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Effects of chronic ouabain treatment on [3H]ouabain binding sites and electrogenic component of membrane potential in cultured rat myotubes
Brain Research, 347 (1985) 121-123 Elsevier
12 l
BRE 21143
Effects of chronic ouabain treatment on [3H]ouabain binding sites and electrogenic component of membrane potential in cultured rat myotubes CHAYA BRODIE and SANFORD R. SAMPSON Department of Life Sciences. Bar-llan University, Ramat-Gan 52 100 (Israeli
(Accepted June 1lth, 1985) Key words: Na,K-ATPase - - ouabain binding site - - membrane potential - - cultured skeletal myotube
The effects of incubation of cultured rat skeletal myotubes in ouabain were studied on the number of [3H]ouabain binding sites and electrogenic component of membrane potential. Ouabain treatment for 2-6 days increased the number of binding sites, resting membrane potential (Era) and the ouabain-sensitive component of E min the muscle cells. The findings strengthen the idea that Na,K-ATPase has an important role in regulation of E m in cultured skeletal muscle and suggest that Na-K pump inhibition during development may be a regulatory mechanism for cellular synthesis of Na,K-ATPase.
N a , K - A T P a s e plays an i m p o r t a n t role in regulating the excitability of skeletal muscle by both maintaining concentration gradients for Na ÷- and K+-ions and contributing to the m e m b r a n e potential (Em) through electrogenic Na/K p u m p i n g 1.3,4,11,12. Little is known about regulation of synthesis of this enzyme. While potassium depletion causes parallel decreases in the n u m b e r of o u a b a i n binding ( N a - p u m p ) sites and in active N a / K - t r a n s p o r t in rat skeletal muscle 5,0, it is not certain whether or not this represents an important regulatory mechanism for enzyme synthesis, or is merely a reflection of a general decrease in protein synthesis. This study examines the possibility of cellular induction of T'-,'a,K-ATPase by incubation of cultured rat skeletal myotubes in ouabain, a specific inhibitor, as has been r e p o r t e d for other cultured cells~,.~. Experiments were p e r f o r m e d on primary cultures of skeletal m y o t u b e s p r e p a r e d from the limbs of neonatal ( 1 - 2 - d a y - o l d ) rats, as already described1,2-4.] i. Twenty-four hours after cells were p l a t e d onto 35-mm plastic dishes (0.8 x 106 cells/ml), the growth medium (83% D u l b e c c o ' s M E M , 15% horse serum, 2% chick e m b r y o extract) was replaced with fresh solution. To halve the cultures, ouabain was a d d e d to a
final concentration of 10 -5 M. Both control and ouabain-containing media were replaced every 3 days thereafter. F o r electrophysiological studies, the medium was changed to p h o s p h a t e - b u f f e r e d saline (pH 7.31-7.35), and recordings were made with fibrefilled glass micropipettes containing 2.8 M KCF.3. The amount of [3H]]ouabain b o u n d by cultured myotubes was d e t e r m i n e d by a modification of the method of V a n d e n b u r g h and Kaufman ~3. as described previously 3,4. The n u m b e r of N a / K - p u m p sites on cultured myotubes increases after fusion to reach maximum values by 6 - 7 days in culture 3. Fig. 1 shows that there was a 2 0 - 3 0 % increase in the n u m b e r of [3H]ouabain binding sites in o u a b a i n - t r e a t e d myotubes over that in control, untreated cells from sister cultures. This effect was detectable by 2 days after addition of ouabain and persisted throughout the time of culture (8 days in vitro). In preliminary experiments, we determined that fibroblasts derived from the muscle cultures bind less than 10% of the ouabain bound by cultures of rat skeletal myotubes. In addition, we found in other experiments that muscle cultures, in which fibroblasts were eliminated by t r e a t m e n t with cytosine arabinoside, did not differ from control cultures
Correspondence: S.R. Sampson, Department of Life Sciences, Bar-Ilan University. Ramat-Gan 52 l/)0, Isracl.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
