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Asthma Research Group, Smooth Muscle Research Group, Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We examined the ionic mechanisms underlying
the responses of canine trachealis to superoxide (generated in vitro by
using xanthine oxidase or added exogenously) and peroxide (generated spontaneously in vitro by the dismutation of superoxide or added exogenously). Although neither had any effect on resting tone, both
triggered relaxations in carbachol-precontracted tissues. These
relaxations were eliminated by catalase but were much less sensitive to
the hydroxyl radical scavenger dimethylthiourea, indicating they were
mediated primarily by peroxide. These relaxations were decreased in
magnitude and/or slowed by nifedipine
(10
6 M), ouabain
(106 M), or tetraethylammonium (25 mM),
but not by 4-aminopyridine (5 mM), and were small or absent in tissues
precontracted with 30 mM KCl. Finally, peroxide triggered membrane
hyperpolarization and elevated cytosolic concentration of
Ca2+ (primarily via release from the internal store). Thus
peroxide-mediated relaxations seem to involve Ca2+ release,
opening of Ca2+-dependent K+ channels,
hyperpolarization, closure of Ca2+ channels, and
relaxation. In addition, some other free radical (hydroxyl radical?)
may activate the Na+-K+ pump, also
hyperpolarizing the membrane and causing relaxation.
reactive oxygen species; free radicals; sarcoplasmic reticulum; contraction/relaxation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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OXYGEN FREE RADICALS (OFRs) and reactive oxygen species (ROS) are generated by the sequential reduction of oxygen (30): transfer of one electron to oxygen (e.g., during the metabolism of xanthine by xanthine oxidase) produces superoxide ion; transfer of two electrons followed by dismutation of superoxide generates hydrogen peroxide; further oxidation of peroxide (e.g., via a Fenton-type reaction catalyzed by a redox-reactive transition metal such as ferrous iron) leads to the highly potent hydroxyl radical.
Inflammatory cells in the airways are capable of producing large quantities of ROS as a defense mechanism against allergens and invading microorganisms (18, 26, 27). Many studies have shown that ROS and OFRs can also alter airway function. For example, the ROS ozone causes airway hyperresponsiveness and accumulation of free radicals (26), both of which are prevented by pretreatment with antioxidants (18). Other ROS and OFRs also alter mechanical activity of airway smooth muscle, but the nature of these changes and the underlying mechanisms vary markedly: they trigger contraction in airway smooth muscle of the cat (1), guinea pig (7, 16), and human (22), but relaxation in rabbit (7) and canine (4, 14) airway smooth muscle. Furthermore, these effects may involve a direct action on the smooth muscle per se (1, 4, 7) or an indirect effect via the epithelium (8, 19, 22), which in turn modulates smooth muscle function. Thus a better understanding of the effects of OFRs and ROS on airway mechanical activity is essential and may lead to new therapies for asthma.
Recent studies using contractions as an indirect index of cytosolic concentration of Ca2+ ([Ca2+]i) have suggested that airway hyperresponsiveness induced by ozone may involve a disruption of Ca2+ handling (20, 23). OFR-induced disruption of Ca2+ handling has been examined in more detail in nonairway smooth muscle cells/tissues, but the underlying mechanisms involved seem to vary from cell type to cell type. For example, OFRs trigger Ca2+ entry through voltage-dependent Ca2+ channels and Ca2+ overload in arterial smooth muscle (17, 24), tracheal epithelial cells (21), and freshly dissociated rat cardiac myocytes (15); in contrast to this, another study of cultured rat cardiac myocytes found that OFR-induced elevation of [Ca2+]i did not involve voltage-dependent Ca2+ channels, Na+-Ca2+ exchange, nor a nonspecific "leakiness" of the plasmalemma (2), whereas in guinea pig ventricular myocytes, OFRs reduce the number of Ca2+ channels and thereby suppress voltage-dependent Ca2+ currents (16). Free radicals also elevate [Ca2+]i in endothelial cells by increasing Ca2+ influx, but this influx is suppressed by Mn2+ but not verapamil, suggesting an ion channel type other than the typical voltage-dependent Ca2+ channel is involved (3). In coronary artery smooth muscle (5), free radicals elevate [Ca2+]i via damage to the sarcoplasmic reticulum Ca2+ pump, leading to a leak of Ca2+ from the sarcoplasmic reticulum. However, the effect(s) of OFRs and ROS on Ca2+ handling in airway smooth muscle is still poorly understood.
