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Pulmonary Division, Department of Medicine, Case Western Reserve University and MetroHealth Medical Center, Cleveland, Ohio 44109
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological conditions. Because the degree of muscle dysfunction observed in some of these conditions appears to be both free radical dependent and modulated by extracellular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations. For this reason, we compared formation of reactive oxygen species (ROS; i.e., free radicals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 trains/s) incubated in organ baths filled with physiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM) calcium levels. Generation of ROS was assessed by measuring the conversion of hydroethidine to ethidium. We found ROS generation with contraction varied with the extracellular calcium level, with low ROS production (3.18 ± 0.40 ng ethidium/mg tissue) for low-calcium studies and with much higher ROS generation for normal-calcium (18.90 ± 2.70 ng/mg) or high-calcium (19.30 ± 4.50 ng/mg) studies (P < 0.001). Control, noncontracting diaphragms (in 2.5 mM calcium) had little ROS production (3.40 ± 0.80 ng/mg; P < 0.001). To further investigate this issue, we added nimodipine (20 µM), an L-type calcium channel blocker, to contracting diaphragms (2.5 mM calcium bath) and found that nimodipine also suppressed ROS formation (2.56 ± 0.85 ng ethidium/mg tissue). These data indicate that ROS generation by the contracting diaphragm is strongly influenced by extracellular calcium concentrations and may be dependent on calcium transport through L-type calcium channels.
skeletal muscle; respiratory muscles
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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EXTRACELLULAR CALCIUM concentrations have been shown to be an important determinant of the degree of cellular injury resulting from a variety of pathophysiological stresses in a number of different types of tissue, including skeletal muscle (12, 14, 18, 26). Jackson et al. (12), for example, found that the amount of cellular injury [gauged by measuring cellular leakage of lactate dehydrogenase (LDH)] observed in mouse extensor digitorum longus (EDL) and soleus muscles in response to exposure to hypoxic solutions was directly proportional to extracellular calcium concentration. Similar observations have been made in cardiac muscle, where the degree of cellular damage induced by periods of ischemia-reperfusion also appears to be dependent on extracellular calcium levels (21).
A number of different hypotheses have been advanced to explain the influence of calcium levels on cellular injury. Intracellular proteolytic systems (e.g., calpain) are highly calcium dependent, and activation of these enzyme systems may promote protein degradation with resultant alterations in cellular metabolism and structural stability (3). Elevations of cellular calcium levels also result in activation of phospholipases, which break down cellular lipids, causing membrane instability and rupture (19). In addition, high calcium levels can adversely affect the function of several enzymes involved in glycolysis and oxidative phosphorylation, reducing cellular energy stores (10).
Recent studies have indicated that free radicals may play an important, if not central, role in the development of many forms of muscle dysfunction, including that resulting from strenuous muscle contractions, that observed after prolonged periods of ischemia-reperfusion, and that associated with endotoxin-induced sepsis (2, 8, 25, 29, 30, 32-35). Because the degree of muscle dysfunction resulting from some of these stresses (e.g., ischemia-reperfusion) appears to be both free radical dependent and modulated by extracellular calcium concentration, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations.
