In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis.
BACKGROUND
Deranged Na(+) homeostasis in skeletal muscle is closely associated with excessive complement activation that is encountered during sepsis. Recent evidence suggests that soluble C5b-9 complexes (SC5b-9), which are readily detected in plasma during sepsis and have long been considered irrelevant nonmembrane binding end products of complement activation, may have numerous biologic effects. The purpose of this study, therefore, was to determine 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 isolated from rats weighing 50 to 70 g and then incubated at 30 degrees C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37 degrees C for 60 minutes) as a source of SC5b-9. Zymosan particles were removed by centrifugation after activation to exclude any noncomplement direct effects. Heat-inactivated rat serum (56 degrees C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 microg/mL), in Ca(2+)-free KHB, with thapsigargin (0.3 or 3 micromol/L), or with ouabain (0.01, 0.1 or 1 micromol/L) before and/or during incubation with 10% zymosan-activated or heat-inactivated rat serum. Intracellular Na(+) and K(+) contents ([Na(+)](i) or [K(+)](i)) of EDL muscle were determined by using flame photometry after washing in ice-cold Na(+)-free Tris-sucrose buffer. SC5b-9 in zymosan-activated human serum was determined 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-twitch EDL muscle, which was completely inhibited by pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) with thapsigargin. The addition of ouabain (at micromolar concentrations) increased myocellular [Na(+)](i) and decreased myocellular [K(+)](i) in both the zymosan-activated and the heat-inactivated rat serum groups. The effects of ouabain on myocellular [Na(+)](i) and [K(+)](i) were equivalent in these 2 groups. Zymosan-activated and heat-inactivated rat serum had similar effects on myocellular [K(+)](i) in the presence or absence of pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+).


CONCLUSIONS
Zymosan-activated rat serum (presumed SC5b-9 enriched) selectively alters Na(+) homeostasis in isolated fast-twitch skeletal muscle. The mechanisms for such effects may be linked to G-proteins, Ca(2+) flux and Na(+),K(+)-adenosine triphosphatase pump binding site blockade.
Wang
W
W
Laboratory for Surgical Metabolism and Nutrition, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
Okamoto
K
K
Rounds
J
J
Chambers
E
E
Jacobs
D O
DO
D000818:Animals; D001769:Blood; D002118:Calcium / Q000378:metabolism; D002414:Cations, Monovalent; D003167:Complement Activation; D003182:Complement C5 / Q000096:biosynthesis* / Q000737:chemistry; D050776:Complement C5b; D006706:Homeostasis; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008297:Male; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D009924:Organ Culture Techniques; D010042:Ouabain / Q000494:pharmacology; D037342:Pertussis Toxin; D011188:Potassium / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000032:analysis / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D015054:Zymosan
D002414:Cations, Monovalent; D003182:Complement C5; D010566:Virulence Factors, Bordetella; D010042:Ouabain; D050776:Complement C5b; D015054:Zymosan; D012964:Sodium; D007770:L-Lactate Dehydrogenase; D037342:Pertussis Toxin; D011188:Potassium; D002118:Calcium