cellca/d0m(1990)11.
343-352
QLongmanGroupUKUdlWO
Effects of thyroid hormone on Ca*+ efflux and Ca*’ transport capacity in rat skeletal muscle M.E. EVERTS Institute of Physiology, University of Aarhus, Arhus, Denmark Abstract The present study was undertaken to investigate the effects of 3,5,3’-triiodothyronine (T3) treatment on passive Ca2+ efflux, Ca2+-dependent Mg2+-ATPase (Ca2+-ATPase) concentration and active Ca2+ transport in isolated rat skeletal muscle. In addition, the question was examined whether changes in Ca2+ efflux at rest and during electrical stimulation in the hyperthyroid state were accompanied by parallel changes in 3-0-methylglucose efflux. The resting Ca2+ efflux from rat soleus muscle was increased by 25% after 8 days of treatment with T3 (20 pg/lOO g body weight). This was associated with a 78% increase in the basal efflux of 3-0-methylglucose. Electrical stimulation resulted in a rapid stimulation of Ca2+ efflux and 3-0-methylglucose efflux in the two groups of rats, and the levels obtained were significantly higher in the THreated group. The stimulating effect of the alkaloid veratridine on Ca2+ efflux was 60% larger in &day hyperthyroid rats. Within 24 h after the start of T3 treatment, a significant (21%) increase in Ca2+-ATPase concentration was detected. Significant increases in active Ca2+ uptake and passive Ca2+ efflux were not observed until after 2 and 3 days of T3 treatment, respectively. It is concluded that T3 stimulates the synthesis of Ca2+ ATPase and augments the intracellular Ca2’ pools (sarcoplasmic reticulum and mitochondria). The latter results in enhancement of the passive Ca2+ leak, which in turn, may lead to activation of substrate transport systems. The suggested increase in intracellular Ca2+ cycling after T3 treatment may, at least partly, explain the T3-induced stimulation of energy metabolism. Skeletal muscle constitutes the major target organ for the thermogenic action of thyroid hormone. It has repeatedly been shown that thyroid hormone stimulates the proliferation of the sarcoplasmic reticulum (SR) in skeletal muscle, thereby increasing the rate as welI as the capacity for calcium accumulation [l-4]. These observations may explain the fact that thyroid hormone increases the rate of relaxation 15, 61 and simultaneously decreases the energetic efficiency of skeletal muscle during work [7-91. The central role of Ca2’ in these changes has been depicted in a previously described model [ 10, 111. The present study was undertaken to
investigate the passive Ca2+ leak from isolated rat muscle at rest as well as under various conditions of depolarization (veratridine, electrical stimulation, The significance of a rise in high Ktoud. cytoplasmic Ca2+ for activation of glucose transport in isolated rat muscles is well documented 112, 131. Therefore, the question was addressed as to whether the thyroid hormone induced changes in Ca2’ efflux were associated with parallel changes-in activation of the glucose transport system. In an earlier study 1141, it was observed that thyroid hormone enhanced the passive leaks of Nat and K+ before any increase in Nat-Kt-dependent 343
344
ATPase concentration could be detected. Therefore, the present study also included investigation of the time-course of the effect of T3 treatment on passive Ca2’ leaks, the synthesis of Ca2+-dependent Mg2+-ATPase and active Ca2+ transport.
Materials and Methods Animals All experiments were carried out using fed female Wistar rats in the weight range 60-70 g (4 week old). The animals had free access to food and water and were kept in a thermostated environment (23°C) with constant humidity (55%) and day length (12 h). Three to four week old rats were injected subcutaneously with T3 (20 pg/lCO g body wt) once daily for periods lasting from 1 up to 8 days. Control rats received an equal volume of the solvent (154 mmolll NaCl containing 1% bovine serum albumin) for a similar period of time. In all animals, body temperature was measured and found to increase progressively with the duration of T3 treatment [14]. T3 treatment for 8 days has earlier been shown to increase body temperature by 1°C [14], the basal oxygen consumption of the perfused hind limb by 25% [lo] and the ratio of heart weight to body weight by 55% [4].
Muscle preparations and incubations Animals were killed by decapitation and the soleus muscle and extensor digitorum longus (EDL) muscles were dissected out as previously described [ 15, 1G].The standard incubation medium was Krebs-Ringer bicarbonate buffer @H 7.4) containing in mmol/l: 120.2 NaCI, 25.1 NaHC03, 4.7 KCl, 1.2 KH2pO4, 1.2 MgS04, 1.3 CaCh and 5 D-glucose. Incubations took place at 30°C under continuous gassing with a mixture of 95% 02 - 5% CO2 in a volume of 2-3 ml. After preparation, the muscles were equilibrated in the standard medium for 20 min and then taken for further incubation. This procedure has been shown to allow the maintenance of constant Kt, Nat and Ca2’ contents for several hours in vitro [4, 171.
CELL CALCIUM
Electrical stimulation An experimental set-up was developed allowing the simultaneous direct stimulation of 12 isolated muscles. Each muscle was stimulated by two platinum electrodes surrounding the central part of the muscle. After equilibration, the muscles were stimulated at a frequency of 50 Hz, with pulses of 1 ms duration and an amplitude of 10 V. Frequency, amplitude and pulse width were checked on a Telequipment DlOl 1 oscilloscope. 45Ca2+eJ54.x These experiments were performed using standard techniques described previously [ 181. Following equilibration, the muscles were loaded for 60 min in Krebs-Ringer bicarbonate buffer containing 45Ca2t (2 @J/ml). This was followed by wash-out through a series of tubes containing non-radioactive buffer without or with the additions indicated. At the end of wash-out, the muscles were blotted, weighed and homogenized in 2 ml of 0.3 moY1 trichloroacetic acid (TCA). After centrifugation, 1 ml of the clear supematant was withdrawn for counting. TCA was added to the wash-out tubes to a final concentration of 0.3 molfl, and 1 ml samples were taken for counting. 45Ca2t activity of the muscles and the wash-out tubes was determined by liquid scintillation counting, and with the use of a computer programme, curves describing the time course of the changes in the fractional loss of isotope from the muscles could be constructed. 3-0-[‘4C]-methylglucose eJT2.u It has ,been shown that activation of the glucose transport system can be quantified by measurement of both the influx as well as the efflux of the non-metabolizable sugar 3-O-methylglucose [ 15, 191. Measurements of 3-O-r’ 4C]-methylglucose efflux were performed exactly as described for 45Ca2t efflux, but with 3-0-[14C]-methylglucose (2 @i/ml and 1 mmol/l) in the loading medium. 45Ca2t uptake This was measured essentially as earlier described
THYROID HORMONE & CALCIUM ION EXCHANGE IN MUSCLE
[4, 201. After equilibration, the muscles were incubated for 4 x 60 min in Krebs-Ringer bicarbonate buffer containing 45Ca2t (1 @i/ml). This was fo”owed by four consecutive washings (each lasting 30 min) at 0°C in 3 ml Ca2+-free Krebs-Ringer bicarbonate buffer containiing EGTA (0.5 mmoY1). Finally, the muscles were blotted, weighed and homogenized in 2 ml of 0.3 mol/l TCA. 45Ca2+ activity was measured in the clear supematant obtained after centrifugation by liquid scintillation counting. 45Ca2t taken up by the muscles and retained after the wash-out in the cold was expressed as nmol/g wet weight. From the time-course of 45Ca2t uptake in isolated rat soleus muscle described previously [4], it was inferred that the 45Ca2+uptake after 4 h of incubation will reflect the exchange of 45Ca2t with the intracellular Ca2’ pools, i.e. the SR and the mitochondria. Ca2“‘-ATPase determination The concentration of Cazt-ATPase was determined by measurement of the Ca2’ -dependent steady state phosphorylation from [y-‘%]ATP in crude muscle homogenates [21]. Individual soleus or EDL muscles were homogenized at 0°C in buffer containing in mmol/l: HEPES 5 and sucrose 300 (pH 7.4). The final tissue content of the homogenates was 10 mg/ml. Two hundred pl ahquots of the homogenates were incubated for 30 s at 0°C in a reaction-mixture containing in mmol/l: imidazole 100, KC1 100, MgClz 5, EGTA 0.5, ATP 0.05., [Y-~%IATP (0.3 @J/ml) pH 7.4) in the absence or presence of CaClz (0.55 mmoY1). The reaction was quenched with TCA. After repeated washings of the pellet with TCA at O”C, the pellet was dissolved in NaOH and neutralized with &S04. [32p] activity was measured by liquid scintillation counting. The Ca2+-ATPase concentration was calculated as the difference in [3aP] incorporation in the presence and absence of CaC12. It was previously shown that 99% of the Ca2’dependent r32p] incorporation could be removed by hydrox lamine after acid precipitation indicating that [3$ ] is bound to an aspartic acid residue [21]. In addition, it was ascertained that measurement of the Ca2+-dependent r3?‘l incorporation in homogenates quantified the total Ca2+-ATPase
345
concentration. This was done by comparing the molecular activity calculated from the 3-0-methylfluorescein phosphatase activity and the Ca2’-dependent [3%‘] incorporation for an isolated SR preparation and the muscle homogenates [21]. Protein determination The protein content of the crude homogenates was measured according to the method of Lowry et al. [22] using bovine serum albumin as a standard Chemicals All chemicals were of analytical grade. Bovine serum albumin, T3 and veratridine were products from the Sigma Co., St Louis, MO, USA. 3-0-methylglucose was obtained from Calbiochem, Los Angeles, USA. Dantrolene was a gift from Eaton, Norwich, NY, USA. 45Ca2t (367 Ci/mol) was obtained from the Danish Atomic Energy Commission, Denmark 3-o-[t4c]ws0, methylglucose (50 Ci/mol) was from The Radiochemical Centre, Amersham, UK, and [Y-~%]ATP (33.8 Wmmol) from NEN, Boston, MA, USA. ]y-‘%]ATP was purified by column chromatography [23]. Statistics All results am given as mean values f SE with the number of observations in parentheses. The significance of difference between group means was calculated using the two-tailed Student’s t-test for non-pained observations. A P value < 0.05 was regarded as statistical significant. This is indicated in the figures and tables by an asterisk.
