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J Appl Physiol 87: 792-800, 1999;
8750-7587/99 $5.00
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Vol. 87, Issue 2, 792-800, August 1999

Formation of reactive oxygen species by the contracting diaphragm is PLA2 dependent

D. Nethery, D. Stofan, L. Callahan, A. DiMarco, and G. Supinski

Pulmonary Division, Department of Medicine, Case Western Reserve University and MetroHealth Medical Center, Cleveland, Ohio 44109


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recent work indicates that respiratory muscles generate superoxide radicals during contraction (M. B. Reid, K. E. Haack, K. M. Francik, P. A. Volberg, L. Kabzik, and M. S. West. J. Appl. Physiol. 73: 1797-1804, 1992). The intracellular pathways involved in this process are, however, unknown. The purpose of the present study was to test the hypothesis that contraction-related formation of reactive oxygen species (ROS) by skeletal muscle is linked to activation of the 14-kDa isoform of phospholipase A2 (PLA2). Studies were performed by using an in vitro hemidiaphragm preparation submerged in an organ bath, and formation of ROS in muscles was assessed by using a recently described fluorescent indicator technique. We examined ROS formation in resting and contracting muscle preparations and then determined whether contraction-related ROS generation could be altered by administration of various PLA2 inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA2; arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of 85-kDa PLA2; and haloenol lactone suicide substrate (HELSS), an inhibitor of calcium-independent PLA2. We found 1) little ROS formation [2.0 ± 0.8 (SE) ng/mg] in noncontracting control diaphragms, 2) a high level of ROS (20.0 ± 2.0 ng/mg) in electrically stimulated contracting diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 s-1), 3) near-complete suppression of ROS generation in manoalide (3.0 ± 0.5 ng/mg, P < 0.001)- and aristolochic acid-treated contracting diaphragms (4.0 ± 1.0 ng/mg, P < 0.001), and 4) no effect of AACOCF3 or HELSS on ROS formation in contracting diaphragm. During in vitro studies examining fluorescent measurement of ROS formation in response to a hypoxanthine/xanthine oxidase superoxide-generating solution, manoalide, aristolochic acid, AACOCF3, and HELSS had no effect on signal intensity. These data indicate that ROS formation by contracting diaphragm muscle can be suppressed by the administration of inhibitors of the 14-kDa isoform of PLA2 and suggest that this enzyme plays a critical role in modulating ROS formation during muscle contraction.

free radicals; skeletal muscle; respiratory muscles


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PREVIOUS STUDIES HAVE demonstrated that reactive oxygen species (ROS, i.e., superoxide and hydroxyl anions) are generated in the respiratory muscles and contribute to the development of muscle fatigue during strenuous, repetitive contraction (6, 14, 28, 34). Although there has been much speculation regarding the mechanisms and pathways by which contraction may elicit heightened free radical production, this phenomenon remains unexplained. In neutrophils, however, recent work indicates that free radical generation by the cell surface NADPH oxidase complex is dependent on the activity of the 14-kDa isoform of phospholipase A2 (PLA2) and that inhibitors of this isoform almost completely suppress superoxide generation by these cells (23). Previous studies have also shown that the development of skeletal muscle injury in some in vitro model systems is PLA2 dependent and can be attenuated by inhibition of this enzyme system (18). It is possible to link these previous observations if one postulates 1) that free radical generation in contracting muscles may, as in white blood cells, be PLA2 dependent and 2) that the protection provided to in vitro muscles by PLA2 inhibitors may result from suppression of free radical-mediated muscle damage.

The purpose of the present study, therefore, was to test the hypothesis that superoxide generation by the contracting diaphragm is dependent on the enzymatic activity of the 14-kDa PLA2 isoform. Studies were done by using an in vitro arterially perfused rat hemidiaphragm muscle preparation, and formation of ROS (including superoxide and its metabolic products) in this muscle was assessed by using a recently described fluorescent assay (1, 9). We compared time-matched diaphragm ROS generation in 1) control, noncontracting diaphragms, 2) contracting diaphragms, and 3) contracting diaphragms treated with PLA2 inhibitors. As a control, we also assessed the effect of administration of Tiron, a potent intracellular superoxide scavenger, during diaphragm contraction to determine the effect of this intervention on our fluorescent index of ROS formation (20).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Care

Adult, male Sprague-Dawley rats (n = 38, 400-600 g) were used in this study. Until the time of study, rats were housed and cared for in the Case Western Reserve University Animal Resource Center according to American Association for Accreditation of Laboratory Animal Care guidelines. The animals were examined daily by university veterinarians; food and water were allowed ad libitum.

