Department of Zoology, La Trobe University, Bundoora, Victoria, 3083 Australia
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ABSTRACT |
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It is unclear whether accumulation of lactate in skeletal muscle fibers during intense activity contributes to muscle fatigue. Using mechanically skinned fibers from rat and toad muscle, we were able to examine the effect of L(+)-lactate on excitation-contraction coupling independently of other metabolic changes. We investigated the effects of lactate on the contractile apparatus, caffeine-induced Ca2+ release from the sarcoplasmic reticulum, and depolarization-induced Ca2+ release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable (20-35%) inhibition of caffeine-induced Ca2+ release, seemingly by a direct effect on the Ca2+ release channels. However, 15 mM lactate had no detectable effect on Ca2+ release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect on normal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.
excitation-contraction coupling; fatigue; exercise; calcium release channel; ryanodine receptor
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INTRODUCTION |
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WHEN SKELETAL MUSCLE undergoes intense activity, force
production eventually declines; this is commonly referred to as fatigue (1, 15, 39). Repeated short contractures induce a type of fatigue with
relatively slow onset and recovery (i.e., over many minutes), which is
thought to be due to metabolic changes within the muscle fibers (1, 15,
18, 35). With vigorous exercise, lactate concentrations in fast-twitch
skeletal muscles can reach 30 mM (20, 22, 30, 34). In many
circumstances, there is a strong inverse correlation between lactate
concentration and force production during the onset of fatigue and
recovery from fatigue (15, 22). It was thought that this correlation was actually due to the concomitant increase in H+
concentration ([H+]) occurring with lactic acid
production rather than to lactate per se (15). However, recent work
indicates that an increase in [H+] is probably
not the primary factor responsible for reductions in Ca2+
release and maximum Ca2+-activated force in fatigue (5, 7,
27). Moreover, other recent studies indicate that lactate itself may
have direct effects on force production and Ca2+ release.
For example, a 15% reduction in force production was observed when
arterial infusion of L(+)-lactate was used to raise muscle
lactate concentration from ~8 to 12 mM without any accompanying pH
change (19). Furthermore, experiments on chemically skinned muscle
fibers show that, with the pH maintained at 7.0, 20 mM lactate in the
cytoplasm causes a small (~5%) reduction in maximum Ca2+-activated force (2). Finally, it has been found that
the presence of 10-20 mM lactate inhibits Ca2+- and
caffeine-activated Ca2+ release and ryanodine binding in
sarcoplasmic reticulum (SR) vesicles and activation of isolated
ryanodine receptor (RyR) Ca2+ release channels, all by
~30-70% (11, 12).
Nevertheless, it is unclear whether lactate inhibits Ca2+ release in functioning muscle fibers. As pointed out by Favero and colleagues (12), the results with SR vesicles and isolated release channels do not show whether lactate interferes with the normal excitation-contraction (E-C) coupling mechanism in muscle, which involves activation of Ca2+ release channels by the voltage sensor/dihydropyridine receptors in the adjacent transverse tubular (T) system (29). Here, we examine this question by investigating the effect of cytoplasmic lactate on Ca2+ release in mechanically skinned muscle fibers in which the normal voltage sensor coupling mechanism is operating (13, 24, 25, 27, 38). We show that lactate appreciably inhibits the ability of caffeine to activate Ca2+ release in muscle fibers but that voltage sensor activation of Ca2+ release is inhibited to only a comparatively small extent (<10%), even at 30 mM lactate. This indicates that lactate accumulation is probably not a major factor in muscle fatigue, even in fast-twitch fibers.
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METHODS |
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Skinned fibers. Mechanically skinned fibers were obtained and used as described previously (25, 27). Briefly, Long-Evans hooded rats were anesthetized with halothane (2% vol/vol) in a bell jar and killed by asphyxiation, and then the extensor digitorum longus (EDL) muscles were removed. Cane toads (Bufo marinus) were stunned by a blow to the head and killed by pithing, and the iliofibularis muscles were removed. Single muscle fibers were mechanically skinned under paraffin oil, and a segment was attached to a force transducer (model AME875, Horten) at 120% of resting length. The skinned fiber (30-50 µm diameter) was placed within a 2-ml Perspex bath containing a potassium hexamethylene diamine tetraacetate (K-HDTA) solution (see below) for 2 min, then the bath was rapidly lowered and a bath with a different solution was substituted to stimulate the fiber. In the experiments on depolarization-induced Ca2+ release, fibers were used with their endogenous level of SR Ca2+ and were not additionally loaded. All experiments were performed at room temperature (23 ± 2°C).
