Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress

The effect of the arbuscular mycorrhizal (AM) fungus, Glomus versiforme, on growth and reactive oxygen metabolism of trifoliate orange (Poncirus trifoliata) seedlings was studied in potted plants under well-watered (WW) and water stressed (WS) conditions. Water stress significantly decreased root colonization. Shoot dry weight, plant height and stem diameter were higher in AM than in non-AM seedlings regardless of the water status. Inoculation with G. versiforme increased root dry weight and leaf number per plant of WW seedlings. There was less malondialdehyde (MDA) concentration in leaves and roots of AM seedlings, as well as lower hydrogen peroxide (H(2)O(2)) and superoxide anion radical (O(2)(-)) concentrations in AM roots under WW and WS conditions. AM inoculation did not affect the H(2)O(2) and O(2)(-) concentrations of WW leaves. Whether WS or not, AM symbiosis notably increased the guaiacol peroxidase (G-POD) activity of leaves, glutathione reductase (GR) activity of leaves and ascorbate peroxidase (APX) activity of roots. AM infection also markedly increased the APX activity of WS leaves. Soluble proteins and glutathione (GSH) in leaves and roots and ascorbate (ASC) in leaves were higher in WW AM than in WW non-AM seedlings. AM infection also enhanced the ASC and GSH contents of leaves and roots in WS seedlings. Cross-tolerance might occur in AM plants and be enhanced by AM symbiosis. Our results suggest that the increased concentrations of antioxidant enzymes and non-enzymatic antioxidants found in AM plants may serve to protect the organism against oxidative damage, enhancing drought tolerance.     

Extracellular glutathione inhibits oxygen-induced permeability changes in alveolar epithelial monolayers.

Exposure to high fractional inspired oxygen for 24 h increases permeability of the alveolar epithelium, contributing to the clinical manifestations of oxygen toxicity. Utilizing a model of the alveolar epithelium in which isolated rat type II cells form polarized monolayers on polycarbonate filters [transepithelial resistance (R(t)) > 1 k Omega x cm(2) by day 4], we evaluated the ability of reduced glutathione (GSH) to ameliorate these changes. On day 4, apical fluid was replaced with culture medium containing 1) no additives, 2) GSH (500 microM), or 3) GSH (500 microM) + glutathione reductase (0.5 U/ml) + nicotinamide adenine dinucleotide phosphate (250 microM). Monolayers were exposed (for 24 h) to room air (control) or 95% O(2), each containing 5% CO(2). After 24 h of hyperoxia, R(t) for condition 1 decreased by 45% compared with control (P < 0.001). In conditions 2 and 3, R(t) did not decrease significantly (P = not significant). Hyperoxia-induced decreases in active ion transport were observed for conditions 1 and 2 (P < 0.05), but not for condition 3 (P = not significant). These findings indicate that extracellular GSH may protect the alveolar epithelium against hyperoxia-induced injury. Addition of glutathione reductase and nicotinamide adenine dinucleotide phosphate may further augment these protective effects of GSH.     

Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains approximately 10% by weight of soluble transition metals, was added (0-200 microg/ml) to BALF or sELF and exposed to 20% (18)O(2) (24 degrees C, 4 h). Oxygen incorporation was quantified as excess (18)O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.     

Morning attenuation in cerebrovascular CO2 reactivity in healthy humans is associated with a lowered cerebral oxygenation and an augmented ventilatory response to CO2.

