1 Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; and 2 Department of Biochemistry, University of Nevada, Reno, Reno, Nevada 89509
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ABSTRACT |
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The purpose of this study was to test the hypothesis that H2O2 decreases the amount of force produced by a given intracellular Ca2+ concentration (i.e., the Ca2+ sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H2O2 and then permeabilized with 10% Triton X-100 to assess the persistent effects of H2O2 on Ca2+ sensitivity. Experiments in which H2O2 was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca2+ sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H2O2 was added to permeabilized strips. H2O2 treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca2+ sensitivity may occur.
regulatory myosin light chain phosphorylation; thiophosphorylation; permeabilized smooth muscle; reactive oxidant species; myosin; actomyosin adenosinetriphosphatase
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INTRODUCTION |
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REACTIVE OXIDANT SPECIES, such as H2O2, play an important physiological role in both muscle (39) and nonmuscle cells (6) and a pathophysiological role in numerous diseases, including acute lung injury, asthma, pulmonary hypertension, ischemia-reperfusion, and arthritis (6). H2O2 reversibly inhibits receptor agonist-induced contraction of both vascular (18, 19, 32) and airway (11, 12, 15, 38, 45) smooth muscle (ASM). Recently, we reported that H2O2-induced relaxation of intact ASM was caused by a reduction in the amount of force produced by a given intracellular Ca2+ concentration ([Ca2+]i, the "Ca2+ sensitivity"), as shown by the fact that the relaxation of canine ASM produced by H2O2 was accompanied by an increase in [Ca2+]i (30). The mechanism for this effect on Ca2+ sensitivity is not known.
Contraction of ASM is controlled largely by the phosphorylation of the 20-kDa regulatory myosin light chain (rMLC), resulting in the cyclic attachment and detachment of the myosin head to actin (i.e., cross-bridge cycling) and the hydrolysis of ATP by actin-activated, myosin ATPase (actomyosin ATPase; see Ref. 43). The level of rMLC phosphorylation depends on the balance between the activities of myosin light chain kinase (MLCK) and smooth muscle protein phosphatase. MLCK activity is regulated by the binding of Ca2+-calmodulin complexes in response to increased [Ca2+]i produced by receptor stimulation, favoring increased rMLC phosphorylation (26). rMLC phosphorylation can also increase if the activity of smooth muscle protein phosphatase is inhibited. The reduction in Ca2+ sensitivity produced by H2O2 in our prior report of H2O2-induced relaxation of intact ASM (30) was accompanied by a decrease in rMLC phosphorylation, suggesting inhibition of Ca2+ and/or activation of smooth muscle protein phosphatases.
H2O2 may also reduce Ca2+ sensitivity by mechanisms not dependent on rMLC phosphorylation, such as by inhibiting actomyosin ATPase activity. The smooth muscle myosin head contains highly reactive cysteine thiols that result in a reduction in ATPase activity when they are covalently modified (4, 28). Illustrating what effects this might have in an analogous system, the mild oxidant nitric oxide reversibly inhibits skeletal muscle actomyosin ATPase activity, decreases Ca2+ sensitivity, and produces relaxation, effects that are fully reversible by a thiol-reducing agent (37). The possibility of a similar effect with a mild oxidant such as H2O2 in smooth muscle has not been evaluated.
The purpose of this study was to test the hypothesis that H2O2 decreases Ca2+ sensitivity in ASM in part by a previously undescribed mechanism independent of changes in rMLC phosphorylation. To achieve this aim, we developed a novel method in which ASM strips were first exposed to H2O2 and then permeabilized with Triton X-100 to assess the persistent effects of H2O2 exposure on Ca2+ sensitivity. We then used purified protein preparations to examine whether H2O2 has a direct inhibitory effect on the actin-activated myosin ATPase activity in a manner consistent with a phosphorylation-independent reduction in Ca2+ sensitivity.
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MATERIALS AND METHODS |
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Tissue preparation. After Institutional Animal Care and Use Committee approval, mongrel dogs of either sex were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated. The trachea was excised and immersed in chilled physiological salt solution (PSS) of the following composition (in mM): 110 NaCl, 26 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. Fat, connective tissue, and the epithelium were removed with tissue forceps and scissors under microscopic observation to make muscle strips.
