Division of Pulmonary, Critical Care & Sleep Medicine, Department of Internal Medicine, College of Medicine and Public Health, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio
Submitted 13 September 2004 ; accepted in final form 17 February 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat
ROS generation has been thought to be more likely during reoxygenation because hypoxia causes a significant accumulation of reducing equivalents in the mitochondria, such as NADH and FADH. Sudden exposure to O2 can promote the formation of O2 through intermediate electron carriers and one electron reduction of molecular O2 in the mitochondrial electron transport chain. However, there is also considerable evidence for ROS formation in hypoxia or ischemia in some tissues, such as the heart. For example, Vanden Hoek et al. (35) and Damerau et al. (4) observed increased ROS production during hypoxia in cardiac myocytes. These data are consistent with earlier observations by Park et al. (20), who used electron spin resonance (ESR) in intact hearts during ischemia, and Kevin et al. (9), who used redox-sensitive fluorescent probes in the intact heart. Liu et al. (13) also identified ROS formation in hypoxic distal pulmonary arteries using ESR. The possibility that both low and high O2 can cause ROS formation in specific circumstances stands as one of the more interesting paradoxes of free radical biology.
In previous studies, we observed that antioxidants administered during hypoxia exposure protected skeletal muscle contractile function (16, 36). On the basis of that previous work, we hypothesized that ROS are produced in skeletal muscle during hypoxia and that they produce a signal that has functional significance with regard to acute adaptations to hypoxia. In the present study, we used a tissue fluorescence system to monitor ROS production and simultaneously monitor changes in intracellular pyridine nucleotides, in real time, during hypoxia in intact, nonperfused diaphragm muscle. The pyridine nucleotides (NADH and FAD) were used as indirect indicators of muscle tissue oxygenation. We then applied this method to evaluate whether tissue hypoxia occurs during fatiguing muscle contractions in in vitro superfused rat diaphragm and whether there are associated elevations in ROS production.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Standard Hfluor-DA or Fluor-DA loading and ebselen treatment. For H2O2 detection, tissue strips were loaded with 50 µM Hfluor-DA at room temperature for 30 min. Hfluor was chosen over other similar redox-sensitive probes because it has considerable advantages with regard to loading and for its relative lack of sensitivity to nitric oxide, Fe2+, and peroxidase activity compared with more commonly used probes, such as dichlorofluorescein or dihydrorhodamine 123 (7, 38). The tissues were washed in fresh buffer for 10 min after the loading period (38). In some cases, the loading time was doubled to increase the detection sensitivity, although this had little effect on the overall outcome of the experiments. For evaluation of the behavior of the oxidized probe within the muscle tissue, 0.05 µM Fluor-DA was loaded for 30 min at room temperature. Ebselen, a glutathione peroxidase mimic used primarily as a scavenger of ROS (30), was applied to the treated tissues at 40 µM in the loading solution and throughout the rest of the experiment. Ebselen was found to be extremely sensitive to degradation in oxygenated buffer solutions, and therefore fresh ebselen stock was added directly to the buffers shortly before use.
Tissue chamber and fluorometer system.
For the majority of hypoxia experiments, after fluorophore loading, tissue strips were transferred to a superfusion chamber. During initial development of this technique, and for some of the data collection, we used a commercially available chamber (model RC-27; Warner Instruments) that allowed 1 mm of space under the tissue for continuous superfusion of both sides of the tissue. This system had a large head space region that was covered loosely with a plastic top, making changes in tissue O2 less rapid and temperature difficult to control. The excitation and emission optical fibers were combined in one cable for epifluorescence measurements. We refer to this technique as "surface fluorometry." For the majority of data collected, a custom-made enclosed chamber was used as shown in Fig. 1, A and B, which allowed for better control of temperature, chamber PO2, more uniform flow, and near-elimination of air bubbles. An O2 electrode (ISO2; World Precision Instruments) was used in some experiments for monitoring chamber PO2 levels, and a thermistor was used to measure and servocontrol the chamber temperature (Warner Instruments). For this configuration, the fluorescent signal was measured on the opposite side of the tissue with independent excitation and emission cables, a configuration that we refer to as "transmission fluorometry." This technique has some advantages at some wavelengths. For example, on the basis of the optical properties of the rat diaphragm, we have modeled the light scattering, absorption, and escape of both the excitation and emission light and have determined that this configuration results in a relatively uniform estimate of fluorescence across the thickness of the tissue for wavelengths used in each Fluor measurement (3).