Results
Erects of veratridine on 45C2t ef/lux T3 treatment for 8 days increased basal 45Ca2’ efflux from rat soleus muscle by around 25% (P < 0.05) (Fig. lA, 60-120 mm). Exposure to veratridine (0.1 mmol/l) which depolarizes the muscle membrane through an action on the
CELL CALCIUM
346
0.03
Fraction of “Co lost per min (min-‘)
0.02
0.01
0.00 0.02
11%xtion
of ‘%I Iost per min (min-‘)
0.01
o.oo- 60
60
100 120 140 Duration of washout (min)
Fig. 1 Effects of veratridine presence
(B) of dantrolene
soleus muscle of euthyroid rats received
without
to the wash-out activity adding
was
injections
buffer
by wash-out
2 ml of unlabelled or with
homogenized
the
containing Krebs-Ringer indicated.
of T33 (20 pg/lOO g
45Ca2’ (2 pCi/ml). bicarbonate The
buffer
muscles
were
in 2 ml TCA (0.3 mol/l) and TCA was also added tubes to a final concentration determined
by liquid
in the wash-out
muscles at the end of the experiment, In the experiments
of 0.3 molll. 4sCa2+
scintillation
counting.
By
tubes to that in the
the fractional
loss of “Ca”
shown in panel B, dantrolene
(10 )rmol/l) was present from the onset of wash-out. Curves
represent
measurements compared
on
the 6-12
to euthyroid
mean
with
muscles.
bars
45Ca2+ and 3-O-[‘4C]-methylglucose [email protected] electricalstimulation
efflux from
rats. Three week old
at 3o’C through a series of tubes
additions
the 45Ca2+ activity
was calculated.
on “Ca*+
the muscles were loaded for 60 min at 3o’C in
bicarbonate
This was followed containing
(10 pmol/l) and hyperthyroid
or the solvent alone for 8 days. After preparation
and equilibration, Krebs-Ringer
160
(0.1 mmoV1) in the absence (A) or
daily subcutaneous
body weight)
160
volta e-dependent Nat channels [24] increased 45Ca8’ efflux considerably in both groups of rats (Fig. lA, 120-180 rnin). The peak response was observed after 30-40 min of exposure. Maximum 45Ca2+’efflux in the presence of veratridine was 60% higher in hyperthyroid soleus than in euthyroid soleus muscle. The relative increase in 45Ca2t efflux induced by veratridine as compared to the basal 45Ca2f efflux was 110% in euthyroid soleus and 169% in hyperthyroid soleus muscle. To see whether release of Ca2’ from intracellular Ca2’ stores was involved in the veratridine-induced increase in 45Ca2+efflux, experiments were repeated in the presence of dantrolene (10 pmol/l) which has earlier been shown to inhibit the high Kf-induced increase in 45Ca2+ efflux [ 101. Dantrolene did not affect the resting 45Ca2t efflux from euthyroid soleus muscle, but reduced 45Ca2t efflux from hyperthyroid soleus muscle and the difference between the two groups was no longer significant (Fig. lB, 60-120 min). The veratridine-induced increase in 45Ca2t efflux was inhibited by dantrolene by 44% in euthyroid soleus (not significant). In hyperthyroid soleus muscle, dantrolene inhibited the response to veratridine by 57% (P < 0.05) and the difference between control and T$-treated rats was no longer significant (Fig. lB, 120-180 min).
denoting
SEM
*P < 0.05,hyperthyroid
of
Possibly due to the attachment to the electrodes, the initial phase of the resting 45Ca2t efflux in the euthyroid and the hyperthyroid group was slightly different in Figure 2A (60-90 min) as compared to Figure 1A (60-120 min). Nevertheless, at t = 85 min. the values for 45Ca2t efflux in Figure 2A were comparable to those of Figure 1A and similarly the effect of T3 treatment amounted to around 22% (P < 0.05) (Fig. 2A, 60-90 min). At the same time, the basal efflux of 3-0-[‘4Clmethylglucose was increased by 78% (P < 0.05) (Fig. 2B, 60-90 min). Electrical stimulation for 1 min at a frequency of 50 Hz induced an increase in the efflux of 45Ca2f (Fig. 2A, 90-140 min). The maximum increase amounted to 83 and 91% in soleus muscle of euthyroid and hyperthyroid rats, respectively. Peak
THYROID
HORMONE
& CALCIUM
ION EXCHANGE
IN MUSCLE
B
sib
IdO Duration
Ng. 2 Effects of electrical
60
Il;o
of washout (mm)
stimulation
at 50 Hz (1 min) on the
efflux
of 45Ca2+ (A) and 3-O-[14C]-methylglucose
soleus
muscle
of euthyroid
treated as described and equilibration, @i/ml)
and hyperthyroid
in the legend to Figure the left muscles
and
3-O-[‘4C]-methylglucose
(2 @i/ml
loss of isotope was measured
were loaded
frequency Each
electrical
were
with ‘%a’+
(2 with
and 1 mmol/l). The fractional
as described
stimulation
from
muscles
in the legend to Figure
1. Each muscle was attached to an electrode wash-out,
(B)
rats. Rats
1. After preparation
contralateral
the
1BO
was
and after 90 min of
applied
for
1 min at a
of 50 Hz (10 V, 1 ms).
curve
measurements
represents
the mean
on 6 muscles.
with bars denoting
*P < 0.05, hyperthyroid
SEM of
347
respectively (Fig. 2B, 90-140 min). Peak values of 3-0-[‘4C]methylglucose efflux during stimulation were significantly (P < 0.05) higher in the hyperthyroid group. Around 25 min after cessation of stimulation, 3-O-[‘4C]methylglucose efflux started to decrease, but it had not returned to the basal level by the end of wash-out in either group. One might expect to underestimate the rise in 45ca2+ efflux due to short stimulation when measurements are performed over 10 min intervals. Therefore, the experiments described in Fi ure 2A were repeated with measurements of 45Ca& efflux over 2 min intervals. When measured during the first 2 min after the onset of stimulation a rapid increase in 45Ca2+ efflux was observed that amounted to 189% in euthyroid (n = 6) and to 167% in hyperthyroid (n = 5) soleus muscle. The peak level of 45Ca2+efflux immediately after the onset of stimulation did not differ between the two groups. The maximum 45Ca2t efflux was followed by a decrease in both groups, and the level in hyperthyroid rats remained around 20% (P < 0.05) higher than in euthyroid rats. 45Ca2t efflux had not returned to the starting level within the four 2 min periods following the cessation of stimulation in either of the groups (data not shown). In a separate series of experiments, the effect of dantrolene (10 pmoY1 on the electrical stimulation 5’ induced increase in 4 Ca2’ efflux was investigated. These measurements, which for practical reasons were performed over 10 min intervals, showed that dantrolene caused no significant suppression of 4sca2+, efflux induced by electrical stimulation, neither in euthyroid nor in hyperthyroid rats (data not shown).
compared
to euthyroid
C~I~~-ATP~~~concentration
values for 45Ca 2+ efflux during stimulation were significantly (P < 0.05) higher in the hyperthyroid group. Durir~~ y+ 40 min of wash-out following stimulation, Ca efflux decreased slowly and had by the end of the experiment completely returned to the starting level in hyperthyroid rats, but not in increase in The relative euthyroid rats. 3-O-[‘4C]methylglucose efflux after the onset of electrical stimulation amounted to 365 and 218% in euthyroid and hyperthyroid soleus muscle,
The effect of T3 treatment for 8 days on the concentration of Ca2’-ATPase was investigated in soleus and EDL muscle of 4 week old rats (Fig. 3). In Ca2+-dependent euthyroid rats, [3+1 incorporation was roughly 5.5 fold higher in EDL than in soleus muscle. T3 treatment increased the Ca2+-dependent [3%] incorporation by around 120% in soleus muscle, and had only a small (7%, not significant) effect in the fast-twitch EDL muscle. These results are in good agreement with
CELL CALCIUM
348
Ca2+ 40
dependent
(nmol/g
32P
content of the soleus muscle homogenates amounted to 147 + 4 mg/g wet weight (n = 5) and 153 + 5 mg/g wet weight (n = 5) in euthyroid and 8 day hyperthyroid rats, respectively. Total protein content of the EDL muscle homogenates amounted to 168 + 5 mg/g wet weight (n = 4) and to 169 + 7 mglg wet weight (n = 4) in euthyroid and 8 day hyperthyroid rats, respectively.
