Life Sciences, Vol. Printed in the USA
50, pp. 109-116
Pergamon P r e s s
SEASONAL CHANGES IN THE RESPONSE OF FAST AND SLOW MAMMALIAN SKELETAL MUSCLE FIBERS TO ZERO POTASSIUM
Simon P. Aiken and Joseph J. McArdle Department of Pharmacology & Toxicology, New Jersey Medical School 0UMDNJ), 185 South Orange Avenue, Newark, New Jersey 07103-2757 (Received in final form November
1, 1991)
Summary While investigating the decline in resting membrane potential (RMP) of rat skeletal muscle fibers in zero potassium solution, we discovered that there is seasonal variation in the response of the extensor digitorum longus muscle (EDL). In January, most EDL fibers hyperpolarize in zero K+; in September, most depolarize; the distribution of RMPs recorded in May is bimodal, with some fibers hyperpolarizing and some depolarizing. Fibers from the soleus muscle depolarize in zero K + irrespective of the season. The ability of EDL fibers to hyperpolarize appears during the 7th and 8th weeks postpartum, and is dependent upon the presence of a functional nerve, since denervation abolished the response. As possible explanations for these findings, inactivation of K+-channels and inhibition of the Na-K pump by zero K + are discussed. A fundamental principle of classical electrophysiology is that the resting membrane potential (RMP) of cells is primarily due to the transmembrane electrochemical gradient for potassium ions (1-4). A consistent experimental finding which provides strong support for this principle is that excitable cells depolarize when they are bathed in solutions containing elevated extracellular potassium (5). In fact, RMP closely follows the predicted Nernst equilibrium potential for external potassium concentrations in excess of 25 raM. However, the recorded values are less negative than predicted at lower levels of extracellular potassium (6-8). This latter non-ideal behavior can be attributed to altered activity of the Na-K ATPase, or Na-K "pump", and/or the resting membrane's permeability to potassium (9,10). Our objective at the outset of the experiments described in this paper was to lower the potassium concentration of solutions bathing rat muscle in order to further investigate the basis of the decline of RMP associated with muscle denervation (11-19). The surprising result of our preliminary experiments was that the normally innervated fast-twitch extensor digitorum longus muscle (EDL) differed dramatically from the corresponding slow-twitch soleus muscle in terms of the change of RMP associated with acute exposure to nominally zero extracellular potassium. Specifically, the EDL exhibited hyperpolarization while the soleus depolarized. When we attempted to reproduce this unexpected finding some months later, both muscles were similarly depolarized by the zero potassium treatment. This led to a more systematic evaluation of the phenomenon that lasted several years. The results of that evaluation, as summarized in this paper, indicate that the differential effect of zero external potassium Correspondence to Dr. J.J. McArdle. 0024-3205/92 $5.00 + .00 Copyright © 1991 Pergamon Press plc All rights reserved.
110
Zero Potassium and Resting Potential
Vol. 50, No. 2, 1992
on the RMP of fast- and slow-twitch mammalian muscle is under seasonal influence, the basis of which is as yet unknown. Furthermore, the hyperpolarizing response of fast muscle appears with maturation. Finally, neither of these muscle types respond to zero potassium with any marked change of RMP 5 days after denervation. Methods
Animals. Rats used were male Wistars from Charles River (North Wilmington, MA) and were housed in our animal facility for at least 2 weeks before use, using a 12/12 light/dark cycle. The temperature was maintained at 21-23°C throughout the year. Where adult rats are specified, animals were over 60 days old. Younger rats (23-57 days), were purpose-bred in our animal facility from Charles River stock. All experiments m one season were conducted within a 2 week period. Some experiments were performed on muscles that had been denervated 5 days previously. Rats were anesthetized lightly with diethyl ether and an incision was made behind the knee. The deep peroneal nerve and the tibial nerve were crushed with forceps approximately 15mm from their entrances into the EDL and the soleus, respectively. The wound was closed with silk sutures, and no animals showed signs of infection. Dissection. Rats were anesthetized with diethyl ether and the EDL and soleus muscles were rapidly removed, being particularly careful to avoid contacting the muscle fibers with dissecting instruments. During dissection the tissues were bathed with a standard physiological medium of the following composition (mM): KC1 (5.0), NaC1 (135.0), NaHCO3 (14.9), MgCI2 (1.0), Na2HPO4 (1.0), CaC12 (2.0), and D-glucose (11.0). This solution was bubbled with an O2:CO 2 mixture (95%:5%) to maintain a pH of 7.2-7.4. Once removed, paired E D L and soleus muscles were placed side-by-side in a Sylgard®-lined Plexiglas ® dish and pinned down at the tendons. These muscles were superfused with the physiological solution (21-23°C) continuously at 5 ml/min. Recording. After a 30 min incubation period in the 5 mM ('normal') potassium solution, RMPs were recorded in 10-12 fibers of each of the adjacent EDL and soleus muscles. The same 3M KCl electrode (5-20 MI~ tip resistance) was used to impale fibers in both muscles, in order to eliminate any contribution of tip junctional potential (6) to measured differences of RMP. The RMP was read from an Axoclamp-2A amplifier. Care was taken to ensure that each sample of RMP readings was taken across the whole width of the muscle; that is, the 10-12 measurements were taken at roughly equal intervals across the muscle. Some variation was found between RMPs in the center, as opposed to at the edges of the muscles, and so this approach kept the variation so induced to a minimum. Recordings were not made from areas of the muscles where endplates were located. Any muscle fiber impalements which did not give an abrupt reading of the RMP were rejected from the analysis. After recording RMPs in normal potassium, the superfusion medium was exchanged for one lacking KCI. After 30 rain in this nominally zero potassium medium, RMPs were recorded in the same manner as above. In most experiments a further change of solution was made. The muscles were superfused with the normal potassium medium for a further 30 minutes, and recovery RMPs were measured.