Fluorescence was detected using a ratiometric tissue fluorometer (Radnoti). The excitation beam from a 150-W Xenon lamp was focused on a 6-mm-diameter fiber optic cable, and the light passed through a filter wheel containing four specific band-pass filters. A second filter wheel with four emission filters was used to isolate emission light at specific wavelengths. This light was focused on the photomultiplier tube (PMT) (model HC 120-05MOD-6308; Hamamatsu PMT Assembly, Japan). Thus the tissue fluorometer could sequentially measure four channels of fluorescence. The three fluorescent channels used for this study were NADH autofluorescence (NADH, excitation 330 BW-80 nm, emission 470 BW-10 nm), fluorescein fluorescence (FAD/Fluor, excitation 490-BW 20 nm, emission 535-BW 35 nm), and FAD autofluorescence (FAD, excitation 455-BW 70 nm, emission 630-BW 50 nm). These three different excitation and emission ranges provided a relatively complete observation window for tissue autofluorescence for both NADH and FAD and for the fluorescence from the Fluor probe. There was no overlap of the Fluor fluorescence signal in the other two channels and no overlap of NADH in any other channel. There was overlap of the FAD autofluorescence signal in the Fluor channel as described herein. To minimize light interference, the tissue chamber was housed in a solid black metal box. The PMT output was collected on a personal computer using an analog-to-digital converter, and measurements from each filter set were signal averaged, recorded, and graphed using a program developed in a modified programming language (Workbench PC; Strawberry Tree). The light shutters were driven by an automated system in which the tissue was exposed for between 3 and 7 s every 30 s. The rotation of the filter wheel exposed the tissue to each excitation wavelength for 8.5 ms every 40 ms. The amount of light reaching the tissue was therefore reduced as much as possible to avoid photobleaching and photooxidation of the fluorophores. The tests of photooxidation and photobleaching that were performed were similar to those described by Murrant et al. (17), and we observed no measurable effects of this exposure time on the rate of Hfluor oxidation or Fluor disappearance (data not shown).
Hypoxia experiments. Most experiments were performed at 23°C because the oxidized probe (Fluor) apparently leaked at a faster rate from tissues maintained at 37°C. However, some entire experiments were performed at 37°C, where noted, and qualitatively similar results were observed at both temperatures. Experiments were generally conducted with groups of four tissues from each animal, with tissues maintained in oxygenated buffer at 23°C until use. Once mounted in the chamber, tissues were superfused with Ringer solution and then prebubbled with 95% O2-5% CO2 for 1015 min or until the fluorescent emission was relatively stabilized. Drift of the Fluor signal was often observed during the experiment, but it was attributed to the balance of background ROS production and leakage or reduction of the oxidized probe. A slow drift upward in the Fluor signal was most evident at 23°C, whereas Fluor signals tended to drift downward at 37°C.
Hypoxia was induced by switching the superfusate to a separate buffer source, preequilibrated at the set temperature and O2 concentration (0%, 5%, 21%, and 40%) and CO2 concentration (5%). Approximately 3 min were required to achieve 90% of the final plateau in PO2 within the chamber as measured using the O2 electrode. After hypoxia, tissues were reoxygenated with 95% O2. The level of O2/CO2/N2 during hypoxia exposure was varied in the superfusate source using a gas mixing system (Reming Bioinstruments).
H2O2 addition on tissues with antioxidant treatments. To determine the sensitivity of the muscle and the detection system to ROS, tissues in the chamber were superfused with 1 or 10 µM H2O2 solution for 10 min. In parallel experiments, ebselen and NAC were administered with H2O2 as antioxidants to block the signal.
