm) and induced by the addition of K+ or an alkali metal cation, such as Rb+, Cs, or Lit, to the extracellular K+-free solution. Alkali metal cations other than K+ were used to eliminate the possibility that a passive membrane K+ current might be altered by changes in the K+ concentration in the vicinity of the membrane due to activation of the Na+-pump. It was concluded that the activity of the electrogenic Natpump current is dependent on the membrane potential. Key words : electrogenic Nay -pump, potential dependency, bullfrog atrial muscles, voltage clamp method, alkali metal cations. The Na+-pump in cell membranes actively extrudes Na+ and concentrates K" to maintain an uneven distribution of these ions between the inside and the outside of cells. It is generally thought that the Na+ and K+ transports driven by the Na+-pump are coupled and that the coupling-ratio of these ions may not be 1:1. In other words, the Na+-pump may be electrogenic (KOKETSU,1971; THOMAS, 1972b). The electrogenicity of the Nat-pump has been recently reported in various cardiac tissues (GLITSCH et al., 1978; GADSBY and CRANEFIELD, 1979; KURACHI et a!., 1981a, b; EISNER and LEDERER, 1980; GLITSCH et a1.,1982). In frog skeletal muscle, the Na+-pump activity is dependent on the intracellular sodium ion concentration ([Na+]i) (MULLINS and FRUMENTO,1963; MULLINS and AWAD,1965). These studies were regarded as evidence supporting the concept that the Nat-pump is dependent on the electrochemical gradient. Indeed, THOMAS (1972a) reported that the activity of the Nat-pump depends primarily Received for publication September 7, 1984 t, 89

2 90 H. HASUO and K. KOKETSU upon [Na+]i, and that changes in external Na+ ([Na+]o) or membrane potential appear to affect the pump indirectly by changing the Na+ influx and thus [Na+]i in snail neurons. On the other hand, the Na+-pump activity appears to be independent of membrane potential in a variety of tissues including squid giant axon (HODGKIN and KEYNES, 1955; BRINLEY and MULLINS, 1974), Anisodoris giant neurone (MARMOR, 1971), human red blood cells (COTTERRELL and WHITTAM,1971), frog skeletal muscle (BEAUGE and SJODIN, 1976), and sheep Purkinje fiber (EISNER and LEDERER, 1980). TAHARA et al. (1973) suggested that the activity of the Nat-pump is directly dependent upon membrane potential in frog skeletal muscle. Experiments were designed to evaluate this possibility. In the present investigation, a cardiac muscle preparation was used, since the electrogenic Na+-pump current can be recorded at different potentials with the voltage clamp method. Electrogenic Nat-pump current was defined as the membrane current which was abolished by ouabain (5 pm) and induced by adding K+ or an alkali metal cation, such as Rb+, Cs, or Lit, to the extracellular K+-free solution. The relationship between Nat-pump current and membrane potential was investigated. The reason for using these alkali metal cations, instead of K+, was to minimize or eliminate the alteration of passive ionic current due to "depletion," which could be caused by the activation of the Nat-pump (cf. EISNER and LEDERER, 1980). METHODS Strips ( x 4 mm in size) of quiescent muscle fiber bundles, excised from the atrium of bullfrog (liana catesbeiana) heart were used. After excising the atrium, the parallel atrial muscle bundles were isolated and the epicardium was removed. The preparations were soaked in normal Ringer solution (112 mm NaCI, 2 mm KCI, 1.8 mm CaC12, and 2.4 mm NaHCO3) for more than 15 min. The cell membranes of damaged atrial muscle fibers were assumed to heal during this period (DELEzE, 1970). The preparations were tightly mounted in a chamber which consisted of three compartments, for the single sucrose-gap experiments (BEELER and REUTER, 1970; AKASU et al., 1978). The right compartment was perfused with Ringer or test solutions. The central compartment (1.0 mm width) was perfused with isotonic sucrose solution. The left compartment was perfused with isotonic KCl solution. The experimental methods for measuring the membrane potential and for voltage clamping have been described elsewhere (AKASU et a!.,1978), and were similar to those reported by BEELER and REUTER (1970). Platinum electrodes were placed in the two outer compartments to apply clamp currents across the sucrose-gap. The fiber bundle length in the compartment with test solution was less than 0.7 mm. The membrane potential was recorded with an intracellular microelectrode filled with 3 M KCl (20-30 MSS) and was clamped using a voltage clamp feedback ampli- Japanese Journal of Physiology

