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Reactive oxygen species contribute to diaphragm dysfunction in certain pathophysiological conditions (i.e., sepsis and fatigue). However, the precise alterations induced by reactive oxygen species or the specific species that are responsible for the derangements in skeletal muscle function are incompletely understood. In this study, we evaluated the effect of the superoxide anion radical (O(2)(-).), hydroxyl radical (.OH), and hydrogen peroxide (H(2)O(2)) on maximum calcium-activated force (F(max)) and calcium sensitivity of the contractile apparatus in chemically skinned (Triton X-100) single rat diaphragm fibers. O(2)(-). was generated using the xanthine/xanthine oxidase system;.OH was generated using 1 mM FeCl(2), 1 mM ascorbate, and 1 mM H(2)O(2); and H(2)O(2) was added directly to the bathing medium. Exposure to O(2)(-). or.OH significantly decreased F(max) by 14.5% (P < 0.05) and 43.9% (P < 0. 005), respectively.OH had no effect on Ca(2+) sensitivity. Neither 10 nor 1,000 microM H(2)O(2) significantly altered F(max) or Ca(2+) sensitivity. We conclude that the diaphragm is susceptible to alterations induced by a direct effect of.OH and O(2)(-)., but not H(2)O(2), on the contractile proteins, which could, in part, be responsible for prolonged depression in contractility associated with respiratory muscle dysfunction in certain pathophysiological conditions.

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This study test the hypothesis that a temporal relationship exists between the production of superoxide anion (O2-) and the contractile activity of perfused rat diaphragm. O2- levels were determined minute to minute by measuring the reduction of cytochrome c in the perfusate as the diaphragms were subjected to various levels of contractile activity. After equilibrating at low contractile rates (one 500 ms 80 Hz train/min), diaphragms were fatigued by increasing their contractile activity for 5 min (one 500 ms 80 Hz train/s) and then allowed to recover for 30 min (one 500 ms 80 Hz train/min). During equilibration, diaphragms did not produce O2- above the background level measured in the presence of superoxide dismutase (SOD). Within the first minute of fatigue-inducing stimulation, however, the rate of O2- production increased to 0.70 +/- 0.17 nmol/min and remained elevated until the recovery period when production returned towards baseline. SOD blocked this stimulation-related increase of O2-. Tension (+/-SOD) fell to 12% of the control value during the fatigue-inducing stimulation. During recovery the contractile response returned to 51% of control, indicating long-lasting effects on the contractile machinery. SOD did not limit fatigue or improve recovery, probably because it is a large protein that cannot cross cell membranes and protect the cells by scavenging O2- at its site of production.

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This study was designed to develop traps for hypochlorous acid (HOCl) which could be used to detect HOCl in the microenvironment of activated neutrophils. Reagent HOCl was found to react with para-aminobenzoic acid (PABA) in aqueous solution to produce a predominant metabolite detectable by high performance liquid chromatography (HPLC). Mass spectroscopy and nuclear magnetic resonance identified this metabolite as the ring addition product 3-chloro PABA. The related compound para-aminosalicylic acid (PAS) was also metabolized by HOCl to 3-chloro PAS. The formation of the 3-chloro metabolite was specific for reactions involving HOCl, since several other oxidants in chloride buffer failed to produce the metabolite. Human blood neutrophils activated by phorbol myristate acetate or zymosan in the presence of PABA (or PAS) used their HOCl to produce large amounts of the 3-chloro metabolite. The formation of 3-chloro PABA was inhibited by azide, catalase, and taurine, which is consistent with the production of the metabolite by the neutrophil myeloperoxidase (MPO) pathway. The reaction of HOCl with PABA and PAS was relatively slow as shown by competitive reactions with endogenous antioxidants like taurine, methionine, and glutathione. This was confirmed in reactions involving PABA/PAS and reagent HOCl or HOCl generated by the MPO enzyme system. In these in vitro systems, glutathione and serum completely inhibited the formation of the 3-chloro metabolite. In contrast, activated neutrophils metabolized PABA/PAS to the 3-chloro metabolite even in the presence of 1% serum. These findings demonstrate that PABA and PAS are specific trapping agents for HOCl produced by neutrophils in complex biological conditions.

