- Email: [email protected]
Influence of various growth factors and conditions on development of resting membrane potential and its electrogenic pump component of cultured rat skeletal myotubes
INFLUENCE OF VARIOUS GROWTH FACTORS AND CONDITIONS ON DEVELOPMENT OF RESTING MEMBRANE POTENTIAL AND ITS ELECTROGENIC PUMP COMPONENT OF CULTURED RAT SKELETAL MYOTUBES CHAYA
Department
BRODIE
and S. R. SAMPSON*
of Life Sciences. Bar-lian
University.
Ramat-Gan
52 100, Israel
(A~~~*pi~li 22 ~~tttttftr~ 19%)
Abstract-The effects of different growth frlctors and growth conditions were studied on the development of resting membrane potential and its electrogenic-ouahain-sensitive-pump component in cultured rilt myotuhes. Resting potential and its electrogenic pump component were dependent on the initial plating density of the myotubes. both values increasing with increasing density. Medium from cells plated at high density. when used to replace the medium of low density cells. increased both the resting potential and its electrogenic pump component of low density myotuhes. Treatment of myotuhes with cytosine-arahinoside delayed the appearance of [‘HI ouahain binding sites and eiectrogenic pump component of resting potential. but by X days in culture there was no difference between treated and control cells. Similarly, cells plated initially in 5% horse serum developed resting potential and its eleetrogenic pump component more slowly than those in IS’% horse serum, hut by X-lOdays ;!I rirro. the differences were no longer apparent. Chick embryo extract was found to have little. if any. influence on development of resting potential and its electroyenic pump component. We conclude that the different growth conditions and factors to the extent that they influence membrane potential, do so by altering the time of appearance of Na-K ATPase. the activity of which contributes a considerable component to resting potentinl. Kq n~~rtl.~: Memhranc myotuhcs.
Na ’ -K ’ ATPase
potential.
plays an important
Growth
factors,
regulatory
Electrogenic
pump.
Na-K
role in the development
ATPase.
Cultured
of properties
of several
different cell types.‘.” Recent studies from this laboratory indicate that activity of this enzyme contributes to resting Em of cultured myotubes and that an increase in level of activity of this enzyme may be involved in the increase in transmembrane resting potential (Em) of cultured myotubes with age.“.” Thus, both the amount of [“Hlouabain binding sites and the ouabainsensitive (electrogenic) pump component of Em, i.e the amount of depolarization produced by ouabain within 3-5 min, increase in parailel with the developmental increase in Em. In addition, we have shown that certain hormones and deficient growth conditions Em to alterations in amount or activity of Nat-K+ ATPase.‘*“‘.2s
may owe their effects on
Little is known about the regulation of appearance and activity of this enzyme in cultured myotubes. Studies so far conducted on the influence of different growth factors and conditions on myotube differentiation have concentrated on morphological properties,‘.‘*““’ or on appearance of muscle specific proteins such as acetylcholine receptor, myosin and creatine phosphokinase.” The few studies on development of Em have either largely ignored the role of Na+-Kf ATPase,“.‘” or deemed it to be relatively unimportant.” The high degree of activity of Na+-K+ ATPAse in cultured
rat myotubes
affords an oppor-
tunity to examine certain aspects of regulation and control of appearance of this enzyme with differentiation. Accordingly, we have investigated effects of certain growth factors and conditions on contribution of Na’-K’ ATPase to Em of myotubes in culture as expressed by the ouabainsensitive component
of Em. In some cases, we have correlated
effects on Em with those on the
number of enzyme sites as measured by the binding of [-‘H]ouabain. The results show that conditions that influence development of Em also influence the contribution of Na-K ATPase activity to Em, and do so in the same direction. There is, thus, a strong correlation differentiation, Em, and amount and activity of Naf-K’ ATPase. Ahhtwiariotts: CEE. chick emhryo extmct: Ara-C. cytosinc ar:lhinosidc: potassium adenosine triphosphatasc: On, membrane potential. * Author to whom correspondence should he addressed.
327
among myotube
HS. horse serum: Na’ -K
’
ATPase.
sodium-
328
C. Brodic and S. R. Sampson
EXPERIMENTAL
PROCEDURES
Skeletal muscle cultures were prepared from thigh muscles obtained from I8 to 2l-day-old rat fetuses, or 1-2 day postnatal, as described previously. ‘.J~~“~~~Pregnant rats were killed by cervical dislocation and the fetuses removed aseptically. The limbs were removed, washed in phosphatebuffered saline (PBS) to remove excess blood cells. and then transferred to a Ca-free, 0.25% trypsin solution containing EDTA (1 mM) for incubation with continuous stirring at 37°C. Cells were collected after successive IO-min periods of incubation until all tissue was dispersed, and then centrifuged for 5 min at 500~. Pellets were resuspended in growth medium and preplated for 20-30 min to reduce the number of fibroblasts. The supernatant was collected and the remaining myoblasts were diluted with growth medium to a concentration of 0.8 x IO” cells/ml for plating in 35 mm collagen-coated plastic tissue culture dishes (I.5 ml/dish, unless otherwise indicated). Cultures were grown in a water-saturated atmosphere of 90% air-IO’% CO? at 37°C. The composition of the growth medium was as follows: Dulbecco’s Minimal Essential Medium (Gibco). 83%; horse serum (Biolab), IS%; chick embryo extract. 2%. For intracellular recording, the growth medium was replaced with a recording medium of the 8; KH?PO_,, 1.4; MgCl?, 0.5; following composition (mM): NaCl, 135; KCI. 2.7; NalHPOJ, CaCl?. 0.7; glucose, 22. The pH of this medium was adjusted to 7.32-7.35. The dishes containing the cultures were placed in a copper-heating sleeve for temperature control by circulating water from a water-bath, and this assembly was placed on the stage of an inverted microscope (Nikon). Recordings were made with glass microelectrodes containing a fiberglass fiber and filled with 2.8 M KCI. The microelectrode was connected by a platinum wire to the input of a microelectrode preamplifier (Winston Electronics), and a second platinum wire inserted into the culture dish served as the indifferent electrode. Resistances of the microelectrodes ranged from 15 to 40 MR. Recordings were displayed on one beam of a storage oscilloscope (Tektronix 5113). Transmembrane resting potential (,&I) was mcasurcd as highest stable potential recorded during an impalement. Tip potentials were between I and 3 mV. The mean value and standard error of resting Et?1 were determined from a minimum of 9 cells from each of 2 dishes under any given experimental condition. Unless stated otherwise, recordings were made at a temperature of 37°C. Recordings were begun routinely 10 min after the changes in medium. Data were analyzed statistically by the Student’s r-test for unpaired samples. The component of Em attributable to activity of the Na-K pump (electrogenic pump component) was determined in the following way. First, the mean Em was determined as described in the previous paragraph, then ouabain was added to a final concentration of IO ’ M. Beginning 5 min after addition of ouabain, we recorded Et?1 again from a minimum of 9 myotubes. We waited this period of time because WC found that ouabain caused depolarization of myotubes within l-2 min of addition to a value which remains constant for nearly 60 min.’ This change in Em is not accompanied by changes in input resistance. The electrogenic pump component (mV) was then calculated as the difference between Etn recorded in the presence of ouabain and that in its absence. The binding of [‘Hlouabain was determined by a modification of the method of Vandenberg and Kaufman.‘-’ Cultures were washed three times with 2 ml K ’ -free phosphate buffer (pH 7.4) and then incubated in I .O ml of buffer containing 12.5 nM [‘HI ouabain (0.4 pCi/ml) and IO ’ FM unlabeled ouabain at room temperature (20-23°C). WC determined in preliminary experiments, from the ratio of specific to non-specific ouabain binding, that saturation of binding sites was obfor 30 min at room temperature tained at a ouabain concentration of IO ’ M. After incubation (which was found to be sufficient for optimal binding). cells were washed five times with cold buffer. They were solubilized in I .O’% SDS, and radioactivity was assayed by standard probinding by non-muscle elements was cedures. Control studies established that [‘H] ouabain < 10% of the total.
