Superoxide scavengers augment contractile but not energetic responses to hypoxia in rat diaphragm.

Acute exposure to severe hypoxia depresses contractile function and induces adaptations in skeletal muscle that are only partially understood. Previous studies have demonstrated that antioxidants (AOXs) given during hypoxia partially protect contractile function, but this has not been a universal finding. This study confirms that specific AOXs, known to act primarily as superoxide scavengers, protect contractile function in severe hypoxia. Furthermore, the hypothesis is tested that the mechanism of protection involves preservation of high-energy phosphates (ATP, creatine phosphate) and reductions of P(i). Rat diaphragm muscle strips were treated with AOXs and subjected to 30 min of hypoxia. Contractile function was examined by using twitch and tetanic stimulations and the degree of elevation in passive force occurring during hypoxia (contracture). High-energy phosphates were measured at the end of 30-min hypoxia exposure. Treatment with the superoxide scavengers 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron, 10 mM) or Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (50 microM) suppressed contracture during hypoxia and protected maximum tetanic force. N-acetylcysteine (10 or 18 mM) had no influence on tetanic force production. Contracture during hypoxia without AOXs was also shown to be dependent on the extracellular Ca(2+) concentration. Although hypoxia resulted in only small reductions in ATP concentration, creatine phosphate concentration was decreased to approximately 10% of control. There were no consistent influences of the AOX treatments on high-energy phosphates during hypoxia. The results demonstrate that superoxide scavengers can protect contractile function and reduce contracture in hypoxia through a mechanism that does not involve preservation of high-energy phosphates.

Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle.

Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degrees C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degrees C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cytochrome c. g dry wt(-1). ml(-1) were reduced in heat compared with 22 +/- 13 nmol. g(-1). ml(-1) in controls (P < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.

Antioxidants protect rat diaphragmatic muscle function under hypoxic conditions.

In hypoxia, mitochondrial respiration is decreased, thereby leading to a buildup of reducing equivalents that cannot be transferred to O2 at the cytochrome oxidase. This condition, called reductive stress, can paradoxically lead to enhanced formation of reactive O2 species, or a decrease in the ability of the cell to defend against an oxidative stress. We hypothesized that antioxidants would protect tissues under conditions of hypoxia. Rat diaphragm strips were incubated in tissue baths containing one of four antioxidants: N-acetyl-L-cysteine, dimethyl sulfoxide, superoxide dismutase, or Tiron. The strips were directly stimulated in an electrical field. Force-frequency relationships were studied under baseline oxygenation (95% O2-5% CO2), after 30 min of hypoxia (95% N2-5% CO2), and 30 min after reoxygenation. In all tissues, antioxidants markedly attenuated the loss of contractile function during hypoxia (P < 0.01) and also significantly improved recovery on reoxygenation (P < 0.05). We conclude that both intracellular and extracellular antioxidants improve skeletal muscle contractile function in hypoxia and facilitate recovery during reoxygenation in an in vitro system. The strong influence of antioxidants during hypoxic exposure suggests that they can be as effective in protecting cell function in a reducing environment as they have been in oxidizing environments.

Biological reactions of peroxynitrite: evidence for an alternative pathway of salicylate hydroxylation.

Salicylate hydroxylation has often been used as an assay of hydroxyl radical production in vivo. We have examined here if hydroxylation of salicylate might also occur by its reaction with peroxynitrite. To test this hypothesis, we exposed salicylate to various concentrations of peroxynitrite, in vitro. We observed the hydroxylation of salicylate at 37 degrees C by peroxynitrite at pH 6, 7 and 7.5, where the primary products had similar retention times on HPLC to 2,3- and 2,5-dihydroxybenzoic acid. The product yields were pH dependent with maximal amounts formed at pH 6. Furthermore, the relative concentration of 2,3- to 2,5-dihydroxybenzoic acid increased with decreasing pH. Nitration of salicylate was also observed and both nitration and hydroxylation reaction products were confirmed independently by mass spectrometry. The spin trap N-t-butyl-alpha-phenylnitrone (PBN), with or without dimethyl sulfoxide (DMSO), was incapable of trapping the peroxynitrite decomposition intermediates. Moreover, free radical adducts of the type PBN/.CH3 and PBN/.OH were susceptible to destruction by peroxynitrite (pH 7, 0.1 M phosphate buffer). These results suggest direct peroxynitrite hydroxylation of salicylate and that the presence of hydroxyl radicals is not a prerequisite for hydroxylation reactions.

Detection of free radicals by electron spin resonance in rat diaphragm after resistive loading.