Experimental Preparation

To carry out the present study, we used a specially developed in vitro diaphragm preparation. This preparation consisted of a large hemidiaphragm strip prepared such that the ribs, intercostal vessels, spinal column, and abdominal aorta remained attached to the diaphragm, making infusion of pharmacological agents and fluorescent indicators into the diaphragm possible. For the purposes of the present study, this preparation was submerged in a glass organ bath (Radnoti, Monrovia, CA) containing physiological solution. Several requirements of the present study made the use of this particular preparation necessary. First, the molecular weights of the various PLA2 inhibitors used in this experiment were extremely high, making diffusion into conventional multifiber strip preparations problematic. Infusion of these agents directly into the diaphragm is possible with our hemidiaphragm preparation, increasing contact with muscle fibers and facilitating achievement of adequate intracellular levels of these agents. In addition, the assay called for in this study (i.e., assessment of tissue ethidium levels) requires a substantial mass of muscle (i.e., 75 mg), which cannot be provided by small, conventional diaphragm muscle strip preparations but is provided by the approach employed here.

On the day of study, rats were anesthetized with inhalational halothane and decapitated. The animals were strapped to a dissecting board, the chest was entered, and the thoracic aorta was cannulated with a 16-gauge angiocatheter. The aorta was then infused with gassed Krebs-Henselheit solution (pH 7.40, 135 mM NaCl, 5 mM KCl, 11.1 mM dextrose, 2.5 mM CaCl2, 1 mM MgSO4, 14.9 mM NaHCO3, 1 mM NaHPO4, and 50 U/l insulin, 95% O2-5% CO2). After cannulation the abdomen was opened, the distal aorta was ligated below the exit of the phrenic arteries, and the heart, lungs, and liver were removed. The arteries supplying the ribs associated with the right hemidiaphragm were ligated, and the ribs were removed. The right hemidiaphragm was then removed, quickly frozen in liquid nitrogen, and stored for later use (see below). The remaining tissue (consisting of the left hemidiaphragm, with the attached lower 9 ribs, the aorta, and a section of the spinal column) was removed en bloc and submerged in an organ bath containing Krebs-Henselheit solution with curare (50 mg/ml) added. At this point, aortic perfusion was discontinued and the aortic catheter was capped. This hemidiaphragm preparation was secured to a support in the base of the organ bath with steel pins. Three lengths of 3-0 silk suture were placed in the central tendon and connected to a rigid steel rod, which was in turn connected to a force transducer (model FT-10, Grass Instruments, Quincy, MA) mounted above the preparation. Electrical stimulation of the muscle preparation was accomplished by using two flat field electrodes. Each electrode consisted of a 2 × 3 cm piece of platinum mesh mounted on a plastic support and placed 5 mm on either side of the diaphragm. Each electrode was connected to an isolated current output stage (Biomedical Technology, Cleveland, OH) that was controlled by a stimulator (model S-48, Grass Instruments). Force output was displayed on a polygraph recorder (model RS3600, Gould, Cleveland, OH).

Assessment of Formation of ROS

This study employed a modification of a recently described fluorescent assay to measure ROS; previous workers have used this assay to examine production of ROS in the lung and in white blood cells (1, 9). This technique makes use of the fact that hydroethidine reacts with several oxygen-derived free radicals to yield ethidium, and the ethidium so produced fluoresces at an emission wavelength of 585 nm when excited with a wavelength of 465 nm. Superoxide anions and peroxynitrite radicals have been shown to react avidly with hydroethidine to form ethidium; in addition, hydrogen peroxide and hydroxyl radicals also oxidize hydroethidine but to a lesser extent than superoxide or peroxynitrite. Because this assay detects all four of these radical species, formation of ethidium in the presence of hydroethidine was taken as an index of formation of this entire group of ROS in the present study.

Because the conversion of hydroethidine to ethidium has only recently been employed to assess the generation of ROS, there are few data concerning the effects of many physiological and environmental variables on this assay. During muscle contraction, for example, many intracellular alterations occur that may influence ethidium measurements: pH changes, muscle temperature rises, a variety of enzyme systems are upregulated, calcium levels fluctuate, and phosphate and creatine phosphokinase concentrations increase. The conditions under which each experiment is conducted (i.e., preparation temperature, ambient light exposure, concentration of hydroethidine infused, and assay techniques) may also have an impact on assay results. Finally, cellular antioxidant status (i.e., cellular levels of glutathione and vitamin E) may also affect or interfere with ethidium formation. It is theoretically possible that physiologically relevant concentrations of these compounds may preferentially react with formed ROS, thereby preventing reaction of these species with hydroethidine and masking detection of ROS generation in tissues.

