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Twitch potentiation and caffeine contractures in isolated rat soleus muscle
Corq~. Biochen~. Physiol. Vol. 74C, No. 2. pp. 349-3.54. Printed in Great Britain.
TWITCH
1983
POTENTIATION IN ISOLATED
0306-4492:83/020349-06$03.00.!0 0 1983 Pergamon Press Ltd.
AND CAFFEINE CONTRACT~RES RAT SOLEUS MUSCLE
R. J. CONNETT,’ L. M. UGOL,’ M. J. HAMMACK’ and E. T. HAYS’ ‘Physiology
Dept., University of Rochester ‘Biology Dept., Nazareth
School of Medicine and Dentistry, Rochester, NY 14642. U.S.A. and College of Rochester, Rochester. NY 14610. U.S.A. (Receive
18 .JIIW 1982)
Abstract-l. Electrically-evoked twitch and tetanic tension were measured in isolated rat soleus muscle after exposure to caffeine. 2. Between 0.01 and 2SmM caffeine twitch tension was potcntiated, reaching a peak of 150% of Resting Tension at 0.5 mM. 3. Biphasic Tension development with relaxation was observed at 2.5 mM caffeine with maximal contractures (110% tetanic tension) occurring at 20 mM. 4. Creatine phosphate and ATP stores were maintained throughout the period of tension development and relaxation. 5. In contrast with amphibian muscle, the isolated soleus is very sensitive to low doses of caffeine and produces biphasic caffeine contractures which relax in the presence of caffeine.
must take into account the direct effect of caffeine on muscle tension development in discussing the effect of caffeine on athletic performance. To date, there have been no systematic dose-response studies done in mammalian muscle. Although there are reports of twitch potentiation in the extensor digitorum longus (EDL) muscle at 2O’C. it has not been demonstrated at 37°C in the soleus, a slow twitch muscle. The specific aims of this study were: (i) to define systematically the effect of caffeine exposure in a mammalian muscle, (ii) to see if the thresholds of twitch potentiation and contracture are comparable to those reported for frog muscle and (iii) to see if contractures were always biphasic in onset, and relaxation would consistently occur in the presence of caffeine. For this purpose we used the soleus muscles from young rats since methods are available in this laboratory for maintaining the muscle in a stable, viable conditions for several hours (Pearce & Connett, 1980). Since Hays & Connett (1978) showed that methylxanthines, including caffeine, lower energy stores in frog muscle and that caffeine doses as low as 5 mM can cause a rapid depletion of energy stores, experiments were also performed to determine if energy depletion would be observed in this preparation when exposed to maximal contracture-producing doses of caffeine.
INTRODUCTION Caffeine has been a useful probe in studies on the mechanism of excitation-contraction coupling in amphibian muscle. It evokes contractures without a change in membrane potential (Axelsson & Thesleff, 1958), potentiates the twitch tension and shifts the mechanical and relation thresholds (Sandow, 1964; Liittgau & Oetliker, 1968). These effects are dosedependent, with twitch potentiation occurring at low concentration (1.0-2.5 mM) and contractures occurring at high concentrations of caffeine (> 2.5 mM). Early experiments with mammalian muscle (Gutmann & Sandow, 1965; Gutmann & Hanziikova. :1966) indicated that rat muscle is apparently less sensitive to caffeine than frog muscle. Later work (Frank & Buss, 1967; Isaacson et al., 1970) showed that when experiments were done at the physiological temperature (37°C) the muscle is more responsive to caffeine. Moulds & Denborough (1974) observed caffeine contractures in isolated human muscle with doses as low as 2 mM. The time course of tension development in the mammalian muscle may differ dramatically from that seen in frog muscie. In rat muscle. caffeine typically produces contractures that have a biphasic rise to peak tension and some reports show relaxation if exposure to caffeine continues. Similar observations have been made with human muscle (Moulds & Denborough, 1974). Recent studies have indicated that athletic performance is improved after ingestion of caffeine (Costill ef al., 1978; Ivy et al., 1979; Toner et al., 1980). It has been suggested that the cause of this improvement is increased metabolic efficiency via fatty acid mobiiization (Costill et al., 1978). If, as was originally sug gested, mammalian muscle is less sensitive to caffeine than amphibian muscle, the exercise results in humans should not involve any direct effect of caffeine on muscle. If, on the other hand, the sensitivity of mammalian and amphibian muscles is similar, one
MATERIALS AND METHODS Soleus muscles (~40 mg) from male Sprague-Dawley rats were isolated and prepared for tension measurements, as described in detail by Pearce & Connett (1980). The incubation medium used had the following composition (mM): NaCl 116, KC1 5.4. CaCI, 1.8, MgClz 3.85, NaH2P04 0.91, NaHCO, 26.6, glucose 4 and tubocurarine chloride 1 fig/ml. All solutions were maintained at 37’C and were gassed with 95% O,-5”/ CO,. Tension measurements Following dissection, each muscle was allowed to recover for about 90 min (until the transient tension charac349
R. J.
350
CONNETT et ul
i-l-L
-
Fig. 1. Twitch in 2.0 mM caffeine. Control twitches are shown at the beginning and end of the record. Caffeine was added at the left arrow and control solution replaced at the right arrow. Stimulation frequency was at 0.1 /sec.
teristic of these muscles was completed (Pearce & Connett, 1980)). The muscle was then given a twitch test of S-10 twitches at a rate of 0.1 per sec. This was followed by a tetanic train of stimuli at a rate of lOt&500 per set until peak tetanus was obtained. This tetanic tension was then used as a reference value for each muscle. Muscles were allowed to recover for 15-20 min, and the twitch retested to make sure there was no residual post-tetanic potentiation. The muscle was then exposed to caffeine and the tnitch was tested at the peak of contracture. For those muscles exposed to doses of caffeine below contracture thresholds. the caffeine twitch was tested about 20min after exposure to caffeine. Muscles were then returned to a solution without caffeine and after 30min the twitch was tested. Potentiation was defined as the increase in twitch tension compared to the initial control twitch and was expressed as a percentage of the control twitch.
