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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We
compared the effects of two redox forms of nitric oxide,
NO+ [liberated by
S-nitroso-N-acetyl-penicillamine (SNAP)] and
NO · [liberated by 3-morpholinosydnonimine (SIN-1) in
the presence of superoxide dismutase], on cytosolic concentration
of Ca2+ ([Ca2+]i;
single cells) and tone (intact strips) obtained from human main stem
bronchi and canine trachealis. SNAP evoked a rise in [Ca2+]i that was unaffected by
removing external Ca2+ but was markedly reduced by
depleting the internal Ca2+ pool using cyclopiazonic acid
(105 M). Dithiothreitol (1 mM) also
antagonized the Ca2+ transient as well as the accompanying
relaxation. SNAP attenuated responses to 15 and 30 mM KCl but not those
to 60 mM KCl, suggesting the involvement of an electromechanical
coupling mechanism rather than a direct effect on the contractile
apparatus or on Ca2+ channels. SNAP relaxations were
sensitive to charybdotoxin (10
7 M) or
tetraethylammonium (30 mM) but not to 4-aminopyridine (1 mM).
Neither SIN-1 nor 8-bromoguanosine 3',5'-cyclic
monophosphate had any significant effect on resting
[Ca2+]i, although both of these
agents were able to completely reverse tone evoked by carbachol
(10
7 M). We conclude that
NO+ causes release of internal Ca2+ in a
cGMP-independent fashion, leading to activation of
Ca2+-dependent K+ channels and relaxation,
whereas NO · relaxes the airways through a cGMP-dependent,
Ca2+-independent pathway.
Ca2+-dependent K+ channels; redox forms of nitric oxide; electromechanical coupling
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INTRODUCTION |
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INHIBITORY NEURAL CONTROL of tracheal smooth muscle
(TSM) of many species is mediated in part by nitrergic nerves (2, 22). Nitric oxide (NO) can also be generated by the epithelium, invading inflammatory cells, and the smooth muscle itself. NO mediates bronchodilation through a variety of mechanisms. For example, it
activates Ca2+-dependent K+ channels in porcine
TSM via guanylate cyclase and G kinase (25) as well as in bovine TSM
through nitrothiosylation of some cellular target molecule (1).
Likewise, NO-mediated relaxations in guinea pig and human airway smooth
muscle are inhibited by blockers of Ca2+-dependent
K+ channels (4). In all of these cases then, the ultimate
effect is hyperpolarization of the membrane and consequent closure of voltage-dependent Ca2+ channels; the mechanism by which
K+ channel activity is increased is not yet entirely clear.
In addition to activating outward K+ currents, NO has been
shown to suppress inward Cl currents (21). NO may
also act through nonelectromechanical coupling mechanisms. For example,
acetylcholine- and caffeine-induced Ca2+ release are
reduced by NO in porcine TSM (16). cGMP, the second messenger through
which NO acts (26), reduces the Ca2+ sensitivity of the
contractile apparatus (15) and modestly reduces cholinergically induced
elevation of cytosolic concentration of Ca2+
([Ca2+]i) (14) in canine TSM.
NO is usually envisaged as an electrically neutral free radical (i.e.,
possessing an unpaired electron; NO ·), which stimulates generation of the second messenger cGMP through guanylate cyclase (19).
However, NO also exists in an oxidized form (NO+) and in a
reduced form (NO), and all three redox forms can
react with other free radicals and molecules
(e.g.,O
2·;
H2O2; CO), producing an increasingly
bewildering variety of reactive species (19). These isoforms of NO, as
well as the reactive species that they generate, can oxidize and
covalently modify target protein(s) on the plasmalemma and/or within
the cells, thereby altering cell function (e.g., Refs. 1 and 23).
In this study, we provide evidence for a novel mechanism whereby NO+, but not NO · evokes relaxation in canine and human airway smooth muscle. In particular, we show that NO+ causes release of internally sequestered Ca2+ through an oxidative process independent of guanylate cyclase and cGMP, leading to activation of Ca2+-dependent K+ channels and relaxation.