Primary cultures of rat skclctal muscle are generally prepared from the limbs of either fetal or neonatal animals. It is not known. however. whether or not myotubes from the two sources differ
Membrane potential and electrogenic pump component in cultured with regard to development Em. and its electrogenic prepared
of certain electrophysiological
pump component
from fetal and neonatal
properties.
muscle
Table 1 compares values of
(E,,) recorded from myotubes at different
rats. Measurements
were obtained
329
culture age
daily from the day of fusion
(day 2 in vitro). There were no striking differences between the two with regard to the various properties, although Em was slightly and consistently higher in myotubes prepared from fetal rats than that from neonatal rats. This,difference could be accounted for nearly entirely by differences in the size of the electrogenic pump component. Table I. Resting membrane potential (Em) and its electrogenic pump component (E,,) at different ages in culture of skeletal myotubes derived from fetal (F) and neonatal (N) rats. Values are mean + SE. of a minimum of 9 determinations on 9 individual myotubes in each of 2 dishes at the age specified. Cell plating density IO” cells/ml (‘P
Age (DIV)’
-76.7 + 0.8 -79.4 5 0.4 -82.620.6 -85.1 kO.9 -84.8’0.6
3 4 6 7 8 * DIV
N
F -74.0 -76.5 -82.1 -82.7 -83.5
* 0.6* + 0.3* 50.6 rt 0.6* + 0.6
F
N
17.4 20.4 22.3 24.4 23.7
15.4 17.9 21.4 21.9 22.3
= Days in vitro.
Cell plating density We have presented evidence elsewhere that cell density may be an important determinant of membrane potential and the expression of its electrogenic pump component in cultured skeletal muscle.’
Because the appearance
of an electrogenic
pump component
may also be influenced
by
the age of the specimen used (fetal or neonatal; Table I), we examined the effects of cell density in Em and its electrogenic pump component in cultures prepared from both fetal and neonatal animals.
Recordings
were
already reached plateau
made on myotubes
after 7 days in culture,
at which time Em has
values.
Figure 1A shows that Em of myotubes from both fetal and neonatal sources are dependent on the density of cells initially plated. Em of fetal myotubes increased with density from -62 mV at 2.5 -84
x 10J
mV
cells/ml to -86 mV at lOh cells/ml, and that of neonatal myotubes increased from -68 at these same densities. The differences were statistically significant (P
densities lower than 10-5 cells/ml.
Figure
1B shows that the size of the electrogenic
to at
pump com-
ponent also increased as a function of cell density. This dependence seemed to be more important for myotubes prepared from fetal than from neonatal material. Thus, the plating at low density resulted in an electrogenic pump component of only about 2 mV in myotubes from fetal rats compared with 8 mV in myotubes derived from neonatal material. At high cell density, the size of the electrogenic pump component was nearly the same in both groups. with that from fetal rats being slightly (2 mV) but consistently higher than that of neonatal animals. Morphologically, there were clear differences in appearance between cells plated at the extremes of cell density, with the low density myotubes being characterized by relatively thin and separated fibers (Fig. 2). In contrast, myotubes plated at densities of 5 x lo’ or lOh cells/ml were large diameter long and branching and seemed to form almost a continuous syncitium of contracting myotubes. As plating cell density increased from the minimum value, the degree of fusion and size of the myotubes
increased progressively.
Effects of high cell density medium The higher Em of cells plated at high density than of those at low density suggests that a factor or factors may be produced by the cells favorable to development of the electrogenic pump component of Em, and that this factor is either produced in insufficient quantities, or is diluted in the low density preparations. Alternatively, cells plated at low density might produce a factor which
C. Brodie
330
2K) % g 8 = 0
25~10~
5x104
and S. R. Sampson
lxx)5
Cell
25x105
>xIv=
IXKJ~
Density
Fig. I. Resting potential (A) and its electrogenic pump component (B) as a function of initial cell density (cells/ml) of cultured fetal (hatched bars) and neonatal (clear bars) rat myotubes. Values represent the mean membrane potential (mV) 5 S.E. of measurements from Y to 12 myotubes in 2 dishes. See text for explanation of values in (B). Recordings were made on myotubes of age
plating in (A) each of 7.DIV..
prevents or inhibits appearance of the electrogenic pump component. To examine these possibilities, we replaced the medium of low density myotubes with that from high density preparations and vice versa after 5 days in culture and every 3 days thereafter.
Em was recorded
daily
from control low density myotubes, low density myotubes with high density medium, and from control high density myotubes. The electrogenic pump component was also determined as the difference
in Em before and 5-10 min after addition
of ouabain.
Figure 3 summarizes
data ob-
tained from three studies of this type. As usual, Em of low density myotubes on any given day in culture is significantly
lower (P
than that of high density myotubes.
Figure 3 shows that
replacement of the growth medium of cells at low plating density with medium from high density myotubes resulted in an increase in Em; the increase was observed as early as 24 hr after medium replacement and persisted as long as low density cells were maintained in high density medium. Figure 3B shows that the increase in Em of low density cells after replacement of the medium was due almost entirely,
if not completely,
to an increase in the electrogenic
pump component
of Em.
Thus. the electrogenic pump component of low density myotubes reached a value of about 5 mV, whereas that of low density cells with high density medium was about 18 mV after 7-9 days in culture compared with about 22 mV for control myotubes plated at high density. Two additional experiments of this type were done, but because the cell plating densities were lower than those for the studies depicted in Fig. 3, the mean Em’s were correspondingly lower and the data not included. The pattern of effect,was, however, identical. In contrast, medium of low density cells had no effect on Em or electrogenic Effects of cytositze arabinoside
(Ara-C)
pump component
of high density cells.
treatment
Cytosine arabinoside is sometimes added to cultured myotubes for 48 hr immediately after fusion (2-3 days after plating) in order to limit proliferation of fibroblasts. We compared effects of this treatment on Em, electrogenic pump component of Em. and number of [-‘Hlouabain binding sites in myotubes at 5 and then again at 7-9 days in vitro. Figure 4 shows that treatment with Ara-C reduces the rate of development of Em and appearance of Na-K ATPase in cultured myotubes. Thus, at day 5 in culture, mean Em of control myotubes was higher than that of AraC-treated myotubes (P
Membrane potential and electrogenic pump component in cultured muscle
Fig. 2. Phase contrast micrographs
of myotubes (7 DIV) plated at initial densities of 5 X 10’ (A) and IO” (B) cells/ml.
