Fig. 3.

Extracellular ATP evoked IL-6 de novo synthesis and secretion. A: ATP increased IL-6 extracellular levels ≤10-fold, depending on transcription and translation processes. Rat myotubes were stimulated with 100 μM ATP after 2-h preincubation, with 30 μM cycloheximide (translation blocker), with 0.5 μM actinomycin D (transcription blocker), or without (w/o) treatment. At different times, 50 μl of supernatant was removed, and IL-6 was quantified by ELISA. B: resting levels of extracellular IL-6 were not affected by 6-h treatment with either cyclohemimide or actinomycin D. C: total protein levels from myotube lysates were significantly reduced by 6-h treatment with cycloheximide but not actinomycin D. D: IL-6 mRNA increase evoked by 100 μM ATP (4 h) was abolished by actinomycin D treatment but largely increased after cycloheximide. Cycloheximide treatment increased IL-6 mRNA levels at rest by 40-fold, suggesting that translation blockade activates a positive loop of IL-6 transcription. IL-6 mRNA was measured by quantitative PCR, normalized to GAPDH, and expressed as fold increase related to nonstimulated condition. E: total mRNA levels form myotube extracts were significantly reduced by 6-h actinomycin D treatment but unaffected by 6-h cycloheximide. Values (n = 3) are expressed as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001, analysis of variance followed by Dunnett's multiple comparison test against each “without treatment” value (A–C and E) or Bonferroni's test (D). NS, not significant.