Vol. 50, No. 2, 1992
Zero Potassium and Resting Potential
111
Results
Effect of zero potassium. It was found that at all times of year the RMP values for the soleus tended to be marginally lower than for the EDL. This could have been due to the more difficult dissection procedure. When RMP was measured in 5 mM K +, both E D L and soleus RMPs appeared to be normally distributed about their respective means. When experiments were conducted in the month of January, RMP values for soleus in zero K ÷ were less negative than in the normal K + (i.e. they depolarized). Fig. 1 shows that although RMPs for soleus were distributed in a regular manner about the mean, there were a few fibers from which considerably more negative RMPs were obtained. The effect of zero K + on E D L fibers was in contrast to this (see Fig. 1). Relatively few fibers were found that had depolarized; the majority of fibers appeared to have hyperpolarized, with the median RMP being -102mV. Means and standard errors are not given for these data, because of the obvious skewness of the distributions; however, means are indicated on the figures by an arrow, in order to emphasize the shifts.
EDL
Soleus 1
ii iI +_,ILo ao
is
1o
1o
"120
-I~o
-100
-1oo
-80
*Ira
-110
-Io
-40
*,tO
-120
-100
*lo
-Iio
-ao
mV
-IO
-40
mV
! 16
10
-120
n .t00
!;tn *10
JANUARY FIG. 1
January response of rat muscle fibers to zero K +. Frequency of occurrence is plotted against RMP on the abscissa. The mean of each distribution is denoted by an arrow. Each set of data is compiled from 160 measurements fil6 muscles). Solid bars represent 5raM K +, hatched bars (lower two gures) are zero K +. Results for EDL (left) and soleus (right) are shown. In May, RMPs in normal K + were comparable to the January data for both E D L and soleus muscles (Fig. 2). Zero K + had the same effect on soleus as in January. The RMPs for E D L fibers in zero K + showed a very different pattern compared to the January data however. It can be seen from Fig. 2 that the May distribution was distinctly bimodal, with roughly half the fibers depolarizing and half hyperpolarizing. Thus the mean RMP is virtually unchanged by the removal of potassium.
112
Zero Potassium and Resting Potential
EDL
Soleus
1o
-11o
Vol. 50, No. 2, 1992
1
-1oo
-¢110
-Do
-40
0 -12o
-$oo
-80
A
-Io
.,o m V
-,o
.,m mV
1 Is
J
lO
-t20
-1O0
-10
-tO
-~'0
-120
n -I00
-et0
MAY FIG. 2 May response of rat muscle fibers to zero K ÷. Details as for Fig. 1, but 192 fibers per data set. Solid bars represent 5 mM K +, hatched bars are zero K ÷.
EDL
Soleus
'°1
A
1
mV
-120
-~00
-80
-eO
-40
-110
-I00
-S¢
-t0
-40
mV
SEPTEMBER FIG. 3 September response of rat muscle fibers to zero K +. Details as for Fig. 1. Solid bars represent 5 mM K +, hatched bars are zero K +.
Vol. 50, No. 2, 1992
Zero Potassium and Reating Potential
In September, the RMPs in normal 5 mM K ÷ were again similar to those January and May, and soleus fibers responded in the same way to zero The majority of E D L fibers depolarized, although there were still fibers negative of -100mV. The distribution appeared to be skewed in the direction, and the median RMP was -61mV.