331
Membrane
potential
and electrogenic
pump component
in cultured
muscle
333
::il;__; 0
I
I
I
3
4(l)
%?I
6. ff31
I
7(4)
85)
I 461
Days In Culture Fig. 3. Effects of high cell density conditioned.medium (HCM) on development of resting potential (A) and its eletrogenic pump component (B) in cultured myotubes. Curves showing the relation of E/n to age in HCM and low density medium (LDM) are represented by open and tilled circles. rcspcctivcly. After 3 DIV. medium from HCM cells was added to LDM cells. and the data from these myotubcs arc represented by the filled triangles. Each point in (A) is the average of the mean membrane potential of 3 experiments. In each experiment the mean value was detcrmincd from measurements of On in Y-12 myotubes in each of 2 dishes. Points in (B) are the average of the values of clectrogenic pump component determined in each of the ?I experiments
5DIV INTROL
B ARA-C
mv
CONTROL
ARA-C
L
Fig. 4. Effects of cytosine arabinoside treatment on membrane potential and number of [-‘Hlouabain binding sites in cultured rat myotubes. Data were obtained at 5 (A) and 7 (B) DIV. Membrane potentials were recorded at 23°C (0). 37°C (QB1,and at 37°C following ouabain (m). To the right of each set of membrane potentials is the plot of number of Na-pump sites. Values as in Fig. I. See text for further explanation.
due to Ara-C treatment were essentially non-existent days after removal of this substance from the growth medium).
differences
Effects
of non-specific
nutritional
by 7 days in culture (at least 4
supplements
Primary cultures of skeletal muscle are frequently grown in media supplemented with animal serum and chick embryo extract, both of which reportedly are required for adequate differentiation. Recent studies have shown that myotubes grown in serum-free, chemically defined medium, display morphological and biochemical properties similar to cells grown in serum.x A recent study from this laboratory has shown, however, that myotubes maintained in serum-free media are not
334
C. Brodie and S. R. Sampson
electrophysiologically fully functional.‘5 In this study we compared Em and its electrogenic component of cells suspended in growth medium supplemented with 15% horse serum (HS) with those in 5% HS. As shown in Fig. 5, the development of Em and the contribution by Na-K+ ATPase is slower when myotubes are grown from the time of initial plating in 5% HS than when in 15% HS, as indicated by the values for mean Em at 37”C, that in 15% HS being significantly higher (PO.l) or in electrogenic pump component between cells grown in the two concentrations of HS (15% HS, 29 mV; 5% HS, 29 mV). The high concentration of HS appears to be important only for early development of Em, as indicated in Fig. 5B. This shows that when the concentration of HS was changed from 15 to 5% immediately after fusion (2-3 days after plating), values for Em of myotubes in both concentrations at 5 days in L’itro were nearly identical (P>O.l).
n BDIV
5DIV
I
15%HS
p+
Fig. 5. Effects
5%HS I
5 DIV
of growth in medium
containing 15 and 5% HS on resting potential ofcultured Each hnr graph as in Fig. 4. See text for further explanation.
myotuhes
Figure 6 shows that there is no difference between myotubes grown in medium supplemented with 1 l-day-old chick embryo extract (CEE) and those in CEE-free growth medium with regard to Em (P>O.5) and its ouabain-sensitive component, whether recorded at 4 days or 10 days in culture. In contrast, CEE from 17- to lPday-old chick embryos caused an increase in both Em at 37°C (P
DlSCUSSlON In recent years, several laboratories have reported on the growth and development of a variety of cultured cells, including skeletal muscle in serum-free, chemically defined media.5.7-“.‘” Cells grown in these media are reported to resemble those grown in conventional. serum-containing medium with regard to general biochemical and morphological properties. Nonetheless, many cultures, particularly skeletal muscle, are still grown in the presence of (horse) serum, as well as apparently because of the fact that myotubes do better under these chick embryo extract,“.“.”
Membrane
potential
and electrogenic
r
pump component
in cultured
muscle
335
A-4DIV
N3
CEE 1
B-10
JO CEE
DIV
CEE 17 DAY
Fig. 6. Effects of chick embryo extract (CEE) on resting potential of J- (A) and IO- (B) day-old myotuhes in culture. Each bar graph as in Fig. 4. See text for further explanation.
rat
Moreover, a recent report from this laboratory” showed that, at least in the case of electrophysiological properties of mammalian skeletal muscle, the development in serum-free medium is incomplete. In this case, the deficiency was accounted for by a decrease in activity of Na-K ATPase in cultures grown in serum-free medium compared to that in conventional medium. We have shown that this enzyme contributes an important component (electrogenic) to the membrane potential of cultured rat myotubes.‘.3 The results of the current study show that the enzyme is also influenced by a variety of specific and non-specific treatments and conditions routinely used in preparation of skeletal muscle cultures. It is, at present, only possible to conjecture as to the possible mechanisms underlying some of the phenomena described here. For example, we found that treatment of cultures with cytosine arabinoside delays the appearance of the enzyme. It is not clear, however. if this effect is due directly to the effects of this substance or is secondary to the decrease in total cell number as a result of the arrest of fibroblast proliferation, as we also found that total cell density at the time of plating (which includes both myoblasts and fibroblasts) markedly affects expression of Na-K ATPase (Fig. 7). In the case of the comparison between high and low serum concentration on expression of Na-K ATPase, we found that high serum concentration was important to Em and its electrogenic component only prior to cell fusion. If the serum concentration was lowered after fusion was complete, there was no difference between cells in low serum and those in high serum. As the rate and extent of fusion, as judged by visual inspection of the cultures, seemed to be about equal for cells plated in different concentrations of serum, the difference in membrane potential would seem to be due to factors unrelated to fusion specifically, although this process does determine the appearance of Na-K ATPase.’ One of the other factors likely to occur for the serum effect on appearance and activity of the Na-K ATPase might be stimulation of Na+ entry, as has been shown to occur in fibroblast and neuroblastoma cultures.‘x.2’ The extent to which other factors may be involved in mediating the effects of serum is not clear as these are not fully understood. I” The studies on effects of chick embryo extract may be considered somewhat surprising in view of the inclusion of this material as a matter of routine in preparation of muscle cultures.” We conditions.
336
C. Brodic and S. R. Sampson
found that there was essentially no difference in Dn or its electrogenic pump component between myotubes in medium with chick embryo extract and those without it. These data appear to be in
et cd. ” and Hooisma
contrast to those of Hagiwara
et al. .I2 who reported that chick embryo ex-
tract was one of several essential factors for growth of chick muscle in culture. Whether or not this represents a fundamental difference between rat and chick muscle in culture is unclear. When extract of older chick embryos was used, we found that electrogenic pump component of Em increased substantially over that of control myotubes with or without extract of young chick embryos. This might possibly be related to the more highly developed nervous system and accompanying neurotropic effects. Clearly, the most important factor regulating the appearance of Na-K ATPase. as expressed by the electrogenic pump component of membrane potential. was plating density. We found that both Et72 and its ouabain-sensitive component were directly related to plating density. Indeed, plating at low density resulted in complete non-expression of an electrogenic pump when cells were prepared from fetal rats. This probably explains the failure of Ritchie and Fambrough” to observe any effects of ouabain in their preparation of myotubes, which were plated at a density of 5 x 10’/ml.
