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

Weiyang Wang, Ken Okamoto, Jan Rounds, Elizabeth Chambers, Danny O. Jacobs

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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°C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37°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°C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 μg/mL), in Ca2+-free KHB, with thapsigargin (0.3 or 3 μmol/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 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 Ca2+ or depletion of intracellular Ca2+ 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 Ca2+ or depletion of intracellular Ca2+. 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, Ca2+ flux and Na+,K+-adenosine triphosphatase pump binding site blockade.

Original languageEnglish (US)
Pages (from-to)209-219
Number of pages11
JournalSurgery
Volume129
Issue number2
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Complement Activation
Zymosan
Homeostasis
Sodium
Serum
Pertussis Toxin
Ouabain
Hot Temperature
Thapsigargin
Muscles
Sepsis
Skeletal Muscle
SC5b-9 protein complex
In Vitro Techniques
Photometry
Tromethamine
Ice
Hindlimb
Immunoenzyme Techniques
Centrifugation

ASJC Scopus subject areas

  • Surgery

Cite this

In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis. / Wang, Weiyang; Okamoto, Ken; Rounds, Jan; Chambers, Elizabeth; Jacobs, Danny O.

In: Surgery, Vol. 129, No. 2, 2001, p. 209-219.

Research output: Contribution to journalArticle

Wang, Weiyang ; Okamoto, Ken ; Rounds, Jan ; Chambers, Elizabeth ; Jacobs, Danny O. / In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis. In: Surgery. 2001 ; Vol. 129, No. 2. pp. 209-219.
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title = "In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis",
abstract = "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°C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10{\%} zymosan-activated rat serum (10 mg/mL at 37°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°C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 μg/mL), in Ca2+-free KHB, with thapsigargin (0.3 or 3 μmol/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 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 Ca2+ or depletion of intracellular Ca2+ 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 Ca2+ or depletion of intracellular Ca2+. 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, Ca2+ flux and Na+,K+-adenosine triphosphatase pump binding site blockade.",
author = "Weiyang Wang and Ken Okamoto and Jan Rounds and Elizabeth Chambers and Jacobs, {Danny O.}",
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pages = "209--219",
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T1 - In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis

AU - Wang, Weiyang

AU - Okamoto, Ken

AU - Rounds, Jan

AU - Chambers, Elizabeth

AU - Jacobs, Danny O.

PY - 2001

Y1 - 2001

N2 - 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°C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37°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°C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 μg/mL), in Ca2+-free KHB, with thapsigargin (0.3 or 3 μmol/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 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 Ca2+ or depletion of intracellular Ca2+ 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 Ca2+ or depletion of intracellular Ca2+. 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, Ca2+ flux and Na+,K+-adenosine triphosphatase pump binding site blockade.

AB - 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°C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37°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°C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 μg/mL), in Ca2+-free KHB, with thapsigargin (0.3 or 3 μmol/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 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 Ca2+ or depletion of intracellular Ca2+ 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 Ca2+ or depletion of intracellular Ca2+. 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, Ca2+ flux and Na+,K+-adenosine triphosphatase pump binding site blockade.

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