Differential effect of oestrogen on post-exercise cardiac muscle myeloperoxidase and calpain activities in female rats.

The effects of oestrogen administration on 1 h post-exercise cardiac muscle myeloperoxidase (MPO) and calpain activities were determined in female rats. Rats were ovariectomized and implanted for 2 weeks with either oestrogen (25 mg 17-oestradiol) or placebo pellets or left with ovaries intact. Rats were then run for 1 h at 21 m min-1, 12% grade, killed 1 h post-exercise and cardiac muscle and blood samples were removed. Control animals from each group were killed without prior exercise. Serum oestrogen levels in the order of the highest to lowest were; ovariectomized oestrogen replaced rats > intact ovaries rats > ovariectomized placebo rats. Oestrogen induced significant (P < 0.05) elevations in cardiac MPO activity at rest and at 1 h post-exercise in ovariectomized rats. No significant elevations in cardiac MPO activity were evident in placebo ovariectomized or normal ovary rats at rest or post-exercise. Cardiac calpain activities were similar in all unexercised groups. Ovariectomized placebo and intact ovary rats had significantly (P < 0.05) elevated cardiac calpain activities 1 h post-exercise while calpain activity was not significantly elevated in hearts from ovariectomized oestrogen rats. These results demonstrate that oestrogen supplementation in ovariectomized rats induces elevations in cardiac muscle MPO activities at rest and at 1 h post-exercise. This is opposite to the effect of oestrogen in post-exercise skeletal muscle and implies a greater neutrophil infiltration into cardiac muscle caused by oestrogen. This effect cannot be explained by changes in 1 h post-exercise cardiac muscle calpain activity, the elevation of which was suppressed by oestrogen administration. Oestrogen influences cardiac calpain activity similarly to its effect in skeletal muscle. Thus, oestrogen administration to ovariectomized rats induces elevations in cardiac MPO activity while suppressing cardiac calpain activity.

Time course of diaphragm injury and calpain activity during resistive loading.

The purpose of this study was to determine the time course of arterial blood gas (ABG) deterioration, increased calpain activity, and diaphragm injury during 4 d of resistive loading. Adult Sprague- Dawley rats were divided into control (C) animals and groups that were tracheally banded (TB) for 1 d (TB1), 2 d (TB2), 3 d (TB3), and 4 d (TB4). In TB rats, the carotid artery was cannulated and the trachea was banded during anesthesia. TB groups (TB1, TB2, TB3, and TB4) had a 67% smaller internal cross-sectional area of the trachea than did C animals. ABG samples from awake rats showed a decreased arterial oxygen tension (Pa(O(2))) and a respiratory acidosis in the TB1, TB2, and TB3 groups. Calpain activity was higher in the diaphragm of TB than of C rats; calpainlike activities in soluble fractions of diaphragm tissue were greater in all TB groups than in C rats, whereas those in bound fractions were greater in the TB2 and TB3 groups. Point counting of hematoxylin and eosin-stained cross-sections showed that the area fraction (A(A)) of normal diaphragm was lower and the A(A) of abnormal muscle and connective tissue was higher in TB3 than in C rats. Increased resistive loading induced by tracheal banding was associated with hypercapnic ventilatory failure, increased calpain activity, and diaphragm injury. Ventilatory failure in response to resistive loading may be due to diaphragm injury and/or to decreased minute ventilation.

Metabolic responses to prolonged work during treadmill and water immersion running.

The primary aim of this study was to compare the physiological responses to prolonged treadmill (TM) and water immersion to the neck (WI) running at threshold intensity. Ten endurance runners performed TM and WI running VO2max tests. Subjects completed submaximal performance tests at ventilatory threshold (Tvent) intensities under TM and WI conditions and responses at 15 and 42 minutes examined. VO2 was lower in WI (p<0.05) at maximal effort and Tvent. The Tvent VO2 intensities interpolated from the TM and WI VO2max tests were performed in both TM (i.e., TM@TM(tvent),TM@WI(tvent), corresponding to 77.6 and 71.3% respectively of TM VO2max) and WI conditions (i.e., WI@TM(tvent), WI@WI(tvent), corresponding to 85.5% and 78.2% respectively of WI VO2max). Each of the dependent variables was analyzed using a 3-way repeated measures ANOVA (2 conditions X 2 exercise intensities X 7 time points during exercise). VO2max values were significantly lower in the WI (52.4(5.1) ml.kg(-1) min(-1)) versus TM (59.7(6.5) ml.kg(-1) min(-1)) condition. VO2 during submaximal tests were similar during the TM and WI conditions. HR and [BLa] responses to exercise at and above WI(tvent) were similar during short-term exercise, but values tended to be lower during prolonged exercise in the WI condition. There were no statistical differences in VE responses in the 2 conditions, however as with HR and [BLa] an upward trend was noted with TM exercise over the 42 minute duration of the tests. RPE at WI(tvent) was similar for TM and WI exercise sessions, however, RPE at TM(tvent) was higher during WI compared to TM running. Cardiovascular drift was observed during prolonged TM but not WI running. Results suggest differences in metabolic responses to prolonged submaximal exercise in WI, however it can be used effectively for cross training.

