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Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
Submitted 4 September 2003 ; accepted in final form 14 March 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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25% at 30 min of exercise compared with rest (168 ± 10 vs. 126 ± 8 µmol·g protein–1·min–1), with no further reductions observed at 90 min (129 ± 6 µmol·g protein–1·min–1). No changes were observed in the Hill coefficient or in the Ca2+ concentration at half-maximal activity. The reduction in maximal Ca2+-dependent ATPase activity at 30 min of exercise was accompanied by oxalate-dependent reductions (P < 0.05) in Ca2+ uptake by 20% (370 ± 22 vs. 298 ± 25 µmol·g protein–1·min–1). Ca2+ release, induced by 4-chloro-m-cresol and assessed into fast and slow phases, was decreased (P < 0.05) by 16 and 32%, respectively, by 90 min of exercise. No differences were found between normoxia and hypoxia for any of the SR properties examined. It is concluded that the disturbances induced in SR Ca2+ cycling with prolonged moderate-intensity exercise in human muscle during normoxia are not modified when the exercise is performed in hypoxia. calcium, cycling; vastus lateralis; prolonged activity
The reductions in Ca2+ uptake and Ca2+ release have been explained to occur as a result of structural modifications to the respective proteins regulating these functions. In the case of Ca2+ uptake, several studies using repetitive contractile activity have found alterations in the region of the nucleotide binding domain that impairs Vmax (9, 26, 28). For Ca2+ release, reductions in [3H]ryanodine binding, which binds specifically to the calcium-release channel (CRC) when it is in the open Ca2+-release state, have also been reported (10). Evidence has been provided to suggest that the structural alterations that occur to the region of the nucleotide-binding site of Ca2+-ATPase, which is induced during contractile activity, occur as a consequence of oxidation and nitrosylation reactions (23). Similarly, oxidation and/or nitrosylation are suspected as being involved in the structural disturbances in the CRC (10). It is of interest that repetitive exercise is known to increase the accumulation of reactive oxygen species (ROS) (8, 37, 40). Although the human studies reported to date involving both submaximal cycling exercise (4, 18) and supramaximal one-leg kicking protocols (16, 21, 25) are all consistent in reporting a disturbance in at least one SR Ca2+-cycling property, it is not clear whether the changes in several properties are coordinated in a time-dependent manner. Moreover, studies have been generally incomplete since only limited characterization of a given property has been attempted. For the Ca2+-ATPase activity, nothing is known about the Ca2+ dependency since only maximal activities have been studied. Although some studies have examined the coupling ratio between Ca2+ uptake and Ca2+-ATPase, the results are suspect because the ratios are unrealistic given the low Ca2+ used to measure Ca2+ uptake (4).
It is known that, when the same submaximal absolute intensity of exercise involving large muscle groups is performed in hypoxia, the time to fatigue is decreased (15). Because depression in Ca2+ cycling appears to be mechanistically linked to fatigue during prolonged exercise (1), it might be expected that the disturbances in SR Ca2+ cycling will be potentiated, given the greater relative percent of peak oxygen consumption (
O2 peak) and the increased metabolic stress that occurs (19, 33). It is possible that damage to specific proteins involved in SR Ca2+ cycling, such as Ca2+-ATPase and the CRC, would be exaggerated during exercise in hypoxia. As a consequence, Ca2+ uptake and Ca2+ release may be depressed to a greater extent when prolonged exercised is performed in hypoxia compared with normoxia.
In this study, our objective was to determine the effects of prolonged exercise, performed both in normoxia and hypoxia, on SR Ca2+-cycling properties in human skeletal muscle. We have hypothesized that prolonged exercise in normoxia would induce a coordinated, time-dependent reduction in Ca2+-ATPase activity, Ca2+ uptake, and Ca2+ release and that these changes would occur in the absence of changes in the coupling ratio between Ca2+-uptake and Ca2+-ATPase activity. In the case of Ca2+-ATPase activity, the exercise-induced effect will be limited to reductions in Vmax. Moreover, we have postulated that, when the same absolute exercise protocol is performed in hypoxia, the disturbances in the SR Ca2+-cycling properties would be exaggerated.
