Favero, Terence G., Anthony C. Zable, David Colter, and Jonathan J. Abramson. Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum. J. Appl. Physiol. 82(2): 447–452, 1997.—Sarcoplasmic reticulum (SR) Ca2+-release channel function is modified by ligands that are generated during about of exercise. We have examined the effects of lactate on Ca2+- and caffeine-stimulated Ca2+ release, [3H]ryanodine binding, and single Ca2+-release channel activity of SR isolated from rabbit white skeletal muscle. Lactate, at concentrations from 10 to 30 mM, inhibited Ca2+- and caffeine-stimulated [3H]ryanodine binding to and inhibited Ca2+- and caffeine-stimulated Ca2+ release from SR vesicles. Lactate also inhibited caffeine activation of single-channel activity in bilayer reconstitution experiments. These findings suggest that intense muscle activity, which generates high concentrations of lactate, will disrupt excitation-contraction coupling. This may lead to decreases in Ca2+ transients promoting a decline in tension development and contribute to muscle fatigue.
- muscle fatigue
- skeletal muscle
skeletal muscle subjected to continuous repetitive contractions will develop fatigue. This can be manifest as a reduction in expected maximal force output or noted as an increase in the effort required to maintain a submaximal force (7). The direct cause(s) of muscle fatigue remain(s) elusive because of the variety of muscular activation patterns and events that have been shown to elicit fatigue. It is generally accepted that fatigue resulting from submaximal continuous exercise arises from a different etiology than fatigue associated with high-intensity maximal efforts. However, in most cases in which well-motivated subjects were used, it is believed that causes of fatigue are related to changes within the muscle itself (3). Two prominent mechanisms have been proposed to explain fatigue in isolated skeletal muscle: 1) decline in contractile force generation via metabolic effects on contractile proteins (5, 10), and 2) depressions in sarcoplasmic reticulum (SR) Ca2+ release (6,11, 14). Most work to date has focused on metabolic effects on contractile proteins from skinned and intact fibers (5, 10). However, the excitation-contraction coupling (ECC) process and SR Ca2+ release have received increased attention (1, 9, 25,26).
ECC is the multistep process whereby t-tubule depolarization results in the release of Ca2+ from the SR, which subsequently binds to troponin to activate cross-bridge cycling (20). The mechanism of ECC in skeletal muscle involves an interaction between the t-tubule dihydropyridine receptor, which functions as a voltage sensor, and the SR Ca2+-release channel (20). Models have been proposed in which charge movement at the t-tubule membrane evokes a conformational change in the voltage sensor, which, in turn, causes the SR Ca2+ channels to open (21). Gyorke (11) has suggested that fatigue might be caused by alterations in any step of the SR Ca2+-release activation sequence, including surface and t-tubule membrane excitability, sensing of the transmembrane potential change by the t-tubule voltage sensor, transmission of the signal across the triadic junction, and/or Ca2+ release from the SR. Considering the many processes involved in this sequence, definitive experiments demonstrating fatigue-induced modification of ECC have proven to be difficult.
The role of Ca2+ ions in ECC and the SR Ca2+-release process have also been subject to considerable interest (28). Ca2+ has long been proposed as an activator in the SR Ca2+-release process (20, 28). Under this scenario, a rapid rise in myoplasmic Ca2+ concentration following activation by depolarization of the t tubule produces a positive-feedback Ca2+-induced Ca2+ release (CICR) from the SR. This mechanism may be critical for the activation of SR Ca2+-release channels not directly coupled to t-tubule voltage sensors.
In this work, we have extended our previous studies examining lactate inhibition of SR Ca2+-release channel function and we have more closely evaluated its effects on Ca2+-activated SR Ca2+ release (8). We demonstrate that lactate inhibits Ca2+- and caffeine-induced Ca2+release from, and [3H]ryanodine binding to, isolated SR vesicles. These results may be important in describing inhibition of SR Ca2+ release and alterations in ECC in vivo that occur during intense bouts of muscle activity in which the lactate concentration is elevated.
Preparation of SR vesicles.
For all studies, SR vesicles were prepared from rabbit hindleg and back white skeletal muscle according to the method of MacLennan (16). The protein concentration was determined by absorption spectroscopy (13).
Measurement of Ca2+ efflux.
