Am J Physiol Cell Physiol 285: C881C890, 2003. First published June 18, 2003; 10.1152/ajpcell.00063.2003.

Effect of indomethacin on force responses and sarcoplasmic reticulum function in skinned skeletal muscle bers and cytosolic [Ca2 ] in myotubes
Renzhi Han,1 Takuya Suizu,1 Miranda D. Grounds,2 and Anthony J. Bakker1
School of Biomedical and Chemical Sciences, and 2School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia 6009, Australia
Submitted 19 February 2003; accepted in nal form 13 June 2003
1

Han, Renzhi, Takuya Suizu, Miranda D. Grounds, and Anthony J. Bakker. Effect of indomethacin on force responses and sarcoplasmic reticulum function in skinned skeletal muscle bers and cytosolic [Ca2 ] in myotubes. Am J Physiol Cell Physiol 285: C881C890, 2003. First published June 18, 2003; 10.1152/ajpcell.00063.2003.In this study, the effects of phospholipase A2 (PLA2) inhibitors on excitation-contraction coupling (ECC) and sarcoplasmic reticulum (SR) function were examined in skinned extensor digitorum longus (EDL) muscle bers of the rat. The nonspecic PLA2 inhibitor indomethacin (200 M) signicantly increased the peak ( 2-fold, P 0.02) and the width ( 6-fold, P 0.008) of depolarization-induced force responses (DIFRs) elicited in the bers (n 4). Exposure of the skinned EDL bers to indomethacin (200 M) (n 7) and another PLA2 inhibitor quinacrine (200 M) (n 5) resulted in the return of large DIFRs after use-dependent rundown. However, aristolochic acid (100 M), an inhibitor of secretory PLA2, failed to return DIFRs after rundown. Indomethacin did not protect against the loss of DIFRs induced by exposure to elevated myobrilar [Ca2 ]. Indomethacin (200 M) produced a small but significant increase in the Ca2 sensitivity of the contractile apparatus of skinned EDL bers and the maximum force production. Indomethacin (200 M) also had signicant effects on SR function, increasing SR Ca2 loading in the skinned bers (117.2 3.0% of controls, P 0.0008, n 8) and inducing intracellular Ca2 release in isolated intact exor digitorum brevis (FDB) bers (n 7) and C2C12 myotubes (n 6). These data suggest that intracellular PLA2 may be an important modulator of ECC in skeletal muscle. phospholipase A2; excitation-contraction coupling

THE PHOSPHOLIPASE A2 (PLA2) family of enzymes hydrolyze the sn-2 fatty acids of glycerophospholipids. They are classied into three major classes: secretory PLA2 (sPLA2), Ca2 -dependent cytosolic PLA2 (cPLA2), and Ca2 -independent cytosolic PLA2 (iPLA2). Activation of PLA2 releases fatty acids such as arachidonic acid (AA) and lysophospholipids that, at low concentrations, act as second messengers but, at high concentrations, are cytotoxic (19). PLA2 activation has been shown in many cell types to play a role in a diverse array of processes such as apoptosis, membrane phospholipid

remodeling, membrane homeostasis, and exocytotic membrane fusion, as well as providing precursors for inammatory factors such as prostaglandins, leukotrienes, and platelet-activating factor (10). Little is presently known about the role of PLA2 in skeletal muscle function, although enzymes from all three PLA2 classes have been reported to be expressed in skeletal muscle (25, 35, 38, 45). Experiments using isolated sarcoplasmic reticulum (SR) indicate that PLA2 activation products such as arachidonic acid (9, 11, 13, 22) and lysophospholipids (1) interfere with SR Ca2 accumulation. Past studies have also implicated PLA2 in Ca2 induced damage of the sarcolemma in skeletal muscle. Raised intracellular Ca2 levels through application of the Ca2 ionophore A-23187 lead to ultrastructural damage (16, 18) and signicant efux of intracellular muscle specic enzymes in isolated skeletal muscle, indicating that A-23187-induced Ca2 inux causes sarcolemmal damage (29). PLA2 inhibitors were found to signicantly reduce the enzyme efux that occurs in response to the damage induced by A-23187, suggesting that much of the damage was mediated by activation of PLA2 (17, 28). How PLA2 acts to cause damage is unknown; however, in other cell types PLA2 activation can lead to changes in membrane phospholipid composition, uidity, and permeability (20, 21). A recent study also demonstrated that administration of aristolochic acid and manoalide, inhibitors of sPLA2, suppressed formation of reactive oxygen species in contracting skeletal muscle (41). Formation of free radicals is also reported to play an important role in the development of fatigue and damage in skeletal muscle (7, 14, 43, 48). Taken together, these results indicate that PLA2 may play an important role in modulating the permeability of the SR and sarcolemmal membranes to diffusible species in skeletal muscle, and inhibition of PLA2 could help to prevent or reverse cell damage in skeletal muscle bers. Mechanically skinned bers provide a unique preparation to study aspects of skeletal muscle function. In these bers, the t-system seals and repolarizes after
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Address for reprint requests and other correspondence: R. Han, Howard Hughes Medical Institute, College of Medicine, Univ. of Iowa, 400 EMRB, Iowa City, IA 52242-1101 (E-mail: HanR@graduate. uwa.edu.au). http://www.ajpcell.org