We conducted a number of preliminary studies to address the above concerns. We first performed studies to determine whether the presence or absence of enzymes, metals, and other cellular constituents might directly interfere with ethidium fluorescence (Table 1). The concentrations of copper, zinc, transferrin, lactoferrin, calpain, phosphorylase kinase, creatine phosphokinase, phosphorylase A, and phosphorylase B tested (Table 1) were chosen on the basis of reports indicating that these concentrations are similar to the actual levels of these substances in normal tissues (3, 7, 10, 15, 16, 21, 22, 26). Briefly, we found that 1) incubation of ethidium in solutions of varying pH and calcium concentration (i.e., pH 5-8 and 0-5 mM calcium were examined) did not substantially alter the fluorescent signals obtained, 2) incubation in solutions containing Fe2+, transferrin, Cu2+/Cu3+, Zn2+, or any one of several other ions and enzymes for 30 min did not substantially alter ethidium determinations, 3) incubation in calpain for 30 min had no significant effect on ethidium fluorescent signals, and 4) addition of high concentrations of free phosphate (i.e., HPO2-4 or HPO-4) had no effect on ethidium signals.

                              
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  		Table 1.   Effect of various agents on ethidium fluorescence

We also studied the effects of a number of experimental variables. We found that 1) tissue samples containing ethidium may be stored for extended periods at -70°C without any alteration in fluorescent signal magnitude, 2) there is a small degree of autoxidation of hydroethidine if this compound is kept in physiological solution at 37°C for protracted periods (i.e., a 10% oxidation over 2 h at 37°C) or exposed to fluorescent lights for long periods of time, 3) there is essentially no autoxidation of hydroethidine to ethidium during incubation at room temperatures in a darkened room for >2 h, and 4) repeated measurement (up to 15 determinations in a spectrophotofluorometer) does not alter ethidium concentrations in a sample, indicating that this level of exposure to light is insufficient to promote substantial photobleaching. We also found that the oxidation of hydroethidine to ethidium by an in vitro superoxide-generating solution (xanthine oxidase/hypoxanthine) was not affected by the addition of physiological levels of reduced glutathione (1,000 nmol/ml) or a water-soluble analog of vitamin E (Trolox, 100 µM). These latter data suggest that physiological muscle antioxidant concentrations should not interfere with reaction of ROS with hydroethidine and, therefore, would also not mask detection of free radical formation as inferred by formation of ethidium in the presence of hydroethidine.

In addition to the preceding investigations, we performed several "in situ" studies to determine how varying infused hydroethidine concentration affects the ethidium formed during diaphragm contractions. For these preliminary studies, hemidiaphragm preparations were used and diaphragms were electrically stimulated with trains of 20-Hz impulses at a train rate of 15 min-1 (train duration 0.5 s) for 10 min. We found no difference in the amount of ethidium formed over a range of hydroethidine concentrations from 5 to 40 µM in these experiments, arguing that doses of hydroethidine in this range are sufficient to prevent substrate limitation of ethidium formation during diaphragm contraction.

We also examined the effect of "spiking" tissue samples with a range of levels of additional ethidium and hydroethidine before sample preparation to determine whether such "internal standards" would quantitatively add to ethidium signals (i.e., if our method of tissue preparation eliminated some ethidium and hydroethidine or caused an interconversion of these species, then we should have difficulty recovering these internal standards). We found good recovery of added internal standards (i.e., 97% recovery of "added" standards), indicating that our assay does not "degrade" or fail to detect ethidium in muscle samples (Fig. 1).


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  		Fig. 1.   Ethidium recovered as a function of amount of ethidium added to diaphragm homogenates.

In view of all the above findings, we performed all experiments at room temperature, minimized ambient light exposure of diaphragm preparations, carried out all tissue preparations for the ethidium assay in the cold in a darkened room, and examined only the ethidium signal in contracting muscle (because hydroethidine was infused in high concentrations, we thought it inaccurate to attempt to assess ROS formation by measuring decrements in the hydroethidine signal). When this assay was performed, 75 mg of each hydroethidine-infused muscle were pulverized under liquid nitrogen, added to 1 ml of ice-cold saline solution, and homogenized by using a Polytron homogenizer. One milliliter of ethanol was added to the homogenate, and the mixture was incubated on ice for 15 min. After incubation, each sample was centrifuged at 10,000 g for 15 min to remove particulate matter. The resulting supernatant was removed and analyzed in an Aminco-Bowman spectrophotofluorometer (American Instrument, Silver Spring, MD) with an excitation wavelength of 465 nm and an emission wavelength of 585 nm. Readings obtained were normalized for tissue weight (in mg).