Indirect evidence supports free radical production in the diaphragm under excessive mechanical loads in both in vitro and in situ preparations. We hypothesized that free radicals are produced in the diaphragm with loads in vivo at a sufficient concentration to be detected by electron spin resonance (ESR) spectroscopy. Anesthetized rats underwent severe inspiratory resistive loading for 2.5-3 h with maintenance of blood oxygenation and arterial blood pressure by breathing 70% oxygen. The ESR spectra of four samples (freeze-clamped at liquid nitrogen temperature) from each experimental animal were compared with the spectra from a control animal breathing air and a control animal breathing 70% oxygen. We observed 1) an approximately 30% increase in intensity of free radical signal in experimental animals (n = 10) compared with control animals breathing oxygen (n = 10; P < 0.01) and control animals breathing air (n = 10; P < 0.05), 2) that oxygen alone had no effect on the ESR spectrum, and 3) the intensity of the ESR signal decreased approximately 25% in the experimental group when samples were taken 10 min postmortem, whereas no difference in signal was observed for control animals. We conclude that the diaphragm shows an increased production of free radicals associated with respiratory failure induced by resistive breathing.

Loss of diaphragm glutathione is associated with respiratory failure induced by resistive breathing.

It has been suggested that oxidant stress may contribute to dysfunction of respiratory muscles undergoing severe work loads. We examined changes in glutathione content and redox status in the diaphragm and intercostal muscles of anesthetized Sprague-Dawley rats exposed to prolonged inspiratory resistive loading while breathing 70% O2. These results were compared with those from control groups breathing air or 70% O2. Changes in liver glutathione were also examined. Freeze-clamping and an enzymatic recycling assay were used. Results show that 1) in controls, glutathione content was higher in the diaphragm than in the intercostals, 2) severe hypercapnic acidosis without hypoxemia was present with loading, 3) total diaphragm glutathione decreased approximately 35% with no increase in glutathione oxidation with resistive breathing, whereas intercostal and liver glutathione remained unchanged, and 4) the drop in diaphragm glutathione correlated significantly with the drop in minute ventilation and the increase in arterial PCO2, whereas it was not directly related to intensity of respiratory muscle activity. In conclusion, although diaphragm susceptibility to oxidant stress may be increased with resistive breathing, it is unlikely that the modest decrease in total glutathione contributed significantly to respiratory failure in this model.

Deficiency of alveolar fluid glutathione in patients with sepsis and the adult respiratory distress syndrome.

The adult respiratory distress syndrome (ARDS) is a devastating clinical illness characterized by refractory hypoxemia and high-permeability pulmonary edema. Reactive oxygen species such as hydrogen peroxide and hypochlorous acid may play a key role in the pathogenesis of the acute lung injury. Glutathione (GSH) is a tripeptide that is able to react with and effectively neutralize oxidants such as hydrogen peroxide and hypochlorous acid. The present study found that the alveolar epithelial lining fluid of patients with ARDS was deficient in total GSH compared to normal subjects (21.7 mumols +/- 7.8 mumols vs 91.8 mumols +/- 14.5 mumols; p = 0.002). In addition, if GSH was measured in unconcentrated bronchoalveolar lavage (BAL) fluid and indexed to total BAL protein, there was also a deficiency in patients with ARDS compared to normal subjects (0.004 +/- 0.003 nmol of GSH per microgram of total protein vs 0.026 +/- 0.005 nmol of GSH per microgram of total protein; p = 0.002). Since patients with ARDS are subjected to an increased burden of oxidants in the alveolar fluid, principally released by recruited neutrophils, this deficiency of GSH may predispose these patients to enhanced lung cell injury.

Depressed aerobic enzyme activity of skeletal muscle in severe chronic heart failure.

A reduction in exercise capacity is a common feature of congestive heart failure. We hypothesized that depressed aerobic enzyme activity of skeletal muscle may contribute to this exercise intolerance. Biopsy samples of vastus lateralis muscle were obtained from seven patients with severe chronic heart failure and analyzed for aerobic enzyme activity. Compared with normal laboratory controls, the patients with heart failure had a moderate reduction (greater than 60%) in skeletal muscle citrate synthase and a marked reduction (greater than 90%) in succinate dehydrogenase and cytochrome oxidase (all p less than 0.001). Depression of aerobic enzyme activity of skeletal muscle is associated with severe chronic heart failure and is likely one of the contributory factors for impaired exercise capacity seen in the advanced stages of this condition.

Early changes in the permeability of the blood-brain barrier produced by toxins associated with liver failure.