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METHODS |
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Preparation of tissues and cell dissociation. Segments of donor (i.e., nondiseased) human main stem bronchi were obtained from the Lung Transplant Program (Toronto, Ontario). Adult mongrel dogs were euthanized with pentobarbital sodium (100 mg / kg); their tracheae were excised and kept in physiological solution. Airway smooth muscle was isolated by removing connective tissue, vasculature, and epithelium and then cut into strips parallel to the muscle fibers (~1 mm wide). For single-cell studies, TSM strips (0.5-1.0 g wet wt) were transferred to dissociation buffer (see Solutions and chemicals for composition) containing collagenase (type IV; 2.7 U / ml), elastase (type IV; 12.5 U / ml), and BSA (1 mg / ml) and 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). To liberate single TSM cells, tissues in enzyme-containing solution were incubated at 37°C for 60-120 min and then gently triturated.
Fura 2 fluorimetry. Single cells were studied 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 fura 2 [fura 2-acetoxymethyl ester (AM), 2 µM for 30 min at 37°C] and 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 using a photomultiplier tube assembly. F340/F380 was converted to [Ca2+] using previously published methods (8). Rmax and Rmin (the fluorescence ratio values under saturating and Ca2+-free conditions, respectively) were obtained previously, and the Ca2+-fura 2 dissociation constant was assumed to be 224 nM (8). Agonists were applied by pressure ejection from a puffer pipette (Picospritzer, General Valve; Fairfield, NJ).
Organ bath studies. TSM strips were mounted vertically in a 3-ml organ baths using silk (Ethicon 4-0) tied to either end of the strip; one end was fastened to a Grass FT03 force transducer, whereas the other was anchored. Isometric changes in tension were digitized and recorded using an on-line program (DigiMed System Integrator, MicroMed; Louisville, KY). Tissues were bathed in Krebs-Ringer buffer (see Solutions and chemicals for composition) containing 10 µM indomethacin 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 first equilibrated for 1 h before the specific experiments were commenced.
Solutions and chemicals. Dissociation buffer contained (in mM) 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 0.25 EDTA, 10 L-taurine, and 10 D-glucose, 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. Intact tissues were studied 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 to maintain pH at 7.4.
Chemicals were obtained from Sigma Chemical, with the exception of fura
2-AM (Calbiochem; La Jolla, CA). All agents were prepared as aqueous
solutions except for cyclopiazonic acid (CPA), fura 2, S-nitroso-N-acetylpenicillamine (SNAP),
3-morpholinosydnonimine (SIN-1), and penicillamine (all prepared in
DMSO) and ryanodine (95% ethanol). SNAP and SIN-1 were prepared as
102 M stock solutions and diluted
appropriately using Ringer or Krebs-Ringer buffer.
Data analysis. The concentration of agonist required to achieve 50% reversal of carbachol tone (EC50) was derived from the concentration-response curve of each tissue. Data are reported as means ± SE and compared using two-tailed Student's t-test (paired or unpaired, as appropriate), with P < 0.05 being considered significant.
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RESULTS |
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SNAP elevates [Ca2+]i by
releasing internally sequestered Ca2+. In freshly
dissociated TSM cells, SNAP (105 M)
triggered sustained elevations of
[Ca2+]i. Figure
1A provides a sample tracing of
such a response as well as that evoked by caffeine (10 mM); the latter
has been described in detail elsewhere (10). In both human and canine
cells, the SNAP-induced elevation of
[Ca2+]i developed much more slowly
than the caffeine response (which reached a peak within seconds) and
was generally much longer lasting; the mean magnitudes of these
responses were 146 ± 42 nM (n = 9) in human cells and 163 ± 12 nM (n = 18) in canine cells (Table 1). Penicillamine
(10
5 M; the carrier molecule for NO in
SNAP) dissolved in vehicle (DMSO) had no effect on
[Ca2+]i (n = 5; data not
shown); previously, we have demonstrated that DMSO alone does not alter
[Ca2+]i in these cells (data not
shown).