incorporation
wet wt) EDL
30
20
Time course Ca2’-ATPase
Soleus *
of the efSect o T3 treatment on d concentration, 5Ca2’ uptake and
45Ca2+eflux
10
0
(9) (6)
(10)(6)
Ca2+-dependent [3%] incorpation in soleus and EDL muscle of euthyroid and hyperthyroid rats. Animals were treated as described in the legend to Figure 1. Ca2’-dependent [‘“PI incorporation was measured in homogenates of individual EDL and soleus muscles as described in Materials and Methods. Results represent means of measurements on 6-10 muscles from different euthyroid (open baxes) and hyperthyroid (hatched boxes) animals. *P < 0.05 hyperthyroid compared to euthyroid Fig. 3
observations on relaxation rate [5] and calcium content [4] and indicate that T3 treatment does not result in a proliferation of the SR and an increase in Ca’+-ATPase concentration in muscles consisting predominantly of fast-twitch fibres. Total protein Table 1 Tie course of the effect rat soleus muscle
In a recent study [14] we have reported that an increase in Nat-Kt-ATPase concentration in muscle after TJ treatment was preceded ,by an increase in the passive fluxes of K and Na . In view of the effects of T3 on 4sCa2’ efflux and Ca2+-ATPase concentration, it was interesting to investigate whether a similar relationship could explain the increase in Ca2+-ATPase concentration in rat soleus muscle. Quantification of the Ca2+-ATPase concentration measurement of Ca2’-dependent 13%] by incorporation showed a significant increase (21%) after 1 day of T3 treatment. Thereafter, the Ca2’-dependent r3%] incorporation increased progressively with the duration of the T3 treatment reaching a level of 139% above the controls after 8 days (Fig. 4A). When the capacity for Ca2+ accumulation into the intracellular organelles of the
of T33 treatment for 1-8 days on basal and 20 mM K+-stimulated “Ca2’ efflux fi-om
Fraction of 45Ca2’ lost per min (min-‘) Basal
+ 20mMp
Controls
0.0063 f 0.0001 (9)
1 day T3 2 days T3 3 days T3 8 days T3
0.0066 If:0.0002 (5)
0.0159 f 0.0008 (5)
0.0064 f 0.0002 (10) 0.0069 f omO3 (10) 0.0079 f 0.0004 (9)*
0.0185 zt 0.0016 (10) 0.0185 fO.OO1O (lo)* N.D.
0.0156 rt 0.0010 (9)
Results show means f SEh4 with the mmber of animals in parentheses. Rats were treated as described in the legend to Figure 4. ‘l%e fmctional loss of%a* fium s&us muscle was detumined as described in the legend to Figure 1. Basal values represent data obtained after 80 min of wash-out, and vahm in the preseme of 20 mm&l K’ were obtained 40 min afk correspmdingtothepeakinmeme.
*P < 0.05, T3-treated BScompared to euthymid controls
the onset of exposure to high IC, i.e.
rHYROID
HORMONE
& CALCIUM
2
0 150
4
Relative 45Ca
ION EXCHANGE
6
increase
uptake
IN MUSCLE
sign&ant increase (16%) was observed after 2 days of T3 treatment. 45Ca2t uptake capacity continued to increase up to 8 days of T3 treatment and reached a level of 119% above the controls (Fig. 4B). The time course of the effect of T3 treatment on 45Ca2t efflux was measured in the absence and in the presence of 20 mmol/l K+ (Table 1). Since the effect of 8 days of T3 treatment on the basal 45Ca2+ efflux amounted to only 25% (Figs 1A and 2A), it 45ca2t might be easier to detect early chan+ges in efflux in the presence of high K (which will stimulate 45Ca2 efflux). As can be seen in the left column, a significant increase (25%) in basal 45Ca2t efflux was observed after 8 days. When 45Ca2t efflux was stimulated by 20 mmoY1 K’, a significant (19%) increase could be detected after 3 days of T3 treatment, indicating that more Ca2’ can be mobilized from the SR after this short period of T3 treatment.
8
in
(percent)
1
*
*
/
Discussion
“1 / 4-&&TB
Duration
Fig.
Time
4
Ca*+-dependent
of T3-treatment
course
of
the
rat soleus muscle. with ‘I’3 (20 p/l00 Ca*+-dependent Materials during
and
individual
Methods.
bicarbonate containing
buffer
all animals
incorporation soleus
incubation
on
were 4 weeks old. was
muscles
containing
period
measured as
with
45Ca2+ (1 @/ml),
EGTA (0.5 mmol/l). ‘%a*’
The results represent controls.
treatment
increase
in
at 30-C
Krebs-Ringer followed
by
buffer
activity was measured
in
counting.
means of determinations
as the relative
in
described
for 4 x 30 min at 0°C in Ca*“-free Krebs-Ringer
TCA extracts by liquid scintillation arc given
T3
45Caz’ uptake was determined
a 4 x 60 min
wash-out
of
(A) and 45Ca2” uptake (B) in
3 week old rats were injected sulxutaneously
[3%‘] of
(days)
g body weight) or the solvent alone for 1 to 8
days. By the time of sacrifice, homogenates
effect
i3%‘] incorporation
on 4-8 animals and
as compared
*P < 0.05, Ts-treated compared
to euthyroid
349
to euthyroid controls
intact soleus muscle was estimated by measurement of 45Ca2+ uptake over a 4 h incubation period, a
The present study confii and extends the observations that thyroid hormone induces a proliferation of the SR leading to an increase in the pool of Ca2’ available for mobilization after depolarization as well as in an increased capacity for tesequestration of Ca2+ from the sarcoplasm [l-4, 10, 111. The major new observation is that a significant increase in the concentration of Ca2’-ATPase can be detected within 24 h of T3 treatment, whereas functional changes in the SR i.e. Ca2’ uptake and Ca2’ release were not observed until after 2-3 days of treatment. To indicate the size of the various calcium pools in soleus muscle from euthyroid and hyperthyroid rats, the results of previous measurements and calculations [4] are shown in Table 2. T3 treatment for 8 days induced a two-fold increase in total calcium content (1st line), without changing the calcium content of the extracellular space (2nd line). The amount of intracellular calcium exchangeable with 4?Ca2’ within the 6 h of the experiment showed a four-fold increase in hyperthyroid rats calcium line). The intracelhllar (3rd non-exchangeable with 45Ca2+ was similar in the two groups (4th line). The data indicate that the
CELL CALCIUM
350
reater part of muscle calcium is exchangeable with $‘Ca2+ and that the increase in total calcium content in hyperthyroid soleus muscle is accounted for by the increase in intracellular calcium pools, i.e. SR and mitochondria. The passive Ca2+ efflux expressed as the fractional loss of 45Ca2+ was increased by around 25% after 8 day of T3 treatment (Figs 1 and 2). This effect would be even larger if it is taken into account that the total calcium content was twice as high in the soleus muscle of hyperthyroid rats as compared to control rats (Table 2). Depolarization b veratridine induced a considerable increase in 4Jca2+ efflux which was 60% larger in 8-day hyperthyroid than in control rats. The response to veratridine could be partly inhibited by dantrolene (Fig. 1). In line with earlier measurements on stimulation of 45Ca2t efflux by high Ktout [lo] the observations indicate that the effect of veratridine is partly due to stimulation of Ca2+ release from the SR. In contrast, the effect of electrical stimulation on 45Ca2t efflux could not be inhibited by dantrolene (see description Fig. 2). It was earlier observed that the effect of dantrolene on the contractile force of rat soleus muscle stimulated at Table 2 Distributionof calcium in soleus muscle from euthyroid and hyperthyroid ratsCalciumcontent (nmoUg wet weight) Euthyroid
1090+40
Total Extracellular
space
(7)
Hyperthyroid
2200 f 320 (7)
446
449
Intracellulaf 45Ca2+exchangeable
354
1513
Iukacellular nonexchangeable with 4sCa2+
290
238
Data are calculated from the experimental results presented in [4]. Total calcium content was determined b%atomic absorption spectrophotometry, the intmcellular *Ca -exchangeable calcium was derived from extmpolation of %a* uptake curves measured overa 6 h incubation period, and the calcium content of the extracellular space was calculated BSthe amount of calcium lost during wy+h-out at 0°C. Inhacellular calcium non-exchangeable with Ca was calculated by subtmcting the calcium content of the extracellular space and the inhacellular 4sCa~-exchangeable calcium 6om the total calcium content (with permission t+om the American physiology Society)
50 Hz was relatively small compared to that observed at 1 Hz [6]. It is also possible that a considerable part of the Ca2+ released into the cytosol during electrical stimulation is derived from the extracellular compartment in a slow-twitch muscle like the soleus. This would imply that it is not inhibitable by dantrolene. The observation that 45Ca2f uptake increased 3-4 fold in soleus muscle of 4 week old euthyroid and hyperthyroid rats during 5 min of stimulation at 1 Hz (M.E. Ever& unpublished results) would support this idea. The increase in fractional loss of 45Ca2t due to electrical stimulation in the experiments with the 2-min intervals amounted to 0.0122 min-’ for euthyroid soleus and to 0.0131 mid’ for hyperthyroid soleus muscle. Again, in view of the increase in total calcium content, these data might reflect a much larger release of Ca2’ upon depolarization in the Ts-treated rat. This does not, however, necessarily imply that the free cytosolic Ca2’ concentration during contraction is higher in the hyperthyroid soleus muscle. Since the capacity for Ca2’ resequestration is also increased in the T3-treated group, the net result of enhanced Ca2’ release and a higher Ca2’ uptake rate might be that the intracellular free Ca2+ concentration is just as high as in control rats. The latter is suggested by the findings that the twitch force is similar in hyperthyroid and euthyroid soleus [6] as well as in ventricular [25, 261 muscle. In addition, measurements of the free cytosolic Ca2+ concentration with either aequorin or Furashowed no change in ventricular papillary muscle [25, 261 nor in isolated myocytes [27] from hyperthyroid animals. Electrical stimulation was associated with a 3-4 fold in increase prom@ 3-O-[‘4C]-methylglucose efflux in euthyroid and hyperthyroid soleus muscle (Fig. 2B). Similar large increases in the permeability of the muscle plasma membrane to glucose during electrical stimulation were earlier observed in rat soleus [15] as well as in frog sartorius muscle [19]. 3-0-[‘4C]-methylglucose efflux was significantly higher both at rest and during stimulation in the soleus muscle of hyperthyroid rats. The question may arise whether this is secondary to the higher 45Ca2t efflux observed in the Ts-treated group. It has been shown
THYROID
HORMONE
& CALCIUM
ION EXCHANGE
IN MUSCLE
that stimulation of 45Ca2+ efflux in rat soleus muscle by a variety of agents was associated with a proportional increase in 3-0-[t4C]-metbylglucose efflux [ 131. Apart from rat soleus muscle, this correlation has been observed in various tissues [12]. Nevertheless, it is still not known, how Ca2’ may activate the glucose transport system and how the conformational changes required for increased glucose transport capacity are brought about. In any case the present data support the view [l 11 that thyroid hormone allows the release of more Ca2’ upon activation, a response that is accompanied by increased mobilization of substrates. In agreement with our earlier observations [4] that T3 treatment for 8 days did not induce an increase in total calcium content or 45Ca2+uptake in EDL, muscle, the present study shows no changes in Ca2+-ATPase concentration in EDL muscle of hyperthyroid rats (Fig. 3) indicating that proliferation of the SR does not occur in the fast-twitch muscle. In a previous study, it was suggested that the increase in the concentration of Nat-Kt-ATPase after T3 treatment occurred secondary to increased passive fluxes of Nat and Kt [14]. The results of the present study do not indicate that an increase in the concentration of Ca2+-ATPase is brought about by a similar mechanism, i.e. an increase in the passive Ca2+ efflux from the SR. A significant increase in Ca2+-dependent [3%] incorporation was observed within 24 h of Ts treatment (Fig. 4A). This is not surprising in view of the observation that the mRNA level for the Ca2’ pump in the hypothyroid rat heart was already increased by 2 h of T3 treatment [28]. The observations that active Ca2+ uptake (Fig. 4B) and passive Ca2’ leak (Table 2) showed a more delayed increase suggest that these func.tionaI changes in Ca2’ transport can not take place until the whole SR membrane system has proliferated. Although it cannot be excluded that an increase in Ca2+-dependent [3%] incorporation is detected earlier than an increase in Ca2’ uptake because of a higher sensitivity of the method used, a discrepancy between changes in Ca2’-ATPase content and Ca2+ uptake has also been reported for the chronically stimulated EDL muscle of the rabbit [29]. Measurements on homogenates showed that the initial Ca2’ uptake rate and the total Ca2’ uptake
351
capacity of the SR were decreased by 50% after 2 days of stimulation, whereas the Ca2+-ATPase content was not changed [29]. This suggests more or less the reverse phenomenon of what is seen during T3 treatment. Although the quantitative significance of the effect of T3 on the SR for the energy consumption during work has been documented [9], the question whether the effect plays a role in the increase in basal metabolic rate still remains open. However, it was recently suggested that considerable Ca2+ recycling occurs in resting skeletal muscle [30]. In the same study it was calculated that the 30 % decrease in SR content upon thyroid hormone depletion could account for around one third of the decrease in basal metabolic rate. The reverse might be the case for Ts-treated skeletal muscle, but remains to be established.