To correct for tissue autofluorescene when carrying out this procedure, we corrected the signal generated by the hydroethidine-infused muscle (as described above) for the signal generated by muscle not infused with hydroethidine. For this latter determination, a portion of diaphragm muscle was excised in each experiment before hydroethidine infusion; this tissue was processed as described above and used as a blank to assess the signal generated by the hydroethidine-infused muscle. The resulting difference was converted to ethidium concentration by means of a standard curve and expressed as nanograms of ethidium per milligram of tissue.

Experimental Protocol

When conducting this study, we thought it important to first demonstrate that the marker of ROS formation chosen for this work (ethidium) increases in response to the repetitive stimulation regimen chosen for these experiments and that this fluorescent signal was reduced by the administration of an intracellular scavenger of free radicals. This work was carried out in part A studies, which consisted of three groups of experiments including 1) examination of ethidium formation in noncontracting hemidiaphragms infused with Krebs-Henselheit solution alone, 2) examination of ethidium formation in preparations electrically stimulated to undergo repetitive isometric contractions for 10 min, and 3) examination of ethidium formed in contracting hemidiaphragms infused with physiological solution containing Tiron, an intracellular superoxide scavenger.

For these studies, hemidiaphragms were first prepared as described above. Hemidiaphragm muscle length was then adjusted to the length at which twitch force generation was maximal, and current was adjusted to a level ~20% greater than that required to achieve maximum force. The preparation was allowed to rest for 10 s, then one twitch and one 100-Hz stimulation (800-ms duration, 0.25 trains/s) were applied for baseline force measurement. The preparation was then allowed to equilibrate for 30 min. With use of a peristaltic pump, 5 ml of saline solution containing 40 µM hydroethidine (for noncontracting and contracting non-Tiron-treated groups) were infused into the preparation. For the Tiron-treated experimental group, diaphragms were infused with solutions containing 40 µM hydroethidine and 10 mM Tiron. After infusion, preparations were incubated for 10 min to allow penetration of hydroethidine into all muscle fibers (to further ensure maintenance of adequate hydroethidine levels in muscle throughout the experimental trials, we did not "flush" the vascular space after this initial hydroethidine infusion). Muscle force-generating capacity was then examined by the construction of a force-frequency curve consisting of sequential stimulation with trains of impulses at stimulation frequencies of 1, 10, 20, 50, and 100 Hz; train duration for these contractions was 800 ms, and 5-s rests were provided between stimulus trains. After a 30-s rest, muscles were stimulated to undergo repetitive contraction for 10 min (this employed repetitive stimulation with 20-Hz trains, 0.25 trains/s, 500-ms train duration). After the conclusion of this repetitive stimulation trial, the hemidiaphragm was removed, quick frozen by using a -51°C spray (Histofreeze, Fisher Scientific), placed in a preweighed Eppendorf tube, and submerged in liquid nitrogen. Muscle weight was determined by reweighing the Eppendorf tube. Samples were stored at -70°C until ethidium analysis (as described above), which was accomplished within 48 h.

In part B we assessed the effect of a variety of PLA2 inhibitors on ethidium formation in the contracting diaphragm. A large number of PLA2 inhibitors have been described, with these various agents differing with regard to their specificity and cell penetrability. The four chosen for study in the present experiment were selected, because 1) this combination of agents tests the effect of inhibition of each of the major groups of PLA2 isoforms (i.e., the 14-kDa isoform, the 85-kDa isoform, and the calcium-independent isoform) and 2) all these agents have good cellular penetrability and have been shown previously to inhibit PLA2 in intact tissues when used in a fashion similar to that employed here. Part B experiments were divided into six groups: 1) noncontracting hemidiaphragms, 2) contracting hemidiaphragms, 3) contracting hemidiaphragms treated with an inhibitor of the 14-kDa isoform of PLA2 [manoalide, 1 µM (23)] 4) contracting hemidiaphragms treated with another inhibitor of 14-kDa PLA2 (aristolochic acid, 1 mM) (29), 5) contracting hemidiaphragms treated with an inhibitor of the 85-kDa isoform of PLA2 [arachidonyltrifluoromethyl ketone (AACOCF3), 40 µM] (4), and 6) contracting hemidiaphragms treated with an inhibitor of the calcium-independent isoform of PLA2 [haloenol lactone suicide substrate (HELSS), 25 µM] (27). The protocol used for part B experiments was identical to that used for part A studies, except one of the above PLA2 inhibitors was infused into the preparation, in conjunction with 40 µM hydroethidine, at the appropriate time.