Muscle contraction experiments. Muscle strips were preloaded with 50 µM Hfluor-DA in oxygenated Ringer solution for 30 min at room temperature and then rinsed for 10 min in buffer. As shown in Fig. 1C, a specially designed tissue bath was used for these experiments. The emission and excitation cables were installed in the bath for transmission fluorometry apposing the tissue 11.5 mm from its surfaces. Once the strip was placed into a tissue bath at 37°C, it was maintained during an equilibration period with slow-twitch contractions at 0.05 Hz for 510 min and 95% O2-5% CO2. The tissue was then stimulated at 1 and 4 Hz for 5 min each, and it was allowed to rest for 10 min between stimulations. At the same time, fluorescence of NADH, Fluor, and FAD channels were monitored, collected, and analyzed.
Fluorescence calculation and statistics. Except where mentioned, the following approach was used to calculate the fluorescence signal. The Fluor signal during hypoxia and H2O2 exposure was characterized by a positive deflection, which returned approximately to baseline after exposures. In each curve, a baseline was established by selecting a stable point before and after the hypoxia or H2O2 administration. The vertical deflection between the extrapolated baseline and the peak point of the signal was divided by the extrapolated baseline signal at the same time point as a way of normalizing the overall fluorescence. For some Fluor and NADH signals, there was no real peak, and the "peak" selected was approximately where the NADH signal reached the maximum. For some FAD determinations, which have a negative deflection during hypoxia exposure, similar vertical distances between the baseline and the minimum were used in calculation. These data were then grouped and analyzed using statistics described in the figure legends.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Sensitivity of the ROS detection system to H2O2. Figure 6 shows the results of H2O2 superfusion onto Hfluor-DA-loaded tissues. Figure 6A shows a typical response to 1 and 10 µM H2O2. Note the rapid reversibility of the H2O2-induced rise in fluorescence that resembles the nature of the hypoxia-induced signal. Changes of both NADH and FAD autofluorescence were essentially no different from noise in response to 1 and 10 µM H2O2. In Fig. 6B, both 1 and 10 µM H2O2 resulted in significant, transient increases in the Fluor fluorescence compared with baseline autofluorescence (n = 5; P < 0.05). Compared with control (H2O2), these signals were greatly attenuated in tissues treated with the antioxidants ebselen (n = 5; P < 0.01), NAC (n = 5; P < 0.05), and ebselen + NAC (n = 4; P < 0.05). The change of Fluor signal induced by 10 µM H2O2 was significantly larger than 1 µM H2O2 (n = 5; P < 0.05), suggesting that the tissue was optimally loaded with probe for ROS detection within this range of ROS exposure. Additional experiments were performed on oxidized probe (Fluor-DA-loaded tissue), which also showed sensitivity to H2O2 compared with autofluorescence (n = 6; P < 0.05). This sensitivity could be blocked significantly by ebselen or NAC treatment (P < 0.05).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At this low range of change in ROS activity, it appears that the fluorescent probe, Hfluor, and related compounds exist in a reversible redox state. This observation is based on the evidence that brief hypoxia, exposure to low concentrations of H2O2, and muscle contractions resulted in only transient increases in fluorescence, which were immediately reversed upon reoxygenation, perfusion with fresh buffer, or discontinuation of stimulation, respectively. The hypothesis is also supported by the observation that when the oxidized probe Fluor-DA is loaded into the tissue, similar responses to both hypoxia and H2O2 can be observed and can be inhibited by cotreatment with ebselen. The apparent ability of tissue to reduce Fluor to Hfluor has not generally been considered in the literature. However, similar findings of the reversibility of the fluorescent signal for several redox-sensitive fluorescent probes have been shown in response to ischemia-reperfusion injury in cardiomyocytes (29, 35) and in intact hearts (9). Interestingly, when diaphragm strips were exposed to high concentrations of H2O2 that are not physiological (i.e., 1 mM), the increase in Fluor fluorescence was very large and was not reversed rapidly by the tissue (data not shown).
Critique of the ROS detection method.