Acknowledgements I thank Torben Clausen for stimulating discussions and critical reading of the manuscript, Tove Lindahl Andersen and Marianne Stiirup-Johansen for skilled technical assistance, and Lis Slcj6t for expert secretarial assistance. The study was supported by the Danish Biomembrane Research Center and the Research Foundation of the University of Aarhus.
References 1. Rim DH. Witzmann FA. Fitts RH. (1982) Effect of thyrotoxicosis on sarcoplasmic reticulum in rat skeletal muscle. Am. J. Physiol., 243. C151-C155. 2 Simonides WS. van Hardeveld C. (1985) The effect of hypothyroidism on sarcoplasmic reticulum in fast-twitch muscle of the rat Biochim. Biophys. Acta, 844, 129-141. 3. Simonides WS. van Hardeveld C. (1986) Effects of the thyroid status on the sarcoplasmic reticulum in slow skeletal muscle of the rat. Cell Calcium, 7, 147-160. 4. Everts ME. Clausen T. (1986) Effects of thyroid hormones on calcium contents and 45Ca exchange in rat skeletal muscle. Am. J. Physiol.. 251, E258E265. 5. Nicol CJM. Bruce DS. (1981) Effect of hyperthymidism on the contractile and histochemical properties of fast and slow twitch skeletal muscle in the rat. Pfliigers Arch., 390, 73-79. 6. Everts ME. van Hardeveld C. (1987) Effects of dantxolene on force development in slow- and fast-twitch muscle of euthyroid, hypothymid, and hyperthyroid rats. Br. J. Pharmacol., 92,41-54. I. Wiles CM. Young A. Jones DA. Edwards RHT. (1979) Muscle relaxation rate, fibre-type composition and energy
CELL CALCIUM
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8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
turnover in hyper- and hypo-thyroid patients. Clin. Sci., 57, 375-384. Everts ME. van Hardeveld C. Ter Keurs HEDJ. Kassenaar AAH. (198 1) Force development and metabolism in skeletal muscle of euthymid and hypothyroid rats. Acts Endocrinol., 97,221-225. Leijendekker WJ. van Hardeveld C. Elxinga G. (1987) Heat production during contraction in skeletal muscle of hypothyroid mice. Am. I. Physiol., 253, E214E220. Van Hardeveld C. Clausen T. (1984) Effect of thyroid status on K+-stimulated metabolism and 4SCa exchange in tat skeletal muscle. Am. J. Physiol., 247, E421-E430. Van Hardeveld C. Clausen T. (1986) Ca” and thyroid hormone action. In: Bader H. Gietzen K. Rosenthal J. Riide.1 R. Wolf HU. (eds). Intmcelluhu calcium regulation. Manchester University Press, pp 355-365. Bihler I. (1988) The role of membrane transport in the control of glucose metabolism and its coupling to cellular function. Can. J. Physiol. Pharmacol.,66, 549-560. Sorensen SS. Christensen F. Clausen T. (1980) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. X. Effect of glucose transport stimuli on the efflux of isotopically labelled calcium and 3-0-methylglucose from soleus muscles and epididymal fat pads of the rat. B&him. Biophys. Acta, 602,433-445. Ever& ME. Clausen T. (1988) Effects of thyroid hormone on Na+-K+ transport in resting and stimulated rat skeletal muscle. Am. J. Physiol., 255, E6OQE612. Kahn PG. Clausen T. (1971) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. VI. The effect of insulin, ouabain, and metabolic inhibitors on the transport of 3-0-methylglucose and glucose in rat soleus muscle. Biochim. Biophys. Acta, 225.277-290. Chinet A. Clausen T. Girardier L. (1977) Microcalorimetric determination of energy expenditure due to active sodium-potassium transport in the soleus muscle and brown adipose tissue of the rat. J. Physiol., 265, 43-61. Clausen T. Flatman JA. (1977) ‘Ihe effect of catecholamines on Na-K transport and membrane potential in rat soleus muscle. J. Physiol., 270, 383-414. Clausen T. Elbtink I. Dahl-Hansen AB. (1975) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. IX. The role of cellular calcium in the activation of the glucose transport system in rat soleus muscle. Biochim. Biophys. Acta, 375, 292-308. Holloszy JO. Narahara HT. (1965) Studies of tissue permeability. X. Changes in permeability to 3-methylglucose associated with contraction of isolated frog muscle. J. Biol. Chem., 240,3493-3500.
20. Clausen T. Dahl-Hansen AB. Elbrink J. (1979) The effect of hyperosmolarity and insulin on resting tension and calcium fluxes in rat soleus muscle. J. Physiol., 292, 505526. 21. Ever@ ME. Andemen JP. Clausen T. Hansen 0. $1989) Quantitative determination of Ca”-dependent Mg +-ATPase from samoplasmic reticulum in muscle biopsies. B&hem. J., 260,443-448. 22. Lowry OH. Rosebrough NJ. Fan AL. Randall RJ. (195 1) Protein measurement with the Folin phenol reagent J. Biol. Cbem., 193,265-275. 23. Norby JG. Jensen J. (1971) Binding of ATP to brain microsomal ATPase. Determination of the ATP-binding capacity and the dissociation constant of the enzyme-ATP complex as a function of KC concentration Biocbim. Biophys. Acta, 233, 104-116. 24. McKimrey LC. Ratzla!TRW. (1987) Sodium permeability of frog skeletal muscle in absence and presence of veratridine. Am. J. Physiol., 252, Cl90-C196. 25. MacKinnon R. Morgan JP. (1986) Influence of the thyroid state on the calcium transient in ventricular muscle. Pfliigers Arch., 407,142-144. 26 MacKinnon R. Gwathmey JK. Allen PD. Briggs GM. Morgan JP. (1988) Modulation by the thyroid state of intraceBular calcium and contractility in ferret ventricular muscle. Circ. Res., 63, 1080-1089. 27. Beekman RE. van Hardeveld C. Simonides WS. (1988) Ef%ct of thyroid state on cytosolic free calcium in resting and electrically stimulated cardiac myocytes. Biochim. Biophys. Acta, 969, 18-27. 28. Rohrer D. Dilbnann WH. (1988) Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca’+-ATPase in the rat heart J. Biol. Chem., 263, 6941-6944. 29. Leberer E. H&tner K-T. Pette D. (1987) Reversible inhibition of ssrcoplasmic reticulum Ca-ATPase by altered neuromuscular activity in rabbit fast-twitch muscle. Eur. J. B&hem., 162,555-561. 30. Simonides WS. van Hardeveld C. (1988) (Ca2+ + M 2+)-ATPase activity associated with the maintenance of a gl+ Ca gradient by sarcoplasmic reticulum at submicromolar external [Ca”]. The effect of hypothyroidism. B&him. Biophys. Acta, 943, 349-359.