In the present study, we sought to characterize the effects of the ROS superoxide, peroxide, and hydroxyl ion on epithelium-denuded canine tracheal smooth muscle function: in particular, we examined the roles of Ca2+-dependent K+ channels, Na+-K+-ATPase activity, release of internally sequestered Ca2+, and voltage-dependent Ca2+ influx in mediating these effects.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation of tissues and cell dissociation. Adult mongrel dogs were euthanized with pentobarbital sodium (100 mg/kg). Tracheae were excised and kept in physiological solution. The smooth muscle was isolated by removing connective tissue, vasculature, and epithelium, then cut into strips parallel to the muscle fibers (1 mm wide). For single-cell studies, tracheal strips (0.5-1.0 g wet wt) were transferred to dissociation buffer (composition given below) containing collagenase (type IV; 2.7 U/ml), elastase (type IV; 12.5 U/ml), and bovine serum albumin (1 mg/ml), then were either used immediately or stored at 4°C for use up to 48 h later; we have previously found that cells used immediately and those used after 48 h of refrigeration exhibit similar functional responses (i.e., contraction and activation of Ca2+-dependent ion conductances; 9, 13). To liberate single tracheal smooth muscle cells, tissues in enzyme-containing solution were incubated at 37°C for 60-120 min, then gently triturated.
Organ bath studies.
Tracheal smooth muscle strips were mounted vertically in 3 ml organ
baths by using silk (Ethicon 4-0) tied to either end of the strip,
one of which was fastened to a Grass FT.03 force transducer whereas the
other was anchored. Isometric changes in tension were digitized (2 Hz)
and recorded by using an on-line program (DigiMed System Integrator,
MicroMed, Louisville, KY). Tissues were bathed in Krebs-Ringer buffer
(see below for composition), bubbled with 95% O2-5%
CO2, and maintained at 37°C. Preload tension was
~1.25 g (determined previously to allow maximal responses). Tissues were equilibrated for 1 h before the specific experiments were started.
Unless indicated otherwise, tissues were precontracted with carbachol
(CCh; 107 M) 20 min before addition of
xanthine/xanthine oxidase; free radical scavengers and/or other
pharmacological agents were also added during this period.
Fura 2 fluorimetry. Single cells were studied by using a Deltascan system (Photon Technology International, South Brunswick, NJ). After settling onto a glass coverslip mounted onto a Nikon inverted microscope, cells were loaded with the membrane-permeant form of fura 2 (fura 2-acetoxymethyl ester, 2 µM for 30 min at 37°C), then superfused continuously with Ringer buffer at 37°C (2-3 ml/min). Cells were illuminated alternately (0.5 Hz) at the excitation wavelengths, and the emitted fluorescence (measured at 510 nm) induced by 340-nm excitation (F340) and that induced by 380-nm excitation (F380) were measured by using a photomultiplier tube assembly. F340/ F380 was converted to cytosolic Ca2+ concentration by using previously published methods (6). Rmax and Rmin (the fluorescence ratio values under saturating and Ca2+-free conditions, respectively) were obtained previously (9, 13); the Ca2+-fura 2 dissociation constant was assumed to be 224 nM (6). Background fluorescence, determined by using cells not loaded with fura 2 but otherwise handled in a similar fashion, was subtracted from the raw data. Agonists were applied by pressure ejection from a puffer pipette (Picospritzer II; General Valve, Fairfield, NJ).
Microelectrode studies.
Intact tissues were carefully pinned out in a chamber having a bath
volume of ~10 ml; Krebs-Ringer buffer (composition given below) was
bubbled with 95% O2-5% CO2, heated to
37°C, and superfused over the tissues at a rate of ~3 ml/min.
Conventional microelectrodes (tip resistance of 30-80 M
when
filled with 3 M KCl) were pulled from borosilicate capillary tubes and
used to impale single smooth muscle cells. Membrane potential changes
were observed on a dual-beam oscilloscope (Tektronix D13; 5A22N
differential amplifier; 5B12 dual time base) and recorded on 1/4-in.
magnetic tape by using a Hewlett-Packard instrumentation recorder.
Portions of the recorded data were played back, digitized (Digidata
1200), and sampled by using pCLAMP 6 software (Axon Instruments, La
Jolla, CA), then fitted by using pCLAMP 6 and/or exported to SigmaPlot
(Jandel, Corte Madera, CA) for graphical presentation.