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mechanical skinning (15, 32, 46) and, therefore, depolarization-induced force responses (DIFRs) via the normal excitation-contraction coupling (ECC) pathway can be elicited by replacing the K -based bath solution with a Na -based solution. The DIFRs are analogous to K contractures in intact bers, and previous studies indicate that the relaxation phase of the force responses is due to voltage-dependent inactivation of the voltage sensors (32, 33). In addition, the skinned bers can be used to quantitatively examine subcellular events occurring within the ber, such as SR Ca2 release, SR Ca2 accumulation, and passive SR Ca2 leak (3, 4). In mechanically skinned skeletal muscle bers, a gradual use-dependent decrease in the size of DIFRs over time has also been observed (27, 30, 31). The cause of this use-dependent rundown is unknown. It could result from Ca2 -induced damage to proteins involved in ECC because raised intracellular Ca2 also results in the uncoupling of ECC in skinned bers (31). Ca2 activated uncoupling may also occur in intact skeletal muscle because interventions that further increase intracellular Ca2 during fatiguing stimulation also prolong fatigue in single intact skeletal muscle bers (12). The most likely way that elevation in free cytosolic Ca2 acts to disrupt ECC in skeletal muscle is via stimulation of certain Ca2 -activated enzymes such as calpains, phospholipase A2 (34), or protein kinase C. Studies using the calpain inhibitors calpeptin and leupeptin have shown that these Ca2 -activated neutral proteases are unlikely to be primarily involved in this process in skinned bers (31) or intact skeletal muscle bers (12). Interestingly, arachidonic acid was recently shown to exacerbate rundown of DIFRs in skinned bers (6). Indomethacin is a nonsteroidal anti-inammatory drug that inhibits all PLA2 activity at high concentrations (IC50 145 M) (20) and nonselectively inhibits cyclooxygenases at low concentrations (IC50 1 M) (24, 47). In this study, we examined the effect of indomethacin on SR function and DIFRs before and after rundown in skinned skeletal muscle bers of the rat.

All experimental procedures and methods undertaken in this study were approved by the University of Western Australia Animal Ethics Committee. In the skinned ber experiments, skeletal muscle bers were isolated from the extensor digitorum longus (EDL) muscles of large male and female Wistar rats (12 wk old, Rattus norvegicus) killed by exposure to a gas mixture of 80% CO2 and 20% O2. The single-muscle bers were dissected and mechanically skinned in parafn oil. To monitor isometric force, the bers were mounted between a xed pair of forceps and a force transducer and placed in a Perspex bath containing 2 ml of a potassium hexamethylene-diamine-tetraacetate (K-HDTA) solution (Table 1) (32). The free Mg2 concentration was 1 mM, and NaN3 was added to inhibit mitochondrial Ca2 uxes. Data were acquired using a PowerLab data acquisition system (ADInstruments, Sydney, Australia) attached to a personal computer. To maximize the force production, the ber was stretched from slack length by 20% to bring the sarcomere length to 2.83.0 m (32). Indomethacin, aristolochic acid, and dexamethasone were dissolved in pure dimethyl sulfoxide (DMSO), whereas quinacrine was dissolved in double-distilled water at a 1,000 times stock of nal concentration used in this study. The nal DMSO concentration in all the test and appropriate control solutions was 0.1%. The effect of indomethacin (200 M) addition on the free [Ca2 ] of the skinned ber solutions was examined using the uorescent Ca2 dye indo 1-K salt. No signicant difference in the uorescence ratio was found between the solutions with and without indomethacin (0.254 0.003 vs. 0.248 0.003, respectively, Students unpaired t-test, P 0.1, n 6), indicating that indomethacin addition does not produce any signicant Ca2 contamination in this study. Depolarization-induced force responses. The mechanically skinned muscle bers used in this study retained normal ECC due to resealing of the t-system after skinning. The bers were maintained in K-HDTA solution for 1 min to repolarize the sealed t-system. DIFRs were elicited by exposing the preparation to a Na-HDTA solution, which was similar in composition to the K-HDTA solution except that Na had been substituted for K (Table 1). The K and Na solutions used with rat bers were isosmotic. The K-HDTA and Na-HDTA solutions were weakly buffered to a pCa of 6.77.0.

Table 1. Solutions used in examination of depolarization-induced force responses, contractile apparatus, and SR function
K-HDTA (depol. Sol.) Na-HDTA (repol. Sol.) Ca2 /Rigor Sol. Ca2 Release/ Depletion Sol. Ca2 leak Sol. Sol. A (Contractile Apparatus) Sol. B (Contractile Apparatus)

Solutions

Rigor Sol.

MgO HEPES HDTA EGTA ATP Creatine phosphate CaCO3 Caffeine NaN3 To pH 7.1 with:

