oka and Stuart (lO)I]. Impairment in excitation- ...... Meissner,. G. Adenine nucleotide stimulation of Ca2'-induced. Ca2' release in sarcoplasmic reticulum. J. BioZ.
Metabolic end products inhibit Ca2’ release and [3H]ryanodine
sarcoplasmic binding
reticulum
TERENCE G. FAVERO, ANTHONY C. ZABLE, MARY BETH BOWMAN, ALISON THOMPSON, AND JONATHAN J. ABRAMSON Department of Biology, University of Portland, Portland 97203; and Department Portland State University, Portland, Oregon 97207 Favero, Terence G., Anthony C. Zable, Mary Beth Bowman, Alison Thompson, and Jonathan J. Abramson. Metabolic end products inhibit sarcoplasmic reticulum Ca2’ release and [3H]ryanodine binding. J. Appl. PhysioZ. 78(5): 16651672, 1995. - Sarcoplasmic reticulum (SR) Ca2’ release channel function is modified by ligands (Mg+, Ca2’, ATP, and H’) that are generated during a bout of exercise. We have examined the effects of changing intracellular metabolites on Ca2’ release, [3H]ryanodine binding, and singleCa2’ release channel activity of SR isolated from white rabbit skeletal muscle. Increasing Mg+ (from 0 to 4 mM) and decreasing pH (7.1-6.5) inhibited SR Ca2’ release and [3H]ryanodine binding. In addition, increasing lactate concentrations from 2 to 20 mM inhibited [3H]ryanodine binding to SR vesicles, inhibited SR Ca2’ release, and decreased the singlechannel open probability. These findings suggest that intracellular modifications that disrupt excitation-contraction coupling and decrease Ca2’ transients will promote a decline in tension development and contribute to muscle fatigue. In addition, we show that hydrogen peroxide induces Ca2’ release and increases [3H]ryanodine binding to its receptor, suggesting that reactive oxygen species produced during exercise may compromise muscle function by altering the normal gating of the SR Ca2’ release channel. muscle fatigue; reactive oxygen species; lactate; skeletal muscle
FATIGUE is a term used to describe a sequence of events in skeletal muscle characterized by a decline in force output during periods of repetitive contraction. This phenomenon has been subject to intensive experimentation to elucidate its cause(s). Although many studies have at tempted to ascribe the reduction in force to various single factors, such as substrate depletion, end produ .ct accumulation, ionic compositi on alterations, and excitation-contraction coupling, it is more likely that impaired muscle function stems from several interrelated elements determined specifically by the nature of the task (e.g., intensity and duration) used to induce fatigue. [For an in-depth and broad discussion of the causes of fatigue, see rev iews by Westerblad et al . (30) an.d Enoka and Stuart (lO)I] Impairment in excitation-contraction coupling has been described by several investigators under a wide variety of experimental conditions during muscle fatigue (2, 10, 12, 13, 15, 29, 30). Coupling of the action potential to cross-bridge cycling involves several steps, each a potential site for failure-and compromised force output (13). Possible alterations in coupling can occur at the levels of 1) sarcolemmal action potential, 2) ttubule (TT) charge movement, 3) coupling of TT charge movement to the sarcoplasmic reticulum (SR) Ca” release channel, 4) Ca2< release from the SR, 5) Ca2’
MUSCLE
0161-7567/95
$3.00
Copyright
of Physics,
binding to troponin, and 6) actomyosin-ATP hydrolysis and cross-bridge cycling. Inhibition of Ca2’ release from the SR has been identified as a likely and relevant factor in the fatiguing process under certain experimental conditions (10, 12, 13, 15, 26, 30). Inadequate or suboptimal delivery of Ca2’ to the myofilaments via SR Ca2’ release could occur under two scenarios: 1) impaired coupling between the voltage sensors in the TT membrane and the SR Ca2’ channel or 2) transient modification of the SR Ca2’ channel, reducing its open probability after activation (15, 30). The intracellular environment of active skeletal muscle exerts a locus of control over SR Ca2’ release, and modification of intracellular elements may result in a decreased Ca2’ transient. Lamb and Stephenson (19), using skinned fibers with intact TT, have shown that after a normal depolarization Ca2’ release and muscle contraction could be completely inhibited by high M$’ concentrations. They argued that the function of the SR Ca2+ channel is controlled by the TT under resting conditions but that it is also sensitive to the changing intracellular milieu. Thus, M$+ and other ligands that modify SR Ca2’ channel function can interfere with the ability of the voltage sensors to open the Ca2’ release channels (19, 22). SR Ca2’ channel function is modified by many ligands (M$‘, Ca2’, ATP, and H’) (22, 23). Many of them are altered during a bout of exercise similar to that observed in muscle fibers during high-frequency stimulation. In this paper, we examine the interactions of several intracellular metabolites and ions on Ca2’ release, [3H] ryanodine binding, and the single-channel characteristics of the SR Ca2’ release channel. We found that increased concentrations of lactate, M$‘, H+, and ATP hydrolysis products inhibit the Ca2’ release mechanism of skeletal muscle SR. RlETHODS