Our study was designed to determine whether substances that appear in the serum during the course of liver failure have a detrimental effect on the passive permeability of the blood-brain [blood-cerebrospinal fluid (CSF)] barrier. Lactic acid, octanoic acid, and ammonia were infused into rabbits for 4 h. The permeability changes of the blood-brain barrier were quantified by infusing polyethylene glycol 400 (PEG 400) and measuring the quantity and average mol wt of the PEG 400 that entered the CSF. The lipid solubility and effective diffusional radius of the PEG molecules were also quantified to provide greater precision for measurements using this probe. None of the animals receiving toxic infusions became seriously ill during the infusions. Low dose infusions of lactic acid, octanoic acid, and ammonia increased the effective pore diameter of the blood-brain barrier from 7.3 A to an average of 8.5 A. The amount of PEG entering the CSF increased from 1.7 to 4.0 (p less than 0.025), 4.7 (p less than 0.025), and 6.7 (p less than 0.001) mmol/L, respectively. Rabbits with galactosamine-induced liver failure had 10.1 mmol/L PEG 400 in the CSF (P less than 0.001) before any evidence of cerebral edema. These changes occur soon after these toxins accumulate in the plasma and may alone or together with other toxins account for the permeability changes that allow neurotoxic substances to enter the brain during hepatic disease and encephalopathies such as Reye's syndrome.

Cellular glutathione and the response of adult rat heart myocytes to oxidant stress.

Freshly isolated adult rat heart myocytes contain total glutathione and reduced glutathione (GSH) at levels quite comparable to those in intact rat heart. Total glutathione can be depleted from 11 to 1 nmol/mg protein or less by treatment with cyclohex-2-ene-1-one without effect on either cellular ATP, rod-cell morphology or the integrity of the sarcolemma. Glutathione levels and redox state are not altered significantly when the Ca-tolerant, quiescent cells are subjected to a period of anoxia followed by reoxygenation. This oxygen paradox protocol results in irreversible hypercontracture of the contractile elements into an amorphous mass in the bulk of the cells, but little loss of sarcolemmal integrity. When the myocytes are subjected to an externally applied oxidant stress by the addition of either diamide or t-butylhydroperoxide, GSH is rapidly depleted with accumulation of oxidized glutathione (GSSG. On continued aerobic incubation both of these reagents promote a slower depletion of cellular ATP and a parallel hypercontracture. Cells treated with t-butylhydroperoxide, but not those with diamide, also generate increasing amounts of thiobarbituric acid reactive species as an indication of lipid peroxidation and show a parallel loss of sarcolemmal integrity. It is concluded that respiring myocytes and those subjected to the oxygen paradox do not produce oxygen radicals in sufficient amounts to displace the GSH/GSSG redox poise and depletion of myocyte glutathione per se is not detrimental to the short term survival of the cells. In addition, aerobic myocytes subjected to external oxidant stress can be damaged irreversibly by two pathways, a hypercontracture that correlates with depletion of ATP and a loss of sarcolemmal integrity that correlates with lipid peroxidation.

Peroxide mediated effects of homocysteine on arterial prostacyclin synthesis.

The effects of homocysteine on the synthesis of arterial prostacyclin (PGI2) were investigated. Homocysteine at 10 mM and 1 mM concentration inhibited PGI2 synthesis from both exogenous and endogenous arachidonic acid. While concentrations of 100 microM and 1 microM stimulated PGI2 synthesis. Similar inhibitory effects of H2O2 on PGI2 synthesis were observed. High concentrations (100 microM and 500 microM) of H2O2 inhibited PGI2 production while low concentrations (1 microM) of H2O2 stimulated it. Catalase overcame the inhibitory effect of H2O2 (100 microM). Homocysteine induced O2 uptake and catalase inhibited the O2 uptake by homocysteine. Thus the inhibitory and stimulatory effect of homocysteine may be dependent on the oxidation of homocysteine and subsequent H2O2 generation.

Prevention of bedrest-induced physical deconditioning by daily dobutamine infusions. Implications for drug-induced physical conditioning.