113
recorded in K + (Fig. 3). with RMPs depolarized
In all instances where the recovery of RMP was recorded by incubating the muscles in 5 mM K + for 30 min after exposure to zero K ÷, RMPs of both E D L and soleus were distributed in a similar manner to the control data. Thus, these effects of zero K + on RMP were reversible. RMP during development. RMP measurements from fibers of rats aged 23 days were approximately 5 mV less negative than equivalent measurements from adult rats. Study of the effect of zero K ÷ during the first 8 weeks postpartum revealed that the unpredicted response of the E D L fibers develops gradually. These experiments were all performed in January, the time when the tendency for EDL fibers to hyperpolarize is greatest (see Fig. 1). Up to 35 days postpartum, no fibers from EDL muscles were observed to hyperpolarize. The adult characteristic of hyperpolarization develops between days 41 and 57, this final age being indistinguishable from the adult. Data from different ages are presented in Fig. 4, by expressing the proportion of fibers in zero K ÷ that deviated from the mean by 1, 2, or 3 standard deviations (SD) in the negative direction, this mean being calculated from fibers of the same group of animals in normal K +. The proximity of the three lines demonstrates that the distribution does not shift gradually over time, rather that any fibers capable of hyperpolarizing can hyperpolarize to the adult extent as soon as they possess the ability. It is simply the proportion of these fibers that increases during development. Soleus fibers from young rats depolarized in response to zero K ÷, as in the adult. Less than 2% of soleus fibers at any age had RMPs in zero K + that were one SD negative to the mean in 5 mM K +. so
1 SD
32sD SD
~ 6o
'~ 4o
0
--
2o
--
3o
4o
8o
Adult
Postpartum age in days
FIG. 4 Hyperpolarization of EDL fibers in zero K* is age related. As age hypabscissa) increases, a higher percentage of fibers (ordinate) significantly erpolarize in zero K + relative to 5 mM K +. The data lines represent the percentage of fibers that in zero K + deviate from the 5 mM K + mean by 1, 2, or 3 standard deviations in the negative direction. Data are derived from 48 measurements (4 muscles) at each age, in January.
114
Zero Potassium and Resting Potential
Vol. 50, No. 2, 1992
Effect of denervation. The effect of zero K ÷ on RMP in muscles that had been denervated 5 days previously was studied in September 0nly. RMPs for EDL and soleus fibers were normally distributed in 5 mM K +, but fibers had depolarized as anticipated. The means for E D L and solens were -55.5 and -60.5mV respectively, compared to -79.9 and -72.2mV for the innervated muscles in September (values from Fig. 3, n = 160 in each case). Zero K ÷ caused little depolarization of E D L or soleus fibers, and no E D L fibers hyperpolarized, as can be seen from Fig. 5. Thus it appears that the capacity of fibers to respond to zero potassium with a change of RMP is lost with denervation.
EDL
Soleus
.
-120
~
-10~
-80
40
-40
lo
o
-1~0
-100
-CO
-loo
4o
40
-40
mV
-ao
-4o
mV
1o
N °I~o
-1~
-~
40
-4o
-1~o
DENERVATED FIG. 5 Response of rat muscle fibers to zero K ÷ 5 days after nerve crush, in September. Details as for Fig. 1, but 60 fibers (6 muscles) per data set. Solid bars represent 5 mM K ÷, hatched bars are zero K ÷.
Discussion
The E D L and soleus muscles of the rat have long served as models of fast- and slow-twitch muscle types on the basis of their histochemical (20,21), pharmacological (22,23), and physiological differences (24-27). The present paper adds a new characteristic which differentiates these two muscles; i.e., their sensitivity to extracellular potassium is dependent upon the season. Specifically, while both muscles are depolarized by zero K + during the Fall, the E D L exhibits hyperpolarization during the Winter. A transition period in the response of muscle fibers to zero K + is clearly detected for the E D L during the Spring. Since housing conditions were identical throughout the year, it would seem that some sort of 'biological clock' is involved in the response. This seasonal plasticity is very intriguing in view of the dynamic nature of muscle fiber types already described by Guth and Yellin (28).
