Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
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ABSTRACT |
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Prolonged exercise followed by a
brief period of reduced activity has been shown to result in an
overshoot in maximal sarcoplasmic reticulum (SR)
Ca2+-ATPase activity [maximal velocity
(Vmax)] in rat locomoter muscles (Ferrington
DA, Reijneveld JC, Bär PR, and Bigelow DJ. Biochim Biophys
Acta 1279: 203-213, 1996). To investigate the functional significance and underlying mechanisms for the increase in
Vmax, we analyzed Ca2+-ATPase
activity and Ca2+ uptake in SR vesicles from the fast rat
gastrocnemius muscles after prolonged running (RUN) and after prolonged
running plus 45 min of low-intensity activity (RUN+) or no activity
(REC45) and compared them with controls (Con). Although no differences were observed between RUN and Con, both Vmax and
Ca2+ uptake were higher (P < 0.05) by 43 and 63%, respectively, in RUN+ and by 35 and 34%, respectively, in
REC45. The increase in Vmax was accompanied by
increases (P < 0.05) in the phosphorylated enzyme
intermediate measured by [-32P]ATP. No differences
between groups for each condition were found for the fluorescent
probes FITC and
(N-cyclohexyl-N1-dimethylamino-
-naphthyl)carbodiimide,
competitive inhibitors of the nucleotide-binding and
Ca2+-binding sites on the enzyme, respectively. Similarly,
no differences for the Ca2+-ATPase were observed between
groups in nitrotyrosine and phosphoserine residues, a measure of
nitrosylation and phosphorylation states, respectively. Western blots
indicated no changes in relative isoform content of sarcoendoplasmic
reticulum (SERCA)1 and SERCA2a. It is concluded that the increase in
Vmax of the Ca2+-ATPase observed in
recovery is not the result of changes in enzyme nitroslyation or
phosphorylation, changes in ATP and Ca2+-binding affinity,
or changes in protein content of the Ca2+-ATPase.
muscle; calcium-adenosinetriphosphatase; calcium uptake; calcium release; vesicles; sarcoplasmic reticulum
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INTRODUCTION |
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IT HAS BEEN REPORTED
THAT, during recovery after dynamic exercise, an overshoot in
maximal Ca2+-ATPase activity [maximal velocity
(Vmax)] of the sarcoplasmic reticulum (SR) in
skeletal muscle occurs (16). The overshoot in
Vmax has not been observed during inactive
recovery (passive recovery) after a brief period of heavy treadmill
exercise in horses (6) but only during reduced intensity
exercise (active recovery) after prolonged moderate-intensity treadmill
exercise in rats (16). The increase in
Vmax was also accompanied by increases in the
steady-state level of the phosphorylated enzyme intermediate formed
from [-32P]ATP during active recovery. On the basis of
these findings, the authors concluded that the increase in
Vmax was the result of an increase in the number
of active Ca2+-ATPase pump units and not the result of
changes in catalytic turnover rates (16). In addition,
kinetic properties of the enzyme, determined from plots of
Ca2+-ATPase activity at varying free Ca2+
concentrations ([Ca2+]f), revealed no changes
in the Hill coefficient (nH) at any time point
during recovery and a small but significant decrease in [Ca2+]f needed to induce half-maximal
activity (pCa50) late in recovery. Both the physiological
significance and the mechanisms underlying the changes in
Ca2+-ATPase kinetics during recovery after exercise remain unclear.
Because no changes were observed in the physical structure of the SR membrane, as assessed by measures of membrane fluidity (16), it might be expected that the increase in Ca2+-ATPase activity observed during recovery would be accompanied by increases in Ca2+ uptake. However, at least after heavy treadmill exercise and passive recovery, this does not appear to be the case, since Ca2+ uptake was elevated and Ca2+-ATPase activity was altered minimally (6). The dissociation between the changes in Ca2+ uptake and Ca2+-ATPase activity would be expected to result in pronounced increases in the coupling ratio and consequently a reduction in the expected energy needed to translocate Ca2+ into the lumen of the SR (27). It is not clear whether a similar alteration in coupling effect occurs after prolonged exercise in active recovery in rats. Conceptually, differences could occur because of differences in species, muscle fiber composition of the muscles examined, the exercise protocol employed, and the form of recovery (active vs. passive).
Although increases in the recruitment of active enzyme may explain the increase in Vmax during active recovery after prolonged exercise, the underlying mechanism remains unclear. A number of possibilities exist. Increases in synthesis of the Ca2+-ATPase enzyme represent the most obvious mechanism; however, given the relatively short time frame for the overshoot in Vmax to occur, this possibility remains remote. Indeed, Ferrington et al. (16) have observed that the increase in Vmax observed during active recovery cannot be explained by alterations in sarcoendoplasmic reticulum (SERCA) levels, either SERCA1 or SERCA2a. Moreover, chronic contractile activity results in a downregulation in Ca2+-ATPase content and not an increase (35). This observation suggests that acute factors are involved in the regulation of the steady-state levels of the active enzyme.
Acute regulatory factors could involve changes in the oligomeric status of the Ca2+-ATPase protein or a phosphorylation-mediated process. Phosphorylation-mediated effects, which increase Vmax, can be induced by direct phosphorylation of the enzyme by a Ca2+/calmodulin-dependent protein kinase (CaM kinase; see Refs. 44 and 45). Phosphorylation can also occur indirectly via phosphorylation of phospholamban (PLB) via either CaM kinase or cAMP-dependent protein kinase A. The effect of PLB phosphorylation is to increase the affinity of the enzyme for Ca2+ (39). However, the effects are most pronounced in the heart and to a smaller degree in slow-twitch-based muscles, both of which contain primarily the SERCA2a isoform (39).