Ca2+ fluxes across actively loaded SR vesicles were monitored by using a dual-wavelength spectrophotometer and recording the differential absorption changes of antipyrylazo III at 720 and 790 nm (8). The standard procedure was as follows: Ca2+ uptake into SR vesicles (0.2 mg/ml) was carried out in a buffer containing 100 mM KCl, 20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 1 mM MgCl2, and 15 μM free Ca2+ and was initiated by the addition of 0.5 mM Mg2+-ATP. When steady-state Ca2+ uptake was achieved, release was induced by the addition of caffeine. After the addition of the effluxing agent, the free extravesicular Ca2+ concentration was recorded as a function of time. At the end of the recording, the Ca2+-antipyrylazo III signal was then calibrated with the addition of 4 μM Ca2+. Ca2+ efflux rates were determined from the maximal slope of the extravesicular Ca2+ concentration vs. time.
For CICR experiments, the procedure was modified. Pyrophosphate (7.5 mM) was included in the reaction medium, and the vesicles were actively loaded with four aliquots of 20 μM Ca2+. Once the vesicles accumulated the added Ca2+, release was induced by adding a single aliquot of 80 μM Ca2+. The free extravesicular Ca2+ concentration was recorded and the release rate calculated as described above.
The use of ryanodine, a plant alkaloid, has significantly enhanced our understanding of SR Ca2+-channel function. Ryanodine binds with nanomolar affinity to open SR Ca2+ channels (18). With few exceptions, compounds that stimulate Ca2+ release via opening of the Ca2+ channel [Ca2+ (μM), ATP, caffeine] stimulate the binding of ryanodine to its receptor, whereas compounds that inhibit Ca2+ release by closing the Ca2+ channel [Ca2+ (mM), Mg2+, ruthenium red] inhibit ryanodine binding. Detailed methods for measuring high-affinity [3H]ryanodine binding have been described previously (18). Briefly, SR membranes (500 μg/ml) were incubated at 37°C for 3 h in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [3H]ryanodine, and 20 mM HEPES, pH 7.1. The binding reaction was quenched by rapid filtration through a Whatman GF/B glass fiber filter, which was then rinsed with 5 ml of ice-cold buffer. The filters were placed in polytubes, filled with 2.5 ml of scintillation cocktail, shaken overnight, and counted the following day. The experiments were repeated at least seven times on two different SR preparations, with essentially identical results. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled ryanodine and subtracted before calculation. Details of individual experiments are described in figure captions.
Single Ca2+-channel analysis.
Single Ca2+-release channel activity was recorded and analyzed after reconstitution of an SR vesicle into a bilayer lipid membrane (BLM), as described previously (8). In a typical experiment, after channel reconstitution, 20 μM Ca2+ was added to thecis chamber, stirred, and the resultant channel activity was recorded for at least 1 min. Caffeine was then added to the cischamber, stirred, and channel activity was recorded. Subsequently, lactate was added to the cis chamber, and activity was recorded again. For analysis, the data were passed through a Krohn-Hite low-pass filter (model 3202) at 1.5 kHz, digitized with a Scientific Solutions analog-to-digital converter, and analyzed by using the pCLAMP software package (version 5.0, Axon Instruments, Burlingame, CA).
All reagents were analytical grade. HEPES was obtained from Research Organics (Cincinnati, OH). [3H]ryanodine was purchased from New England Nuclear. All other chemicals were obtained from Sigma Chemical (St. Louis, MO).
SR Ca2+ release.
Ca2+ release was evaluated after active loading of the SR vesicles. The addition of lactate before initiating release significantly inhibited both Ca2+- and caffeine-induced Ca2+ efflux. CICR was inhibited by >50%, as the rate of Ca2+ release diminished from 5.89 ± 0.60 to 2.58 ± 0.58 in the presence of 20 mM lactate (P < 0.05), whereas the maximal rate of caffeine-stimulated Ca2+ release was inhibited 27 and 37% by lactate at 10 and 20 mM, respectively (P < 0.05) (Fig. 1).
