leupeptins and Shock--Septic

Excessive activation of calpains has been implicated in the pathophysiology of inflammation, trauma, and ischemia reperfusion injury. Here, we investigated the effects of calpain inhibition on myocardial dysfunction and inflammation induced by endotoxin in rats. Rats were treated i.v. with endotoxin (10 mg/kg) or endotoxin plus calpain inhibitors and were then prepared after 4 h for myocardial contractility assessment, detection of endothelium leukocyte interactions, and plasma TNF-alpha, nitrite/nitrate, and endocan levels. Compared with vehicle-treated rats, hearts from endotoxin-treated rats had reduced systolic performance that was partially prevented by calpain inhibitors, i.e., acetyl-leucyl-leucyl-arginal (leupeptin), carbobenzoxy-valyl-phenylalanial (calpain inhibitor III), and N-acetyl-leucinyl-leucinyl-norleucinal (ALLN). Leupeptin and calpain inhibitor III reduced plasma TNF-alpha levels in endotoxin-treated rats. ALLN reduced plasma TNF-alpha and nitrite/nitrate levels in endotoxin-treated rats. Endotoxin treatment increased mesenteric venule leukocyte rolling (10 +/- 3 leukocytes/min vs. 44 +/- 10 leukocytes/min; P < 0.01) and adhesion (2 +/- 2 leukocytes/min vs. 15 +/- 3 leukocytes/min; P < 0.01), which was reduced by calpain inhibitors. Attenuation of leukocyte endothelium interactions observed in calpain inhibitor-treated rats with sepsis was associated with increases in plasma anti-adhesion molecule endocan. In conclusion, calpain inhibitors improved endotoxin-induced cardiac dysfunction, which may be attributed to the modulation of endothelium leukocyte interactions in the inflamed vasculature.

Topics: Animals; Cardiomyopathies; Endotoxins; Glycoproteins; Heart; In Vitro Techniques; Inflammation; Injections, Intravenous; Leukocyte Rolling; Leupeptins; Male; Myocardial Contraction; Myocardium; Nitrates; Nitrites; Rats; Rats, Sprague-Dawley; Shock, Septic; Tumor Necrosis Factor-alpha

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