For the measurements of energy stores in these muscles. the methods used were similar to those used previously for frog muscle (Connett & Hays. 197X; Hays & Connett, 1978). All results are expressed as high energy phosphate content (PE) where PE = creatine phosphate + 2 ATP.
evokes a twitch with tension which is larger than that seen in the absence of caffeine, i.e. potentiation occurs. A typical result using the standard protocol outlined in Materials and Methods is shown in Fig. 1. The twitch potentiation is stable and the twitch tension returns to control levels when the caffeine is washed out. The effect of several doses of caffeine on the size of the potentiation were studied. A summary of the results is given in Fig. 2. Potentiation of the twitch becomes apparent at caffeine concentrations as low as 10nM and seems to be maximal in the range of 0.552.5 mM. At higher doses. contractures appear and the size of the twitch that can be evoked in the presence of caffeine falls sharply to 80:: of the control twitch at 3.5 mM caffeine. In general. if a contracture develops with a maximal tension > loo/, of the tetanic tension, then the size of the twitch in the presence of caffeine is below control values. If the maximal contracture tension is >50:1 tetanic tension. then no detectable twitch occurs in the presence of caffeine, Thus these slow twitch muscles appear to be sensitive to caffeine at concentrations well below the contracture threshold.
RESULTS Twitch
poter7liutior7
In the presence of low concentrations of caffeine (~3 mM). direct electrical stimulation of the muscle
Concentrations evokes contractures
I I I
0 0.01
of caffeine > 5 mM regularly in the isolated soleus muscle. The
1
0.05
0.1
0.5
I.o
5.0
LCAFFEINE~ mM Fig. 2. Twitch potentiation by caffeine. The percentage twitch potentiation was defined as the percentage increase in twttch tension using the twitch before addition of caffeine as the reference point. The dashed line. --. is the result of connecting the 2.0 mM value to the 3.5 mM value which would be below 0 on this scale.
Caffeine
contractures
I
351
in rat soleus
25Q
H
5min
Fig. 3. Time course of caffeine contractures. The figure shows the time course of the contracture induced by two different doses of caffeine (A = 5 mM; B = 20 mM). The plateau at the left is the tetanic stimulus. Caffeine was added at the left arrow. In A caffeine was removed when the tension reached the baseline. The right arrow in B indicates the time at which caffeine was removed.
of relaxation and whether the relaxation is complete were not addressed in the study reported here. The size of the initial rapid peak appears to be dependent on the dose of caffeine. However, no clear analysis can be carried out at this time since, as the dose of caffeine increases, an increasing fraction of the tension measured at the inflection point is derived from the second peak. At this time we have no clear model for the form of maximal peak and therefore we cannot correct for its contribution to the initial tension measurement. The dependence of the height of the maximal contracture tension on the caffeine dose is shown in Fig. 4. Contractures appear at caffeine doses of 2.0-3.0 mM, show a half-maximal effect around 7.5 mM and saturate by 20 mM caffeine.
time course of these contractures is illustrated by typical results from two muscles at different caffeine doses shown in Figs 3A and B. Four phases were always seen: (i) a fast sharp rise in tension (2-4min). the height of which appears to be dose dependent. The rate of rise appears to be independent of the dose and is probably a diffusion limited process in these muscles; (ii) an inflection in the rise of tension; (iii) a slower increase in tension which leads to the maximal contracture tension, which at high doses reaches tetanic or slightly higher tension. The rate of rise of the second tension peak is dose dependent, as is the size of the final peak tension. Finally (iv), the relaxation phase in which tension falls from the maximal peak either back to resting (3A) or perhaps to a low level of sustained tension (3B). The issues of the rate
0.4L I
o
_
q/c41 1.0
/I 2.5
5.0
[CAFFEINE]
IO
20
100
mM
Fig. 4. Caffeine effect on contracture tension. The tension is expressed as fraction of tetanic tension and plotted against the caffeine concentration. All points are plotted as mean & SEM. The numbers in parentheses are the number of muscles included in the average.
R. J. CONNETTet d.
352
[CAFFEINE]mM Fig. 5. Caffeine effect on rate of tension increase. The rate of rise is defined as (time to peak tension)- I_ Each point is the average of 4-6 measurements and is plotted as mean + SEM. The line shown is a least squares fit to the data.
The time after caffeine addition at which the maximal tension occurs is also dependent on the dose of caffeine. The average rate of the tension development can be approximated by the reciprocal of the time after caffeine addition at which the peak occurs. This approximation is used since the rate of increase in tension is neither simply linear with time nor is there a clear set of exponential components. Both the initial rise in tension and the relaxation phase complicate a more detailed kinetic analysis. Figure 5 shows the relationship between the average rate of tension development and the caffeine dose. The effect of caffeine on the rate appears to be linearly related to the dose and, unlike the maximal tension. shows negligible saturation by 50 mM. Energy stores
Since our results indicates that maximal contracture tensions are obtained with caffeine doses of 20mM. we measured creatine phosphate, ATP and total creatine in muscles which had been exposed to 20mM caffeine for varying lengths of time (Table 1). Exposure to caffeine results in a significant reduction in energy content within 5 min, but for the next halfhour no further significant change in energy content occurs. Only phases (i) and (ii) of the contracture are completed in the first 5 min (Fig. 3). Thus it appears that the final peak tension development and the relaxation phases occur in the absence of any further change in energy stores, implying that the energy supply is keeping pace with demand. In addition, since there is a negligible change in ATP level throughout the entire contracture, it is unlikely that any of the processes are affected by ATP availability.