Cell plating density has been shown to influence the expression of properties
of other
cells in primary culture, such as rat heart and granulosa cells from porcine ovaries. “.” The higher Em and electrogenic pump activity in myotubes plated at high density rather than low density appears to be due to the release of a factor (or factors) density “conditioned”
medium
added to myotubes
by the former,
as we found that high
plated at low density caused their Err? and
electrogenic pump component to increase. Other studies have shown that myotubes as well as other primary cells synthesize and release factors that influence growth of other cell types in culture. ‘t’.zJ As far as we are aware, there is only one other report of factors released by myotubes in cuiture
to influence
other myotubes,
this by Vandenburgh,”
who reported
such effects for
chick myotubes in culture, The present study thus appears to be the first description of a factor (or factors) released by cultured mammalian muscle. The nature of this factor. and regulation of its synthesis and release. remain to be investigated. The results presented here demonstrate that there is a strong correlation
between
the level of
resting membrane potential and the size of its electrogenic pump component. This correlation is strengthened by the additional observation that in those conditions in which the electrogenic pump component is not yet fully expressed (treatment with Ara-C, plating in low serum concentration, low cell density), changes in temperature from 23 to 37°C are relatively ineffective in altering
the level of Em. These findings provide
ATPase
plays an important
role in regulation
additional
support
of &rz in cultured
to the idea that Na+-K’
rat skeletal muscle.-?.3
WC Arkrrna,/~,cl~c,rtlctlrs-Supported in part by the Rescarch Authority. Bar-llan University. and Yad Hanadiv, T&Aviv. w*ish to acknowledge the technical assistance of Mrs Asia Bak in preparing the cultures, and to thank Mrs Bluma Ledcrhendlcr for expert typing of the manuscript.
REFERENCES 1, Bannett R. R.. Sampson S. R. and Shainberg A. (1983) lnflucnce of thyroid hormones on some electrophysiological properties of developing rat skeletal muscle cells in culture. Rrcri~t Rex. 294, 75-82. 2. Brodie C.. Bak A. and Sampson S. R. (1985) Dependenee of Nit+- K’ ATPase and electrogenic component of Etn in cultured myotubes on cell fusion. Brc~in Rev. 36, 384386. 3. Brodie C. and Sampson S. R. (1985) Contribution of electrogenic s~~diurn-p‘~tassium ATPase to resting membrane potential of cultured rat skeletal myotuhes. Rruitt Res. 37, 28-35. J. Castro J.. Maqued~~n~~ A. and Olive M. (19X-t) Lipid synthesis in isolated rat hcpatocytes: ~i~tiv~Iti‘~ll by insulin and vanadatc and inhibition by ouahain. Rio&ertr. ltzf. 9. Jl3-t20. 5. Claycomh W. C. (1980) Culture of cardiac muscle cells in serum-free media. fC.x-pi( ‘M Rt_;. 131, 7.1 l-736. 6. Dryden W. F.. Erulkar S. D. and de la Haha G. (1974) Properties of the cell membrane of developing skclctal muscle fihres in culture and its sensitivity to acetyicholine. c‘liiz. r.r,~. Phanrroc. Physiol. I ( X19-3X7. 7. Ewton D. Z. and Florini J. R. (19X1) Effects of the somatomedins and insulin in myohlast diffcrentiatirm i!r t,i/ro. L)cjt,/ Biol. 86, 3 I-3‘. 8. Florini J. R. and Roherts S. F. (lY79) A serum-free medium for the growth of muscle ct’lls in culture. /!I Vi/w 15,
983-002. 0. Florini J. R. nnd Ewton D. Z. (1981) Imulin acts as a sorn~~torneclitl-;ln~~l‘~~in stimulattng myohlas~ growth in serumfree medium. I/t Virro 17, 7h.F-76X. IO. Gospodarowicz 55s.
D. and Moran
J. S. (1976)
Growth
factors in mammalian
cell culture.
.4\,rrr. Kcr,. Hiochcrn. 45, 531-
Membrane
potential
and electrogenic
pump component
in cultured
muscle
337
I I.
Hagiwara Y.. Kimura I. and Ozawa E. (19X1) Chick embryo extract. muscle trophic factor and chick and horse sera as environments for chick myogenic cell growth. Det~I Grow/h Differ. 23, 249-254. 12. Hooisma J.. De Drijger J.. De Groot D. M. G.. Magchietse T. and Muijser H. (IYXI) Effects of extracts of denervated muscles on the morphology of cultured muscle cells. Nerrrosci. Lett. 22. 47-50. 13. lnestrosa N. C. (19X2) Differentiation of skeletal muscle cells in culture. Cc// Sfrm-f. F~rrc/. 7, 91-109. 14. Kaplan J. G. (1978) Membrane cation transport and the control of proliferation of mammalian cells. Awr. Rell. Physiol. 40. 19--11. IS. Kidokoro Y. (1975) Developmental changes in membrane electrical properties in a rat skeletal muscle cell line. J. Physiol. 244. I2Y- 143. In. Mather J. P. and Sato G. H. (1979) The use of hormone-supplemented serum-free media in primary cultures. Expl Cell Rev. 124. 2 15-221. 17. May J. V. and Schomberg D. W. (1984) The effects of plating density on granulosa cell growth and differentiation in
vitro. IX. 19.
20. 21. 22.
23. 24.
25.
Molec.
cell. En&x-r.
34, 201-213.
Moolenaar W. H.. Mummery C. L., van der Saag P. T. and de Laat S. W. (19X1) Rapid ion events and the initiation of growth on serum-stimulated neurohlastoma cells. Cell 23, 7X9-79X. Ritchie A. K. and Fambrough H. M. (1975) Electrophysiological properties of the membrane and acetylcholine receptor in developing rat and chick myotubes. J. gcn. Physiol. 66, 327-355. Sampson S. R.. Bannett R. R. and Shainberg A. (19X2) Effects of thyroxine on transmembrane resting potentials of skeletal muscle cells in culture. J. Nerrrosci. Rev. 8, 5YS-601. Smith J. B. and Rosengurt E. (197X) Serum stimulates the Na ’ . K + pump in quiescent fibroblasts by increasing Na’ entry. Proc~. trnnr. Accrd. Sci. U.S.A. 75, SS60--5.564. Speicher D. W.. Peace J. N. and McCarl R. L. (19X1) Effects of plating density and age in culture on growth and cell division of neonatal rat heart primary cultures. I/r Vifro 17. X63-X70. Vandenburg H. H. and Kaufman S. (19X1) Stretch-induced growth of skeletal myotubes correlates with activation of the sodium pump. J. cdl. camp. Physiol. 109, 20.5-214. Vandenburg H. H. (19X3) Cell shape and growth regulation in skeletal muscle: exogenous versus endogenous factors. J. cell. amp. Physiol. 116. 363-371 Yoles E.. Bak A. and Sampson S. R. (10X4) Some electrophysiological properties of developing rat skeletal myotubes grown in serum-free. chemically detined medium. /!I/. J. t/e~d Nmtrosci. 2, JX3-4YO.