8.5 90 50 0.025 8 10

8.5 90 50 0.025 8 10

8.5 90 68 0.1 0 0

8.5 90 68 0.1 0 0 0.4 CaCl2 (free Ca2 70 M) 1 KOH

0.1275 90 50 0.75 8 10

8.5 90 50 0.875 8 10

10.3 90 50 8 10

8.12 90 50 8 10 48.5

1 KOH

1 NaOH

1 KOH

30 1 KOH

1 KOH

1 KOH

1 KOH

Values are concentrations (in mM). Sol., solution. AJP-Cell Physiol VOL
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High Ca2 /rigor experiments. In experiments to examine the effect of indomethacin on the uncoupling of ECC induced by exposure to elevated intracellular Ca2 , the bers were rst exposed to a control rigor solution (Table 1) for 1 min and then transferred to the identical rigor solution with 400 M Ca2 (free [Ca2 ]: 70 M) for 30 s (31). Fibers were exposed to high-Ca2 solutions under rigor conditions to deplete myoplasmic ATP and ensure that the SR Ca2 pumps did not reduce the free Ca2 concentration at the level of the myobrils. Force responses upon depolarization were then measured under control conditions and in the presence of indomethacin. In a similar experiment, indomethacin was also added to the Ca2 /rigor solution, and control DIFRs were measured and compared with control responses made before exposure to this modied Ca2 /rigor solution. Contractile apparatus. Mechanically skinned EDL bers were exposed to Triton X-100 for 1015 min at the beginning of the experiment to destroy all membranous compartments. The force-[Ca2 ] relationship in each ber was determined by exposing the ber to a sequence of strongly Ca2 -buffered solutions at progressively higher free [Ca2 ], allowing the ber to reach close to a steady force level in each solution before moving to the next. The strongly Ca2 -buffered solutions were prepared by mixing specic proportions of EGTA2 (Sol. A) and CaEGTA (Sol. B) solutions (32). The free [Ca2 ] of the solutions was calculated using a Kapp for EGTA of 4.78 106 M 1 (23). Maximal force was determined by exposure of the ber to Sol. B (pCa 4.5). The effect of indomethacin on the Ca2 sensitivity of the contractile apparatus and maximal force production in the EDL bers was determined by exposing bers to a solution sequence with or without 200 M indomethacin. Measurements in the presence of indomethacin were compared with control measurements made before and after indomethacin exposure to compensate for the small progressive decline in force that occurs during these experiments. The plateaus of the force responses elicited by exposure to solutions of increasing free [Ca2 ] were expressed as a percentage of maximum Ca2 activated force and plotted as a function of pCa. The data were tted with sigmoidal curves using the curve-tting software package GraphPad Prizm (GraphPad Software). The slope and pCa50 values (pCa value corresponding to 50% of maximum force) of the individual curves derived from data from each ber were determined for both indomethacin and control data, and the values were compared statistically. Note that in Fig. 7 curves have been tted to the mean data. Effect of indomethacin on SR Ca2 loading and SR Ca2 leak. In the experiments carried out to determine the effect of indomethacin on SR Ca2 loading, the SR of the ber was rst depleted of all releasable Ca2 by exposure to a depletion solution (Table 1) and then partly reloaded with Ca2 in a weakly buffered Ca2 load solution for a xed period. The ber was again depleted of all releasable Ca2 , and the integral of the force response elicited was used as an indicator of the amount of Ca2 loaded during the loading period (5). Depletion measurements made after loading in the presence of 200 M indomethacin were compared with control measurements made before and after loading with the drug to minimize errors associated with any deterioration in the size of the control responses. Before exposure to the load solution containing indomethacin, the bers were exposed to a K-HDTA solution containing indomethacin for 30 s to allow time for indomethacin to equilibrate within the ber. Skinned EDL bers exhibit a small SR Ca2 leak under resting conditions (5). To examine the effect of indomethacin on this passive SR Ca2 leak in the skinned bers, the SR of
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the bers was rst depleted of Ca2 and then reloaded with Ca2 for a specic time. The bers were then exposed to a Ca2 leak solution for 70 s. This solution contained a relatively high concentration of EGTA (Table 1) to chelate all leaked Ca2 . The bers were then exposed to the depletion solution (Table 1), and the integral of the resulting force response was measured. Under control conditions, this integral was less than previous control measurements made using a similar protocol without exposure to the leak solution, indicating the existence of the SR Ca2 leak pathway in the skinned bers. The measurement of the leak under control conditions was then compared with subsequent measurements made using an identical leak experiment protocol, with the exception that the leak solution also contained 200 M indomethacin. Note that the control leak solution contained DMSO at the same nal concentration to the leak solution containing indomethacin. Resting membrane potential measurement. The resting membrane potential (RMP) was monitored in randomly selected bers from intact EDL muscles. The RMPs were measured before and after incubation with 200 M indomethacin for 30 min (27). RMPs were recorded using a glass microelectrode lled with 3 M KCl in conjunction with a NeuroProbe Amplier system (model 1600, A-M Systems). The data were compared using a paired t-test. Isolation of FDB bers. Single-muscle bers were enzymatically dissociated from predominantly fast-twitch exor digitorum brevis (FDB) muscles of the rat (45 wk old). The FDB muscles were incubated in -minimum essential medium (MEM; GIBCO BRL) with 10% fetal bovine serum (GIBCO BRL) containing 0.2% type I collagenase (Sigma) for 2 h and then triturated to release single bers. Cell culture. C2C12 myoblasts were seeded on collagencoated glass coverslips in 35-mm culture disks. Cells were grown to around 80% of conuence in DMEM medium supplemented with 10% fetal bovine serum (GIBCO), 2.0 mM glutamine, 100 IU/ml penicillin, 100 g/ml streptomycin, and 1% fungizone in a humidied atmosphere of 5% CO2 and 95% air at 37C. Myoblasts fusion was induced by replacing 10% fetal bovine serum with 2% horse serum (GIBCO). The cultured cells grew into contractile myotubes within 3 to 6 days. Intracellular [Ca2 ] measurement. The C2C12 myotubes and isolated FDB bers were loaded with fura 2-AM (3 M) (TEFLABS) in the presence of 0.0125% (wt/vol) Pluronic F-127 (Molecular Probes, Eugene, OR) for 45 min at room temperature (2223C). The cells were maintained in physiological rodent saline (composition in mM: 138 NaCl, 2.7 KCl, 1.8 CaCl2, 1.06 MgCl2, 12.4 HEPES, and 5.6 glucose, pH 7.3) in a chamber ( 2 ml) mounted on the stage of an inverted microscope (Nikon TE2000, Japan) connected to a spectrophotometer (Cairn). The ratio of uorescence emission (510 nm) at 340- and 380-nm excitation (F340/F380) was used as an indicator of cytosolic [Ca2 ]. Ratio data were acquired, stored, and analyzed using the Cairn software package (Cairn). The Ca2 -free solution was similar to physiological rodent saline with the exception that 1.8 mM CaCl2 was replaced with 1 mM EGTA and 3.6 mM MgCl2 (pH 7.3). All experiments were conducted at room temperature (21 22C), and all data are expressed as means SE. Unless otherwise stated, all force responses in the presence of indomethacin were converted to a percentage of the control response and compared using a two-tail, one-sample Students t-test. All statistical analysis was undertaken using the statistics software package GraphPad INSTAT.
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Fig. 1. Effect of indomethacin (200 M) on depolarization-induced force responses (DIFRs). Indomethacin signicantly increased the peak and the width of the DIFRs. After removal of indomethacin, DIFRs could not be elicited under control conditions.