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To ascertain the degree to which Ca2+ influx contributed to this fluorimetric response, canine TSM cells were studied in Ca2+-free medium containing 0.1 mM EGTA. Under these conditions, SNAP still evoked large elevations of [Ca2+]i, the mean magnitudes of which were not significantly different from control (Fig. 1, C and D). When the internal Ca2+ store was depleted using CPA (30 µM; see Refs. 10, 12, and 13), however, the fluorimetric responses to SNAP were markedly and significantly smaller than control (Fig. 1, B and D).
SNAP-induced elevation of
[Ca2+]i
is nonetheless accompanied by relaxation. Elevation of
[Ca2+]i in smooth muscle is usually
thought to result in contraction. We therefore established that SNAP
did in fact act as a relaxant in these tissues. In canine TSM
preconstricted with carbachol (107 M),
SNAP evoked relaxations at concentrations ranging from
10
8 to
10
4 M (Fig.
2A); these were transient at
concentrations less than 10
6 M but
tended to be sustained at higher concentrations (Fig.
2A). The mean dose-response relationship for SNAP under these
control conditions is given in Fig. 2B; the mean
EC50 was 0.2 ± 0.02 µM (n = 6).
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NO+ acts via an oxidative process. NO can exist as a cation, which acts through oxidation and covalent modification of certain target protein(s) (19). We used the reducing agent dithiothreitol (DTT, 1 mM) to test whether this was involved in the relaxations seen in these airway muscle preparations; SNAP was used as a source of NO+ (19).
Figure 2C shows a typical SNAP-induced elevation of [Ca2+]i as well as the complete reversal of this response on addition of 1 mM DTT. In 11 canine and 7 human cells tested in this way, the SNAP response was reversed 115 ± 9 and 53 ± 17%, respectively, by DTT. In other cells, which had first been pretreated with DTT (1 mM), subsequent addition of SNAP evoked only a small rise in [Ca2+]i, which was markedly and significantly smaller than the control response (Table 1).
Likewise, in intact tissues pretreated with DTT (1 mM), the SNAP concentration-response relationship was significantly displaced a full log unit to the right, with an EC50 of 1.2 ± 0.3 µM (n = 6).
NO · acts through stimulation of
guanylate cyclase. The electrically neutral free radical form of NO
acts through stimulation of guanylate cyclase to generate the second
messenger molecule cGMP (26). We tested whether this pathway was also
involved using SIN-1 because decomposition of this agent tends to
produce NO ·; unless indicated otherwise, superoxide
dismutase (3 U/ml) was used to prevent reaction of NO · with
O2, leading to the more reactive
peroxynitrite species.
Surprisingly, SIN-1 (104 M) had no
significant effect on [Ca2+]i
in single cells (Fig. 3A; Table
1). Despite having little effect on
[Ca2+]i, cholinergic contractions
could be fully reversed by SIN-1 in a dose-dependent fashion, with an
EC50 of 1.0 ± 0.3 µM (n = 6; Fig. 3, B
and C). DTT (1 mM) had no statistically significant effect on
SIN-1-evoked relaxations; the EC50 for SIN-1 in the presence of DTT was 2.0 ± 0.1 µM (n = 5; Fig. 3C).
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Similarly, 8-bromoguanosine 3',5'-cyclic monophosphate
(104 M) had very little effect on
[Ca2+]i but markedly reversed
cholinergic tone (Fig. 4, A and
B, respectively; Table 1).
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SNAP relaxations involve electromechanical coupling. The
signaling pathway(s) through which NO mediates its relaxant effect was
examined first by comparing cholinergic contractions in the absence and
presence of 105 M SNAP; carbachol was
applied at concentrations ranging from 10
9 to
10
4 M, added in cumulative fashion,
because different excitation-contraction coupling mechanisms
predominate at different degrees of cholinergic stimulation (6). SNAP
was most effective against the lower concentrations of carbachol, where
electromechanical coupling mechanisms predominate (6) but had
relatively little effect against the higher concentrations of carbachol
(Fig. 5).
