In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis

Background. Deranged Na+ homeostasis in skeletal muscle is closely associat ed with excessive complement activation that is encountered during sepsis. Recent evidence suggests that soluble C5b-9 complexes (SC5b-9), which are r eadily detected in plasma during sepsis and have lon been considered irrele vant nonmembrane binding end products of complement activation, may have nu merous biologic effects. The purpose of this study, therefore, was to deter mine the effects of SC5b-9 on myocellular ion homeostasis and its mechanism (s) of action. Methods. Hindlimb fast-twitch extensor digitorum longus (EDL) was freshly i solated from rats weighing 50 to 70 g and then incubated at 30 degreesC for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% z ymosan-activated rat serum (10 mg/mL at 37 degreesC for 60 minutes) as a so urce of SC5b-9. Zymosan particles were removed by centrifugation after acti vation to exclude any noncomplement direct effects. Heat-inactivated rat se rum (56 degreesC for 30 minutes) was used as control. EDL muscle was also i ncubated with pertussis toxin (1 mug/mL), in Ca2+-free KHB, with thapsigarg in (0.3 or 3 mu mol/L), or with ouabain (0.01, 0.1 or 1 mmol/L) before and/ or during incubation with 10% zymosan-activated or heat-inactivated rat ser um. Intracellular Na+ and K+ contents ([Na+](i) or [K+](i)) of EDL muscle w ere determined by using flame photometer after washing in ice-cold Na+-free Tris-sucrose buffer. SC5b-9 in zymosan-activated human serum was determine d by SC5b-9 enzyme-linked immunoassay. Results. SC5b-9 in zymosan-activated human serum significantly increased by 400% as compared with nonactivated, normal human serum. Zymosan-activated rat serum markedly increased [NA(+)](i) without affecting [K+](i) in fast-t witch EDL muscle, which was completely inhibited by pertussis toxin, remova l of extracellular Ca2+ or depletion of intracellular Ca2+ with thapsigargi n. The addition of ouabain (at micromolar concentrations) increased myocell ular [Na+](i) and decreased myocellular [K+](i) in both the zymosan-activat ed and the heat-inactivated rat serum groups. The effects of ouabain on myo cellular [Na+](i) and [K+](i) were equivalent in these 2 groups. Zymosan-ac tivated and heat-inactivated rat serum had similar effects on myocellular [ K+](i) in the presence or absence of pertussis toxin, removal of extracellu lar Ca2+ or depletion of intracellular Ca2+. Conclusions. Zymosan-activated rat serum (presumed SC5b-9 enriched) selecti vely alters Na+ homeostasis in isolated fast-twitch skeletal muscle. The me chanisms for such effects may be linked to G-proteins, Ca2+ flux and Na+,K-adenosine triphosphatase pump binding site blockade.