to caffeine, and second, to compare the response of mammalian muscle to that seen in amphibian muscle. Twitch
potentiation
Twitch potentiation occurs in frog muscles at caffeine concentrations of l-2mM and higher doses (> 2.5 mM) lead to contractures (Sandow et al., 1964; Ltittgau & Oetliker, 1968; Axelsson & Thesleff, 1958). Previous studies on soleus muscle from young rats showed that 20 and 40mM caffeine could potentiate twitch at 20°C while contractures were reliably seen only at 40 mM. Frank & Buss (1967) showed that raising the temperature to 37°C increased the sensitivity of the preparation to caffeine so that contractures were observed at 5 mM caffeine. They did not study twitch potentiation at 37°C. The results (Figs 2 and 4) demonstrate a clear potentiation of twitch at low doses of caffeine and show the continuum from potentiation through contracture. Twitch potentiation is seen with doses of caffeine in the range of 0.01&2.5 mM, whereas at higher doses twitch tension decreases and is absent at 10mM caffeine, a concentration of caffeine that proTable 1. Effects of exposure caffeine
on
time to 20mM energy stores in isolated rat soleus muscle
Time of exposure (min) 0 5 10 15 20 30
(,nmol/~Zol 0.809 0.601 0.524 0.563 0.596 0.516
+ + jI k + +
Cr,)
0.081 0.075 0.094 0.109 0.107 0.086
(27) (4) (4) (5) (4) (4)
DISCUSSION
The studies reported here were undertaken with two general purposes in mind. First, to use the stable soleus preparation to resolve some of the previous controversy about the response of mammalian muscle
All values were mean k S.E.M. with number of muscles given in parentheses, * PE = Potential phosphate-bond energy = creatine phosphate + 2 ATP. Cr, = total creatine content,
Caffeine contractures in rat soleus duces nearly maximal tension. Thus, at the normal physiological temperature of 37°C the sensitivity of the soleus muscle to caffeine, as measured by twitch potentiation, is about forty times greater than that observed at 20°C. When these results are compared with the results obtained in the frog sartorious preparation. it appears that the slow twitch rat soleus muscle is much more sensitive to the potentiating effect of low doses of caffeine. The potentiating doses of caffeine are in the range of those observed in humans ingesting caffeine. Blood levels obtained z 1 hr after I-2 cups of coffee range from 440pM (Axelrod & Reichenthal, 1953; Robertson et u/., 1978). Therapeutic doses gave levels in body water as high as 75 /tM (Khanna ef al.. 1980). Increases in exercise performance are observed with caffeine doses giving blood levels near 40 LAM(Costill et al., 1978; Ivy et ul.. 1979). All these blood concentrations are in the range of caffeine causing twitch potentiation in the isolated soleus (Fig. 2). Thus our data suggest that some of the effects of caffeine in humans could be due to direct effects on muscle.
Cont,.uc’t1rr.c Mammalian muscle was long suggested to be less sensitive to the contracture producing effects of caffeine than amphibian muscle (see e.g. Gutmann & Hanzlikova. 1966). Frank & Buss (1967). and Isaacson et ul. (1970) suggested that this apparent insensitivity of soleus muscle was a temperature effect. With 20 and 50mM caffeine, Isaacson et ul. found contractures of 50 and 65”;, tetanic tension, respectively, but these differences were not significant. We observe measurable contractures at 2.5 mM caffeine, with maximal contractures occurring in the range of 10 20mM caffeine. Unlike the results of Isaacson er trl. (1970) but similar to the results of Frank and Buss. our maximal contractures are IlO”<, tetanic tension, significantly higher than any obtained by them with the exception of studies on a split soleus with a dose of 20 mM caffeine where they obtained contractures that were 900/,, tetanic. Our observation of a higher maximal contracture tension may be the consequence of using only muscles that weighed less than 40 mg, so that we could be assured that size did not limit substrate availability to the individual muscle fibres (Chaudry & Gould. 1969). With larger muscles, caffeine diffusion may be limited so that while the surface fibers are relaxing, the internal muscle fibers are developing tension. This would result in an overall decrease in measured peak tension. Isaacson & Sandow (1967) did find that in the EDL with weights 3@100mg the diffusion of caffeine was two times slower than it is in frog sartorius. In the thicker soleus it may be even slower. The fact that higher contracture to tension ratios could be obtained by Isaacson ct d. (1970) with split soleus muscles would support this conclusion. Secondly. all our measurements are performed after the resting tension transient characteristic of these muscles is complete. thereby assuring that the twitch has recovered from the decrease that occurs during the tension transient (Pearce & Connett. 1980). Thus we feel that our results reflect the sensitivity of the slow twitch mammalian muscle to caffeine contractures. This conclusion is further supported by the observation that caffeine contractures
353
occur in human muscle in the concentration range’of 2230 mM (Moulds & Denborough. 1974). The threshold for caffeine contractures in frog sartorius muscle is -2.5 mM and maximal values are attained by 10 mM caffeine. Thus. although the sensitivity of the twitch tension of mammalian muscle to low doses of caffeine is greater than that of amphibian muscle, the sensitivity to a contracture-producing dose of caffeine is very similar in both mammalian slow muscle and frog sartorius.