The purpose of the present experiment was, therefore, to test the hypothesis that generation of reactive oxygen species (ROS) by contracting skeletal muscle is dependent on extracellular calcium concentrations. Studies were performed by using in vitro rat diaphragms incubated in physiological solutions containing low, medium, or high concentrations of calcium, and formation of ROS in these muscles was assessed by using a recently described fluorescent indicator (1, 5). To test the specificity of the fluorescent assay used in these studies, we also examined the effect of Tiron, an intracellular superoxide scavenger; superoxide dismutase (SOD); and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on the fluorescent signal generated by contracting muscle.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Care
Studies were performed by using 25 adult male Sprague-Dawley rats. These animals were housed in the Animal Resource Center of Case Western Reserve University. As per Accreditation of Laboratory Animal Care guidelines, rats were examined daily by veterinarians. Food and water were allowed to the animals ad libitum before experimental use.Experimental Preparation
The present study employed an in vitro diaphragm preparation recently developed in our laboratory (22). This consists of a large hemidiaphragm strip prepared in such a way that the ribs, spinal column and aorta remain attached to the diaphragm, making it possible to arterially infuse pharmacological agents into this muscle. For the purposes of the present study, this preparation was submerged in an organ bath containing physiological solution bubbled with oxygen and carbon dioxide. Use of this particular experimental preparation has several advantages for a study, such as the following: 1) intramuscular delivery of a fluorescent indicator can be accomplished effectively because indicators can be infused into the vascular supply of the diaphragm, permitting rapid and uniform diffusion into muscle cells; 2) penetration of pharmacological agents (e.g., nimodipine in this study) into muscle cells is facilitated, allowing rapid equilibration and achievement of high tissue levels; and 3) compared with single-fiber preparations, which are an alternative means of ensuring that indicators and drugs have adequate contact with muscle cells, our hemidiaphragm preparation allows a large muscle mass to be studied (up to 300 mg), facilitating performance of complex biochemical analyses (e.g., ethidium assessment) that require substantial amounts of tissue.As a first step when preparing the diaphragm in this fashion, animals
were anesthetized with halothane and killed by
decapitation. The thoracic aorta was cannulated with a
16-gauge angiocatheter and infused with gassed physiological solution
(the electrolyte composition of solutions will be provided in
Experimental Protocols). The abdomen
was subsequently opened; the distal abdominal aorta was ligated; and
the heart, lungs, and liver were removed. The left hemidiaphragm was
then isolated, with special care taken to ensure that the intercostal
and phrenic arteries perfusing this portion of the diaphragm were left
in continuity with the aorta. The right hemidiaphragm was removed,
frozen in liquid nitrogen, stored at 
70°C, and used for
subsequent biochemical analysis. The intercostal vessels and associated
ribs connected to the right hemidiaphragm were ligated, and these ribs
were removed. The remaining tissue (left hemidiaphragm, the attached
aorta, a section of the spinal column, and the lower 9 ribs) was placed
in a large organ bath containing physiological solution containing
curare (50 mg/ml). Aortic perfusion was then discontinued, and the
aortic catheter was capped. After the preparation was secured to the
base of the organ bath, two ties were placed in the central tendon.
These ties were connected to a rigid steel rod suspended from Grass FT10 force transducer (Grass Instruments) mounted above the organ bath.
The transducer was attached to an adjustable transducer positioner
(Radnoti Glass, Monrovia, CA); diaphragm muscle length was adjusted by
raising or lowering the transducer. Platinum mesh field electrodes were
placed on each side of the hemidiaphragm preparation so that the
distance between each electrode and the muscle was 5 mm. These
electrodes were connected to an isolated current output stage
(Biomedical Technology Corporation of America, Cleveland, OH) attached,
in turn, to a Grass S48 stimulator (Grass Instruments).
Assessment of Formation of ROS
This study employed a derivation of a recently described fluorescent assay to measure ROS (5). Previous studies (1, 5) have used this assay to examine the role of ROS in the lung and in white blood cells. Hydroethidine, the reduced form of ethidium, reacts with oxygen-derived free radicals to yield ethidium. The ethidium so produced fluoresces at an emission wavelength of 585 nm when excited with a wavelength of 465 nm. Superoxide and peroxynitrite radicals have been shown to react avidly with hydroethidine. Comparatively, hydrogen peroxide also oxidizes hydroethidine but to a lesser extent than does either superoxide or peroxynitrite. Because these radicals are all derived from the production of superoxide, the formation of ethidium from the reaction of hydroethidine and free radicals is taken to indicate the presence of ROS. On the basis of the above rationale, we took the conversion of hydroethidine to ethidium in the present study to indicate the production of ROS.Because the conversion of hydroethidine to ethidium has only recently been employed to assess the generation of ROS, few data exist concerning the effects of many physiological and environmental variables on this assay. During muscle contraction, for example, many intracellular alterations occur that may influence either hydroethidine or ethidium fluorescence (i.e., pH changes, muscle temperature increases, a variety of enzyme systems are upregulated, calcium levels fluctuate, phosphate and creatine phosphokinase concentrations rise). The conditions under which each experiment is conducted may also have an impact on assay results (i.e., preparation temperature, ambient light exposure, the concentration of hydroethidine infused). Last, cellular antioxidant status may interfere with ethidium formation. The two most important antioxidants contained in cells are glutathione and vitamin E (25, 26). Physiologically relevant concentrations of these compounds may alter the assessment of ROS generation by using hydroethidine.