Vol. 50, No. 2, 1992
Zero Potassium and ReBting Potential
115
The data we derived from immature rats clearly demonstrate that the unique Winter response of the EDL to zero K + requires maturation. While we are completely ignorant of the mechanism involved, our present data suggest that some neural influence may be involved, since neither the EDL nor the soleus are capable of responding to zero K + with a change of RMP after denervation. It is possible that there is atrophic influence on skeletal muscle (29) which may be seasonally modulated. Our data raise several important questions. For example, what is the membrane basis for the response to zero potassium? There are at least two possible answers. The first is that reduction of potassium may inhibit the Na-K pump and thus bring about depolarization (30). The second is that reduction of extracellular potassium inactivates a K÷-channel which helps to maintain a negative RMP. Duval and L6oty (26) showed that rat soleus muscle has both fast and slow inactivating outward sarcolemmal K÷-channels. The former is lacking in fast-twitch rat muscle and this contributes to the greater frequency response of such muscle types (27). It remains to be determined whether the activity of the Na-K pump and/or K÷-channels of the EDL and soleus respond in a qualitatively different manner to zero potassium, and therefore we do not draw any conclusions about the mechanisms involved with these effects. Regardless of what the basis of the differential sensitivity to zero potassium proves to be, it is essential to understand the nature of the seasonal and neural influences on this mechanism. Although data are presented here for only one species, our preliminary findings suggest that there is a similar seasonal variation in the mouse. If this phenomenon does extend to species other than the rat, as well as to excitable cells besides skeletal muscle, the implications may be very important. Acknowledgments This work was supported by grants NS-11055 from the National Institutes of Neurological Diseases and Communicative Disorders and Stroke, and 5 R01 AA08025 from the National Institute on Alcohol Abuse and Alcoholism. References
1. J. BERNSTEIN, Pfliigers Arch. Ges. Physiol. 92, 521-562 (1902). 2. R. HOBER, Pfliigers Arch. Ges. Physiol. 106, 599-635 (1905). 3. A.L. HODGKIN, Biol. Rev. Camb. Philos. Soe. 26, 339-409 (1951). 4. B. KATZ, Nerve, Muscle, and Synapse, McGraw-Hill, New York (1966). 5. A.L. HODGKIN and P. HOROWICZ, J. Physiol. 148, 127-160 (1959). 6. R.H. ADRIAN, J. Physiol. 133, 631-658 (1956). 7. H.P. JENERICK, J. Cell. Comp. Physiol. 42, 427-448 (1953). 8. G. LING and R.W. GERARD, Nature 165, 113-114 (1950). 9. T.C. RUCH and H.D. PATTON, Physiology and Biophysics, W.B. Saunders Company, Philadelphia (1965). 10. L.C. SELLIN and N. SPERELAKIS, Exp. Neurol. 62, 605-617 (1978). 11. E.X. ALBUQUERQUE and R.J. McISAAC, Life Sci. 8, 409-416 (1969). 12. E.X. ALBUQUERQUE, F.T. SCHUH and F.C. KAUFFMAN, Pfliigers Arch. 328, 36-50 (1971). 13. S. LOCKE and H.C. SOLOMON, J. Exp. Zool. 166, 377-386 (1967). 14. J.J. McARDLE and E.X. ALBUQUERQUE, J. Gen. Physiol. 61. 1-23 (1973). 15. J3. McARDLE and E.X. ALBUQUERQUE, Exp. Neurol. 47, 353-356 (1975). 16. J.J. McARDLE and A.J. D'ALONZO, Exp. Neurol. 7_!,1134-143 (1981). 17. P. REDFERN and S. TI-IESLEFF, Acta Physiol. Scand. 8_!,1557-564 (1971).
116
Zero Potassium and Resting Potential
Vol. 50, No. 2, 1992
18. L.C. SELLIN and J.J. McARDLE, Exp. Neurol. 55, 483-492 (1977). 19. F. WARE, Jr., A.L. BENNETT and A.R. McINTYRE, Am. J. Physiol. 177, 115-118 (1954). 20. L. GUTH, F.J. SAMAHA and R.W. ALBERS, Exp. Neurol. 26, 126-135 (1970). 21. E. GUTMANN and B.M. CARLSON, Pfliigers Arch. 353, 227-239 (1975). 22. E.X. ALBUQUERQUE and R.J. McISAAC, EXP. Neurol. 26, 183-202 (1970). 23. E. GUTMANN and V. HANZLIKOV,~, Physiol. Bohem. 15, 404-414 (1966). 24. D.J. CHIARANDINI and E. STEFANI, J. Physiol. 335, 29-40 (1983). 25. R. CLOSE, J. Physiol. 173, 74-95 (1983). 26. A. DUVAL and C. LEOTY, J. Physiol. 307, 43-57 (1980). 27. J.J. McARDLE, L. MICHELSON and A.J. D'ALONZO, J. Gen. Physiol. 75, 655-672 (1980). 28. L. GUTH and H. YELLIN, EXP. Neurol. 31, 277-300 (1971). 29. J.J. McARDLE, Prog. Neurobiol. 21, 135-198 (1983). 30. L.C. SELLIN and J.J. McARDLE, Eur. J. Pharmacol. 41, 337-340 (1977).