The effects of intermittent infusions of dobutamine were studied in young normal male subjects during a period of bedrest deconditioning to determine whether this synthetic catechol affects physical conditioning processes in humans. 24 volunteers were placed at bedrest and randomized to daily 2-h treatments of saline infusions (control), dobutamine infusions, or maintenance exercise (control). Exercise, hemodynamic, and metabolic studies were performed at base line and at the termination of the 3-wk treatment period. Maximal exercise (duration, oxygen consumption, and workload) fell for the saline group and remained unchanged for the dobutamine and exercise groups. Hemodynamics during exercise were maintained the same as pretreatment base line for the dobutamine and exercise groups, whereas stroke volume and cardiac output dropped and heart rate rose for the saline group. The metabolic profile showed an increased blood lactate response at rest and during submaximal exercise after 3 wk of bedrest for the saline group, and essentially no change for the exercise and the dobutamine groups. Extraction of oxygen across the exercising lower limb rose for the dobutamine group, as did the activity of the skeletal muscle oxidative enzymes, citrate synthetase, and succinate dehydrogenase. In contrast to the exercise control group, the saline and dobutamine groups developed orthostatic hypotension, tachycardia, and accentuation of the renin-aldosterone response over the 3-wk treatment period; for the saline group, this is best explained by the observed fall in blood volume and for the dobutamine group, by the blunting of vascular vasoconstrictive responses. During a period of bedrest deconditioning in humans, infusions of dobutamine maintain many of the physiologic expressions of physical conditioning.

Effects of mannitol on the postischemic kidney. Biochemical, functional, and morphologic assessments.

UNLABELLED: To determine the effects of mannitol on the postischemic kidney rats were subjected to 25 minutes of renal artery occlusion and immediately after vascular clamp release they received a 2-ml intravenous mannitol bolus (20%). Equimolar urea-injected rats and sham-injected rats served as controls. Postischemic renal blood flow, tubular metabolic work (renal O2 consumption), adenine nucleotide pools, renal oxidant stress (tissue glutathione, malondialdehyde levels), and tubular cell/mitochondrial swelling (histomorphometry) were assessed at variable times during the early vascular reflow period (15 to 60 minutes). The severity of acute renal failure was determined by serial blood urea nitrogen and serum creatinine studies (24, 48 hours), and by renal histology (48 hours). Mannitol increased postischemic renal blood flow (2-fold), renal O2 consumptions (3-fold), and urine flow compared to urea-injected and sham-injected controls. Postischemic glutathione levels were equally depressed (reduced 33%) in all three treatment groups. Malondialdehyde did not rise. Mannitol significantly lowered total adenine nucleotide content without changing ATP at 15 minutes post renal artery occlusion. At 60 minutes post renal artery occlusion, mannitol- and urea-treated groups had comparable ATP levels, 25% higher than the noninjected controls. Mannitol and urea induced comparable decrements in proximal tubular cell swelling, returning cell volumes to normal values. However, mitochondrial swelling was unabated. Mannitol and urea caused significant and nearly identical degrees of functional and morphologic amelioration of renal injury. CONCLUSIONS: Mannitol administered after renal ischemia ameliorates both functional and morphologic aspects of acute tubular injury despite dramatically increasing tubular aerobic work. This protection appears not to be due to early postischemic improvements in adenine nucleotide content, to increased renal blood flow, to increased urine flow, or to a lessening of oxidant stress. The data are consistent with the view that protection results from acute hypertonic solute loading which either directly or indirectly decreases tubular cell but not mitochondrial swelling.

Effect of exercise training on antioxidant enzymes and cardiotoxicity of doxorubicin.

The purpose of this study was to correlate the exercise-induced changes of oxidant stress enzymes with possible modification of the response to the putative oxidant stressor doxorubicin. Enzymatic and histological changes were studied in mice placed on a 21-wk swim training program (1 h/day, 5 days/wk) with and without anthracycline administration. Doxorubicin (4 mg/kg) was administered intravenously through a tail vein on 10 separate days over a 7-wk period (twice weekly during weeks 10, 11, 14, 15, and 16). Blood, liver, and heart levels of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GP) were measured following the 9th and 21st wk. Myocardial histomorphological observations were made by light microscopy after 21 wk. Following 9 wk of training swim-trained animals had significantly elevated levels of CAT, SOD, and GP in blood, as well as elevated GP in liver. After 21 wk, trained animals, regardless of drug status, had elevated blood CAT and SOD activity and increased liver CAT and GP. Training also produced increases in blood GP, liver SOD, and heart CAT; however, in conjunction with doxorubicin these changes were not seen. The degree of cardiotoxicity was significantly greater in the sedentary drug-treated animals than in the swim-trained drug-treated animals. The results suggest a correlation between antioxidant enzyme levels in blood and liver and the degree of damage caused by an anthracycline drug. It was concluded that exercise ameliorates severe toxic damage caused by doxorubicin administration, possibly by increasing enzymes that combat free radical damage.

Effects of immediate blood flow enhancement on the postischemic kidney: functional, morphologic, and biochemical assessments.

