D. Stofan

Free radicals alter maximal diaphragmatic mitochondrial oxygen consumption in endotoxin-induced sepsis

Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State 3 respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State 3 diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-SOD, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-SOD or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-SOD. We found: (a) no alteration in maximal State 3 mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-SOD or L-NAME (but neither denatured PEG-SOD nor D-NAME) prevented endotoxin-mediated reductions in State 3 respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-SOD prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State 3 mitochondrial oxygen consumption are free radical-mediated.

Effects of protein kinase A inhibition on rat diaphragm force generation.

Although protein kinases are known to play a role in modulating a variety of intracellular functions, the direct effect of inhibition of these enzymes on skeletal muscle force production has not been studied. The purpose of the present study was to examine this issue by determining the effects produced on diaphragm force generation by two protein kinase inhibitors: (a) H7, an inhibitor of both cAMP-dependent protein kinase (PKA) and of protein kinase C, and (b) H89, a selective inhibitor of PKA. Experiments (n=15) were performed using isolated, arterially perfused, electrically stimulated rat diaphragms. Perfusate temperature was adjusted to maintain muscle temperature at 27 degrees C and arterial pressure was kept at 150 Torr. Animals were divided into three groups: (a) a control group perfused with Krebs-Henselheit solution equilibrated with 95% O(2)/5% CO(2), (b) a group in which H7 (2 microM) was added to the perfusate, and (c) a group perfused with solution containing H89 (4 microM). In all three groups, we assessed diaphragm twitch kinetics, force-frequency relationships and in vitro fatiguability. We found that both H7 and H89 administration slowed twitch relaxation, augmented force generation in response to low frequency stimulation, and increased the rate of development of fatigue. Specifically, for control, H7 and H89 groups, respectively, we found: (a) 1/2 relaxation time averaged 64+/-2 S.E.M., 87+/-6 and 90+/-2 ms, P<0. 003, (b) force production during 10-Hz stimulation averaged 12.6+/-1. 1, 20.1+/-2.3, and 20.3+/-2.1 N/cm(2), P<0.035, and (c) force fell to 14.3+/-2.0, 9.5+/-0.5 and 8.7+/-0.2% of its initial value after 20 min of fatiguing stimulation, P<0.035. These data show that it is possible to produce large increases in low frequency skeletal muscle force generation by directly inhibiting PKA. We speculate that it may be possible to pharmacologically augment respiratory muscle force and pressure generation in clinical medicine by administration of PKA inhibitors.