50, pp. 109-116
Pergamon P r e s s
SEASONAL CHANGES IN THE RESPONSE OF FAST AND SLOW MAMMALIAN SKELETAL MUSCLE FIBERS TO ZERO POTASSIUM
Simon P. Aiken and Joseph J. McArdle Department of Pharmacology & Toxicology, New Jersey Medical School 0UMDNJ), 185 South Orange Avenue, Newark, New Jersey 07103-2757 (Received in final form November
1, 1991)
Summary While investigating the decline in resting membrane potential (RMP) of rat skeletal muscle fibers in zero potassium solution, we discovered that there is seasonal variation in the response of the extensor digitorum longus muscle (EDL). In January, most EDL fibers hyperpolarize in zero K+; in September, most depolarize; the distribution of RMPs recorded in May is bimodal, with some fibers hyperpolarizing and some depolarizing. Fibers from the soleus muscle depolarize in zero K + irrespective of the season. The ability of EDL fibers to hyperpolarize appears during the 7th and 8th weeks postpartum, and is dependent upon the presence of a functional nerve, since denervation abolished the response. As possible explanations for these findings, inactivation of K+-channels and inhibition of the Na-K pump by zero K + are discussed. A fundamental principle of classical electrophysiology is that the resting membrane potential (RMP) of cells is primarily due to the transmembrane electrochemical gradient for potassium ions (1-4). A consistent experimental finding which provides strong support for this principle is that excitable cells depolarize when they are bathed in solutions containing elevated extracellular potassium (5). In fact, RMP closely follows the predicted Nernst equilibrium potential for external potassium concentrations in excess of 25 raM. However, the recorded values are less negative than predicted at lower levels of extracellular potassium (6-8). This latter non-ideal behavior can be attributed to altered activity of the Na-K ATPase, or Na-K "pump", and/or the resting membrane's permeability to potassium (9,10). Our objective at the outset of the experiments described in this paper was to lower the potassium concentration of solutions bathing rat muscle in order to further investigate the basis of the decline of RMP associated with muscle denervation (11-19). The surprising result of our preliminary experiments was that the normally innervated fast-twitch extensor digitorum longus muscle (EDL) differed dramatically from the corresponding slow-twitch soleus muscle in terms of the change of RMP associated with acute exposure to nominally zero extracellular potassium. Specifically, the EDL exhibited hyperpolarization while the soleus depolarized. When we attempted to reproduce this unexpected finding some months later, both muscles were similarly depolarized by the zero potassium treatment. This led to a more systematic evaluation of the phenomenon that lasted several years. The results of that evaluation, as summarized in this paper, indicate that the differential effect of zero external potassium Correspondence to Dr. J.J. McArdle. 0024-3205/92 $5.00 + .00 Copyright © 1991 Pergamon Press plc All rights reserved.
110
Zero Potassium and Resting Potential
Vol. 50, No. 2, 1992
on the RMP of fast- and slow-twitch mammalian muscle is under seasonal influence, the basis of which is as yet unknown. Furthermore, the hyperpolarizing response of fast muscle appears with maturation. Finally, neither of these muscle types respond to zero potassium with any marked change of RMP 5 days after denervation. Methods
Animals. Rats used were male Wistars from Charles River (North Wilmington, MA) and were housed in our animal facility for at least 2 weeks before use, using a 12/12 light/dark cycle. The temperature was maintained at 21-23°C throughout the year. Where adult rats are specified, animals were over 60 days old. Younger rats (23-57 days), were purpose-bred in our animal facility from Charles River stock. All experiments m one season were conducted within a 2 week period. Some experiments were performed on muscles that had been denervated 5 days previously. Rats were anesthetized lightly with diethyl ether and an incision was made behind the knee. The deep peroneal nerve and the tibial nerve were crushed with forceps approximately 15mm from their entrances into the EDL and the soleus, respectively. The wound was closed with silk sutures, and no animals showed signs of infection. Dissection. Rats were anesthetized with diethyl ether and the EDL and soleus muscles were rapidly removed, being particularly careful to avoid contacting the muscle fibers with dissecting instruments. During dissection the tissues were bathed with a standard physiological medium of the following composition (mM): KC1 (5.0), NaC1 (135.0), NaHCO3 (14.9), MgCI2 (1.0), Na2HPO4 (1.0), CaC12 (2.0), and D-glucose (11.0). This solution was bubbled with an O2:CO 2 mixture (95%:5%) to maintain a pH of 7.2-7.4. Once removed, paired E D L and soleus muscles were placed side-by-side in a Sylgard®-lined Plexiglas ® dish and pinned down at the tendons. These muscles were superfused with the physiological solution (21-23°C) continuously at 5 ml/min. Recording. After a 30 min incubation period in the 5 mM ('normal') potassium solution, RMPs were recorded in 10-12 fibers of each of the adjacent EDL and soleus muscles. The same 3M KCl electrode (5-20 MI~ tip resistance) was used to impale fibers in both muscles, in order to eliminate any contribution of tip junctional potential (6) to measured differences of RMP. The RMP was read from an Axoclamp-2A amplifier. Care was taken to ensure that each sample of RMP readings was taken across the whole width of the muscle; that is, the 10-12 measurements were taken at roughly equal intervals across the muscle. Some variation was found between RMPs in the center, as opposed to at the edges of the muscles, and so this approach kept the variation so induced to a minimum. Recordings were not made from areas of the muscles where endplates were located. Any muscle fiber impalements which did not give an abrupt reading of the RMP were rejected from the analysis. After recording RMPs in normal potassium, the superfusion medium was exchanged for one lacking KCI. After 30 rain in this nominally zero potassium medium, RMPs were recorded in the same manner as above. In most experiments a further change of solution was made. The muscles were superfused with the normal potassium medium for a further 30 minutes, and recovery RMPs were measured.