Exercise promotes a subcellular redistribution of calcium-stimulated protease activity in striated muscle.

The aims of this study were (i) to investigate whether the contractile activity associated with running increases calcium-stimulated, calpastatin-inhibited protease activity (calpain-like) in a time-dependent manner and (ii) to determine whether the changes, if any, are proportionately distributed between soluble (cytosolic) and particulate (bound) fractions of striated muscle in vivo. Calcium-dependent, calpastatin-inhibited caseinolysis (i.e., calpain-like activity) was measured in control and exercised rats (25 m/min, 0% grade) at 2, 5, 15, 30, and 60 min. Total calpain-like activity in skeletal muscle increased by 26% (13.2 +/- 1.3 vs. 17.9 +/- 2.2 U/g wet wt.) (p < 0.05) after running (60 min), accompanied by an increased activity in the particulate fraction. In cardiac muscle, exercise (60 min) increased total calpain-like activity by 33% (p < 0.05), which was attributable to increases in both the cytosolic and particulate fractions. Both tissues responded with an early (2-5 min) activation of total calpain-like activity (p < 0.05), supported by early increases for particulate fractions from skeletal muscle; whereas for cardiac muscle, a noticeable early drop (p < 0.05) occurred in the particulate fraction. Minimal changes were observed for total, cytosolic, and particulate fractions of noncontracting tissue (i.e., liver). The results of this study support the hypothesis that the total calpain-like activity increases associated with level running occur early on with exercise and that the increases are accompanied by changes in the redistribution of soluble to particulate fractions. The changes would set the stage for enhanced rates of protein degradation known to occur in striated muscle with exercise.

Chronic resistive loading induces diaphragm injury and ventilatory failure in the hamster.

The purpose of this study was to examine the effects of tracheal banding for 30 days on arterial blood gases, and diaphragm structure and function. Hamsters were tracheal banded (TB) or underwent a sham procedure (C) (n = 16 and 18, respectively). After 30 days, arterial blood gases from awake TB hamsters showed hypoxemia and a respiratory acidosis. Histochemical analysis of diaphragm cross-sections showed a five-fold greater area fraction of abnormal muscle; a greater variation in fiber size; and a 3% higher proportion of type 1 fibers in TB than C hamsters. In vitro physiologic studies of costal strips from TB hamsters showed lower stress (45-70% over 10-100 Hz) than C values. Maximal esophageal pressure during occlusion was 45% higher and normalized diaphragm mass was 10% higher in TB hamsters than C hamsters. We conclude that the lower stress in vitro was attributable, at least in part, to diaphragm injury. Hypercapnea was present in spite of the higher diaphragm mass and maximal esophageal pressures in banded hamsters.

Exercise-induced muscle injury: a calpain hypothesis.

It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca2+ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca2+] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.

Effect of energy restriction on muscle function and calcium stimulated protease activity in recreationally active women.

The purpose of this study was to investigate whether changes in substrate oxidation that are caused by energy restriction influenced muscle function and skeletal muscle calcium stimulated protease activity in female athletes. Endurance athletes were randomly assigned to maintenance energy (100% kcal) or energy restricted (75% kcal) diet treatment groups for 14 days while maintaining regular activity. Body weight significantly decreased in the 75% diet group (-1.7 +/- 0.3 kg; p < .05), while fat oxidation increased (p < .05). Minimal changes in quadriceps function (assessed using the Kin/Com isokinetic dynamometer) were observed following diet treatment, except selected loss of muscle function in the 75% diet group at a movement velocity of 120 deg/s. These results suggest that increased fat oxidation that is induced by an acute energy restriction does not promote loss of general muscle function and activation of calcium-sensitive muscle proteases.