Please send reprint request to : Dr Ma+a E. Everts, Institute of Physiology, University of Aarhus, 8000 Arhus C, Denmark. Received : 15 August 1989 Revised 19 December 1989 Accepted : 8 January 1990
343-352
QLongmanGroupUKUdlWO
Effects of thyroid hormone on Ca*+ efflux and Ca*’ transport capacity in rat skeletal muscle M.E. EVERTS Institute of Physiology, University of Aarhus, Arhus, Denmark Abstract The present study was undertaken to investigate the effects of 3,5,3’-triiodothyronine (T3) treatment on passive Ca2+ efflux, Ca2+-dependent Mg2+-ATPase (Ca2+-ATPase) concentration and active Ca2+ transport in isolated rat skeletal muscle. In addition, the question was examined whether changes in Ca2+ efflux at rest and during electrical stimulation in the hyperthyroid state were accompanied by parallel changes in 3-0-methylglucose efflux. The resting Ca2+ efflux from rat soleus muscle was increased by 25% after 8 days of treatment with T3 (20 pg/lOO g body weight). This was associated with a 78% increase in the basal efflux of 3-0-methylglucose. Electrical stimulation resulted in a rapid stimulation of Ca2+ efflux and 3-0-methylglucose efflux in the two groups of rats, and the levels obtained were significantly higher in the THreated group. The stimulating effect of the alkaloid veratridine on Ca2+ efflux was 60% larger in &day hyperthyroid rats. Within 24 h after the start of T3 treatment, a significant (21%) increase in Ca2+-ATPase concentration was detected. Significant increases in active Ca2+ uptake and passive Ca2+ efflux were not observed until after 2 and 3 days of T3 treatment, respectively. It is concluded that T3 stimulates the synthesis of Ca2+ ATPase and augments the intracellular Ca2’ pools (sarcoplasmic reticulum and mitochondria). The latter results in enhancement of the passive Ca2+ leak, which in turn, may lead to activation of substrate transport systems. The suggested increase in intracellular Ca2+ cycling after T3 treatment may, at least partly, explain the T3-induced stimulation of energy metabolism. Skeletal muscle constitutes the major target organ for the thermogenic action of thyroid hormone. It has repeatedly been shown that thyroid hormone stimulates the proliferation of the sarcoplasmic reticulum (SR) in skeletal muscle, thereby increasing the rate as welI as the capacity for calcium accumulation [l-4]. These observations may explain the fact that thyroid hormone increases the rate of relaxation 15, 61 and simultaneously decreases the energetic efficiency of skeletal muscle during work [7-91. The central role of Ca2’ in these changes has been depicted in a previously described model [ 10, 111. The present study was undertaken to
investigate the passive Ca2+ leak from isolated rat muscle at rest as well as under various conditions of depolarization (veratridine, electrical stimulation, The significance of a rise in high Ktoud. cytoplasmic Ca2+ for activation of glucose transport in isolated rat muscles is well documented 112, 131. Therefore, the question was addressed as to whether the thyroid hormone induced changes in Ca2’ efflux were associated with parallel changes-in activation of the glucose transport system. In an earlier study 1141, it was observed that thyroid hormone enhanced the passive leaks of Nat and K+ before any increase in Nat-Kt-dependent 343
344
ATPase concentration could be detected. Therefore, the present study also included investigation of the time-course of the effect of T3 treatment on passive Ca2’ leaks, the synthesis of Ca2+-dependent Mg2+-ATPase and active Ca2+ transport.
Materials and Methods Animals All experiments were carried out using fed female Wistar rats in the weight range 60-70 g (4 week old). The animals had free access to food and water and were kept in a thermostated environment (23°C) with constant humidity (55%) and day length (12 h). Three to four week old rats were injected subcutaneously with T3 (20 pg/lCO g body wt) once daily for periods lasting from 1 up to 8 days. Control rats received an equal volume of the solvent (154 mmolll NaCl containing 1% bovine serum albumin) for a similar period of time. In all animals, body temperature was measured and found to increase progressively with the duration of T3 treatment [14]. T3 treatment for 8 days has earlier been shown to increase body temperature by 1°C [14], the basal oxygen consumption of the perfused hind limb by 25% [lo] and the ratio of heart weight to body weight by 55% [4].
Muscle preparations and incubations Animals were killed by decapitation and the soleus muscle and extensor digitorum longus (EDL) muscles were dissected out as previously described [ 15, 1G].The standard incubation medium was Krebs-Ringer bicarbonate buffer @H 7.4) containing in mmol/l: 120.2 NaCI, 25.1 NaHC03, 4.7 KCl, 1.2 KH2pO4, 1.2 MgS04, 1.3 CaCh and 5 D-glucose. Incubations took place at 30°C under continuous gassing with a mixture of 95% 02 - 5% CO2 in a volume of 2-3 ml. After preparation, the muscles were equilibrated in the standard medium for 20 min and then taken for further incubation. This procedure has been shown to allow the maintenance of constant Kt, Nat and Ca2’ contents for several hours in vitro [4, 171.
CELL CALCIUM
Electrical stimulation An experimental set-up was developed allowing the simultaneous direct stimulation of 12 isolated muscles. Each muscle was stimulated by two platinum electrodes surrounding the central part of the muscle. After equilibration, the muscles were stimulated at a frequency of 50 Hz, with pulses of 1 ms duration and an amplitude of 10 V. Frequency, amplitude and pulse width were checked on a Telequipment DlOl 1 oscilloscope. 45Ca2+eJ54.x These experiments were performed using standard techniques described previously [ 181. Following equilibration, the muscles were loaded for 60 min in Krebs-Ringer bicarbonate buffer containing 45Ca2t (2 @J/ml). This was followed by wash-out through a series of tubes containing non-radioactive buffer without or with the additions indicated. At the end of wash-out, the muscles were blotted, weighed and homogenized in 2 ml of 0.3 moY1 trichloroacetic acid (TCA). After centrifugation, 1 ml of the clear supematant was withdrawn for counting. TCA was added to the wash-out tubes to a final concentration of 0.3 molfl, and 1 ml samples were taken for counting. 45Ca2t activity of the muscles and the wash-out tubes was determined by liquid scintillation counting, and with the use of a computer programme, curves describing the time course of the changes in the fractional loss of isotope from the muscles could be constructed. 3-0-[‘4C]-methylglucose eJT2.u It has ,been shown that activation of the glucose transport system can be quantified by measurement of both the influx as well as the efflux of the non-metabolizable sugar 3-O-methylglucose [ 15, 191. Measurements of 3-O-r’ 4C]-methylglucose efflux were performed exactly as described for 45Ca2t efflux, but with 3-0-[14C]-methylglucose (2 @i/ml and 1 mmol/l) in the loading medium. 45Ca2t uptake This was measured essentially as earlier described
THYROID HORMONE & CALCIUM ION EXCHANGE IN MUSCLE
[4, 201. After equilibration, the muscles were incubated for 4 x 60 min in Krebs-Ringer bicarbonate buffer containing 45Ca2t (1 @i/ml). This was fo”owed by four consecutive washings (each lasting 30 min) at 0°C in 3 ml Ca2+-free Krebs-Ringer bicarbonate buffer containiing EGTA (0.5 mmoY1). Finally, the muscles were blotted, weighed and homogenized in 2 ml of 0.3 mol/l TCA. 45Ca2+ activity was measured in the clear supematant obtained after centrifugation by liquid scintillation counting. 45Ca2t taken up by the muscles and retained after the wash-out in the cold was expressed as nmol/g wet weight. From the time-course of 45Ca2t uptake in isolated rat soleus muscle described previously [4], it was inferred that the 45Ca2+uptake after 4 h of incubation will reflect the exchange of 45Ca2t with the intracellular Ca2’ pools, i.e. the SR and the mitochondria. Ca2“‘-ATPase determination The concentration of Cazt-ATPase was determined by measurement of the Ca2’ -dependent steady state phosphorylation from [y-‘%]ATP in crude muscle homogenates [21]. Individual soleus or EDL muscles were homogenized at 0°C in buffer containing in mmol/l: HEPES 5 and sucrose 300 (pH 7.4). The final tissue content of the homogenates was 10 mg/ml. Two hundred pl ahquots of the homogenates were incubated for 30 s at 0°C in a reaction-mixture containing in mmol/l: imidazole 100, KC1 100, MgClz 5, EGTA 0.5, ATP 0.05., [Y-~%IATP (0.3 @J/ml) pH 7.4) in the absence or presence of CaClz (0.55 mmoY1). The reaction was quenched with TCA. After repeated washings of the pellet with TCA at O”C, the pellet was dissolved in NaOH and neutralized with &S04. [32p] activity was measured by liquid scintillation counting. The Ca2+-ATPase concentration was calculated as the difference in [3aP] incorporation in the presence and absence of CaC12. It was previously shown that 99% of the Ca2’dependent r32p] incorporation could be removed by hydrox lamine after acid precipitation indicating that [3$ ] is bound to an aspartic acid residue [21]. In addition, it was ascertained that measurement of the Ca2+-dependent r3?‘l incorporation in homogenates quantified the total Ca2+-ATPase
345
concentration. This was done by comparing the molecular activity calculated from the 3-0-methylfluorescein phosphatase activity and the Ca2’-dependent [3%‘] incorporation for an isolated SR preparation and the muscle homogenates [21]. Protein determination The protein content of the crude homogenates was measured according to the method of Lowry et al. [22] using bovine serum albumin as a standard Chemicals All chemicals were of analytical grade. Bovine serum albumin, T3 and veratridine were products from the Sigma Co., St Louis, MO, USA. 3-0-methylglucose was obtained from Calbiochem, Los Angeles, USA. Dantrolene was a gift from Eaton, Norwich, NY, USA. 45Ca2t (367 Ci/mol) was obtained from the Danish Atomic Energy Commission, Denmark 3-o-[t4c]ws0, methylglucose (50 Ci/mol) was from The Radiochemical Centre, Amersham, UK, and [Y-~%]ATP (33.8 Wmmol) from NEN, Boston, MA, USA. ]y-‘%]ATP was purified by column chromatography [23]. Statistics All results am given as mean values f SE with the number of observations in parentheses. The significance of difference between group means was calculated using the two-tailed Student’s t-test for non-pained observations. A P value < 0.05 was regarded as statistical significant. This is indicated in the figures and tables by an asterisk.