A biphasic rise in tension and relaxation was seen at all contracture-producing doses where the contracture was greater than lo”,, of tetanic tension. Biphasic tension development has been reported rarely in frog muscle (Bianchi & Bolton. 1967; Gebert, 1968; Ltittgau & Oetliker. 1968; Konya c’t al., 1978) whereas it is always present in mammalian muscles at 37’C (Isaacson et cd.. 1970; Moulds & Denborough. 1974; Fig. 3). The relaxation or inactivation of the contracture has been reported previously for rat soleus (Isaacson ct cd.. 1970) but has not been observed in caffeine contractures in frog muscle. although there is some indication in single fiber records that a relaxing process may be occurring (Axelsson & Thesleff, 1958; Liittgau & Oetliker. 1968). Two problems may have prevented this process from being regularly observed in the frog studies. First. caffeine contractures are poorly reversible and most studies have involved short exposures to caffeine at contracture-producing doses. thus perhaps missing a relaxation phase. Second. there is an almost complete depletion of energy supplies on exposure of frog sartorius muscle to contracture-producing (5 mM) doses of caffeine (Hays & Connett. 1978). This could result in a sustained tension due to the production of rigor by the ATP depletion. In contrast. the rat muscles maintain ATP levels throughout both the development of tension and the relaxation of the caffeine-induced contracture (Table I). When compared with the well-studied frog preparations, these slowtwitch mammalian muscles: (a) are more sensitive to the potentiating effects of caffeine; (ii) show similar sensitivity to the contracture-producing effects of caffeine; (iii) give contractures which are always biphasic during the tension development; and (iv) relax after peak tension is reached. The first two observations could indicate that the potentiating effect and contracture effect result from two different processes, both of which occur in both species. The rat has a higher sensitivity for the potentiating process but a similar sensitivity to caffeine for the contracture producing process than frog muscle. The relaxation, which is not seen in frog muscle. may be due to a process which is unique to mammalian muscle or may simply be a response which cannot be observed in the ATP-depleted frog muscle. Detailed studies on isolated rat muscle fibers would be necessary to resolve these issues. Acknowledgcmmts~This work was supported in part by grant in aid from the Muscular Dystrophy Association and by NIH grant No. AM22124. The authors would like to thank Carrie Golliger for excellent technical assistance. R. J. Connett is the recipient of USPHS RCDA 5K04AM00155.
R. J. CONNETT et ul
354 REFERENCES
AXLEROD J. & REICHENTHAL J. (1953). The fate of caffeine
in man and a method for its estimation in biological material. J. Pharmac. rxp. Thw. 107, 517-523. AXELSSON J. & THESLEFF S. (1958) Activation of the contractile mechanism in striated muscle. Acta physiol. stand. 44, 55-66. BIANCHI C. P. & BOLTON T. C. (1967) Action of local anesthetics on coupling systems in muscles. J. Pharmuc. rxp. Thu. 157, 388-405. CHAUDRY F. H. & GOULD M. K. (1969). Kinetics of glucose uptake in isolated soleus muscle. Biochwn. hiophys. Acta 177. 527-536. CONNETT R. J. & HAYS E. T. (1978). Energy coupling to sodium transport in frog skeletal muscle. Am. J. Ph,siol. 235, C25X34. COSTILL D. L.. DALSKY G. P. & FINK W. J. (1978) Effects of caffeine ingestion on metabolism and exercise performance. Med. Sci. Sport 10, 155-158. FRANK G. B. & Buss W. C. (1967) Caffeine-induced contractures in mammalian skeletal muscle. Archs inr. Pharmacodyn. The+. 170, 343-349. GERERT G. (1968) Caffeine contracture of frog skeletal muscle and of single fibers. Am. J. Pllysiol. 215, 296298. GUTMANN E. & HANZLIKOVA V. (1966) Contracture responses of fast and slow mammalian muscles. Physiol. Bohcm. 15, 404-414. GUTMANN E. & SANDOW A. (1965) Caffeine-induced contracture and potentiation of contracture in normal and denervated rat muscle. I& Sci. 4, 1149-l 156. HAYS E. T. & CONNETT R. J. (1978) Effect of methylxanthines and elevated external potassium on high energy phosphate content in frog skeletal muscle. Biochem. Phurmac. 27. 2965-2968. ISAACSONA.. HINKES M. J. & TAYLOR S. (1970) Contracture and twitch potentiation of fast and slow muscles of the rat at 20. and 37’C. Am. J. PhJsiol. 218, 33-41.
ISAACSONA. & SANDOW A. (1967) Caffeine
effects on radiocalcium movement in normal and denervated rat muscle. J. Pharmuc. cup. Thrr. 155, 376388. IVY J. L., COSTILL D. L.. FINK W. J. and LOWER R. W. (1979) Influence of caffeine and carbohydrate feedings on endurance performance. Med. Sci. Sports 11, 61 I. KHANNA N. N.. BADA H. S. & SOMANI S. M. (1980) Use of salivary concentrations in the prediction of serum caffeine and theophylline concentrations in premature infants. J. Pediat. 96, 494 499. KENYA L.. J~NA I.. K&CR A. & ARADI F. (197X) Determination of dose and time dependence of muscle contractures elicited by caffeine. Actor hiol. Acad. Sci. Inmq. 29, 129 137. Liirrc;~r~ H. C. & OFTLIK~K H. (1968) The action of caffeine on the activation of the contractile mechanism in striated muscle fibres. J. Physiol. 194. 51-74. MOULDS R. F. W. & DLNBOKOUC;HM. A. (1974) A study of the action of caffeine. halothane. potassium chloride and procaine on normal human skeletal muscle. C/in. cp. Pharmuc. PhJsiol. 1, 197-209. PEAKC~,F. J. & COUN~TT R. J. (1980) Effect of lactate and palmitate on substrate utilization of isolated soleus muscle. Am. J. Physiol. 238, Cl49 159. RORFKTSON D.. FR~~LICHJ. C., CARR R. K.. WATSOU J. T.. HOLLIEIELD J.. SHAND D. & OATCS J. (1978) Effects of caffeine on plasma rcnin activity, catecholamines and blood pressure. Neir’ Engl. J. Med. 298, 181 186. SAEZDOWA. (1964) Potentiation of muscular contraction. Archs Pkv. Med. Rehahil. 45. 62-81. SANDOW A.. TAYLOR S. R.. ISAACSONA. & STQLIIN J. J. (1964) Electromechanical coupling in potentiation of muscular contraction. Scicww 143, 577-579. TONER M. M.. KIRCKENDALL D., CHASE J. M.. D~LIO D. J.. CLEARY P. & Fox E. L. (1980) The effect of caffeine on the metabolic and cardiovascular responses to exercise. Mc,d. Sci. Sports Ewr. 12, 109.