Solutions.
All chemicals were obtained from Sigma Chemical (St. Louis, MO), unless
specified otherwise. The standard K-HDTA solutions used for rat and
toad fibers contained (mM) 117 (toad) or 126 (rat) K+, 37 Na+, 50 HDTA2 (Fluka, Buchs,
Switzerland), 8 total ATP, 8.6 total Mg2+, 10 phosphocreatine, 0.05 total EGTA, 60 (toad) or 90 (rat) HEPES, and 1 N
3, with pH adjusted to 7.10 ± 0.01 and pCa to 7.0. Skinned fibers were depolarized by substitution of an Na-HDTA solution, which was identical to the corresponding K-HDTA
solution, except all K+ was replaced with Na+.
All solutions had an osmolality of 255 ± 5 (toad) or 295 ± 5 mosmol/kg (rat) and a free Mg2+ concentration
([Mg2+]) of 1 mM, with the assumption of
apparent Mg2+ affinity constants of 6.9 × 103 M
1 for ATP, 8 M
1 for HDTA, and 15 M
1 for phosphocreatine (14, 37).
Corresponding solutions with 15 or 30 mM L(+)-lactate were
made by mixing the above solutions with an appropriate amount of a
corresponding lactate stock solution in which all HDTA (50 mM,
K2 or Na2) was replaced with 120 mM lactate
(K+ or Na+). The other constituents in the
lactate stock solution were unchanged, except the total
Mg2+ (added as MgO) was increased by an additional 0.7 mM
to maintain the free [Mg2+] at 1 mM on the
basis of an apparent Mg2+ affinity constant of 9 M
1 for lactate (32). The substitution of
a divalent anion (HDTA) with a monovalent anion (lactate) meant that
the osmolarity was slightly increased (<8%) and the ionic strength
slightly decreased (<3.2%) in solutions for rat fibers at the
highest lactate concentration used (30 mM). The net effect of these
changes on maximum Ca2+-activated force and
depolarization-induced force responses would have been very small
(<2% increase) (28) and, furthermore, should be of even less
importance when the relative changes of the two parameters are compared
(e.g., the percent decline in both parameters in the presence of
lactate). In all solutions used for depolarization-induced Ca2+ release, total EGTA concentration was 50 µM and pCa
was 7.0, as measured with a Ca2+-sensitive electrode (Orion
Research, Boston, MA).
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Contractile apparatus measurements. In the experiments on the properties of the contractile apparatus, the skinned fiber was exposed to a sequence of solutions, each with 10 mM total EGTA and progressively higher Ca2+ concentration ([Ca2+]), made by appropriate mixture of the 50 mM Ca-EGTA and 50 mM free EGTA solutions, together with four times the volume of the standard K-HDTA solution or the corresponding K-HDTA solution with lactate (to give a final lactate concentration of 15 or 30 mM). Force measurements in the presence of lactate were always bracketed by similar sequences in the absence of lactate. The force produced by the fiber at each pCa under a given condition was expressed as a percentage of the corresponding maximum Ca2+-activated force and plotted against pCa. The scientific analysis program GraphPad Prism (GraphPad Software, San Diego, CA) was used to fit Hill curves to the force-pCa data obtained under each condition.