We hypothesized that, in healthy subjects without pharmacological intervention, an overnight reduction in cerebrovascular CO(2) reactivity would be associated with an elevated hypercapnic ventilatory [ventilation (VE)] responsiveness and a reduction in cerebral oxygenation. In 20 healthy male individuals with no sleep-related disorders, continuous recordings of blood velocity in the middle cerebral artery, arterial blood pressure, VE, end-tidal gases, and frontal cortical oxygenation using near infrared spectroscopy were monitored during hypercapnia (inspired CO(2), 5%), hypoxia [arterial O(2) saturation (Sa(O(2))) approximately 84%], and during a 20-s breath hold to investigate the related responses to hypercapnia, hypoxia, and apnea, respectively. Measurements were conducted in the evening (6-8 PM) and in the early morning (6-8 AM). From evening to morning, the cerebrovascular reactivity to hypercapnia was reduced (5.3 +/- 0.6 vs. 4.6 +/- 1.1%/Torr; P < 0.05) and was associated with a reduced increase in cerebral oxygenation (r = 0.39; P < 0.05) and an elevated morning hypercapnic VE response (r = 0.54; P < 0.05). While there were no overnight changes in cerebrovascular reactivity or VE response to hypoxia, there was greater cerebral desaturation for a given Sa(O(2)) in the morning (AM, -0.45 +/- 0.14 vs. PM, -0.35 +/- 0.14%/Sa(O(2)); P < 0.05). Following the 20-s breath hold, in the morning, there was a smaller surge middle cerebral artery velocity and cerebral oxygenation (P < 0.05 vs. PM). These data indicate that normal diurnal changes in the cerebrovascular response to CO(2) influence the hypercapnic ventilatory response as well as the level of cerebral oxygenation during changes in arterial Pco(2); this may be a contributing factor for diurnal changes in breathing stability and the high incidence of stroke in the morning.     

Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages

Murine alveolar macrophages (AM) were shown to have proliferative ability and to form colonies in vitro. The factors in lung-conditioned medium (CM) and L929-CM which stimulate the proliferation of AM were considered to be granulocyte-macrophage colony-stimulating factor (GM-CSF) and CSF-1, respectively, because recombinant murine (rm)GM-CSF and recombinant human (rh)CSF-1 could replace the activities of lung-CM and L929-CM, respectively. The phenotype of the cells in the colonies formed by AM incubated with rmGM-CSF or lung-CM was AM-like; more than 90% of the cells were stained by anti-asialo GM1 but not by FITC-LPS, and had AM-like morphology. Expression of Mac-1 Ag determined by M1/70HL in these cells as well as original AM was low. However, the phenotype of the cells in the colonies formed by AM incubated with rhCSF-1 or L929-CM was peritoneal macrophage (PM)-like; more than 90% of the cells were stained by FITC-LPS and M1/70HL, but not by anti-asialo GM1, and showed PM-like morphology. The cells in the colonies formed by AM incubated with rmGMCSF changed their phenotype after treatment with rhCSF-1; the percentage of cells stained by anti-asialo GM1 decreased, and that of cells stained by FITC-LPS increased. The cells in the colonies formed by AM incubated with rhCSF-1 never changed their phenotype after incubation with rmGM-CSF. In contrast to AM, more than 90% of the cells in all colonies formed by PM incubated with either rmGM-CSF, rhCSF-1, lung-CM, or L929-CM were stained by FITC-LPS but not by anti-asialo GM1. These results show that although AM and PM can proliferate, AM, in contrast to PM, are bipotential cells that can differentiate into two types of macrophages responding to distinct types of CSF, and that one of the molecular mechanisms controlling macrophage heterogeneity may be based on the type of CSF produced at distinct tissues.     

Effects of antioxidants on contracting spinotrapezius muscle microvascular oxygenation and blood flow in aged rats

Aged rats exhibit a decreased muscle microvascular O(2) partial pressure (Pmv(O(2))) at rest and during contractions compared with young rats. Age-related reductions in nitric oxide bioavailability due, in part, to elevated reactive O(2) species, constrain muscle blood flow (Qm). Antioxidants may restore nitric oxide bioavailability, Qm, and ameliorate the reduced Pmv(O(2)). We tested the hypothesis that antioxidants would elevate Qm and, therefore, Pmv(O(2)) in aged rats. Spinotrapezius muscle Pmv(O(2)) and Qm were measured, and oxygen consumption (Vm(O(2))) was estimated in anesthetized male Fisher 344 x Brown Norway hybrid rats at rest and during 1-Hz contractions, before and after antioxidant intravenous infusion (76 mg/kg vitamin C and 52 mg/kg tempol). Before infusion, contractions evoked a biphasic Pmv(O(2)) that fell from 30.6 +/- 0.9 Torr to a nadir of 16.8 +/- 1.2 Torr with an "undershoot" of 2.8 +/- 0.7 Torr below the subsequent steady-state (19.7 +/- 1.2 Torr). The principal effect of antioxidants was to elevate baseline Pmv(O(2)) from 30.6 +/- 0.9 to 35.7 +/- 0.8 Torr (P < 0.05) and reduce or abolish the undershoot (P < 0.05). Antioxidants reduced Qm and Vm(O(2)) during contractions (P < 0.05), while decreasing force production 16.5% (P < 0.05) and elevating the force production-to-Vm(O(2)) ratio (0.92 +/- 0.03 to 1.06 +/- 0.6, P < 0.05). Thus antioxidants increased Pmv(O(2)) by altering the balance between muscle O(2) delivery and Vm(O(2)) at rest and during contractions. It is likely that this effect arose from antioxidants reducing myocyte redox below the level optimal for contractile performance and directly (decreased tension) or indirectly (altered balance of vasoactive mediators) influencing O(2) delivery and Vm(O(2)).     