RESULTS

Effect of indomethacin on DIFRs. To gain an understanding of the role of PLA2 in ECC of skeletal muscle, an examination of the effects of the general PLA2 inhibitor indomethacin on DIFRs was undertaken in skinned EDL bers of the rat. Depolarization of bers in the presence of the indomethacin (200 M) resulted in DIFRs that were signicantly larger (95.1 4.1% vs. 50.8 9.3% of maximum force, P 0.02) and broader (half-peak width: 38.4 5.6 vs. 6.2 1.9 s, P 0.008) than initial control responses (n 4) (Fig. 1). After depolarization in indomethacin, the bers later failed to respond to depolarization under control conditions (Fig. 1). However, incubation of bers in a repolarization (K-HDTA) solution containing indomethacin (200 M) (1.52 min) had no marked effect on later control DIFRs (98.7 4.3% of initial control, P 0.13, n 4) (Fig. 2). Furthermore, if the ber was initially depolarized in the presence of indomethacin and then quickly returned to the repolarization solution with indomethacin so as to produce only a brief response (e.g., sixth depolarization, Fig. 2), the ber was still able to later respond to depolarization under control conditions (n 3) (Fig. 2). Therefore, the presence of indomethacin per se had little effect on the ability of the bers to contract in response to depolarization under control conditions, and it is likely that it is the size and duration of DIFRs elicited in the presence of indomethacin that leads to the uncoupling of depolarization and force in these bers, possibly due to damage of the ECC system. Effect of PLA2 inhibitors on DIFRs after rundown. Rundown of the DIFRs usually started after 1015 responses had been elicited and resulted in a gradual decline in force (e.g., rst ve responses; Fig. 3). This is

similar to previous studies in this and other laboratories (27, 31). In skinned bers in which DIFRs had eventually run down to zero under control conditions, depolarization in the presence of the PLA2 inhibitor indomethacin (200 M) resulted in restoration of DIFRs. The DIFRs elicited in the presence of indomethacin after rundown were again signicantly larger (initial controls, 54.5 10.6% of maximum force; indomethacin, 91.3 1.7% of the maximum force, P 0.02) and broader (half-peak width of initial controls, 5.1 1.5 s; indomethacin, 51.1 11.3 s, P 0.01, paired t-test, n 7) than the initial control DIFRs (Fig. 3). The responses elicited in the presence of indomethacin after rundown were similar in peak (unpaired t-test, P 0.67) and half-peak width (unpaired t-test, P 0.08) to those elicited in indomethacin before rundown (see Figs. 1 and 3). To provide information concerning the dose dependence of the effects of indomethacin on restoration of DIFRs after rundown, four bers were exposed to depolarization solutions containing a range of indomethacin concentrations after rundown had commenced. Out of a total of four bers, no bers responded to depolarization with a force response in 2 or 20 M indomethacin, one ber had a force response in 100 M indomethacin, two bers had a force response in 150 M indomethacin, and all bers produced DIFRs in the presence of 200 M and 300 M indomethacin. After washout of indomethacin, no DIFRs could be elicited under control conditions, indicating that the continual presence of the inhibitor is required for DIFR restoration. Spontaneous force responses (79 5% of maximum force, n 3) were also observed in some bers when they were maintained in K repolarization solution in the presence of indomethacin (200 or 300 M). The effects of other PLA2 inhibitors were also examined to ensure that the effects of indomethacin were PLA2 related. Depolarization in the presence of the nonspecic PLA2 inhibitor, quinacrine (200 M), (IC50 30 and 150 M for cPLA2 and iPLA2, respectively) also resulted in the return of large DIFRs (90.8 7.3% of maximum force) after rundown had occurred (initial responses, 46.8 18.5% of maximum force, n 5) (Fig. 4). The DIFRs in the presence of quinacrine were also signicantly broadened by 4fold (half-peak width of control, 5.1 0.7 s; quinacrine, 19.8 1.5 s). Interestingly, when the bers were transferred from control repolarization solution to a repolarization solution containing 200 M quinacrine, a large

Fig. 2. Pretreatment of the ber with indomethacin (200 M) in the repolarization solution or brief depolarization (the fth response) in the presence of indomethacin had no signicant effect on control DIFRs.

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Fig. 3. Effect of indomethacin (IND, 200 M) and dexamethasone (DEX, 200 M) on the DIFRs after rundown in skinned extensor digitorum longus (EDL) bers of the rat.