Compared with most other fluorescein derivatives, Hfluor is insensitive to nitric oxide, has low sensitivity to Fe2+, and has relatively low sensitivity to changes in peroxidase activity; yet, it maintains excellent loading characteristics and good sensitivity to H2O2 (7). We speculate that the predominant ROS signal measured during hypoxia in this study was H2O2, in part because the signal was inhibited by ebselen. Although ebselen may scavenge other ROS such as peroxynitrite (31), its primary targets are thought to be peroxides. Experiments in which nitric oxide synthase was inhibited have shown no inhibition of the hypoxia-induced fluorescence signal (data not shown), thus making it likely that peroxynitrite is not a predominant species in this setting. The effectiveness of ebselen as a scavenger of peroxides arises from its ability to use glutathione as an electron acceptor, which is highly concentrated in muscle cells (0.8 mM).
In contrast, the inability of NAC to attenuate the endogenous ROS formation during hypoxia is not surprising, because it is considered a relatively weak and slow scavenger of H2O2 (1). It did attenuate the H2O2-induced Hfluor oxidation, but this is likely due to the fact that the NAC was coincubated in the buffer solution with H2O2. Consider the fact that the concentration of NAC was 1,000-fold that of H2O2 in the superfusion medium during these experiments.
The lack of specificity of the fluorescein-based redox probes is an important consideration as described recently (38). These probes are sensitive to a variety of oxidants and produce O2 after oxidation by H2O2 (27) and during photooxidation (28). However, on the basis of direct measurements of spontaneous Fluor formation in the presence and absence of light, we do not think that photooxidation was significant in this preparation. Furthermore, whereas the formation of O2
after oxidation of fluorescein-based redox probes has been described as a potentially autocatalytic process (27, 28), it can be argued that the reaction results in the net conversion of one H2O2 molecule (2-electron reduction of O2) to one O2
molecule (1-electron reduction of O2), which should not be autocatalytic with an intact antioxidant defense network in vivo. Regardless, measurements of ROS formation using fluorescent probes such as Hfluor should be interpreted with caution, because their redox chemistry is complex and dependent on numerous variables.
Another consideration is the pH dependence of the emission intensity of fluorescein. We can presume that the hypoxic muscles in the present study became acidotic during hypoxia as a result of increases in glycolysis. However, a reduction in pH results in a net decrease in the fluorescence intensity of Fluor (12), and therefore we ruled out this possibility as a potential source of the reversible signal observed in hypoxia and assume that this effect underestimated our net measured responses during hypoxia. Finally, we could not identify significant O2 quenching effects on Fluor fluorescence in physiological buffer systems and could identify no such effect in both Hfluor- and Fluor-loaded dead tissues.
The H2O2 addition experiments provided an estimate of the range of sensitivity of our system. Although we were able to detect a clear signal with as low as 1 µM H2O2 in the superfusion medium, and because the intracellular H2O2 concentration has been estimated to be 7- to 10-fold lower than the extracellular H2O2 in such experiments (33), we estimate that our system has a threshold sensitivity to intracellular H2O2 of 100200 nM.
There are a number of ways to measure fluorescence in intact tissues. Previously, we used laser-scanning confocal microscopy to detect intracellular ROS formation (37), and many other investigators have used this technique or other, more conventional types of epifluorescence microscopy. Tissue fluorometry has three major advantages over microscopy for some applications. 1) It obtains fluorescent signals from hundreds of cells simultaneously because of the large diameter of its detection platform. 2) Because of the relatively large observation area, most artifacts due to tissue or cell movements can be greatly minimized; movement can result in substantial problems in microscopy experiments. 3) As mentioned earlier, the light source has only minimal effects of photobleaching or photodamage, whereas this phenomenon is common in microscopy experiments. Therefore, the tissue fluorometer can be used as an effective spectroscopy tool for investigations in which details at the cellular level can be sacrificed.
The intracellular levels of NADH and FAD greatly affect autofluorescence in their respective emission ranges. Our results illustrate the critical importance of measuring their autofluorescence when studying redox behavior during hypoxia using fluorescent probes, because these signals are large and extend through much of the visible spectrum. Of particular importance is the fact that Fluor has excitation and emission ranges that lie within those of FAD, and when loading is poor, the emission signal of Fluor does not provide reliable information about ROS formation, owing to the FAD interference. However, we were able to overcome this problem by measuring and documenting autofluorescence in unloaded muscles and by measuring FAD autofluorescence in an accompanying channel that did not overlap the one used for Fluor measurements. The other great advantage of measuring FAD and NADH in these experiments is that they provide insights into the time course and extent of tissue hypoxia during the experiment. Because O2 availability becomes limiting for cytochrome c oxidase, reducing equivalents in the form of cytosolic or mitochondrial NADH and mitochondrial FADH buildup and can be considered fluorescent sentinels of local hypoxia.