Data Analysis

Hemidiaphragm force was normalized for muscle cross-sectional area as follows (9)
Force/cm<SUP>2</SUP> = force × muscle length × 1.06/muscle weight

Statistical Analysis

A one-way ANOVA was used to compare single variables (e.g., ethidium levels) across animal groups, with post hoc testing used to determine statistical differences between individual groups.

A repeated-measures ANOVA was used for comparisons in which repeated measurements of a given variable were made under different conditions (e.g., force-frequency curves from different groups).

Values are means ± SE. P < 0.05 was taken to indicate statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Part A

Diaphragm force generation during Tiron experiments. The force-generating ability of preparations in part A studies was determined before the infusion of hydroethidine and/or Tiron. Baseline twitch forces were similar for the Tiron- and non-Tiron-treated groups. Specifically, the twitch forces of the non-Tiron- and the Tiron-treated groups averaged 7.48 ± 0.21 and 7.65 ± 0.36 N/cm2, respectively. Likewise, there was no difference in baseline 100-Hz force between these groups of experiments: 100-Hz force was 24.07 ± 0.56 and 25.70 ± 0.75 N/cm2 for non-Tiron- and Tiron-treated groups, respectively.

Tiron also had no significant effect on twitch kinetics or on the diaphragm force-frequency relationship (Table 2, Fig. 2). Tiron did, however, have a small effect on diaphragm force generation during repetitive contraction trials (i.e., during 10 min of repetitive electrical stimulation). As seen in Table 3, diaphragm muscles infused with Tiron displayed a slightly greater force at the 5-min point (i.e., the midpoint) and the conclusion of repetitive contraction trials (P < 0.02 for comparison at 5 min and P < 0.03 for comparison at 10 min).

                              
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  		Table 2.   Twitch kinetics



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  		Fig. 2.   Diaphragm force as a function of electrical stimulation frequency for diaphragm preparations studied in absence (open circle ) and presence () of Tiron. Error bars, SE.


                              
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  		Table 3.   Force during repetitive contraction trials

Effect of Tiron on diaphragm ethidium concentrations. The results of the ethidium analyses for part A studies are displayed in Fig. 3. Noncontracting hemidiaphragm preparations had very low ethidium levels, averaging 2.0 ± 0.8 ng/mg tissue. In contrast, ethidium concentrations were much higher (9.5-fold) for non-Tiron-treated diaphragm preparations that underwent repetitive contraction trials (P < 0.001 for comparison of ethidium in non-Tiron-treated contracting and noncontracting muscles). This contraction-related increase in ROS generation, as assessed from ethidium formation, was entirely ablated by the infusion of Tiron before muscle stimulation. Specifically, ethidium concentration for Tiron-treated contracting diaphragm preparations was 2.0 ± 1.0 ng ethidium/mg tissue, a value much lower than that achieved in non-Tiron-treated contracting muscle (P < 0.001 for this comparison) and similar to that observed in noncontracting diaphragms.


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  		Fig. 3.   Ethidium levels measured in muscle homogenates from 3 groups of diaphragm preparations: noncontracting diaphragms, repetitively contracting diaphragms studied in absence of Tiron, and repetitively contracting diaphragms treated with Tiron.

Part B

Diaphragm force generation during PLA2 inhibitor experiments. As expected, the twitch and 100-Hz force generation of part B diaphragm preparations before hydroethidine and PLA2 inhibitor infusion were similar. Twitch forces were 7.50 ± 0.20, 7.97 ± 0.38, 7.02 ± 0.48, 7.41 ± 0.56, and 6.60 ± 0.38 N/cm2 for contracting nontreated preparations, contracting preparations treated with manoalide, contracting preparations treated with aristolochic acid, contracting preparations treated with AACOCF3, and contracting preparations treated with HELSS, respectively. Force generation in response to 100-Hz stimulation was 25.40 ± 0.78, 27.05 ± 0.62, 26.00 ± 1.47, 26.93 ± 0.81, and 25.00 ± 1.18 N/cm2, respectively, for these same groups.

Twitch kinetics measured after the infusion of hydroethidine and/or PLA2 inhibitors were the same in these groups (Table 2). The infusion of inhibitors of different PLA2 isoforms into hemidiaphragm preparations also did not produce appreciable effects on the diaphragm force-frequency relationship, as shown in Fig. 4.