We performed a number of preliminary studies to address the above
concerns (Table 1). When examining a number
of cellular and physiological variables, we found that
1) incubation of ethidium under
conditions of varying pH and calcium concentration (i.e., a pH range of
5-8 and calcium concentrations from 0 to 5 mM) did not
substantially alter the ethidium fluorescent signal;
2) incubation of ethidium in
solutions containing Fe2+,
transferrin,
Cu2+/Cu3+,
Zn2+, or calpain for 30 min did
not substantially alter ethidium determinations, 3) addition of high concentrations
of free phosphate (i.e., either HPO+4 or
HPO
4) had no
effect on ethidium signals; 4)
tissue samples may be stored for extended periods at 70°C
without an alteration in the magnitude of ethidium fluorescent signals;
5) hydroethidine undergoes
autooxidation if it is kept in physiological solution at 37°C for
protracted periods (i.e., a 10% oxidation over 2 h at 37°C) or if
it exposed to fluorescent light for long periods; and
6) there is no autooxidation of
hydroethidine to ethidium during incubation at room temperatures in a
darkened room for 2 h. We also found that the oxidation of hydroethidine to ethidium by a superoxide generating solution (xanthine
oxidase/hypoxanthine) was not affected by the addition of
physiologically relevant levels of either GSH (1,000 nmol/ml) or a
water-soluble analog of vitamin E (Trolox; 100 µM).
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We also performed a series of experiments to assess the potential role of superoxide in generating ethidium during muscle contraction. These investigations comprise protocol C and are described in detail below.
In view of the above findings, we performed all experiments at room temperature, minimized ambient light exposure of diaphragm preparations, and carried out all tissue preparations for the ethidium assay in the cold in a darkened room. Because we used high hydroethidine concentrations, we thought it best to assess ROS formation by measuring ethidium formation rather than disappearance of hydroethidine (the latter is difficult to assess by using this particular type of protocol). We also examined the effect of nimodipine on hydroethidine and ethidium fluorescence in vitro. This substance had no effect on either hydroethidine or ethidium fluorescence, and, moreover, addition of nimodipine to a mixture of hydroethidine and xanthine oxidase/hypoxanthine superoxide-generating solution did not affect ethidium formation rates.
Two muscle samples were obtained from each experimental preparation. The first sample (the right hemidiaphragm, which was obtained during the initial rat dissection and not infused with hydroethidine or nimodipine) was used to measure tissue autofluorescence at the wavelengths utilized for ethidium analysis. The second muscle sample (left hemidiaphragm) was obtained after the completion of the experimental protocol (including the infusion of hydroethidine), as will be described in detail below. A portion (75 mg) of each muscle was 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) by using an excitation wavelength of 465 nm and an emission wavelength of 585 nm. Readings obtained were normalized for tissue weight (in mg). For each experiment, the normalized value of the muscle sample obtained during preparation dissection (right hemidiaphragm) was subtracted from the value of the sample (left hemidiaphragm) frozen at the conclusion of the experimental protocol (in this way, the inclusion of background tissue fluorescence unrelated to ethidium formation was eliminated). The resulting difference was converted to ethidium concentration by means of a standard curve and expressed as nanograms of ethidium per milligram tissue.
Experimental Protocols
Three groups of experiments were carried out (protocols A, B, and C). Our basic study design was to 1) infuse hydroethidine into hemidiaphragm preparations, 2) electrically stimulate diaphragms to undergo repetitive isometric contractions for 10 min, and 3) assay the diaphragm for ethidium and use this measure as an index of formation of ROS. The details of each of these three protocols are described below.Protocol A. Four groups of isolated hemidiaphragm preparations were examined in this protocol: 1) hemidiaphragms studied while submerged in Krebs-Henselheit buffer containing 2.5 mM calcium (we will define this as our "normal" calcium condition; Krebs-Henselheit contains 135 mM NaCl, 5 mM KCl, 11.1 mM dextrose, 2.5 mM CaCl2, and 1 mM MgSO4, adjusted to a pH of 7.40) and receiving no electrical stimulation, 2) hemidiaphragms studied while submerged in Krebs-Henselheit containing 2.5 mM calcium and made to rhythmically contract in response to repetitive electrical stimulation, 3) hemidiaphragms studied while submerged in Krebs-Henselheit modified to contain 1 mM calcium ("low" calcium) and receiving electrical stimulation, and 4) hemidiaphragms studied while submerged in modified Krebs-Henselheit containing 5 mM calcium ("high" calcium) and receiving stimulation. When preparing solutions of 1 mM calcium and 5 mM calcium, we modified the composition of Krebs-Henselheit, increasing the NaCl to 136.5 mM for the 1 mM calcium to maintain osmolarity constant and decreasing the amount of NaCl to 132.5 mM for the 5 mM calcium solution.