spontaneous force response was also observed (n 3). Incubation of the bers after rundown with a specic inhibitor of cPLA2, dexamethasone (200 M), failed to restore fully the DIFRs (n 5), although small responses were evident in three of the ve bers (14.7 6.1% of maximum force) (Fig. 3). Aristolochic acid (100 M) (IC50 40 M), a specic secretory PLA2 inhibitor, failed to restore DIFRs after rundown in any bers tested (n 5, data not shown). The lipoxygenase (LO) inhibitor NDGA (50 M) (IC50 0.2, 30 and 30 M for 5-LO, 12-LO, and 15-LO, respectively) also did not restore DIFRs after rundown (n 4, data not shown). This suggests that reactivation of the DIFRs by inhibition of PLA2 is not via inhibition of the LO system. In this study, indomethacin had no signicant effect on the mean resting membrane potential of randomly chosen intact EDL bers (before 200 M indomethacin, 82.9 1.8 mV; after 30 min indomethacin exposure, 82.3 1.5 mV, P 0.05, n 10), suggesting that indomethacin is not reversing the rundown of DIFRs or triggering spontaneous SR Ca2 release (Fig. 3) through effects on the membrane potential. Effect of PLA2 inhibitors on damaging Ca2 exposure-induced uncoupling. Previous studies (27, 30, 31) have shown that exposure of skinned bers to an elevated free cytosolic Ca2 levels results in failure of DIFRs. This raises the possibility that continual exposure to elevated Ca2 during repeated depolarizations is also responsible for the rundown of DIFRs in skinned bers. In the present study, DIFRs could be restored after rundown in the presence of indomethacin and quinacrine, raising the possibility that indomethacin might prevent Ca2 -induced uncoupling of ECC. We have shown previously that the benzophenanthridine drug chelerythrine (12 M) can restore DIFRs after high-Ca2 exposure, although the mechanism remains unknown (27). Therefore, it was of interest to examine the effects of PLA2 inhibition on Ca2 -induced uncou-

Fig. 4. Effect of quinacrine (QUIN, 20 M; 200 M) on the DIFRs in skinned EDL bers of the rat. AJP-Cell Physiol VOL

pling in skinned bers. In these experiments, the bers were rstly exposed to a rigor solution containing elevated Ca2 (free [Ca2 ]: 70 M) to abolish the DIFRs, and the bers were then incubated in the repolarization solution with indomethacin (200 M) or quinacrine (200 M). In ve bers, the DIFRs did not recover at all within 10 min after exposure to elevated cytosolic Ca2 levels. However, in three bers, DIFRs did recover to some extent ( 10% of the initial control responses) (Fig. 5). In all bers, exposure to chelerythrine (12 M) resulted in the return of large DIFRs (Fig. 5). The possible protective effects of PLA2 inhibitors on the Ca2 -induced uncoupling in skinned EDL bers was also investigated. In this experiment, the rigor solutions, both with and without Ca2 , also contained indomethacin (200 M) or quinacrine (200 M). Fibers in which substantial initial control DIFRs had been measured were exposed to a rigor solution with 70 M free [Ca2 ] before any force rundown was observed. The bers were then washed in a repolarization solution for 2 min and transferred to a depolarization solution. No DIFRs were observed after exposure to the rigor/high-Ca2 solution with indomethacin or quinacrine (n 5 for indomethacin; n 2 for quinacrine) (Fig. 6). Note that the rigor solution alone had no marked effect on control DIFRs. These results suggest that PLA2 inhibitors have no protective or marked restorative effects against Ca2 -induced loss of DIFRs in skinned EDL bers. Effect of indomethacin on the contractile apparatus. The rise in basal force levels in the presence of indomethacin (Figs. 1 and 2) suggests that indomethacin was having effects on the contractile apparatus. Therefore, the effect of indomethacin on the contractile apparatus was examined. In the presence of 200 M indomethacin, the maximum force production in the skinned EDL bers was 6.6 1.1% greater than under control conditions (P 0.0007, n 8). The contractile apparatus was also more sensitive to Ca2 in the presence of 200 M indomethacin (mean pCa50 value: 200 M indomethacin, 6.219 0.019; control, 6.082 0.016, paired t-test, P 0.0001, n 8). The mean slope of the force-pCa curves was not signicantly affected by the presence of indomethacin (indomethacin, 4.28 0.32; control, 4.33 0.34, paired t-test, P 0.67, n 8) (Fig. 7). The effects of indomethacin on contractile apparatus must be taken into consideration when interpreting the effects of this drug on the DIFRs (see DISCUSSION).
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Fig. 5. Effect of indomethacin (200 M), quinacrine (200 M), and chelerythrine (CHEL, 12 M) on the rigor/high-Ca2 -activated loss of depolarization-induced force in the skinned bers.

Effect of indomethacin on SR function. The larger and markedly prolonged DIFRs elicited in the presence of indomethacin (Figs. 1 and 3) could be due to the increased SR Ca2 uptake and/or SR Ca2 release in the presence of these drugs. The effect of indomethacin (200 M) on SR Ca2 uptake was examined in skinned bers. In these experiments, the integral of force responses elicited after exposure of (previously Ca2 depleted) bers to a SR Ca2 loading solution in the presence and absence of 200 M indomethacin was measured. The presence of indomethacin in the Ca2 loading solution was found to increase signicantly SR Ca2 loading by 17.2 3.0% (P 0.0008, n 8) compared with control levels (mean of control measurements made before and after exposure to indomethacin) in skinned EDL bers (Fig. 8). A comparison of the SR Ca2 loading in the presence of indomethacin and the initial control measurement alone yielded a similar difference (14.7 2.5%, P 0.0006, n 8). This result suggests that indomethacin signicantly increases SR Ca2 accumulation in skinned EDL bers. Increases in SR Ca2 accumulation can be due to either increased SR Ca2 pump activity or decreased SR Ca2 leak. Therefore, the effect of indomethacin (200 M) on the SR Ca2 leak in the skinned EDL bers was also investigated. However, force responses elicited after exposure to a leak solution containing 200 M indomethacin were not signicantly different from the mean control response (control measurements made after exposure to the control leak solution, both before and after the indomethacin response) (indomethacin responses: 94.4 7.1% of controls, P 0.47, n 8) (data not shown). This result suggests that the increased SR Ca2 accumulation in the presence of

