benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Shock--Septic

We have developed a novel LPS probe using a highly purified and homogenous preparation of [(3)H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The alpha-subunit (PSMA1 C2, 29.5 kDa) and the beta-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome's chymotrypsin activity as well as macrophage TNF-alpha secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.

Topics: Acetylcysteine; Acute-Phase Proteins; Animals; Carrier Proteins; Cell Line; Chymotrypsin; Cross-Linking Reagents; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Activation; Escherichia coli; Glutamate Synthase; Leupeptins; Lipopolysaccharides; Macrophages; Macrophages, Peritoneal; Membrane Glycoproteins; Membrane Proteins; Methanosarcina; Mice; Mice, Inbred C3H; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Shock, Septic; Signal Transduction; Tritium

NF-kappaB is a ubiquitous transcription factor that mediates the inflammatory response. Inhibition of NF-kappaB may be of potential therapeutic benefit in the treatment of septic shock. The antioxidant pyrrolidine dithiocarbamate (PDTC) has been shown in previous work to selectively inhibit NF-kappaB activation. Likewise, the proteasome inhibitor MG132 inhibits NF-kappaB formation and degradation of its inhibitor I-kappaB. The goal of this study was to determine if PDTC and MG-132 could inhibit resistance arteriole vasodilation in response to endotoxin and to determine PDTC's site of action in our isolated vessel preparation. Male Sprague-Dawley rats were given an intraperitoneal injection of PDTC, an intravenous injection of MG132, or a sham injection. First-order cremasteric arterioles were isolated, cannulated, and pressurized. A segment of thoracic aorta was then placed in series with the microvascular preparation. Vessels were allowed to achieve spontaneous myogenic tone in a bath of buffer over 1 h (t = 0). Internal vessel diameters were measured and the response to endotoxin (ET) or continued infusion of buffer was measured over 1 h (t = 60). Group 1 (n = 7) was a time-control group. Group 2 (n = 7) was exposed to ET only, Group 3 (n = 5) received PDTC and was exposed to ET, Group 4 (n = 5) received PDTC only, Group 5 (n = 4) received MG132 only, and Group 6 (n = 5) received MG132 and was exposed to ET. To determine the site of action of PDTC, a segment of aorta from an animal treated with PDTC was placed in series with a cremasteric arteriole from an animal receiving a sham injection. The preparation was then exposed to ET, and this is Group 7 (n = 4). Group 8 (n = 4) received ET and was composed of thoracic aorta from an animal receiving a sham injection and a cremasteric arteriole from a PDTC-treated animal. Spontaneous tone was similar in the eight groups at the end of the equilibration period (t = 0). After 1 h (t = 60), Group 2 (vessels exposed to ET only) had significantly less tone (26.1%+/-2.6%; P < 0.01) than Group 1 (39.0%+/-2.4%), Group 3 (39.3%+/-3.1%), Group 4 (41.2%+/-1.6%), Group 5 (39.2%+/-2.9%), Group 6 (41.0%+/-2.7%), Group 7 (45.1%+/-6.5%), and Group 8 (41.1%+/-4.5%). We conclude that PDTC and MG132, inhibitors of NF-kappaB, block ET-induced vasodilation in isolated rat skeletal muscle arterioles. PDTC has effects at both the level of the aortic segment as well as the resistance arteriole. Inhibitors of NF-kappaB may potentially

Topics: Animals; Arterioles; In Vitro Techniques; Leupeptins; Male; Muscle, Skeletal; NF-kappa B; Pyrrolidines; Rats; Rats, Sprague-Dawley; Shock, Septic; Thiocarbamates; Vascular Resistance; Vasodilation