Caffeine-induced Ca2+ release experiments. The Ca2+ content of the SR was assayed (Fig. 1) by preequilibrating the fiber for 10 s in the standard K-HDTA solution with 0.5 mM EGTA (pCa 8; preequilibration solution) and then totally depleting the SR in the 30 mM caffeine-low [Mg2+] solution with 0.5 mM EGTA (pCa 8; total release solution) (3). It is necessary to have 0.5 mM EGTA present in the 30 mM caffeine-low [Mg2+] solution to rapidly chelate the released Ca2+ in order that the resulting force response can be related to the amount of Ca2+ released. The area under the force response on first depletion of the SR was indicative of the level of SR Ca2+ present endogenously. (Note that the fiber was skinned under oil and could neither gain nor lose Ca2+ during the skinning procedure.) The fiber was left in the total release solution for 2 min to ensure complete Ca2+ depletion (17), washed for 30 s, and then reloaded for a set time (e.g., 30 or 40 s) in the load solution. The fiber was then immediately depleted again with the above procedure (preequilibration and 30 mM caffeine-low [Mg2+]; total release in Fig. 1) or was first equilibrated for 15 s in a weakly Ca2+-buffered K-HDTA solution (pCa 7.0, 50 µM EGTA) with or without lactate, exposed to the same solution with 7 mM caffeine for 15 s, and then totally depleted of Ca2+ (Fig. 1). Additional caffeine release experiments were carried out with all solutions containing Na-HDTA (i.e., zero K+) to keep the T system chronically depolarized and the voltage sensors inactivated.
Force traces and presentation and analysis of results. In all traces showing depolarization-induced force responses, unless indicated otherwise, the skinned muscle fiber was bathed in the standard K-HDTA solution (pCa 7.0, 50 µM EGTA) and depolarized in the matching Na+ solution. Values are means ± SE, and n indicates the number of fibers examined. Statistical significance was determined with Student's t-test (1-tailed, paired or unpaired, as appropriate), with mean values considered significantly different if P < 0.05.
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RESULTS |
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Effect of lactate on the contractile apparatus in rat EDL fibers. We first examined the effect of lactate on the properties of the contractile apparatus in the skinned fibers. Each rat EDL fiber was exposed to sequences of solutions, with and without lactate, in which the free [Ca2+] was heavily buffered (10 mM total EGTA) at various levels. Force was plotted against pCa, and Hill curves were fitted for each fiber individually. In the absence of lactate, the pCa producing half-maximal force (pCa50) had a mean of 5.97 ± 0.03, with a mean Hill coefficient of 4.6 ± 0.4 (n = 17). Maximum Ca2+-activated force was reduced in the presence of 15 and 30 mM lactate to 97.0 ± 1.3% (n = 7, P < 0.05) and 97.9 ± 0.8% (n = 16, P < 0.05), respectively, of the average of the bracketing control level in the absence of lactate (HDTA concentration was reduced when lactate was added; see METHODS). [Normalizing data to the average of the bracketing controls shows the effect of lactate independently of the small decline (2-3%) in maximum force that occurs with each successive sequence irrespective of exposure to lactate. The maximum for the control sequence after lactate exposure in the above experiments was 94.8 ± 0.9% of the initial control (n = 33).] The Ca2+ sensitivity of the contractile apparatus, however, was not significantly altered at either concentration of lactate [mean change in pCa50 = 0.008 ± 0.005 (n = 7) and 0.006 ± 0.006 (n = 12) and mean change in Hill coefficient = 0.10 ± 0.06 and 0.09 ± 0.08 in 15 and 30 mM lactate, respectively]. Thus the effect of lactate on the contractile apparatus was very small.
Caffeine-induced Ca2+ release. Lactate inhibits caffeine-induced Ca2+ release in isolated SR vesicles (11, 12). We wished to examine whether lactate showed a similar inhibitory action on caffeine-induced Ca2+ release in skinned fibers with the normal voltage sensor coupling mechanism still intact and with the cytoplasmic [Mg2+] and SR luminal [Ca2+] at close to physiological levels. In these experiments, each skinned fiber was subjected to repeated cycles in which the SR was fully depleted of Ca2+ (by exposure to a solution with 30 mM caffeine-low [Mg2+] and 0.5 mM EGTA, pCa 8; see METHODS) and then reloaded with Ca2+ to particular levels. The [Mg2+] was lowered in the depleting solution from the physiological level of 1 mM to 0.05 mM, because this makes caffeine far more effective at eliciting total Ca2+ release in mammalian fibers (17). Such load-release cycles gave highly reproducible results in each fiber. As found previously (3, 10), the "area" (i.e., time integral) of the force response produced on full depletion of the SR (e.g., 1st total release in Fig. 1) was approximately linearly related to the loading time (not shown), which meant that it could be used as an indicator of the amount of Ca2+ present in the SR. Because fibers were skinned under oil (see METHODS), the amount of Ca2+ present in the SR endogenously could be ascertained, and then the SR could be repeatedly depleted of Ca2+ and reloaded to that level or some other level as desired.