indomethacin is the result of increased Ca2 SR uptake rather than decreased SR Ca2 leak. The effect of indomethacin (200 M) on SR Ca2 release could not be examined in the skinned bers because indomethacin was shown to increase the Ca2 sensitivity of the contractile apparatus, rendering any results inconclusive. Therefore, to examine the effect of indomethacin on SR Ca2 release, resting free cytosolic Ca2 concentrations ([Ca2 ]i) were directly monitored in single isolated FDB skeletal muscle bers using the uorescent Ca2 indicator fura 2. In the isolated FDB bers, the resting free [Ca2 ]i increased to a high level ( 2.3 times the peak of the Ca2 transients evoked by electrical eld stimulation) after addition of indomethacin (200 M) and did not return to the initial baseline level within 610 min (n 7). This result is consistent with the hypothesis that indomethacin promotes SR Ca2 release in mammalian skeletal muscle. We also examined the effect of indomethacin in cultured C2C12 myotubes. Indomethacin also increased the resting free [Ca2 ], in a dose-dependent manner in these cells (Fig. 9B). Unlike adult skeletal muscle bers, myotubes have been reported to partially rely on the inux of extracellular Ca2 for ECC (44). Therefore, we also examined the effect of indomethacin on resting free [Ca2 ]i in the myotubes in the absence of external Ca2 . Removal of external Ca2 in the bath solution did not abolish the Ca2 elevation induced by 200 M indomethacin (n 4), although the peak value of the uorescence ratio increase in the absence of

Fig. 6. Indomethacin (200 M) did not protect the mechanically skinned EDL bers from the high-[Ca2 ] exposure-induced loss of DIFRs. AJP-Cell Physiol VOL

Fig. 7. Effect of indomethacin (200 M) on the force-[Ca2 ] relationship of the contractile apparatus in skinned EDL bers of the rat. Hill plots were tted to the mean force measurements (as a percentage of maximum force) made in a set of highly buffered Ca2 solutions of different known free Ca2 concentration, in the absence (control) and presence of indomethacin. www.ajpcell.org

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Fig. 8. Effect of indomethacin (200 M) on the sarcoplasmic reticulum (SR) Ca2 loading in skinned EDL bers of the rat.

extracellular Ca2 was signicantly decreased by 43.6% (P 0.01, unpaired Students t-test), suggesting the indomethacin-induced Ca2 elevation may also directly or indirectly induce Ca2 inux in myotubes. The peak value of the uorescence ratio (F340/F380) induced by 200 M indomethacin was comparable to the peak of a 60-Hz electrical eld stimulation-induced Ca2 transient or larger (Fig. 10). The effect of indomethacin on [Ca2 ]i could be repeated after the drug had been washed out of the preparation (Fig. 10). These results indicate that indomethacin can induce a large rapid Ca2 release from an internal Ca2 store, most likely the SR in C2C12 myotubes.
DISCUSSION

of indomethacin on the contractile apparatus would explain much of the difference in the force amplitudes observed in the presence and absence of the inhibitors. The peaks of the DIFRs reactivated before rundown by indomethacin were around 55% of maximum force. From Fig. 7, a Ca2 level producing 55% of maximum force in controls should produce DIFRs peaks of around 82% of maximum force in the presence of indomethacin, which is close to the mean peak of 91% of maximum force found in the presence of indomethacin. However, the effect of indomethacin (200 M) on restoration of DIFRs after rundown is unlikely to be due solely to the effects of the drug on the contractile apparatus (Fig. 7). From Fig. 7, if peak Ca2 during release dropped to a level producing little force under control conditions after rundown (e.g., pCa 6.6; Fig. 7), the force in the presence of indomethacin at the same [Ca2 ] would only produce around 2% of maximum force, far below the 91% of maximum force level observed in this study. In this study, the PLA2 inhibitors indomethacin and quinacrine could restore a form of DIFRs after rundown in the skinned EDL bers; however, they produced no protection against the damaging effects of exposure to elevated intracellular [Ca2 ] restore and did not DIFRs after disruption of ECC via exposure to high Ca2 . These ndings suggest, rst, that PLA2 activation is not primarily involved in Ca2 -induced

In this study, we found that 200 M indomethacin, a concentration that is reported to inhibit PLA2 activity in cells, substantially increased the peak and duration of the DIFRs in fully functional skinned EDL bers. Quinacrine, another general PLA2 inhibitor, also increased the peak and duration of the DIFRs. The similar effects of two structurally different general PLA2 inhibitors indicate that inhibition of PLA2 has marked effects on ECC in mammalian skinned skeletal muscle bers. Another important nding of this study was that indomethacin and quinacrine, and to some extent dexamethasone, restored DIFRs after rundown in rat skinned EDL bers. This suggests that PLA2 activation may be an important step in the rundown of DIFRs that occurs in skinned bers. Again, the fact that the DIFRs returned (at least partially) in the presence of three structurally different PLA2 inhibitors would argue against this result being due to a nonspecic effect of either drug. The DIFRs that were restored by the PLA2 inhibitors after rundown were considerably larger and much broader than the initial control responses and similar to those activated in the presence of the PLA2 inhibitors before rundown occurred. Indomethacin (200 M) was also found to increase the sensitivity of the contractile apparatus to Ca2 in the skinned bers. This explains the increase in the resting tension in the repolarization solution in the presence of indomethacin (Figs. 1 and 2) and raises the possibility that the products of PLA2 activation can alter the function of the contractile proteins. This effect
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Fig. 9. Effect of indomethacin on the resting free cytosolic Ca2 concentration ([Ca2 ]i) in collagenase-digested exor digitorum brevis (FDB) bers and C2C12 myotubes. A: a typical example of traces recorded from a FDB ber and a C2C12 myotube exposed to indomethacin (200 M). The arrows indicate the time when indomethacin was added into the incubation bath. B: peak [Ca2 ]i evoked by indomethacin in C2C12 myotubes (20200 M) and in FDB bers (peak values after 10 min, n 4). Elimination of external Ca2 in the bath solution did not abolish the [Ca2 ]i increase induced by indomethacin (200 M) in C2C12 myotubes, although the amplitude of the [Ca2 ]i increase in the absence of extracellular Ca2 was signicantly decreased. www.ajpcell.org