Mechanical measurements. For studies of permeabilized muscle strips (5 mm long × 0.3 mm diameter) and simultaneous measurement of force and [Ca2+]i in intact (nonpermeabilized) tissue, strips were mounted in 0.1-ml cuvettes and continuously superfused with PSS (37°C) aerated with 94% O2-6% CO2. One end of the strips was anchored via stainless steel microforceps attached to a micrometer, and the other end was attached via stainless steel microforceps to a force transducer (model KG4; Scientific Instruments, Heidelberg, Germany). For rMLC phosphorylation measurements in intact tissue, muscle strips were suspended in 25-ml water-jacketed tissue baths that were filled with PSS (37°C) aerated with 94% O2-6% CO2. One end of the strips was anchored to a metal hook at the bottom of the tissue bath; the other end was attached to a calibrated force transducer (model FT03D; Grass Instrument, Quincy, MA).
In both cases, during a 3-h equilibration period, the strips were repeatedly contracted isometrically for 2 min every 10 min by maximal stimulation with 1 µM ACh. The length of the strips was increased after each stimulation until active force was maximal (optimal length, Lo). Each strip was maintained at this Lo for the remainder of the experiment.Isometric force and fura 2 fluorescence measurements. Muscle strips were incubated with PSS (25°C) containing 5 µM fura 2-AM and aerated with 96% O2-4% CO2 for 3 h (23). Fura 2-AM was dissolved in DMSO and 0.02% cremophor. After fura 2 loading, the strips were washed with PSS (37°C) for 30-50 min to remove extracellular fura 2-AM and DMSO and to allow de-esterification of any remaining cytosolic fura 2-AM.
Fura 2 fluorescence intensity was measured by a photometric system that measures optical and mechanical parameters of isolated tissue simultaneously (16). Light from a mercury high-pressure lamp was passed through rotating bandpass filters to restrict excitation light to 340 and 380 nm. Fluorescence emitted from the strips was filtered at 450 ± 5 nm and detected by a photomultiplier (Scientific Instruments). The emission fluorescence intensities due to excitation at 340-nm (F340) and 380-nm (F380) wavelengths were measured, and the F340-to-F380 ratio was used as an index of [Ca2+]i. Absolute values of [Ca2+]i were not calculated since the dissociation constant of fura 2 for Ca2+ within the smooth muscle cytosol cannot be determined (29). In preliminary work, we found that H2O2 alone did not affect F340 and F380 fluorescence intensities when added to solutions containing Ca2+ and fura 2 (data not shown).Permeabilization of muscle strips.
The strips were either superfused (in studies of force) or incubated
(in studies of rMLC phosphorylation and thiophosphorylation) for 20 min
with relaxing solution containing 10% Triton X-100 (at 25°C; see
Ref. 24). The composition of the relaxing solution was as
follows (in mM): 85 K+, 2.1 disodium ATP
(Na2ATP), 4 EGTA, and 20 imidazole. After permeabilization, the strips were washed with relaxing solution for 5 min to remove excess Triton X-100. Solutions of varying free Ca2+
concentration were prepared using a previously described algorithm (8). Rigor solutions contained (in mM) 85 K+,
0 Na2ATP, 0 free Mg2+, 4 EGTA, 20 imidazole,
and either 1 nM (low-Ca2+ rigor solution) or 10 µM
(high-Ca2+ rigor solution) free Ca2+. The rigor
solutions also contained 2.1 mM adenosine
5'-O-(3-thio)triphosphate (ATPS) for selected experiments
in which the rMLC was thiophosphorylated (22). The pH of
all solutions was buffered to 7.0 with propionic acid, and the ionic
strength was kept constant at 0.2 M by adjusting the concentration of
potassium propionate.
Determination of extent of myosin regulatory light chain phosphorylation in muscle strips. After experimental interventions, muscle strips were flash-frozen with dry ice-cooled acetone containing 10% (wt/vol) TCA and 10 mM dithiothreitol (DTT). For intact muscle, strips were frozen immediately after removal from organ baths while maintaining Lo, because we found in preliminary work that length affects rMLC phosphorylation in intact strips. Permeabilized strips for rMLC phosphorylation measurements were prepared separately according to the same procedures as for force measurements but were incubated in wells (without determination of Lo) instead of being superfused. The strips were pinned at both ends to maintain isometric conditions. After permeabilization, all conditions were identical to those present in the strips used to measure isometric force responses. In preliminary data obtained for prior studies (3, 17, 25, 48), we have shown that muscle length does not affect rMLC phosphorylation in permeabilized ASM in which [Ca2+]i is controlled.