Vol. 50, No. 2, 1992
Zero Potassium and Resting Potential
111
Results
Effect of zero potassium. It was found that at all times of year the RMP values for the soleus tended to be marginally lower than for the EDL. This could have been due to the more difficult dissection procedure. When RMP was measured in 5 mM K +, both E D L and soleus RMPs appeared to be normally distributed about their respective means. When experiments were conducted in the month of January, RMP values for soleus in zero K ÷ were less negative than in the normal K + (i.e. they depolarized). Fig. 1 shows that although RMPs for soleus were distributed in a regular manner about the mean, there were a few fibers from which considerably more negative RMPs were obtained. The effect of zero K + on E D L fibers was in contrast to this (see Fig. 1). Relatively few fibers were found that had depolarized; the majority of fibers appeared to have hyperpolarized, with the median RMP being -102mV. Means and standard errors are not given for these data, because of the obvious skewness of the distributions; however, means are indicated on the figures by an arrow, in order to emphasize the shifts.
EDL
Soleus 1
ii iI +_,ILo ao
is
1o
1o
"120
-I~o
-100
-1oo
-80
*Ira
-110
-Io
-40
*,tO
-120
-100
*lo
-Iio
-ao
mV
-IO
-40
mV
! 16
10
-120
n .t00
!;tn *10
JANUARY FIG. 1
January response of rat muscle fibers to zero K +. Frequency of occurrence is plotted against RMP on the abscissa. The mean of each distribution is denoted by an arrow. Each set of data is compiled from 160 measurements fil6 muscles). Solid bars represent 5raM K +, hatched bars (lower two gures) are zero K +. Results for EDL (left) and soleus (right) are shown. In May, RMPs in normal K + were comparable to the January data for both E D L and soleus muscles (Fig. 2). Zero K + had the same effect on soleus as in January. The RMPs for E D L fibers in zero K + showed a very different pattern compared to the January data however. It can be seen from Fig. 2 that the May distribution was distinctly bimodal, with roughly half the fibers depolarizing and half hyperpolarizing. Thus the mean RMP is virtually unchanged by the removal of potassium.
112
Zero Potassium and Resting Potential
EDL
Soleus
1o
-11o
Vol. 50, No. 2, 1992
1
-1oo
-¢110
-Do
-40
0 -12o
-$oo
-80
A
-Io
.,o m V
-,o
.,m mV
1 Is
J
lO
-t20
-1O0
-10
-tO
-~'0
-120
n -I00
-et0
MAY FIG. 2 May response of rat muscle fibers to zero K ÷. Details as for Fig. 1, but 192 fibers per data set. Solid bars represent 5 mM K +, hatched bars are zero K ÷.
EDL
Soleus
'°1
A
1
mV
-120
-~00
-80
-eO
-40
-110
-I00
-S¢
-t0
-40
mV
SEPTEMBER FIG. 3 September response of rat muscle fibers to zero K +. Details as for Fig. 1. Solid bars represent 5 mM K +, hatched bars are zero K +.
Vol. 50, No. 2, 1992
Zero Potassium and Reating Potential
In September, the RMPs in normal 5 mM K ÷ were again similar to those January and May, and soleus fibers responded in the same way to zero The majority of E D L fibers depolarized, although there were still fibers negative of -100mV. The distribution appeared to be skewed in the direction, and the median RMP was -61mV.