To test the effect of lactate on caffeine-induced Ca2+ release, the SR was loaded with Ca2+ to a particular level, and then the fiber was equilibrated for 15 s in a weakly Ca2+-buffered solution (50 µM EGTA) at pCa 7.0 with 1 mM free Mg2+ and then exposed to the same solution with 7 mM caffeine (e.g., Fig. 1). Provided that the SR was loaded above some threshold level (between ~25 and 50% of the endogenous level), exposure to the 7 mM caffeine solution invariably elicited Ca2+ release and a force response in every EDL fiber. After a total of 15 s of exposure, the caffeine was washed out and the SR was fully depleted of Ca2+ with the 30 mM caffeine-low [Mg2+] solution (e.g., 2nd total release in Fig. 1) to ascertain how much Ca2+ had remained in the SR after the 15-s exposure to 7 mM caffeine. The entire procedure was then repeated with lactate in the 7 mM caffeine solution (and during the preceding 15-s equilibration) and then again without lactate. Because there was very little Ca2+ buffer in the 7 mM caffeine solutions, the area of the resulting force response is not simply related to the amount of Ca2+ released from the SR and cannot be directly compared with the area of the force response to the total release solution (30 mM caffeine-low [Mg2+]-0.5 mM EGTA). It is likely that, during the Ca2+ release in the 7 mM caffeine solution, a substantial amount of Ca2+ was simultaneously being taken back up by the SR. Consequently, the reduction in the area of the response on subsequent total release indicates the net loss of Ca2+ from the fiber over the period in 7 mM caffeine. The finding that the force response to 7 mM caffeine ceased even though the SR evidently still contained substantial Ca2+ (Fig. 1) appears broadly consistent with the observation that the SR had to be loaded to some minimum level to induce any Ca2+ release to 7 mM caffeine (see above), with both results possibly suggesting that the SR loading level regulates the responsiveness of the Ca2+ release channels to caffeine. More studies need to be done on the detailed characteristics and basis of this regulation. When the SR was loaded with Ca2+ at approximately the level present endogenously, the force response to 7 mM caffeine was significantly smaller and slower rising in the presence of 30 mM lactate than in the absence of lactate (reduction to 75 and 64%, respectively; Table 1). Less marked effects were observed in the presence of 15 mM lactate (Table 1). These reduced force responses cannot be explained by effects of lactate on the contractile apparatus (see above) and imply that caffeine-induced Ca2+ release was inhibited to some extent in lactate. The reduction in Ca2+ release in lactate was further apparent from the extent of depletion of SR Ca2+ in these experiments. In the absence of lactate, exposure to the 7 mM caffeine solution resulted in a decrease in the area of the force response on subsequent emptying of the SR to 79 ± 9% (n = 11) of that found without any exposure to 7 mM caffeine (cf. 1st and 2nd total release in Fig. 1). In other words, there had been a net loss of ~21% of the total SR Ca2+ from the fiber over the equilibration and 7 mM caffeine exposure periods in the absence of lactate. When the same procedure was performed with lactate, the force response on emptying the SR was significantly larger than that in the absence of lactate (110 ± 3% of non-lactate case, n = 11, P < 0.05, Student's paired t-test; cf. 3rd total release with 2nd and 4th in Fig. 1), indicating that there had been less net Ca2+ loss from the fiber in 7 mM caffeine when lactate was present, which in turn suggests that there had been less Ca2+ release. All the above experiments were performed using K-HDTA solutions, with the T system polarized and the voltage sensors activatable (see below). Virtually identical results were also obtained (not shown) when all solutions were made with Na-HDTA (see METHODS), so that the T system was kept depolarized and the voltage sensors were inactivated.
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Depolarization-induced responses in skinned fibers.
As described previously with rat and toad muscle (25, 27), the E-C
coupling mechanism remains functional after muscle fibers are
mechanically skinned under paraffin oil. Importantly, the fibers also
retain the endogenous level of SR Ca2+ and require no
additional loading. On skinning, the T system seals over (24) and
repolarizes if the fiber segment is bathed in a high
[K+] solution and can then be rapidly
depolarized by substitution of a solution in which all K+
is replaced with Na+ (see METHODS). Such
stimulation elicits a rapidly rising force response that lasts for only
2-4 s, owing to inactivation of the voltage sensors and reuptake
of released Ca2+ into the SR (25, 26) (Fig.