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  		Fig. 4.   Diaphragm force as a function of electrical stimulation frequency for diaphragm preparations studied in absence (open circle , n = 7) and presence of several different phospholipase A2 inhibitors: manoalide (, n = 7), aristolochic acid (, n = 5), arachidonyltrifluoromethyl ketone (, n = 5), and haloenol lactone suicide substrate (triangle , n = 5).

Diaphragm force generation during repetitive stimulation trials performed in the absence and presence of the different PLA2 inhibitors is shown in Table 3, which displays the diaphragm force produced in response to 20-Hz stimulation at the beginning (0 min), middle (5 min), and end (10 min) of these trials. Force generation at the initiation of trials (0 min) was similar across all experimental groups, as was force generation at the trial midpoint (i.e., at 5 min). At trial conclusion (10 min), the force generated by the manoalide-treated group was slightly higher than the force generated by the non-PLA2 inhibitor-treated group (P < 0.05), but the forces generated by groups treated with the other three PLA2 inhibitors (aristolochic acid, AACOCF3, and HELSS) were similar to those generated by the non-PLA2 inhibitor-treated group.

Effect of PLA2 inhibitors on diaphragm ethidium concentrations. As for part A studies, ethidium concentrations for diaphragm preparations undergoing repetitive contraction but not treated with any of the PLA2 inhibitors were much higher than ethidium levels for noncontracting muscles: ethidium concentrations were 2.0 ± 0.8 ng/mg tissue for noncontracting preparations and 20.0 ± 2.0 ng/mg tissue for contracting hemidiaphragms not treated with a PLA2 inhibitor (P < 0.001; Fig. 5). Ethidium levels were also high for contracting, AACOCF3-treated muscles and for contracting, HELSS-treated muscles: 14.0 ± 2.3 and 20.9 ± 9.0 ng/mg, respectively. Treatment with manoalide (a 14-kDa PLA2 inhibitor), however, prevented ethidium formation in contracting hemidiaphragm preparations (P < 0.001 for this comparison; Fig. 5). Aristolochic acid also inhibited contraction-related formation of ethidium (P < 0.001 for comparison of contracting diaphragms treated with aristolochic acid and muscles contracting but receiving no PLA2 inhibitor).


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  		Fig. 5.   Ethidium levels measured in muscle homogenates from several groups of diaphragm preparations, including (from left to right) noncontracting diaphragms, repetitively contracting diaphragms studied in absence of phospholipase A2 inhibitor administration (n = 7) and repetitively contracting diaphragms treated with manoalide (n = 7), aristolochic acid (n = 5), arachidonyltrifluoromethyl ketone (AACOCF3, n = 5), or haloenol lactone suicide substrate (HELSS, n = 5).

In Vitro Conversion of Hydroethidine to Ethidium

The various inhibitors employed in this study (manoalide, aristolochic acid, HELSS, AACOCF3, and Tiron) were incubated in vitro with hydroethidine or ethidium for 10 min to determine whether any of these substances could degrade ethidium or interfere with signals from these compounds. We found that none of these chemicals altered hydroethidine fluorescence or resulted in degradation of the ethidium signal. In addition, when each of these substances was added to hydroethidine and a xanthine oxidase/hypoxanthine superoxide-generating solution, none of these agents interfered with the conversion of hydroethidine to ethidium.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that administration of manoalide and aristolochic acid, inhibitors of the 14-kDa PLA2 isoform, ablates contraction-related production of ROS by the diaphragm. We also found that AACOCF3, an inhibitor of the 85-kDa cytosolic PLA2 isoform, and HELSS, an inhibitor of the calcium-independent PLA2 isoform, had no effect on contraction-related diaphragm ROS formation.

This study made use of a recently described fluorescent indicator assay to monitor intracellular ROS generation by the diaphragm, and the potential limitations of this assay should be considered (1, 9). This assay is predicated on the fact that hydroethidine, a molecule that easily penetrates cell membranes, reacts with relatively high avidity with several free radical species (superoxide anions, peroxynitrite, hydrogen peroxide, and hydroxyl radicals) to form ethidium, a highly polar compound that does not cross membranes. As a result, the ethidium formed by reaction of hydroethidine with intracellular ROS species remains "trapped" intracellularly.