After preparation of hemidiaphragms and submersion in the appropriate calcium solution, diaphragm muscle length was adjusted to L0 (defined as the length at which twitch force was maximal). At that time, current was also adjusted to achieve supramaximal levels (i.e., to 20% greater than that required to elicit maximal twitch force). After a 30-min equilibration period, diaphragmatic preparations were then infused with 40 µM hydroethidine and dissolved in 5 ml of Krebs-Henselheit solution containing the same calcium concentration as that in which the hemidiaphragm was submerged. Diaphragms were incubated for an additional 10 min to allow thorough penetration of hydroethidine into muscle fibers. We subsequently assessed diaphragm force-generating capacity by constructing a force-frequency curve; this was done by sequentially stimulating the diaphragm with trains of stimuli at 1, 10, 20, 50, and 100 Hz (with an 800-ms train duration) and allowing 5-s rests between adjacent trains. A repetitive contraction trial was then carried out by electrically stimulating muscles with trains of stimuli (20 Hz, 500-ms train duration) for 10 min. At the conclusion of the contraction trial, muscle length was measured, and the diaphragm was cut down, flash frozen in a preweighed tube, and then reweighed to obtain the strip weight. Diaphragm samples were stored at70°C until analysis
of ethidium content, as described above.
Protocol B. In this protocol, we examined five additional isolated hemidiaphragm preparations which were infused with nimodipine, a blocker of L-type calcium channels (36). This intervention was added once the results of protocol A studies were known and suggested that alterations in calcium concentrations affect free radical formation. The purpose of this latter group of studies was to determine whether similar alterations in generation of ROS could be achieved by the administration of a calcium channel blocking agent. The hemidiaphragm preparations studied with this second protocol were submerged in normal-calcium Krebs-Henselheit solution (i.e., 2.5 mM). After a 30-min equilibration period (as in protocol A), 40 µM hydroethidine and 20 µM nimodipine were infused into the diaphragm. The remainder of this second protocol was identical to that used for protocol A. Diaphragm function and ROS formation in this group of experiments were subsequently compared with results for protocol A studies of noncontracting and contracting diaphragm preparations bathed in 2.5 mM calcium solutions.
Protocol C. Protocol C studies were also undertaken once the results of protocol A studies were known. These studies examined whether superoxide (or one of its degradation species) or nitric oxide was responsible for the altered ethidium signals observed in response to differing calcium concentrations. The hemidiaphragm preparations studied in protocol C were submerged in normal-calcium Krebs-Henselheit solution (i.e., 2.5 mM). After a 30-min equilibration period (as in protocol A), 40 µM hydroethidine and either 1) Tiron (10 mg/ml), 2) SOD (4 U/ml), or 3) L-NAME (1 µg/ml) in 5 ml normal-calcium Krebs-Henselheit solution were infused into the diaphragm. Studies were then carried out in the same manner as those in protocol A. If superoxide (or one of its degenerative products) was responsible for influencing ethidium generation in response to diaphragm contraction in 2.5 mM calcium-containing Krebs-Henselheit solution, both SOD and Tiron should affect the ethidium signal. If, on the other hand, nitric oxide plays a role in the formation of ethidium from hydroethidine, L-NAME should alter ethidium generation by the contracting diaphragm.