3 POTENTIAL DEPENDENCY OF Nat-PUMP 91 fier (Nihon Kohden, CEZ 1100). To avoid the effects of the voltage and time dependent outward current (Ix) or the slow inward current (IS1) (cf. NOBLE, 1979), the current voltage relationships (I -V relationships) were obtained at hyperpolarized potentials (more than - 50 mv). The steady-state currents were obtained by stepping the membrane potential at 10 mv increments for durations of 3 sec. The ionic compositions of the solutions used in the present experiment were : sucrose solution (240 mm sucrose), Ringer solution (112 mm NaCI, 2 mm KCI, 0.18 mm CaCl2, 6 mm MgCl2, 2.4 mm NaHCO3, and 2.5 mm glucose). The Ca2+ concentration of the Ringer solution was reduced to 0.18 mm, and 6 mm MgCl2 was added to minimize spontaneous contraction and facilitate the prolonged maintenance of the microelectrode in individual cells. The K+-free Ringer solution was prepared by omitting KCl from the Ringer solution. To prepare solutions with the alkali metal cations (K+, Rb+, Cs, Lit), the chloride salt of the alkali metal cation was added to the K+-free Ringer solution and the NaCI concentration was reduced to maintain the tonicity (cf. EISNER and LEDERER, 1980). The ph of all the solutions was The test solution compartment had a volume of 0.2 ml and was perfused at the rate of 4 ml/min. All the experiments were carried out at room temperature (20-22 C). Ouabain was obtained from Merck. RESULTS Preparations were initially superfused with the K+-free Ringer solution in the test solution compartment for more than 60 min to increase the [Na+]i. Thereafter, K+-activated hyperpolarizations were measured by changing to a Ringer solution (2 mm K+); these hyperpolarizations were attributed to activation of the electrogenic Nat-pump. (e.g. NoMA and IRISAWA, 1974; AKASU et al., 1978; KURACHI et a!., 1981b). The K+-activated hyperpolarization could be reproduced with little change in amplitude, provided there was a 15 min interval between the 1 min applications of the Ringer solution (2 mm K+). The hyperpolarization was completely blocked by ouabain (5 /2M). A small depolarization remained which can be assumed to represent a diffusional membrane potential induced by the increase in external K+, as shown in Fig. 1. When replacing the K+-free Ringer solution with solutions containing Rb+, Cs, or Lit, similar hyperpolarizations were recorded in the atrium (e.g. EISNER and LEDERER,1980; KURACHI et a!., 1981a; GoTo et a!., 1982). When the membrane potential was clamped at the resting membrane potential (-50 mv) in the K+-free Ringer solution, a large outward current was induced by the Ringer solution (2 mm K+) (Fig. IA). The preparations were thereafter exposed for 20 min to a K+-free Ringer solution containing ouabain (5 /LM). After this treatment, a small inward current could be recorded when the superfusing solution was changed to a Ringer solution (2 mm K+) with 5, tm ouabain (Fig. 1B). The ouabain-sensitive electrogenic Na+-pump current was estimated by adding the Vol. 35, No. 1, 1985

4 92 H. HASUO and K. KOKETSU Fig. 1. Estimation of the ouabain-sensitive electrogenic Na+-pump current. Record A is the outward current induced at a holding potential of -50 mv by adding 2 mm K} to the external K+-free Ringer solution. The upward and downward arrows indicate the period of K+ application. Record B is the inward current induced by adding 2 mm K+ in the presence of ouabain (5 flm). Records A and B were obtained from the same preparation. Record C superimposes records A and B. The amount of current indicated by p is the ouabain-sensitive electrogenic Na+-pump current as defined for these experiments. Fig. 2. Relationship between ouabain-sensitive electrogenic Nat-pump current and membrane potential. The control I -V relationship (.) was obtained in a K+-free Ringer solution. The Nat-pump was activated by adding 2 mm K+ to the external solution, and the second I -V relationship (E) was obtained. After a 20 min exposure to ouabain in the K+-free Ringer solution, the third I -V relationship (v) was obtained. Two mm K+ was then added to the external solution (with ouabain) and the fourth I -V relationship (o) was obtained. The theoretical I -V relationship (S) was obtained from the previous four I V relationships by estimating the electrogenic ouabain-sensitive Nay-pump current (CA) from the sum of the differences between C and B, and C and D at each potential. See details in the text. Japanese Journal of Physiology

