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 myofibrillar proteolysis was determined in incubated extensor digitorum longus 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 proteolysis was determined in the absence or presence of Ca2" or by blocking the Ca2+-dependent proteases calpain I and II. Energy-dependent proteolysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of free and conjugated ubiquitin were determined by Northern and Western blots, respectively, to assess the role of the ATP-ubiquitin-dependent proteolytic pathway. Total and myofibrillar protein breakdown was increased during sepsis by 50 and 440%, respectively. Lysosomal and Ca2"-dependent proteolysis was similar in control and septic rats. In contrast, energy-dependent total and myofibrillar protein breakdown was increased by 172% and more than fourfold, respectively, in septic muscle. Ubiquitin mRNA was increased severalfold in septic muscle. The results suggest 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. (J. Clin. Invest. 1994. 94:2255-2264

Recent studies suggest that sepsis-induced increase in muscle proteolysis mainly reflects energy-ubiquitin-dependent protein breakdown. We tested the hypothesis that glucocorticoids activate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis. Rats underwent induction of sepsis by cecal ligation and puncture or were sham-operated and muscle protein breakdown rates were measured 16 h later. The glucocorticoid receptor antagonist RU 38486 or vehicle was administered to groups of septic and sham-operated rats. In other experiments, dexamethasone (2.5 or 10 mg/kg) was injected subcutaneously in normal rats. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Energydependent proteolysis was determined in incubated muscles depleted of energy with 2-deoxyglucose and 2,4-dinitrophenol. Levels of muscle ubiquitin mRNA and free and conjugated ubiquitin were determined by Northern and Western blot, respectively. RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates and blunted the increase in ubiquitin mRNA levels and free ubiquitin. Some, but not all, sepsisinduced changes in ubiquitin protein conjugates were inhibited by RU 38486. Injection of dexamethasone in normal rats increased energy-dependent proteolysis and ubiquitin mRNA levels. The results suggest that glucocorticoids regulate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis.

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The purpose of this study was to establish the existence of Na/H exchange in cardiac muscle and to evaluate the contribution of Na/H exchange to pHi regulation . The kinetics of pH i changes in cultured chick heart cells were monitored microfluorometrically with 6-carboxyfluorescein and correlated with Nai content changes analyzed by atomic absorption spectrophotometry ; transmembrane H+ movements were evaluated under pH stat conditions . After induction of an intracellular acid load by pretreatment with NH 4C1, a regulatory cytoplasmic alkalinization occurred with a t1/2 of 2.9 min. pHi regulation required external Na' and was concomitant with transmembrane H+ extrusion as well as a rapid rise in Nai content in an Na/H ratio of 1 :1 . Microelectrode recordings of membrane potential demonstrated directly the electroneutral character of pHi regulation . Acid-induced net Na' uptake could be either stimulated by further decreasing pHi or inhibited by decreasing pH .; Na' uptake was unaffected by tetrodotoxin (10 ug/ml), quinidine (10-3 M), DIDS (10-4 M), C16-free solution, or HC03-free solution . Amiloride (10-3 M) maximally inhibited both pHi regulation and Na' uptake ; the ID 3a for amiloride inhibition of Na* uptake was 3 I.M . Na.-dependent H+ extrusion showed halfmaximal activation at 15 mM Na.; Li+, but not K+ or choline', could substitute for Na' to support H+ extrusion. Ca.-free solution also stimulated acid-induced Na* uptake . We conclude that pHi regulation following an acid load in cardiac muscle cells is by an amiloride-sensitive, electroneutral Na/H exchange. Stimulation of Na/H exchange up to 54 pmol/cm2 .s indicates the rapidity of this exchange across cardiac cell membranes. Na/H exchange may also participate in steady state maintenance of pHi.

Cytosolic free magnesium concentration [Mg2+]; and its regulation were studied in cultured embryonic chicken heart cells by use of the fluorescent indicator 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,Otriacetic acid (Furaptra). The intracellular location ofFuraptra was confirmed by its complete release from cells upon addition of saponin. The basal [Mg2+J], which averaged 0.48 ± 0.03 mM (n = 31), increased 3-fold on perfusion with sodium-free solution. This increase could not simply be attributed to intracellular sodium-extracellular magnesium exchange be- Indeed, even the basal [Mg2+] level is controversial (7,8). Page and Polimeni (9) showed that the rate of 28Mg uptake in perfused rat heart is increased by increasing extracellular Mg2+ ([Mg2+]0) and that 28Mg efflux is also stimulated by increasing [Mg2+]0 (Mg2+-Mg2' exchange). In frog skeletal muscle, 28Mg uptake is increased in alkaline solution and depressed in acid solution (10). The properties of sodiummagnesium exchange have been studied in squid axon (11,12) and barnacle muscle fibers (13 tTo whom reprint requests should be addressed. 2981The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.