SEPSIS STIMULATES NONLYSOSOMAL, ENERGY-DEPENDENT PROTEOLYSIS AND INCREASES UBIQUITIN MESSENGER-RNA LEVELS IN RAT SKELETAL-MUSCLE

We tested the role of different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Sepsis was induced in rats by cecal ligation and puncture; controls were sham operated. Total and myofibr illar proteolysis was determined in incubated extensor digitorum longu s muscles as release of tyrosine and 3-methylhistidine, respectively. Lysosomal proteolysis was assessed by using the lysosomotropic agents NH4Cl, chloroquine, leupeptin, and methylamine. Ca2+-dependent proteol ysis was determined in the absence or presence of Ca2+ or by blocking the Ca2+-dependent proteases calpain I and II. Energy-dependent proteo lysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of fre e and conjugated ubiquitin were determined by Northern and Western blo ts, respectively, to assess the role of the ATP-ubiquitin-dependent pr oteolytic pathway. Total and myofibrillar protein breakdown was increa sed during sepsis by 50 and 440%, respectively. Lysosomal and Ca2+-dep endent proteolysis was similar in control and septic rats. In contrast , energy-dependent total and myofibrillar protein breakdown was increa sed by 172% and more than fourfold, respectively, in septic muscle. Ub iquitin mRNA was increased severalfold in septic muscle. The results s uggest that the increase in muscle proteolysis during sepsis is due to an increase in nonlysosomal energy-dependent protein breakdown, which may involve the ubiquitin system.