19
J. Physiol. (1977), 265, pp. 19-42 With 6 text-ftgure8 Printed in Great Britain
THE EFFECT OF INSULIN ON THE TRANSPORT OF SODIUM AND POTASSIUM IN RAT SOLEUS MUSCLE
BY TORBEN CLAUSEN AND PETER GEORGE KOHN From the Institute of Physiology, University of Arhus, Universitet8parken, DK-8000 Arhus C, Denmark and the Department of Physiology, University of Sheffield, Sheffield S10 2TN
(Received 19 December 1975) SUMMARY
1. The action of insulin on the transport and the distribution of Na and K has been studied in rat soleus muscles incubated at 300 C in glucose-free Krebs-Ringer bicarbonate buffer. 2. Measurements of the uptake and the wash-out of 22Na indicate that the muscles contain an intracellular pool of Na available for transport which is confined to the water space not available to sucrose. Ouabain (10-4-10-3M) inhibited 22Na efflux by 69 % (0.287 #tmole/g tissue wet weight per minute) and 42K-influx by 40 % (0.196 gmole/g tissue wet weight per minute). When all extracellular Na was replaced by Li, both 22Na-efflux and 42K-influx were inhibited to about the same extent and ouabain produced very little further inhibition. 2,4-dinitrophenol decreased the ouabain-resistant component of 22Na-efflux by 39 %, 3. Insulin (from 0.1 to 100 mu./ml.) increased the rate coefficient of 22Na-efflux by from 11 to 46 % within 15 min. In the presence of ouabain (10-3M), the same relative increase was obtained, indicating that the hormone stimulates the glycoside-sensitive and the glycoside-insensitive Na transport to a similar extent. The effect of insulin on 22Na-efflux was not abolished by tetracaine (0 5 x 10-3M), phlorizin (0.5 x 10-2M) or by the substitution of Na, K, Mg or Ca. In the presence of 2,4-dinitrophenol (0.5 X 10-4M) or at temperatures below 150 C, the hormone produced no detectable change in 22Na-efflux. 4. Insulin increased 42K-influx from 0*525 to 0 664 ,umole/g tissue wet weight per minute. This effect was entirely blocked by ouabain but not by tetracaine. Insulin produced a 14 % transient decrease in 42K-efflux. 5. The continued exposure to insulin led to a new steady state, in which the intracellular Na pool was decreased from around 10 to around 5,almole/g tissue wet weight and the K content increased by an equivalent amount. In the presence of ouabain or at low extracellular concentrations of K,
T. CLAUSEN AND P. G. KOHN 20 insulin increased the rate of 22Na-influx by around 35 %. This effect was blocked by 2,4-dinitrophenol but not by tetracaine. 6. It is concluded that insulin stimulates the active coupled transport of Na and K, possibly by increasing the relative Na-affinity of the system mediating this process. INTRODUCTION
Several studies with isolated preparations of muscle tissue indicate that the classical hypokalaemic effect of insulin (Harrop & Benedict, 1924) can for a major part be accounted for as the result of increased net accumulation of K in the cytoplasm of muscle cells (Kamminga, Willebrands, Groen & Blickman, 1950; Zierler, 1960; Creese & Northover, 1961; Gourley, 1965). It has repeatedly been demonstrated that this is associated with a considerable decrease in the intracellular Na concentration (Creese & Northover, 1961; Kernan, 1962; Moore, 1973; Zierler, Rogus & Hazlewood, 1966), and the observation that insulin stimulated the efflux of isotopic Na (Creese, 1968; Moore, 1973) suggests that the effect of the hormone on the distribution of Na and K is the result of an activation of the system mediating the active coupled transport of Na and K (Skou, 1965). Indeed, cardiac glycosides were found to produce almost complete (Moore, 1973) or partial suppression (Grinstein & Erlij, 1974) of the insulininduced increase in 22Na efflux from frog muscles and, recently, insulin was reported to augment the activity of Na-K-activated ATPase isolated from rat muscle (Brodal, Jebens, Oy & Iversen, 1974) and frog muscle (Gavryck, Moore & Thompson, 1975). However, in the extensor digitorum longus muscle of the rat, the effect of insulin on Na extrusion was reported to be resistant to ouabain (Zierler, 1966), and in a detailed study of Na-K-activated ATPase isolated from rat muscle, it was not possible to detect any significant effect of insulin (Rogus, Price & Zierler, 1969). These disagreements together with the fact that little information is available about the acute effects of insulin on the transport of Na and K in mammalian skeletal muscle prompted the present investigation, which at the same time represents an attempt to establish some basic parameters of the movements of these ions in peripheral muscle. Soleus muscles have recently been introduced for the study of insulin action (Chaudry & Gould, 1969) and measurements of the fluxes of hexoses and ions indicate that this preparation is suitable for the characterization of factors controlling the transport properties of the plasma membrane (Kohn & Clausen, 1971; Clausen & Hansen, 1974; Clausen, Elbrink & Dahl-Hansen, 1975).