Striated muscle calcium-stimulated cysteine protease (calpain-like) activity promotes myeloperoxidase activity with exercise.

An inflammatory response triggered by neutrophil accumulation into muscle tissue is thought to occur with exercise-induced muscle damage. To investigate the relationship between Ca2+-stimulated proteolysis (calpain-like activity) and neutrophil accumulation [myeloperoxidase (MPO) activity], cardiac and plantaris muscles from rats (n = 10) completing 1 h exercise (25 m/min) were investigated. Exercise promoted increases (P<0.05) in both calpain-like and MPO activities; ranging from 2.79 to 58.9 U/g wet weight (ww) and 0.03 to 4.88 U/g ww respectively. Pearson's correlational analysis (r) on calpain-like and MPO activities for cardiac and plantaris muscle data were 0.97 (P<0.001) and 0.68 (P<0.05) respectively, with a combined r of 0.83 (P<0.001) for both muscles across all conditions. To investigate further the extent to which calpain-like activity may promote neutrophil accumulation, another exercise group (n = 5) was pre-injected with the cysteine protease inhibitor, E64c, 1 h before exercise. Administration of E64c lowered calpain-like and MPO activities by 66% and 56% respectively (average from both muscles). From these results it is concluded that a relationship exists between Ca2+-stimulated proteolysis and neutrophil accumulation into striated muscle with exercise, and that the calpain system is involved in localizing the neutrophilic response with exercise.

A calcium stimulated cysteine protease involved in isoproterenol induced cardiac hypertrophy.

The purpose of this study was to test the relationship between biochemical and functional changes accompanying beta-agonist induced cardiac hypertrophy and the activation of a calcium stimulated cysteine protease. Because the ultrastructural and ionic changes accompanying beta-agonist induced cardiac hypertrophy are reminiscent of the actions of the calcium activated neutral protease, calpain, it was hypothesized that lowering calpain activity (by the use of an exogenous inhibitor(s)) would reduce the extent of hypertrophy. Rats (275-300 g) were randomly assigned to either a control, beta-agonist (iso) or cysteine protease inhibitor (E64c) group. Isoproterenol administration (1 mg/kg) resulted in changes for ventricular weight to body weight ratio (increases 19%), ventricular [RNA] (increases 105.6%), rate of pressure development (increases 22% for +dP/dt) and maximum developed left ventricular pressure (increases 19%) (p < 0.05) after 3 days. Calpain-like activity (assessed by microplate method) increased by 45% (p < 0.05), while [cAMP] returned to control levels (following a transient rise at 1 day; 606.03 +/- 124.1 pmol/g/wet/wt to 937.9 +/- 225 (p < 0.05)). E64c (administered 1 h prior to iso) reduced the extent of hypertrophy, from 19 to 12%, and prevented the increases in; total [RNA], left ventricular function, the initial [cAMP] increase and calpain-like activity. It is concluded that a calcium stimulated cysteine protease(s), such as calpain, may be involved in the biochemical and functional changes associated with isoproterenol induced cardiac hypertrophy.

The effect of diazepan and exercise training on selected biochemical and histochemical properties of rat skeletal muscle.

The effects of chronic diazepam (D) treatment and exercise training on total body mass (TBM), microsomal protein yield (MPY), calcium uptake by fragmented sarcoplasmic reticulum (SR), muscle fibre cross-sectional area, and both PFK and SDH activities were investigated in the tibialis anterior (TA), soleus (Sol), and plantaris (Plt) muscles of 50 male albino Sprague-Dawley rats. Rats were assigned randomly to control (C), sprint-trained (S), or endurance-trained (E) groups. Training was of 12 weeks duration. One-half of each group received daily intraperitoneally D doses of 5 mg kg-1 of TBM. Exercise reduced TBM (p < 0.05); increased the relative BM of the TA (E = 2.02 +/- 0.02, p < 0.01) and Plt (E = 1.15 +/- 0.02, p < 0.01; S = 1.13 +/- 0.03, p < 0.01), as well as the Ca++ uptake of the Sol SR (C = 0.08 +/- 0.02, E = 0.16 +/- 01, p < 0.05). MPY was elevated in S-Sol (C = 1.12 +/- 0.6, S = 1.52 +/- 0.1, p < 0.01). D elevated Sol MPY as well as TA PFK. S-trained animals had lower mean fibre areas than the E-trained (D-treated and untreated) animals. The elevated relative masses of TA and Plt are explained by a decreased TBM with exercise. The increased Ca++ uptake of the Sol indicates that E enhances this function, and the increased MPY probably implies an increased SR. The D could be responsible for the D-elevated Sol MPY as well as the TA PFK. El D did not reduce neuromuscular activity to a level adversely affecting oxidative enzyme activity, but in the case of PFK activity in the TA muscle, such a reduction was evident.