5 POTENTIAL DEPENDENCY OF Nat-PUMP 93 maximums for these two currents (AKASU et al., 1978) (Fig. 1C). The current obtained by this method should only be electrogenic Na+-pump current, if it is assumed that the Na+-pump was completely inhibited by this concentration of ouabain (5,uM)(cf. AKASU et al., 1978). To determine the relationship between the magnitude of the electrogenic Na+pump current and membrane potential, the membrane potential was initially clamped at a resting membrane potential of - 50 mv. The steady-state I V relationship was then estimated by applying step command pulses to the membrane. The control I V relationship obtained in the K+-free Ringer solution was almost a straight line (Fig. 2, closed circles). When Ringer (2 mm K+) was applied, an outward current (about 0.5,uA in amplitude) was induced by activation of the electrogenic Nat-pump. The I -V relationship which was obtained during initiation of the potassium induced outward current in a steady-state (about 60 sec after applying Ringer solution) was non-linear, as seen in Fig. 2 (open squares). No significant difference was observed between the I V relationships in K+-free Ringer solution with and without ouabain (5,uM), illustrated in Fig. 2 with closed triangles and closed circles, respectively. The I -V relationship obtained after the addition of 2 mm K+ to the external solution in the presence of ouabain had an increased slope conductance (open triangles in Fig. 2). From this series of experiments, four types of I V relationships were obtained, as shown in Fig. 2. The current expressed as CB (the difference between points C and B) could be assumed to be the sum of the Na+-pump current and the passive inward current. The passive inward current expressed as CD (the difference between points C and D) was added to CB to obtain CA (the difference between points C and A). CA was assumed to represent the virtual ouabain-sensitive electrogenic Nat-pump current at different membrane potentials. A theoretical I -V relationship, closed squares in Fig. 2, was then obtained. Figure 3 shows the K+activated outward current recorded at different potentials. This is in good agreement with the current expressed by CB in Fig. 2. The I V relationship was also observed in the presence of Rb, Cs, or Lit, Fig. 3. The membrane current induced by adding K+ to a K+-free Ringer solution at several membrane potentials. A, B, and C are responses induced by adding 2 mm K+ to the external K+-free Ringer solution at holding potentials of -50, -80, and -110 mv, respectively. The upward and downward arrows indicate the periods of K+ application. The amplitude of the induced current was similar to CB in Fig. 2 (see text). Vol. 35, No. 1, 1985

6 94 H. HASUO and K. KOKETSU which are less permeable to the membrane than K+ (cf. SJODIN, 1959). The passive membrane current due to these ions would be a much smaller part of the total membrane current than with K+. In fact, the inward currents induced by Rb+ or Cs~ in the presence of ouabain were smaller than those induced by the same concentration of K~, i.e. about 70 and 50 %, respectively. The inward current produced by Li+ was small enough (less than 1>< 10-$ A) to be ignored. As a preliminary experiment, dose-response relationships were obtained to evaluate the ability of these "activator cations," Rb+, Cs, and Lit, to activate the Na+-pump. Ouabain-sensitive electrogenic Na+-pump currents were measured by the same procedure as described for Fig. 1. The ouabain-sensitive electrogenic Nat-pump current induced by 2 mm K+ at the resting membrane potential (-50 mv) was taken as the control value (100 %) and the current induced by a different concentration of "activator cations" was measured relative to this control. The results for each concentration were obtained from 6-15 preparations. As seen in Fig. 4A, the relationships between the Nat-pump currents and the logarithm of concentration of extracellular "activator cations" seems to be expressed by a sig- Fig. 4. Activation of the Nat-pump by alkali metal cations. A. The concentration-relationship between the concentrations of the external alkali metal cations and the ouabainsensitive electrogenic Nat-pump current is illustrated. The ordinate is a relative value of the pump current (R) and the abscissa is an extracellular logarithmic scale of the concentration of alkali metal cation. The K+ (2 mm) induced pump current (C) is considered to be 100%. The vertical lines at each point are standard deviations (n=6-15) obtained from different preparations. B. Double reciprocal plots (Lineweaver-Burk's plots) were constructed from diagram A. The Hill number and the apparent dissociation constant are expressed as n and Km, respectively. Japanese Journal of Physiology