122 with regard to the amount of ouabain bound (Brodie and Sampson, in preparation). We conclude, therefore, that the increase in ouabain binding as a result of prolonged incubation in ouabain is due mainly, if not entirely, to an increase in number of Na/K-pump sites on the myotubes. The resting E m of cultured skeletal muscle increases with age, the increase being most dramatic following fusion (day 2-3), and reaches a plateau by day 6 in vitro 1,3,4,11. Myotubes incubated in the presence of ouabain had a higher resting E m than untreated sister cultures (Fig. 2A). The difference in E m also was detectable by 2 days after addition of ouabain and persisted for the duration of the culture period. At any given day in culture, the difference was accounted for by the electrogenic component of E m (Fig. 2B), addition of ouabain (10 -3 M) having resulted in a fall in E mto about -60 mV within 5 min in each case 3,4. Analysis of the dose-effect relation of ouabain on E m of control and ouabain-treated rat myotubes showed that there was a parallel shift to the right as a result of incubation with ouabain (Fig. 3). Thus, the dose of ouabain to reduce the electrogenic component of E m by 50% was 1.2 x 10-5 M in control cells and 2.5 x 10-5 M in ouabain-treated cultures. In addition, the maximum effect of ouabain was obtained at concentrations of 10-4 M and 10-3 M for control
and ouabain-treated cells, respectively. This study is consistent with other work showing that prolonged exposure of cultured cells to ouabain leads to an increase in number of Na/K-pump sites. Thus, Girardi (heart) and Hela cells 2 and chick heart cells 8 in culture have been reported to display an increase in ouabain binding similar to that reported
-100,
A
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-8C >
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OC----~,, Days in Culture
30
B
25 20
@
18
-
Q,
20
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16
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o
0 Ca 0
"6
~o =
10 8
UJ
~ 6 ~o 4
/f
J
g
~ 2
o
;
~
~
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3
4
5
6
7
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Days in Culture Days in Culture
Fig. 1. [3HJouabain binding as a function of age in control (O) and ouabain-treated ( 0 ) rat skeletal myotubes in culture. Ouabain was added to the cultures (final concentration = 10 5 M) on day 2, and m e a s u r e m e n t s of b o u n d ouabain were made daily beginning 24 h later. The data are from 1 of 3 experiments with qualitatively similar results. Each point represents the mean of 3 different samples for each day.
Fig. 2, Effects of ouabain treatment on E m (A) and its electrogenic c o m p o n e n t (B) in cultured rat skeletal muscle. A: values for E m in control (©) and ouabain-treated ( 0 ) cultures, each point being the mean (_+ S.E.M.) of 9 m e a s u r e m e n t s of E m in each of 2 dishes on the day of recording. B: plotted values representing the electrogenic c o m p o n e n t of E,,, in control (o) and ouabain-treated (C)) myotubes. Conditions of ouabain treatment as in Fig. I.
123 thus, represents the first report of cellular regulation of amount of Na,K-ATPase in skeletal muscle. In the previous reports increased ouabain binding was accompanied by increased Na/K-pump activity when ouabain was removed. In the experiments reported here, we found that the elevated number of pump sites was accompanied by an increase in the
2
electrogenic component of E m, indicative of a higher level of Na/K pumping. An elevated level of N a - K exchange by individual pump sites may occur concomitant with the increase in the number of pump units, and contributes to the electrogenic component. In any case, the results strengthen the idea that Na,K-ATPase has an important role in the regulation of E m in cultured myotubes~& In addition, the results indicate that Na-pump inhibition during development and maturation, as might occur by endogenous ouabain-like compounds<7, may be a regulatory mechanism for cellular synthesis of Na.K-ATPase.