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ontivation of neutrophils by phorbol-12-myristate-13-acetate (PMA) causes rapid production of superoxide radical (O2-), leading to the formation of additional reactive oxygen species, including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and possibly hydroxyl radical (.OH). These reactive oxygen species have been associated with the oxidation of some drugs. We investigated the metabolism of phenytoin (5,5-diphenylhydantoin) and the covalent binding of reactive intermediates to cellular macromolecules in activated neutrophils. In incubations with 100 microM phenytoin, PMA-stimulated neutrophils from six human subjects produced p-, m-, and o-isomers of 5-(hydroxyphenyl)-5-phenylhydantoin (HPPH) in a ratio of 1.0:2.1:2.8, respectively, as well as unidentified polar products. Analysis of cell pellets demonstrated that phenytoin was bioactivated to reactive intermediates that bound irreversibly to macromolecules in neutrophils. Glutathione, catalase, superoxide dismutase, azide, and indomethacin all diminished the metabolism of phenytoin and the covalent binding of its reactive intermediates. The iron-inactivating chelators desferrioxamine and diethylenetriaminepentaacetic acid had little or no effect on the metabolism of phenytoin by neutrophils, demonstrating that adventitious iron was not contributing via Fenton chemistry. In an .OH-generating system containing H2O2 and Fe2+ chelated with ADP, phenytoin was oxidized rapidly to unidentified polar products and to p-, m-, and o-HPPH (ratio 1.0:1.7:1.5, respectively). Reagent HOCl and human myeloperoxidase (MPO), in the presence of Cl- and H2O2, both formed the reactive dichlorophenytoin but no HPPH. However, no chlorinated phenytoin was detected in activated neutrophils, possibly because of its high reactivity. These findings, which demonstrated that activated neutrophils biotransform phenytoin in vitro to hydroxylated products and reactive intermediates that bind irreversibly to tissue macromolecules, are consistent with phenytoin hydroxylation by .OH generated by a transition metal-independent process, chlorination by HOCl generated by MPO, and possibly cooxidation by neutrophil hydroperoxidases. Neutrophils activated in vivo may similarly convert phenytoin to reactive intermediates, which could contribute to some of the previously unexplained adverse effects of the drug.

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Neutrophils adhered to biologic surfaces exhibit proteolytic cleavage of surface proteins even in the presence of proteinase inhibitors. Such proteolysis is restricted to the pericellular space and appears to require the dual action of proteinases and reactive oxygen species. The present study was designed to investigate the mechanism by which tumor necrosis factor-alpha (TNF) stimulates neutrophil proteolysis. Tissue culture wells were coated with insoluble 3H-labeled elastin substrate. Human blood neutrophils (0.5 to 2.0 x 10(6) cells/ml/well) were incubated in the coated wells for 4 to 18 h at 37 degrees C in the presence of varying concentrations of serum or purified alpha 1-antitrypsin (A1AT). TNF (0 to 1,000 U/ml) was also present in the incubations. Elastin degradation was determined as soluble 3H-elastin fragments released into the supernatants. As previously reported, cells (no TNF) exhibited spontaneous elastolysis even in the presence of 1% serum or 4 microM AlAT. Compared with cells incubated alone (no TNF), TNF increased elastolysis 3-fold in the 4-h incubations and 83% in 18-h incubations. TNF also significantly increased proteolysis when neutrophils were concurrently treated with phorbol myristate acetate or N-formylmethionylleucylphenylalanine. Since TNF is known to prime neutrophils for hypochlorous acid (HOCl) release, the present study hypothesized that the enhancement of proteolysis by TNF was related to increased release of HOCl. First, TNF caused a 4-fold increase in HOCl release by neutrophils adhered to elastin surfaces. Second, the effect of methionine on elastolysis by adherent neutrophils was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