Solutions and chemicals. Dissociation buffer contained the following (in mM): 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 0.25 EDTA, 10 D-glucose, and 10 L-taurine, 10; pH 7.0.
Single cells were studied in Ringer buffer containing (in mM): 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose; pH 7.4. Nominally Ca2+-free Ringer buffer was prepared by omitting CaCl2 and adding 1 mM EGTA. Intact tissues were studied by using Krebs-Ringer buffer containing (in mM) 116 NaCl, 4.2 KCl, 2.5 CaCl2, 1.6 NaH2PO4, 1.2 MgSO4, 22 NaHCO3, and 11 D-glucose, bubbled continuously to maintain pH at 7.4. Chemicals were obtained from Sigma Chemical, with the exception of fura 2 acetoxymethyl ester (Calbiochem, La Jolla, CA). All agents were prepared as aqueous solutions except for cyclopiazonic acid, fura 2, and KO2 (DMSO), and nifedipine (absolute EtOH).Data analysis. As described above, tension recordings for each tissue were sampled at a frequency of 2 Hz and saved in digital format as a string of data pairs [i.e., (time:tension)]. For graphical presentation, tracings from several tissues were "averaged" in the following way: 1) the string of data pairs for each tissue was synchronized and standardized by identifying the datum pair collected 5 min before addition of xanthine/xanthine oxidase, then re-defining the time of that datum pair as zero and the tension of that datum pair as 100%; 2) all subsequent data from each tissue were then rescaled appropriately; and 3) the average tension at each point in time (500-ms intervals) was calculated. For statistical analysis of the data from these experiments (i.e., values in text and Table 1), the mean tone for each individual trace was determined, then the overall mean (±SE) was calculated. Responses are reported as means ± SE. Comparisons were made by using a two-tailed paired Student's t-test, with P < 0.05 being considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Free radicals reverse CCh-induced tone, but not resting tone, in
canine airway smooth muscle.
Resting tone was unaffected by either xanthine
(105 and
104 M) or xanthine oxidase (10 and 100 mU), added individually or in combination (data not shown). Similarly,
neither of these agents, when added alone, had any effect on tone
induced by CCh (107 M; not shown).
However, the combined addition of xanthine and xanthine oxidase in
CCh-precontracted tissues triggered a small and transient increase in
tension followed by marked reversal of tone that was sustained in some
tissues (e.g., Fig. 1A) but oscillated periodically in others (e.g., Fig. 1B); in either
case, the overall relaxant effect stabilized within 10-15 min. To
portray graphically the overall degree of inhibition produced by
addition of ROS, we averaged the traces obtained under
each experimental condition by using the approach outlined in
METHODS. Figure 2 shows the
average effect of 10 and 100 mU xanthine oxidase in all tissues studied
(in the presence of 104 M xanthine);
ultimately, CCh-induced tone was reversed 25.3 ± 7.4 and 47.7 ± 6.7% (n = 5), respectively, by this free-radical-generating system.
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5 M, and nearly
complete reversal of CCh-induced tone after addition of
104 M KO2 (n = 4).
Free radical-induced relaxations are mediated by peroxide. Peroxide is formed by the dismutation of superoxide (either spontaneously or catalyzed by cytosolic and/or mitchondrial superoxide dismutase) and can in turn be further oxidized to produce hydroxyl free radical (30; see the beginning of this study). We therefore examined the relative contributions of these different OFRs in mediating xanthine oxidase-triggered effects.
Pretreatment of the tissues with the hydroxyl radical scavenger dimethylthiourea (DMTU; 103 M; Ref. 29)
had no marked effect on the time course of the relaxations triggered by
xanthine oxidase plus xanthine (Fig. 2), and the mean magnitude of
these relaxations was not significantly different from control (Table
1).
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4 M, and
nearly complete reversal of tone with
103 M H2O2
(n = 8). Oscillations of tone were rarely seen in tissues exposed to H2O2. DMTU
(103 M) also had no effect on the peak
magnitude of relaxations evoked by H2O2 (Fig.
3B); however, in contrast to the sustained relaxation generally
seen in control tissues, H2O2-induced
relaxations reversed over time in tissues pretreated with DMTU
(103 M; Fig. 3B).
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Peroxide induces membrane hyperpolarization.
Free radicals may reverse CCh-induced tone via membrane
hyperpolarization (17). We impaled intact tissues with microelectrodes to record membrane potential changes. Cholinergic stimulation led to
depolarization of the membrane from a resting potential of 62.3 ± 1.1 mV to one of 41.2 ± 2.2 mV (n = 19; Fig.