Potential physiological significance of ROS production in hypoxia.
Normal intracellular PO2 in perfused skeletal muscle is thought to be 10 Torr at rest, dropping to a mean value of 35 Torr during intense exercise (25) and not reaching a critical level until cellular PO2 drops to
1.5 Torr (26). Whether this deoxygenation of skeletal muscle can be defined as hypoxia depends on one's definition of hypoxia. The transient deoxygenation associated with acute intense exercise would be a likely example of tissue hypoxia in skeletal muscle, where mismatches between local O2 consumption and perfusion have been shown to occur over a time frame of 23 min at the onset of moderate-intensity exercise (5). This rapid deoxygenation may be sufficient to induce a transient reactive oxygen signal as described in this study. Furthermore, it is reasonable to predict that if the signal exists in intact organisms, it might be amplified in hypoxic patients with chronic heart or lung disease or during exercise at high altitude.
The muscle stimulation experiments shown in Figs. 7 and 8 were performed to demonstrate a direct and practical application of this technology and to investigate whether tissue hypoxia and ROS formation are likely to occur in isolated nonperfused muscles of this thickness. On the basis of the changes in autofluorescence within the NADH and FAD ranges, it is clear that isolated rat diaphragm does become hypoxic at this level of stimulation and in this in vitro setting. In normally perfused, steady-state, moderately exercising muscles, NADH generally decreases, indicating that in steady-state exercise, skeletal muscles do not generally become critically hypoxic at the tissue level, except under conditions of compromised O2 delivery (19) or during transient states of high exercise intensity (5) as discussed earlier. It is possible that the experimental configuration used in this study, which required the electrodes to be relatively close to the muscle surfaces (1 mm), compromised O2 availability to the tissue underneath the fiberoptic probes, thus amplifying the hypoxia. However, it is just as likely to be due to the thickness of the rat diaphragm and the long diffusion gradients necessary to deliver O2 to the core of the tissue (
0.50.6 mm in diameter). Although reactive oxygen signals associated with contraction have been described extensively in the literature (6, 11, 18, 22, 23, 32), it is interesting to speculate that at least part of the ROS signal observed in previous studies may be secondary to local hypoxia induced by inadequate O2 supply during high O2 demand in isolated or compromised preparations.
What possible function could a transient ROS signal provide to exercising muscle? In heart muscle, ROS is thought to play an important role in the phenomenon of preconditioning (see, e.g., Ref. 9), resulting in the protection of heart muscle and vasculature during subsequent exposure to ischemia. The exact cell signaling pathway that is stimulated by ROS has been studied extensively, but it is complex and still poorly understood. Kohin et al. (10) demonstrated a similar phenomenon in single frog skeletal muscles in which brief exposures to hypoxia appear to precondition the fibers, protecting them from subsequent hypoxia. At this time, we can only speculate that the ROS signal that we have observed could have a functional role in preconditioning.
Paradoxically, our laboratory previously observed that antioxidants have a protective influence on skeletal muscle function during 30-min exposure to near anoxia (16, 36), suggesting that oxidants have a negative influence on contractile function. However, the role of oxidants at very low metabolic states may also be thought of as protective of the muscle in the same way that muscle fatigue is thought of as an inherently protective mechanism to prevent injury as a result of overstimulation. Therefore, oxidants in severe or prolonged hypoxia might provide a different kind of functional role from those observed in brief exposures.