The frozen strips were then allowed to warm to room temperature in the same solution. After TCA was washed out with acetone containing 10 mM DTT, strips were allowed to dry. rMLC was extracted as described by Gunst et al. (14), and phosphorylation was determined by glycerol-urea gel electrophoresis followed by Western blotting with a polyclonal affinity-purified rabbit anti-20-kDa rMLC antibody, as previously described (3). Unphosphorylated and phosphorylated bands of rMLC were visualized by PhosphorImage analysis (Cyclone storage phosphor system; Packard Instrument, Downers Grove, IL) using 125I-labeled protein A (New England Nuclear) to bind to the rMLC antibody. Fractional phosphorylation was calculated as the density ratio of the sum of mono- and diphosphorylated rMLC to total rMLC using OptiQuant software (version 3.0; Packard Instrument).Purified protein preparations.
Smooth muscle myosin was prepared from frozen chicken gizzards
(20) obtained from Pel-Freeze (Rogers, AR). Heavy
meromyosin (HMM) was obtained from a Staphylococcus aureus
protease digestion of 200-400 mg of smooth muscle myosin, as
described previously (21). SDS gel analysis showed that
>95% pure HMM was obtained. HMM concentration was determined using an
extinction coefficient (1% at 280 nm) of 6.5. Ca2+ was prepared from frozen turkey gizzards by the method
of Adelstein and Klee (1). Actin was prepared from frozen
leg and back muscles of rabbits by the method of Spudich and Watt
(44).
Experimental protocols. Each experimental protocol was conducted using muscle strips obtained from a different set of animals. Previous studies, including those from this laboratory, have shown that smooth muscle relaxation induced by H2O2 is partly mediated by cyclooxygenase products, which activate adenylate cyclase, and that this mechanism is completely inhibited by indomethacin (11, 12, 38). Thus, for each protocol, all strips were incubated with 10 µM indomethacin to prevent the formation of prostanoids and the increase in intracellular cAMP levels induced by H2O2 (11, 12, 30).
We found in preliminary studies, and confirmed as described below, that H2O2 did not affect Ca2+ sensitivity when added to permeabilized smooth muscle. Thus, to examine the proposed hypothesis, we developed an experimental protocol in which intact nonpermeabilized strips were briefly exposed to H2O2. H2O2 was then washed from the strips and subsequently permeabilized with Triton X-100 to allow for control of [Ca2+]i. This experimental approach was first validated by three protocols to demonstrate that the effect of H2O2 on Ca2+ sensitivity persisted after complete washout from the intact tissue and after subsequent permeabilization. Next, two protocols were conducted to determine whether the persistent effect of H2O2 on Ca2+ sensitivity after permeabilization was because of inhibition of proteins that regulated rMLC phosphorylation levels, regulated force independent of rMLC phosphorylation levels, or a combination of both processes.Validation protocols.
The first protocol (Fig. 1) determined
whether H2O2 inhibited Ca2+
sensitivity when applied to quiescent (unstimulated), intact muscle
strips. After strips were loaded with fura 2 and set at Lo for force development, they were maximally
activated with 40 mM isotonic KCl for 10 min, inducing stable increases
in both F340/F380 and force. After complete
washout of isotonic KCl for ~20 min and recovery of
F340/F380 and isometric force to the
unstimulated baseline values, the strips were exposed to 1 mM
H2O2 for 10 min. The increase in
F340/F380 and isometric force values induced by H2O2 was expressed as a percentage of the
steady-state values measured during maximal activation with 40 mM
isotonic KCl. In a separate set of three strips mounted in organ baths,
rMLC phosphorylation levels were determined during basal conditions or
after stimulation with either 40 mM isotonic KCl or 1 mM
H2O2 for 10 min.
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Dependence of H2O2-induced effects in
permeabilized tissue on rMLC phosphorylation.
Two experimental protocols were performed to determine if the effects
of H2O2 observed in the permeabilized
preparation depended on changes in rMLC phosphorylation. In the first
protocol, strips were pinned in wells to maintain isometric conditions
for rMLC phosphorylation measurements and then treated using a similar protocol to that represented in Fig. 3. After stimulation with 1 µM
ACh, strips were exposed to 0 (control), 1, or 10 mM
H2O2 for 10 min and then permeabilized
(requiring 25 min) after complete H2O2 washout.