TWITCH
1983
POTENTIATION IN ISOLATED
0306-4492:83/020349-06$03.00.!0 0 1983 Pergamon Press Ltd.
AND CAFFEINE CONTRACT~RES RAT SOLEUS MUSCLE
R. J. CONNETT,’ L. M. UGOL,’ M. J. HAMMACK’ and E. T. HAYS’ ‘Physiology
Dept., University of Rochester ‘Biology Dept., Nazareth
School of Medicine and Dentistry, Rochester, NY 14642. U.S.A. and College of Rochester, Rochester. NY 14610. U.S.A. (Receive
18 .JIIW 1982)
Abstract-l. Electrically-evoked twitch and tetanic tension were measured in isolated rat soleus muscle after exposure to caffeine. 2. Between 0.01 and 2SmM caffeine twitch tension was potcntiated, reaching a peak of 150% of Resting Tension at 0.5 mM. 3. Biphasic Tension development with relaxation was observed at 2.5 mM caffeine with maximal contractures (110% tetanic tension) occurring at 20 mM. 4. Creatine phosphate and ATP stores were maintained throughout the period of tension development and relaxation. 5. In contrast with amphibian muscle, the isolated soleus is very sensitive to low doses of caffeine and produces biphasic caffeine contractures which relax in the presence of caffeine.
must take into account the direct effect of caffeine on muscle tension development in discussing the effect of caffeine on athletic performance. To date, there have been no systematic dose-response studies done in mammalian muscle. Although there are reports of twitch potentiation in the extensor digitorum longus (EDL) muscle at 2O’C. it has not been demonstrated at 37°C in the soleus, a slow twitch muscle. The specific aims of this study were: (i) to define systematically the effect of caffeine exposure in a mammalian muscle, (ii) to see if the thresholds of twitch potentiation and contracture are comparable to those reported for frog muscle and (iii) to see if contractures were always biphasic in onset, and relaxation would consistently occur in the presence of caffeine. For this purpose we used the soleus muscles from young rats since methods are available in this laboratory for maintaining the muscle in a stable, viable conditions for several hours (Pearce & Connett, 1980). Since Hays & Connett (1978) showed that methylxanthines, including caffeine, lower energy stores in frog muscle and that caffeine doses as low as 5 mM can cause a rapid depletion of energy stores, experiments were also performed to determine if energy depletion would be observed in this preparation when exposed to maximal contracture-producing doses of caffeine.
INTRODUCTION Caffeine has been a useful probe in studies on the mechanism of excitation-contraction coupling in amphibian muscle. It evokes contractures without a change in membrane potential (Axelsson & Thesleff, 1958), potentiates the twitch tension and shifts the mechanical and relation thresholds (Sandow, 1964; Liittgau & Oetliker, 1968). These effects are dosedependent, with twitch potentiation occurring at low concentration (1.0-2.5 mM) and contractures occurring at high concentrations of caffeine (> 2.5 mM). Early experiments with mammalian muscle (Gutmann & Sandow, 1965; Gutmann & Hanziikova. :1966) indicated that rat muscle is apparently less sensitive to caffeine than frog muscle. Later work (Frank & Buss, 1967; Isaacson et al., 1970) showed that when experiments were done at the physiological temperature (37°C) the muscle is more responsive to caffeine. Moulds & Denborough (1974) observed caffeine contractures in isolated human muscle with doses as low as 2 mM. The time course of tension development in the mammalian muscle may differ dramatically from that seen in frog muscie. In rat muscle. caffeine typically produces contractures that have a biphasic rise to peak tension and some reports show relaxation if exposure to caffeine continues. Similar observations have been made with human muscle (Moulds & Denborough, 1974). Recent studies have indicated that athletic performance is improved after ingestion of caffeine (Costill ef al., 1978; Ivy et al., 1979; Toner et al., 1980). It has been suggested that the cause of this improvement is increased metabolic efficiency via fatty acid mobiiization (Costill et al., 1978). If, as was originally sug gested, mammalian muscle is less sensitive to caffeine than amphibian muscle, the exercise results in humans should not involve any direct effect of caffeine on muscle. If, on the other hand, the sensitivity of mammalian and amphibian muscles is similar, one
MATERIALS AND METHODS Soleus muscles (~40 mg) from male Sprague-Dawley rats were isolated and prepared for tension measurements, as described in detail by Pearce & Connett (1980). The incubation medium used had the following composition (mM): NaCl 116, KC1 5.4. CaCI, 1.8, MgClz 3.85, NaH2P04 0.91, NaHCO, 26.6, glucose 4 and tubocurarine chloride 1 fig/ml. All solutions were maintained at 37’C and were gassed with 95% O,-5”/ CO,. Tension measurements Following dissection, each muscle was allowed to recover for about 90 min (until the transient tension charac349
R. J.
350
CONNETT et ul
i-l-L
-
Fig. 1. Twitch in 2.0 mM caffeine. Control twitches are shown at the beginning and end of the record. Caffeine was added at the left arrow and control solution replaced at the right arrow. Stimulation frequency was at 0.1 /sec.
teristic of these muscles was completed (Pearce & Connett, 1980)). The muscle was then given a twitch test of S-10 twitches at a rate of 0.1 per sec. This was followed by a tetanic train of stimuli at a rate of lOt&500 per set until peak tetanus was obtained. This tetanic tension was then used as a reference value for each muscle. Muscles were allowed to recover for 15-20 min, and the twitch retested to make sure there was no residual post-tetanic potentiation. The muscle was then exposed to caffeine and the tnitch was tested at the peak of contracture. For those muscles exposed to doses of caffeine below contracture thresholds. the caffeine twitch was tested about 20min after exposure to caffeine. Muscles were then returned to a solution without caffeine and after 30min the twitch was tested. Potentiation was defined as the increase in twitch tension compared to the initial control twitch and was expressed as a percentage of the control twitch.
