[Experimental approach to the prophylaxis and treatment of acute lung injury syndrome with proteinase inhibitors and corvitin]. / Eksperymental'ni pidkhody do profilaktyky ta likuvannia syndromu hostroho ushkodzhennia lehen' z vykorystanniam inhibitoriv proteïnaz i korvitynu.

The results of a combined study of the proteolysis on a model of post-ischemic toxemia in rats showed a decrease in antiproteinase potential and an activation of proteolysis. The activation of proteolysis and inhibition of antiproteinases was observed not only in the blood, but also in the bronchoalveolar secretion. Those changes were accompanied with the changes in the morphological structure of the lungs. The data obtained have shown a high effectiveness of proteinase inhibitor (contrical) and an antioxidant of flavonoid group (corvetine). Those drugs decreased the morphological changes in the lungs and prevented the development of imbalance in proteinase-inhibitor system. The prophylactic effect was more considerable when both drugs were used in a combined way.

Endotoxemia in rats is associated with induction of the P4504A subfamily and suppression of several other forms of cytochrome P450.

Bacterial lipopolysaccharide (LPS) has been previously shown to down-regulate the mRNA and protein expression of the hepatic cytochrome P450 (P450) isozymes 2C11 and 2C12. In this study, we examined the effects of LPS on the constitutive expression of P4503A2, P4502E1, and the P4504A subfamily in the rat. Fischer 344 and Sprague-Dawley rats were each administered 1 mg/kg LPS intraperitoneally and killed for hepatic RNA and microsome isolation at different times. LPS treatment was found to suppress P4502C11, P4503A2, and P4502E1 protein and mRNA expression in both strains of rat. Total microsomal P450 levels decreased by 30%, which was smaller than the effects on the levels of individual isozymes. The magnitude of suppression exhibited in the Sprague-Dawley rats, however, seemed to be more variable than that in the F344 strain. The mRNAs of all three of the P4504A subfamily members were induced 2- to 6-fold in the F344 rat livers after LPS administration. P4504A3 protein expression increased 2-fold, whereas P4504A1/2 protein levels decreased by 30%. Lauric acid omega-hydroxylase activity increased 1.6-fold in LPS-treated Fischer 344 rats and omega-1-hydroxylase activity decreased by 38%. In the Sprague-Dawley strain, however, decreases were seen in both omega- and omega-1-hydroxylase activities after LPS treatment. Our data demonstrate that LPS administration induces P4504A subfamily mRNA and P4504A3 protein expression. Furthermore, our findings also suggest strain differences in both suppression and induction of P450s between the Sprague-Dawley and F344 rats.

Effects of inhibition of nitric oxide synthesis on TNF alpha serum levels in E. coli endotoxemic rats.

We investigated the effects of nitric oxide (NO) synthesis inhibition on mortality rate and TNF alpha serum levels in rats inoculated with E. Coli endotoxin (30 mg/kg i.v.) Pre-treatment of endotoxemic rats with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis by both the constitutive and the inducible isoforms of the NO synthase, did not change the mortality rate but significantly reduced TNF alpha serum levels. By contrast, administration of aminoguanidine, a more specific inhibitor of the inducible NO synthase, did not modify serum TNF alpha. These results suggest that, in E. Coli endotoxemic rats, NO synthetized by the constitutive isoform of the NO synthase positively modulates TNF alpha synthesis.

Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.

Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-gamma (IFN-gamma) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-gamma and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung.

Modulation of glyceraldehyde-3-phosphate dehydrogenase in Salmonella abortus equi lipopolysaccharide-treated mice.

Nitric oxide (NO) is known to inhibit glyceraldehyde-3-phosphate dehydrogenase enzyme activity caused by an NAD(+)-dependent posttranslational protein modification mechanism. In order to study a possible similar protein modification under in vivo conditions, mice were injected with bacterial endotoxin known to endogenously generate NO. In endotoxin-treated mice glyceraldehyde-3-phosphate dehydrogenase enzyme activity was significantly reduced in cytosolic fractions of heart and spleen, compared to 100,000 x g supernatants of untreated control animals. Enzyme activity was unaffected in lung and kidney cytosol of the endotoxin-treated group. Employing the differential NAD(+)-dependent labelling method, glyceraldehyde-3-phosphate dehydrogenase in heart and spleen cytosol of the endotoxin-treated group, versus the control group, had been endogenously modified. These changes were not observed in lung and kidney cytosol of endotoxin-challenged animals. Using Western blot analysis no significant changes in the amount of protein (glyceraldehyde-3-phosphate dehydrogenase) in control versus endotoxin-treated animals was detectable. Since an endogenously NAD(+)-modified glyceraldehyde-3-phosphate dehydrogenase occurred in endotoxin-treated mice, at least in some organs, this NO-stimulated posttranslational protein modification mechanism seems to function under in vivo conditions. A covalent protein modification mechanism, rather than differences in the amount of the protein is likely to cause changes in enzyme activity.