After permeabilization, rMLC phosphorylation was measured 10 min after stimulation with either 0.9 or 10 µM free Ca2+
(see data for Fig. 4).
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Treatment of thiophosphorylated HMM with peroxide and ATPase
measurements in the presence of actin.
To further characterize rMLC-independent H2O2
effects on this system, we directly measured actomyosin ATPase
activity. We studied this using purified HMM, which is a soluble
proteolytic fragment of myosin containing two myosin heads adjoined by
the hinge region. The regulatory light chains of HMM were
thiophosphorylated by incubation in 10 mM MOPS (pH 7.0), 0.1 mM EGTA,
50 mM NaCl, 2.5 mM MgCl2, 2.5 mM CaCl2, 4 µg/ml calmodulin, 1 mM ATPS, and 30 µg/ml MLCK at 25°C for
1 h, followed by overnight incubation on ice (7).
Thiophosphorylated HMM (HMM-P) was separated from reagents by spinning
the sample through a 5-ml buffer exchange column (36)
prepared with Sephadex G-50-80 resin in nonreducing buffer [50 mM
NaCl and 10 mM MOPS (pH 7.0)]. The extent of thiophosphorylation before and after H2O2 treatment was verified by
8 M urea gel electrophoresis, which can detect 5% or greater
unphosphorylated rMLC (7). The ratio of thiophosphate to
HMM was at least 1.9:1 (gels not shown) for all samples studied.
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(1) |
Materials. The polyclonal affinity-purified rabbit anti-20-kDa rMLC antibody was a generous gift of Dr. Susan J. Gunst (Dept. of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN). Na2ATP was purchased from Research Organics (Cleveland, OH). All other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO).
Statistical analysis. Data are expressed as means ± SE; n represents the number of dogs. Paired t-tests were used to compare two groups. Repeated-measures ANOVA was used to compare multiple groups, and the Dunnett's test was used for post hoc comparisons. A value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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Validation of preparation. Exposure of intact quiescent strips to 1 mM H2O2 produced a greater increase in F340/F380 and a smaller increase in isometric force compared with prior maximal responses to 40 mM isotonic KCl (Fig. 1). This was accompanied by a smaller increase in rMLC phosphorylation than that observed for a maximal KCl response that was not significantly different from baseline unstimulated values (Fig. 1). Thus, when applied to unstimulated strips, H2O2 inhibited Ca2+ sensitivity, in part by inhibiting the increase in rMLC phosphorylation produced for a given [Ca2+]i.
When added to intact strips contracted with 25 mM isotonic KCl, 1 mM H2O2 increased F340/F380 and caused an initial decrease in isometric force (Fig. 2, top). However, at 10 min of exposure to H2O2, force had recovered such that it was not significantly different from that measured before H2O2 exposure. When H2O2 was washed from the muscle strips during continuous stimulation with 25 mM isotonic KCl, the F340/F380 rapidly returned to pre-H2O2 values. However, force decreased to nearly baseline values measured before stimulation with isotonic KCl. At this point (10 min after H2O2 washout), the level of rMLC phosphorylation measured in a separate set of strips was significantly less in strips that had been exposed to H2O2 compared with time control strips that had not been exposed to H2O2 (11.0 ± 5.3 and 23.2 ± 2.4%, respectively, P = 0.03, n = 5). In fact, this level of rMLC phosphorylation 10 min after H2O2 washout was not significantly different from the baseline level measured in the unstimulated strips before stimulation with isotonic KCl (4.1 ± 3.2%, P = 0.16). Thus the effect of H2O2 on Ca2+ sensitivity and rMLC phosphorylation persists in intact strips 10 min after complete H2O2 washout. When added to strips before permeabilization, H2O2 induced a small increase in isometric force (Fig. 3), as in the first experiment (Fig. 1). This exposure decreased the force response to 10 µM free Ca2+ after permeabilization by 44% compared with control strips that were not exposed to H2O2. When added to strips that were stimulated with 10 µM free Ca2+ after permeabilization (i.e., the control strips for this experiment), 1 mM H2O2 did not affect isometric force (Fig. 6). Thus the effect of H2O2 on Ca2+ sensitivity that occurred in the intact tissue (Fig. 1) and persisted after H2O2 washout for 10 min (Fig. 2) also persisted 25 min after permeabilization. By contrast, H2O2 did not affect Ca2+ sensitivity when added after the muscle strips had been permeabilized.