7 POTENTIAL DEPENDENCY OF Nat-PUMP 95 Fig. 5. The I -V relationship between membrane potential and ouabain-sensitive electrogenic Nat-pump current induced by 2 mm K+ (A) and 10 ms Rb+ (C). The relationships between Nat-pump current and membrane potential are shown in B and D. The I -V relationships were obtained by the experimental procedure described in Fig. 2. The control I -V relationship obtained in K+-free Ringer solution and the theoretical l V relationship between the Nat-pump current and membrane potential are expressed by 0 and, respectively, in A and C. The curves in B and D were obtained by measuring CA (see Fig. 2) at different potentials, indicating the Na+-pump current at each potential. moid curve. The electrogenic Na+-pump currents induced by Rb+, Cs, and Li+ at the concentration of 5 mm were approximately 100, 42, and 11 % of the currents induced by Kt These results are comparable qualitatively with those reported by RANG and RITCHIE (1968) in the nerve fiber and by EISNER and LEDERER (1980) in the sheep Purkinje fiber. The double reciprocal plot (Lineweaver-Burk's plot) between the reciprocal of the current and the n'th power of [M]o values produced nearly straight lines when n (Hill number) was 1.3 (Fig. 4B). The values of the apparent dissociation constants (Km) for K+, Rb+, Cs, and Li+ were 2.7, 2.5, 9.1, and 30, respectively. The Vmax for Rb+ was almost the same as the Vmax for K+ but the Vmax for Cs~ or Li+ was somewhat lower. A similar finding has been reported for the squid axon (BAKER et al., 1969), frog skeletal muscle (BEAUGE,1975), and sheep Purkinje fiber (EISNER and LEDERER, 1980). Vol. 35, No. 1, 1985

8 96 H. HASUO and K. KOKETSU Fig, 6. The I -V relationship between membrane potential and ouabain-sensitive electrogenic Nat-pump current induced by 10 mni Cs+ (A) and 40 mm Li+ (C). The relationships between Nat-pump current and membrane potential are shown in B and D. The I -V relationships were obtained by the experimental procedure described in Fig. 2, The control 1 V relationship obtained in K~-free Ringer solution and the theoretical 1 V relationship between the Na+-pump current and membrane potential are expressed by 0 and, respectively, in A and C. The curves in B and D were obtained by measuring CA (see Fig. 2) at different potentials, indicating the Nat-pump current at each potential. I -V relationships obtained by the procedure described in Fig. 2 are shown in Figs. 5 and 6. In these figures the theoretical I -V relationship, which is illustrated by closed squares in Fig. 2, is shown to clarify the potential dependency of the ouabain-sensitive electrogenic Na+-pump current. These figures demonstrate that the Nat-pump current was apparently potential dependent. With K+, the maximum value of the electrogenic Na+-pump current was consistently observed at about -80 mv (Fig. 5B). For the other alkali metal cations, Rb, Cs, or Lit, the electrogenic Na+-pump current decreased almost proportionately with increased membrane potential between - 50 mv and -120 mv (Figs. SD, 6B, D). DISCUSSION ship. The effects of "depletion" of extracellular activator cations on the I V relation- There are two mechanisms by which "depletion" may occur during experi- Japanese Journal of Physiology