It has been proposed that the effects of exercise and recovery may be
to relieve a preexisting inhibition, allowing the recruitment of
enzymes from the inactive to the active state (16). It is possible that, under such circumstances, alterations in the folding state may occur, allowing greater access of substrates such as ATP
and/or Ca2+ to the nucleotide-binding site and/or the
Ca2+-binding site in the enzyme. Under such circumstances,
increases in FITC and/or
N-cyclohexyl-N1-(dimethylamino--naphthyl)carbodiimide
(NCD-4), competitive inhibitors of the nucleotide-binding site and the
Ca2+-binding site, respectively, might be expected. It is
of interest that some studies report reductions in FITC binding after
exercise (14), a finding that would suggest that
alterations in the nucleotide-binding site may account for the
reduction in Vmax that accompanies the reductions in FITC binding.
Alterations in the nucleotide-binding site observed with chronic contractile activity have been attributed to oxidation and tyrosine nitration via a direct attack on selected amino acids by reactive oxygen species (ROS) and/or peroxynitrate generated from nitric oxide (NO) and superoxide (21). Accordingly, relief of a preexisting inhibition as observed during recovery could be mediated at the nucleotide-binding site and could be accompanied by decreases in nitrosylation. Accordingly, it could be argued that passive recovery compared with active recovery would allow for an even greater increase in Vmax activity of the Ca2+-ATPase.
If increases in Ca2+ uptake accompany the increase in Ca2+-ATPase activity, increased rates of muscle relaxation would be expected to occur during recovery (11). The increased rate of relaxation could have several physiological consequences. In the absence of changes in SR Ca2+ release kinetics, force may be compromised and/or a greater oscillation in force may result at a given activation frequency. If, on the other hand, Ca2+ release is altered in conjunction with Ca2+ uptake, both the magnitude and kinetics of [Ca2+]f could be affected, leading to changes in myofibrillar activation and force transients. For these reasons, measurements of the functional behavior of the SR both during exercise and recovery are important in providing insight into the mechanical alterations that occur.
In this study, we have investigated the effects of both passive and active recovery after prolonged exercise on the alterations in Ca2+-ATPase and Ca2+ uptake in SR vesicles prepared from muscle of predominantly fast-fiber composition. We have hypothesized that increases in both Ca2+-ATPase activity and Ca2+ uptake would occur and that the increases would be more pronounced with passive recovery. Moreover, we have postulated that the increase in Vmax is the result of increases in the steady-state level of the active enzyme, which is mediated by increased accessibility of the nucleotide-binding site for the substrate, ATP. The increase in substrate binding allows greater activation of the nucleotide-binding site, which is mechanistically linked to reduced nitrosylation.
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METHODS |
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Animal description and exercise protocol. Two experiments consisting of different exercise and recovery conditions were employed in this study. In both experiments, mature untrained female Sprague-Dawley rats were housed in a climate-controlled animal care facility on alternating 12:12-h light-dark cycles and were fed laboratory chow and water ad libitum.
In experiment 1, rats weighing 266 ± 16 g were randomly assigned to sedentary control (Con) or one of two exercise groups (n = 11 for each group). For experiment 1, the exercise protocol was very similar to the protocol used by Ferrington et al. (16). It consisted of continuous treadmill running at 21 m/min and 8% grade until fatigue or for a maximum of 2 h (RUN). The second exercise group (RUN+) performed the same exercise as RUN but then exercised at a lower intensity (10 m/min and 8% grade) for 45 min after RUN. This was done to assess the effects of active recovery after exhaustive exercise on SR function, as previously reported (16). To further probe the effects of exercise and recovery on SR structure and function, an additional experiment (experiment 2) was conducted. In experiment 2, rats weighing 258 ± 18 g ran on a treadmill at 21 m/min and 8% grade until fatigue or for a maximum of 2 h, which was followed by 45 min (REC45) of sedentary activity. The exercise was identical to that of the RUN group in experiment 1. Sedentary activity was accomplished by removing the rats from the treadmill and placing them in cages designed to hold a single rat. The REC45 group was compared with a Con group that was separate from the Con in experiment 1. The passive recovery conditions were done to limit activity after the exercise and to assess whether the effects differ from those of active recovery. Animals (n = 8 for each group) were randomly assigned to each group. Enriched SR membrane fractions (SR vesicles) were prepared from both gastrocnemius muscles of each animal for each experiment. The entire gastrocnemius muscle including both the red and white portions was used to give an adequate amount of tissue for analysis. Each group of rats was anesthetized with pentobarbital sodium (6 mg/100 g body wt) and prepared for muscle sampling. After sampling, muscle preparations were kept on ice or refrigerated (0-4°C) at all times. Experimental protocols were initiated at approximately the same time each day to prevent diurnal variations in physiological parameters, including muscle glycogen (8). The Animal Care Committee of the University of Waterloo approved all experimental protocols.Sample preparation.
While the animals were anesthetized, both gastrocnemius muscles were
excised from each hindlimb and placed in ice-cold homogenization buffer. A small piece of white gastrocnemius was rapidly sampled from
each muscle from each animal and frozen in liquid nitrogen for later
analysis of muscle metabolites. The remainder of the gastrocnemius
muscles were used to prepare SR vesicles utilizing a combination of two
SR isolation protocols (13, 19), as previously used in our
laboratory (41). The two muscles from each animal were
combined and diluted 1:5 (wt/vol) in ice-cold homogenizing buffer (pH
7.5) containing (in mM) 250 sucrose, 5 HEPES, 0.2 phenylmethylsulfonyl fluoride (PMSF), and 0.2% sodium azide (NaN3). No
dithiothreitol was used in the preparation of SR vesicles. The muscles
were homogenized mechanically with a Polytron homogenizer (PT 3100) at
16,500 rpm for 2 × 30 s bursts separated by a 30-s break. SR
isolation was accomplished by sucrose gradient and differential
centrifugation to remove unwanted cellular organelles, fat, and debris.