Aging potentiates the effect of congestive heart failure on muscle microvascular oxygenation.

Congestive heart failure (CHF) is most prevalent in aged individuals and elicits a spectrum of cardiovascular and muscular perturbations that impairs the ability to deliver (Qo(2)) and utilize (Vo(2)) oxygen in skeletal muscle. Whether aging potentiates the CHF-induced alterations in the Qo(2)-to-Vo(2) relationship [which determines microvascular Po(2) (Pmv(O(2)))] in resting and contracting skeletal muscle is unclear. We tested the hypothesis that old rats with CHF would demonstrate a greater impairment of skeletal muscle Pmv(O(2)) than observed in young rats with CHF. Phosphorescence quenching was utilized to measure spinotrapezius Pmv(O(2)) at rest and across the rest-to-contractions (1-Hz, 4-6 V) transition in young (Y) and old (O) male Fischer 344 Brown-Norway rats with CHF induced by myocardial infarction (mean left ventricular end-diastolic pressure >20 mmHg for Y(CHF) and O(CHF)). In CHF muscle, aging significantly reduced resting Pmv(O(2)) (32.3 +/- 3.4 Torr for Y(CHF) and 21.3 +/- 3.3 Torr for O(CHF); P < 0.05) and in both Y(CHF) and O(CHF) compared with their aged-matched counterparts, CHF reduced the rate of the Pmv(O(2)) fall at the onset of contractions. Moreover, across the on-transient and in the subsequent steady state, Pmv(O(2)) values in O(CHF) vs. Y(CHF) were substantially lower (for steady-state, 20.4 +/- 1.7 Torr for Y(CHF) and 16.4 +/- 2.0 Torr for O(CHF); P < 0.05). At rest and during contractions in CHF, the pressure driving blood-muscle O(2) diffusion (Pmv(O(2))) is substantially decreased in old animals. This finding suggests that muscle dysfunction and exercise intolerance in aged CHF patients might be due, in part, to the failure to maintain a sufficiently high Pmv(O(2)) to facilitate blood-muscle O(2) exchange and support mitochondrial ATP production.     

O(2)-evoked regulation of HIF-1alpha and NF-kappaB in perinatal lung epithelium requires glutathione biosynthesis

To test the genetic capacity of the perinatal lung to respond to O(2) shifts that coincide with the first respiratory movements, rat fetal alveolar type II (fATII) epithelial cells were cultured at fetal distal lung PO(2) (23 Torr) and then exposed to postnatal (23 --> 76 Torr; mild hyperoxic shift), moderate (23 --> 152 Torr; moderate hyperoxic shift), or severe (23 --> 722 Torr; severe hyperoxic shift) oxygenation. Nuclear abundance and consensus binding characteristics of hypoxia-inducible factor (HIF)-1alpha and nuclear factor (NF)-kappaB (Rel A/p65) plus glutathione biosynthetic capacity were determined. Maximal HIF-1alpha activation at 23 Torr was sustained over the postnatal shift in (Delta) PO(2) and was elevated in vivo throughout late gestation. NF-kappaB was activated by the acute postnatal DeltaPO(2) in fATII cells, becoming maximal with moderate and severe oxygenation in vitro and within 6 h of birth in vivo, declining thereafter. fATII cell and whole lung glutathione and GSH-to-GSSG ratio increased fourfold with a postnatal DeltaPO(2) and were matched by threefold activity increases in gamma-glutamylcysteine synthetase and glutathione synthase. GSH concentration depletion by L-buthionine-(S, R)-sulfoximine abrogated both HIF-1alpha and NF-kappaB activation, with HIF-1alpha showing a heightened sensitivity to GSH concentration. We conclude that O(2)-linked genetic regulation in perinatal lung epithelium is responsive to developmental changes in glutathione biosynthetic capacity.     