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METHODS |
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O2 peak, as assessed during a progressive cycle task to fatigue in normoxia, was 3.09 ± 2.5 1/min. As required, each participant was fully informed of all experimental procedures and risks before signing a consent form. As a condition of entry into the study, none of the volunteers engaged in vigorous exercise more than once per week. This study, including the consent form, was approved by Office of Research Ethics at the University of Waterloo, where the study was conducted. Experimental design. To investigate the role of exercise and environmental condition on SR function, a randomized cross-over design was employed where the participants exercised in both normoxic and hypoxic conditions. A standardized protocol consisting of 90 min of cycling performed at moderate intensity was used as the exercise challenge. The exercise tests were conducted using two different inspired O2 fractional (FIO2) concentrations, namely normoxia (FIO2 = 0.21) and hypoxia (FIO2 = 0.14). The hypoxic condition selected was based on previous research, which demonstrated that prolonged exercise at moderate intensity could be performed despite disturbances in energy metabolism (19). Before and at selected time points during the exercise (30, 60, 90 min), ventilatory, gas exchange, and heart rate responses were measured. As well, tissue samples were obtained from the vastus lateralis muscle from preprepared sites before exercise and at 30 and 90 min of exercise. A period of 3–4 wk separated the exercise tests. All tests were conducted under conditions of temperature and relative humidity, which averaged 24°C for temperature and 50–60% for relative humidity. There were no differences in these conditions between normoxia and hypoxia.
All participants were scheduled for laboratory sessions on at least four different occasions. During the first visit, a progressive cycle exercise protocol to fatigue was performed for measurement of O2 peak and related properties. This test was repeated on another occasion if it appeared, on the basis of representative values of respiratory gas exchange and heart rate, that a maximal response was not observed. During the second visit, the submaximal exercise protocol was performed for a brief period of time (15–20 min) at the exercise intensity prescribed for each individual. The intent was to have each individual perform at power output (PO) designed to elicit 50% O2 peak, based on the O2 peak measured during normoxia. Appropriate adjustments were made to PO on subsequent tests to ensure that this relative percentage of O2 peak occurred. During hypoxia, subjects cycled at the same absolute PO as in normoxia. It was estimated that the relative percentage of O2 peak in hypoxia was 64% (19). During the third and fourth visits, participants performed the 90 min of cycling during normoxia and hypoxia in randomized order.
For all exercise tests, the participants sat in the upright position on an electrically braked cycle ergometer (Quinton 870), which was calibrated on a daily basis. The protocol used to measure O2 peak consisted of 4 min of a baseline period of cycling at 25 W followed by 15-W step increases in PO each minute. Volunteers pedalled at 60 cycles/min until volitional fatigue. Fatigue was defined as an inability to maintain at least 50 cycles/min. Ventilation, gas exchange, and heart rate were monitored both before exercise and throughout exercise by previous published methods (22). Ventilatory volume and gas-fraction signals were integrated to produce 30-s windows of minute ventilation (E), oxygen uptake (O2), and CO2 uptake (CO2).
For the prolonged exercise tests, volunteers reported to the laboratory 60 min before the beginning of exercise. During this period, preparations were made for tissue sampling. For tissue sampling, three sites over the vastus lateralis (randomized between legs) were prepared during each exercise test. The preparation for tissue sampling included making three separate incisions after local anaesthesia was applied. These incisions were used to extract samples using the biopsy technique (3) during rest before exercise and at 30 and 90 min of exercise. Two separate samples were extracted from each site to secure sufficient tissue (100 mg) for the analytical measurements. Ventilation and gas-exchange measurements were made over a 4- to 5-min period during rest and beginning at 25, 55, and 85 min of exercise with techniques identical to those described previously (22). Heart rate was also recorded during the gas-collection periods with standard electrocardiographic techniques.
The prolonged exercise tests were performed at approximately the same time of day for each participant and 3–4 h after the ingestion of a liquid supplement consisting of one can of Ensure (1.045 kJ, 14.8% protein, 3.15% fat, and 53.7% carbohydrates; Ross Laboratories, Montreal, Canada). All subjects were requested to refrain from any supplement, including coffee, on the day of testing. Moreover, coffee or alcohol was not permitted the day before testing, and vigorous exercise was not allowed during the 3–4 days before the submaximal tests.