Our assay for ethidium involves disruption of diaphragm cells and precipitation of cellular proteins with an ethanol technique; the resulting supernatant is then assessed spectrophotofluorometrically for ethidium content. We have found that exogenous ethidium spiking of biological samples results in quantitative recovery of added ethidium (i.e., the ethidium measured equals that of the exogenous standard plus the tissue level when measured separately) and that in biological membranes remaining after our ethanol extraction technique no ethidium is present, as assessed by using fluorescent microscopy. In addition, we have found that addition of various concentrations of a variety of organic molecules and electrolytes does not interfere with measurement of ethidium assessed in this manner. Moreover, neither manoalide, aristolochic acid, AACOCF3, nor HELSS alters measurements of ethidium in vitro, nor do these substances alter generation of ethidium during incubation of a free radical-generating solution (xanthine oxidase/hypoxanthine) with hydroethidine.

A potential criticism of this approach is that the fluorescent indicator chosen does not detect a single species of free radical but measures a variety of ROS products (i.e., peroxynitrite, superoxide, hydrogen peroxide, and hydroxyl radicals). In a sense, however, this can also be viewed as an advantage. All the species previously shown to react with hydroethidine in biological systems are derived from superoxide; i.e., peroxynitrite is generated by reaction of superoxide with nitric oxide, hydrogen peroxide is formed by the dismutation of superoxide, and hydroxyl radical is formed by a metal-catalyzed reaction of superoxide with hydrogen peroxide (17). As a result, this assay can be viewed as an index of formation of all the important reactive biological species formed from superoxide.

One problem with our approach, however, is the fact that hydroethidine administered in the quantities used (we used excess hydroethidine so that formation of ethidium would not be substrate limited by hydroethidine) acts, in effect, as an ROS scavenger. As a result, ROS formed in diaphragm during contraction in all experimental groups examined in this study may well have been scavenged before having access to any cellular constituents. This fact makes it impossible to determine the extent to which PLA2 inhibition of free radical formation has functional consequences by using the present experimental approach. Although it is true that Tiron administration resulted in a diminution in the fall of diaphragm force during repetitive contraction trials in the present study, this effect was very small. Moreover, there was no physiologically important difference between the fall of diaphragm force over time during repetitive contraction trials carried out in the presence and absence of the four PLA2 inhibitors tested in this study. This similarity of rate of fall of force over time across these various experimental groups is likely due to the fact that diaphragms in all experimental groups contained relatively high concentrations of hydroethidine.

One might ask whether the failure of HELSS or AACOCF3 administration to reduce ethidium formation in the contracting diaphragm was due to an insufficient dose of these agents (i.e., a type II error). The doses of HELSS and AACOCF3 chosen for the present study are based on previous work demonstrating a clear efficacy of these agents in these doses to suppress calcium-independent PLA2 and 85-kDa PLA2 isoform activity (4, 27). If anything, the doses used for this testing are in what is considered a "high" range for these agents; doses any higher than those used here are associated with nonspecific effects that are not related to PLA2 antagonism. We therefore believe that it is unlikely that the failure of these agents to alter ROS generation was on the basis of insufficient dose.

Comparison to Previous Studies

The present study demonstrates production of ROS in the contracting diaphragm by use of a probe that preferentially detects superoxide and its metabolic products. This finding is consistent with a number of previous studies that have provided direct and indirect evidence of free radical generation in the respiratory muscles in response to strenuous contractions. Specifically, electrically induced contractions of in vitro respiratory muscle preparations have previously been shown to result in the release of superoxide anions from myocytes (as assessed by measuring superoxide dismutase-suppressible cytochrome c reduction rates) and to increase intramuscular hydroxyl anion concentrations (assessed by measuring 2,3-dihydroxybenzoic acid formation in salicylate-superfused preparations) (14, 28). In addition, lipid peroxidation by-products (thiobarbituric acid-reactive products and 8-isoprostane) and protein oxidation products (glutathione oxidation and formation of protein carbonyls) have been detected in the respiratory muscles of intact animals in which the respiratory workload was increased through the application of external inspiratory resistive loads (2, 32, 33).

Potential Mechanisms of Free Radical Formation in Contracting Skeletal Muscle

The metabolic pathways responsible for generating free radicals in contracting respiratory muscles have not been determined in the past studies referenced above. Some authors have suggested, however, that likely sources for free radical generation by contracting muscle include mitochondrial (i.e., superoxide generation by the electron transport chain) and nonmitochondrial (membrane-bound NADPH oxidase, membrane-bound NADH oxidase, xanthine oxidase, cyclooxygenase, lipooxygenase, and cytochrome P-450) enzyme systems (5, 8, 30). With regard to the former possibility, it is known that superoxide radicals are normally generated in mitochondria as a by-product of respiration due to one-electron transfers to oxygen by the NADH dehydrogenase and semiquinone components of the electron transport chain (11). With resting levels of respiration, it is thought that intramitochondrial scavenging systems are sufficient to prevent "leakage" of superoxide or its reaction products from the mitochondria. It has been postulated that exercise leads to an increase in this "normal" superoxide formation by the electron transport chain, overwhelming local scavenging defense systems and causing damage to cellular organelles (13). In further support of this potential mechanism of contraction-induced free radical generation, several studies have indicated that measures that increase concentrations of mitochondrial antioxidants (e.g., vitamin E supplementation) blunt muscle oxidative damage during strenuous exercise (19, 37).