The effect of diazepan and exercise training on selected biochemical and histochemical properties of rat skeletal muscle

The effects of chronic diazepan (D) treatment and exercise training on total body mass (TBM), microsomal protein yield (MPY), calcium uptake by fragmented sarcoplasmic reticulum (SR), muscle fibre cross-sectional area, and both PFK and SDH activities were investigated in the tibialis anterior (TA), soleus (Sol), and plantaris (Plt) muscles of 50 male albino Sprague-Dawley rats. Rats were assigned randomly to control (C), sprint-trained (S), or endurance-trained (E) groups. Training was of 12 weeks duration. One-half of each group received daily intraperitoneally D doses of 5 mg kg(-1) of TBM. Exercise reduced TBM (p<0.05); increased the relative BM of the TA (E=2.02+0.02, p<0.01) and Plt (E=1.15+0.02, p<0.01; S=1.13+0.03, p<0.01), as well as the Ca++ uptake of the Sol SR (C=0.08+0.02, E=0.16+01, p<0.05). MPY was elevated in S-Sol (C=1.12+0.6, S=1.52+0.1, p<0.01). Delevated Sol MPY as well as TA PFK. S-trained animals had lower mean fibre areas than the E-trained (D-treated and untreated) animals. The elevated relative masses of TA and Plt are explained by a decreased TBM with exercise. The increased Ca++ uptake of the Sol indicates that E enhances this function, and the increased MPY probably implies an increased SR. The D could be responsible for the D-elevated Sol MPY as well as the TA PFK. El D did not reduce neuromuscular activity to a level adversely affecting oxidative enzyme activity, but in the case of PFK activity in the TA muscle, such a reduction was evident.

Role of calcium-activated neutral protease (calpain) with diet and exercise.

Although the proteolytic events accompanying acute and chronic perturbations in striated muscle protein turnover remain to be fully elucidated, the purpose of this paper is to (a) review the chemistry of the nonlysosomal calpain-calpastatin system, and (b) provide evidence for the involvement of a nonlysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to altered nutritional status (diet composition; energy restriction) and increased periods of contractile activity (exercise). In reviewing the literature, it is apparent that calpain is involved in the protein catabolism which accompanies alterations in diet composition and/or energy restriction. The precise mechanism of calpain action remains to be elucidated; however, the role of altered metabolic status contributing to calcium imbalances is discussed relative to increasing protein degradation. Hypotheses for further investigation are provided in regard to identifying the targeting of selected proteins (and organelles) for degradation by calpain.

Heart, liver, and skeletal muscle myeloperoxidase activity during exercise.

The purpose of this study was to determine whether contractile activity associated with running exercise was a prerequisite for neutrophil infiltration into rat tissues. H2O2-dependent myeloperoxidase (MPO) activity for rat (n = 8) liver, heart, and gastrocnemius muscles was assayed after 58 +/- 11 min of running to voluntary exhaustion (25 m/min; 0% grade). MPO activity values measured with 0.6 mM H2O2 were 0.988 +/- 0.331 (SD) U/g (skeletal muscle), 1.563 +/- 0.303 U/g (heart), and 1.652 +/- 0.510 U/g (liver) for control samples, compared with 1.690 +/- 0.321, 3.128 +/- 1.221, and 2.752 +/- 0.437 U/g, respectively, for the exercise group (P < or = 0.05). Kinetic analysis revealed that maximum velocity for all tissues increased as a result of the exercise (P < 0.05). The Michaelis constant (Km) values at rest for all tissues were similar (range 0.53-0.57 mM H2O2; P > or = 0.05). Exercise did not alter the Km values for cardiac and liver samples; however, for skeletal muscle, the Km was 28% lower than control (P < or = 0.05). The results of this study show that, with prolonged running, MPO activity is elevated in most rat tissues and not exclusively in skeletal muscle. Moreover, the metabolic status of the tissues may be an important factor for neutrophil infiltration with exercise and not exclusively the type of muscle contraction, as previously hypothesized.

Calcium-supported calpain degradation rates for cardiac myofibrils in diabetes. Sulfhydryl and hydrophobic interactions.