Data Analysis
Hemidiaphragm force was normalized for muscle cross-sectional area by using the following formula (6)
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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).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Protocol A: Effect of Extracellular Calcium Concentration on Diaphragm Force Generation
The twitch kinetics of hemidiaphragms exposed to different extracellular calcium concentrations were similar. Specifically, contraction times were 80.0 ± 2.0, 80.7 ± 1.7, and 76.3 ± 3.2 ms for muscles submerged in low, normal, and high calcium, respectively. One-half relaxation times were 63.8 ± 2.4, 63.6 ± 1.8, and 60.0 ± 2.0 ms in the same groups.Similarly, altering the level of extracellular calcium had no effect on
initial diaphragm force generation, as assessed by force-frequency
curves. Figure 1 displays mean
force-frequency relationships for low-, normal-, and high-calcium
groups; forces generated in response to the entire range of applied
frequencies were similar in these three groups of experiments. For
example, twitch tensions were 5.86 ± 0.48, 5.90 ± 0.51, and
5.93 ± 0.69 N/cm2
for low-, normal-, and high-calcium studies, respectively. Forces produced in response to 100 Hz stimulation were 21.86 ± 0.39, 22.36 ± 0.99, and 23.77 ± 1.65 N/cm2 for low-, normal-, and
high-calcium groups.
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Diaphragmatic responses to 10 min of repetitive stimulation are plotted
in Fig. 2. Again, no significant
differences were observed between the different calcium groups with
regard to the absolute forces generated at each point in time during
these repetitive contraction trials.
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Protocol A: Effect of Extracellular Calcium Concentration on Diaphragm Ethidium Formation
Although alteration of organ bath calcium concentration did not affect force generation, this intervention had a large effect on ROS generation, as assessed by the amount of ethidium formed during repetitive contraction trials (Fig. 3). Specifically, repetitive contraction in low-calcium physiological solution resulted in less diaphragm ethidium production than stimulation in normal-calcium media, with ethidium levels being 3.2 ± 0.4 and 18.9 ± 2.7 ng ethidium/mg tissue, respectively (P < 0.002). Diaphragms contracting in high-calcium baths had ethidium concentrations (19.3 ± 4.5 ng/mg) similar to that of the normal-calcium group experiments. As expected, control, noncontracting diaphragms in 2.5 mM calcium baths had very little ROS generation (i.e., 3.4 ± 0.8 ng/mg tissue).
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Protocol B: Effect of Nimodipine on Diaphragm Force Generation
The administration of nimodipine, an L-type calcium channel blocker, had no effect on twitch kinetics or the diaphragm force-frequency curves (see Table 2) compared with data from hemidiaphragms studied while submerged in normal-calcium Krebs-Henselheit solution containing no nimodipine. Nimodipine also had no effect on the diaphragmatic forces generated during repetitive contraction (Fig. 4), with identical forces at each point in time during these trials when comparing contracting diaphragms in normal-calcium solutions with and without added nimodipine.
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Protocol B: Effects of Nimodipine on Diaphragm Ethidium Formation
Nimodipine did have a considerable effect on contraction-related ROS production. Hemidiaphragms to which nimodipine was administered before repetitive contraction had ethidium levels approximately times times lower than nonnimodipine-treated contracting preparations (Fig. 5). Specifically, mean ethidium concentrations were 19.0 ± 4.0 ng/mg tissue for nonnimodipine-treated contracting diaphragms and 2.6 ± 0.9 ng/mg tissue for the nimodipine-treated group (P < 0.002).
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Protocol C: Effects of Tiron, SOD, and L-NAME on Diaphragm Ethidium Formation
We assessed the effects of Tiron, SOD, and L-NAME on ethidium formation to provide additional insight into the specific ROS responsible for ethidium generation during diaphragm contraction. We found that Tiron, an intracellular superoxide scavenger, totally suppressed contraction-related ethidium formation in the diaphragm (Fig. 6; P < 0.001 for comparison of ethidium generation with and without Tiron). We also found that SOD, an extracellular superoxide scavenger, also affected contraction-related ethidium formation, reducing this signal by two-thirds (P < 0.01; Fig. 6). L-NAME, a NOS inhibitor, in contrast, had no effect on contraction-related ethidium formation by the diaphragm.
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To further assess the potential role of nitric oxide in generating an
ethidium signal, we also incubated a nitric oxide-generating solution
with hydroethidine in vitro. In this last experiment, we found that
addition of 5 mM sodium nitroprusside (this compound generates nitric
oxide at a rate of 2.8 mmol/min, a rate ~1,000 times greater than the
rate at which nitric oxide is generated in skeletal muscle; Ref. 16) to
hydroethidine resulted in the formation of very small amounts of
ethidium (0.2 ng ethidium/min, a rate ~
the rate of signal
formation in contracting muscle). These latter data argue that
physiological concentrations of nitric oxide in muscle are unlikely to
elicit formation of significant amounts of ethidium.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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These data indicate that it is possible to suppress free radical generation (i.e., production of ROS) by the contracting diaphragm by decreasing extracellular calcium concentration. We found similar reductions in free radical generation also resulted when an L-type calcium channel blocker was administered to the contracting diaphragm. These latter results suggest that free radical formation by contracting muscles may be dependent on calcium influx through L-type calcium channels.