INSULIN AND Na-K TRANSPORT IN MUSCLE
21
METHODS
Experimental procedures. The procedures for the preparation and incubation of isolated soleus muscles have been described in detail elsewhere (Kohn & Clausen, 1971). All the rats were weaned for at least 6 days on to a diet of laboratory pellets and, except when indicated, only fed animals weighing from 60 to 70 g were used. These yielded muscles weighing around 30 mg. The incubations took place in centrifuge tubes containing from 1-5 to 4 ml. buffer which was kept agitated by continuous bubbling with an appropriate gas mixture. Unless otherwise indicated, all experiments were performed at 300 C using Krebs-Ringer bicarbonate buffer (Cohen, 1951) containing 1 mm sodium pyruvate and equilibrated to pH 7-35 by gassing with a mixture of O2 (95%) and CO2 (5%). Tris-buffers were prepared by replacing all the NaHCO3 by isosmotic amounts of Tris-(hydroxymethyl)-aminomethane titrated to pH 7-35 with HCl. In this case, gassing was performed using 100% 02. In some experiments, NaCl was replaced by isosmotic amounts of LiCl or Tris chloride (pH 7.35). A Na-free Li-bicarbonate buffer was prepared by replacing NaHCO3 by LiHCO3 and Na pyruvate by Li pyruvate. The measurements of the uptake and the wash-out of isotopic Na and K were performed as described in our studies of the hexose transport system in this muscle (Kohn & Clausen, 1971, 1972) and in a brief preliminary report (Dahl-Hansen & Clausen, 1973). Further details are given, where appropriate, in the results section. 22Na- and 42K-radioactivity was in most cases determined by gamma-spectrometry using aliquots of the incubation media and trichloroacetic acid extracts of the muscles (Kohn & Clausen, 1971). In some experiments, the amount of 22Na and ['4C]sucrose taken up by the muscles was determined using a liquid scintillation spectrometer with channels adjusted so as to allow the measurements of 14C- and 22Na-radioactivity in the same tissue extract. For this purpose, a Triton-X-100/ toluene scintillation mixture was used (Kohn & Clausen, 1971). The Na and K contents were determined by flame photometry of the clear supernatant obtained by centrifugation (at 2000 g for 10 min) of trichloroacetic acid extracts. A series of standards containing trichloroacetic acid, Na and K in the same concentration range as the tissue extracts were used for the calibration. Tissue dry weight was determined following 2 hr of desiccation at 1100 C. Chemicals and isotope8. All chemicals used (except the toluene and the Triton-X100) were of analytical grade. Bovine serum albumin was purchased from Sigma and used after 24 hr of dialysis against distilled water in the cold. 22Na (3000 mc/mmole) and [U-14C]sucrose (400 mc/mmole) were obtained from the Radiochemical Centre, Amersham, Bucks and 42K (100 mc/mmole) from the Danish Atomic Energy Commission, Isotope Laboratory, Riso; mono-component pork insulin (purified by chromatography, 25 i.u./mg) was a gift from the Novo Research Laboratories, Copenhagen. RESULTS
Experiments with 22Na The effect of insulin on the transport of Na and its distribution among cellular compartments was assessed in four ways: (1) measurements of the uptake of 22Na in the tissue space not available to [14C]sucrose; (2) analysis of efflux curves for 22Na; (3) determinations of the acute changes in the rate coefficient for the efflux of 22Na; (4) measurements of the amount of 22Na taken up during the early phase of exposure to this isotope.
T. CLAUSEN AND P. G. KOHN Fig. 1 shows the effects of insulin (100 mu./ml.) and ouabain (10-3M) on the uptake of 22Na into the space not available to [14C]sucrose. In the absence of ouabain, the amount of isotopic sodium taken up in this com partment approaches a steady level after between 60 and 120 min and 22
50 r
4' 110
-o0
40
6 0 1530 0
0
4. W
) 0 IU
S0. 020'I M
.0 0. C
z34,0. 10 I.-
0
0
01
L0
L
120 60 Duration of incubation (min) Fig. 1. Effect of insulin and ouabain on 22Na uptake. Isolated rat soleus muscles were incubated in Krebs-Ringer bicarbonate buffer containing 22Na (0.2 psc/ml.) and 1 mM [U-_4C]sucrose (0-2 ,uc/ml.) without or with the additions indicated. The spaces available to the two isotopes were determined by liquid scintillation counting of a trichloroacetic acid extract of the muscles. The amount of 22Na taken up into the space not available to [U-14C]sucrose was calculated and expressed as #umole/g tissue wet weight with bars denoting 5.E. of mean. Each point represents the mean of from four to six observations. Control, 0-0; insulin (100 mu./ml.), V-V; ouabain (10-3M), 0-A; ouabain (10-AM) and insulin (100 mu./ml.),
-V .
insulin induces a decrease of around 50 % in this steady-state level. In the presence of ouabain, the 22Na uptake was markedly increased, which is consistent with an inhibition of active Na-expulsion. Under these conditions, insulin produced no detectable change during the first 60 min of
INSULIN AND Na-K TRANSPORT IN MUSCLE
23 incubation but, after 120 min, a significant rise in uptake (P < 0.02) was found. For comparison, the effects of insulin and ouabain were assessed in a series of wash-out experiments performed under similar conditions. Fig. 2 shows the time course of 22Na release from muscles which were loaded and 100 385
1-:
29-0 4,
0
10
50
8-9
44A_~ K~4
K1_
._
E 0 (4
1*0
0 .0
05
zW(4
0.1
V
\'
Y
0*05
-
L
0
I
I
30 90 60 Duration of wash-out (min)
-
120
Fig. 2. Effect of insulin and ouabain on 22Na release. Isolated rat soleus muscles were incubated in Krebs-Ringer bicarbonate buffer containing 22Na (6-7 ,uc./ml.). The wash-out of isotope was then followed by transferring the muscles through a series of tubes containing 3 ml. unlabelled buffer without or with the additions indicated. At the end of the wash-out, the amount of 22Na retained in the muscles was determined and the time course of the decrease in 22Na content was obtained by adding the amount of 22Na released into each tube. On the basis of the specific activity of the loading media, all results are expressed as molee 22Na per gram wet weight. Each point represents the mean of three to four observations with bars denoting S.E. of mean. The straight portions of the curves have been extrapolated back to the onset of wash-out and the intercept values are indicated. Muscles loaded for 60 min and washed for 70 min in buffer without (0-0) or with insulin (100 mu./ml.) (V-V ). Muscles loaded for 120 min in the presence of ouabain (10-3M) and washed for 100 min in buffer containing ouabain (10-3M) without (-*) or with insulin (100 mu./ml.)