4), often accompanied by sinusoidal
oscillations of the membrane potential; these effects have been
described in detail elsewhere (10). Subsequent addition of
10-4 M H2O2 caused a slowly
developing hyperpolarization to a mean value of 60.8 ± 1.7 mV
(n = 19; Fig. 4).
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Role of K+ channel and Na+-K+-ATPase activity in free radical-mediated relaxations. Membrane hyperpolarization can involve opening of K+ channels and/or activation of the electrogenic Na+-K+-ATPase, leading to cessation of Ca2+ influx through voltage-dependent Ca2+ channels, which otherwise maintains CCh-evoked tone (25). We examined these possibilities by using a variety of approaches.
First, tone induced by KCl (30 mM) alone was not reversed by 10 mU xanthine oxidase, and was only marginally reduced (11.1 ± 4.0%) by 100 mU xanthine oxidase (Fig. 5A). In tissues precontracted with CCh in the presence of tetraethylammonium (TEA; 25 mM; a nonselective blocker of Ca2+-dependent K+ channels), xanthine plus xanthine oxidase induced a long-lasting contractile response that eventually resolved and was followed by partial reversal of tone (Fig. 5B); ultimately, the magnitudes of the relaxations were not significantly different from control (25.3 ± 7.4 and 21.4 ± 12.7%, respectively). This marked slowing of the relaxation was not seen in tissues pretreated with 4-aminopyridine (4-AP; 5 mM; blocker of voltage-dependent K+ channels) before induction of CCh tone (Fig. 5B).
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6 M), a selective blocker of
Na+-K+-ATPase activity, prevented the xanthine
oxidase-induced reversal of CCh tone (Fig. 6A; Table 1). Similarly, in the
presence of ouabain, 103 M
H2O2 reversed CCh-induced tone by 34.4 ± 3.5% (n = 4), compared with the 75.5 ± 1.2% (n = 6) reversal seen under control conditions (Fig. 6B).
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7 M) to prevent voltage-dependent
Ca2+ influx, the relaxations triggered by xanthine oxidase
or H2O2 followed a much slower time course than
did those seen in control tissues (Fig. 6).
Effects of peroxide on Ca2+ handling. Previous studies using other cell types have shown that free radicals can interfere with Ca2+ handling (see the beginning of this study). We investigated this possibility directly by using fura 2 fluorimetry.
In airway smooth muscle cells loaded with the Ca2+-indicator dye fura 2 and superfused with Ringer buffer, H2O2 elevated [Ca2+]i in a dose-dependent fashion (Fig. 7). In contrast to the spikelike Ca2+-transient response typically evoked by agents such as acetylcholine (Fig. 7; Ref. 13), this response to H2O2 developed more slowly, reaching a plateau within 2 min that persisted despite washing out of H2O2 (Fig. 7, A and C). In addition to its action as a strong oxidant and source of free radicals, H2O2 may alter the osmolarity of the media surrounding the cell, which could alter membrane-associated processes or even lead to cell lysis. To test whether the effects of H2O2 were simply an osmotic effect, we applied distilled H2O to the cells (Fig. 7B, left); there was no change in basal F340/F380 nor in the magnitude or time course of cholinergic responses (n = 3). Furthermore, cholinergic responses could still be evoked from cells preexposed to H2O2 (n = 8; Fig. 7, A and B), also suggesting that cell lysis had not occurred.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Peroxide mediates relaxation in canine airway smooth muscle. In this study, we investigated the relaxant effects of ROS on epithelium-denuded canine tracheal smooth muscle. Coadministration of xanthine oxidase and xanthine that leads to liberation of a variety of ROS caused marked reversal of cholinergic-stimulated tone. These relaxations seemed to be mediated predominantly by peroxide, because they were 1) not prevented by the hydroxyl-radical scavenger DMTU (Fig. 2); 2) blocked by catalase (Fig. 2); and 3) mimicked by exogenously added peroxide in a dose-dependent fashion (Fig. 3).