Effect of extracellular PO2 on the fall in intracellular PO2 in contracting single myocytes.

The purpose of this investigation was to study the effects of altered extracellular Po(2) (Pe(O(2))) on the intracellular Po(2) (Pi(O(2))) response to contractions in single skeletal muscle cells. Single myocytes (n = 12) were dissected from lumbrical muscles of adult female Xenopus laevis and injected with 0.5 mM Pd-meso-tetra(4-carboxyphenyl)porphine for assessment of Pi(O(2)) via phosphorescence quenching. At a Pe(O(2)) of approximately 20 (low), approximately 40 (moderate), and approximately 60 (high) Torr, tetanic contractions were induced at a frequency of 0.67 Hz for approximately 2 min with a 5-min recovery between bouts (blocked order design). The Pi(O(2)) response to contractions was characterized by a time delay followed by a monoexponential decline to steady-state (SS) values. The fall in Pi(O(2)) to SS values was significantly greater at each progressively greater Pe(O(2)) (all P < 0.05). The mean response time (time delay + time constant) was significantly faster in the low (35.2 +/- 5.1 s; P < 0.05 vs. high) and moderate (43.3 +/- 6.4 s; P < 0.05 vs. high) compared with high Pe(O(2)) (61.8 +/- 9.4 s) and was correlated positively (r = 0.965) with the net fall in Pi(O(2)). However, the initial rate of change of Pi(O(2)) (calculated as net fall in Pi(O(2))/time constant) was not different (P > 0.05) among Pe(O(2)) trials. These latter data suggest that, over the range of 20-60 Torr, Pe(O(2)) does not play a deterministic role in setting the initial metabolic response to contractions in isolated frog myocytes. Additionally, these results suggest that oxidative phosphorylation in these myoglobin-free myocytes may be compromised by Pe(O(2)) at values nearing 60 Torr.     

Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione

Pseudomonas aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis (CF) whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin triggers tissue damage mainly by its redox cycling and induction of reactive oxygen species (ROS). The reactions between reduced glutathione (GSH) and pyocyanin were observed using absorption spectra from spectrophotometry and the reaction products analysed by nuclear magnetic resonance imaging. Pyocyanin reacted with GSH non-enzymatically at 37 degrees C resulting in the production of red-brown products, spectophotometrically visible as a 480 nm maximum absorption peak after 24 h of incubation. The reaction was concentration-dependent on reduced glutathione but not on pyocyanin. Minimizing the accessibility of oxygen to the reaction decreased its rate. The anti-oxidant enzyme catalase circumvented the reaction. Proton-NMR analysis demonstrated the persistence of the original aromatic ring and the methyl-group of pyocyanin in the red-brown products. Anti-oxidant agents having thiol groups produced similar spectophotometrically visible peaks. The presence of a previously unidentified non-enzymatic GSH-dependent metabolic pathway for pyocyanin has thus been identified. The reaction between pyocyanin and GSH is concentration-, time-, and O(2)-dependent. The formation of H(2)O(2) as an intermediate and the thiol group in GSH seem to be important in this reaction.     

Cerebral and myocardial blood flow responses to hypercapnia and hypoxia in humans

In humans, cerebrovascular responses to alterations in arterial Pco(2) and Po(2) are well documented. However, few studies have investigated human coronary vascular responses to alterations in blood gases. This study investigated the extent to which the cerebral and coronary vasculatures differ in their responses to euoxic hypercapnia and isocapnic hypoxia in healthy volunteers. Participants (n = 15) were tested at rest on two occasions. On the first visit, middle cerebral artery blood velocity (V(P)) was assessed using transcranial Doppler ultrasound. On the second visit, coronary sinus blood flow (CSBF) was measured using cardiac MRI. For comparison with V(P), CSBF was normalized to the rate pressure product [an index of myocardial oxygen consumption; normalized (n)CSBF]. Both testing sessions began with 5 min of euoxic [end-tidal Po(2) (Pet(O(2))) = 88 Torr] isocapnia [end-tidal Pco(2) (Pet(CO(2))) = +1 Torr above resting values]. Pet(O(2)) was next held at 88 Torr, and Pet(CO(2)) was increased to 40 and 45 Torr in 5-min increments. Participants were then returned to euoxic isocapnia for 5 min, after which Pet(O(2)) was decreased from 88 to 60, 52 and 45 Torr in 5-min decrements. Changes in V(P) and nCSBF were normalized to isocapnic euoxic conditions and indexed against Pet(CO(2)) and arterial oxyhemoglobin saturation. The V(P) gain for euoxic hypercapnia (%/Torr) was significantly higher than nCSBF (P = 0.030). Conversely, the V(P) gain for isocapnic hypoxia (%/%desaturation) was not different from nCSBF (P = 0.518). These findings demonstrate, compared with coronary circulation, that the cerebral circulation is more sensitive to hypercapnia but similarly sensitive to hypoxia.     

Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis.

Cystic fibrosis (CF) is characterized by accumulation of activated neutrophils and macrophages on the respiratory epithelial surface (RES); these cells release toxic oxidants, which contribute to the marked epithelial derangements seen in CF. These deleterious consequences are magnified, since reduced glutathione (GSH), an antioxidant present in high concentrations in normal respiratory epithelial lining fluid (ELF), is deficient in CF ELF. To evaluate the feasibility of increasing ELF GSH levels and enhancing RES antioxidant protection, GSH aerosol was delivered (600 mg twice daily for 3 days) to seven patients with CF. ELF total, reduced, and oxidized GSH increased (P < 0.05, all compared with before GSH therapy), suggesting adequate RES delivery and utilization of GSH. Phorbol 12-myristate 13-acetate-stimulated superoxide anion (O2-.) release by ELF inflammatory cells decreased after GSH therapy (P < 0.002). This paralleled observations that GSH added in vitro to CF ELF inflammatory cells suppressed O2-. release (P < 0.001). No adverse effects were noted during treatment. Together, these observations demonstrate the feasibility of using GSH aerosol to restore RES oxidant-antioxidant balance in CF and support the rationale for further clinical evaluation.     

Effect of contractile duration on intracellular PO2 kinetics in Xenopus single skeletal myocytes.

It has been suggested that skeletal muscle O(2) uptake (Vo(2)) kinetics follow a first-order control model. Consistent with that, Vo(2) should show both 1) similar onset kinetics and 2) an on-off symmetry across submaximal work intensities regardless of the metabolic perturbation. To date, consensus on this issue has not been reached in whole body studies due to numerous confounding factors associated with O(2) availability and fiber-type recruitment. To test whether single myocytes demonstrate similar intracellular Po(2) (Pi(O(2))) on- and off-transient kinetics at varying work intensities, we studied Xenopus laevis single myocyte (n = 8) Pi(O(2)) via phosphorescence quenching during two bouts of electrically induced isometric muscle contractions of 200 (low)- and 400 (high)-ms contraction duration (1 contraction every 4 s, 15 min between trials, order randomized). The fall in Pi(O(2)), which is inversely proportional to the net increase in Vo(2), was significantly greater (P < 0.05) during the high (24.1 +/- 3.2 Torr) vs. low (17.4 +/- 1.6 Torr) contraction bout. However, the mean response time (MRT; time to 63% of the overall change) for the fall in Pi(O(2)) from resting baseline to end contractions was not different (high, 77.8 +/- 11.5 vs. low, 76.1 +/- 13.6 s; P > 0.05) between trials. The initial rate of change at contraction onset, defined as DeltaPi(O(2))/MRT, was significantly greater (P < 0.05) in high compared with low. Pi(O(2)) off-transient MRT from the end of the contraction bout to initial baseline was unchanged (high, 83.3 +/- 18.3 vs. low, 80.4 +/- 21.6 s; P > 0.05) between high and low trials. These data revealed that Pi(O(2)) dynamics in frog isolated skeletal myocytes were invariant despite differing contraction durations and, by inference, metabolic demands. Thus these findings demonstrate that mitochondria can respond more rapidly at the initial onset of contractions when challenged with an augmented metabolic stimulus in accordance with an apparent first-order rate law.     

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.     

Intermittent hypoxia induces phrenic long-term facilitation in carotid-denervated rats.