Results
Erects of veratridine on 45C2t ef/lux T3 treatment for 8 days increased basal 45Ca2’ efflux from rat soleus muscle by around 25% (P < 0.05) (Fig. lA, 60-120 mm). Exposure to veratridine (0.1 mmol/l) which depolarizes the muscle membrane through an action on the
CELL CALCIUM
346
0.03
Fraction of “Co lost per min (min-‘)
0.02
0.01
0.00 0.02
11%xtion
of ‘%I Iost per min (min-‘)
0.01
o.oo- 60
60
100 120 140 Duration of washout (min)
Fig. 1 Effects of veratridine presence
(B) of dantrolene
soleus muscle of euthyroid rats received
without
to the wash-out activity adding
was
injections
buffer
by wash-out
2 ml of unlabelled or with
homogenized
the
containing Krebs-Ringer indicated.
of T33 (20 pg/lOO g
45Ca2’ (2 pCi/ml). bicarbonate The
buffer
muscles
were
in 2 ml TCA (0.3 mol/l) and TCA was also added tubes to a final concentration determined
by liquid
in the wash-out
muscles at the end of the experiment, In the experiments
of 0.3 molll. 4sCa2+
scintillation
counting.
By
tubes to that in the
the fractional
loss of “Ca”
shown in panel B, dantrolene
(10 )rmol/l) was present from the onset of wash-out. Curves
represent
measurements compared
on
the 6-12
to euthyroid
mean
with
muscles.
bars
45Ca2+ and 3-O-[‘4C]-methylglucose [email protected] electricalstimulation
efflux from
rats. Three week old
at 3o’C through a series of tubes
additions
the 45Ca2+ activity
was calculated.
on “Ca*+
the muscles were loaded for 60 min at 3o’C in
bicarbonate
This was followed containing
(10 pmol/l) and hyperthyroid
or the solvent alone for 8 days. After preparation
and equilibration, Krebs-Ringer
160
(0.1 mmoV1) in the absence (A) or
daily subcutaneous
body weight)
160
volta e-dependent Nat channels [24] increased 45Ca8’ efflux considerably in both groups of rats (Fig. lA, 120-180 rnin). The peak response was observed after 30-40 min of exposure. Maximum 45Ca2+’efflux in the presence of veratridine was 60% higher in hyperthyroid soleus than in euthyroid soleus muscle. The relative increase in 45Ca2t efflux induced by veratridine as compared to the basal 45Ca2f efflux was 110% in euthyroid soleus and 169% in hyperthyroid soleus muscle. To see whether release of Ca2’ from intracellular Ca2’ stores was involved in the veratridine-induced increase in 45Ca2+efflux, experiments were repeated in the presence of dantrolene (10 pmol/l) which has earlier been shown to inhibit the high Kf-induced increase in 45Ca2+ efflux [ 101. Dantrolene did not affect the resting 45Ca2t efflux from euthyroid soleus muscle, but reduced 45Ca2t efflux from hyperthyroid soleus muscle and the difference between the two groups was no longer significant (Fig. lB, 60-120 min). The veratridine-induced increase in 45Ca2t efflux was inhibited by dantrolene by 44% in euthyroid soleus (not significant). In hyperthyroid soleus muscle, dantrolene inhibited the response to veratridine by 57% (P < 0.05) and the difference between control and T$-treated rats was no longer significant (Fig. lB, 120-180 min).
denoting
SEM
*P < 0.05,hyperthyroid
of
Possibly due to the attachment to the electrodes, the initial phase of the resting 45Ca2t efflux in the euthyroid and the hyperthyroid group was slightly different in Figure 2A (60-90 min) as compared to Figure 1A (60-120 min). Nevertheless, at t = 85 min. the values for 45Ca2t efflux in Figure 2A were comparable to those of Figure 1A and similarly the effect of T3 treatment amounted to around 22% (P < 0.05) (Fig. 2A, 60-90 min). At the same time, the basal efflux of 3-0-[‘4Clmethylglucose was increased by 78% (P < 0.05) (Fig. 2B, 60-90 min). Electrical stimulation for 1 min at a frequency of 50 Hz induced an increase in the efflux of 45Ca2f (Fig. 2A, 90-140 min). The maximum increase amounted to 83 and 91% in soleus muscle of euthyroid and hyperthyroid rats, respectively. Peak
THYROID
HORMONE
& CALCIUM
ION EXCHANGE
IN MUSCLE
B
sib
IdO Duration
Ng. 2 Effects of electrical
60
Il;o
of washout (mm)
stimulation
at 50 Hz (1 min) on the
efflux
of 45Ca2+ (A) and 3-O-[14C]-methylglucose
soleus
muscle
of euthyroid
treated as described and equilibration, @i/ml)
and hyperthyroid
in the legend to Figure the left muscles
and
3-O-[‘4C]-methylglucose
(2 @i/ml
loss of isotope was measured
were loaded
frequency Each
electrical
were
with ‘%a’+
(2 with
and 1 mmol/l). The fractional
as described
stimulation
from
muscles
in the legend to Figure
1. Each muscle was attached to an electrode wash-out,
(B)
rats. Rats
1. After preparation
contralateral
the
1BO
was
and after 90 min of
applied
for
1 min at a
of 50 Hz (10 V, 1 ms).
curve
measurements
represents
the mean
on 6 muscles.
with bars denoting
*P < 0.05, hyperthyroid
SEM of
347
respectively (Fig. 2B, 90-140 min). Peak values of 3-0-[‘4C]methylglucose efflux during stimulation were significantly (P < 0.05) higher in the hyperthyroid group. Around 25 min after cessation of stimulation, 3-O-[‘4C]methylglucose efflux started to decrease, but it had not returned to the basal level by the end of wash-out in either group. One might expect to underestimate the rise in 45ca2+ efflux due to short stimulation when measurements are performed over 10 min intervals. Therefore, the experiments described in Fi ure 2A were repeated with measurements of 45Ca& efflux over 2 min intervals. When measured during the first 2 min after the onset of stimulation a rapid increase in 45Ca2+ efflux was observed that amounted to 189% in euthyroid (n = 6) and to 167% in hyperthyroid (n = 5) soleus muscle. The peak level of 45Ca2+efflux immediately after the onset of stimulation did not differ between the two groups. The maximum 45Ca2t efflux was followed by a decrease in both groups, and the level in hyperthyroid rats remained around 20% (P < 0.05) higher than in euthyroid rats. 45Ca2t efflux had not returned to the starting level within the four 2 min periods following the cessation of stimulation in either of the groups (data not shown). In a separate series of experiments, the effect of dantrolene (10 pmoY1 on the electrical stimulation 5’ induced increase in 4 Ca2’ efflux was investigated. These measurements, which for practical reasons were performed over 10 min intervals, showed that dantrolene caused no significant suppression of 4sca2+, efflux induced by electrical stimulation, neither in euthyroid nor in hyperthyroid rats (data not shown).
compared
to euthyroid
C~I~~-ATP~~~concentration
values for 45Ca 2+ efflux during stimulation were significantly (P < 0.05) higher in the hyperthyroid group. Durir~~ y+ 40 min of wash-out following stimulation, Ca efflux decreased slowly and had by the end of the experiment completely returned to the starting level in hyperthyroid rats, but not in increase in The relative euthyroid rats. 3-O-[‘4C]methylglucose efflux after the onset of electrical stimulation amounted to 365 and 218% in euthyroid and hyperthyroid soleus muscle,
The effect of T3 treatment for 8 days on the concentration of Ca2’-ATPase was investigated in soleus and EDL muscle of 4 week old rats (Fig. 3). In Ca2+-dependent euthyroid rats, [3+1 incorporation was roughly 5.5 fold higher in EDL than in soleus muscle. T3 treatment increased the Ca2+-dependent [3%] incorporation by around 120% in soleus muscle, and had only a small (7%, not significant) effect in the fast-twitch EDL muscle. These results are in good agreement with
CELL CALCIUM
348
Ca2+ 40
dependent
(nmol/g
32P
content of the soleus muscle homogenates amounted to 147 + 4 mg/g wet weight (n = 5) and 153 + 5 mg/g wet weight (n = 5) in euthyroid and 8 day hyperthyroid rats, respectively. Total protein content of the EDL muscle homogenates amounted to 168 + 5 mg/g wet weight (n = 4) and to 169 + 7 mglg wet weight (n = 4) in euthyroid and 8 day hyperthyroid rats, respectively.
incorporation
wet wt) EDL
30
20
Time course Ca2’-ATPase
Soleus *
of the efSect o T3 treatment on d concentration, 5Ca2’ uptake and
45Ca2+eflux
10
0
(9) (6)
(10)(6)
Ca2+-dependent [3%] incorpation in soleus and EDL muscle of euthyroid and hyperthyroid rats. Animals were treated as described in the legend to Figure 1. Ca2’-dependent [‘“PI incorporation was measured in homogenates of individual EDL and soleus muscles as described in Materials and Methods. Results represent means of measurements on 6-10 muscles from different euthyroid (open baxes) and hyperthyroid (hatched boxes) animals. *P < 0.05 hyperthyroid compared to euthyroid Fig. 3
observations on relaxation rate [5] and calcium content [4] and indicate that T3 treatment does not result in a proliferation of the SR and an increase in Ca’+-ATPase concentration in muscles consisting predominantly of fast-twitch fibres. Total protein Table 1 Tie course of the effect rat soleus muscle
In a recent study [14] we have reported that an increase in Nat-Kt-ATPase concentration in muscle after TJ treatment was preceded ,by an increase in the passive fluxes of K and Na . In view of the effects of T3 on 4sCa2’ efflux and Ca2+-ATPase concentration, it was interesting to investigate whether a similar relationship could explain the increase in Ca2+-ATPase concentration in rat soleus muscle. Quantification of the Ca2+-ATPase concentration measurement of Ca2’-dependent 13%] by incorporation showed a significant increase (21%) after 1 day of T3 treatment. Thereafter, the Ca2’-dependent r3%] incorporation increased progressively with the duration of the T3 treatment reaching a level of 139% above the controls after 8 days (Fig. 4A). When the capacity for Ca2+ accumulation into the intracellular organelles of the
of T33 treatment for 1-8 days on basal and 20 mM K+-stimulated “Ca2’ efflux fi-om
Fraction of 45Ca2’ lost per min (min-‘) Basal
+ 20mMp
Controls
0.0063 f 0.0001 (9)
1 day T3 2 days T3 3 days T3 8 days T3
0.0066 If:0.0002 (5)
0.0159 f 0.0008 (5)
0.0064 f 0.0002 (10) 0.0069 f omO3 (10) 0.0079 f 0.0004 (9)*
0.0185 zt 0.0016 (10) 0.0185 fO.OO1O (lo)* N.D.