For the measurements of energy stores in these muscles. the methods used were similar to those used previously for frog muscle (Connett & Hays. 197X; Hays & Connett, 1978). All results are expressed as high energy phosphate content (PE) where PE = creatine phosphate + 2 ATP.
evokes a twitch with tension which is larger than that seen in the absence of caffeine, i.e. potentiation occurs. A typical result using the standard protocol outlined in Materials and Methods is shown in Fig. 1. The twitch potentiation is stable and the twitch tension returns to control levels when the caffeine is washed out. The effect of several doses of caffeine on the size of the potentiation were studied. A summary of the results is given in Fig. 2. Potentiation of the twitch becomes apparent at caffeine concentrations as low as 10nM and seems to be maximal in the range of 0.552.5 mM. At higher doses. contractures appear and the size of the twitch that can be evoked in the presence of caffeine falls sharply to 80:: of the control twitch at 3.5 mM caffeine. In general. if a contracture develops with a maximal tension > loo/, of the tetanic tension, then the size of the twitch in the presence of caffeine is below control values. If the maximal contracture tension is >50:1 tetanic tension. then no detectable twitch occurs in the presence of caffeine, Thus these slow twitch muscles appear to be sensitive to caffeine at concentrations well below the contracture threshold.
RESULTS Twitch
poter7liutior7
In the presence of low concentrations of caffeine (~3 mM). direct electrical stimulation of the muscle
Concentrations evokes contractures
I I I
0 0.01
of caffeine > 5 mM regularly in the isolated soleus muscle. The
1
0.05
0.1
0.5
I.o
5.0
LCAFFEINE~ mM Fig. 2. Twitch potentiation by caffeine. The percentage twitch potentiation was defined as the percentage increase in twttch tension using the twitch before addition of caffeine as the reference point. The dashed line. --. is the result of connecting the 2.0 mM value to the 3.5 mM value which would be below 0 on this scale.
Caffeine
contractures
I
351
in rat soleus
25Q
H
5min
Fig. 3. Time course of caffeine contractures. The figure shows the time course of the contracture induced by two different doses of caffeine (A = 5 mM; B = 20 mM). The plateau at the left is the tetanic stimulus. Caffeine was added at the left arrow. In A caffeine was removed when the tension reached the baseline. The right arrow in B indicates the time at which caffeine was removed.
of relaxation and whether the relaxation is complete were not addressed in the study reported here. The size of the initial rapid peak appears to be dependent on the dose of caffeine. However, no clear analysis can be carried out at this time since, as the dose of caffeine increases, an increasing fraction of the tension measured at the inflection point is derived from the second peak. At this time we have no clear model for the form of maximal peak and therefore we cannot correct for its contribution to the initial tension measurement. The dependence of the height of the maximal contracture tension on the caffeine dose is shown in Fig. 4. Contractures appear at caffeine doses of 2.0-3.0 mM, show a half-maximal effect around 7.5 mM and saturate by 20 mM caffeine.
time course of these contractures is illustrated by typical results from two muscles at different caffeine doses shown in Figs 3A and B. Four phases were always seen: (i) a fast sharp rise in tension (2-4min). the height of which appears to be dose dependent. The rate of rise appears to be independent of the dose and is probably a diffusion limited process in these muscles; (ii) an inflection in the rise of tension; (iii) a slower increase in tension which leads to the maximal contracture tension, which at high doses reaches tetanic or slightly higher tension. The rate of rise of the second tension peak is dose dependent, as is the size of the final peak tension. Finally (iv), the relaxation phase in which tension falls from the maximal peak either back to resting (3A) or perhaps to a low level of sustained tension (3B). The issues of the rate
0.4L I
o
_
q/c41 1.0
/I 2.5
5.0
[CAFFEINE]
IO
20
100
mM
Fig. 4. Caffeine effect on contracture tension. The tension is expressed as fraction of tetanic tension and plotted against the caffeine concentration. All points are plotted as mean & SEM. The numbers in parentheses are the number of muscles included in the average.
R. J. CONNETTet d.
352
[CAFFEINE]mM Fig. 5. Caffeine effect on rate of tension increase. The rate of rise is defined as (time to peak tension)- I_ Each point is the average of 4-6 measurements and is plotted as mean + SEM. The line shown is a least squares fit to the data.
The time after caffeine addition at which the maximal tension occurs is also dependent on the dose of caffeine. The average rate of the tension development can be approximated by the reciprocal of the time after caffeine addition at which the peak occurs. This approximation is used since the rate of increase in tension is neither simply linear with time nor is there a clear set of exponential components. Both the initial rise in tension and the relaxation phase complicate a more detailed kinetic analysis. Figure 5 shows the relationship between the average rate of tension development and the caffeine dose. The effect of caffeine on the rate appears to be linearly related to the dose and, unlike the maximal tension. shows negligible saturation by 50 mM. Energy stores
Since our results indicates that maximal contracture tensions are obtained with caffeine doses of 20mM. we measured creatine phosphate, ATP and total creatine in muscles which had been exposed to 20mM caffeine for varying lengths of time (Table 1). Exposure to caffeine results in a significant reduction in energy content within 5 min, but for the next halfhour no further significant change in energy content occurs. Only phases (i) and (ii) of the contracture are completed in the first 5 min (Fig. 3). Thus it appears that the final peak tension development and the relaxation phases occur in the absence of any further change in energy stores, implying that the energy supply is keeping pace with demand. In addition, since there is a negligible change in ATP level throughout the entire contracture, it is unlikely that any of the processes are affected by ATP availability.