Episodic hypoxia elicits a long-lasting augmentation of phrenic inspiratory activity known as long-term facilitation (LTF). We investigated the respective contributions of carotid chemoafferent neuron activation and hypoxia to the expression of LTF in urethane-anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats. One hour after three 5-min isocapnic hypoxic episodes [arterial Po(2) (Pa(O(2))) = 40 +/- 5 Torr], integrated phrenic burst amplitude was greater than baseline in both carotid-denervated (n = 8) and sham-operated (n = 7) rats (P < 0.05), indicating LTF. LTF was reduced in carotid-denervated rats relative to sham (P < 0.05). In this and previous studies, rats were ventilated with hyperoxic gas mixtures (inspired oxygen fraction = 0.5) under baseline conditions. To determine whether episodic hyperoxia induces LTF, phrenic activity was recorded under normoxic (Pa(O(2)) = 90-100 Torr) conditions before and after three 5-min episodes of isocapnic hypoxia (Pa(O(2)) = 40 +/- 5 Torr; n = 6) or hyperoxia (Pa(O(2)) > 470 Torr; n = 6). Phrenic burst amplitude was greater than baseline 1 h after episodic hypoxia (P < 0.05), but episodic hyperoxia had no detectable effect. These data suggest that hypoxia per se initiates LTF independently from carotid chemoafferent neuron activation, perhaps through direct central nervous system effects.     

Relationship of ovarian hormones to hypoxemia in women residents of 4,300 m

Prevalence of excessive erythrocytosis, the main sign of chronic mountain sickness (CMS), is greater in postmenopausal Andean women than in premenopausal women. It is uncertain whether this greater prevalence is related to the decline in female hormones and ventilatory function after the occurrence of the menopause. To study this, we compared the physiological variables involved in the physiopathology of CMS [end-tidal CO(2) (PET(CO(2)), Torr) and end-tidal O(2) (PET(O(2)), Torr), arterial oxygen saturation (Sa(O(2)), %), and Hb concentration (g/dl)] and progesterone and estradiol levels between postmenopausal and premenopausal women, both in the luteal and follicular phases. Women residing in Cerro de Pasco (n = 33; 4,300 m) aged 26--62 yr were studied. Postmenopausal women compared with premenopausal women in the luteal phase had lower PET(O(2)) (48 +/- 4 vs. 53 +/- 2 Torr, P = 0.005) and Sa(O(2)) levels (82 +/- 12 vs. 88 +/- 12%, P < 0.005) and higher PET(CO(2)) (34 +/- 2 vs. 29 +/- 3 Torr, P = 0.005) and Hb concentration (19 +/- 1 vs. 14 +/- 2 g/dl, P < 0.005). In addition, plasma progesterone was negatively correlated with PET(CO(2)) and positively correlated with PET(O(2)) and Sa(O(2)). No clear relationship was found among the cycle phases between estradiol and the variables studied. In conclusion, our results reveal that, before menopause, there is better oxygenation and lower Hb levels in women long residing at altitude, and this is associated with higher levels of progesterone in the luteal phase of the cycle.     

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.     

Inhibition of AMPK expression in skeletal muscle by systemic inflammation in COPD rats

Background

Analysis of exhaled breath condensates (EBC) is a non-invasive technique to evaluate biomarkers such as antioxidants in the pediatric population, but limited data exists of its use in intubated patients, particularly newborns. Currently, tracheal aspirate (TA) serves as the gold standard collection modality in critically ill newborns, but this method remains invasive. We tested the hypothesis that glutathione status would positively correlate between EBC and TA collections in intubated newborns in the Newborn Intensive Care Unit (NICU). We also hypothesized that these measurements would be associated with alveolar macrophage (AM) glutathione status in the newborn lung.

Methods

Reduced glutathione (rGSH), glutathione disulfide (GSSG), and total GSH (rGSH + (2 X GSSG)) were measured in sequential EBC and TA samples from 26 intubated newborns via high performance liquid chromatography (HPLC). Additionally, AM glutathione was evaluated via immunofluorescence. Pearson’s correlation coefficient and associated 95% confidence intervals were used to quantify the associations between raw and urea-corrected concentrations in EBC and TA samples and AM staining. Statistical significance was defined as p ≤ 0.05 using two-tailed tests. The sample size was projected to allow for a correlation coefficient of 0.5, with 0.8 power and alpha of 0.05.

Results

EBC was obtainable from intubated newborns without adverse clinical events. EBC samples demonstrated moderate to strong positive correlations with TA samples in terms of rGSH, GSSG and total GSH. Positive correlations between the two sampling sites were observed in both raw and urea-corrected concentrations of rGSH, GSSG and total GSH. AM glutathione staining moderately correlated with GSSG and total GSH status in both the TA and EBC.