0.0156 rt 0.0010 (9)
Results show means f SEh4 with the mmber of animals in parentheses. Rats were treated as described in the legend to Figure 4. ‘l%e fmctional loss of%a* fium s&us muscle was detumined as described in the legend to Figure 1. Basal values represent data obtained after 80 min of wash-out, and vahm in the preseme of 20 mm&l K’ were obtained 40 min afk correspmdingtothepeakinmeme.
*P < 0.05, T3-treated BScompared to euthymid controls
the onset of exposure to high IC, i.e.
rHYROID
HORMONE
& CALCIUM
2
0 150
4
Relative 45Ca
ION EXCHANGE
6
increase
uptake
IN MUSCLE
sign&ant increase (16%) was observed after 2 days of T3 treatment. 45Ca2t uptake capacity continued to increase up to 8 days of T3 treatment and reached a level of 119% above the controls (Fig. 4B). The time course of the effect of T3 treatment on 45Ca2t efflux was measured in the absence and in the presence of 20 mmol/l K+ (Table 1). Since the effect of 8 days of T3 treatment on the basal 45Ca2+ efflux amounted to only 25% (Figs 1A and 2A), it 45ca2t might be easier to detect early chan+ges in efflux in the presence of high K (which will stimulate 45Ca2 efflux). As can be seen in the left column, a significant increase (25%) in basal 45Ca2t efflux was observed after 8 days. When 45Ca2t efflux was stimulated by 20 mmoY1 K’, a significant (19%) increase could be detected after 3 days of T3 treatment, indicating that more Ca2’ can be mobilized from the SR after this short period of T3 treatment.
8
in
(percent)
1
*
*
/
Discussion
“1 / 4-&&TB
Duration
Fig.
Time
4
Ca*+-dependent
of T3-treatment
course
of
the
rat soleus muscle. with ‘I’3 (20 p/l00 Ca*+-dependent Materials during
and
individual
Methods.
bicarbonate containing
buffer
all animals
incorporation soleus
incubation
on
were 4 weeks old. was
muscles
containing
period
measured as
with
45Ca2+ (1 @/ml),
EGTA (0.5 mmol/l). ‘%a*’
The results represent controls.
treatment
increase
in
at 30-C
Krebs-Ringer followed
by
buffer
activity was measured
in
counting.
means of determinations
as the relative
in
described
for 4 x 30 min at 0°C in Ca*“-free Krebs-Ringer
TCA extracts by liquid scintillation arc given
T3
45Caz’ uptake was determined
a 4 x 60 min
wash-out
of
(A) and 45Ca2” uptake (B) in
3 week old rats were injected sulxutaneously
[3%‘] of
(days)
g body weight) or the solvent alone for 1 to 8
days. By the time of sacrifice, homogenates
effect
i3%‘] incorporation
on 4-8 animals and
as compared
*P < 0.05, Ts-treated compared
to euthyroid
349
to euthyroid controls
intact soleus muscle was estimated by measurement of 45Ca2+ uptake over a 4 h incubation period, a
The present study confii and extends the observations that thyroid hormone induces a proliferation of the SR leading to an increase in the pool of Ca2’ available for mobilization after depolarization as well as in an increased capacity for tesequestration of Ca2+ from the sarcoplasm [l-4, 10, 111. The major new observation is that a significant increase in the concentration of Ca2’-ATPase can be detected within 24 h of T3 treatment, whereas functional changes in the SR i.e. Ca2’ uptake and Ca2’ release were not observed until after 2-3 days of treatment. To indicate the size of the various calcium pools in soleus muscle from euthyroid and hyperthyroid rats, the results of previous measurements and calculations [4] are shown in Table 2. T3 treatment for 8 days induced a two-fold increase in total calcium content (1st line), without changing the calcium content of the extracellular space (2nd line). The amount of intracellular calcium exchangeable with 4?Ca2’ within the 6 h of the experiment showed a four-fold increase in hyperthyroid rats calcium line). The intracelhllar (3rd non-exchangeable with 45Ca2+ was similar in the two groups (4th line). The data indicate that the
CELL CALCIUM
350
reater part of muscle calcium is exchangeable with $‘Ca2+ and that the increase in total calcium content in hyperthyroid soleus muscle is accounted for by the increase in intracellular calcium pools, i.e. SR and mitochondria. The passive Ca2+ efflux expressed as the fractional loss of 45Ca2+ was increased by around 25% after 8 day of T3 treatment (Figs 1 and 2). This effect would be even larger if it is taken into account that the total calcium content was twice as high in the soleus muscle of hyperthyroid rats as compared to control rats (Table 2). Depolarization b veratridine induced a considerable increase in 4Jca2+ efflux which was 60% larger in 8-day hyperthyroid than in control rats. The response to veratridine could be partly inhibited by dantrolene (Fig. 1). In line with earlier measurements on stimulation of 45Ca2t efflux by high Ktout [lo] the observations indicate that the effect of veratridine is partly due to stimulation of Ca2+ release from the SR. In contrast, the effect of electrical stimulation on 45Ca2t efflux could not be inhibited by dantrolene (see description Fig. 2). It was earlier observed that the effect of dantrolene on the contractile force of rat soleus muscle stimulated at Table 2 Distributionof calcium in soleus muscle from euthyroid and hyperthyroid ratsCalciumcontent (nmoUg wet weight) Euthyroid
1090+40
Total Extracellular
space
(7)
Hyperthyroid
2200 f 320 (7)
446
449
Intracellulaf 45Ca2+exchangeable
354
1513
Iukacellular nonexchangeable with 4sCa2+
290
238
Data are calculated from the experimental results presented in [4]. Total calcium content was determined b%atomic absorption spectrophotometry, the intmcellular *Ca -exchangeable calcium was derived from extmpolation of %a* uptake curves measured overa 6 h incubation period, and the calcium content of the extracellular space was calculated BSthe amount of calcium lost during wy+h-out at 0°C. Inhacellular calcium non-exchangeable with Ca was calculated by subtmcting the calcium content of the extracellular space and the inhacellular 4sCa~-exchangeable calcium 6om the total calcium content (with permission t+om the American physiology Society)
50 Hz was relatively small compared to that observed at 1 Hz [6]. It is also possible that a considerable part of the Ca2+ released into the cytosol during electrical stimulation is derived from the extracellular compartment in a slow-twitch muscle like the soleus. This would imply that it is not inhibitable by dantrolene. The observation that 45Ca2f uptake increased 3-4 fold in soleus muscle of 4 week old euthyroid and hyperthyroid rats during 5 min of stimulation at 1 Hz (M.E. Ever& unpublished results) would support this idea. The increase in fractional loss of 45Ca2t due to electrical stimulation in the experiments with the 2-min intervals amounted to 0.0122 min-’ for euthyroid soleus and to 0.0131 mid’ for hyperthyroid soleus muscle. Again, in view of the increase in total calcium content, these data might reflect a much larger release of Ca2’ upon depolarization in the Ts-treated rat. This does not, however, necessarily imply that the free cytosolic Ca2’ concentration during contraction is higher in the hyperthyroid soleus muscle. Since the capacity for Ca2’ resequestration is also increased in the T3-treated group, the net result of enhanced Ca2’ release and a higher Ca2’ uptake rate might be that the intracellular free Ca2+ concentration is just as high as in control rats. The latter is suggested by the findings that the twitch force is similar in hyperthyroid and euthyroid soleus [6] as well as in ventricular [25, 261 muscle. In addition, measurements of the free cytosolic Ca2+ concentration with either aequorin or Furashowed no change in ventricular papillary muscle [25, 261 nor in isolated myocytes [27] from hyperthyroid animals. Electrical stimulation was associated with a 3-4 fold in increase prom@ 3-O-[‘4C]-methylglucose efflux in euthyroid and hyperthyroid soleus muscle (Fig. 2B). Similar large increases in the permeability of the muscle plasma membrane to glucose during electrical stimulation were earlier observed in rat soleus [15] as well as in frog sartorius muscle [19]. 3-0-[‘4C]-methylglucose efflux was significantly higher both at rest and during stimulation in the soleus muscle of hyperthyroid rats. The question may arise whether this is secondary to the higher 45Ca2t efflux observed in the Ts-treated group. It has been shown
THYROID
HORMONE
& CALCIUM
ION EXCHANGE
IN MUSCLE
that stimulation of 45Ca2+ efflux in rat soleus muscle by a variety of agents was associated with a proportional increase in 3-0-[t4C]-metbylglucose efflux [ 131. Apart from rat soleus muscle, this correlation has been observed in various tissues [12]. Nevertheless, it is still not known, how Ca2’ may activate the glucose transport system and how the conformational changes required for increased glucose transport capacity are brought about. In any case the present data support the view [l 11 that thyroid hormone allows the release of more Ca2’ upon activation, a response that is accompanied by increased mobilization of substrates. In agreement with our earlier observations [4] that T3 treatment for 8 days did not induce an increase in total calcium content or 45Ca2+uptake in EDL, muscle, the present study shows no changes in Ca2+-ATPase concentration in EDL muscle of hyperthyroid rats (Fig. 3) indicating that proliferation of the SR does not occur in the fast-twitch muscle. In a previous study, it was suggested that the increase in the concentration of Nat-Kt-ATPase after T3 treatment occurred secondary to increased passive fluxes of Nat and Kt [14]. The results of the present study do not indicate that an increase in the concentration of Ca2+-ATPase is brought about by a similar mechanism, i.e. an increase in the passive Ca2+ efflux from the SR. A significant increase in Ca2+-dependent [3%] incorporation was observed within 24 h of Ts treatment (Fig. 4A). This is not surprising in view of the observation that the mRNA level for the Ca2’ pump in the hypothyroid rat heart was already increased by 2 h of T3 treatment [28]. The observations that active Ca2+ uptake (Fig. 4B) and passive Ca2’ leak (Table 2) showed a more delayed increase suggest that these func.tionaI changes in Ca2’ transport can not take place until the whole SR membrane system has proliferated. Although it cannot be excluded that an increase in Ca2+-dependent [3%] incorporation is detected earlier than an increase in Ca2’ uptake because of a higher sensitivity of the method used, a discrepancy between changes in Ca2’-ATPase content and Ca2+ uptake has also been reported for the chronically stimulated EDL muscle of the rabbit [29]. Measurements on homogenates showed that the initial Ca2’ uptake rate and the total Ca2’ uptake
351
capacity of the SR were decreased by 50% after 2 days of stimulation, whereas the Ca2+-ATPase content was not changed [29]. This suggests more or less the reverse phenomenon of what is seen during T3 treatment. Although the quantitative significance of the effect of T3 on the SR for the energy consumption during work has been documented [9], the question whether the effect plays a role in the increase in basal metabolic rate still remains open. However, it was recently suggested that considerable Ca2+ recycling occurs in resting skeletal muscle [30]. In the same study it was calculated that the 30 % decrease in SR content upon thyroid hormone depletion could account for around one third of the decrease in basal metabolic rate. The reverse might be the case for Ts-treated skeletal muscle, but remains to be established.