Department
BRODIE
and S. R. SAMPSON*
of Life Sciences. Bar-lian
University.
Ramat-Gan
52 100, Israel
(A~~~*pi~li 22 ~~tttttftr~ 19%)
Abstract-The effects of different growth frlctors and growth conditions were studied on the development of resting membrane potential and its electrogenic-ouahain-sensitive-pump component in cultured rilt myotuhes. Resting potential and its electrogenic pump component were dependent on the initial plating density of the myotubes. both values increasing with increasing density. Medium from cells plated at high density. when used to replace the medium of low density cells. increased both the resting potential and its electrogenic pump component of low density myotuhes. Treatment of myotuhes with cytosine-arahinoside delayed the appearance of [‘HI ouahain binding sites and eiectrogenic pump component of resting potential. but by X days in culture there was no difference between treated and control cells. Similarly, cells plated initially in 5% horse serum developed resting potential and its eleetrogenic pump component more slowly than those in IS’% horse serum, hut by X-lOdays ;!I rirro. the differences were no longer apparent. Chick embryo extract was found to have little. if any. influence on development of resting potential and its electroyenic pump component. We conclude that the different growth conditions and factors to the extent that they influence membrane potential, do so by altering the time of appearance of Na-K ATPase. the activity of which contributes a considerable component to resting potentinl. Kq n~~rtl.~: Memhranc myotuhcs.
Na ’ -K ’ ATPase
potential.
plays an important
Growth
factors,
regulatory
Electrogenic
pump.
Na-K
role in the development
ATPase.
Cultured
of properties
of several
different cell types.‘.” Recent studies from this laboratory indicate that activity of this enzyme contributes to resting Em of cultured myotubes and that an increase in level of activity of this enzyme may be involved in the increase in transmembrane resting potential (Em) of cultured myotubes with age.“.” Thus, both the amount of [“Hlouabain binding sites and the ouabainsensitive (electrogenic) pump component of Em, i.e the amount of depolarization produced by ouabain within 3-5 min, increase in parailel with the developmental increase in Em. In addition, we have shown that certain hormones and deficient growth conditions Em to alterations in amount or activity of Nat-K+ ATPase.‘*“‘.2s
may owe their effects on
Little is known about the regulation of appearance and activity of this enzyme in cultured myotubes. Studies so far conducted on the influence of different growth factors and conditions on myotube differentiation have concentrated on morphological properties,‘.‘*““’ or on appearance of muscle specific proteins such as acetylcholine receptor, myosin and creatine phosphokinase.” The few studies on development of Em have either largely ignored the role of Na+-Kf ATPase,“.‘” or deemed it to be relatively unimportant.” The high degree of activity of Na+-K+ ATPAse in cultured
rat myotubes
affords an oppor-
tunity to examine certain aspects of regulation and control of appearance of this enzyme with differentiation. Accordingly, we have investigated effects of certain growth factors and conditions on contribution of Na’-K’ ATPase to Em of myotubes in culture as expressed by the ouabainsensitive component
of Em. In some cases, we have correlated
effects on Em with those on the
number of enzyme sites as measured by the binding of [-‘H]ouabain. The results show that conditions that influence development of Em also influence the contribution of Na-K ATPase activity to Em, and do so in the same direction. There is, thus, a strong correlation differentiation, Em, and amount and activity of Naf-K’ ATPase. Ahhtwiariotts: CEE. chick emhryo extmct: Ara-C. cytosinc ar:lhinosidc: potassium adenosine triphosphatasc: On, membrane potential. * Author to whom correspondence should he addressed.
327
among myotube
HS. horse serum: Na’ -K
’
ATPase.
sodium-
328
C. Brodic and S. R. Sampson
EXPERIMENTAL
PROCEDURES
Skeletal muscle cultures were prepared from thigh muscles obtained from I8 to 2l-day-old rat fetuses, or 1-2 day postnatal, as described previously. ‘.J~~“~~~Pregnant rats were killed by cervical dislocation and the fetuses removed aseptically. The limbs were removed, washed in phosphatebuffered saline (PBS) to remove excess blood cells. and then transferred to a Ca-free, 0.25% trypsin solution containing EDTA (1 mM) for incubation with continuous stirring at 37°C. Cells were collected after successive IO-min periods of incubation until all tissue was dispersed, and then centrifuged for 5 min at 500~. Pellets were resuspended in growth medium and preplated for 20-30 min to reduce the number of fibroblasts. The supernatant was collected and the remaining myoblasts were diluted with growth medium to a concentration of 0.8 x IO” cells/ml for plating in 35 mm collagen-coated plastic tissue culture dishes (I.5 ml/dish, unless otherwise indicated). Cultures were grown in a water-saturated atmosphere of 90% air-IO’% CO? at 37°C. The composition of the growth medium was as follows: Dulbecco’s Minimal Essential Medium (Gibco). 83%; horse serum (Biolab), IS%; chick embryo extract. 2%. For intracellular recording, the growth medium was replaced with a recording medium of the 8; KH?PO_,, 1.4; MgCl?, 0.5; following composition (mM): NaCl, 135; KCI. 2.7; NalHPOJ, CaCl?. 0.7; glucose, 22. The pH of this medium was adjusted to 7.32-7.35. The dishes containing the cultures were placed in a copper-heating sleeve for temperature control by circulating water from a water-bath, and this assembly was placed on the stage of an inverted microscope (Nikon). Recordings were made with glass microelectrodes containing a fiberglass fiber and filled with 2.8 M KCI. The microelectrode was connected by a platinum wire to the input of a microelectrode preamplifier (Winston Electronics), and a second platinum wire inserted into the culture dish served as the indifferent electrode. Resistances of the microelectrodes ranged from 15 to 40 MR. Recordings were displayed on one beam of a storage oscilloscope (Tektronix 5113). Transmembrane resting potential (,&I) was mcasurcd as highest stable potential recorded during an impalement. Tip potentials were between I and 3 mV. The mean value and standard error of resting Et?1 were determined from a minimum of 9 cells from each of 2 dishes under any given experimental condition. Unless stated otherwise, recordings were made at a temperature of 37°C. Recordings were begun routinely 10 min after the changes in medium. Data were analyzed statistically by the Student’s r-test for unpaired samples. The component of Em attributable to activity of the Na-K pump (electrogenic pump component) was determined in the following way. First, the mean Em was determined as described in the previous paragraph, then ouabain was added to a final concentration of IO ’ M. Beginning 5 min after addition of ouabain, we recorded Et?1 again from a minimum of 9 myotubes. We waited this period of time because WC found that ouabain caused depolarization of myotubes within l-2 min of addition to a value which remains constant for nearly 60 min.’ This change in Em is not accompanied by changes in input resistance. The electrogenic pump component (mV) was then calculated as the difference between Etn recorded in the presence of ouabain and that in its absence. The binding of [‘Hlouabain was determined by a modification of the method of Vandenberg and Kaufman.‘-’ Cultures were washed three times with 2 ml K ’ -free phosphate buffer (pH 7.4) and then incubated in I .O ml of buffer containing 12.5 nM [‘HI ouabain (0.4 pCi/ml) and IO ’ FM unlabeled ouabain at room temperature (20-23°C). WC determined in preliminary experiments, from the ratio of specific to non-specific ouabain binding, that saturation of binding sites was obfor 30 min at room temperature tained at a ouabain concentration of IO ’ M. After incubation (which was found to be sufficient for optimal binding). cells were washed five times with cold buffer. They were solubilized in I .O’% SDS, and radioactivity was assayed by standard probinding by non-muscle elements was cedures. Control studies established that [‘H] ouabain < 10% of the total.