Role of epithelium and prostanoids. Our study, together with those of others, highlights the species-related and tissue-related variability in the response of airway smooth muscle to ROS. For example, Gao and Vanhoutte (4) showed that peroxide reverses cholinergic tone ~50% in canine bronchial smooth muscle and that this response is epithelium dependent; the peroxide-mediated relaxations that we describe in canine tracheal smooth muscle are much larger (nearly complete reversal of cholinergic tone) and occur in the absence of an epithelium. Despite these differences, the dose dependence of peroxide-induced relaxations in tracheal tissues (this study) and in bronchial tissues (4) were similar, with threshold and half-maximal concentrations of ~1 µM and ~100 µM, respectively. Similarly, peroxide induces partial relaxation (again, ~50%) of rabbit tracheal smooth muscle, and this effect is partially epithelium dependent and mediated by inhibitory arachidonic acid metabolites (7). In human airway smooth muscle, on the other hand, peroxide evokes an epithelium-dependent contractile response (22).
Role of K+ channels in OFR-induced relaxations. Cholinergic tone involves several excitation-contraction coupling mechanisms, including release of internally sequestered Ca2+, voltage-dependent Ca2+ influx, and increased sensitivity of the contractile apparatus to Ca2+ (25). Presumably, then, peroxide may act by interfering with one or more of these mechanisms.
We found that peroxide evoked a marked membrane hyperpolarization in tissues that had been depolarized by cholinergic stimulation (Fig. 4), to a potential below that which is necessary for opening of voltage-dependent Ca2+ channels. We tested whether this hyperpolarization involved K+ channels by exposing tissues to 30 mM KCl to elevate the K+ equilibrium potential to approximately
35 mV, to both contract the tissues and make it
impossible for K+ channel activation to cause any
significant hyperpolarization: in these tissues, the inhibitory effect
of xanthine oxidase was markedly impaired (Fig. 5A). The type
of K+ channel involved in this response seems not to be the
delayed rectifier K+ channel, because 4-AP did not either
alter the time course or decrease the effect of xanthine oxidase on
CCh-tone. TEA, however, markedly delayed these relaxations (Fig.
5B), suggesting that Ca2+-dependent K+
channels are involved: these may have become activated by
peroxide-induced release of internally sequestered Ca2+,
because we found that H2O2 triggered a
sustained elevation of [Ca2+]i
(Fig. 7), which was sensitive to CPA (Fig. 7D). At least six regions of the sarcoplasmic reticulum Ca2+-ATPase have been
shown to be modified by H2O2 (28).
Thus it seems that superoxide/peroxide acts on canine airway smooth
muscle by releasing internally sequestered Ca2+, thereby
interfering with cholinergic responses in two ways. First, the
Ca2+ that is liberated activates Ca2+-dependent
K+ channels, which hyperpolarize the membrane and thereby
abrogate the ongoing voltage-dependent Ca2+ influx that
maintains cholinergic tone (i.e., electromechanical coupling). Although
it might be expected that Ca2+ release would increase tone
(because contraction is also Ca2+ dependent), we have shown
that the sarcoplasmic reticulum in airway smooth muscle divides the
cytosol into two functionally distinct regions, a region immediately
underneath the sarcolemma and its ion channels and a deeper region in
which the contractile apparatus is found, and that
[Ca2+]i in these two regions can
change independently (9). The data from that earlier study show that
Ca2+ release can be preferentially directed into the
subplasmalemmal space (leading to activation of
Ca2+-dependent K+ channels), whereas
[Ca2+]i in the deeper cytosol is
not elevated sufficiently to elicit a contraction.
Second, peroxide-mediated Ca2+ release into the
subsarcolemmal space with subsequent extrusion out of the cell leads to
depletion of the internal Ca2+ pool through which
cholinergic agonists trigger contraction (i.e., pharmacomechanical coupling).
Involvement of a second free radical and/or additional mechanism of action? Our data also suggest that an additional, delayed mechanism also contributes to the reversal of cholinergic responses by ROS. First, although TEA markedly delays the onset and slows the time course of the relaxation evoked by xanthine oxidase, ultimately the magnitude of the reversal of tone produced by xanthine oxidase is not different (Fig. 5B). Second, although H2O2-evoked relaxations developed rapidly and were sustained in the absence of DMTU, these reversed (i.e., cholinergic tone was restored) over the course of 10-15 min in the presence of DMTU (Fig. 3) (DMTU did not have this effect on relaxations evoked by xanthine plus xanthine oxidase, possibly because the latter cause a continuous production of ROS that exceeds the scavenging ability of DMTU). These findings suggest that the initial (i.e., DMTU-insensitive) component of the relaxation is mediated by H2O2, whereas the subsequent DMTU-sensitive component may involve hydroxyl radicals. The time delay may be related to the time required for hydroxyl radicals to be generated (e.g., via oxidation of H2O2) and accumulate above some critical threshold, because 1) catalase completely eliminated the relaxant response, whereas other blockers tested were only effective against the earlier component of the response (e.g., TEA, ouabain, nifedipine); and 2) the lower concentration of xanthine oxidase that we tested did not reverse KCl-induced tone, whereas a 10-fold higher concentration of the enzyme did.