(v-v).
T. CLAUSEN AND P. G. KOHN 24 washed in the absence or in the continued presence of insulin (100 mu./ml.), without or with ouabain (10-3M). Under all the conditions tested, the 22Na content shows a rapid initial decrease followed by a much slower and approximately exponential fall. In the untreated control muscles, the zero time intercept of the straight portion of the curve corresponds to 8-9 ,umole/g wet weight. The muscles which had been exposed to insulin (100 mu./ml.) during both the loading and the wash-out period yielded an efflux curve whose linear portion had the same slope but the intercept corresponded to 4-4 gnmole/g wet weight, which is 51 % lower than that of the controls. Neither of these values are significantly different from those obtained in the measurements of the amount of 22Na taken up into the space not available to [14C]sucrose within a period of 60 min (Fig. 1). When the muscles were loaded for 120 min and washed in the continued presence of ouabain (10-3M), the linear portion of the efflux curve could be extrapolated back to an intercept value of 29*0, lmole/g wet weight. Under these conditions, the addition of insulin (100 mu./ml.) to both the loading and the wash-out media gave a slightly steeper efflux curve with a somewhat higher intercept value (38.5 lsmole/g wet weight). Again, there is fairly good agreement with the figures obtained in the uptake experiments and it seems reasonable to assume that both in the absence and in the presence of ouabain, the continued exposure to insulin leads to a change in the Na content of a cellular compartment. In the absence of ouabain, it is possible to obtain a steady state where this Na pool is constant and located in a space not available to a compound (sucrose) which seems not to penetrate the plasma membrane at an appreciable rate (Clausen & Hansen, 1974). The wash-out experiments are compatible with the view that the linear portion of the 22Na efflux curves represents the transport of isotopic Na out of the same pool. In the presence of ouabain, the situation is more complex since the size of the Na pool is con-
tinuously changing. In the following, an attempt is made to account for the above-mentioned changes in terms of alterations in the relative rates of efflux and influx of 22Na. In order to identify the mechanisms by which insulin may influence Na transport, some of the basal characteristics of this process were investigated. Since the uptake of 22Na into the space not available to [14C]sucrose had reached an almost constant level within 60 min, this interval of time was chosen as an appropriate loading period for studies of 22Na-efflux. Following the acute exposure to ouabain (10-3M), the rate coefficient of 22Nawashout (i.e. the fraction of 22Na lost from the muscles per minute) was decreased by two thirds within 20 min. Assuming that the cellular pool from which 22Na is transported remains constant within this interval of
INSULIN AND Na-K TRANSPORT IN MUSCLE
25 time, it could be calculated that the ouabain-suppressible component of 22Na-efflux corresponds to 0-29 /Zmole/g tissue wet weight per minute. In the presence of ouabain the pool size is continuously increasing (Fig. 1). However, as can be seen from Fig. 2 and Table 1, the rate coefficient of 22Na wash-out remains constant for an appreciable length of time and there seems to be a linear relationship between the 22Na-efflux and the Na concentration in the pool from which the ion is transported. TABLE 1. The effect of ouabain and of K on the rate coefficient and the rate of 22Na efflux from rat soleus muscle Rate of 22Na taken up 22Nainto the space efflux not available to (jumole/ [14C]sucrose g wet wt x per Rate coefficient of (,cumole/g minute) wet wt.) 22Na-washout Experimental conditions 0-42 7.5 ± 1.2 (5) 0-055 + 0-002 (7) 120 min in Krebs-Ringer without additions 0-59 0-015+ 0-001 (3) 36-1+ 0-2 (3) 120 min in Krebs-Ringer 0-017 + 0-001 (3) containing 10-3M ouabain 0-72 44.7 ± 2-9 (4) 240 min in Krebs-Ringer 0-016±+ 0-001 (3) 0-016 + 0-001 (3) containing 10-3M ouabain 0-042 + 0-002 (3) 17-4+ 2-7 (6) 0-79 130 min in K-free Krebs-Ringer 0-049 + 0-004 (3) 0-092 + 0-005 (3) 1-52 17-4+ 2-7 (6) 120 min in K-free Krebs-Ringer 0-083 + 0-005 (3) + 10 xnin in Krebs-Ringer containing 6 mM-K Experimental conditions as described in the legends to Figs. 1 and 2. The amount of 22Na taken up into the space not available to [14C]sucrose was determined in separate series of experiments. The rate of 22Na efflux was calculated by multiplying these values with the fraction of 22Na lost per minute in wash-out experiments. The results are given as mean + S.E. of mean with the number of observations in brackets.