Primary cultures of rat skclctal muscle are generally prepared from the limbs of either fetal or neonatal animals. It is not known. however. whether or not myotubes from the two sources differ
Membrane potential and electrogenic pump component in cultured with regard to development Em. and its electrogenic prepared
of certain electrophysiological
pump component
from fetal and neonatal
properties.
muscle
Table 1 compares values of
(E,,) recorded from myotubes at different
rats. Measurements
were obtained
329
culture age
daily from the day of fusion
(day 2 in vitro). There were no striking differences between the two with regard to the various properties, although Em was slightly and consistently higher in myotubes prepared from fetal rats than that from neonatal rats. This,difference could be accounted for nearly entirely by differences in the size of the electrogenic pump component. Table I. Resting membrane potential (Em) and its electrogenic pump component (E,,) at different ages in culture of skeletal myotubes derived from fetal (F) and neonatal (N) rats. Values are mean + SE. of a minimum of 9 determinations on 9 individual myotubes in each of 2 dishes at the age specified. Cell plating density IO” cells/ml (‘P
Age (DIV)’
-76.7 + 0.8 -79.4 5 0.4 -82.620.6 -85.1 kO.9 -84.8’0.6
3 4 6 7 8 * DIV
N
F -74.0 -76.5 -82.1 -82.7 -83.5
* 0.6* + 0.3* 50.6 rt 0.6* + 0.6
F
N
17.4 20.4 22.3 24.4 23.7
15.4 17.9 21.4 21.9 22.3
= Days in vitro.
Cell plating density We have presented evidence elsewhere that cell density may be an important determinant of membrane potential and the expression of its electrogenic pump component in cultured skeletal muscle.’
Because the appearance
of an electrogenic
pump component
may also be influenced
by
the age of the specimen used (fetal or neonatal; Table I), we examined the effects of cell density in Em and its electrogenic pump component in cultures prepared from both fetal and neonatal animals.
Recordings
were
already reached plateau
made on myotubes
after 7 days in culture,
at which time Em has
values.
Figure 1A shows that Em of myotubes from both fetal and neonatal sources are dependent on the density of cells initially plated. Em of fetal myotubes increased with density from -62 mV at 2.5 -84
x 10J
mV
cells/ml to -86 mV at lOh cells/ml, and that of neonatal myotubes increased from -68 at these same densities. The differences were statistically significant (P
densities lower than 10-5 cells/ml.
Figure
1B shows that the size of the electrogenic
to at
pump com-
ponent also increased as a function of cell density. This dependence seemed to be more important for myotubes prepared from fetal than from neonatal material. Thus, the plating at low density resulted in an electrogenic pump component of only about 2 mV in myotubes from fetal rats compared with 8 mV in myotubes derived from neonatal material. At high cell density, the size of the electrogenic pump component was nearly the same in both groups. with that from fetal rats being slightly (2 mV) but consistently higher than that of neonatal animals. Morphologically, there were clear differences in appearance between cells plated at the extremes of cell density, with the low density myotubes being characterized by relatively thin and separated fibers (Fig. 2). In contrast, myotubes plated at densities of 5 x lo’ or lOh cells/ml were large diameter long and branching and seemed to form almost a continuous syncitium of contracting myotubes. As plating cell density increased from the minimum value, the degree of fusion and size of the myotubes
increased progressively.
Effects of high cell density medium The higher Em of cells plated at high density than of those at low density suggests that a factor or factors may be produced by the cells favorable to development of the electrogenic pump component of Em, and that this factor is either produced in insufficient quantities, or is diluted in the low density preparations. Alternatively, cells plated at low density might produce a factor which
C. Brodie
330
2K) % g 8 = 0
25~10~
5x104
and S. R. Sampson
lxx)5
Cell
25x105
>xIv=
IXKJ~
Density
Fig. I. Resting potential (A) and its electrogenic pump component (B) as a function of initial cell density (cells/ml) of cultured fetal (hatched bars) and neonatal (clear bars) rat myotubes. Values represent the mean membrane potential (mV) 5 S.E. of measurements from Y to 12 myotubes in 2 dishes. See text for explanation of values in (B). Recordings were made on myotubes of age
plating in (A) each of 7.DIV..
prevents or inhibits appearance of the electrogenic pump component. To examine these possibilities, we replaced the medium of low density myotubes with that from high density preparations and vice versa after 5 days in culture and every 3 days thereafter.
Em was recorded
daily
from control low density myotubes, low density myotubes with high density medium, and from control high density myotubes. The electrogenic pump component was also determined as the difference
in Em before and 5-10 min after addition
of ouabain.
Figure 3 summarizes
data ob-
tained from three studies of this type. As usual, Em of low density myotubes on any given day in culture is significantly
lower (P
than that of high density myotubes.
Figure 3 shows that
replacement of the growth medium of cells at low plating density with medium from high density myotubes resulted in an increase in Em; the increase was observed as early as 24 hr after medium replacement and persisted as long as low density cells were maintained in high density medium. Figure 3B shows that the increase in Em of low density cells after replacement of the medium was due almost entirely,
if not completely,
to an increase in the electrogenic
pump component
of Em.
Thus. the electrogenic pump component of low density myotubes reached a value of about 5 mV, whereas that of low density cells with high density medium was about 18 mV after 7-9 days in culture compared with about 22 mV for control myotubes plated at high density. Two additional experiments of this type were done, but because the cell plating densities were lower than those for the studies depicted in Fig. 3, the mean Em’s were correspondingly lower and the data not included. The pattern of effect,was, however, identical. In contrast, medium of low density cells had no effect on Em or electrogenic Effects of cytositze arabinoside
(Ara-C)
pump component
of high density cells.
treatment
Cytosine arabinoside is sometimes added to cultured myotubes for 48 hr immediately after fusion (2-3 days after plating) in order to limit proliferation of fibroblasts. We compared effects of this treatment on Em, electrogenic pump component of Em. and number of [-‘Hlouabain binding sites in myotubes at 5 and then again at 7-9 days in vitro. Figure 4 shows that treatment with Ara-C reduces the rate of development of Em and appearance of Na-K ATPase in cultured myotubes. Thus, at day 5 in culture, mean Em of control myotubes was higher than that of AraC-treated myotubes (P
Membrane potential and electrogenic pump component in cultured muscle
Fig. 2. Phase contrast micrographs
of myotubes (7 DIV) plated at initial densities of 5 X 10’ (A) and IO” (B) cells/ml.