Lo3 Ouabain-
M
Fig. 3. Dose-effect relation of ouabain on resting E mof control (©) and ouabain-treated (O) cultured rat myotubes. The maximum effect is defined on the depolarization produced within 5-10 rain by 10 3 M ouabain. Studies were done on 7-day-old myotubes. First, control E mwas measured (9 determinations in each of 2 dishes) and the ouabain added to the appropriate concentration and another set of measurements made. Each point was derived from the differences in mean E m before and after addition of ouabain.
h e r e . S k e l e t a l m u s c l e cells in c u l t u r e h a v e n o t h e r e t o -
We thank Asia Bak for technical assistance and
f o r e b e e n s t u d i e d in this r e g a r d . E f f e c t s o f p o t a s s i u m
Bluma Lederhendler for typing the manuscript. The
d e p l e t i o n h a v e b e e n s t u d i e d a n d b e e n f o u n d to r e s u l t
s t u d y was s u p p o r t e d by f u n d s f r o m t h e R e s e a r c h A u -
in a d e c r e a s e in n u m b e r o f p u m p sites, b u t this s e e m s
t h o r i t y , B a r - I l a n U n i v e r s i t y a n d Y a d H a n a d i v , Tel-
to be d u e to a g e n e r a l m e t a b o l i c e f f e c t 5,9. T h i s s t u d y ,
A v i v , Israel.
1 Bannett, R.R., Sampson, R.R. and Shainberg, A., Influt_,nce of thyroid hormones on some electrophysiologieal properties of developing rat skeletal muscle cells in culture, Brain Research, 294 (1984) 75-82. 2 Boardman, L.J., Lamb, J.F. and McCall, D., Uptake of [~H]ouabain and Na pump turnover rates in cells cultured in ouabain, J. Physiol. (London), 225 (1972)619-635. 3 Brodie, C., Bak A. and Sampson, S.R., Dependence of Na,K-ATPase and electrogenic components of E m in cultured myotubes on cell fusion. Brain Research, 336 (1985) 384-386. 4 Brodie, C. and Sampson, S.R. (1985). Contribution ofelectrogenic Na.K-ATPase to resting membrane potential of cultured rat myotubes, Brain Research, in press. 5 Clauscn, T., Kjeldsen, K. and Norgaard, A., Effects of denervation on sodium, potassium and [3H]ouabain binding in muscles of normal and potassium-depleted rats, J. Physiol. (London), 345 ( 19831 123-134. 6 Fishman. M.C. (1979), Endogenous digitalis-like activity in mammalian brain, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 46f~1- 4663. 7 Flier, J.. Edwards, M.W., Daly, J.W. and Myers, C.W., Widespread occurrence in frogs and toads of skin c o r n -
8
9
10
11
12 13
pounds interacting with the ouabam site of Na,K-ATPasc, Science. 208 (19811) 5/13-,51/5. Kim, D., Marsh, J.D., Barry, W.H. and Smith, T.W., Effects of growth in low potassium medium or ouabain on membrane Na,K-ATPase. cation transport, and contractility in cultured chick heart cells, Circ'. Res.. 55 (1984) 39-48. Norgaard, A., Kjeldsen, K. and Clausen, T., Potassium depletion decreases the number of [3H]ouabain binding sites and active Na-K transport in skeletal muscle, Nature (London), 293 (1981) 739-741. Pollack, L.R., Tate, E.H. and Cook, J.S., Turnover and regulation of Na,K-ATPase in Hela cells. J. (ell. Physiol.. 106 (1981) 85-97. Sampson, SR..Bannett, R.R. and Shainberg, A., Effects of thyroxine on transmembrance resting potentials of skeletal muscle cells in culture. J. Neurosci. Res., 8 11982) 595-601. Thomas, R.C.. Electrogenic pump in ncrvc and muscle cells, Physiol. Rev., 52(1972)563-594. Vandenburgh, H.H. and Kaufman. S.. Stretch-induced growth of skeletal myotubes correlates with activation of the sodium pump, J. (?ell. Ph;'siol., 1119( 1981 ) 205 214.
