Fig. 5.

Hyperammonemia increases tyrosine nitration of skeletal muscle proteins that localize to the autophagosome. Tyrosine nitration of muscle proteins was quantified on immunoblots. A: representative immunoblots from controls and cirrhotic patients (n = 12 each). B: densitometry of all of the bands in each lane showed significant increase (**P < 0.01) in nitration of muscle proteins in cirrhosis. C: representative immunoblots from PCA and sham rats (n = 6 each). D: densitometric quantification of all of the bands in each lane showed significant increase (**P < 0.01) in nitrated proteins in the muscle of the PCA rat compared with the sham-operated control animals. Murine C₂C₁₂ myotubes exposed to Am.Ac for different times (experiments in triplicate). E: representative immunoblots at different times probed using 3-nitrotyrosine antibody. F: densitometric quantification of all of the bands in each lane showed increased protein nitration by hyperammonemia compared with controls. **P < 0.01 compared with controls. G: significant increase in concentration of nitrotyrosine/tyrosine ratio in cell lysates in response to Am.Ac. *P < 0.01 compared with controls; **P < 0.001 compared with controls and P < 0.05 compared with 30-m sample. H: murine C₂C₁₂ myotubes stably transfected with GFP-LC3 were exposed to Am.Ac for different time points. Representative immunostain for 3-nitrotyrosine (red) in these GFP-LC3 stably transfected C₂C₁₂ myotubes showed an initial rapid red staining of nitrated proteins that subsequently begin to colocalize with green LC3 puncta in the autophagosomes (yellow). I: quantification of the colocalization of the nitrated proteins to the GFP-LC3⁺ puncta. *P < 0.01 and **P < 0.001 compared with control. J: no significant change in intracellular pH in murine myotubes exposed to Am.Ac compared with controls. Nigericin exposure in the presence of a high-potassium buffer with alteration of extracellular pH was used to calibrate the system and demonstrate the appropriate fluorescence responses. A separate set of C₂C₁₂ murine myotubes was exposed to 10 mM ammonium chloride to demonstrate the anticipated changes in intracellular pH that have been published by others (4) and are different from the response to Am.Ac used in the present studies. BSS, balanced salt solution.