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.

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.

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.

Independent preclerkship study: a five year experience.

An independent study program (ISP) was instituted in 1970 for a group of 32 preclerkship medical students picked randomly from a group of 64 volunteers. The academic achievements of these classes, as measured by mean National Board scores, is compared with their classmates in the lecture discussion (LD) program where possible. The significant differences noted obtain even when the ISP students are compared only with LD students matched for premedical point-hour ratios and Medical College Admissions Test scores. These data attest to the overall success of the ISP program. The differences appear to us to be due mainly to factors related to motivation and maturity. The ease of preparations, convenience, and economy of the associated computer-assisted instruction make these methods attractive for use in residency training and continuing medical education where maturity and motivation may well be presumed.

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.

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.

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.

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.

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.

Enzyme adaptations in rat skeletal muscle after two intensities of treadmill training.

The effects of high intensity, short duration chronic exercise (HSD, N = 9) and low intensity, long duration chronic exercise (LLD, N = 9) on the selected enzyme activities of three muscles of rats were studied. After 12 wk of treadmill training, the LLD group showed 15-23% decreases in lactate dehydrogenase activity in all muscles (P less than 0.05), while succinic oxidase activity increased in the soleus (49%) and the vastus lateralis profundus (42%) (P less than 0.05). No change in creatine kinase activity was found in either group. The HSD group showed no change in lactate dehydrogenase activity; however, phosphofructokinase activity increased (greater than 50%) in both of the fast-twitch muscles (P less than 0.01), while only the vastus lateralis superficialis showed increased succinic oxidase activity (30-40%) (P less than 0.05). Following training no difference was observed in mean heart weights; however, the mean body weight of the sedentary control group (SC, N = 9) was greater than both exercise groups (P less than 0.05). No difference was observed between the exercise groups. The mean heart weight/body weight ratios were significantly different between all groups (P less than 0.01). These results indicated that the HSD group selectively increased its glycolytic power, while the LLD group increased only its oxidative ability. Furthermore, both exercise groups exhibited less gains in body weight than the non-exercise group. This change was independent of any work performed.