12 l
BRE 21143
Effects of chronic ouabain treatment on [3H]ouabain binding sites and electrogenic component of membrane potential in cultured rat myotubes CHAYA BRODIE and SANFORD R. SAMPSON Department of Life Sciences. Bar-llan University, Ramat-Gan 52 100 (Israeli
(Accepted June 1lth, 1985) Key words: Na,K-ATPase - - ouabain binding site - - membrane potential - - cultured skeletal myotube
The effects of incubation of cultured rat skeletal myotubes in ouabain were studied on the number of [3H]ouabain binding sites and electrogenic component of membrane potential. Ouabain treatment for 2-6 days increased the number of binding sites, resting membrane potential (Era) and the ouabain-sensitive component of E min the muscle cells. The findings strengthen the idea that Na,K-ATPase has an important role in regulation of E m in cultured skeletal muscle and suggest that Na-K pump inhibition during development may be a regulatory mechanism for cellular synthesis of Na,K-ATPase.
N a , K - A T P a s e plays an i m p o r t a n t role in regulating the excitability of skeletal muscle by both maintaining concentration gradients for Na ÷- and K+-ions and contributing to the m e m b r a n e potential (Em) through electrogenic Na/K p u m p i n g 1.3,4,11,12. Little is known about regulation of synthesis of this enzyme. While potassium depletion causes parallel decreases in the n u m b e r of o u a b a i n binding ( N a - p u m p ) sites and in active N a / K - t r a n s p o r t in rat skeletal muscle 5,0, it is not certain whether or not this represents an important regulatory mechanism for enzyme synthesis, or is merely a reflection of a general decrease in protein synthesis. This study examines the possibility of cellular induction of T'-,'a,K-ATPase by incubation of cultured rat skeletal myotubes in ouabain, a specific inhibitor, as has been r e p o r t e d for other cultured cells~,.~. Experiments were p e r f o r m e d on primary cultures of skeletal m y o t u b e s p r e p a r e d from the limbs of neonatal ( 1 - 2 - d a y - o l d ) rats, as already described1,2-4.] i. Twenty-four hours after cells were p l a t e d onto 35-mm plastic dishes (0.8 x 106 cells/ml), the growth medium (83% D u l b e c c o ' s M E M , 15% horse serum, 2% chick e m b r y o extract) was replaced with fresh solution. To halve the cultures, ouabain was a d d e d to a
final concentration of 10 -5 M. Both control and ouabain-containing media were replaced every 3 days thereafter. F o r electrophysiological studies, the medium was changed to p h o s p h a t e - b u f f e r e d saline (pH 7.31-7.35), and recordings were made with fibrefilled glass micropipettes containing 2.8 M KCF.3. The amount of [3H]]ouabain b o u n d by cultured myotubes was d e t e r m i n e d by a modification of the method of V a n d e n b u r g h and Kaufman ~3. as described previously 3,4. The n u m b e r of N a / K - p u m p sites on cultured myotubes increases after fusion to reach maximum values by 6 - 7 days in culture 3. Fig. 1 shows that there was a 2 0 - 3 0 % increase in the n u m b e r of [3H]ouabain binding sites in o u a b a i n - t r e a t e d myotubes over that in control, untreated cells from sister cultures. This effect was detectable by 2 days after addition of ouabain and persisted throughout the time of culture (8 days in vitro). In preliminary experiments, we determined that fibroblasts derived from the muscle cultures bind less than 10% of the ouabain bound by cultures of rat skeletal myotubes. In addition, we found in other experiments that muscle cultures, in which fibroblasts were eliminated by t r e a t m e n t with cytosine arabinoside, did not differ from control cultures
Correspondence: S.R. Sampson, Department of Life Sciences, Bar-Ilan University. Ramat-Gan 52 l/)0, Isracl.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