OBJECTIVE: The purpose was to investigate the calcium required for calpain-mediated degradation of selected cardiac myofibril proteins modified by diabetes, sulfhydryl (SH) and hydrophobic reagents. METHODS: After 20 weeks of streptozotocin-induced (55 mg.kg-1) diabetes, calcium sensitive calpain (1.5 U.ml-1) degradation rates of purified cardiac myofibrillar proteins (1 mg.ml-1) were measured, in vitro, and compared to degradation rates for N-ethylmaleimide (NEM) and 2-p-toluidinylnapthalene-6-sulfonate (TNS) treated samples. RESULTS: Diabetes (blood glucose of 550 +/- 32 mg.dl-1) reduced the yield of purified myofibrillar protein with minimal change in fibril protein composition. Total SH group reactivities (nmol.mg-1.30min) were 220 +/- 21, 163 +/- 17 and 156 +/- 24 for control, diabetic and NEM-treated (0.5 mM) myofibrils (p < or = 0.05). Calpain degradation rates were faster for all diabetic and SH modified myofibrillar proteins (p < or = 0.05), with a 45 and 35% reduction in the pCa50 for a 37 kDa protein of diabetic and NEM-treated fibril complexes. For control myofibrils, both 100 and 200 uM TNS, reduced calpain degradation rates to a similar extent for all substrate proteins. In contrast, diabetic and NEM-treated samples showed a further reduction in calpain degradation rates with increasing TNS from 100 to 200 uM. CONCLUSION: Our results support the hypothesis that in diabetes the calcium requirements for calpain degradation rates are reduced and dependent upon sulfhydryl group status and Ca(2+)-induced hydrophobic interactions, implicating a 37 kDa myofbillar-complexed protein.

Diaphragm injury and myofibrillar structure induced by resistive loading.

The purpose of this study was to determine whether ventilatory failure is associated with muscle fiber damage and myofibrillar protein alterations. Ventilatory failure was induced by tightening a polyvinyl band around the trachea of hamsters (TB; n = 14) for 6 days, which resulted in severe respiratory acidosis (PCO2: 97.9 +/- 29.6 vs. 51.6 +/- 19.6 Torr; pH: 7.16 vs. 7.35), hypoxemia (PO2: 42.8 +/- 16.8 vs. 65.9 +/- 25.8 Torr), and increased pulmonary resistance (1.89 +/- 1.61 vs. 0.29 +/- 0.27 cmH2O.ml-1 x min; P < 0.05). The point-counting technique of hematoxylin- and eosin-stained cross sections showed a higher area fraction of abnormal muscle and inflammatory cells in the costal [0.133 +/- (SE) 0.33 vs. 0.040 +/- 0.010] and crural regions (0.069 +/- 0.020 vs. 0.012 +/- 0.003) of the diaphragm in TB hamsters than in control hamsters. Electron micrographs revealed sarcomeric disruption and Z band streaming in the diaphragm of TB hamsters. Myofibrillar changes of the diaphragm associated with ventilatory failure were quantitative (i.e., a lower yield of purified myofibrils) but not qualitative (similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles); however, sulfhydryl group reactivities were reduced (P < 0.05). Proteolysis of purified myofibrils from the diaphragm digested with calpain showed faster degradation rates for tropomyosin and alpha-actinin but not for all proteins for the TB animals. Ventilatory failure induced by resistive loading was associated with diaphragm injury; some of this injury was linked to changes in myofibrillar complexes, specifically their susceptibility to calpain-mediated degradation.

Function of skeletal muscle sarcoplasmic reticulum vesicles with exercise.

In this study the response of sarcoplasmic reticulum (SR) to prolonged moderate-intensity exercise was examined in highly purified native vesicles isolated from rat gastrocnemius muscle. Maximal Ca(2+)-dependent ATP hydrolysis was reduced by 12.6% within 2 min after the onset of exercise. The reduction in Ca(2+)-dependent adenosinetriphosphatase activity progressed to 18% at 30 min of exercise and was maintained throughout the subsequent 90-100 min of exercise. Oxalate stimulation of unidirectional Ca2+ transport (Ca2+ loading) was unaffected by exercise. However, in the absence of anion stimulation, steady-state Ca2+ uptake (bidirectional flux) was 51.2 +/- 7.3 nmol Ca2+/mg SR after exercise compared with 36.2 +/- 2.5 nmol Ca2+/mg SR for the control period (P < 0.05). Anion-induced Ca2+ release increased from a control value of 33.9 +/- 4.3 to 55.9 +/- 9.8 nmol Ca2+/mg SR after exercise (P < 0.05). The mechanistic basis for the increase in apparent Ca(2+)-ATP coupling is unclear, although the early onset of the changes suggests the potential for functional adaptation for the SR in response to increased contractile activity.