113
recorded in K + (Fig. 3). with RMPs depolarized
In all instances where the recovery of RMP was recorded by incubating the muscles in 5 mM K + for 30 min after exposure to zero K ÷, RMPs of both E D L and soleus were distributed in a similar manner to the control data. Thus, these effects of zero K + on RMP were reversible. RMP during development. RMP measurements from fibers of rats aged 23 days were approximately 5 mV less negative than equivalent measurements from adult rats. Study of the effect of zero K ÷ during the first 8 weeks postpartum revealed that the unpredicted response of the E D L fibers develops gradually. These experiments were all performed in January, the time when the tendency for EDL fibers to hyperpolarize is greatest (see Fig. 1). Up to 35 days postpartum, no fibers from EDL muscles were observed to hyperpolarize. The adult characteristic of hyperpolarization develops between days 41 and 57, this final age being indistinguishable from the adult. Data from different ages are presented in Fig. 4, by expressing the proportion of fibers in zero K ÷ that deviated from the mean by 1, 2, or 3 standard deviations (SD) in the negative direction, this mean being calculated from fibers of the same group of animals in normal K +. The proximity of the three lines demonstrates that the distribution does not shift gradually over time, rather that any fibers capable of hyperpolarizing can hyperpolarize to the adult extent as soon as they possess the ability. It is simply the proportion of these fibers that increases during development. Soleus fibers from young rats depolarized in response to zero K ÷, as in the adult. Less than 2% of soleus fibers at any age had RMPs in zero K + that were one SD negative to the mean in 5 mM K +. so
1 SD
32sD SD
~ 6o
'~ 4o
0
--
2o
--
3o
4o
8o
Adult
Postpartum age in days
FIG. 4 Hyperpolarization of EDL fibers in zero K* is age related. As age hypabscissa) increases, a higher percentage of fibers (ordinate) significantly erpolarize in zero K + relative to 5 mM K +. The data lines represent the percentage of fibers that in zero K + deviate from the 5 mM K + mean by 1, 2, or 3 standard deviations in the negative direction. Data are derived from 48 measurements (4 muscles) at each age, in January.
114
Zero Potassium and Resting Potential
Vol. 50, No. 2, 1992
Effect of denervation. The effect of zero K ÷ on RMP in muscles that had been denervated 5 days previously was studied in September 0nly. RMPs for EDL and soleus fibers were normally distributed in 5 mM K +, but fibers had depolarized as anticipated. The means for E D L and solens were -55.5 and -60.5mV respectively, compared to -79.9 and -72.2mV for the innervated muscles in September (values from Fig. 3, n = 160 in each case). Zero K ÷ caused little depolarization of E D L or soleus fibers, and no E D L fibers hyperpolarized, as can be seen from Fig. 5. Thus it appears that the capacity of fibers to respond to zero potassium with a change of RMP is lost with denervation.
EDL
Soleus
.
-120
~
-10~
-80
40
-40
lo
o
-1~0
-100
-CO
-loo
4o
40
-40
mV
-ao
-4o
mV
1o
N °I~o
-1~
-~
40
-4o
-1~o
DENERVATED FIG. 5 Response of rat muscle fibers to zero K ÷ 5 days after nerve crush, in September. Details as for Fig. 1, but 60 fibers (6 muscles) per data set. Solid bars represent 5 mM K ÷, hatched bars are zero K ÷.
Discussion
The E D L and soleus muscles of the rat have long served as models of fast- and slow-twitch muscle types on the basis of their histochemical (20,21), pharmacological (22,23), and physiological differences (24-27). The present paper adds a new characteristic which differentiates these two muscles; i.e., their sensitivity to extracellular potassium is dependent upon the season. Specifically, while both muscles are depolarized by zero K + during the Fall, the E D L exhibits hyperpolarization during the Winter. A transition period in the response of muscle fibers to zero K + is clearly detected for the E D L during the Spring. Since housing conditions were identical throughout the year, it would seem that some sort of 'biological clock' is involved in the response. This seasonal plasticity is very intriguing in view of the dynamic nature of muscle fiber types already described by Guth and Yellin (28).