122 with regard to the amount of ouabain bound (Brodie and Sampson, in preparation). We conclude, therefore, that the increase in ouabain binding as a result of prolonged incubation in ouabain is due mainly, if not entirely, to an increase in number of Na/K-pump sites on the myotubes. The resting E m of cultured skeletal muscle increases with age, the increase being most dramatic following fusion (day 2-3), and reaches a plateau by day 6 in vitro 1,3,4,11. Myotubes incubated in the presence of ouabain had a higher resting E m than untreated sister cultures (Fig. 2A). The difference in E m also was detectable by 2 days after addition of ouabain and persisted for the duration of the culture period. At any given day in culture, the difference was accounted for by the electrogenic component of E m (Fig. 2B), addition of ouabain (10 -3 M) having resulted in a fall in E mto about -60 mV within 5 min in each case 3,4. Analysis of the dose-effect relation of ouabain on E m of control and ouabain-treated rat myotubes showed that there was a parallel shift to the right as a result of incubation with ouabain (Fig. 3). Thus, the dose of ouabain to reduce the electrogenic component of E m by 50% was 1.2 x 10-5 M in control cells and 2.5 x 10-5 M in ouabain-treated cultures. In addition, the maximum effect of ouabain was obtained at concentrations of 10-4 M and 10-3 M for control
and ouabain-treated cells, respectively. This study is consistent with other work showing that prolonged exposure of cultured cells to ouabain leads to an increase in number of Na/K-pump sites. Thus, Girardi (heart) and Hela cells 2 and chick heart cells 8 in culture have been reported to display an increase in ouabain binding similar to that reported
-100,
A
_9o I
-8C >
E - 7C
OC----~,, Days in Culture
30
B
25 20
@
18
-
Q,
20
E
16
0
o
0 Ca 0
"6
~o =
10 8
UJ
~ 6 ~o 4
/f
J
g
~ 2
o
;
~
~
~
~
~
0 ---'¢
3
4
5
6
7
8
Days in Culture Days in Culture
Fig. 1. [3HJouabain binding as a function of age in control (O) and ouabain-treated ( 0 ) rat skeletal myotubes in culture. Ouabain was added to the cultures (final concentration = 10 5 M) on day 2, and m e a s u r e m e n t s of b o u n d ouabain were made daily beginning 24 h later. The data are from 1 of 3 experiments with qualitatively similar results. Each point represents the mean of 3 different samples for each day.
Fig. 2, Effects of ouabain treatment on E m (A) and its electrogenic c o m p o n e n t (B) in cultured rat skeletal muscle. A: values for E m in control (©) and ouabain-treated ( 0 ) cultures, each point being the mean (_+ S.E.M.) of 9 m e a s u r e m e n t s of E m in each of 2 dishes on the day of recording. B: plotted values representing the electrogenic c o m p o n e n t of E,,, in control (o) and ouabain-treated (C)) myotubes. Conditions of ouabain treatment as in Fig. I.
123 thus, represents the first report of cellular regulation of amount of Na,K-ATPase in skeletal muscle. In the previous reports increased ouabain binding was accompanied by increased Na/K-pump activity when ouabain was removed. In the experiments reported here, we found that the elevated number of pump sites was accompanied by an increase in the
2
electrogenic component of E m, indicative of a higher level of Na/K pumping. An elevated level of N a - K exchange by individual pump sites may occur concomitant with the increase in the number of pump units, and contributes to the electrogenic component. In any case, the results strengthen the idea that Na,K-ATPase has an important role in the regulation of E m in cultured myotubes~& In addition, the results indicate that Na-pump inhibition during development and maturation, as might occur by endogenous ouabain-like compounds<7, may be a regulatory mechanism for cellular synthesis of Na.K-ATPase.