Summary. This report provides evidence for hypoxia-induced ROS generation in skeletal muscle using tissue fluorometry. The possibility of internal conversion of Hfluor and Fluor was also noted in this research. We speculate that ROS formation during these transient hypoxic exposures may have an important role as a mediator of O2 sensing and cell adaptation. Such ROS signals may also work in synchrony with those produced during exercise-induced temperature elevation in muscle (37). Future studies to confirm these experiments in intact muscle using other complementary methods are needed to address the molecular source of hypoxia-induced ROS, to evaluate its potential role as an efferent arm of an O2-sensing system, and to determine its functional role in adaptation to exercise and exposure to stressful environments.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Boveris A and Chance B. The mitochondrial generation of hydrogen peroxide: general properties and effect of hyperbaric oxygen. Biochem J 134: 707716, 1973.[ISI][Medline]
3. Clanton TL and Zuo L. Measurement of intracellular reactive oxygen species (ROS) using transmission fluorescence detection across thin tissue sections (Abstract). Biophys J 86: 609A, 2004.
4. Damerau W, Ibel J, Thurich T, Assadnazari H, and Zimmer G. Generation of free radicals in Langendorff and working hearts during normoxia, hypoxia and reoxygenation. Basic Res Cardiol 88: 141149, 1993.[ISI][Medline]
5. DeLorey DS, Kowalchuk JM, and Paterson DH. Relationship between pulmonary O2 uptake kinetics and muscle deoxygenation during moderate-intensity exercise. J Appl Physiol 95: 113120, 2003.
6. Diaz PT, She ZW, Davis WB, and Clanton TL. Hydroxylation of salicylate by the in vitro diaphragm: evidence for hydroxyl radical production during fatigue. J Appl Physiol 75: 540552, 1993.[Abstract]
7. Hempel SL, Buettner GR, O'Malley YQ, Wessels DA, and Flaherty DM. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic Biol Med 27: 146159, 1999.[CrossRef][ISI][Medline]
8. Keston AS and Brandt R. The fluorometric analysis of ultramicro quantities of hydrogen peroxide. Anal Biochem 11: 15, 1965.[CrossRef][ISI][Medline]
9. Kevin LG, Camara AK, Riess ML, Novalija E, and Stowe DF. Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion. Am J Physiol Heart Circ Physiol 284: H566H574, 2003.
10. Kohin S, Stary CM, Howlett RA, and Hogan MC. Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation. Am J Physiol Cell Physiol 281: C142C146, 2001.
11. Kolbeck RC, She ZW, Callahan LA, and Nosek TM. Increased superoxide production during fatigue in the perfused rat diaphragm. Am J Respir Crit Care Med 156: 140145, 1997.
12. Lakowicz JR. Fluorescence sensing. In: Principles of Fluorescence Spectroscopy (2nd ed.). New York: Kluwer Academic/Plenum, 1999, p. 531572.
13. Liu JQ, Sham JS, Shimoda LA, Kuppusamy P, and Sylvester JT. Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 285: L322L333, 2003.
14. López-Barneo J, del Toro R, Levitsky KL, Chiara MD, and Ortega-Sáenz P. Regulation of oxygen sensing by ion channels. J Appl Physiol 96: 11871195, 2004.
15. Michelakis ED, Hampl V, Nsair A, Wu X, Harry G, Haromy A, Gurtu R, and Archer SL. Diversity in mitochondrial function explains differences in vascular oxygen sensing. Circ Res 90: 13071315, 2002.
16. Mohanraj P, Merola AJ, Wright VP, and Clanton TL. Antioxidants protect rat diaphragmatic muscle function under hypoxic conditions. J Appl Physiol 84: 19601966, 1998.
17. Murrant CL, Andrade FH, and Reid MB. Exogenous reactive oxygen and nitric oxide alter intracellular oxidant status of skeletal muscle fibres. Acta Physiol Scand 166: 111121, 1999.[CrossRef][ISI][Medline]
18. Nethery D, Stofan D, Callahan L, DiMarco A, and Supinski G. Formation of reactive oxygen species by the contracting diaphragm is PLA2 dependent. J Appl Physiol 87: 792800, 1999.
19. Nioka S, McCully K, McClellan G, Park J, and Chance B. Oxygen transport and intracellular bioenergetics on stimulated cat skeletal muscle. Adv Exp Med Biol 510: 267272, 2003.[ISI][Medline]
20. Park Y, Kanekal S, and Kehrer JP. Oxidative changes in hypoxic rat heart tissue. Am J Physiol Heart Circ Physiol 260: H1395H1405, 1991.