When the muscles had been loaded with Na by incubation in a K-free buffer, the re-establishment of the normal extracellular concentration of K (5.9 mM) led to a prompt and marked rise in 22Na-efflux (Table 1). This effect was completely suppressed when the experiment was performed in the presence of ouabain (10-3 M). When all Na in the wash-out medium was replaced by an equivalent amount of Li, the rate coefficient of 22Na-wash-out was rapidly decreased to a level not very different from that obtained in the presence of ouabain. The addition of ouabain did not produce any significant further inhibition (data not presented). When the wash-out was performed at 1-5' C, the rate coefficient was reduced to around a tenth of the control level. It should be noted that even at this low
T. CLAUSEN AND P. G. KOHN temperature, ouabain produced a further reduction (44 %) in the rate of 22Na wash-out (see also Fig. 5). Fasting for 24 hr produced no significant change in the rate coefficient of 22Na release and the addition of glucose (5 mM) or pyruvate (1 mM) had no immediate effect, neither in muscles obtained from fed nor from fasted animals. In several series of experiments, it was found that the 26
2,4-Dinitrophenol (0-1 mM 0 06
i"
-
i
E~~~ C
E 004 0.
z
0- 0.02 0
4
V
0*00
20
80 60 40 Duration of wash-out (min)
I 100
Fig. 3. The effect of 2,4-dinitrophenol on 22Na release. Experimental conditions as described in the legend to Fig. 2. At the arrow, 2,4-dinitrophenol (0.1 mm) was added to the wash-out medium. controls 0-0; 2,4-dinitrophenol (0.1 mM), *-; ouabain (10-3M), A-A; ouabain (10-3M) and 2,4-dinitrophenol (0.1 mM) A-A. The fraction of 22Na released per minute was calculated as previously described (Clausen, 1969). Each point represents the mean of four observations with bars denoting s.E. of mean.
omission of pyruvate caused no significant change in the basal rate of 22Na-efflux or its response to ouabain, K, Li, 2,4-dinitrophenol or insulin. When glucose (10 mM) was present during both the loading and the washout, the rate coefficient of muscles from fed rats was increased by 1 2 1 %
INSULIN AND Na-K TRANSPORT IN MUSCLE 27 (P < 0-025). These results indicate that 22Na efflux is not markedly dependent on the availability of metabolizable substrate in the incubation medium. The addition of cyanide (2 mm) or the total replacement of 02 by N2 in the mixture used for gassing gave little or no reduction in the rate coefficient of 22Na efflux. Insulin
0.10
(100 mu./ml.)
E~~~~~ 0-08 0-08
a-0 6 , ,\J /J6
0
0-0
tU 004 Z\ 0 C
0
0 02.
-
U
LL.
0*00I
10
l
l
l
20 30 40 50 60 Duration of wash-out (min)
70
Fig. 4. The effect of insulin on 22Na release. Experimental conditions as described in the legend to Fig. 2. At the arrow, insulin (100 mu./ml.) was added to the wash-out medium. Controls, 0-0; insulin, @-O; ouabain (10-3M), V-V; ouabain (10-3M) and insulin, V-V. Each point represents the mean of three observations with bars denoting s.E. of mean.
In contrast, 2,4-dinitrophenol was a very potent inhibitor of 22Na efflux. This effect could be detected at concentrations down to 106M and at 5 X 10-4M the inhibition was significantly greater than that exerted by ouabain (10-3M). From Fig. 3 it can be seen that even in the presence of ouabain (10-3M), 2,4-dinitrophenol (10-4M) produced a significant decrease in 22Na-efflux (P < 0-001). On the basis of this characterization of 22Na release, we have made an attempt to analyse in more detail the mechanism by which insulin influences Na-K transport in soleus muscles. The immediate effects of the hormone on 22Na transport have been investigated in the absence or in the presence of the factors tested above. Fig. 4 shows the changes in the time course of 22Na release produced by
T. CLAUSEN AND P. G. KOHN
28
TABLE 2. A, the effect of insulin on 22Na-release Fraction of 22Na released per minute
Experimental conditions No additions
Ouabain (10-3M) All K replaced by Na.
Without insulin 0-045 + 0-003 (3) 0-016+0-001 (3) 0-012+0-0001 (3)
With insulin 0-081 ± 0-005 (3) 0-024+0-001 (3) 0-020+ 0001 (3)
P < 0-005 < 0-005 0-05 >0-05 < 0-01
Ouabain (I10-3M) All Na replaced by Li All Na replaced by Li.
Ouabain (10-3M) All Na replaced by Tris. Ouabain (10-3 M) All Ca replaced by Na. EGTA 0-5 mm, Ouabain (10-3M) All Ca and Mg replaced by Na. EGTA (0-5 mm). Ouabain (10-3M) Tetracaine (0.5 nBM) Phlorizin (5 mm) 2,4-Dinitrophenol 0-005 mM 0-020 mM 0-050 mm 0-50 mM Bovine serum albumin
(3) (3) (3) (3) (3)
(2)
(3) (3) (3) (3) (3)
(0-1% ) Experimental conditions as described in the legend to Fig. 2. The fraction of 22Naradioactivity lost per minute as determined in the interval from 50 to 60 min after the onset of wash-out is presented with S.E. of mean and the number of observations in brackets. Insulin, when added, was present from 40 min after the start of washout. For each experimental condition, values obtained in the absence and the presence of insulin (100 mu/ml.) within the same experiment are given B, effect of insulin on 22Na-release Fraction of 22Na released per minute
Experimental conditions Without ouabain Control Insulin (0-1 mu./ml.) Insulin (1-0 mu./ml.) Insulin (5.0 mu./ml.)