The final pellet, an enriched SR membrane fraction, was stored at
70°C for future analysis of SR function. Isolation of SR vesicles
was done on the same day as muscle extraction. Protein determination of
homogenates and SR vesicles was made by the method of Lowry as modified
by Schacterle and Pollock (37). For protein
determinations, all samples from both experiments were analyzed in triplicate.
SR Ca2+-ATPase activity. Measurements of Ca2+-induced SR Ca2+-ATPase activity were made on enriched SR membrane fractions. Ca2+-ATPase enzyme kinetics were assessed by progressively increasing the [Ca2+]f and measuring the Ca2+-ATPase activity at numerous [Ca2+]f. The markers of Ca2+-ATPase enzyme kinetics included the Vmax, [Ca2+]f required for half-maximal activity (pCa50), and the nH, which is a measure of the cooperative Ca2+-binding affinity of the Ca2+-ATPase enzyme. Ca2+-induced Ca2+-ATPase activity was measured according to the methods of Leberer et al. (24). The reaction buffer contained (in mM) 100 KCl, 20 HEPES, 10 MgCl2, 10 NaN3, 10 phosphoenolpyruvate (PEP), 5 ATP, and 1 EGTA. The pH of the reaction buffer was adjusted to 7.0 at 37°C. Immediately before the reaction was started, 18 U/ml lactate dehydrogenase (LDH), 18 U/ml pyruvate kinase (PK), 0.3 mM NADH, 1 µM Ca2+ ionophore A-23187 (C-7522; Sigma), and 5 µl of SR were added to 1 ml of reaction buffer. Assays were performed at 37°C and 340 nm (UV 160; Shimadzu). After the recording of baseline absorbance and fluorescence of NADH, the reaction was initiated by adding 2 µl of 100 mM CaCl2 and monitored for ~2 min. At the end of this period, 15 additional 0.5-µl additions of 100 mM CaCl2 were made to allow assessment of Ca2+-dependent Ca2+-ATPase activity kinetics and to ensure the activity was maximal. Maximal Ca2+-ATPase activity was established by progressively raising the [Ca2+]f until a plateau and subsequent decline in the Ca2+-ATPase activity occurred. Basal or Mg2+-ATPase activity was determined by adding 1 µl of the specific inhibitor of the Ca2+-ATPase, cyclopiazonic acid. The [Ca2+]f corresponding to the additions of 100 mM Ca2+ were measured on a separate tissue sample using the fluorescent dye indo 1 (33).
Ca2+-ATPase activity was then plotted against the negative logarithm of [Ca2+]f (pCa). Nonlinear regression with computer software (GraphPad Prism) using the following sigmoidal dose-response relationship allowed assessment of nH and pCa50
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(1) |
SR Ca2+ uptake and release.
Oxalate-supported Ca2+ uptake was measured using the
Ca2+ fluorescent dye indo 1 according to the methods of
O'Brien et al. (32, 33), as modified by Ruell et al.
(36). Fluorescence measurements were made on a
spectrofluorometer (Ratiomaster system; Photon Technology
International) equipped with dual-emission monochromators. The
measurement of [Ca2+]f using this procedure
is based on the difference in maximal emission wavelengths between the
Ca2+ bound to indo 1 and the free form. The excitation
wavelength was 355 nm and the emission maxima were 485 and 405 nm for
Ca2+ free (G) and Ca2+ bound (F) to indo 1, respectively. The ratio (R) of F to G decreases during SR
Ca2+ uptake and was used to calculate
[Ca2+]f. Felix software (Photon Technology
International) was used to calculate Ca2+ concentration by
the following equation (18)
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(2) |
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Fluorescence measurements.
The fluorescent probes, FITC (Sigma) and NCD-4 (Molecular Probe), were
used to assess structural alterations in the nucleotide and
Ca2+-binding domains of the Ca2+-ATPase enzyme,
respectively. FITC and NCD-4 were stored at a concentration of 5 mM in
ethanol at 20°C. SR vesicles were prepared for FITC labeling after
each sample was washed two times in wash buffer. After the same amount
of protein was added to centrifuge tubes for each sample, 7.5 ml of
wash buffer were added to each tube. Wash buffer contained (pH 7.5) 5 mM HEPES, 0.2 mM PMSF, and 0.2% NaN3. Samples were then
centrifuged at 23,400 rpm for 15 min. After two washes, the pellets for
each sample were resuspended in FITC-labeling buffer and vortexed
gently in darkness for 20 min at 25°C. The FITC-labeling buffer was
wash buffer plus 2.5 µM FITC (pH 8.8). Fluorescence measurements
using the FITC probe were made according to Lalonde et al.
(23) on a spectrofluorometer (Molecular Devices) in
triplicate, and relative fluorescence units (RFUs) were recorded.
Excitation of samples at 490 nm allowed FITC emission spectra ranging
from 500 to 550 nm to be collected in 1-nm increments. Only the maximum
RFU value is reported. NCD-4 labeling was done by washing the SR
samples two times in wash buffer and then resuspending the samples in
NCD-4-labeling buffer. NCD-4-labeling buffer was wash buffer plus 150 µM NCD-4 (pH 6.2). NCD-4 fluorescence measures were made by exciting
samples at 340 nm and scanning the emission intensity from 400 to 430 nm in 1-nm increments on a spectrofluorometer (Shimadzu). The maximum
fluorescence intensity is reported.