331
Membrane
potential
and electrogenic
pump component
in cultured
muscle
333
::il;__; 0
I
I
I
3
4(l)
%?I
6. ff31
I
7(4)
85)
I 461
Days In Culture Fig. 3. Effects of high cell density conditioned.medium (HCM) on development of resting potential (A) and its eletrogenic pump component (B) in cultured myotubes. Curves showing the relation of E/n to age in HCM and low density medium (LDM) are represented by open and tilled circles. rcspcctivcly. After 3 DIV. medium from HCM cells was added to LDM cells. and the data from these myotubcs arc represented by the filled triangles. Each point in (A) is the average of the mean membrane potential of 3 experiments. In each experiment the mean value was detcrmincd from measurements of On in Y-12 myotubes in each of 2 dishes. Points in (B) are the average of the values of clectrogenic pump component determined in each of the ?I experiments
5DIV INTROL
B ARA-C
mv
CONTROL
ARA-C
L
Fig. 4. Effects of cytosine arabinoside treatment on membrane potential and number of [-‘Hlouabain binding sites in cultured rat myotubes. Data were obtained at 5 (A) and 7 (B) DIV. Membrane potentials were recorded at 23°C (0). 37°C (QB1,and at 37°C following ouabain (m). To the right of each set of membrane potentials is the plot of number of Na-pump sites. Values as in Fig. I. See text for further explanation.
due to Ara-C treatment were essentially non-existent days after removal of this substance from the growth medium).
differences
Effects
of non-specific
nutritional
by 7 days in culture (at least 4
supplements
Primary cultures of skeletal muscle are frequently grown in media supplemented with animal serum and chick embryo extract, both of which reportedly are required for adequate differentiation. Recent studies have shown that myotubes grown in serum-free, chemically defined medium, display morphological and biochemical properties similar to cells grown in serum.x A recent study from this laboratory has shown, however, that myotubes maintained in serum-free media are not
334
C. Brodie and S. R. Sampson
electrophysiologically fully functional.‘5 In this study we compared Em and its electrogenic component of cells suspended in growth medium supplemented with 15% horse serum (HS) with those in 5% HS. As shown in Fig. 5, the development of Em and the contribution by Na-K+ ATPase is slower when myotubes are grown from the time of initial plating in 5% HS than when in 15% HS, as indicated by the values for mean Em at 37”C, that in 15% HS being significantly higher (P
n BDIV
5DIV
I
15%HS
p+
Fig. 5. Effects
5%HS I
5 DIV
of growth in medium
containing 15 and 5% HS on resting potential ofcultured Each hnr graph as in Fig. 4. See text for further explanation.
myotuhes
Figure 6 shows that there is no difference between myotubes grown in medium supplemented with 1 l-day-old chick embryo extract (CEE) and those in CEE-free growth medium with regard to Em (P>O.5) and its ouabain-sensitive component, whether recorded at 4 days or 10 days in culture. In contrast, CEE from 17- to lPday-old chick embryos caused an increase in both Em at 37°C (P
DlSCUSSlON In recent years, several laboratories have reported on the growth and development of a variety of cultured cells, including skeletal muscle in serum-free, chemically defined media.5.7-“.‘” Cells grown in these media are reported to resemble those grown in conventional. serum-containing medium with regard to general biochemical and morphological properties. Nonetheless, many cultures, particularly skeletal muscle, are still grown in the presence of (horse) serum, as well as apparently because of the fact that myotubes do better under these chick embryo extract,“.“.”
Membrane
potential
and electrogenic
r
pump component
in cultured
muscle
335
A-4DIV
N3
CEE 1
B-10
JO CEE
DIV
CEE 17 DAY
Fig. 6. Effects of chick embryo extract (CEE) on resting potential of J- (A) and IO- (B) day-old myotuhes in culture. Each bar graph as in Fig. 4. See text for further explanation.
rat
Moreover, a recent report from this laboratory” showed that, at least in the case of electrophysiological properties of mammalian skeletal muscle, the development in serum-free medium is incomplete. In this case, the deficiency was accounted for by a decrease in activity of Na-K ATPase in cultures grown in serum-free medium compared to that in conventional medium. We have shown that this enzyme contributes an important component (electrogenic) to the membrane potential of cultured rat myotubes.‘.3 The results of the current study show that the enzyme is also influenced by a variety of specific and non-specific treatments and conditions routinely used in preparation of skeletal muscle cultures. It is, at present, only possible to conjecture as to the possible mechanisms underlying some of the phenomena described here. For example, we found that treatment of cultures with cytosine arabinoside delays the appearance of the enzyme. It is not clear, however. if this effect is due directly to the effects of this substance or is secondary to the decrease in total cell number as a result of the arrest of fibroblast proliferation, as we also found that total cell density at the time of plating (which includes both myoblasts and fibroblasts) markedly affects expression of Na-K ATPase (Fig. 7). In the case of the comparison between high and low serum concentration on expression of Na-K ATPase, we found that high serum concentration was important to Em and its electrogenic component only prior to cell fusion. If the serum concentration was lowered after fusion was complete, there was no difference between cells in low serum and those in high serum. As the rate and extent of fusion, as judged by visual inspection of the cultures, seemed to be about equal for cells plated in different concentrations of serum, the difference in membrane potential would seem to be due to factors unrelated to fusion specifically, although this process does determine the appearance of Na-K ATPase.’ One of the other factors likely to occur for the serum effect on appearance and activity of the Na-K ATPase might be stimulation of Na+ entry, as has been shown to occur in fibroblast and neuroblastoma cultures.‘x.2’ The extent to which other factors may be involved in mediating the effects of serum is not clear as these are not fully understood. I” The studies on effects of chick embryo extract may be considered somewhat surprising in view of the inclusion of this material as a matter of routine in preparation of muscle cultures.” We conditions.
336
C. Brodic and S. R. Sampson
found that there was essentially no difference in Dn or its electrogenic pump component between myotubes in medium with chick embryo extract and those without it. These data appear to be in
et cd. ” and Hooisma
contrast to those of Hagiwara
et al. .I2 who reported that chick embryo ex-
tract was one of several essential factors for growth of chick muscle in culture. Whether or not this represents a fundamental difference between rat and chick muscle in culture is unclear. When extract of older chick embryos was used, we found that electrogenic pump component of Em increased substantially over that of control myotubes with or without extract of young chick embryos. This might possibly be related to the more highly developed nervous system and accompanying neurotropic effects. Clearly, the most important factor regulating the appearance of Na-K ATPase. as expressed by the electrogenic pump component of membrane potential. was plating density. We found that both Et72 and its ouabain-sensitive component were directly related to plating density. Indeed, plating at low density resulted in complete non-expression of an electrogenic pump when cells were prepared from fetal rats. This probably explains the failure of Ritchie and Fambrough” to observe any effects of ouabain in their preparation of myotubes, which were plated at a density of 5 x 10’/ml.