0-054+0-001 0-060 + 0-001 0-065 + 0-002 0-076 ± 0-004
(6) (6) (5) (3)
With ouabain (10-3M) 0-0165 + 0-0003 (6)
J. Physiol. (1977), 265, pp. 19-42 With 6 text-ftgure8 Printed in Great Britain
THE EFFECT OF INSULIN ON THE TRANSPORT OF SODIUM AND POTASSIUM IN RAT SOLEUS MUSCLE
BY TORBEN CLAUSEN AND PETER GEORGE KOHN From the Institute of Physiology, University of Arhus, Universitet8parken, DK-8000 Arhus C, Denmark and the Department of Physiology, University of Sheffield, Sheffield S10 2TN
(Received 19 December 1975) SUMMARY
1. The action of insulin on the transport and the distribution of Na and K has been studied in rat soleus muscles incubated at 300 C in glucose-free Krebs-Ringer bicarbonate buffer. 2. Measurements of the uptake and the wash-out of 22Na indicate that the muscles contain an intracellular pool of Na available for transport which is confined to the water space not available to sucrose. Ouabain (10-4-10-3M) inhibited 22Na efflux by 69 % (0.287 #tmole/g tissue wet weight per minute) and 42K-influx by 40 % (0.196 gmole/g tissue wet weight per minute). When all extracellular Na was replaced by Li, both 22Na-efflux and 42K-influx were inhibited to about the same extent and ouabain produced very little further inhibition. 2,4-dinitrophenol decreased the ouabain-resistant component of 22Na-efflux by 39 %, 3. Insulin (from 0.1 to 100 mu./ml.) increased the rate coefficient of 22Na-efflux by from 11 to 46 % within 15 min. In the presence of ouabain (10-3M), the same relative increase was obtained, indicating that the hormone stimulates the glycoside-sensitive and the glycoside-insensitive Na transport to a similar extent. The effect of insulin on 22Na-efflux was not abolished by tetracaine (0 5 x 10-3M), phlorizin (0.5 x 10-2M) or by the substitution of Na, K, Mg or Ca. In the presence of 2,4-dinitrophenol (0.5 X 10-4M) or at temperatures below 150 C, the hormone produced no detectable change in 22Na-efflux. 4. Insulin increased 42K-influx from 0*525 to 0 664 ,umole/g tissue wet weight per minute. This effect was entirely blocked by ouabain but not by tetracaine. Insulin produced a 14 % transient decrease in 42K-efflux. 5. The continued exposure to insulin led to a new steady state, in which the intracellular Na pool was decreased from around 10 to around 5,almole/g tissue wet weight and the K content increased by an equivalent amount. In the presence of ouabain or at low extracellular concentrations of K,
T. CLAUSEN AND P. G. KOHN 20 insulin increased the rate of 22Na-influx by around 35 %. This effect was blocked by 2,4-dinitrophenol but not by tetracaine. 6. It is concluded that insulin stimulates the active coupled transport of Na and K, possibly by increasing the relative Na-affinity of the system mediating this process. INTRODUCTION
Several studies with isolated preparations of muscle tissue indicate that the classical hypokalaemic effect of insulin (Harrop & Benedict, 1924) can for a major part be accounted for as the result of increased net accumulation of K in the cytoplasm of muscle cells (Kamminga, Willebrands, Groen & Blickman, 1950; Zierler, 1960; Creese & Northover, 1961; Gourley, 1965). It has repeatedly been demonstrated that this is associated with a considerable decrease in the intracellular Na concentration (Creese & Northover, 1961; Kernan, 1962; Moore, 1973; Zierler, Rogus & Hazlewood, 1966), and the observation that insulin stimulated the efflux of isotopic Na (Creese, 1968; Moore, 1973) suggests that the effect of the hormone on the distribution of Na and K is the result of an activation of the system mediating the active coupled transport of Na and K (Skou, 1965). Indeed, cardiac glycosides were found to produce almost complete (Moore, 1973) or partial suppression (Grinstein & Erlij, 1974) of the insulininduced increase in 22Na efflux from frog muscles and, recently, insulin was reported to augment the activity of Na-K-activated ATPase isolated from rat muscle (Brodal, Jebens, Oy & Iversen, 1974) and frog muscle (Gavryck, Moore & Thompson, 1975). However, in the extensor digitorum longus muscle of the rat, the effect of insulin on Na extrusion was reported to be resistant to ouabain (Zierler, 1966), and in a detailed study of Na-K-activated ATPase isolated from rat muscle, it was not possible to detect any significant effect of insulin (Rogus, Price & Zierler, 1969). These disagreements together with the fact that little information is available about the acute effects of insulin on the transport of Na and K in mammalian skeletal muscle prompted the present investigation, which at the same time represents an attempt to establish some basic parameters of the movements of these ions in peripheral muscle. Soleus muscles have recently been introduced for the study of insulin action (Chaudry & Gould, 1969) and measurements of the fluxes of hexoses and ions indicate that this preparation is suitable for the characterization of factors controlling the transport properties of the plasma membrane (Kohn & Clausen, 1971; Clausen & Hansen, 1974; Clausen, Elbrink & Dahl-Hansen, 1975).