Methodological Issues
In this study, we varied extracellular calcium concentrations simply by altering the amount of calcium added to a conventional physiological solution (Krebs-Henselheit solution) and increasing or decreasing the amount of sodium chloride contained in these solutions to maintain solution molarity constant. For our low-calcium solution, we added 1 mM calcium when making up our medium; for a normal-calcium solution we added 2.5 mM calcium; and for our high-calcium solution experiments we added 5 mM. We chose to vary calcium concentrations in this manner in large part because we wished to reproduce the approach employed by Jackson et al. (12) in their study examining the influence of extracellular calcium concentration on muscle injury.As in the study by Jackson et al. (12), we did not add calcium chelating agents to our various solutions to "buffer" calcium levels to a specific level, and, as a result, it is likely that the "actual" concentrations of calcium present along the sarcolemmal membrane of muscle fibers and in the t tubules differed to some degree from the nominal calcium levels in the added bathing solutions. In fact, even if chelating agents were used, it is difficult to ensure complete equilibration of extracellular medium concentrations with ion levels in the invaginated t-tubular system. For these reasons, we interpret our findings as simply indicating that, qualitatively, reductions in extracellular calcium levels reduce contraction related ROS formation in the isolated diaphragm.
Another issue that should be addressed is our use of high concentrations of hydroethidine as a means of detecting generation of ROS within the diaphragm. We chose to use high levels of this agent to ensure that formation of ethidium, the product of the chemical reaction of hydroethidine with diaphragmatic ROS, would not be substrate limited by hydroethidine. As a result, however, it is likely that hydroethidine acted as a free radical scavenger in the present study, eliminating ROS generated in the diaphragm during contraction before these molecular species had a chance to react with and alter cellular constituents. Although it appears that nimodipine and low-calcium-containing solutions abolished generation of ROS during diaphragmatic contraction, reaction of hydroethidine with the ROS generated in the other experimental groups (i.e., high-calcium studies, studies performed in the absence of nimodipine) may have eliminated ROS in these latter groups as well, with the result that no ROS-related muscle dysfunction may have occurred in any of the experimental groups in this study. This fact may account for the finding that force generation over time during repetitive contraction trials was similar across all experimental groups in this study [i.e., across all levels of calcium concentrations (Fig. 2) and in both the presence and absence of nimodipine (Fig. 4)].
One might ask whether electrical stimulation might be responsible for or be a contributor to ethidium formation in our preparation. We think this unlikely for several reasons: 1) direct electrical stimulation of buffer containing hydroethidine formation generates no ethidium formation; 2) if electrical stimulation per se were responsible for generating an ethidium signal, then addition of nimodipine would be unlikely to block the ethidium formed during contraction; and 3) if electrical stimulation per se were responsible for ethidium formation, altering calcium concentrations would not be expected to affect ethidium formation during contraction. For all of these reasons, we think it unlikely that electrical stimulation per se could be a source of significant ethidium formation under the conditions examined in this study.
Role of Extracellular Calcium and Free Radicals In Modulating Muscle Dysfunction
A number of previous studies have suggested that extracellular calcium concentrations influence the rate at which tissue dysfunction develops in pathophysiological situations (3, 9, 15, 19, 20, 21, 27, 28). For skeletal muscle, Jackson et al. (12) found that the degree of muscle cellular injury, assessed by measuring LDH release, in incubated soleus and EDL muscle strips is strongly influenced by the concentration of calcium bathing these strips. When the medium bathing muscles was replaced with a low-calcium-containing solution (0 mM calcium), muscle LDH fell by 80% from that recorded when muscles were incubated with physiological solutions containing normal-calcium concentrations.Recent work also indicates that free radicals contribute to muscle dysfunction in several pathophysiological conditions (i.e., sepsis, ischemia-reperfusion, fatigue induced by strenuous concentric or isometric contraction, eccentric contraction-induced injury) (2, 8, 25, 29, 32-35). More recently, it has been suggested that free radical formation by muscle in several of these conditions (i.e., contraction, sepsis) is highly dependent on the activity of the 14-kDa isoform of phospholipase A2 (PLA2), a calcium-dependent enzyme preferentially located in mitochondrial and microsomal compartments (4, 11, 23, 24, 31). Evidence supporting this contention is provided by a recent study in which administration of inhibitors of the 14-kDa PLA2 isoform (i.e., manoalide and aristolochic acid) was found to completely ablate generation of ROS in skeletal muscle during in vitro contraction (23). There are two potential mechanisms by which PLA2 activation could lead to free radical formation in muscle: 1) arachidonic acid formed by active PLA2 could provide substrate for the cyclooxygenase pathway, which generates superoxide as a by-product of the formation of PGH from PGG, and 2) arachidonic acid generated in mitochondria can interact with the electron transport pathway to augment superoxide formation by complexes I and III.