Our purpose was to assess the influence of blood flow enhancement to the immediate postischemic kidney on tubular cell energetics and on the severity of the resulting ischemic acute renal failure (IARF). Female Sprague-Dawley rats were subjected to 40 minutes of bilateral renal artery occlusion (RAO). Half of the rats received a 5% body weight infusion of iso-oncotic saline solution (IOS; over 50 minutes) to acutely increase renal blood flow (RBF) immediately after vascular clamp release. The remaining half of the rats served as controls. The short-term effects (0 to 1 hour after vascular reflow) of IOS infusion on RBF, clearance iothalamate sodium (Cioth), urine Na excretion (UNaE), urine flow rate, tubular metabolic work (renal oxygen consumption, Qo2), adenine nucleotide concentrations, and renal histologic findings were assessed. The severity of the IARF (Cioth histologic findings) was also compared between the IOS-treated and the control groups 24 hours later. Postischemic (0 to 1 hour) RBF in control IARF rats and IOS-treated IARF rats was 2.3 +/- 0.3 and 13.6 +/- 0.4 ml/min, respectively (P less than 0.01) (normal RBF 6.1 +/- 0.4 ml/min). At 0 to 1 hour after reflow IOS-treated IARF rats had significantly higher Cioth (13 X), UNaE (18 X), urine flow (18 X), and Qo2 (4 X) than the control IARF group. Despite the fourfold increase in aerobic tubular work induced by IOS infusion, renal adenosine triphosphate (ATP) content did not decrease. At 24 hours after vascular reflow the severity of IARF was the same in the control and IOS-treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of citrulline synthesis by octanoate and its modulation by adenine nucleotides.

Liver mitochondria from octanoate-treated rabbits showed an impaired ability to synthesize citrulline. Two methods were used to evaluate citrulline synthesis in rat liver mitochondria. Under these conditions octanoate inhibited citrulline synthesis by over 50%. When ATP was included in the assay medium the inhibitory effect of octanoate was prevented. In the absence of ATP in the suspending medium, octanoate did not significantly lower total adenine nucleotides in rat liver mitochondria. However, under these conditions octanoate caused a change in the adenine nucleotide profile such that ATP content was decreased and AMP content was increased. When ATP was present in the assay medium, octanoate caused a similar increase in AMP content. However, ATP decreased only slightly. The alterations in mitochondrial adenine nucleotide profile by octanoate and the reversal of the effect by exogenous ATP suggests that octanoate inhibits citrulline synthesis via reduced intramitochondrial ATP levels. The ability of octanoate to lower mitochondrial ATP and elevate mitochondrial AMP may be related to its intramitochondrial activation by the medium chain fatty acid activating enzyme.

Responses of the normal rat kidney to sequential ischemic events.

UNLABELLED: This study was undertaken to help define how one episode of renal ischemia, insufficient to cause acute renal failure, influences the susceptibility of the kidney to a second more severe ischemic event. Female Sprague-Dawley rats underwent either 15 min of bilateral renal artery occlusion (RAO) or sham RAO. They were subjected 30 min, 3.5 h, or 24 h later to 25 min of RAO. Renal function (GFR, BUN, creatinine), histology, and adenine nucleotide concentrations were compared before and after the 25-min ischemic event. Only the rats with a 30-min hiatus between the 15- and 25-min bouts of RAO had significantly worse renal failure than controls subjected to a single 25-min ischemic event. Three findings were noted only in the rats with increased susceptibility: tubular cell swelling and luminal membrane injury prior to 25 min of RAO and a relative failure of ATP formation immediately following 25 min of RAO. Susceptibility to 25 min of RAO did not correlate with preischemia ATP content. CONCLUSION: prior mild ischemic injury transiently lowers renal resistance to a second ischemic event. Normal resistance is rapidly restored once improvements in prior cell membrane injury, cell volume regulation, and cellular energetics occur. However, resistance to additional ischemia can be normal despite persisting depressions in renal ATP content.

Peroxidase-mediated oxidation of isoniazid.

Oxidation of isonicotinic acid hydrazide (isoniazid) by horseradish peroxidase at the expense of H2O2 yielded reactive species which were able to reduce nitroblue tetrazolium and bleach p-nitrosodimethylaniline. Nicotinic acid hydrazide oxidation did not cause these effects. At slightly alkaline pH, oxidation of isonicotinic acid hydrazide by horseradish peroxidase proceeded at the expense of molecular O2, and the reaction was oxygen consuming. The addition of H2O2 abolished O2 consumption. Bovine liver catalase enhanced the rate of nitroblue tetrazolium reduction and decreased the maximal velocity of the reaction proportionately to catalase concentration. During oxidation of isonicotinic acid hydrazide by horseradish peroxidase-H2O2, splitting of the heme group of horseradish peroxidase took place as shown by the disappearance of the Soret and minor bands in the visible region of the spectrum.