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These observations suggest that NO is acting largely through a
mechanism involving electromechanical coupling, that is, triggering membrane hyperpolarization leading to closure of voltage-dependent Ca2+ channels and subsequent cessation of contraction. To
test this, we examined the ability of SNAP to alter contractions evoked
by KCl, which acts largely through depolarization and opening of Ca2+ channels. Contractile responses in intact tissues
exposed to KCl at concentrations ranging from 15 to 60 mM (increased in
15 mM increments) were recorded both before and after exposure to SNAP
(105 M) (Fig.
6A). SNAP markedly and
significantly attenuated the contractile response evoked by 15 mM
KCl but had much less effect against the higher concentrations of
KCl (Fig. 6). These observations are consistent with a mechanism
involving SNAP-induced opening of K+ channels and
rule out the possibilities that NO is acting directly on
voltage-dependent Ca2+ channels or on the contractile
apparatus; however, they do not necessarily rule out the possible
additional involvement of other pathways such as enhanced
Ca2+ uptake in the sarcoplasmic reticulum.
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Finally, we examined the sensitivity of SNAP-evoked relaxations to the
Ca2+-dependent K+ channel blockers
charybdotoxin (107 M) and
tetraethylammonium chloride (TEA; 30 mM) as well as to the
voltage-dependent K+ channel blocker 4-aminopyridine (4-AP;
1 mM). Canine TSM tissues were treated with these agents for 15 min,
which tended to lead to elevation of tone, and then further
preconstricted with carbachol (10
7 M).
SNAP (10
6 M) evoked substantial
relaxations in control tissues as well as in those pretreated with
4-AP, whereas those evoked in tissues pretreated with charybdotoxin or
TEA were markedly and significantly reduced (Fig.
7). The mean magnitude of the relaxant
response (expressed as percent reversal of overall tone produced by
K+ channel blocker plus carbachol) evoked by
10
6 M SNAP was 62 ± 13% under control
conditions and 68 ± 14% in the presence of 4-AP compared with 25 ± 6 and 20 ± 7% in the presence of charybdotoxin and TEA, respectively
(n = 5 animals for all 4 groups). At higher concentrations,
SNAP was able to completely reverse tone under all four conditions
(Fig. 7).
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DISCUSSION |
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Although there already have been several studies of the mechanisms
underlying NO-mediated relaxation of airway smooth muscle (see the
introduction), we report here for the first time that this effect is
accompanied by elevation of [Ca2+]i
in human and canine airway smooth muscle. Although this finding might
be counter-intuitive (elevation of
[Ca2+]i is usually thought to
result in contraction), it has been shown previously in bovine TSM
cells that -adrenergic agonists also elevate
[Ca2+]i in the peripheral regions
of the cytosol but decrease that in the deeper cytosol (24).
Moreover, we have shown that this Ca2+ response is mediated
by NO+ but not by NO · nor by cGMP (which
actually cause a net decrease in
[Ca2+]i) and involves oxidation of
some target molecule, leading to membrane hyperpolarization and
consequent suppression of voltage-dependent Ca2+ influx.
Our observation that the SNAP-induced Ca2+ release was
markedly reduced by CPA but was relatively unaffected by removal of
external Ca2+ suggests that the source of this
Ca2+ was the sarcoplasmic reticulum. The cellular target
which NO+ modifies to produce these changes might include
the ryanodine receptor on the sarcoplasmic reticulum, as is the case in
canine cardiac muscle (23); the ryanodine receptor in that tissue
contains 84 thiol groups and nitrosylation of up to 12 of these
(i.e., 3 thiols on each of the 4 subunits) causes progressive channel activation, which can be reversed using a reducing agent. Xu et al.
(23) also showed that oxidation of the ryanodine receptor under certain
conditions was not reversible; this might explain why we found that DTT
was much less able to reverse the SNAP-induced Ca2+
responses in human ASM cells compared with those of the dog.