SR properties. Immediately after extraction of the tissue samples, whole muscle homogenates were prepared, rapidly frozen in liquid N2, and stored at –80°C, pending measurements of Ca2+-ATPase activity, Ca2+ uptake, and Ca2+ release. Homogenates were prepared in ice-cold homogenizing buffer (11:1 vol/wt dilution) containing (in mM) 250 sucrose, 5 HEPES, 10 NaN3, and 0.2 PMSF (pH 7.5) by using a hand-held glass homogenizer (Kontes, Duall 20) (30). Each homogenate was divided into a number of aliquots before freezing and storage. Protein content in homogenates was measured by the method of Lowry, as modified by Schacterle and Pollock (34).
Ca2+-uptake and Ca2+-release measurements were performed during a single assay according to procedures previously detailed from our laboratory (35, 41). These measurements involve assessing the changes in free Ca2+ concentration ([Ca2+]f) in the buffer. The measurement of [Ca2+]f is obtained using the fluorescent Ca2+ indicator indo 1 as described earlier (7, 32) with a spectrofluorometer (Ratiomaster, Photon Technology Internationale, Brunswick, NJ) 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 indo1 and the free form. The excitation wavelength was 355 nm, and the Ca2+ free and Ca2+ bound to indo1 have emission maxima of 485 and 405 nm, respectively. Changes in the ratio are used to calculate [Ca2+]f according to method at Grynkiewicz et al. (20). For whole muscle homogenates, we have used a dissociation constant of 250 nM for the binding of indo1 and Ca2+ (20). It should be emphasized that in homogenates the actual dissociation constant is significantly affected by protein composition, and, consequently, the values obtained for Ca2+ uptake and Ca2+ release may vary between experimental conditions when alterations in protein composition occur. For these reasons, the dissociation constant employed is not a true value but only an estimate, and, therefore, the pCa values indicated throughout the paper are only apparent.
For the measurements of Ca2+ uptake and Ca2+ release, the reaction buffer contained (in mM) 200 KC1, 20 HEPES, 10 NaN3, 0.005 N,N,N1,N1-tetrakis(2-pyridylmethyl)-ethylenediame, 5 oxalate, 15 MgCl2, and 10 phosphoenolpyruvate (pH 7.0). Before emission spectra were collected, 18 U/ml lactate dehydrogenase, 18 U/ml pyruvate kinase, and 1.5 µM of indo 1 were added to the cuvette containing 2 ml of the reaction buffer. In addition, 3 µl of CaCl2 (10 mM) was added at each trial to achieve an initial [Ca2+]f of 3.0 µM. Immediately after data collection was initiated, 90 µl of homogenate were added to the cuvette. The reaction was initiated by the addition of 5 mM ATP. Ca2+ uptake was followed until it plateaued. Average rates of Ca2+ uptake were determined at 500, 1,000, 1,500, and 2,000 nM of [Ca2+]f obtained during a single assay.
After active loading of the SR when [Ca2+]f declined to a plateau, Ca2+ release was initiated by adding 10 µM of 4-chloro-m-cresol (4-CMC) to the cuvette. We have found that 4-CMC induces a biphasic Ca2+-release pattern, which is characterized by a rapid early release, which we have labeled phase 1, and a slower, more delayed release, which we have labeled phase 2 (41).
The maximal rate of Ca2+ uptake at each of the four [Ca2+]f concentrations as well as the maximal rate of Ca2+ release for phase 1 and phase 2 were obtained by smoothing the [Ca2+]f vs. time curve over 21 points (Savitsky-Golay algorithm) and differentiating the linear-fit curves to determine maximal rates.