Glucocorticoids regulate intestinal glutamine synthetase gene expression in endotoxemia.

PURPOSE: Although glutamine is required to maintain gut mucosal metabolism and function, intestinal glutamine uptake from the gut lumen and from the bloodstream is decreased during sepsis. We hypothesized that endogenous mucosal glutamine biosynthesis is increased during endotoxemia, and we attempted to define the "stress" mediators that regulate the activity of small intestinal glutamine synthetase (GS), the principal enzyme of de novo glutamine biosynthesis in the gut. METHODS: Adult rats received Escherichia coli lipopolysaccharide (LPS) (7.5 mg/kg intraperitoneally), RU 38486 (a glucocorticoid antagonist; 10 mg/kg by gavage) 2 hours prior to LPS administration, antibody to tumor necrosis factor (TNF) (4 mg/kg intraperitoneally) prior to LPS administration, or ketorolac tromethamine (a prostaglandin synthesis inhibitor; 1 mg/kg intraperitoneally) followed by LPS administration. Mucosal GS activity was assayed 12 hours after LPS administration. In a separate set of studies, cultured intestinal mucosal cells (Caco-2) were exposed to LPS, interleukin 1 (IL-1), IL-6, TNF-alpha, interferon-gamma, prostaglandin E2, or dexamethasone. Twelve hours later, GS activity was assayed and messenger RNA was extracted. The GS transcripts were labeled with a GS complementary DNA probe radiolabeled with phosphorus 32, were quantitated by phosphoimaging, and were normalized to beta-actin. RESULTS: In vivo LPS treatment increased mucosal GS activity by 250%. Pretreatment with antibody to TNF or ketorolac did not inhibit the LPS-induced increase in mucosal GS, whereas pretreatment with RU 38486 attenuated the increase in gut GS activity by 60%. Lipopolysaccharide, IL-1, IL-6, TNF-alpha, gamma-interferon, and prostaglandin E2 did not increase GS activity in Caco-2 cells, whereas dexamethasone increased GS activity and messenger RNA 2.5-fold and threefold, respectively. These data indicate that cytokines and prostaglandins (prostaglandin E2) do not regulate mucosal GS expression during endotoxemia. Glucocorticoids, however, stimulate GS gene expression directly. CONCLUSIONS: This hormonally mediated response may support de novo mucosal GS during septic states when uptake of glutamine from the lumen and blood is decreased.

[Alterations of protein kinase C activity in rat myocardium and aorta smooth muscle during endotoxemia].

A decline of cytosol protein kinase C activity was observed in rat myocardial cells at 4 and 8 h after endotoxin administration, and membrane-associated protein kinase C activity rose at the same time. The activity of membrane protein kinase C in aortic smooth muscle cells at 0.5 and 4 h after endotoxin injection was higher than that in control, while cytosol protein kinase C activity was lower. The results indicate that protein kinase C was activated in myocardial cells and aortic smooth muscle cells during various phases of endotoxemia.

Induction of early-phase tolerance to endotoxin-induced mucosal injury, xanthine oxidase activation, and bacterial translocation by pretreatment with endotoxin.

The goal of this study was to determine whether tolerance would develop to endotoxin-induced mucosal injury, xanthine oxidase activation, and bacterial translocation. To accomplish this goal, four groups of mice were studied: 1) mice receiving ip injections of saline 96 and 24 hr prior to sacrifice, 2) mice receiving ip injections of saline 96 and endotoxin (0.1 mg) 24 hr prior to sacrifice, 3) mice receiving ip injections of endotoxin 96 and 24 hr prior to sacrifice, and 4) mice receiving ip injections of endotoxin 96 hr and saline 24 hr prior to sacrifice. In contrast to the saline control animals or mice sacrificed 96 hr after a single dose of endotoxin, mice sacrificed 24 hr after receiving a single dose of endotoxin had evidence of mucosal injury, elevated levels of ileal xanthine oxidase activity, and an 81% incidence of bacterial translocation. Mice sacrificed 24 hr after a second dose of endotoxin were largely protected against the toxic effects of endotoxin. Thus tolerance to endotoxin-induced bacterial translocation does develop and is associated with tolerance to endotoxin-induced ileal mucosal injury and xanthine oxidase activation.

