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Calcium activated neutral protease (calpain) and the neutrophil: their relationship and association with the acute inflammatory response to exercise Raj, Daniel Adelbert


The purpose of this study was to determine whether exercise-induced neutrophil accumulation into striated muscle could be assisted by locally produced myogenic factors. It is hypothesized that a relationship exists between the processes of calpain-mediated muscle injury and the recruitment of neutrophils to the injured sites. To test this hypothesis, male Sprague-Dawley rats (-325 g) were randomly assigned to either a control (n = 10) or an experimental (n = 10) group, where the experimental protocol involved 1 hour of -14% grade running at 25 m/min on a motorized treadmill. Neutrophil chemotaxis assays were conducted on extracts of whole tissue samples of the heart, soleus, and plantaris; processed in phosphate buffered saline for the membrane soluble and cytosolic protein fractions. Chemotaxis was quantified in triplicate across all conditions and normalized to the maximal chemotactic response at 10"7M/MLP. In the membrane soluble fraction, neutrophil chemotaxis was increased in the skeletal muscles following exercise: Condition Control (% max chemotaxis) Exercise (% max chemotaxis) Significance Soleus 9.03 ± 3.03 46.70 ±1.91 p < 0.05 Plantaris 9.31 ± 1.25 22.31 ±4.06 p < 0.05 Heart 7.74 ± 2.73 4.28 ± 1.83 p > 0.05 However, all muscle extracts from the cytosolic fraction reflected a significant decrease in neutrophil chemotaxis following exercise: Condition Control (% max chemotaxis) Exercise (% max chemotaxis) Significance Soleus 31.79 ±14.41 9.26 ±4.63 p < 0.05 Plantaris 16.16 ±4.68 4.97 ± 2.49 p < 0.05 Heart 47.14 ±13.69 10.92 ± 4.74 p < 0.05 These results indicate that there exists a neutrophil chemoattract(s) in muscle. It is plausible that this chemoattactant(s), initially contained within the cytoplasm, could feasibly leak out into the interstitium/circulation mainstream during exercise. To investigate a relationship between calpain-like activity (Ca2+-stimulated proteolysis) and neutrophil accumulation as measured by myeloperoxidase (MPO) activity, these activites were compared in cardiac and plantaris muscles from male Wistar rats (n = 10) completing 1 hour of running exercise (25 m/min). Exercise promoted increases (p < 0.05) in both calpain-like and MPO activities; ranging from 2.79 to 58.9 U/g wet wt and 0.03 to 4.88 U/g wet wt respectively. Pearson's correlational analysis (r) on calpain-like and MPO activities for cardiac and plantaris data were 0.97 (p < 0.001) and 0.68 (p < 0.05) respectively; with a combined r = 0.83 (p < 0.001) for both muscles across all conditions. To further investigate 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 hour before exercise. Administration of E64c lowered calpain-like and MPO activities by 66% and 56% respectively (average from cardiac and plantaris muscles). From these results it is concluded that: 1) a relationship exists between Ca2 + - stimulated proteolysis and neutrophil accumulation into striated muscle with exercise; and 2) the calpain system is involved in localizing the neutrophilic response associated with exercise.

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