Preparation of SR vesicles. For all studies, SR vesicles were prepared from rabbit hindleg and back white skeletal muscles according to the method of MacLennan (21). The protein concentration was determined by absorption spectroscopy (18). Measurement of Ca2’ efflux. Ca2’ fluxes across SR vesicles were monitored using a Ca2+- selective electrode interfaced to an IBM XT computer (1). The system is calibrated by adding known amounts of Ca2’ to a buffer of 100 mM KCl, 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanestionic acid (HEPES), and 1 mM M$’ at pH 7.0. Millivolt readings from the Ca2’selective electrode, referenced to an Ag-AgCl electrode (Mere-l, World Precision Instruments), are read into the computer. The response of the electrode is sensitive to only Ca2’ and is not altered by changing conditions noted below. A computer program internally generates linear regression of the poten0 1995 the American Physiological Society 1665
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tial difference vs. the logarithm (base 10) of the Ca2’ concentration. The procedure corrects for Ca2’ present in the buffer (2-3 ,uM Ca2’) before the addition of known aliquots of Ca2’ and displays the slope of the plot (29 t 1 mV per lo-fold increase in Ca2+ concn). 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 mM HEPES, 1 mM MgC12, and 20 PM free Ca2’. Uptake was initiated by the addition of 0.5 mM Mg2’-ATP. On achievement of steadystate Ca2+ uptake, release was induced by the addition of the effluxing agent. Extravesicular Ca2+ concentration was recorded (10 Hz) as a function of time and was stored in the computer. An analysis program displays the data as a function of time and calculates the maximal Ca2+ efflux rate (in nmol mg-l s-l). Spectrophotomeric determination of Ca2+ efflux. Ca2’ fluxes across SR vesicles were also monitored using a dual-wavelength spectrophotometer by monitoring the differential absorption changes of antipyralazo III at 720-790 nm. Ca2+ uptake and efflux were carried out as described in the above section. After the addition of the effluxing agent, free extravesicular Ca2+ concentration was recorded as a function of time. The Ca2’-antipyralazo III signal was then calibrated with the addition of 4 PM Ca2’. Ca2’ efflux rates were determined from the maximal slope of the extravesicular Ca2’ concentration vs. time. (3H]ryanodine binding. Detailed methods for measuring high-affinity [3H]ryanodine binding have been described elsewhere (12,23). Briefly, SR membranes (100 pg/ml) were incubated at 37°C for 3 h in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [3H]ryanodine (sp act 4 @/mmol), and 20 mM HEPES at pH 7.1. Depending on the conditions of the assay, various channel modifiers [O-4 mM M$+, AMPphencyclidine (PCP), ADP, 1 mM AMP, 2-20 mM lactate, 20 PM doxorubicin, 1 mM hydrogen peroxide (H202), or H+ (pH 6.1-7.1)] were present during the incubation procedure. The binding reaction was quenched by rapid filtration through Whatman GF/B glass fiber filters, which were then rinsed with 5 ml of ice-cold buffer. The filters were placed in polytubes (Fischer), filled with 3 ml of scintillation cocktail (Beckman, ReadySafe), shaken overnight, and counted the next day. The experiments were repeated at least twice on two different SR preparations with essentially identical results. Nonspecific binding was measured in the presence of a lOOfold excess of unlabeled ryanodine and was subtracted before calculation. For details of individual experiments refer to the figure legends. Single-Ca2+ channeZ analysis. Ca2+ release channel reconstitution into a bilayer membrane was carried out by the addition of SR vesicles to the cis side of a planar bilayer lipid membrane. Bilayers, made with a 5:3 mixture of phosphatidylethanolamine-phosphatidylserine at 50 mg/ml in decane, were formed across a 150~pm hole drilled in a polystyrene cup separating two chambers of 0.7 ml each. The cis chamber contained 500 mM CsCl, 200 PM CaC12, and 10 mM HEPES at pH 7.0, and the trans side contained 100 mM CsCl and 10 mM HEPES at pH 7.0. SR vesicles, suspended in 0.3 M sucrose, were added to the cis side. After fusion of a single vesicle, 200 PM ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) at pH 7.0 was added to the cis chamber to stop further fusions. The cis chamber was then perfused with an identical buffer containing no added Ca2’ or EGTA. Channel activity was then measured at a holding potential of +25 mV with respect to the trans (ground) side. A bilayer clamp amplifier (model BC-525A, Warner Instruments) was used to amplify picoampere currents. The data were processed with a digital data recorder (model VR-10, Instratech), stored unfiltered on a videocasl