INSULIN AND Na-K TRANSPORT IN MUSCLE
21
METHODS
Experimental procedures. The procedures for the preparation and incubation of isolated soleus muscles have been described in detail elsewhere (Kohn & Clausen, 1971). All the rats were weaned for at least 6 days on to a diet of laboratory pellets and, except when indicated, only fed animals weighing from 60 to 70 g were used. These yielded muscles weighing around 30 mg. The incubations took place in centrifuge tubes containing from 1-5 to 4 ml. buffer which was kept agitated by continuous bubbling with an appropriate gas mixture. Unless otherwise indicated, all experiments were performed at 300 C using Krebs-Ringer bicarbonate buffer (Cohen, 1951) containing 1 mm sodium pyruvate and equilibrated to pH 7-35 by gassing with a mixture of O2 (95%) and CO2 (5%). Tris-buffers were prepared by replacing all the NaHCO3 by isosmotic amounts of Tris-(hydroxymethyl)-aminomethane titrated to pH 7-35 with HCl. In this case, gassing was performed using 100% 02. In some experiments, NaCl was replaced by isosmotic amounts of LiCl or Tris chloride (pH 7.35). A Na-free Li-bicarbonate buffer was prepared by replacing NaHCO3 by LiHCO3 and Na pyruvate by Li pyruvate. The measurements of the uptake and the wash-out of isotopic Na and K were performed as described in our studies of the hexose transport system in this muscle (Kohn & Clausen, 1971, 1972) and in a brief preliminary report (Dahl-Hansen & Clausen, 1973). Further details are given, where appropriate, in the results section. 22Na- and 42K-radioactivity was in most cases determined by gamma-spectrometry using aliquots of the incubation media and trichloroacetic acid extracts of the muscles (Kohn & Clausen, 1971). In some experiments, the amount of 22Na and ['4C]sucrose taken up by the muscles was determined using a liquid scintillation spectrometer with channels adjusted so as to allow the measurements of 14C- and 22Na-radioactivity in the same tissue extract. For this purpose, a Triton-X-100/ toluene scintillation mixture was used (Kohn & Clausen, 1971). The Na and K contents were determined by flame photometry of the clear supernatant obtained by centrifugation (at 2000 g for 10 min) of trichloroacetic acid extracts. A series of standards containing trichloroacetic acid, Na and K in the same concentration range as the tissue extracts were used for the calibration. Tissue dry weight was determined following 2 hr of desiccation at 1100 C. Chemicals and isotope8. All chemicals used (except the toluene and the Triton-X100) were of analytical grade. Bovine serum albumin was purchased from Sigma and used after 24 hr of dialysis against distilled water in the cold. 22Na (3000 mc/mmole) and [U-14C]sucrose (400 mc/mmole) were obtained from the Radiochemical Centre, Amersham, Bucks and 42K (100 mc/mmole) from the Danish Atomic Energy Commission, Isotope Laboratory, Riso; mono-component pork insulin (purified by chromatography, 25 i.u./mg) was a gift from the Novo Research Laboratories, Copenhagen. RESULTS
Experiments with 22Na The effect of insulin on the transport of Na and its distribution among cellular compartments was assessed in four ways: (1) measurements of the uptake of 22Na in the tissue space not available to [14C]sucrose; (2) analysis of efflux curves for 22Na; (3) determinations of the acute changes in the rate coefficient for the efflux of 22Na; (4) measurements of the amount of 22Na taken up during the early phase of exposure to this isotope.
T. CLAUSEN AND P. G. KOHN Fig. 1 shows the effects of insulin (100 mu./ml.) and ouabain (10-3M) on the uptake of 22Na into the space not available to [14C]sucrose. In the absence of ouabain, the amount of isotopic sodium taken up in this com partment approaches a steady level after between 60 and 120 min and 22
50 r
4' 110
-o0
40
6 0 1530 0
0
4. W
) 0 IU
S0. 020'I M
.0 0. C
z34,0. 10 I.-
0
0
01
L0
L
120 60 Duration of incubation (min) Fig. 1. Effect of insulin and ouabain on 22Na uptake. Isolated rat soleus muscles were incubated in Krebs-Ringer bicarbonate buffer containing 22Na (0.2 psc/ml.) and 1 mM [U-_4C]sucrose (0-2 ,uc/ml.) without or with the additions indicated. The spaces available to the two isotopes were determined by liquid scintillation counting of a trichloroacetic acid extract of the muscles. The amount of 22Na taken up into the space not available to [U-14C]sucrose was calculated and expressed as #umole/g tissue wet weight with bars denoting 5.E. of mean. Each point represents the mean of from four to six observations. Control, 0-0; insulin (100 mu./ml.), V-V; ouabain (10-3M), 0-A; ouabain (10-AM) and insulin (100 mu./ml.),
-V .
insulin induces a decrease of around 50 % in this steady-state level. In the presence of ouabain, the 22Na uptake was markedly increased, which is consistent with an inhibition of active Na-expulsion. Under these conditions, insulin produced no detectable change during the first 60 min of
INSULIN AND Na-K TRANSPORT IN MUSCLE
23 incubation but, after 120 min, a significant rise in uptake (P < 0.02) was found. For comparison, the effects of insulin and ouabain were assessed in a series of wash-out experiments performed under similar conditions. Fig. 2 shows the time course of 22Na release from muscles which were loaded and 100 385
1-:
29-0 4,
0
10
50
8-9
44A_~ K~4
K1_
._
E 0 (4
1*0
0 .0
05
zW(4
0.1
V
\'
Y
0*05
-
L
0
I
I
30 90 60 Duration of wash-out (min)
-
120
Fig. 2. Effect of insulin and ouabain on 22Na release. Isolated rat soleus muscles were incubated in Krebs-Ringer bicarbonate buffer containing 22Na (6-7 ,uc./ml.). The wash-out of isotope was then followed by transferring the muscles through a series of tubes containing 3 ml. unlabelled buffer without or with the additions indicated. At the end of the wash-out, the amount of 22Na retained in the muscles was determined and the time course of the decrease in 22Na content was obtained by adding the amount of 22Na released into each tube. On the basis of the specific activity of the loading media, all results are expressed as molee 22Na per gram wet weight. Each point represents the mean of three to four observations with bars denoting S.E. of mean. The straight portions of the curves have been extrapolated back to the onset of wash-out and the intercept values are indicated. Muscles loaded for 60 min and washed for 70 min in buffer without (0-0) or with insulin (100 mu./ml.) (V-V ). Muscles loaded for 120 min in the presence of ouabain (10-3M) and washed for 100 min in buffer containing ouabain (10-3M) without (-*) or with insulin (100 mu./ml.)