SDS-PAGE and Western blotting. SDS-PAGE was performed to separate and isolate the SR Ca2+-ATPase (110 kDa) by size. Immunoblotting was performed using the primary monoclonal antibodies specific for rat SERCA1 (IIH11; Affinity Bioreagents) and SERCA2a (7E6; Affinity Bioreagents), which were diluted 1:6,000 and 1:4,000 in 10% nonfat milk, respectively. In addition, immunoblotting was performed using polyclonal antibodies specific for nitrotyrosine (Molecular Probes) and phosphoserine (Chemicon), which were diluted 1:1,000 in 10% nonfat milk and 1:250 in 5% BSA, respectively. A suspension of 0.5 mg/ml protein from SR vesicles in 40 µl of buffer (1.25 M sucrose, 0.25 M Tris · HCl, pH 6.8, 5% SDS, and 0.01% bromphenol) was brought to 200 µl with distilled water. Five micrograms of each sample were loaded for SDS-PAGE. All samples were analyzed in duplicate on separate 7% polyacrylamide SDS gels (Mini-PROTEAN II; Bio-Rad), with a 3.75% stacking gel. Every SDS gel contained an equal-number sample for each condition from both experiments 1 and 2 to reduce bias between gels. SERCA1 and SERCA2a immunoblotting and anti-nitrotyrosine and anti-phosphoserine immunoblotting were all measured on SR vesicles.
After SDS-PAGE and a 5-min equilibration on cold transfer buffer [25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol], the proteins were transferred to a polyvinylidene difluoride membrane (PVDF membrane; Bio-Rad) by placing the gel in transfer buffer and applying a low voltage (23 V) for 35 min (Trans-Blot Cell; Bio-Rad). With the exception of immunoblotting for anti-phosphoserine, nonspecific binding sites were blocked with 10% nonfat skim milk powder in Tris-buffered saline (pH 7.5), applied for 1 h at room temperature. Before anti-phosphoserine immunoblotting, the nonspecific binding sites were blocked with 5% BSA and Tris-buffered saline (pH 7.5) applied for 1 h. With the exception of immunoblotting for anti-phosphoserine, incubation of the PVDF membrane with the primary antibodies was performed for 1 h at 25°C. Anti-phosphoserine immunoblotting required the PVDF membrane to be incubated with the primary antibody for 4 h at 25°C. After the wash, a secondary antibody was applied for 1 h at room temperature. The secondary antibody used for SERCA1 and SERCA2a was anti-mouse IgG1 conjugated to horseradish peroxidase. The secondary antibody used for nitrotyrosine and phosphoserine was anti-rabbit IgG1 conjugated to horseradish peroxidase. After application of the appropriate secondary antibody, protein quantification was performed using an enhanced chemiluminescence immunodetection procedure (ECL-RPN2106P1; Amersham). After exposure to photographic film (Hyperfilm-ECL; Kodak), the blot was developed for 90 s in Kodak GBX developing solution, washed in distilled water, and fixed in Kodak GBX fixer. Relative protein levels were determined by scanning densitometry, and values were expressed as a percentage of Con. Western immunoblotting allowed determination of the relative concentrations of the SERCA1 isoform, SERCA2a isoform, and level of nitrotyrosine or phosphoserine in SERCA. For all Western immunoblots, each sample was compared with the same standard, which comprised SR vesicles from a subsample of Con rats. For each antibody, the linearity of the signal with progressive increases in protein content covering the range used in the experiment was established before any experiments were conducted (data not shown). In our SR vesicle preparation, we used only the proteolytic inhibitor PMSF. In our hands, we found only a single band for the Ca2+-ATPase corresponding to the 110-kDa standard. On a given analytical day, four samples from both experiments 1 and 2 were analyzed. Analyses for all animals were done on consecutive days to minimize variability.Enzyme phosphate formation.
Stable levels of phosphorylated enzyme intermediate formed in SR
vesicles from [-32P]ATP were determined in the absence
and presence of 2.25 mM CaCl2. Scintillation counting of
[
-32P]ATP was performed to calculate the concentration
of the phosphoenzyme, where the Ca2+-dependent
phosphoenzyme intermediate was obtained from the difference between
trials in the presence and absence of 2.25 mM CaCl2.
Similar to Ferrington et al. (16), we have used this
technique to determine if the exercise-induced activation of the SR
Ca2+-ATPase was the result of an increase in the number of
active pumps or an increase in the catalytic rate of each pump.
[3H]ryanodine binding. Measurement of the concentration of [3H]ryanodine-binding sites was made on SR vesicles according to the methods of Damiani et al. (9). Maximal ryanodine binding in response to Ca2+ was used as a measure of functional Ca2+ release channels (CRCs). This technique has been used as a measure of the number of functional channels that are responsive to Ca2+ (15). Assessment of the concentration of [3H]ryanodine binding was used to complement measures of Ca2+ release.