l
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sette recorder tape, and subsequently analyzed for channel activity. In a typical experiment, 50 PM Ca2’ was added to the cis chamber and stirred, and the resultant channel activity was recorded for at least 1 min. Lactate (5 mM) was then added to the cis chamber and stirred, and the channel activity was recorded. Additional aliquots of 5 mM lactate were added to the cis chamber, and, after each addition, the channel activity was again recorded. For analysis, the data were passed through a low-pass filter (model 3202, Krohn-Hite) at 1.5 kHz, digitized with an analog-to-digital converter (Scientific Solutions), and analyzed using the pCLAMP software package (version 5.0, Axon Instruments, Burlingame, CA). Materials. All reagents were analytic grade. HEPES was obtained from Research Organics (Cincinnati, OH). [3H] ryanodine was purchased from New England Nuclear, and ryanodine-dehydroryanodine was purchased from Agrisysterns International (Wind Gap, PA). All other chemicals were obtained from Sigma Chemical. RESULTS
During intense exercise, muscle cell metabolism is rapidly activated to produce the high-energy phosphates necessary to sustain maximal contractions. To maintain adequate ATP production, lactate ions and H+ are generated, reducing the pH within the active cell. The resulting increase in H+ can drop the pH of the muscle cell to a value ~6.5 (6). Ca2’ efflux experiments, using actively loaded SR vesicles, were carried out over the pH range of 7.1-6.5. The amount of active Ca2’ uptake mediated by the Ca2’ adenosinetriphosphatase (ATPase) has been shown to be pH dependent. However, if the vesicles are submaximally loaded at low Ca2’ (20 uM), then the amount of Ca2’ actively accumulated does not vary significantly with pH over the range of 7.1-6.5 (100 nmol/mg SR). The Ca2’ release rate is proportional to the Ca2’ gradient across the SR vesicles. Independent of the influence of pH on the Ca2’ uptake, if the SR is submaximally loaded to the same extent at different values of pH, then any change in Ca2’ release that is observed should reflect only modification to the Ca2’ release mechanism. After ATPstimulated Ca2’ uptake into SR vesicles, Ca2’ release was induced by the addition of the reactive oxygen species H202 (5 mM). The maximal rate of Ca2’ release was inhibited as the pH of the release medium decreased (Fig. 1). A similar pH-dependent inhibition of Ca2’ release was observed using the potent Ca2’ releasing agent doxorubicin (data not shown). A more direct assay for monitoring the interaction between the SR Ca2’ channel and various metabolites involves the highly specific Ca2’ channel probe [3H]ryanodine. Ryanodine binds with nanomolar affinity to open SR Ca2’ channels (23). With few exceptions, compounds that stimulate Ca2’ release via opening of the Ca2’ channel (e.g., Ca2’, ATP, and doxorubicin) stimulate the binding of ryanodine to its receptor, whereas compounds that inhibit Ca2’ release by closing the Ca2’ channel (e.g., M$’ and ruthenium red) inhibit ryanodine binding. [3H] ryanodine binding was measured as a function of pH in the presence of the Ca2+ channel activators H202 and doxorubicin. Decreasing the pH of the incubation medium reduced the amount
SR cA2+ RELEASE
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T
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0 Control l
Doxorubicin
* 2.00 it # 1.75 0 1.50 % - 1.25 s d
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3II 0.75 p1 ;sp 0.50 u 0.25 0.00 0.0
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1.0
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FIG. 1. Hydrogen peroxide (H202)-induced Ca2’ release is inhibited by decreasing pH. Sarcoplasmic reticulum (SR) vesicles at 0.2 mg/ml were actively loaded in a buffer containing 100 mM KCl, 20
FIG. 3. Effect of Mg’ concn on [3H]ryanodine binding stimulated by 50 PM Ca2’ (control) and 20 ,uM doxorubicin. Binding assays were conducted as detailed in Fig. 2 with increasing Mg2f concn (from
mM
0 to 4 mM)
HEPES,
20 PM
CaC12,
and
1 mM
M$’
by the addition
of 0.5
mM Mg-ATP. On completion of Ca2’ uptake (free Ca2+ concn = l2 PM), 5 mM H202 was added. Ca2’ concn was continuously monitored
with
a Ca2+-selective
electrode
as a function
of time
and was
recorded. Ca2’ release rate was calculated from the initial slope of free Ca2’ concn vs. time after addition of H202. Data are means + SD of 4 independent expts. *P < 0.05.
of E3H]ryanodine bound regardless of the modulating conditions (Fig. 2). In control conditions (50 PM free Ca2’), the amount of ryanodine bound was reduced from a value of 3.41 pmol/mg at pH 7.1 to 1.12 pmoI/
at pH
7.1. Data
are averages
of representative
expts
performed in duplicate. For each condition, error is typically