This DMTU-sensitive mechanism may involve a cellular target other than K+ channels, because some relaxation could still be evoked by xanthine oxidase in tissues precontracted with KCl (Fig. 5A): with the K+ equilibrium potential elevated in this way, further activation of K+ channels cannot have an inhibitory effect. The target may be Na+-K+-ATPase, with resultant membrane hyperpolarization and relaxation, because ouabain significantly suppressed the relaxations evoked by xanthine oxidase (Fig. 6A) or by H2O2 (Fig. 6B). It should be noted, however, that these tissues were exposed to ouabain for up to 30 min (one-half of this time during cholinergic excitation) before challenge with free radicals; these conditions may have led to dissipation of the K+ concentration gradient, thereby also interfering with Ca2+-dependent K+ channel hyperpolarization referred to above. In a previous study (12), we have shown that airway smooth muscle cells that have been depleted of internal Ca2+ by cyclopiazonic acid exhibit phasic relaxations that are triggered by Na+-K+-ATPase activity; the same may be happening in this study, because H2O2 also triggers release of internal Ca2+ (Fig. 7).Relevance. Inflammatory cells play a central, causal role in asthma and experimentally induced airway hyperresponsiveness. In both, there is an accumulation of OFRs and ROS (26, 27); furthermore, the latter is prevented by pretreatment with antioxidants and seems to involve changes in Ca2+ handling by the airway smooth muscle (11, 23). Thus a better understanding of the mechanisms by which free radicals modulate airway smooth muscle function is essential and may lead to novel therapies for asthma. The present findings, showing the inhibitory effects of OFRs on smooth muscle function, may account for the reduced in vitro responsiveness sometimes reported in isolated airway segments removed from animals exposed to allergen in vivo (11); although PGE2 contributes in part to this, a portion of it seems to involve some unidentified nonprostanoid relaxant agent, possibly free radicals generated by allergen-activated inflammatory cells. In another study (14), we described relaxations triggered by free radicals generated by electrical stimulation: the present study corroborates this ability of free radicals to interfere with excitatory signaling events within airway smooth muscle and further elaborates on the mechanisms involved.
Conclusions. Peroxide (added exogenously or generated by dismutation of superoxide derived from xanthine oxidase activity) reverses cholinergic tone in canine tracheal smooth muscle through several mechanisms. On the one hand, it activates Ca2+-dependent K+ channels, leading to hyperpolarization and cessation of voltage-dependent Ca2+ influx that otherwise maintains cholinergic tone; the K+ channels may be triggered directly by Ca2+, because we also found that H2O2 causes release of internally sequestered Ca2+ in smooth muscle cells at rest (i.e., not stimulated with cholinergic agonist). On the other hand, peroxide seems to deplete the Ca2+ store through which cholinergic agonists act, further inhibiting contraction. In addition, the data suggest that there may be accumulation of some other free radical (hydroxyl?) that leads to activation of Na+-K+-ATPase activity, hyperpolarization of the membrane and relaxation.
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ACKNOWLEDGEMENTS |
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The authors acknowledge Matt Ostrowski, who performed the intracellular microelectrode electrophysiological studies.
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FOOTNOTES |
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These studies were supported by a grant from the Medical Research Council of Canada, as well as a Career Award (to L. J. Janssen) from the Pharmaceutical Manufacturers' Association of Canada (Health Research Foundation) and the Medical Research Council of Canada.
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: L. J. Janssen, Dept. of Medicine, L-314 St. Joseph's Hospital, 50 Charlton Ave. East., Hamilton, Ontario, Canada L8N 4A6.
Received 1 March 1999; accepted in final form 29 September 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) in the ratio of fluorescence induced by 340-nm (F340)
and that induced by 380-nm excitation (F380;
F340/F380). D: pretreatment of cells
with cyclopiazonic acid (CPA; 60 M for 10 or 15 min, as indicated)
significantly reduced magnitude of H2O2-induced
change in F340/F380.