In parallel assays, 50 µg (~25 µl) of sample were reacted with 500 µl of buffer 1 in the presence and absence of a high concentration (20 µM) of unlabeled ryanodine for 3 h at 37°C. Buffer 1 (pH 8.0) contained 150 mM KCl, 10 mM Tris, 3 mM ATP, 31.6 µM CaCl2, and 20 nM [3H]ryanodine. After the incubation, all samples were diluted with 5 ml of ice-cold ethanol. The samples were then filtered through both a glass microfiber filter (1.2 µm pore size; Whatman GF/C) and a cellulose acetate-cellulose nitrate filter (GSWP 0.22 µm pore size; Millipore) using a sampling manifold (Millipore). The filters were washed three times with ice-cold ethanol. The acetate-cellulose nitrate filters were placed in scintillation vials, and 10 ml of Triton-based toluene scintillation fluid were added before they were read on a scintillation counter. The difference between trials in the presence and absence of a high concentration (20 µM) of unlabeled ryanodine was used as the concentration of [3H]ryanodine binding. The concentration of [3H]ryanodine-binding sites was assessed in duplicate for all samples. On a given analytical day, two complete sets of samples from either experiment 1 or 2 were analyzed. Analyses for all animals were done on consecutive days to minimize variability.Data analyses. Experiments 1 and 2 each had their own Con group and were analyzed separately. For Ca2+ uptake measures in experiments 1 and 2, a two-way ANOVA was used to discriminate between differences resulting from the exercise condition and the [Ca2+]f used for analysis in SR vesicles. For Ca2+ release measures in both experiments, a two-way ANOVA was used to discriminate between differences resulting from the exercise condition and the phase of Ca2+ release. For all other measures, a one-way ANOVA or an unpaired t-test (for SR vesicles in experiment 2 only) was used to test for differences between means. Where significant differences are found, Bonferroni's post hoc tests were used to compare specific means. Statistical significance was accepted at P < 0.05.
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RESULTS |
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Ca2+ uptake.
The rate of Ca2+ uptake (nmol · mg
protein1 · min
1) was
assessed at five different [Ca2+]f levels in
purified SR vesicles. In experiment 1, the rate of Ca2+ uptake, analyzed at [Ca2+]f
of 2,000 nM, was 63% higher (P < 0.05) in RUN+
compared with Con (Fig. 2A).
Increases (P < 0.05) in Ca2+ uptake for
RUN+ ranged from 32 to 62% compared with Con for the lower
[Ca2+]f values used for analysis. For
experiment 2, 34, 52, and 47% higher (P < 0.05) rates of Ca2+ uptake occurred for REC45 compared with
Con when analyzed at [Ca2+]f of 2,000, 1,500, and 1,000 nM, respectively (Fig. 2B). No differences were
observed between Con and REC45 at [Ca2+]f of
500 and 250 nM.
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Ca2+ release.
Both AgNO3 and 4-CMC were used as releasing agents in this
study. Because both releasing agents produced the same results, only
the values for 4-CMC are provided. Two distinct phases of the
Ca2+ release rate (nmol · mg
protein1 · min
1) were
assessed based on the biphasic response to either releasing agent after
active Ca2+ loading (Fig. 1). In both experiments,
phase 1 of Ca2+ release was higher (main effect,
P < 0.05) compared with phase 2 for both
releasing agents. Exercise or exercise plus recovery (active or
inactive) did not affect the rate of SR Ca2+ release for
either phase 1 or phase 2 (Fig.
3). In addition, the type of releasing
agent employed did not affect these findings.
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Ca2+-ATPase activity.
For experiment 1, the Vmax of
Ca2+-ATPase activity in SR vesicles was 43% higher
(P < 0.05) in RUN+ compared with Con (Table 1). No difference in
Vmax was found between Con and RUN. For experiment 2, the Vmax of
Ca2+-ATPase activity in SR vesicles was 36% higher
(P < 0.05) in REC45 compared with Con. Basal or
Mg2+-ATPase was not different between conditions in either
experimental group. The Vmax of
Ca2+-ATPase activity occurred at a
[Ca2+]f of ~6-10 µM in all groups
(data not shown). Neither running nor recovery (active vs. passive)
resulted in changes in nH or pCa50
(Table 1).
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Enzyme phosphate formation.
Stable levels of phosphorylated enzyme intermediate formed from
[-32P]ATP were determined in the absence and
presence of 2.0 mM CaCl2 in SR vesicles (Fig
4). In Experiment 1,
scintillation counting of [
-32P]ATP showed
that RUN+ had 52% higher (P < 0.05) values compared with Con and RUN. For experiment 2, REC45 had 51% higher
(P < 0.05) values of phosphorylated enzyme
intermediate compared with Con.
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Fluorescence measurements.
In SR vesicles, the fluorescent probes FITC and NCD-4 were used to
assess structural alterations of the nucleotide and
Ca2+-binding sites of the Ca2+-ATPase enzyme,
respectively. The maximum fluorescence measured in RFUs was not
different between any of the groups for FITC trials (Fig.
5). Similarly, the maximum emission
intensity was unchanged in all conditions using NCD-4 (Fig.
6).
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Western blotting: SERCA isoforms.
The relative content of SR Ca2+-ATPase protein was measured
using Western blotting with monoclonal antibodies specific for the SERCA1 and SERCA2a isoforms (Figs. 7 and
8). Exercise or exercise plus
recovery (active or inactive) did not change the relative content of
SERCA1 or SERCA2a protein, since no significant differences were found
for Western immunoblots in SR vesicles when all conditions were
compared within experiment 1 or 2.
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Western blotting: Anti-nitrotyrosine and anti-phosphoserine.
The relative contents of nitrotyrosine (Figs.
9 and 10)
and phosphoserine (Figs. 11 and
12) were measured in SR
vesicles by Western immunoblotting using polyclonal antibodies for
nitrotyrosine and phosphoserine residues. Exercise or exercise plus
recovery (active or inactive) did not change the relative content of
nitrosylated or phosphorylated SERCA protein, since no significant
differences were found for Western immunoblots at the molecular mass of
SERCA (110 kDa) in SR vesicles when all experimental conditions were compared within experiment 1 or 2.
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[3H]ryanodine binding.
The dependence of [3H]ryanodine incorporation on
[Ca2+]f was assessed in a subsample of Con SR
vesicles to ensure that the optimal [Ca2+]f
value was used for maximal [3H]ryanodine binding (Fig.