21. Pattwell D, McArdle A, Griffiths RD, and Jackson MJ. Measurement of free radical production by in vivo microdialysis during ischemia/reperfusion injury to skeletal muscle. Free Radic Biol Med 30: 979985, 2001.[CrossRef][ISI][Medline]
22. Reid MB, Shoji T, Moody MR, and Entman ML. Reactive oxygen in skeletal muscle. II. extracellular release of free radicals. J Appl Physiol 73: 18051809, 1992.
23. Reid MB, Haack KE, Franchek KM, Valberg PA, Kobzik L, and West MS. Reactive oxygen in skeletal muscle I. intracellular oxidant kinetics and fatigue in vitro. J Appl Physiol 73: 17971804, 1992.
24. Reid MB and Moody MR. Dimethyl sulfoxide depresses skeletal muscle contractility. J Appl Physiol 76: 21862190, 1994.
25. Richardson RS, Noyszewski EA, Kendrick KF, Leigh JS, and Wagner PD. Myoglobin O2 desaturation during exercise. J Clin Invest 96: 19161926, 1995.[ISI][Medline]
26. Richmond KN, Burnite S, and Lynch RM. Oxygen sensitivity of mitochondrial metabolic state in isolated skeletal and cardiac myocytes. Am J Physiol Cell Physiol 273: C1613C1622, 1997.
27. Rota C, Chignell CF, and Mason RP. Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescein to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radic Biol Med 27: 873881, 1999.[CrossRef][ISI][Medline]
28. Rota C, Fann YC, and Mason RP. Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. J Biol Chem 274: 2816128168, 1999.
29. Shao ZH, Li CQ, Vanden Hoek TL, Becker LB, Schumacker PT, Wu JA, Attele AS, and Yuan CS. Extract from Scutellaria baicalensis Georgi attenuates oxidant stress in cardiomyocytes. J Mol Cell Cardiol 31: 18851895, 1999.[CrossRef][ISI][Medline]
30. Sies H. Ebselen, a selenoorganic compound as glutathione peroxidase mimic. Free Radic Biol Med 14: 313323, 1993.[CrossRef][ISI][Medline]
31. Sies H and Masumoto H. Ebselen as a glutathione peroxidase mimic and as a scavenger of peroxynitrite. In: Advances in Pharmacology: Antioxidants in Disease Mechanisms and Therapy, edited by Sies H (vol. ed.). San Diego, CA: Academic, 1997, vol. 38, p. 229246.
32. Stofan DA, Callahan LA, DiMarco AF, Nethery DE, and Supinski GS. Modulation of release of reactive oxygen species by the contracting diaphragm. Am J Respir Crit Care Med 161: 891898, 2000.
33. Stone JR. An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems. Arch Biochem Biophys 422: 119124, 2004.[CrossRef][ISI][Medline]
34. Tsan MF. Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice. Int J Mol Med 7: 1319, 2001.[ISI][Medline]
35. Vanden Hoek TL, Li C, Shao Z, Schumaker PT, and Becker LB. Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia before reperfusion. J Mol Cell Cardiol 29: 25712583, 1997.[CrossRef][ISI][Medline]
36. Wright VP, Klawitter PF, Iscru DF, Merola AJ, and Clanton TL. Superoxide scavengers augment contractile but not energetic responses to hypoxia in rat diaphragm. J Appl Physiol 98: 17531760, 2005. First published January 7, 2005; doi:10.1152/japplphysiol.01022.2004.
37. Zuo L, Christofi FL, Wright VP, Liu CY, Merola AJ, Berliner LJ, and Clanton TL. Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle. Am J Physiol Cell Physiol 279: C1058C1066, 2000.
38. Zuo L and Clanton TL. Detection of reactive oxygen and nitrogen species in tissues using redox-sensitive fluorescent probes. Methods Enzymol 352: 307325, 2002.[ISI][Medline]
39. Zuo L, Pasniciuc S, Wright VP, Merola AJ, and Clanton TL. Sources for superoxide release: lessons from blockade of electron transport, NADPH oxidase, and anion channels in diaphragm. Antioxid Redox Signal 5: 667675, 2003.[CrossRef][ISI][Medline]