Cell plating density has been shown to influence the expression of properties
of other
cells in primary culture, such as rat heart and granulosa cells from porcine ovaries. “.” The higher Em and electrogenic pump activity in myotubes plated at high density rather than low density appears to be due to the release of a factor (or factors) density “conditioned”
medium
added to myotubes
by the former,
as we found that high
plated at low density caused their Err? and
electrogenic pump component to increase. Other studies have shown that myotubes as well as other primary cells synthesize and release factors that influence growth of other cell types in culture. ‘t’.zJ As far as we are aware, there is only one other report of factors released by myotubes in cuiture
to influence
other myotubes,
this by Vandenburgh,”
who reported
such effects for
chick myotubes in culture, The present study thus appears to be the first description of a factor (or factors) released by cultured mammalian muscle. The nature of this factor. and regulation of its synthesis and release. remain to be investigated. The results presented here demonstrate that there is a strong correlation
between
the level of
resting membrane potential and the size of its electrogenic pump component. This correlation is strengthened by the additional observation that in those conditions in which the electrogenic pump component is not yet fully expressed (treatment with Ara-C, plating in low serum concentration, low cell density), changes in temperature from 23 to 37°C are relatively ineffective in altering
the level of Em. These findings provide
ATPase
plays an important
role in regulation
additional
support
of &rz in cultured
to the idea that Na+-K’
rat skeletal muscle.-?.3
WC Arkrrna,/~,cl~c,rtlctlrs-Supported in part by the Rescarch Authority. Bar-llan University. and Yad Hanadiv, T&Aviv. w*ish to acknowledge the technical assistance of Mrs Asia Bak in preparing the cultures, and to thank Mrs Bluma Ledcrhendlcr for expert typing of the manuscript.
REFERENCES 1, Bannett R. R.. Sampson S. R. and Shainberg A. (1983) lnflucnce of thyroid hormones on some electrophysiological properties of developing rat skeletal muscle cells in culture. Rrcri~t Rex. 294, 75-82. 2. Brodie C.. Bak A. and Sampson S. R. (1985) Dependenee of Nit+- K’ ATPase and electrogenic component of Etn in cultured myotubes on cell fusion. Brc~in Rev. 36, 384386. 3. Brodie C. and Sampson S. R. (1985) Contribution of electrogenic s~~diurn-p‘~tassium ATPase to resting membrane potential of cultured rat skeletal myotuhes. Rruitt Res. 37, 28-35. J. Castro J.. Maqued~~n~~ A. and Olive M. (19X-t) Lipid synthesis in isolated rat hcpatocytes: ~i~tiv~Iti‘~ll by insulin and vanadatc and inhibition by ouahain. Rio&ertr. ltzf. 9. Jl3-t20. 5. Claycomh W. C. (1980) Culture of cardiac muscle cells in serum-free media. fC.x-pi( ‘M Rt_;. 131, 7.1 l-736. 6. Dryden W. F.. Erulkar S. D. and de la Haha G. (1974) Properties of the cell membrane of developing skclctal muscle fihres in culture and its sensitivity to acetyicholine. c‘liiz. r.r,~. Phanrroc. Physiol. I ( X19-3X7. 7. Ewton D. Z. and Florini J. R. (19X1) Effects of the somatomedins and insulin in myohlast diffcrentiatirm i!r t,i/ro. L)cjt,/ Biol. 86, 3 I-3‘. 8. Florini J. R. and Roherts S. F. (lY79) A serum-free medium for the growth of muscle ct’lls in culture. /!I Vi/w 15,
983-002. 0. Florini J. R. nnd Ewton D. Z. (1981) Imulin acts as a sorn~~torneclitl-;ln~~l‘~~in stimulattng myohlas~ growth in serumfree medium. I/t Virro 17, 7h.F-76X. IO. Gospodarowicz 55s.
D. and Moran
J. S. (1976)
Growth
factors in mammalian
cell culture.
.4\,rrr. Kcr,. Hiochcrn. 45, 531-
Membrane
potential
and electrogenic
pump component
in cultured
muscle
337
I I.
Hagiwara Y.. Kimura I. and Ozawa E. (19X1) Chick embryo extract. muscle trophic factor and chick and horse sera as environments for chick myogenic cell growth. Det~I Grow/h Differ. 23, 249-254. 12. Hooisma J.. De Drijger J.. De Groot D. M. G.. Magchietse T. and Muijser H. (IYXI) Effects of extracts of denervated muscles on the morphology of cultured muscle cells. Nerrrosci. Lett. 22. 47-50. 13. lnestrosa N. C. (19X2) Differentiation of skeletal muscle cells in culture. Cc// Sfrm-f. F~rrc/. 7, 91-109. 14. Kaplan J. G. (1978) Membrane cation transport and the control of proliferation of mammalian cells. Awr. Rell. Physiol. 40. 19--11. IS. Kidokoro Y. (1975) Developmental changes in membrane electrical properties in a rat skeletal muscle cell line. J. Physiol. 244. I2Y- 143. In. Mather J. P. and Sato G. H. (1979) The use of hormone-supplemented serum-free media in primary cultures. Expl Cell Rev. 124. 2 15-221. 17. May J. V. and Schomberg D. W. (1984) The effects of plating density on granulosa cell growth and differentiation in
vitro. IX. 19.
20. 21. 22.
23. 24.
25.
Molec.
cell. En&x-r.
34, 201-213.
Moolenaar W. H.. Mummery C. L., van der Saag P. T. and de Laat S. W. (19X1) Rapid ion events and the initiation of growth on serum-stimulated neurohlastoma cells. Cell 23, 7X9-79X. Ritchie A. K. and Fambrough H. M. (1975) Electrophysiological properties of the membrane and acetylcholine receptor in developing rat and chick myotubes. J. gcn. Physiol. 66, 327-355. Sampson S. R.. Bannett R. R. and Shainberg A. (19X2) Effects of thyroxine on transmembrane resting potentials of skeletal muscle cells in culture. J. Nerrrosci. Rev. 8, 5YS-601. Smith J. B. and Rosengurt E. (197X) Serum stimulates the Na ’ . K + pump in quiescent fibroblasts by increasing Na’ entry. Proc~. trnnr. Accrd. Sci. U.S.A. 75, SS60--5.564. Speicher D. W.. Peace J. N. and McCarl R. L. (19X1) Effects of plating density and age in culture on growth and cell division of neonatal rat heart primary cultures. I/r Vifro 17. X63-X70. Vandenburg H. H. and Kaufman S. (19X1) Stretch-induced growth of skeletal myotubes correlates with activation of the sodium pump. J. cdl. camp. Physiol. 109, 20.5-214. Vandenburg H. H. (19X3) Cell shape and growth regulation in skeletal muscle: exogenous versus endogenous factors. J. cell. amp. Physiol. 116. 363-371 Yoles E.. Bak A. and Sampson S. R. (10X4) Some electrophysiological properties of developing rat skeletal myotubes grown in serum-free. chemically detined medium. /!I/. J. t/e~d Nmtrosci. 2, JX3-4YO.