Lo3 Ouabain-
M
Fig. 3. Dose-effect relation of ouabain on resting E mof control (©) and ouabain-treated (O) cultured rat myotubes. The maximum effect is defined on the depolarization produced within 5-10 rain by 10 3 M ouabain. Studies were done on 7-day-old myotubes. First, control E mwas measured (9 determinations in each of 2 dishes) and the ouabain added to the appropriate concentration and another set of measurements made. Each point was derived from the differences in mean E m before and after addition of ouabain.
h e r e . S k e l e t a l m u s c l e cells in c u l t u r e h a v e n o t h e r e t o -
We thank Asia Bak for technical assistance and
f o r e b e e n s t u d i e d in this r e g a r d . E f f e c t s o f p o t a s s i u m
Bluma Lederhendler for typing the manuscript. The
d e p l e t i o n h a v e b e e n s t u d i e d a n d b e e n f o u n d to r e s u l t
s t u d y was s u p p o r t e d by f u n d s f r o m t h e R e s e a r c h A u -
in a d e c r e a s e in n u m b e r o f p u m p sites, b u t this s e e m s
t h o r i t y , B a r - I l a n U n i v e r s i t y a n d Y a d H a n a d i v , Tel-
to be d u e to a g e n e r a l m e t a b o l i c e f f e c t 5,9. T h i s s t u d y ,
A v i v , Israel.
1 Bannett, R.R., Sampson, R.R. and Shainberg, A., Influt_,nce of thyroid hormones on some electrophysiologieal properties of developing rat skeletal muscle cells in culture, Brain Research, 294 (1984) 75-82. 2 Boardman, L.J., Lamb, J.F. and McCall, D., Uptake of [~H]ouabain and Na pump turnover rates in cells cultured in ouabain, J. Physiol. (London), 225 (1972)619-635. 3 Brodie, C., Bak A. and Sampson, S.R., Dependence of Na,K-ATPase and electrogenic components of E m in cultured myotubes on cell fusion. Brain Research, 336 (1985) 384-386. 4 Brodie, C. and Sampson, S.R. (1985). Contribution ofelectrogenic Na.K-ATPase to resting membrane potential of cultured rat myotubes, Brain Research, in press. 5 Clauscn, T., Kjeldsen, K. and Norgaard, A., Effects of denervation on sodium, potassium and [3H]ouabain binding in muscles of normal and potassium-depleted rats, J. Physiol. (London), 345 ( 19831 123-134. 6 Fishman. M.C. (1979), Endogenous digitalis-like activity in mammalian brain, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 46f~1- 4663. 7 Flier, J.. Edwards, M.W., Daly, J.W. and Myers, C.W., Widespread occurrence in frogs and toads of skin c o r n -
8
9
10
11
12 13
pounds interacting with the ouabam site of Na,K-ATPasc, Science. 208 (19811) 5/13-,51/5. Kim, D., Marsh, J.D., Barry, W.H. and Smith, T.W., Effects of growth in low potassium medium or ouabain on membrane Na,K-ATPase. cation transport, and contractility in cultured chick heart cells, Circ'. Res.. 55 (1984) 39-48. Norgaard, A., Kjeldsen, K. and Clausen, T., Potassium depletion decreases the number of [3H]ouabain binding sites and active Na-K transport in skeletal muscle, Nature (London), 293 (1981) 739-741. Pollack, L.R., Tate, E.H. and Cook, J.S., Turnover and regulation of Na,K-ATPase in Hela cells. J. (ell. Physiol.. 106 (1981) 85-97. Sampson, SR..Bannett, R.R. and Shainberg, A., Effects of thyroxine on transmembrance resting potentials of skeletal muscle cells in culture. J. Neurosci. Res., 8 11982) 595-601. Thomas, R.C.. Electrogenic pump in ncrvc and muscle cells, Physiol. Rev., 52(1972)563-594. Vandenburgh, H.H. and Kaufman. S.. Stretch-induced growth of skeletal myotubes correlates with activation of the sodium pump, J. (?ell. Ph;'siol., 1119( 1981 ) 205 214.