Measurement of Ca2+-dependent SR Ca2+-ATPase activity in whole muscle homogenates was performed using spectrophotometric techniques as described by Simonides and van Hardeveld (39) with minor modification by our laboratory (43). In this procedure, total Ca2+-Mg2+-ATPase and basal ATPase are assessed, and Ca2+-ATPase is calculated as the difference between the two measures. Other potentially contaminating ATPases are selectively inhibited by different pharmacological agents. It has been shown that the measurement is highly specific to Ca2+-ATPase as indicated by the near total loss of activity with the addition of cyclopiazonic acid, known to selectively inhibit the enzyme (32, 39). We have also been able to confirm this result (43). The reaction buffer contained (in mM) 200 KC1, 20 HEPES, 15 MgCl2, 1 EGTA, 10 NaN3, and 5 ATP (pH 7.0). In addition, 18 U/ml lactate dehydrogenase, 18 U/ml pyruvate kinase, 0.3 mM NADH, and 1 µM of the ionophore A-23187 (Sigma C-7522) were added. Assays were performed at 37°C and at 340 nm (Shimadzu UV 160) using 1 mg wet weight of tissue (25-µl homogenate) in 1 ml of reaction buffer. After the baseline absorbance of NADH was recorded for 1 min, the reaction was initiated by adding 1 µl of 100 mM CaCl2 and monitored for 2 min. Maximal Ca2+-ATPase activity and Ca2+-dependent Ca2+-ATPase activity was measured using 0.5-µl additions of 100 mM CaCl2. Basal or background ATPase activity was determined in the presence of 40 µM of cyclopiazonic acid. Measurement of [Ca2+]f used to assess Ca2+ dependency of the Ca2+-ATPase reaction was assessed as described earlier (44).
Because the measurement of Ca2+-ATPase activity is based on the regeneration of ATP, a potential limitation is the loss of ADP to AMP via the myokinase reaction. However, we have shown using a myokinase inhibitor (AP5A) that Ca2+-ATPase activity is unaffected (unpublished observations).
The kinetic properties of the Ca2+-ATPase activity that were measured included Vmax, the [Ca2+]f needed to obtain half-Vmax (Ca50) and the Hill coefficient (nH). These properties were obtained by plotting Ca2+-ATPase activity against the negative logarithm of [Ca2+]f (pCa). Vmax represented the peak value, Ca50 represented the [Ca2+]f obtained from a sigmoid fit of the data that yields 50% of Vmax, and nH was obtained through nonlinear regression with computer software (Graph Pad Software) by using a portion of the curve that corresponded to between 20 and 80% of Vmax.
All SR properties assessed were measured in duplicate. For any given analytical session, care was taken to ensure that all samples for a given subject for both conditions were analyzed together. All measurements were made in aliquots that had been allowed to thaw only once. The coefficient of variation for duplicate trials for Ca2+-ATPase and Ca2+ uptake was 7.9 ± 0.9 and 7.6 ± 0.9%, respectively. Only a single trial was used for Ca2+ release.
Statistics. The data were analyzed by two- and three-way ANOVA procedures for repeated measures. For two-way ANOVA applications, exercise time and oxygen condition represented the independent variables. These procedures were applicable to the gas exchange, ventilation, and heart rate data, and all SR properties were assessed with the exception of Ca2+ uptake. For Ca2+ uptake, a three-way ANOVA was utilized (Ca2+ concentration, exercise time and, oxygen condition). Where significant differences were found, the Newman-Keuls technique was applied to determine which means were different. Significance was set at the 0.05 level. Throughout the text, data are expressed as means ± SE.
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RESULTS |
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O2 (Table 1). The steady-state O2 that was achieved represented 47.5 ± 2.2% of the O2 peak obtained during normoxia. Although O2 increased from rest to exercise, no changes in O2 were observed throughout the 90-min exercise protocol. Similarly, no differences were observed between normoxia and hypoxia either before exercise or during exercise. As with O2, CO2 increased from rest to exercise and then remained stable as time progressed. As well, like O2, a main effect of condition was not observed for CO2. The respiratory exchange ratio (RER), defined as the ratio of CO2 to O2, was different during exercise in both conditions compared with rest. In the case of exercise in normoxia, the increase in RER was only noted at 15 min. For hypoxia, RER was lower than before exercise at all exercise time points. Higher RER levels were also observed in hypoxia compared with normoxia during both rest and exercise. Hypoxia also resulted in a higher E during exercise compared with normoxia but not at rest. As expected, E increased with exercise in normoxia and then remained stable. The same pattern was observed with hypoxia, except that E at 90 min was greater than at 30 min. Heart rate was persistently elevated during hypoxia compared with normoxia both before and during exercise. For both normoxia and hypoxia, a drift in heart rate was observed during exercise. For normoxia, heart rate was higher at 90 min of exercise compared with 30 and 60 min. For hypoxia, heart rate was higher at 90 min compared with 30 min.