It would be possible to explain all these previous findings if elevations in extracellular calcium concentrations lead to activation of muscle PLA2, which, in turn, induces free radical formation. Free radicals so produced could react with lipid and protein cellular constituents, altering membrane structure, affecting contractile protein function, and modifying cellular high-energy-phosphate-generating systems. The present findings are consistent with this mechanism, and provide the first evidence of a direct link between extracellular calcium concentrations and the generation of potentially damaging ROS in skeletal muscle during contraction. The fact that this finding was reproduced by administration of an L-type calcium channel blocker (i.e., administration of this blocker in the presence of extracellular calcium produced the same effect as that evoked by decreasing extracellular calcium levels) suggests that influx of extracellular calcium into one or more intracellular compartments is required for free radical generation during muscle contractions. This finding also argues against a cell surface "free radical generator" or a direct effect of extracellular calcium on cellular processes, because administration of a calcium channel-transport inhibitor would not be expected to alter free radical formation under such circumstances.
In addition, the fact that administration of both L-type calcium channel blockers and inhibitors of 14-kDa PLA2 block ROS formation by contracting muscle suggests that calcium entry into a cellular compartment containing 14-kDa PLA2 may be an important factor in regulating free radical formation during contraction. As mentioned earlier, this PLA2 isoform is found attached to mitochondrial and microsomal membranes (37), and it seems reasonable to speculate that calcium concentrations at these sites may be influenced by alterations in extracellular calcium concentrations.
We should also point out that constitutive NOS is another calcium-dependent enzyme contained in muscle mitochondria. It is possible that alterations in extracellular calcium concentrations may also affect the activity of NOS and thereby influence nitric oxide generation intracellularly. Our data indicate, however, that ethidium formation in our experiments is unlikely to be a result of heightened nitric oxide formation. Specifically, we found that nitric oxide per se has only a limited ability to oxidize hydroethidine to ethidium in vitro. In addition, L-NAME, a NOS inhibitor, had no effect on ethidium formation by contracting muscle (see Fig. 6). In contrast, both Tiron (an intracellular superoxide scavenger) and SOD (an extracellular superoxide scavenger) substantially reduced ethidium formation by contracting muscle. These latter findings suggest that the superoxide anion, or its degenerative species, was largely or entirely responsible for the formation of ethidium during contraction in the present group of experiments. The lesser ability of SOD, an extracellular agent, compared with Tiron, an intracellular agent, in suppressing ethidium formation would be consistent with the possibility that superoxide generation in contracting muscle largely occurs in an intracellular compartment (e.g., mitochondria) or microsomal compartments.
Potential Implications
Because excessive generation of free radical species in muscle is thought to induce dysfunction, and because calcium appears to influence free radical generation, it should be possible, theoretically, to influence the development of free radical-mediated muscle dysfunction by manipulating cellular calcium metabolism. It is neither feasible nor desirable to do this clinically by altering extracellular calcium levels. The observation, however, that free radical formation in muscle can be minimized by administration of a calcium channel transport inhibitor provides an alternative means of accomplishing this goal. It is conceivable that a therapeutic approach on the basis of this concept could be used to prevent muscle damage in certain pathophysiological conditions.| |
ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-54825 and HL-38926.
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FOOTNOTES |
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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 24 July 1998; accepted in final form 18 August 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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), normal-calcium (
) and high-calcium (
)
experimental groups. Error bars, SE.
) and presence (
) of
nimodipine.