2). The voltage dependence of activation
and inactivation and modulation by pharmacological agents (e.g., D600)
and Cl (9, 25, 26, 27) strongly indicate that the
responses in these skinned fibers are controlled by the same basic E-C
coupling mechanism as in intact fibers. Although the precise membrane
potentials are not known, the above characteristics indicate that the T
system of EDL fibers is polarized to more negative levels than
80 mV in the standard 126 mM K+ solution, and this
is largely or fully dissipated by Na+ substitution (9).
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Effect of prolonged exposure to lactate.
When the total duration of the lactate exposure was increased to
3-5 min, the force response to depolarization after washout of
lactate was actually reduced considerably, rather than returned to the
original control level (e.g., Fig.
3A). The mean
depolarization-induced force response on washout of lactate under such
circumstances was 76.2 ± 4.9% of the original control response in
rat EDL fibers (n = 23) and 31.7 ± 10.4% in toad
iliofibularis fibers (n = 10). This reduction in the response
was not simply due to fiber "rundown," because it was largely
reversed if the fiber was returned to the lactate solution (e.g., end
of Fig. 3A). Furthermore, the reduction in the response was
simply not due to the Na+ substitution becoming less
effective at fully depolarizing the T system, because depolarization by
ChCl substitution was similarly reduced in effectiveness (not shown),
even though such substitution should be a very potent depolarizing
stimulus (9, 25, 27). It was also found that, after prolonged exposure
to lactate, fibers frequently gave a spontaneous force response ~10 s
after the lactate was washed out (Fig. 3B). This was observed
in 6 of 10 toad fibers and 7 of 23 rat fibers, with the response
lasting from ~2 to 30 s and the peak size ranging from 1 to 34%
(mean 9 ± 5%) of the control depolarization-induced response in the
toad fibers and from 1 to 16% (mean 6 ± 3%) in the rat fibers. This
force response was due to Ca2+ release from the SR, and the
resulting partial depletion of SR Ca2+ in these cases
probably exacerbated the reduction in the response to the following
depolarization (Fig. 3B).
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DISCUSSION |
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In this study, mechanically skinned skeletal muscle fibers were used to investigate the effect of L(+)-lactate on the sequence of E-C coupling from depolarization to muscle contraction, as well as on individual aspects within that sequence. With this preparation we were able to rapidly apply and wash out lactate in the cytoplasm and quantify its effects on the contractile apparatus and on SR Ca2+ release evoked directly with caffeine or by the physiological coupling mechanism involving the voltage sensors in the T system. Lactic acid is virtually fully dissociated into lactate and H+ at physiological pH, and here we held the pH constant at 7.1 to examine the effects of lactate alone.
Effects of lactate on contractile apparatus and caffeine-induced Ca2+ release. Under the conditions used here, lactate had only very small effects on the properties of the contractile apparatus. In rat EDL fibers, Ca2+ sensitivity was not significantly affected, and the maximum Ca2+-activated force in 15 and 30 mM lactate only decreased to 97.0 and 97.9%, respectively, of that in the absence of lactate. These values are quite comparable to those obtained previously in chemically skinned psoas fibers from rabbit (2), where, depending on the exact ion substitution used, the maximum Ca2+-activated force was 92.3-97.8% of control in 15 mM lactate and 95.9-101.6% of control in 30 mM lactate.