Vol. 50, No. 2, 1992
Zero Potassium and ReBting Potential
115
The data we derived from immature rats clearly demonstrate that the unique Winter response of the EDL to zero K + requires maturation. While we are completely ignorant of the mechanism involved, our present data suggest that some neural influence may be involved, since neither the EDL nor the soleus are capable of responding to zero K + with a change of RMP after denervation. It is possible that there is atrophic influence on skeletal muscle (29) which may be seasonally modulated. Our data raise several important questions. For example, what is the membrane basis for the response to zero potassium? There are at least two possible answers. The first is that reduction of potassium may inhibit the Na-K pump and thus bring about depolarization (30). The second is that reduction of extracellular potassium inactivates a K÷-channel which helps to maintain a negative RMP. Duval and L6oty (26) showed that rat soleus muscle has both fast and slow inactivating outward sarcolemmal K÷-channels. The former is lacking in fast-twitch rat muscle and this contributes to the greater frequency response of such muscle types (27). It remains to be determined whether the activity of the Na-K pump and/or K÷-channels of the EDL and soleus respond in a qualitatively different manner to zero potassium, and therefore we do not draw any conclusions about the mechanisms involved with these effects. Regardless of what the basis of the differential sensitivity to zero potassium proves to be, it is essential to understand the nature of the seasonal and neural influences on this mechanism. Although data are presented here for only one species, our preliminary findings suggest that there is a similar seasonal variation in the mouse. If this phenomenon does extend to species other than the rat, as well as to excitable cells besides skeletal muscle, the implications may be very important. Acknowledgments This work was supported by grants NS-11055 from the National Institutes of Neurological Diseases and Communicative Disorders and Stroke, and 5 R01 AA08025 from the National Institute on Alcohol Abuse and Alcoholism. References
1. J. BERNSTEIN, Pfliigers Arch. Ges. Physiol. 92, 521-562 (1902). 2. R. HOBER, Pfliigers Arch. Ges. Physiol. 106, 599-635 (1905). 3. A.L. HODGKIN, Biol. Rev. Camb. Philos. Soe. 26, 339-409 (1951). 4. B. KATZ, Nerve, Muscle, and Synapse, McGraw-Hill, New York (1966). 5. A.L. HODGKIN and P. HOROWICZ, J. Physiol. 148, 127-160 (1959). 6. R.H. ADRIAN, J. Physiol. 133, 631-658 (1956). 7. H.P. JENERICK, J. Cell. Comp. Physiol. 42, 427-448 (1953). 8. G. LING and R.W. GERARD, Nature 165, 113-114 (1950). 9. T.C. RUCH and H.D. PATTON, Physiology and Biophysics, W.B. Saunders Company, Philadelphia (1965). 10. L.C. SELLIN and N. SPERELAKIS, Exp. Neurol. 62, 605-617 (1978). 11. E.X. ALBUQUERQUE and R.J. McISAAC, Life Sci. 8, 409-416 (1969). 12. E.X. ALBUQUERQUE, F.T. SCHUH and F.C. KAUFFMAN, Pfliigers Arch. 328, 36-50 (1971). 13. S. LOCKE and H.C. SOLOMON, J. Exp. Zool. 166, 377-386 (1967). 14. J.J. McARDLE and E.X. ALBUQUERQUE, J. Gen. Physiol. 61. 1-23 (1973). 15. J3. McARDLE and E.X. ALBUQUERQUE, Exp. Neurol. 47, 353-356 (1975). 16. J.J. McARDLE and A.J. D'ALONZO, Exp. Neurol. 7_!,1134-143 (1981). 17. P. REDFERN and S. TI-IESLEFF, Acta Physiol. Scand. 8_!,1557-564 (1971).
116
Zero Potassium and Resting Potential
Vol. 50, No. 2, 1992
18. L.C. SELLIN and J.J. McARDLE, Exp. Neurol. 55, 483-492 (1977). 19. F. WARE, Jr., A.L. BENNETT and A.R. McINTYRE, Am. J. Physiol. 177, 115-118 (1954). 20. L. GUTH, F.J. SAMAHA and R.W. ALBERS, Exp. Neurol. 26, 126-135 (1970). 21. E. GUTMANN and B.M. CARLSON, Pfliigers Arch. 353, 227-239 (1975). 22. E.X. ALBUQUERQUE and R.J. McISAAC, EXP. Neurol. 26, 183-202 (1970). 23. E. GUTMANN and V. HANZLIKOV,~, Physiol. Bohem. 15, 404-414 (1966). 24. D.J. CHIARANDINI and E. STEFANI, J. Physiol. 335, 29-40 (1983). 25. R. CLOSE, J. Physiol. 173, 74-95 (1983). 26. A. DUVAL and C. LEOTY, J. Physiol. 307, 43-57 (1980). 27. J.J. McARDLE, L. MICHELSON and A.J. D'ALONZO, J. Gen. Physiol. 75, 655-672 (1980). 28. L. GUTH and H. YELLIN, EXP. Neurol. 31, 277-300 (1971). 29. J.J. McARDLE, Prog. Neurobiol. 21, 135-198 (1983). 30. L.C. SELLIN and J.J. McARDLE, Eur. J. Pharmacol. 41, 337-340 (1977).