Conclusions

GSH status in EBC samples of intubated newborns significantly correlated with the GSH status of the TA sample and was reflective of cellular GSH status in this cohort of neonatal patients. Non-invasive EBC sampling of intubated newborns holds promise for monitoring antioxidant status such as GSH in the premature lung. Further studies are necessary to evaluate the potential relationships between EBC biomarkers in the intubated premature newborn and respiratory morbidities.     

Dissociation between skeletal muscle microvascular PO2 and hypoxia-induced microvascular inflammation.

Systemic hypoxia (SHx) produces microvascular inflammation in mesenteric, cremasteric, and pial microcirculations. In anesthetized rats, SHx lowers arterial blood pressure (MABP), which may alter microvascular blood flow and microvascular Po(2) (Pm(O(2))) and influence SHx-induced leukocyte-endothelial adherence (LEA). These experiments attempted to determine the individual contributions of the decreases in Pm(O(2)), venular blood flow and shear rate, and MABP to the hypoxia-induced increase in LEA. Cremaster microcirculation of anesthetized rats was visualized by intravital microscopy. Pm(O(2)) was measured by a phosphorescence-quenching method. SHx [inspired Po(2) of 70 Torr for 10 min, MABP of 65 +/- 3 mmHg, arterial Po(2) (Pa(O(2))) of 33 +/- 1 Torr] and cremaster ischemia (MABP of 111 +/- 7 mmHg, Pa(O(2)) of 86 +/- 3 Torr) produced similar Pm(O(2)): 7 +/- 2 and 6 +/- 2 Torr, respectively. However, LEA increased only in SHx (1.9 +/- 0.9 vs. 11.2 +/- 1.1 leukocytes/100 microm, control vs. SHx, P < 0.05). Phentolamine-induced hypotension (MABP of 55 +/- 4 mmHg) in normoxia lowered Pm(O(2)) to 26 +/- 6 Torr but did not increase LEA. Cremaster equilibration with 95% N(2)-5% CO(2) during air breathing (Pa(O(2)) of 80 +/- 1 Torr) lowered Pm(O(2)) to 6 +/- 1 Torr but did not increase LEA. On the other hand, when cremaster Pm(O(2)) was maintained at 60-70 Torr during SHx (Pa(O(2)) of 35 +/- 1 Torr), LEA increased from 2.1 +/- 1.1 to 11.1 +/- 1.5 leukocytes/100 microm (P < 0.05). The results show a dissociation between Pm(O(2)) and LEA and support the idea that SHx results in the release of a mediator responsible for the inflammatory response.     

Pulmonary gas exchange during exercise in highly trained cyclists with arterial hypoxemia.

The causes of exercise-induced hypoxemia (EIH) remain unclear. We studied the mechanisms of EIH in highly trained cyclists. Five subjects had no significant change from resting arterial PO(2) (Pa(O(2)); 92.1 +/- 2.6 Torr) during maximal exercise (C), and seven subjects (E) had a >10-Torr reduction in Pa(O(2)) (81.7 +/- 4.5 Torr). Later, they were studied at rest and during various exercise intensities by using the multiple inert gas elimination technique in normoxia and hypoxia (13.2% O(2)). During normoxia at 90% peak O(2) consumption, Pa(O(2)) was lower in E compared with C (87 +/- 4 vs. 97 +/- 6 Torr, P < 0.001) and alveolar-to-arterial O(2) tension difference (A-aDO(2)) was greater (33 +/- 4 vs. 23 +/- 1 Torr, P < 0. 001). Diffusion limitation accounted for 23 (E) and 13 Torr (C) of the A-aDO(2) (P < 0.01). There were no significant differences between groups in arterial PCO(2) (Pa(CO(2))) or ventilation-perfusion (VA/Q) inequality as measured by the log SD of the perfusion distribution (logSD(Q)). Stepwise multiple linear regression revealed that lung O(2) diffusing capacity (DL(O(2))), logSD(Q), and Pa(CO(2)) each accounted for approximately 30% of the variance in Pa(O(2)) (r = 0.95, P < 0.001). These data suggest that EIH has a multifactorial etiology related to DL(O(2)), VA/Q inequality, and ventilation.