The present work extends these previous studies by indicating a role for PLA2 as a modulator of free radical generation in contracting muscle. In fact, we found that pharmacological PLA2 inhibition resulted in complete suppression of free radical generation by contracting muscle, arguing that contraction-related ROS generation in muscle is largely derived from PLA2-dependent reaction pathways.

There are several potential mechanisms by which PLA2 may exert such an effect. One possibility is that activation of PLA2 influences free radical generation by the mitochondrial electron transport chain. In support of this theory, PLA2 inhibitors have been shown to ablate calcium-stimulated free radical generation by isolated mitochondrial suspensions, whereas addition of arachidonic acid to mitochondrial electron transport chain components stimulates free radical generation (36). It is also known that muscle mitochondrial calcium concentrations rise with strenuous exercise, providing a possible stimulus for activation of mitochondrial PLA2 (35). Taking these various observations together, one could account for all these findings by postulating that contraction elicits free radical formation by the following mechanistic sequence: 1) contraction results in increases in cytosolic calcium levels and reductions in cytosolic ATP concentrations, 2) reductions in ATP lead to preferential uptake of calcium by mitochondria, 3) rising mitochondrial calcium concentrations activate mitochondrial PLA2, leading to arachidonic acid formation, and 4) arachidonic acid interaction with the electron transport chain elicits an increase in free radical formation (19, 35-37).

PLA2 could also modulate free radical generation by increasing substrate (arachidonic acid) for eicosinoid formation. Specifically, PLA2 catalyzes the hydrolysis of sn-2 fatty acyl chains of phospholipid substrates to generate fatty acids (including arachidonic acid) and other lipid products (e.g., lysophospholipids) (24) that act as substrate, in turn, for cyclooxygenase- and lipooxygenase-catalyzed reaction pathways that can generate free radicals as a by-product. One argument against this mechanism of free radical generation in muscles, however, is the previous observation that administration of cyclooxygenase inhibitors does not blunt superoxide release from contracting muscle in vitro (31).

Another possibility is that PLA2 may modulate free radical generation at a cell surface site in muscle that contains a free radical-generating complex akin to that present in neutrophils. In neutrophils, recent studies have indicated that PLA2 activation triggers free radical generation by stimulating a cell surface NADPH oxidase complex (23). This work has shown that manoalide administration reduces white cell NADPH complex-mediated superoxide generation by 95%, a finding reminiscent of the effectiveness of this inhibitor in suppressing superoxide generation by the contracting diaphragm in the present study (23). It has also been shown recently that an NADPH-dependent membrane-associated free radical-generating enzyme system is present in endothelial cells (38). It is conceivable that a similar membrane-associated enzyme system exists in muscle and accounts for the PLA2 dependence of contraction-related superoxide production or, alternatively, that shear stresses resulting from muscle contraction activate an NADPH-dependent free radical-forming cell surface system in endothelial cells.

The present study was not designed to determine whether one or all of the above potential mechanisms are responsible for the PLA2 dependence of free radical generation in contracting muscle, and additional studies are required to sort out these possibilities. One additional clue as to the potential mechanism and site of PLA2-dependent free radical formation is provided, however, by the pattern of response to the various PLA2 inhibitors tested in this study. The 14-kDa isoforms of this enzyme are located preferentially in the mitochondria and the inner face of the sarcolemmal membrane (39). On the other hand, the 85-kDa isoform of PLA2 is found in the cytosol, whereas the calcium-independent PLA2 isoform is thought to be localized to the cytosol and sarcoplasmic reticulum (39). Because only inhibition of the 14-kDa isoform reduced contraction-related free radical formation in the present study, it is reasonable to postulate that contraction-related free radical formation in muscle may be localized to muscle mitochondria or to the sarcolemmal membrane, the two sites at which the 14-kDa PLA2 isoform is concentrated.


    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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Supinski, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109.

Received 8 June 1998; accepted in final form 20 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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