(v-v).
T. CLAUSEN AND P. G. KOHN 24 washed in the absence or in the continued presence of insulin (100 mu./ml.), without or with ouabain (10-3M). Under all the conditions tested, the 22Na content shows a rapid initial decrease followed by a much slower and approximately exponential fall. In the untreated control muscles, the zero time intercept of the straight portion of the curve corresponds to 8-9 ,umole/g wet weight. The muscles which had been exposed to insulin (100 mu./ml.) during both the loading and the wash-out period yielded an efflux curve whose linear portion had the same slope but the intercept corresponded to 4-4 gnmole/g wet weight, which is 51 % lower than that of the controls. Neither of these values are significantly different from those obtained in the measurements of the amount of 22Na taken up into the space not available to [14C]sucrose within a period of 60 min (Fig. 1). When the muscles were loaded for 120 min and washed in the continued presence of ouabain (10-3M), the linear portion of the efflux curve could be extrapolated back to an intercept value of 29*0, lmole/g wet weight. Under these conditions, the addition of insulin (100 mu./ml.) to both the loading and the wash-out media gave a slightly steeper efflux curve with a somewhat higher intercept value (38.5 lsmole/g wet weight). Again, there is fairly good agreement with the figures obtained in the uptake experiments and it seems reasonable to assume that both in the absence and in the presence of ouabain, the continued exposure to insulin leads to a change in the Na content of a cellular compartment. In the absence of ouabain, it is possible to obtain a steady state where this Na pool is constant and located in a space not available to a compound (sucrose) which seems not to penetrate the plasma membrane at an appreciable rate (Clausen & Hansen, 1974). The wash-out experiments are compatible with the view that the linear portion of the 22Na efflux curves represents the transport of isotopic Na out of the same pool. In the presence of ouabain, the situation is more complex since the size of the Na pool is con-
tinuously changing. In the following, an attempt is made to account for the above-mentioned changes in terms of alterations in the relative rates of efflux and influx of 22Na. In order to identify the mechanisms by which insulin may influence Na transport, some of the basal characteristics of this process were investigated. Since the uptake of 22Na into the space not available to [14C]sucrose had reached an almost constant level within 60 min, this interval of time was chosen as an appropriate loading period for studies of 22Na-efflux. Following the acute exposure to ouabain (10-3M), the rate coefficient of 22Nawashout (i.e. the fraction of 22Na lost from the muscles per minute) was decreased by two thirds within 20 min. Assuming that the cellular pool from which 22Na is transported remains constant within this interval of
INSULIN AND Na-K TRANSPORT IN MUSCLE
25 time, it could be calculated that the ouabain-suppressible component of 22Na-efflux corresponds to 0-29 /Zmole/g tissue wet weight per minute. In the presence of ouabain the pool size is continuously increasing (Fig. 1). However, as can be seen from Fig. 2 and Table 1, the rate coefficient of 22Na wash-out remains constant for an appreciable length of time and there seems to be a linear relationship between the 22Na-efflux and the Na concentration in the pool from which the ion is transported. TABLE 1. The effect of ouabain and of K on the rate coefficient and the rate of 22Na efflux from rat soleus muscle Rate of 22Na taken up 22Nainto the space efflux not available to (jumole/ [14C]sucrose g wet wt x per Rate coefficient of (,cumole/g minute) wet wt.) 22Na-washout Experimental conditions 0-42 7.5 ± 1.2 (5) 0-055 + 0-002 (7) 120 min in Krebs-Ringer without additions 0-59 0-015+ 0-001 (3) 36-1+ 0-2 (3) 120 min in Krebs-Ringer 0-017 + 0-001 (3) containing 10-3M ouabain 0-72 44.7 ± 2-9 (4) 240 min in Krebs-Ringer 0-016±+ 0-001 (3) 0-016 + 0-001 (3) containing 10-3M ouabain 0-042 + 0-002 (3) 17-4+ 2-7 (6) 0-79 130 min in K-free Krebs-Ringer 0-049 + 0-004 (3) 0-092 + 0-005 (3) 1-52 17-4+ 2-7 (6) 120 min in K-free Krebs-Ringer 0-083 + 0-005 (3) + 10 xnin in Krebs-Ringer containing 6 mM-K Experimental conditions as described in the legends to Figs. 1 and 2. The amount of 22Na taken up into the space not available to [14C]sucrose was determined in separate series of experiments. The rate of 22Na efflux was calculated by multiplying these values with the fraction of 22Na lost per minute in wash-out experiments. The results are given as mean + S.E. of mean with the number of observations in brackets.