13). Subsequently, all samples were
assayed at a pCa of 4.5 to obtain maximal [3H]ryanodine
binding. A full dependency curve of [3H]ryanodine binding
on pCa, which would allow calculations of the nH
and pCa50, was not done on every sample because of
restrictions on the amount of tissue available. In experiment
1, the number of functional CRCs in SR vesicles, as measured by
[3H]ryanodine binding, was 40% lower (P < 0.05) in RUN+ compared with Con (Fig.
14A). For experiment
2, no differences in [3H]ryanodine binding were
shown between Con and REC45 (Fig. 14B).
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DISCUSSION |
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In this study, we have confirmed the findings of others (16), namely that prolonged exercise of moderate intensity was without effect in altering the kinetic properties of Ca2+-ATPase, namely Vmax, nH, and pCa50 when measured in SR vesicles. We have also been able to confirm that increases in Vmax occur during active recovery and are accompanied by an increase in the phosphorylated enzyme intermediate. As was suggested previously (16), we have indications that the catalytic turnover rate was unchanged, as assessed from the ratio of Ca2+-ATPase activity to phosphorylated enzyme concentration.
In addition, our study also contains several new findings. We have demonstrated that, similar to Ca2+-ATPase, exercise also failed to alter Ca2+ uptake and that the overshoot in Ca2+-ATPase activity observed during active recovery is also accompanied by an overshoot in Ca2+ uptake. Moreover, the changes in Ca2+ uptake occur in isolation, since changes in Ca2+ release were not observed. In this study, we have also shown that the increase in Vmax occurs in the absence of alterations in the region of the nucleotide-binding site on the enzyme or to changes in the nitrosylated or phosphorylated state of the enzyme. Evidence is presented, however, to indicate that changes may occur to the Ca2+-binding region of the enzyme, which might be mechanistically linked to the increased enzyme recruitment underlying the increase in Vmax. Finally, we demonstrate that similar results occur during passive and active recovery, indicating that the overshoot observed in Vmax is not affected by continued low-intensity activity.
Based on the fact that the -32P-labeled phosphorylated
enzyme intermediate showed a similar increase in
Vmax of the Ca2+ pump, it has been
suggested that increased recruitment of inactive pumps and not
increases in the catalytic turnover rates of existing pumps occurred.
Because no changes were observed in the relative contents of either the
SERCA1 or SERCA2a in gastrocnemius, as assessed by Western immunoblot
techniques, the increase in Vmax that we have
observed in recovery appears not to be the result of increased protein
content but rather because of posttranslational modifications to the
enzyme itself or to related proteins. Because increased enzyme
recruitment could be associated with increased availability of
nucleotide-binding sites or Ca2+-binding sites on the
enzyme, we have used the fluorescent probes FITC and NCD-4 to examine
whether changes occurred. Our results indicate that structural changes,
at least in the region of the nucleotide-binding site of the enzyme,
could not be implicated in the overshoot in Ca2+- ATPase
activity that was observed in recovery. However, there is evidence that
changes occurred in the region of the Ca2+-binding site,
since, when both active and passive recovery conditions were combined,
a significant increase over preexercise and exercise was observed.
Although increased enzyme recruitment has been suggested as the mechanism involved (16) to explain the increase in Vmax, changes in catalytic turnover rate, defined as the maximum number of moles of substrate converted to a product per second per mole of enzyme active site, could conceivably be involved. Conceivably, a downregulation in phosphatase activity could occur, resulting in a decreased rate of dephosphorylation and an increased phosphorylated enzyme formation (31). Alternatively, any mechanism that increases the rate of phosphorylated enzyme formation could increase the catalytic turnover rate. Changes in the regulatory phosphorylation state of the enzyme or in the accumulation of nitrotyrosine residues also appear to be insignificant in this regard, since we could find no changes in these properties, as assessed by Western blotting using polyclonal antibodies specific for phosphoserine and nitrotyrosine. Because none of our probes was able to identify potential mechanisms underyling the increases in Vmax and phosphorylated enzyme formation, other approaches appear necessary. As an example, an additional study examining specialized sites on the Ca2+ enzyme that would allow more detailed examination of the characteristics of each site may be helpful. In this regard, the Ca2+-binding sites may be particularly interesting, since our evidence indicates modifications during recovery.
The effects of prolonged running exercise and recovery on Ca2+-ATPase activity in rat muscle when measured in SR-enriched fractions continue to remain perplexing. Our inability to detect reductions in Vmax of the Ca2+ pump with our prolonged exercise protocol is consistent with our earlier findings (7) and with those of Ferrington et al. (16); however, they are at odds with other studies (2, 5, 46). Moreover, the increase in Vmax that we have observed in recovery in the absence of changes in nH and pCa50 are also in agreement with Ferrington et al. (16). Because different protocols were employed for isolation of enriched SR vesicles and because we have demonstrated previously that similar results are obtained with muscle homogenates (38), a selective retention of damaged or undamaged Ca2+ pumps after exercise and/or recovery would not appear to account for our findings.
There is evidence that the inactivation in Ca2+-ATPase activity that has been reported previously in rats after prolonged exercise may be specific to muscles with a predominance of high oxidative fibers, both type I (46) and type II (5, 17), and not to muscles with a low oxidative potential (5, 17). In the current study, we have investigated only the gastrocnemius muscle, which in the rat is dominated by type II fibers of low to moderate oxidative potential (10). Differences in sensitivities between muscles of different fiber-type characteristics to the effects of prolonged exercise on SR Ca2+ handling could occur because of differences in recruitment and activation profiles (1), differences in isoform susceptibility to structural alteration (43), and differences in the intracellular environment of the contracting cells (30). As a consequence, all muscles, regardless of fiber type characteristics, could experience modifications to SR Ca2+-handling proteins if the contractile demands were prolonged sufficiently and were intense. This is most evident with the chronic low-frequency stimulation model, where inactivation of the Ca2+-ATPase enzyme can be observed in muscles of predominantly type II composition and low oxidative potential (12, 20). The mechanism underlying the reduction in Ca2+-ATPase activity with exercise appears to be similar in type II fibers with high oxidative (25) and low oxidative potential (12, 29), namely an alteration in structure in the region of the nucleotide-binding site. The alteration in the nucleotide-binding site has been shown to result from protein oxidation and peroxynitrate-mediated tyrosine nitration, which appears to occur secondarily to the accumulation of ROS and NO (21).