[3H]ryanodine binding was measured after incubation of SR vesicles with a range of Ca2+ (from 10 nM to 3 mM ± 20 mM lactate) concentrations, caffeine, and caffeine+lactate. In Fig.2, we demonstrate that lactate inhibits Ca2+-dependent binding of ryanodine to its receptor. Lactate causes the activation curve to be shifted to the right, reducing the bound [3H]ryanodine at all free Ca2+ concentrations. In additional experiments in which caffeine was used as a channel agonist, caffeine (1.5 mM) enhanced binding by 11% compared with the untreated SR (50 μM free Ca2+), whereas caffeine+lactate inhibited binding by 26 and 33%, when compared with untreated and caffeine-stimulated binding, respectively (Table 1). These results concur with the reduction in Ca2+-release rates shown in Fig. 1, suggesting that Ca2+ activation of the SR Ca2+ channel/ryanodine receptor was inhibited by lactate.
Single Ca2+-channel analysis.
In a previous paper (8), we demonstrated that lactate inhibits Ca2+-activated single-channel gating activity. In the present experiments, both Ca2+ and caffeine were used to activate single Ca2+ channels, and the effects of lactate were observed. In Fig. 3, the release channel was activated by adding Ca2+ (20 μM) (cis) and 1.5 mM caffeine (cis) and was subsequently inhibited by 30 mM lactate (cis). Trace A in Fig. 3demonstrates normal channel gating in the absence of added Ca2+ [open probability (P o) = 0.01]. In Fig. 3, trace B, 20 μM of added Ca2+ stimulated channel-gating activity, increasing the P o to 0.25. The addition of 1.5 mM caffeine further stimulated channel gating (P o = 0.45). Subsequently, 30 mM of lactate were added to the cis chamber, and channel gating was inhibited. Lactate inhibited the Ca2+-release channel by reducingP o to 0.10 without affecting the single-channel conductance. As viewed in Fig. 4, A andB, caffeine and lactate, respectively, open and close the channel in a concentration-dependent manner. An increase in caffeine concentrations from 0 to 1.5 mM increased the P o from 0.25 to 0.45 (Fig.4 A), whereas subsequent additions of lactate decreased theP o from 0.45 to 0.11 (Fig. 4 B).
The results of these experiments indicate that lactate inhibits the SR Ca2+-release channel when activated by Ca2+and/or caffeine. We show on both a macro- and microscopic scale (vesicle flux and single-channel experiments, respectively) that lactate, in the concentration range that would be operative during high-intensity exercise, significantly inhibits Ca2+-stimulated Ca2+ flux through the SR release channel.
The physiological trigger for SR Ca2+ release has yet to be completely defined; thus it becomes critical to evaluate the inhibitory effects of lactate by using different probes that represent competing hypotheses for activation of Ca2+ release. In our previous work, we documented similar inhibitory effects of lactate on the release channel using Ag+ and H2O2(8). Although neither of these pharmacological probes could be considered as physiologically relevant as is Ca2+, they have been used in describing and characterizing sulfhydryl oxidation-induced Ca2+ release. It has been proposed that oxidation of endogenous sulfhydryl groups, localized on the SR release protein, open the SR Ca2+ channel and induce Ca2+ release. Reduction of these thiol groups inhibits channel function and inhibits or reduces SR Ca2+ release (15, 24). Sulfhydryl oxidation-induced Ca2+ release is sensitive to all known in vivo modifiers of SR Ca2+release.
In contrast, although CICR is believed to be the likely release mechanism in cardiac muscle, it does not appear to be the relevant mechanism in skeletal muscle. Buffering of the extracellular Ca2+ concentrations to very low levels does not interfere with normal coupling, suggesting that Ca2+ is not required for signal transmission between the dihydropyridine receptor and the SR Ca2+-release channel (23). Release of Ca2+ from SR vesicles, high-affinity [3H]ryanodine binding, and single-channel activity are all Ca2+ dependent. Although it is unlikely that Ca2+ is the primary trigger responsible for opening the Ca2+-release channel, it is likely that the release process is Ca2+ dependent or is modulated by the cytosolic Ca2+ concentration.
Although caffeine is not a physiological stimulus for SR Ca2+ release, it has been used extensively in single-muscle fiber and SR Ca2+-release experiments (1, 14, 18, 22, 27). In single-fiber experiments, caffeine induces Ca2+-activated tension by stimulating Ca2+release from the SR (1, 14). The use of caffeine is important because it acts directly on the [3H]ryanodine receptor and bypasses many of the steps in ECC. This drug sensitizes the release channel to activation by Ca2+ and thus stimulates CICR (18). Inhibition of caffeine activation of the Ca2+ channel by lactate provides compelling evidence that the effect of lactate is localized to the SR release channel and may be relevant to the muscle fatigue process during conditions when high concentrations of lactate are present in active muscle.