When the muscles had been loaded with Na by incubation in a K-free buffer, the re-establishment of the normal extracellular concentration of K (5.9 mM) led to a prompt and marked rise in 22Na-efflux (Table 1). This effect was completely suppressed when the experiment was performed in the presence of ouabain (10-3 M). When all Na in the wash-out medium was replaced by an equivalent amount of Li, the rate coefficient of 22Na-wash-out was rapidly decreased to a level not very different from that obtained in the presence of ouabain. The addition of ouabain did not produce any significant further inhibition (data not presented). When the wash-out was performed at 1-5' C, the rate coefficient was reduced to around a tenth of the control level. It should be noted that even at this low
T. CLAUSEN AND P. G. KOHN temperature, ouabain produced a further reduction (44 %) in the rate of 22Na wash-out (see also Fig. 5). Fasting for 24 hr produced no significant change in the rate coefficient of 22Na release and the addition of glucose (5 mM) or pyruvate (1 mM) had no immediate effect, neither in muscles obtained from fed nor from fasted animals. In several series of experiments, it was found that the 26
2,4-Dinitrophenol (0-1 mM 0 06
i"
-
i
E~~~ C
E 004 0.
z
0- 0.02 0
4
V
0*00
20
80 60 40 Duration of wash-out (min)
I 100
Fig. 3. The effect of 2,4-dinitrophenol on 22Na release. Experimental conditions as described in the legend to Fig. 2. At the arrow, 2,4-dinitrophenol (0.1 mm) was added to the wash-out medium. controls 0-0; 2,4-dinitrophenol (0.1 mM), *-; ouabain (10-3M), A-A; ouabain (10-3M) and 2,4-dinitrophenol (0.1 mM) A-A. The fraction of 22Na released per minute was calculated as previously described (Clausen, 1969). Each point represents the mean of four observations with bars denoting s.E. of mean.
omission of pyruvate caused no significant change in the basal rate of 22Na-efflux or its response to ouabain, K, Li, 2,4-dinitrophenol or insulin. When glucose (10 mM) was present during both the loading and the washout, the rate coefficient of muscles from fed rats was increased by 1 2 1 %
INSULIN AND Na-K TRANSPORT IN MUSCLE 27 (P < 0-025). These results indicate that 22Na efflux is not markedly dependent on the availability of metabolizable substrate in the incubation medium. The addition of cyanide (2 mm) or the total replacement of 02 by N2 in the mixture used for gassing gave little or no reduction in the rate coefficient of 22Na efflux. Insulin
0.10
(100 mu./ml.)
E~~~~~ 0-08 0-08
a-0 6 , ,\J /J6
0
0-0
tU 004 Z\ 0 C
0
0 02.
-
U
LL.
0*00I
10
l
l
l
20 30 40 50 60 Duration of wash-out (min)
70
Fig. 4. The effect of insulin on 22Na release. Experimental conditions as described in the legend to Fig. 2. At the arrow, insulin (100 mu./ml.) was added to the wash-out medium. Controls, 0-0; insulin, @-O; ouabain (10-3M), V-V; ouabain (10-3M) and insulin, V-V. Each point represents the mean of three observations with bars denoting s.E. of mean.
In contrast, 2,4-dinitrophenol was a very potent inhibitor of 22Na efflux. This effect could be detected at concentrations down to 106M and at 5 X 10-4M the inhibition was significantly greater than that exerted by ouabain (10-3M). From Fig. 3 it can be seen that even in the presence of ouabain (10-3M), 2,4-dinitrophenol (10-4M) produced a significant decrease in 22Na-efflux (P < 0-001). On the basis of this characterization of 22Na release, we have made an attempt to analyse in more detail the mechanism by which insulin influences Na-K transport in soleus muscles. The immediate effects of the hormone on 22Na transport have been investigated in the absence or in the presence of the factors tested above. Fig. 4 shows the changes in the time course of 22Na release produced by
T. CLAUSEN AND P. G. KOHN
28
TABLE 2. A, the effect of insulin on 22Na-release Fraction of 22Na released per minute
Experimental conditions No additions
Ouabain (10-3M) All K replaced by Na.
Without insulin 0-045 + 0-003 (3) 0-016+0-001 (3) 0-012+0-0001 (3)
With insulin 0-081 ± 0-005 (3) 0-024+0-001 (3) 0-020+ 0001 (3)
P < 0-005 < 0-005 0-05 >0-05 < 0-01
Ouabain (I10-3M) All Na replaced by Li All Na replaced by Li.
Ouabain (10-3M) All Na replaced by Tris. Ouabain (10-3 M) All Ca replaced by Na. EGTA 0-5 mm, Ouabain (10-3M) All Ca and Mg replaced by Na. EGTA (0-5 mm). Ouabain (10-3M) Tetracaine (0.5 nBM) Phlorizin (5 mm) 2,4-Dinitrophenol 0-005 mM 0-020 mM 0-050 mm 0-50 mM Bovine serum albumin
(3) (3) (3) (3) (3)
(2)
(3) (3) (3) (3) (3)
(0-1% ) Experimental conditions as described in the legend to Fig. 2. The fraction of 22Naradioactivity lost per minute as determined in the interval from 50 to 60 min after the onset of wash-out is presented with S.E. of mean and the number of observations in brackets. Insulin, when added, was present from 40 min after the start of washout. For each experimental condition, values obtained in the absence and the presence of insulin (100 mu/ml.) within the same experiment are given B, effect of insulin on 22Na-release Fraction of 22Na released per minute
Experimental conditions Without ouabain Control Insulin (0-1 mu./ml.) Insulin (1-0 mu./ml.) Insulin (5.0 mu./ml.)
0-054+0-001 0-060 + 0-001 0-065 + 0-002 0-076 ± 0-004
(6) (6) (5) (3)
With ouabain (10-3M) 0-0165 + 0-0003 (6)