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Fig. 10. Repeatable indomethacin (200 M)induced [Ca2 ]i responses. The 3 [Ca2 ]i responses were recorded from 1 single C2C12 myotube. Between each response, there was a 5-min wash. The dashed line indicates the presence of 200 M indomethacin.

uncoupling in skinned bers and, second, that Ca2 induced uncoupling is unlikely to be involved in the use-dependent rundown of DIFRs observed in skinned bers of the rat. Interestingly, depolarization in the presence of indomethacin (or quinacrine) under control conditions in healthy bers actually resulted in the loss of the ability of the bers to respond to depolarization with a force response. This was not simply due to exposure of the bers to indomethacin, because exposure of the bers to indomethacin in the repolarization solution alone or brief depolarization in the presence of indomethacin did not cause loss of the DIFRs. Therefore, the loss of ECC in skinned bers depolarized in the presence of indomethacin and quinacrine is likely to be a direct consequence of the large force responses that occur upon depolarization in the presence of indomethacin and quinacrine. It is possible that the force responses lead to depolarization of the sealed t-system. Eccentric contractions have been shown to cause signicant and persistent depolarization of the resting membrane potential in rat skeletal muscle bers (37). Although the skinned bers have been stimulated to contract isometrically in this study, the skinning process may make bers susceptible to damage normally only produced after eccentric exercise. This hypothesis would explain both the use-dependent rundown and the immediate rundown caused by the large extended contractions elicited in the presence of indomethacin. In this study, indomethacin signicantly increased SR Ca2 accumulation in the skinned bers. This is consistent with the reported effects of the products of PLA2 activity, lysophospholipids and arachidonic acid, both of which have demonstrated roles in cell signaling (8, 39). In SR vesicles prepared from canine masseter skeletal muscle, arachidonic acid was shown to decrease SR calcium uptake by 50% and indomethacin reversed the reduction of SR calcium uptake by arachidonic acid (42). This is consistent with the ndings of the present study that indomethacin increased SR calcium loading in the skinned bers and suggests that interruption of arachidonic acid production may be one of the mechanisms responsible for the effects of the PLA2 inhibitors used in this study. Furthermore, in skinned bers, arachidonic acid has recently been shown to increase the rate of use-dependent rundown of DIFRs (6), which is consistent with the ability of indomethacin to reverse rundown in this study.
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Indomethacin was also found to induce a spontaneous elevation in [Ca2 ]i in both intact isolated adult rat FDB bers and C2C12 myotubes in this study, indicating that PLA2 inhibition promotes SR Ca2 release in intact skeletal muscle cells. In the intact FDB bers, the Ca2 release induced by indomethacin must have been substantial as the cytosolic [Ca2 ] rose slowly and steadily and eventually resulted in cell contracture and death, indicating that the sarcolemmal and SR Ca2 pumps were completely overwhelmed by the amount of Ca2 release that occurred (even in the presence of increased SR loading in the presence of indomethacin). The precise mechanism responsible for this Ca2 release is unknown; however, it is unlikely to be via membrane depolarisation because indomethacin did not affect the membrane potential in intact EDL bers in this study. The drug may be affecting the function of the voltage sensors, slowing inactivation of the voltage sensors, and prolonging SR Ca2 release. However, indomethacin caused Ca2 release in intact polarized (twitched in response to electrical eld stimulation) bers where a vast majority of the voltage sensors would not be in the inactivated state. The most likely mechanism is that indomethacin directly increased the activity of the SR Ca2 release channels. Many effects of indomethacin in the skinned bers were also consistent with indomethacin activating the SR Ca2 release channels. The effect of indomethacin on SR Ca2 release could not be investigated directly (e.g., Ref. 3) in the skinned bers in this study due to the effects of indomethacin on the contractile apparatus (Fig. 7). However, increased SR Ca2 release in response to voltage sensor activation would explain the dramatically increased duration of the DIFRs observed in the presence of indomethacin in this study. In addition, indomethacin was found to induce spontaneous Ca2 release in some skinned EDL bers when the ber was initially transferred to the repolarization solution containing indomethacin (e.g., Fig. 3), which is also consistent with indomethacin directly activating SR Ca2 release in EDL bers. Increased SR Ca2 release channel activity in the presence of indomethacin in the skinned bers would also explain the restoration of the DIFRs after rundown upon exposure to the drug. Indomethacin may increase the sensitivity of the SR to respond (with Ca2 release) to upstream signaling events (voltage sensor or intermediate proteins) that may have been weakened during the runwww.ajpcell.org