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20%, were fully manifested by 30 min of exercise. Exercise in hypoxia did not have any additional effect on Ca2+ uptake over that observed in normoxia. Two phases of Ca2+-release kinetics were assessed, a fast phase (phase 1) and a slow phase (phase 2) (Fig. 1). Both phase 1 and phase 2 were reduced with exercise (Fig. 3). In the case of phase 1, the reduction, which amounted to 16%, was only found at the end of exercise. For phase 2, the decrease was progressive, amounting to 18% at 30 min and 32% at 90 min. As with Ca2+ uptake, we could find no additional effect of hypoxia compared with normoxia.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A number of previous studies on humans using a variety of exercise protocols other than prolonged cycling have reported reductions in Ca2+-uptake (12, 16, 21, 42), Ca2+-release (21, 25), and Ca2+-ATPase activity (25, 42) in the SR of vastus lateralis. Prolonged submaximal exercise has also been reported to alter SR Ca2+ cycling in human skeletal muscle. As an example, Booth et al. (4) have found that cycling exercise performed at 75% O2 peak to exhaustion (72-min duration) resulted in a reduction in Ca2+-uptake and Ca2+-ATPase activity of 17 and 21%, respectively. Our group has reported reductions in Ca2+-ATPase activity of 14% during 30 min of cycle exercise at 58% O2 peak, which was followed by another 19% reduction during exercise at 72% O2 peak (18). Several studies using prolonged treadmill exercise in rats have also detected reductions in muscle Ca2+ uptake (5, 14), Ca2+-ATPase activity (2, 5, 26, 49), and Ca2+ release (11, 31). These studies in conjunction with a variety of other studies using different species and different contractile schedules (46, 47) contribute to the growing consensus that exercise can substantially disrupt SR Ca2+-cycling homeostasis.
However, not all studies report exercise-induced disturbances in SR function with prolonged exercise in running rats. Work from our laboratory (6, 35) as well as others (13, 14) has failed to find reductions in Ca2+-ATPase activity after treadmill exercise in rats. Although the underlying reasons for the contradictory results remain unclear, differences in exercise protocol, the tissues examined, and fractionation procedures all remain of possible importance. Time of tissue sampling after the exercise has also emerged as another potentially significant consideration. Compared with preexercise and immediate postexercise samples, large increases in maximal Ca2+-ATPase activity have been described relatively early in the recovery period (13). We have recently confirmed this finding, and we have been able to demonstrate that the increase in Vmax is accompanied by increases in Ca2+ uptake but not in Ca2+ release (35).
A distinguishing feature of our present study, in contrast to most earlier studies investigating exercise in both humans and animals, which have generally only measured selected SR properties, is the comprehensive nature of our analytical assessment. The collective measurements that we have made, in addition to providing Ca2+-dependent examination of Ca2+-uptake and Ca2+-ATPase activity, have also allowed examination of the interrelations between Ca2+-uptake, Ca2+-ATPase, and Ca2+-release characteristics. These measurements are important for a number of reasons but particularly because a dissociation between changes in Ca2+-ATPase activity and Ca2+ uptake during prolonged exercise has previously been reported (2, 14). Based on these properties, we have been able to conclude that prolonged moderate exercise in humans resulted in reductions in Ca2+ uptake that were independent of [Ca2+]f. In addition, we found that, although Vmax of Ca2+-ATPase activity was depressed, no changes were found in the affinity of the enzyme for [Ca2+]f, as indicated by a lack of change in nH and Ca50. Moreover, the efficiency of Ca2+ transport was unchanged, as shown by the coupling ratio, which is defined as the ratio between Ca2+-uptake and Ca2+-ATPase activity. These results have important functional implications, because repetitive sustained contractile activity, given the changes in intracellular temperature, substrate, and metabolite milieu, could conceivably alter SR membrane composition and fluidity in addition to enzyme behavior (17, 24, 36).
Based on previous studies employing prolonged exercise in rats (26) and chronic low-frequency stimulation in rats and rabbits (9), it would appear that the reduction in Vmax we have observed with exercise is due to structural alterations in the region of the nucleotide binding site of the enzyme. Moreover, it appears that the structural alterations in enzymes occur as a result of oxidation and nitrosylation, which occur as a consequence of elevations in ROS (23). If such is the case, recovery of normal enzyme catalytic activity may take 2–3 days (27).