Our finding that SNAP releases internal Ca2+ and thereby elevates [Ca2+]i in single canine TSM cells is not inconsistent with a previous report that SIN-1 reduced cholinergically evoked [Ca2+] transients in intact canine TSM strips (14). NO · (produced by SIN-1), acting through cGMP, could promote uptake of Ca2+ from the deep cytosol (16), whereas NO+ (produced by SNAP) could trigger its release into the subplasmalemmal space for subsequent extrusion and activation of Ca2+-dependent K+ channels in the plasmalemma. We have shown that Ca2+ release through ryanodine-gated channels in canine TSM is preferentially directed toward the plasmalemma (where the Ca2+-dependent K+ channels are found) rather than the deep cytosol (10). This then would explain in part the membrane hyperpolarizing effect of NO in airway smooth muscle (1, 4, 25). This mechanism is also consistent with that proposed to account for vasodilation (18); that is, relaxants act in vascular smooth muscle by triggering localized bursts of Ca2+ via ryanodine receptors on the sarcoplasmic reticulum ("Ca2+ sparks"), with consequent brief openings of Ca2+-dependent K+ channels on the plasmalemma (spontaneous transient outward currents). SNAP did not elevate [Ca2+]i in porcine TSM cells (16); this may be related to the exposure of those cells to DTT for 2 h (to activate papaine) during enzymatic dissociation, or it may indicate a species-related difference.
Thus it may be that relaxants such as NO (and -agonists) act
by increasing [Ca2+]i (in the
subplasmalemmal space) and altering Ca2+-dependent
K+ channel function directly much in the same way that many
spasmogens act by both elevating
[Ca2+]i (in the deep cytosol) and
increasing the Ca2+ sensitivity of the contractile apparatus.
Our data do not speak against the dogma that NO also evokes relaxation
in a cGMP-dependent fashion (4, 14, 25). In fact, the data indicate
that NO is acting through more than one pathway. For example, SIN-1
(which produces NO ·) and cGMP both triggered relaxation but
did not elevate [Ca2+]i. Also, SNAP
in the presence of DTT (which might reduce NO+ to
NO ·) was still able to fully relax the tissues even though the rise in [Ca2+]i was prevented.
Finally, whereas charybdotoxin and TEA were effective against low
concentrations of SNAP (106 M), they
were much less effective against the higher concentrations of SNAP
used. The parallel contributions of these two distinct pathways would
rationalize previously paradoxical findings such as the differential
effects of guanylate cyclase inhibitors (methylene blue and hemoglobin)
on relaxations triggered by different NO donors (7, 20, 26).
These findings may also shed light on the changes accompanying airway hyperresponsiveness. Our laboratory and several others have been puzzled by the finding that allergen inhalation produces airway hyperresponsiveness, which can be measured in vivo, but that airway tissues excised from these same animals and studied in vitro exhibit hyporesponsiveness (11, 17). Although PGE2 contributes to part of this reduced responsiveness, a considerable portion is evidently due to some nonprostanoid factor (11). More recently, NO synthase has been localized to the narrow space between the sarcoplasmic reticulum and the plasmalemma in canine TSM cells (5); this is the same cytosolic region in which we have documented the preferentially directed release of Ca2+ toward the plasmalemma rather than toward the deep cytosol (10). It may be then that allergen-induced airway inflammation somehow results in altered Ca2+ handling, which in turn would alter the activity of NO synthase, generation of NO, and the inhibitory effects described in the present study.
In conclusion, we have shown that in addition to the well-described cGMP-mediated inhibitory actions of NO, there is an additional cGMP-independent pathway, which involves oxidation of some target molecule by NO+, leading to release of internally sequestered Ca2+ (3). The cellular target, which is modified by NO+ to produce these changes in airway smooth muscle, is as yet unclear. Together with our earlier study of the superficial buffer barrier in airway smooth muscle (10), these data suggest that this Ca2+ flux is preferentially directed toward the plasmalemma, resulting in activation of Ca2+-dependent K+ channels and electromechanically coupled inhibition as well as extrusion of Ca2+ from the cell by the plasmalemmal Ca2+ pump.
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ACKNOWLEDGEMENTS |
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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 Manufacturer's Association of Canada and the Medical Research Council of Canada.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. J. Janssen, Dept. of Medicine, McMaster Univ., 50 Charlton Ave. East, Hamilton, Ontario, Canada L8N 4A6 (E-mail: janssenl{at}fhs.csu.mcmaster.ca).
Received 27 August 1999; accepted in final form 30 November 1999.
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