We further found that lactate inhibited caffeine-induced Ca2+ release in the skinned EDL fibers in a concentration-dependent manner. The relative effects were slightly smaller than those found previously with SR vesicles and isolated RyR Ca2+ release channels (11, 12). In the present experiments, the coupling mechanism between the Ca2+ release channels and the voltage sensors remained intact and functional, and the [Mg2+] and ATP concentration were at physiological levels. We found that the peak and the rate of rise of the force response to caffeine were reduced in the presence of lactate (Fig. 1) but that the effects were less marked when the SR was loaded with Ca2+ at the physiological level than when the SR was partially depleted of Ca2+ (Table 1). This shows that the modulatory effects of various ligands on the Ca2+ release channels depend on the exact conditions used, and this highlights the importance of keeping conditions as close to physiological as possible. At physiological SR loading, 30 mM lactate caused a 25% reduction in the peak of the force response to caffeine and a 36% reduction in the rate of rise of force (Table 1), with the latter value possibly being the better estimate of the relative reduction in rate of Ca2+ release in lactate. This inhibitory effect of lactate on caffeine-induced Ca2+ release is probably due to a direct action of lactate on the RyR Ca2+ release channel, as evidently occurs in isolated RyRs (12). Nevertheless, we cannot entirely exclude the possibility that lactate exerted its effects more indirectly in the experiments here. We previously showed that addition of ClEffect of lactate on depolarization-induced Ca2+ release. Although lactate evidently inhibits caffeine-induced Ca2+ release, this does not necessarily mean that it has a similar inhibitory effect on Ca2+ release evoked by the normal voltage sensor coupling mechanism. This latter mechanism is considerably more efficacious at inducing release and is also known to be able to largely overcome or bypass the strong inhibitory effects of other ligands, such as Mg2+ and H+, on the release channels (27). Importantly, we found here that the presence of 15 mM lactate had no significant effect on depolarization-induced force responses in EDL fibers and that 30 mM lactate caused only a small decrease in the response (~9% in peak size and ~4% in half-width; Table 2). Only about one-fifth of the reduction in the peak force response observed with 30 mM lactate was due to the direct effects of lactate on the contractile apparatus (i.e., ~2% decline; see above). We previously used a fiber-lysing technique to quantify the relationship between SR Ca2+ content and the size of the depolarization-induced force response and showed that even small decreases in the total amount of Ca2+ release in rat EDL fibers produce a measurable reduction in the peak of the force response to depolarization and a considerably larger reduction in the area of the response (31). From this we can conclude that 30 mM lactate caused <10% reduction in the total amount of Ca2+ release by depolarization in rat EDL fibers. Similar consideration of the data obtained with toad iliofibularis muscle fibers (Table 2) suggests that the decrease is even smaller in such fibers (0-5%). Here we are assessing the total amount of Ca2+ released by a depolarization, not the rate of release, and we would not detect a change in the initial rate of release unless it was substantial. Thus it is still possible that lactate reduces the initial rate of depolarization-induced Ca2+ release and that this might mean that the twitch response of an intact fiber would be reduced. Nevertheless, the contractures found here should be quite comparable to tetanic responses in a muscle fiber and so should indicate the normal physiological response to lactate.
Thus the presence of lactate in the cytoplasm had a far smaller inhibitory effect on depolarization-induced Ca2+ release than on caffeine-induced Ca2+ release. This might be the result of voltage sensor stimulation largely overcoming or bypassing the inhibitory effects of lactate on the Ca2+ release channel or might simply be due to lactate only exerting a mild degree of inhibition on the release channels, because this would appear proportionately more important with a comparatively weakly activating stimulus, such as caffeine, than with the far more potent stimulating effect of the voltage sensors. We cannot exclude the possibility that lactate simply interfered with caffeine binding to the Ca2+ release channels, but there is no obvious similarity in chemical structure of lactate and caffeine, and also such an explanation would not account for the ability of lactate to inhibit both Ca2+- and caffeine-induced Ca2+ release in SR vesicles (11, 12). In line with our conclusions on caffeine-induced Ca2+ release (see above), we assume that the small inhibitory effect of lactate on depolarization-induced Ca2+ release is not due to lactate addition causing a transient change in SR potential. If we are wrong in this assumption, lactate would not exert even this small inhibitory effect on depolarization-induced Ca2+ release in vivo, because lactate would equilibrate across the SR as its concentration slowly rose in the cytoplasm. Our experiments with longer exposures to lactate (Fig. 3) indicated that lactate also gradually accumulated in the sealed T system of the skinned fibers. This was concluded from the fact that when lactate had been present in the cytoplasm for 3-5 min, removal of the lactate actually inhibited the response to