Accompanying the exercise-induced reductions in Ca2+ uptake we have observed were reductions in Ca2+ release. Unlike many previous studies, which have employed AgNO3 to measure functional CRC, we have employed 4-CMC. The use of 4-CMC instead of AgNO3 was based on a recent study by our group (41), which demonstrated that AgNO3 but not 4-CMC induced Ca2+ release from the lumen of the SR by acting both on the CRC and Ca2+-ATPase. We have also found that two kinetic phases of Ca2+ release can be determined with 4-CMC, a rapid phase (phase 1) and a more delayed phase (phase 2). Although the characteristics of Ca2+ release would be expected to be influenced by the technique employed, a multiphase release pattern has also been demonstrated previously in other types of preparations (29, 38). Prolonged exercise resulted in reductions in both phase 1 and phase 2. It should be emphasized that the physiological significance of each of these phases remains unclear. In our system, those phases were measured in homogenates prepared from whole muscle. It is to be expected, given the protein complexity of the homogenate preparation, that differences in both the time and magnitude of the Ca2+-release phases would differ between enriched SR fractions and single-channel preparations (10). It is of interest that previous studies using [3H]ryanodine binding to assess channel integrity have shown that exercise-induced reductions in Ca2+ release are also accompanied by reductions in [3H]ryanodine binding (11). Although definitive evidence is lacking, there is a consensus that the structural alterations in CRC are mediated by oxidation secondary to ROS accumulation (10). It is noteworthy that the reductions in Ca2+-cycling behavior we have found, both Ca2+ uptake and phase 2 Ca2+ release, were both initially observed early in exercise, suggesting a common mechanism. However, unlike Ca2+ uptake, which remained unchanged over the remainder of the exercise, the impairment is Ca2+ release increased. In addition, phase 1 was only reduced at 90 min of exercise. It is unclear whether the impairment in Ca2+ uptake and Ca2+ release are mediated by common mechanisms.
We could find no additional effect of hypoxia on SR function during exercise. This was unexpected, given the significance of altered SR Ca2+ cycling in fatigue in submaximal exercise (1) and the effects of hypoxia on increasing fatigue (15). As shown by our E, RER, and heart rate data, exercise in hypoxia compared with normoxia clearly increased the responses. The increases in E, RER, and heart rate that we have observed during exercise in hypoxia compared with normoxia have all been previously documented (45, 48). Although greater increases in CO2 appeared to have occurred in hypoxia compared with normoxia, as might be expected (45), these differences were not significant. Hypoxia did not result in an altered O2 during either rest or exercise compared with normoxia. These results indicate that, for the submaximal exercise intensity employed, compensatory adjustments are capable of protecting oxidative phosphorylation during steady-state exercise. Based on previous research (48), it would appear that oxidative phosphorylation is protected primarily via increases in blood flow to the working muscle, which maintains arterial oxygen delivery in the face of decrease in arterial content.
The maintenance of oxidative phosphorylation during hypoxia may be important in preventing additional disturbances in SR Ca2+-cycling properties because the intracellular environment would not be dramatically altered even though the volunteers were working at a greater percentage of O2 peak. It has been estimated that 2–4% of oxygen transport through the mitochondria escapes in the form of ROS (37). However, increased ROS accumulation would be expected to occur as a result of a greater dependence on AMP-to-inosine 5'-monophosphate formation in hypoxia (19, 33) and consequent degradation to inosine via the xanthine oxidase system (37). Alternatively, given the relatively light nature of the prolonged exercise protocol (i.e., 48% of O2 peak), ROS scavenger mechanisms may have been sufficient to minimize further changes in the oxidative environment during exercise in hypoxia. In this regard, it would appear productive to examine the effects of heavier exercise, which is thought to result in greater production of ROS (40) on the SR Ca2+-cycling function in working muscle during both normoxia and hypoxia.
In summary, we have found that prolonged exercise of moderate intensity results in a disturbance in SR Ca2+ homeostasis, as indicated by the reductions in Ca2+ uptake and Ca2+ release that occurs. The reduction in Ca2+ uptake appears to be due to the inhibition of Ca2+-ATPase activities because coupling ratios were unaltered by exercise. Published evidence suggests that the reduction in in vitro Ca2+-ATPase activity is mediated by structural alterations in the region of the nucleotide binding region of the enzyme, an effect mediated by ROS accumulation. The performance of the same exercise protocol in hypoxia (FIO2 = 0.14) does not further increase the disturbance in SR Ca2+ homeostasis.
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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.
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