The increase in Vmax that we have observed is the specific result of changes in Ca2+-ATPase activity, since basal or Mg2+-ATPase was unchanged from rest and exercise during both active and passive recovery. The functional significance of the increase in Ca2+-ATPase activity is demonstrated by the associated increases that we have observed in Ca2+ uptake. This was expected, since Ferrington et al. (16) have demonstrated that there is no change in the physical structure of the SR membrane, as might be expected if changes occurred in lipid composition and/or protein aggregation state.
Several possibilities exist to explain the increase in Vmax and phosphorylated enzyme formation of the Ca2+-ATPase. These include increased phosphorylation, decreased oxidation and nitrosylation, and an altered aggregation state of enzymes. We had reasoned that the increase in phosphoenzyme formation during recovery could occur because of an increase in the ability of ATP and Ca2+ to access the nucleotide-binding site or the Ca2+-binding site as a result of protein folding and/or aggregation state. However, because we found no changes in FITC binding during recovery when an overshoot in Ca2+-ATPase activity was observed, accessibility and binding of these substrates to their respective sites would not appear to be involved. Our inability to detect changes in FITC binding immediately after the exercise is as expected, given the lack of an effect of our exercise protocol on Ca2+-ATPase activity. Previous studies have shown that reductions in Ca2+-ATPase activity are accompanied by alterations in the region of the nucleotide-binding site (12, 29). The trend that we have observed in NCD-4 binding during active recovery deserves special comment. In SR vesicles, there was a trend toward an increase (P = 0.19), which coincided with a similar trend (P < 0.12) toward an increase in nH. A similar trend was observed for passive recovery. Although the results are not significant when analyzed individually in the context of the probability levels (P < 0.05) established for this study, significance was found when analyzed together. Further investigation is warranted to determine if the cooperative binding affinity of Ca2+ to the Ca2+-ATPase is modified during recovery. In addition, increased accessibility of the Ca2+-binding sites as a result of changes in protein folding may be related to the increases in Vmax that we have observed in recovery.
Increased phosphorylation could occur either as a result of direct phosphorylation to the enzyme itself or indirectly via phosphorylation of a related protein such as PLB (4). However, because the gastrocnemius muscle contains primarily the SERCA1 isoform (4) and the phosphorylation of PLB has been shown to increase the affinity of the Ca2+ pump for Ca2+ in cardiac muscle, which contains the SERCA2a isoform (39) and, to a modest degree, the affinity in skeletal muscles expressing the SERCA2a isoform, such as the soleus (44), an increase in Vmax mediated by this mechanism was not expected. Direct phosphorylation of the enzyme via CaM kinase, which can increase Vmax (44, 45), would also be of questionable significance, since this effect appears to be limited to muscle expressing the SERCA2a isoform (44). The fact that we found no change in the phosphorylation of serine-38 of the Ca2+ pump, previously shown to be phosphorylated by CaM kinase (44), is further evidence that direct enzyme phosphorylation of the enzyme in increasing Vmax during recovery is unimportant.
Although direct phosphorylation of the enzyme is without consequence in enzyme regulation in SERCA1-based skeletal muscle, other mechanisms appear capable of regulating the activity of this isoform. Sarcolipin, as one example, appears to be able to increase Vmax in fast muscles that contain the SERCA1 isoform (28, 34). However, sarcolipin is not a phosphorylatable substrate; consequently, its regulation is probably through altered expression. Given the acute nature of our experiment, it is unlikely that altered expression occurred during the recovery phase (40).
We have also measured the level of nitrotyrosine, a direct measure of enzyme nitrosylation, using a polyclonal antibody specific for anti-nitrotyrosine. As hypothesized, the relative concentration of nitrotyrosine in the SR Ca2+-ATPase was unchanged after exercise and during recovery. It is well established that NO is a vital second messenger that can modulate contractile performance in skeletal muscle (22). In skeletal muscle, it appears that NO exerts its effects by either direct nitrosylation of target proteins or through cGMP-mediated signaling cascades (26). Both mechanisms seem important in modulating SR function. It appears that, in skeletal muscle, direct nitrosylation of target proteins mediated through NO or NO-derived species act to depress Ca2+ resequestration. Conversely, the cGMP-mediated effects induced by lower concentrations of NO appear to act in the opposite manner by increasing the SR Ca2+ pump activity of the SERCA1 isoform (3). The increased Ca2+ pump activity correlated with decreases in the half-relaxation time in skeletal muscle (26). Our results indicate that nitrosylation cannot account for the altered Ca2+-ATPase behavior observed in recovery. However, the effects of exercise and recovery on the cGMP-mediated NO effects were not measured in this study. The increase in SR Ca2+-ATPase and Ca2+ uptake demonstrated during active and passive recovery in this study could be caused by a time-dependent exercise-induced activation of this pathway.
In the current study, we have investigated the effects of a fixed period of both active and passive recovery after a standardized session of exercise on SR Ca2+ handling. We have found that the exercise results in an ~31% depletion of glycogen in the gastrocnemius muscle, indicating recruitment during the exercise (unpublished observation). During active recovery, there was no change in muscle glycogen level. Similarly, passive recovery failed to result in a repletion of glycogen. Contrary to our hypothesis, we could find no difference in SR Ca2+-cycling properties between recovery conditions. This would indicate that a period of reduced exercise intensity does not impede enzyme recruitment.