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down process (e.g., via contraction-induced depolarization). It has recently been shown that the benzophenanthridine drug chelerythrine also has the ability to restore DIFRs after rundown in skinned bers, and this drug also increased SR Ca2 loading and release in these bers (27). It should be noted that the increased SR loading that occurs in the presence of indomethacin could also be playing an indirect role in enhancing SR Ca2 release in the EDL bers. It had been shown in skinned bers that SR Ca2 overload can promote Ca2 -induced Ca2 release from the SR (33). Therefore, SR Ca2 release in EDL bers may be augmented in the presence of indomethacin by increased SR Ca2 release channel activity, an increase in the amount of SR Ca2 available for release, and an increase in the sensitivity of the SR Ca2 -induced Ca2 release. The exact isozyme(s) of PLA2 targeted by indomethacin and quinacrine in this study to restore the reactivation of DIFRs after rundown is(are) uncertain. However, sPLA2 inhibition is unlikely to be involved, rst, because the specic sPLA2 inhibitor aristolochic acid (IC50 40 M) at a concentration of 100 M did not restore DIFRs after rundown in this study, and second, because it is highly unlikely that the millimolar Ca2 concentrations required for sPLA2 activation (40) would have occurred under the experimental conditions used in this study. The downstream pathways responsible for restoration of DIFRs in the presence of PLA2 inhibition are also unknown. However, neither the cyclooxygenase (COX) nor the LO pathways appear to be involved because neither indomethacin (20 M), a potent nonspecic COX inhibitor at nanomolar concentrations (IC50 740 nM for COX-1, 970 nM for COX-2), nor the LO inhibitor NDGA (50 M) was able to restore the DIFRs after rundown in this study. It is possible that arachidonic acid and/or lysophospholipids are acting directly on membrane integrity, because both these substances can have a detergent effect and decrease the stability of cell membrane structures (10). The results of this study show that PLA2 inhibition by indomethacin plays an important role in modulation of SR function in mammalian skeletal muscle bers that have full functional integrity. The ability of PLA2 inhibitors to restore DIFRs in the skinned bers suggests that PLA2 may also be a factor contributing to the rundown of DIFRs in skinned bers. In intact skeletal muscle, PLA2 activation has been implicated in the effects of contraction-induced muscle damage (2, 26) and long-term fatigue (34). However, the precise in vivo role of PLA2 in skeletal muscle contractile function remains to be elucidated. From the results of this study, PLA2 activation could play a protective role during strenuous exercise, acting to dampen SR function after damaging contractile activity to minimize further damage and prevent ber necrosis. Further investigation of the mechanism of action of PLA2 in skeletal muscle could also have important ramications for our understanding of the mechanisms of muscle disease, because PLA2 activity has recently been
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shown to be elevated in patients with the Duchenne and Becker forms of muscular dystrophy (36).
We thank Dr. Jason White for providing us with C2C12 myoblasts. DISCLOSURES This work was supported by a grant from Australian Research Council. REFERENCES 1. Ambudkar IS, Abdallah ES, and Shamoo AE. Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum. Mol Cell Biochem 79: 8189, 1988. 2. Armstrong RB. Initial events in exercise-induced muscular injury. Med Sci Sports Exerc 22: 429435, 1990. 3. Bakker AJ and Berg HM. Effect of taurine on sarcoplasmic reticulum function and force in skinned fast-twitch skeletal muscle bres of the rat. J Physiol 538: 185194, 2002. 4. Bakker AJ, Head SI, Wareham AC, and Stephenson DG. Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle bers. Am J Physiol Cell Physiol 274: C1718C1726, 1998. 5. Bakker AJ, Lamb GD, and Stephenson DG. The effect of 2,5-di-(tert-butyl)-1,4-hydroquinone on force responses and the contractile apparatus in mechanically skinned muscle bres of the rat and toad. J Muscle Res Cell Motil 17: 5567, 1996. 6. Barnes M and Stephenson DG. Arachidonic acid decreases depolarisation induced force in mechanically skinned skeletal muscle bers from the rat. Proc Aust Physiol Pharmacol Soc 2000 31: 21P, 2000. 7. Borzone G, Zhao B, Merola AJ, Berliner L, and Clanton TL. Detection of free radicals by electron spin resonance in rat diaphragm after resistive loading. J Appl Physiol 77: 1218, 1994. 8. Brash AR. Arachidonic acid as a bioactive molecule. J Clin Invest 107: 13391345, 2001. 9. Cardoso CM and De Meis L. Modulation by fatty acids of Ca2 uxes in sarcoplasmic reticulum vesicles. Biochem J 296: 4952, 1993. 10. Chang J, Musser JH, and McGregor H. Phospholipase A2: function and pharmacological regulation. Biochem Pharmacol 36: 24292436, 1987. 11. Cheah AM. Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum. Biochim Biophys Acta 648: 113119, 1981. 12. Chin ER and Allen DG. The role of elevations in intracellular [Ca2 ] in the development of low frequency fatigue in mouse single muscle bres. J Physiol 491: 813824, 1996. 13. Dettbarn C and Palade P. Arachidonic acid-induced Ca2 release from isolated sarcoplasmic reticulum. Biochem Pharmacol 45: 13011309, 1993. 14. Diaz PT, She ZW, Davis WB, and Clanton TL. Hydroxylation of salicylate by the in vitro diaphragm: direct evidence for hydroxyl radical production during fatigue. J Appl Physiol 175: 540545, 1993. 15. Donaldson SKB. Peeled mammalian skeletal muscle bres. Possible stimulation of Ca2 release via a transverse tubule sarcoplasmic reticulum mechanism. J Gen Physiol 86: 501525, 1985. 16. Duncan CJ. Role of intracellular calcium in promoting muscle damage: a strategy for controlling the dystrophic condition. Experientia 34: 15311535, 1978. 17. Duncan CJ and Jackson MJ. Different mechanisms mediate structural changes and intracellular enzyme efux following damage to skeletal muscle. J Cell Sci 87: 183188, 1987. 18. Duncan CJ and Smith JL. The action of caffeine in promoting ultrastructural damage in frog skeletal muscle bers. Evidence for the involvement of the calcium-induced release of calcium from the sarcoplasmic reticulum. Naunyn Schmiedebergs Arch Pharmacol 305: 159166, 1978. www.ajpcell.org

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