Changes in catalase activity in lung and liver after endotoxemia in sheep.

Endotoxemia in the sheep produces oxidant-induced cardiopulmonary dysfunction and lung and liver lipid peroxidation, which can be prevented with exogenous catalase, indicating a role for hydrogen peroxide. We determined whether endotoxin-induced oxidant release altered endogenous catalase activity to help explain the lipid peroxidation. Unanesthetized sheep were given 2 micrograms/kg Escherichia coli endotoxin and killed at 5 hr or 24 hr. Lung and liver lipid peroxidation, measured as malondialdehyde, and catalase activity were determined after endotoxin and compared with controls. Lung tissue MDA increased by 100% at 5 hr and was still elevated by 50% at 24 hr, while catalase activity decreased by 50% at 5 hr and remained decreased, suggesting irreversible inactivation. Liver MDA was also doubled at both 5 and 24 hr, but catalase activity remained unchanged. We conclude that endotoxemia results in a significant inactivation of endogenous catalase activity in lung, but not in liver. The lung may be more prone to a subsequent H2O2 injury before restoration of catalase activity.

[The effect of triamcinolone acetonide on the liver mitochondria in endotoxemia]. / Vliianie triamtsinolona atsetonida na mitokhondrii pecheni pri éndotoksemii.

Endotoxin Salmonella typhimurium (LD50) was administered intraperitoneally to mice. It was shown that triamcinolone acetonide in a dose of 1 mg/kg living weight administered to mice 1 hour before endotoxin administration completely prevents the death of the animals and decreases the level of changes in the activities of enzymes of glutamate dehydrogenase, succinate dehydrogenase, monoaminoxidase, cytochrome oxidase in the liver mitochondria in endotoxemia. The level of lipid peroxidation in mitochondria during endotoxemia against the background of triamcinolone acetonide action is close to control. The use of triamcinolone acetonide in the absence of the effect of endotoxin results in an insignificant damage of mitochondrial membranes.

[Lactate dehydrogenase isoenzymes in radiation therapy of cancer patients]. / Izofermenty laktatdegidrogenazy pri luchevoi terapii u onkologicheskikh bol'nykh.

Changes in the lactate dehydrogenase (LDH) isozymic spectrum have been examined in the patients with Stages I-III malignant tumors of the uterus before radiation therapy, during radiation reactions, and after these reactions have been remedied with polydesum and aminocaproic acid. A comparative analysis has shown that the blood serum LDH isozymic spectrum is changed in oncologic patients mainly at the expense of increased content of LDH4 and LDH5. Radiation reactions are associated with a considerable reduction of LDH1 activity and an essential rise of LDH3 and LDH4 activities, and an insignificant elevation of LDH2. The detected shifts in LDH isozymic spectrum during radiation reactions appear to be due to radiation toxemia. Detoxifying therapy with polydesum and epsilon-aminocaproic acid during 3-5 days helped normalize the LDH isozymic ratio.

[Effect of kontrikal on the dynamics of proteolytic system indices in postischemic toxemia]. / Vliianie kontrikala na dinamiku pokazatelei sistemy proteoliza pri postishemicheskoi toksemii.

It was shown in Wistar male rats that the development of tourniquet shock was followed by an increase of proteolytic activity in the blood by 3 times, activity of aspartate aminotransferase (AST) by 3 times, that of alanine aminotransferase (ALT) by 6 times, contents of urea and residual nitrogen by 2.5-3 times; level of alpha 1-protease inhibitor (alpha 1-PI) decreased by 4 times and that of alpha 2-macroglobulin (alpha 2MG) by 2.5 times. At administration of contrykal (10,000 U/kg) proteolytic activity increased only by 32.5%, content of alpha 1-PI decreased only by 10-20% and level of alpha 2-MG did not differ from that in healthy animals. Activity of AST and ALT remained high, and contents of urea and residual nitrogen were near-normal.