Diaphragm caspase-3 activation. Diaphragm caspase-3 activation was assessed, both by assessing caspase-3 enzymatic activity from the rate of cleavage of a caspase fluorogenic substrate by diaphragm homogenates (A), and by employing Western blotting to determine intact and active cleaved caspase-3 levels for diaphragm homogenates (B) from control, cecal ligation perforation (CLP), CLP plus calpain inhibitor (CI) III, and CLP plus zVAD-fmk groups. A: diaphragm samples from CLP animals (solid bar) had higher caspase activities than samples from control animals (open bar) (P < 0.02). Administration of CI III to CLP animals (light shaded bars) did not reduce caspase activation, but administration of zVAD-fmk, a caspase inhibitor, did reduce caspase activation in CLP animals (dark shaded bars; P < 0.01 for the comparison of CLP to CLP plus zVAD-fmk). B: CLP also induced an increase in active caspase-3 protein (P < 0.02 for comparison to control). Administration of zVAD-fmk to CLP animals reduced caspase-3 protein levels (P < 0.02), whereas administration of CI III did not. *Significant statistical difference compared with the control group, P < 0.05. Error bars for this and all other figures represent ±1 SE. AMC, 7-amino-4-methylcoumarin; AU, arbitrary units.