Acknowledgements I thank Torben Clausen for stimulating discussions and critical reading of the manuscript, Tove Lindahl Andersen and Marianne Stiirup-Johansen for skilled technical assistance, and Lis Slcj6t for expert secretarial assistance. The study was supported by the Danish Biomembrane Research Center and the Research Foundation of the University of Aarhus.
References 1. Rim DH. Witzmann FA. Fitts RH. (1982) Effect of thyrotoxicosis on sarcoplasmic reticulum in rat skeletal muscle. Am. J. Physiol., 243. C151-C155. 2 Simonides WS. van Hardeveld C. (1985) The effect of hypothyroidism on sarcoplasmic reticulum in fast-twitch muscle of the rat Biochim. Biophys. Acta, 844, 129-141. 3. Simonides WS. van Hardeveld C. (1986) Effects of the thyroid status on the sarcoplasmic reticulum in slow skeletal muscle of the rat. Cell Calcium, 7, 147-160. 4. Everts ME. Clausen T. (1986) Effects of thyroid hormones on calcium contents and 45Ca exchange in rat skeletal muscle. Am. J. Physiol.. 251, E258E265. 5. Nicol CJM. Bruce DS. (1981) Effect of hyperthymidism on the contractile and histochemical properties of fast and slow twitch skeletal muscle in the rat. Pfliigers Arch., 390, 73-79. 6. Everts ME. van Hardeveld C. (1987) Effects of dantxolene on force development in slow- and fast-twitch muscle of euthyroid, hypothymid, and hyperthyroid rats. Br. J. Pharmacol., 92,41-54. I. Wiles CM. Young A. Jones DA. Edwards RHT. (1979) Muscle relaxation rate, fibre-type composition and energy
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turnover in hyper- and hypo-thyroid patients. Clin. Sci., 57, 375-384. Everts ME. van Hardeveld C. Ter Keurs HEDJ. Kassenaar AAH. (198 1) Force development and metabolism in skeletal muscle of euthymid and hypothyroid rats. Acts Endocrinol., 97,221-225. Leijendekker WJ. van Hardeveld C. Elxinga G. (1987) Heat production during contraction in skeletal muscle of hypothyroid mice. Am. I. Physiol., 253, E214E220. Van Hardeveld C. Clausen T. (1984) Effect of thyroid status on K+-stimulated metabolism and 4SCa exchange in tat skeletal muscle. Am. J. Physiol., 247, E421-E430. Van Hardeveld C. Clausen T. (1986) Ca” and thyroid hormone action. In: Bader H. Gietzen K. Rosenthal J. Riide.1 R. Wolf HU. (eds). Intmcelluhu calcium regulation. Manchester University Press, pp 355-365. Bihler I. (1988) The role of membrane transport in the control of glucose metabolism and its coupling to cellular function. Can. J. Physiol. Pharmacol.,66, 549-560. Sorensen SS. Christensen F. Clausen T. (1980) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. X. Effect of glucose transport stimuli on the efflux of isotopically labelled calcium and 3-0-methylglucose from soleus muscles and epididymal fat pads of the rat. B&him. Biophys. Acta, 602,433-445. Ever& ME. Clausen T. (1988) Effects of thyroid hormone on Na+-K+ transport in resting and stimulated rat skeletal muscle. Am. J. Physiol., 255, E6OQE612. Kahn PG. Clausen T. (1971) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. VI. The effect of insulin, ouabain, and metabolic inhibitors on the transport of 3-0-methylglucose and glucose in rat soleus muscle. Biochim. Biophys. Acta, 225.277-290. Chinet A. Clausen T. Girardier L. (1977) Microcalorimetric determination of energy expenditure due to active sodium-potassium transport in the soleus muscle and brown adipose tissue of the rat. J. Physiol., 265, 43-61. Clausen T. Flatman JA. (1977) ‘Ihe effect of catecholamines on Na-K transport and membrane potential in rat soleus muscle. J. Physiol., 270, 383-414. Clausen T. Elbtink I. Dahl-Hansen AB. (1975) The relationship between the transport of glucose and cations across cell membranes in isolated tissues. IX. The role of cellular calcium in the activation of the glucose transport system in rat soleus muscle. Biochim. Biophys. Acta, 375, 292-308. Holloszy JO. Narahara HT. (1965) Studies of tissue permeability. X. Changes in permeability to 3-methylglucose associated with contraction of isolated frog muscle. J. Biol. Chem., 240,3493-3500.
20. Clausen T. Dahl-Hansen AB. Elbrink J. (1979) The effect of hyperosmolarity and insulin on resting tension and calcium fluxes in rat soleus muscle. J. Physiol., 292, 505526. 21. Ever@ ME. Andemen JP. Clausen T. Hansen 0. $1989) Quantitative determination of Ca”-dependent Mg +-ATPase from samoplasmic reticulum in muscle biopsies. B&hem. J., 260,443-448. 22. Lowry OH. Rosebrough NJ. Fan AL. Randall RJ. (195 1) Protein measurement with the Folin phenol reagent J. Biol. Cbem., 193,265-275. 23. Norby JG. Jensen J. (1971) Binding of ATP to brain microsomal ATPase. Determination of the ATP-binding capacity and the dissociation constant of the enzyme-ATP complex as a function of KC concentration Biocbim. Biophys. Acta, 233, 104-116. 24. McKimrey LC. Ratzla!TRW. (1987) Sodium permeability of frog skeletal muscle in absence and presence of veratridine. Am. J. Physiol., 252, Cl90-C196. 25. MacKinnon R. Morgan JP. (1986) Influence of the thyroid state on the calcium transient in ventricular muscle. Pfliigers Arch., 407,142-144. 26 MacKinnon R. Gwathmey JK. Allen PD. Briggs GM. Morgan JP. (1988) Modulation by the thyroid state of intraceBular calcium and contractility in ferret ventricular muscle. Circ. Res., 63, 1080-1089. 27. Beekman RE. van Hardeveld C. Simonides WS. (1988) Ef%ct of thyroid state on cytosolic free calcium in resting and electrically stimulated cardiac myocytes. Biochim. Biophys. Acta, 969, 18-27. 28. Rohrer D. Dilbnann WH. (1988) Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca’+-ATPase in the rat heart J. Biol. Chem., 263, 6941-6944. 29. Leberer E. H&tner K-T. Pette D. (1987) Reversible inhibition of ssrcoplasmic reticulum Ca-ATPase by altered neuromuscular activity in rabbit fast-twitch muscle. Eur. J. B&hem., 162,555-561. 30. Simonides WS. van Hardeveld C. (1988) (Ca2+ + M 2+)-ATPase activity associated with the maintenance of a gl+ Ca gradient by sarcoplasmic reticulum at submicromolar external [Ca”]. The effect of hypothyroidism. B&him. Biophys. Acta, 943, 349-359.
Please send reprint request to : Dr Ma+a E. Everts, Institute of Physiology, University of Aarhus, 8000 Arhus C, Denmark. Received : 15 August 1989 Revised 19 December 1989 Accepted : 8 January 1990