We have demonstrated that lactate inhibits Ca2+ release activated by caffeine, Ca2+, H2O2, and Ag+ (8). The fact that these activators are chemically so different from one another strongly suggests that they interact with different sites on the SR Ca2+-release channel. Given that lactate inhibits Ca2+ release induced by such a diverse group of activators, it is likely that the site at which lactate binds is critical to normal function. It is, therefore, also likely that, independent of what the physiological trigger is, it is probably inhibited by lactate.
Although it appears that lactate may interfere with the ECC process and inhibit SR Ca2+ release, extrapolation of these findings to intact muscle is difficult. Experiments must be performed examining the interaction between lactate and SR Ca2+ channels that are coupled to intact voltage sensors. However, in a recent report (12), lactate was shown to reduce tension development in dog muscle. Lactate infused into arterial blood significantly increased muscle lactate concentrations without any decline in arterial or muscle pH. A corresponding decrease in tension development was observed after lactate infusion. Although no concrete mechanism was proposed for the reduction in tension, the authors did not rule out the SR as a site of interaction. Subsequently, after muscle perfusion in the absence of lactate, the inhibition of tension development was alleviated. This reversible effect is consistent with our observations. Pretreatment of SR vesicles with 30 mM lactate failed to inhibit Ca2+release when the pretreated SR was diluted into a lactate-free flux medium and release experiments were conducted (final lactate concentration <1 mM, unpublished observations). Alternatively, it was recently reported that lactate inhibits cross-bridge cycling and force production (2).
It is recognized that under certain muscle activation patterns tension development in muscle can recover in advance of lactate restoration (19) and pH (4). Thus no simplistic relationship exists between the individual ligands or metabolites that modify ECC or contractile activity and muscle fatigue. This underscores the importance of evaluating the effects of lactate by using a wide variety of reagents. The development of fatigue is most likely a complex process where any number of events keyed by the particular activation pattern and/or tension requirements leads to a decline in force production. Some, but not all, of the contractile scenarios that produce fatigue may be related to Ca2+ movements within the active muscle cell. However, the wisdom of focusing the dialogue concerning muscle fatigue on ECC and SR Ca2+ handling was recently outlined by Williams and Klug (26). In their Ca2+-exchange hypothesis, they suggest that control of intracellular Ca2+ concentration may be a critical link between muscle activity and fatigue (26). They argue that the majority of energy consumed during muscle contraction is directly related to Ca2+ ions [cross-bridge cycling myosin adenosinetriphosphatase (ATPase) activity] and SR Ca2+sequestration (SR Ca2+-ATPase activity). Inhibition of SR Ca2+ release and peak intracellular Ca2+concentration would significantly decrease both myosin and Ca2+-ATPase activities, downregulating the energy cost of contractility. Under these conditions, fatigue becomes a circuit breaker regulated by the SR. The Ca2+-mediated reduction in force production and energy usage may be a transient trade off to maintain the structural and functional integrity of the cell.
In summary, we show that lactate significantly inhibits SR Ca2+-channel activity, as indicated by SR Ca2+flux measurements, [3H]ryanodine-binding experiments, and single-channel analysis. Lactate inhibition of Ca2+-activated Ca2+-channel activity should reduce the amount of Ca2+ released after normal muscle activation. Although disruptions in the ECC process and inhibition of the SR Ca2+-release mechanism may not fully account for the tension decline observed during continuous muscle activation, disruptions in Ca2+ transients contribute to and promote muscle fatigue. Continued research detailing the steps of the ECC process and how they may be altered by increased muscle activity should provide a clearer description of the mechanisms of muscle fatigue.
This work was supported by a grant from the Murdock Foundation to T. G. Favero; by American Heart Association (AHA), Oregon Affiliate, grant-in-aid to J. J. Abramson; and by an AHA Summer Research Fellowship to D. Colter.
Address for reprint requests: T. G. Favero, Dept. of Biology, Univ. of Portland, 5000 N. Willamette Dr., Portland, OR 97203.
- Copyright © 1997 the American Physiological Society