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There is increasing evidence that oxygen-derived free radicals produced during strenuous work by the diaphragm may contribute to diaphragm fatigue and/or injury. However, the precise identity of these oxygen radicals remains unknown, inasmuch as oxygen free radicals are extremely short lived and their detection in biologic systems is quite difficult. There is recent evidence that the salicylate-trapping method may be a useful means of monitoring tissue production of hydroxyl radical (.OH). This method is predicated on the fact that salicylate's phenolic ring can be attacked by .OH at the 3 or 5 position to yield 2,3- or 2,5-dihydroxybenzoic acid (DHB). These metabolites are stable and can be identified by high-performance liquid chromatography (HPLC) coupled with electrochemical or ultraviolet detection. To test the hypothesis that hydroxylated salicylates are produced during diaphragm fatigue, we exposed in vitro rat diaphragm strips to a physiological saline solution containing 2.0 mM sodium salicylate for approximately 15 min. The solution was then removed, and the strips were fatigued (20 Hz, 200-ms train duration, 1 train/s) via phrenic nerve stimulation for 30 s-10 min. The diaphragm strips were subsequently homogenized, and the homogenate was analyzed by HPLC coupled with ultraviolet detection. Levels of 2,3-DHB were significantly higher in fatigued than in control nonfatigued strips. There was also a significant correlation between the amount of 2,3-DHB in the fatigued muscle and the accumulated tension-time product developed during fatigue. 2,5-DHB was not consistently identified in control or experimental strips.(ABSTRACT TRUNCATED AT 250 WORDS)

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Cross-linking receptors for the Fc region of immunoglobulin G (IgG) (Fc gamma R) induces tumor necrosis factor-alpha (TNF-alpha) release; however, there is controversy about release of interleukin (IL)-1 beta. The purpose of this study was to investigate the role of endotoxin priming on the ability of monocytes to release these cytokines after Fc gamma R cross-linking. Monocytes were incubated with plated or soluble human IgG or albumin with or without endotoxin priming. Monocytes isolated by Percoll, containing low concentrations of endotoxin, and incubated on plated IgG released 4.5 +/- 1.6 ng/ml TNF-alpha and 1.6 +/- 0.6 ng/ml IL-1 beta. Monocytes isolated by a "clumping" technique released 1.0 +/- 0.4 ng/ml TNF-alpha but no IL-1 beta. Priming with endotoxin, which did not affect Fc gamma R expression, resulted in augmented release of TNF-alpha (4.3 +/- 1.3 vs. 0.1 +/- 0.0 ng/ml, P < 0.05) and IL-1 beta (4.0 +/- 1.0 vs. 0.6 +/- 0.3 ng/ml, P < 0.01) when clumped monocytes were incubated on plated IgG vs. plated albumin.

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Alveolar epithelial lining fluid (ELF) contains several antioxidant substances that may provide in vivo protection. We studied the ability of ELF and ELF components to inhibit the neutrophil oxidant hypochlorous acid (HOCI). Normal bronchoalveolar lavage fluid containing ELF was incubated with physiologically relevant concentrations of HOCI (0.04 mM). After incubation, residual HOCI activity was titered by the iodide method. The inhibitory activity of lavage fluid was unexpectedly strong. For example, lavage fluid diluted 20-fold in the assay system quenched 49% of starting HOCI. We initially postulated that ELF total protein and glutathione would account for most of the inhibition of HOCI. However, several experimental approaches demonstrated that the total protein and glutathione concentrations in diluted lavage fluid were too low to explain the observed inhibition. Instead, the majority of HOCI inhibition was due to the lidocaine used for upper airway anesthesia. Reagent lidocaine exhibited strong reactivity in the HOCI assay system. Furthermore, the lavage fluid lidocaine concentration (32.4 +/- 6.9 micrograms/ml) was sufficient to explain most of the observed quenching activity. Additional experiments explored the hypothetical quenching activity of ELF components devoid of lidocaine. These findings demonstrate the technical problems posed by lidocaine in antioxidant studies involving lavage fluid or ELF.