to caffeine, and second, to compare the response of mammalian muscle to that seen in amphibian muscle. Twitch
potentiation
Twitch potentiation occurs in frog muscles at caffeine concentrations of l-2mM and higher doses (> 2.5 mM) lead to contractures (Sandow et al., 1964; Ltittgau & Oetliker, 1968; Axelsson & Thesleff, 1958). Previous studies on soleus muscle from young rats showed that 20 and 40mM caffeine could potentiate twitch at 20°C while contractures were reliably seen only at 40 mM. Frank & Buss (1967) showed that raising the temperature to 37°C increased the sensitivity of the preparation to caffeine so that contractures were observed at 5 mM caffeine. They did not study twitch potentiation at 37°C. The results (Figs 2 and 4) demonstrate a clear potentiation of twitch at low doses of caffeine and show the continuum from potentiation through contracture. Twitch potentiation is seen with doses of caffeine in the range of 0.01&2.5 mM, whereas at higher doses twitch tension decreases and is absent at 10mM caffeine, a concentration of caffeine that proTable 1. Effects of exposure caffeine
on
time to 20mM energy stores in isolated rat soleus muscle
Time of exposure (min) 0 5 10 15 20 30
(,nmol/~Zol 0.809 0.601 0.524 0.563 0.596 0.516
+ + jI k + +
Cr,)
0.081 0.075 0.094 0.109 0.107 0.086
(27) (4) (4) (5) (4) (4)
DISCUSSION
The studies reported here were undertaken with two general purposes in mind. First, to use the stable soleus preparation to resolve some of the previous controversy about the response of mammalian muscle
All values were mean k S.E.M. with number of muscles given in parentheses, * PE = Potential phosphate-bond energy = creatine phosphate + 2 ATP. Cr, = total creatine content,
Caffeine contractures in rat soleus duces nearly maximal tension. Thus, at the normal physiological temperature of 37°C the sensitivity of the soleus muscle to caffeine, as measured by twitch potentiation, is about forty times greater than that observed at 20°C. When these results are compared with the results obtained in the frog sartorious preparation. it appears that the slow twitch rat soleus muscle is much more sensitive to the potentiating effect of low doses of caffeine. The potentiating doses of caffeine are in the range of those observed in humans ingesting caffeine. Blood levels obtained z 1 hr after I-2 cups of coffee range from 440pM (Axelrod & Reichenthal, 1953; Robertson et u/., 1978). Therapeutic doses gave levels in body water as high as 75 /tM (Khanna ef al.. 1980). Increases in exercise performance are observed with caffeine doses giving blood levels near 40 LAM(Costill et al., 1978; Ivy et ul.. 1979). All these blood concentrations are in the range of caffeine causing twitch potentiation in the isolated soleus (Fig. 2). Thus our data suggest that some of the effects of caffeine in humans could be due to direct effects on muscle.
Cont,.uc’t1rr.c Mammalian muscle was long suggested to be less sensitive to the contracture producing effects of caffeine than amphibian muscle (see e.g. Gutmann & Hanzlikova. 1966). Frank & Buss (1967). and Isaacson et ul. (1970) suggested that this apparent insensitivity of soleus muscle was a temperature effect. With 20 and 50mM caffeine, Isaacson et ul. found contractures of 50 and 65”;, tetanic tension, respectively, but these differences were not significant. We observe measurable contractures at 2.5 mM caffeine, with maximal contractures occurring in the range of 10 20mM caffeine. Unlike the results of Isaacson er trl. (1970) but similar to the results of Frank and Buss. our maximal contractures are IlO”<, tetanic tension, significantly higher than any obtained by them with the exception of studies on a split soleus with a dose of 20 mM caffeine where they obtained contractures that were 900/,, tetanic. Our observation of a higher maximal contracture tension may be the consequence of using only muscles that weighed less than 40 mg, so that we could be assured that size did not limit substrate availability to the individual muscle fibres (Chaudry & Gould. 1969). With larger muscles, caffeine diffusion may be limited so that while the surface fibers are relaxing, the internal muscle fibers are developing tension. This would result in an overall decrease in measured peak tension. Isaacson & Sandow (1967) did find that in the EDL with weights 3@100mg the diffusion of caffeine was two times slower than it is in frog sartorius. In the thicker soleus it may be even slower. The fact that higher contracture to tension ratios could be obtained by Isaacson ct d. (1970) with split soleus muscles would support this conclusion. Secondly. all our measurements are performed after the resting tension transient characteristic of these muscles is complete. thereby assuring that the twitch has recovered from the decrease that occurs during the tension transient (Pearce & Connett. 1980). Thus we feel that our results reflect the sensitivity of the slow twitch mammalian muscle to caffeine contractures. This conclusion is further supported by the observation that caffeine contractures
353
occur in human muscle in the concentration range’of 2230 mM (Moulds & Denborough. 1974). The threshold for caffeine contractures in frog sartorius muscle is -2.5 mM and maximal values are attained by 10 mM caffeine. Thus. although the sensitivity of the twitch tension of mammalian muscle to low doses of caffeine is greater than that of amphibian muscle, the sensitivity to a contracture-producing dose of caffeine is very similar in both mammalian slow muscle and frog sartorius.