depolarization, rather than restored it, and also frequently induced Ca2+ release. Because this latter effect only occurred when the T system was polarized and the voltage sensors were operable, we conclude that, when the lactate was washed out, the T system became sufficiently depolarized to trigger Ca2+ release. Such partial depolarization would also explain the reduced response to T system depolarization by Na+ substitution (Fig. 3A) and ChCl substitution (not shown), because some voltage sensors would not recover from inactivation, and normal coupling would be hindered (25). Evidently, when lactate was present in the cytoplasm, it gradually accumulated in the sealed T system, probably as a result of a specific transport mechanism and passive diffusion (21). Consequently, when lactate was rapidly removed from the cytoplasm, it would still have been present in the T system at first, and this concentration difference apparently caused depolarization of the T system membrane. This might have occurred because of ion movements directly or indirectly accompanying lactate transport back into the cytoplasm. Whatever the basis of the effect may be, we were unable to prevent it with quercetin, an inhibitor of lactate transport (21), or 9-AC, an inhibitor of the major anion channel in the T system (9). Clearly then, lactate was moving into the T system during even the brief (30- to 45-s) exposures to lactate, even though it apparently did not reach high levels in the T system over this period, because, on removal of lactate from the cytoplasm, depolarization-induced responses recovered to the control level and spontaneous responses were not observed (Figs. 2A and 3A). In view of this, it is possible that even the small degree of inhibition of the depolarization-induced responses seen in the presence of 30 mM lactate (Fig. 2, Table 2) was caused by chronic partial depolarization of the T system arising in some way from the transport of lactate into the T system. We have no evidence that this was occurring, but if it was, it would again mean that even the small level of inhibition of depolarization-induced Ca2+ release found here overestimated the effect occurring in vivo, where lactate concentrations would not change so rapidly and, hence, become so disparate between the inside and the outside of the fiber. The possible indirect effects of lactate on T system potential observed here (Fig. 3) may have contributed to the substantial level of inhibition of tetanic force production seen by Spangenburg and colleagues (33) with application of up to 50 mM lactate to the extracellular solution bathing whole muscles. They found that the inhibitory effect was maximal within 3 min of lactate exposure, and this seems more easily explained by the transport of lactate across the surface and T system membranes, causing some degree of partial depolarization and action potential failure, than by some effect of cytoplasmic lactate itself, because it seems unlikely that the concentration within the fibers would reach a high level in such a brief time, even in the case of superficial fibers.Relevance to muscle fatigue. The presence of lactate in the cytoplasm has only a very small inhibitory effect directly on the contractile apparatus (see RESULTS) (2). We further found here that lactate has only a minor inhibitory effect on depolarization-induced Ca2+ release, even at 30 mM (Fig. 2, Table 2). Thus it appears that the build-up of lactate per se could contribute only a small degree to the development of fatigue, even in fast-twitch muscles. In view of this, the inverse correlation observed between muscle lactate concentration and force (see the introduction) is likely to be the result of other metabolic changes occurring during heavy exercise, such as increases in Pi concentration and [Mg2+] and, to a lesser degree, [H+] (1, 16, 18, 27, 35), and also possibly glycogen depletion (6, 36). The production of lactic acid and other metabolic changes occurring in these circumstances will increase the number of osmotically active particles in the muscle fiber (15, 30), and the influx of extracellular water will result in swelling of the fiber to maintain osmotic equilibrium (1, 39). In total, there will probably be relatively little change in intracellular ionic strength, and any such change is unlikely to substantially affect E-C coupling (28). It is nevertheless possible that, in some circumstances, fiber swelling might affect action potential propagation in the T system or communication between the T system and the SR and, hence, might contribute to muscle fatigue. In summary, although it is possible that lactate accumulation contributes to muscle fatigue in some indirect manner, the findings of this study show that the presence of a high cytoplasmic concentration of lactate itself has only a relatively minor inhibitory effect on normal E-C coupling.
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ACKNOWLEDGEMENTS |
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We are grateful to Aida Yousef for technical assistance.
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FOOTNOTES |
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This study was supported by a grant from the National Health and Medical Research Council of Australia (9936582).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. D. Lamb, School of Zoology, La Trobe University, Bundoora, Victoria 3083, Australia (E-mail: zoogl{at}zoo.latrobe.edu.au).
Received 26 July 1999; accepted in final form 21 October 1999.
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