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Five cancer patients undergoing intravenous infusions of human recombinant tumor necrosis factor (TNF)alpha were evaluated for the effects these infusions had on the priming of circulating neutrophils for hypochlorous acid (HOCl) production. These patients were also studied for changes in temperature, circulating white blood cell counts, blood pressure, and spontaneous monocyte interleukin 1 beta (IL-1 beta) and TNF production. As predicted by previous in vitro studies, patient neutrophils increased their HOCl production to unopsonized zymosan from a baseline of 29.2 +/- 5.9 nmol I- oxidized/4 x 10(6) cells to a peak of 64.2 +/- 9.8 nmol I- oxidized/4 x 10(6) cells at 4 h after TNF infusion (P less than 0.01). Similar increases were also seen at 4 h with phorbol myristic acetate and opsonized zymosan as the stimuli. The priming effect could be reproduced in neutrophils from a normal individual by incubating them with the 30-min serum samples from the infused patients. The ability of this serum to prime neutrophils was completely blocked by a monoclonal anti-TNF alpha-antibody but not by an anti-IL-1 beta antibody. In addition to the priming of their neutrophils, patients also experienced fever, marked hypotension, and an initial fall, followed by rebound to an elevation, in circulating white blood cell counts. The TNF infusions did not produce detectable circulating IL-1 beta nor did they induce significant production of TNF or IL-1 beta by circulating blood monocytes. These studies confirm the role of TNF in producing the signs of sepsis such as hypotension, fever, and leukopenia followed by leukocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

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Tumor necrosis factor (TNF) has a weak direct effect on neutrophil oxidative metabolism and primes neutrophils for oxidant release in response to other stimuli. We examined the effect of recombinant human TNF alpha (rTNF alpha) on production of hypochlorous acid (HOCl) by human neutrophils. TNF alone, even at concentrations of 1,000 U/ml, did not stimulate HOCl production. In contrast, rTNF alpha, in a dose-dependent manner, primed neutrophils for HOCl production in response to the weak agent unopsonized zymosan. rTNF alpha concentrations as low as 10 U/ml resulted in a fivefold increase in HOCl in this system. rTNF alpha-primed cells also exhibited increased phagocytosis. Priming in this model system occurred regardless of whether cells were preincubated with rTNF alpha before addition of zymosan or coincubated with both rTNF alpha and zymosan. rTNF alpha priming for HOCl production could not be washed away and required a lag period of approximately 10 min. rTNF alpha priming was not dependent on extracellular Ca2+ and Mg2+. Preincubation experiments demonstrated that rTNF alpha priming was not inhibited by the microfilament blocker cytochalasin B. Although the mechanism remains unclear, these findings demonstrate that rTNF alpha has an important priming effect on the neutrophil myeloperoxidase pathway.

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Salicylates are metabolized in vivo to hydroxylated compounds, including 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid). The present study hypothesized that activated neutrophils represent one pathway for salicylate hydroxylation. Human neutrophils were incubated in medium containing 10 mM salicylate and stimulated with phorbol myristate acetate (PMA) for 1 hr. The cell-free supernatant fractions were analyzed by HPLC. Neutrophils (1 x 10(6) cells) produced 55 +/- 11 ng of gentisic acid. Neutrophils also produced smaller quantities of 2,3-dihydroxybenzoic acid. Antioxidant inhibitor experiments indicated that superoxide dismutase (SOD), heme protein inhibitors, and glutathione blocked gentisic acid formation, whereas catalase, mannitol, and deferoxamine failed to inhibit. Experiments with the reagent hypochlorous acid (HOCl) and the model myeloperoxidase (MPO) enzyme system did not support a role for the MPO pathway in gentisic acid formation. These findings demonstrate that activated neutrophils can hydroxylate salicylate by an unknown pathway. This pathway may contribute to the increased recovery of hydroxylated salicylates in patients with inflammatory disorders.

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