9 POTENTIAL DEPENDENCY OF Na+-PUMP 97 ments measuring I V relationships. 1) Depletion may occur during the hyperpolarizing pulses, which could decrease the concentration of extracellular cation close to the cell membrane necessary for carrying inward current (NOBLE, 1976). 2) Depletion may also occur by activation of the Na+-pump, which could increase the active uptake of the external activator cations and thus lower the concentrations of these ions immediately outside of the cell membrane (cf. EISNER and LEDERER, 1980). The effect of the first type of depletion upon the I V relationship would be very small, because the ouabain-sensitive electrogenic Na+-pump current was estimated by subtracting passive membrane currents, including those caused by hyperpolarizing pulses, from the total membrane current at different membrane potentials (see Fig. 2). Indeed, depletion due to hyperpolarizing pulses would occur both in the absence and the presence of ouabain. Thus the passive membrane current due to depletion should be eliminated by subtraction. The effect of the second type of depletion upon the I V relationships could be minimized by using Rb+, Cs, or Li+ instead of K+, since these alkali metal cations are less permeable to the membrane than K+. Rb+ depletion should occur to some extent when the pump is activated by Rb+, because Rb+ appears to be pumped at the same rate as K+ (see Fig. 4). EISNER and LEDERER (1980), however, obtained evidence indicating that the effect of Rb+ depletion on membrane currents is much smaller than the effect of K+ depletion. Therefore the contribution of the passive membrane current of Rb+ to the total membrane current would be much smaller than for Kt Furthermore, since Cs+ and Li+ are less potent activators of the Na+-pump than K+, a smaller fractional depletion will occur when these ions are used to activate the Nat-pump. This is expected because higher concentrations of Cs+ and Li+ are required than K+ to activate the Na+-pump to the same extent. Consequently, the effect of "depletion" on the I V relationships can almost be ignored in these experiments, when Rb+, Cs, and particularly Li+ were used instead of Kt Potential dependency of the ouabain-sensitive electrogenic Nat -pump. As already discussed, many investigators using different preparations have reported that the activity of the electrogenic Nat-pump does not depend upon the membrane potential. However, the results obtained in the present experiment show that the ouabain-sensitive electrogenic Na+-pump current, recorded with the voltage clamp method, depends upon the membrane potential and decreases with hyperpolarization (see Figs. 5D, 6B, D). RAPOPORT (1970) proposed a model based on an equation which suggests that the Na+-pump is potential dependent unless the coupling ratio of Na+-K+ is 1:1. In many exitable membranes, the Na+-pump has been demonstrated to be electrogenic and the coupling ratio of Na+-K+ is approximately 3: 2 or 3:1 (KOKETSU, 1971; THOMAS, 1972b). The potential dependency observed in the present experiment could be therefore explained according to his equation. Vol. 35, No. 1, 1985

10 98 H. HASUO and K. KOKETSU The potential dependency of the Na+-pump in the present experiments does not seem to be due to changes in [Na+]i, which would be increased during hyperpolarization, because these results indicate that the Nat-pump current was decreased when the membrane was hyperpolarized. If the Na+-pump activity is not directly dependent on the membrane potential, the present results demonstrating dependency of the Na+-pump on the membrane potential may be interpreted as follows. The Nat-pump is activated by external K+ or other activator cations in the present experiment. An interaction between the Nat-pump site at the membrane surface and the external activator cation can be expressed by the following equation : kl ~ C+P~CP;CP*, (1) k2 a where P and C represent Nat-pump sites and K+ or other activator cation, respectively, and CP and CPX represent resting and active states of the pump site-ion complex, respectively. k1, k2, a, and 3 are the velocity constants for each reaction. Assuming that the intracellular conditions, such as intracellular Na+ concentration, are constant during the experiment, one would expect that the potential dependency of the Na+-pump would be due to a potential dependency of, at least, one of the following four factors: 1) the affinity (k1/k2) of the Nat-pump sites, 2) the total number of the Na+-pump sites, 3) the probability (18/a) for activation of a pump site-ion complex (CP), 4) the coupling ratio of Na+-Kt It is impossible to dismiss entirely the possibility of a "specific membrane conductance change" as proposed by KoNONENKO and KOSTYUK (1976) for snail neurons. A special type of ionic channel may appear in the membrane when the Na+-pump is in a state of high activity. If this applies to the cardiac preparation, these channels would allow not only K+ to pass through but also Rb+, Cs, and Lit Consequently, the potential dependency should have been observed even when the Nat-pump was activated by Rb+, Cs, or Lit Pump equilibrium potential. TAHARA et al. (1973) suggested that a Natpump equilibrium potential exists in frog skeletal muscles. It was not determined in the present experiments whether an equilibrium potential exists in bullfrog cardiac muscles. If a Na+-pump equilibrium potential exists, it may fall between -130 and -150 mv, as determined by extrapolation of the relationship between the Nat-pump current and membrane potential (Fig. 6B, D). It may be noted that the pump reversal potential is predicted by computer simulation study of the Nat-pump (CHAPMAN et al., 1983). This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES AKASU, T., OHTA, Y., and KoKETSU, K. (1978) The effect of adrenaline on the electrogenic Japanese Journal of Physiology

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