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Dependence of H2O2 effects on rMLC phosphorylation in permeabilized muscle. rMLC phosphorylation was measured in permeabilized strips after exposing the intact tissue to 0 (control), 1, or 10 mM H2O2 before permeabilization. H2O2 did not affect rMLC phosphorylation under conditions of low free Ca2+ concentration (Fig. 4). H2O2 (10 mM but not 1 mM) significantly inhibited the increase in rMLC phosphorylation induced by either 0.9 or 10 µM free Ca2+. Thus the persistent effect of 1 mM H2O2 on Ca2+ sensitivity in permeabilized strips observed in the previous experiment (Fig. 3; as assessed by isometric force responses) was produced by mechanisms independent of changes in rMLC phosphorylation. For 10 mM H2O2, inhibition of the Ca2+-dependent increase in rMLC phosphorylation was also present.
To confirm the existence of H2O2 effects on Ca2+ sensitivity that are independent of changes in rMLC phosphorylation, ATP
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DISCUSSION |
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The findings of this study confirm prior observations that H2O2 relaxes intact ASM by decreasing Ca2+ sensitivity, in part by decreasing rMLC phosphorylation maintained for a given [Ca2+]i (Fig. 2). However, results in the permeabilized smooth muscle preparation used in these studies also demonstrate for the first time that H2O2 also inhibits Ca2+ sensitivity by mechanisms independent of changes in rMLC phosphorylation. Although we were unable to demonstrate inhibition of actomyosin ATPase using an in situ ATPase assay, we demonstrate that a purified subfragment of smooth muscle myosin is modified by H2O2 treatment and that this modification results in inhibition of its ATPase activity in the presence of F-actin. These results suggest that an oxidant species may decrease force at a given [Ca2+]i in part by a direct effect on a key contractile protein, specifically myosin.
In a prior report, we found that H2O2-induced relaxation of canine ASM contracted by muscarinic stimulation or membrane depolarization was caused entirely by decreases in Ca2+ sensitivity, since isometric force decreased while [Ca2+]i increased. These effects on isometric force, Ca2+ sensitivity, and [Ca2+]i were spontaneously reversible after H2O2 washout, demonstrating that these observations do not reflect permanent damage to smooth muscle myocytes in ASM (30). In the current study, we also found a dramatic, reversible increase in [Ca2+]i when H2O2 was added to quiescent, unstimulated strips. This is consistent with prior reports in other cell types (18, 31, 40, 41). Proposed mechanisms include lipid peroxidation (31), stimulation of sarcoplasmic reticulum Ca2+ channels (9), direct activation of intracellular kinases (47), and activation of membrane Ca2+ channels (40). These increases in [Ca2+]i probably account for prior reports that H2O2 increases tone in resting ASM (2, 12, 15, 34, 38, 45). However, the increase in isometric force associated with the increase in [Ca2+]i induced by H2O2 is much less than that expected from the force and F340/F380 responses induced by isotonic KCl. These data indicate that H2O2 decreases Ca2+ sensitivity when applied to quiescent, unstimulated tissue.
We found that H2O2 had no effect on Ca2+ sensitivity when added to strips that were already permeabilized. This finding suggests that components of the solutions bathing the permeabilized muscle inhibited the ability of H2O2 to oxidize intracellular protein targets that regulate isometric force or that a factor important for H2O2 actions was lost during permeabilization. A number of factors, including the chelation of divalent metal ions that may catalyze peroxide-mediated thiol oxidation (35, 46) and the presence of the buffer imidazole, which inefficiently reacts with peroxide (42, 49), may also contribute to this lack of peroxide effect in the permeabilized preparation. This finding has implications regarding the mechanism by which peroxide mediates its effects in this smooth muscle tissue that deserve further exploration.
The results of the experiment in which H2O2 was administered and then removed from isotonic KCl-depolarized strips showed that increases in [Ca2+]i produced by H2O2 can offset H2O2-induced decreases in Ca2+ sensitivity such that at 10 min of exposure there was no net change in isometric force (Fig. 2). However, complete H2O2 washout during continuous activation with isotonic KCl (Fig. 3) uncovered an underlying, persistent effect on Ca2+ sensitivity that was at least partially mediated by decreases in rMLC phosphorylation. This decrease in rMLC phosphorylation is consistent with that observed in the presence of H2O2 found in our previous study (30). The persistence of this effect on Ca2+ sensitivity made it possible to pursue a strategy of permeabilization after treatment of intact strips with H2O2, an exposure that occurs under conditions allowing relevant chemical intermediates to be generated.