Circadian rhythm of glycogen in the hamster diaphragm.

The purpose of this study was to determine the diurnal fluctuation of glycogen stores for the whole hemidiaphragm and within a specific myofibrillar ATPase (M-ATPase) fibre type and diaphragmatic region. Fifty-six golden Syrian hamsters were randomly divided into six groups according to the time of sampling biopsies from the diaphragm: 03:00, 07:00, 11:00, 15:00, 19:00, and 23:00. The right hemidiaphragm was quick frozen and biochemically assayed for glycogen levels. Biopsies from the left hemidiaphragm of the same animal were cut from the anterior costal and crural regions, and stained with periodic acid--Schiff (PAS) and for M-ATPase. Optical density measures of PAS-stained fibres were determined to quantitate glycogen in different M-ATPase fibre types and diaphragmatic regions. Biochemical assay of the entire hemidiaphragm showed slightly greater glycogen content of biopsies taken at 11:00 and 15:00 than at 03:00, 19:00, and 23:00 (range of differences: 6.4-10.0%). However, glycogen levels within a specific M-ATPase fibre type and diaphragm region were not different in biopsies sampled at different times. Because the hamster has a small diurnal variation of glycogen in the diaphragm, which is similar to the small diurnal variation of glycogen in human skeletal muscle, this species may be a good animal model for metabolic studies of the diaphragm that could be affected by diurnal glycogen variability.

Skeletal muscle calcium-activated neutral protease (calpain) with exercise.

The purpose of this study was to investigate whether exercise could induce calpain activation by altering the Ca2+ required for half-maximal activity (pCa50) and/or susceptibility of digestible muscle protein substrates. Rats (225 g) were assigned to control, exercise (25 m/min, 0% grade), and 24-h recovery groups. Exercise resulted in a generalized 48 +/- 18% loss of muscle glycogen and a twofold increase in plasma creatine kinase levels (P < or = 0.05). Exercise increased total caseinolysis of diethylaminoethyl Sepharose-prepared low (u) and high (m) Ca2+ calpain isoforms by 22 and 30%, respectively (P < or = 0.05). The pCa50 of u- and m-calpain with exercise increased from 5.98 +/- 0.12 to 6.20 +/- 0.15 (P > or = 0.05) and from 3.63 +/- 0.10 to 3.90 +/- 0.16 (P > or = 0.05), respectively. In vitro, calpain-mediated degradation/disappearance rates (i.e., percentage of protein degraded in 10 min) for control tropomyosin and alpha-actinin were 69 and 30% compared with 92 and 61% after exercise (P < or = 0.05). The results of this study confirm that level running increases total nonlysosomal Ca2+ specific protease activity, which may promote exercise-induced muscle damage or fatigue.

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.

Calcium-activated neutral protease effects upon skeletal muscle sarcoplasmic reticulum protein structure and calcium release.

In this study, the effects of Ca(2+)-activated neutral protease (CANP) upon skeletal muscle heavy sarcoplasmic reticulum (HSR) structure and function were investigated. CANP was immunolocalized to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent-insoluble fraction of purified HSR membranes. Ca2+ activation of the endogenous membrane-bound CANP produced a characteristic partial fragmentation of the HSR 565-kDa Ca2+ release channel. Similarly, the major substrate for both micromolar and millimolar Ca(2+)-sensitive isoforms of exogenous CANP was the Ca2+ release channel with proteolysis of a 88-kDa HSR protein also observed. Ca2+ release channel proteolysis was initiated at a single cleavage site with coincidental production of 410- and 150-kDa peptide fragments. Appearance of 160- and 137-kDa limiting peptides accompanied secondary proteolysis of the primary 410- and 150-kDa fragments, respectively. Despite extensive proteolysis of the Ca2+ release channel, CANP did not dramatically alter the Ca2+ handling and ryanodine binding properties of HSR membranes. The association of CANP with isolated HSR membranes suggests that, in vivo, this protease may modify an additional property of the Ca2+ release channel. This may be related to the CANP-susceptible structural association of the Ca2+ release channel with dihydropyridine receptors at T-tubule/sarcoplasmic reticulum junctions.