A biphasic rise in tension and relaxation was seen at all contracture-producing doses where the contracture was greater than lo”,, of tetanic tension. Biphasic tension development has been reported rarely in frog muscle (Bianchi & Bolton. 1967; Gebert, 1968; Ltittgau & Oetliker. 1968; Konya c’t al., 1978) whereas it is always present in mammalian muscles at 37’C (Isaacson et cd.. 1970; Moulds & Denborough. 1974; Fig. 3). The relaxation or inactivation of the contracture has been reported previously for rat soleus (Isaacson ct cd.. 1970) but has not been observed in caffeine contractures in frog muscle. although there is some indication in single fiber records that a relaxing process may be occurring (Axelsson & Thesleff, 1958; Liittgau & Oetliker. 1968). Two problems may have prevented this process from being regularly observed in the frog studies. First. caffeine contractures are poorly reversible and most studies have involved short exposures to caffeine at contracture-producing doses. thus perhaps missing a relaxation phase. Second. there is an almost complete depletion of energy supplies on exposure of frog sartorius muscle to contracture-producing (5 mM) doses of caffeine (Hays & Connett. 1978). This could result in a sustained tension due to the production of rigor by the ATP depletion. In contrast. the rat muscles maintain ATP levels throughout both the development of tension and the relaxation of the caffeine-induced contracture (Table I). When compared with the well-studied frog preparations, these slowtwitch mammalian muscles: (a) are more sensitive to the potentiating effects of caffeine; (ii) show similar sensitivity to the contracture-producing effects of caffeine; (iii) give contractures which are always biphasic during the tension development; and (iv) relax after peak tension is reached. The first two observations could indicate that the potentiating effect and contracture effect result from two different processes, both of which occur in both species. The rat has a higher sensitivity for the potentiating process but a similar sensitivity to caffeine for the contracture producing process than frog muscle. The relaxation, which is not seen in frog muscle. may be due to a process which is unique to mammalian muscle or may simply be a response which cannot be observed in the ATP-depleted frog muscle. Detailed studies on isolated rat muscle fibers would be necessary to resolve these issues. Acknowledgcmmts~This work was supported in part by grant in aid from the Muscular Dystrophy Association and by NIH grant No. AM22124. The authors would like to thank Carrie Golliger for excellent technical assistance. R. J. Connett is the recipient of USPHS RCDA 5K04AM00155.
R. J. CONNETT et ul
354 REFERENCES
AXLEROD J. & REICHENTHAL J. (1953). The fate of caffeine
in man and a method for its estimation in biological material. J. Pharmac. rxp. Thw. 107, 517-523. AXELSSON J. & THESLEFF S. (1958) Activation of the contractile mechanism in striated muscle. Acta physiol. stand. 44, 55-66. BIANCHI C. P. & BOLTON T. C. (1967) Action of local anesthetics on coupling systems in muscles. J. Pharmuc. rxp. Thu. 157, 388-405. CHAUDRY F. H. & GOULD M. K. (1969). Kinetics of glucose uptake in isolated soleus muscle. Biochwn. hiophys. Acta 177. 527-536. CONNETT R. J. & HAYS E. T. (1978). Energy coupling to sodium transport in frog skeletal muscle. Am. J. Ph,siol. 235, C25X34. COSTILL D. L.. DALSKY G. P. & FINK W. J. (1978) Effects of caffeine ingestion on metabolism and exercise performance. Med. Sci. Sport 10, 155-158. FRANK G. B. & Buss W. C. (1967) Caffeine-induced contractures in mammalian skeletal muscle. Archs inr. Pharmacodyn. The+. 170, 343-349. GERERT G. (1968) Caffeine contracture of frog skeletal muscle and of single fibers. Am. J. Pllysiol. 215, 296298. GUTMANN E. & HANZLIKOVA V. (1966) Contracture responses of fast and slow mammalian muscles. Physiol. Bohcm. 15, 404-414. GUTMANN E. & SANDOW A. (1965) Caffeine-induced contracture and potentiation of contracture in normal and denervated rat muscle. I& Sci. 4, 1149-l 156. HAYS E. T. & CONNETT R. J. (1978) Effect of methylxanthines and elevated external potassium on high energy phosphate content in frog skeletal muscle. Biochem. Phurmac. 27. 2965-2968. ISAACSONA.. HINKES M. J. & TAYLOR S. (1970) Contracture and twitch potentiation of fast and slow muscles of the rat at 20. and 37’C. Am. J. PhJsiol. 218, 33-41.
ISAACSONA. & SANDOW A. (1967) Caffeine
effects on radiocalcium movement in normal and denervated rat muscle. J. Pharmuc. cup. Thrr. 155, 376388. IVY J. L., COSTILL D. L.. FINK W. J. and LOWER R. W. (1979) Influence of caffeine and carbohydrate feedings on endurance performance. Med. Sci. Sports 11, 61 I. KHANNA N. N.. BADA H. S. & SOMANI S. M. (1980) Use of salivary concentrations in the prediction of serum caffeine and theophylline concentrations in premature infants. J. Pediat. 96, 494 499. KENYA L.. J~NA I.. K&CR A. & ARADI F. (197X) Determination of dose and time dependence of muscle contractures elicited by caffeine. Actor hiol. Acad. Sci. Inmq. 29, 129 137. Liirrc;~r~ H. C. & OFTLIK~K H. (1968) The action of caffeine on the activation of the contractile mechanism in striated muscle fibres. J. Physiol. 194. 51-74. MOULDS R. F. W. & DLNBOKOUC;HM. A. (1974) A study of the action of caffeine. halothane. potassium chloride and procaine on normal human skeletal muscle. C/in. cp. Pharmuc. PhJsiol. 1, 197-209. PEAKC~,F. J. & COUN~TT R. J. (1980) Effect of lactate and palmitate on substrate utilization of isolated soleus muscle. Am. J. Physiol. 238, Cl49 159. RORFKTSON D.. FR~~LICHJ. C., CARR R. K.. WATSOU J. T.. HOLLIEIELD J.. SHAND D. & OATCS J. (1978) Effects of caffeine on plasma rcnin activity, catecholamines and blood pressure. Neir’ Engl. J. Med. 298, 181 186. SAEZDOWA. (1964) Potentiation of muscular contraction. Archs Pkv. Med. Rehahil. 45. 62-81. SANDOW A.. TAYLOR S. R.. ISAACSONA. & STQLIIN J. J. (1964) Electromechanical coupling in potentiation of muscular contraction. Scicww 143, 577-579. TONER M. M.. KIRCKENDALL D., CHASE J. M.. D~LIO D. J.. CLEARY P. & Fox E. L. (1980) The effect of caffeine on the metabolic and cardiovascular responses to exercise. Mc,d. Sci. Sports Ewr. 12, 109.