At least one component of H2O2-induced inhibition of Ca2+ sensitivity survived permeabilization, as indicated by decreases in the isometric force induced by 10 µM free Ca2+ in permeabilized strips treated before permeabilization with H2O2 (Fig. 3). However, with 1 mM H2O2, this effect was not associated with a decreased level of rMLC phosphorylation (Fig. 4). Thus the effect of this concentration of H2O2 on rMLC phosphorylation was either reversed by permeabilization or had dissipated over the 30-40 min required by the protocol. After exposure to 10 mM H2O2, the effect on rMLC phosphorylation did persist. Thus two mechanisms were responsible for the decrease in Ca2+ sensitivity induced by H2O2 in the intact strips: 1) a component dependent on a decrease in rMLC phosphorylation, which was present after permeabilization only after exposure to a high concentration of H2O2 (10 mM) and 2) a component that did not depend on changes in rMLC phosphorylation, which persisted after permeabilization observed after exposure to 1 mM H2O2. The presence of the latter mechanism was confirmed by the thiophosphorylation experiments (Figs. 5 and 7), which support the study hypothesis.
The mechanisms of H2O2 effects on Ca2+ sensitivity remain speculative. There are several examples of H2O2-induced oxidation of intracellular proteins, with resultant changes in the activity of the protein, including creatine kinase (13, 27) and the ryanodine receptor Ca2+ release channel (9). Many proteins are susceptible to oxidative stress by selective modification of sulfur-containing amino acid residues, such as cysteine (9) and methionine (10), which can impact enzyme and cellular function. Cysteine thiols can be oxidized by H2O2 to several states, depending on the duration of exposure to H2O2, H2O2 concentration, and the reactivity of the thiol (9). Potential targets responsible for the effects dependent on changes in rMLC phosphorylation include calmodulin, smooth muscle protein phosphatase, and Ca2+. The latter is known to contain reactive thiols that are potential targets of H2O2 (33).
Effects independent of changes in rMLC phosphorylation could be related to effects on smooth muscle actomyosin ATPase. The myosin head contains reactive cysteine thiols, which when oxidized or covalently modified inhibit actomyosin ATPase activity (4, 28). Such a modification would decrease isometric force independent of [Ca2+]i or rMLC phosphorylation, an effect noted for myosin oxidation produced by sodium nitroprusside in skeletal muscle (37). Using a purified protein preparation, we have demonstrated that 1 mM H2O2, under conditions similar to those used in intact tracheal smooth muscle experiments (30), inhibited smooth muscle actin-activated myosin ATPase activity by ~50% (Fig. 8). The studies were performed on maximally thiophosphorylated HMM, indicating that the observed reduction in actomyosin ATPase activity occurred independently of rMLC phosphorylation. Although allowing for the fact that H2O2 treatment of intact tracheal smooth muscle occurs in a different microenvironment, we find it plausible that the smooth muscle myosin itself is an H2O2-sensitive target. Inhibition of actomyosin ATPase activity on this basis would account for the phosphorylation-independent reduction in Ca2+ sensitivity. Other possible explanations for this novel observation, including H2O2 oxidation of actin, actin-associated proteins, or of enzymes regulating actin-associated proteins, have not yet been addressed.
In summary, we confirm that H2O2 decreases Ca2+ sensitivity in ASM in part by decreasing the rMLC phosphorylation produced by a given Ca2+ concentration. This effect depends on factors not present or active under the study conditions used in permeabilized ASM. However, experiments in which H2O2 is administered before permeabilization have made it possible for the first time to observe a distinct mechanism that is independent of changes in rMLC phosphorylation that also contributes to H2O2-induced decreases in Ca2+ sensitivity. We provide evidence that oxidation of myosin may provide an explanation for this mechanism of H2O2-mediated reduction in Ca2+ sensitivity.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-45532 and HL-54757 and by funds from Mayo Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. A. Jones, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: jones.keith{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 18, 2002;10.1152/ajplung.00159.2002
Received 22 May 2002; accepted in final form 2 October 2002.
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