In this study, we have also examined the effects of exercise and exercise plus recovery on the functional and structural properties of the CRC. We have employed two releasing agents to investigate Ca2+ release and the [3H]ryanodine-binding technique to examine structural changes. Recent work from our laboratory has shown that a commonly used releasing agent, AgNO3, causes Ca2+ release by activating the CRC and by reversing ion flow through the Ca2+-ATPase (42). It was also found that another Ca2+-releasing agent, 4-CMC, causes SR Ca2+ release without reversing ion flow through the Ca2+-ATPase, although it does inhibit its activity (42). Because we have found an increase in the Ca2+-ATPase activity with active and passive recovery, the use of AgNO3 as a releasing agent may confound the Ca2+ release results. To distinguish between the confounding effects of AgNO3 on the Ca2+-ATPase during Ca2+ release measures, both releasing agents were used in this study.
Our findings did not support a reduction in SR Ca2+ release immediately after prolonged exercise. Contrary to our hypotheses, both phase 1 and phase 2 of SR Ca2+ release, assessed by in vitro measures in the gastrocnemius muscle of rats, were not changed immediately after prolonged exercise. This finding is at odds with a previous report by Favero et al. (15), who showed that prolonged treadmill exercise in rats reduced the rate of AgNO3-induced SR Ca2+ release in SR vesicles. However, the confounding effects of AgNO3 on Ca2+- ATPase during Ca2+ release measures previously mentioned (42) must be considered in their conclusions (15). Contrary to our hypotheses, SR Ca2+ release was not changed during active or passive recovery. The additional exercise during active recovery failed to decrease Ca2+ release, suggesting that an additional volume of exercise was not important in causing a reduction in Ca2+ release.
Our results indicate that [3H]ryanodine binding assessed in SR vesicles was reduced during active recovery. However, [3H]ryanodine binding was unchanged immediately after prolonged exercise and during passive recovery. This finding is partially consistent with a previous report, which demonstrated a 20% depression in [3H]ryanodine binding after prolonged treadmill running in rats (15). It appears that the initial exercise intensity and/or volume was insufficient to reduce the number of functional CRCs. The additional exercise during active recovery may have resulted in the depression in [3H]ryanodine binding. With passive recovery, no alteration in [3H]ryanodine binding was observed.
In recovery, a dissociation between SR Ca2+ release measures and the [3H]ryanodine-binding measures was observed. Ryanodine, in low concentrations, binds exclusively to open (or active) CRCs. Therefore, the [3H]ryanodine-binding assay supposedly measures the number of functional CRCs that respond to Ca2+. If the number of active CRCs was reduced, it would be expected that SR Ca2+ release would be depressed. In this study, [3H]ryanodine binding was reduced during active recovery, but no changes in Ca2+ release were reported. A number of factors could account for these differences. Because Ca2+ release and uptake do not occur independently and the net rate of Ca2+ flow through the SR was measured in this study, differences in Ca2+ release may be masked by changes in Ca2+ uptake. In addition, alterations to the SR membrane composition and fluidity, which could affect the net rate of Ca2+ flow through the SR, were not assessed in this study and are not known.
In summary, we have shown that a single bout of prolonged moderate-intensity exercise followed by a period of reduced exercise intensity (active recovery) or no activity (passive recovery) causes an increase in SR Ca2+ uptake and the Vmax of Ca2+-ATPase activity in skeletal muscle of predominantly type II (fast-twitch) composition, compared with Con. In addition, we have demonstrated that the increases in Ca2+ uptake and Ca2+-ATPase activity are associated with an increase in phosphorylated enzyme intermediate formation, suggesting that, during active and passive recovery, there is an increase in the number of active Ca2+ pumps resequestering Ca2+. It appears that structural alterations in the region of the nucleotide-binding site of the Ca2+-ATPase enzyme did not cause this, since FITC binding was unaltered. Increases in Ca2+-binding sites of the Ca2+-ATPase enzyme, as determined by NCD-4, were observed, which could explain the increase in Vmax. An inviting hypothesis is that a transient exercise-induced disruption of Ca2+ homeostasis could activate intracellular signaling cascades, leading to a recruitment of latent enzyme and/or alteration in the SR Ca2+-ATPase oligimeric or folding state and resulting in an upregulation in Vmax and the rate of SR Ca2+ resequestration in recovery. The increase in Ca2+ uptake could have several important consequences. The increase in Ca2+ uptake could restore an exercise-induced disturbance in Ca2+ cycling and prevent a detrimental long-lasting accumulation of cytosolic Ca2+. Contractile function during recovery could also be affected. Because an increase in Ca2+ uptake occurred in the absence of changes in Ca2+ release, the rate of relaxation should be increased. This would allow for more rapid recovery, which could be particularly important in dynamic activity where rapid changes in [Ca2+]f are required. It is of interest that the overshoot in Ca2+-ATPase activity and Ca2+ uptake during recovery was observed in a muscle composed of primarily type II fibers, which is fast contracting and in which [Ca2+]f is regulated rapidly.
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ACKNOWLEDGEMENTS |
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We are appreciative of the capable technical support provided by Dr. Jing Ouyang.
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FOOTNOTES |
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Financial support for the research was received from the National Sciences and Engineering Research Council of Canada.
Address for reprint requests and other correspondence: H. Green, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, ON Canada N2L 3G1 (E-mail: green{at}healthy.uwaterloo.ca).
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. Section 1734 solely to indicate this fact.
First published October 29, 2002;10.1152/ajpendo.00190.2002
Received 6 May 2002; accepted in final form 24 October 2002.
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