Ionic mechanisms underlying electrical slow waves in canine
airway smooth muscle
Luke J.
Janssen,
Chris
Hague, and
Roopung
Nana
Asthma Research Group and Smooth Muscle Research Program, Department
of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
In canine bronchial smooth muscle (BSM),
spasmogens evoke oscillations in membrane potential ("slow
waves"). The depolarizing phase of the slow waves is mediated by
voltage-dependent Ca2+ channels;
we examined the roles played by
Cl
and
K+ currents and
Na+-K+-ATPase
activity in mediating the repolarizing phase. Slow waves were evoked
using tetraethylammonium (25 mM) in the presence or absence of niflumic
acid (100 µM; Cl
channel
blocker) or ouabain (10 µM; block
Na+-K+-ATPase)
or after elevating external K+
concentration ([K+])
to 36 mM (to block K+ currents);
curve fitting was performed to quantitate the rates of rise/fall and
frequency under these conditions. Slow waves were markedly slowed, and
eventually abolished, by niflumic acid but were unaffected by ouabain
or high [K+].
Electrically evoked slow waves were also blocked in similar fashion by
niflumic acid. We conclude that the repolarization phase is mediated by
Ca2+-dependent
Cl
currents. This
information, together with our earlier finding that the depolarizing
phase is due to voltage-dependent
Ca2+ current, suggests that slow
waves in canine BSM involve alternating opening and closing of
Ca2+ and
Cl
channels.
voltage-dependent calcium currents; calcium-dependent chloride
currents; calcium-dependent potassium currents; airway
hyperresponsiveness
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INTRODUCTION |
AIRWAY SMOOTH MUSCLE (ASM) exhibits several ionic
currents that are activated during membrane depolarization. At least
two different voltage-dependent
Ca2+ currents (L type and T type)
and two different K+ currents
(delayed rectifier and Ca2+
dependent) are activated in direct response to the change in membrane
potential (10, 15, 25, 26, 31). Opening of the
Ca2+ channels results in
Ca2+ influx, which in turn
contributes further to
Ca2+-dependent
K+ channel activity and activates
Ca2+-dependent
Cl
currents (22). These
membrane currents play key roles in many physiological responses. For
example, voltage-dependent Ca2+
channels are involved in excitation-contraction coupling by mediating voltage-dependent Ca2+ influx
(electromechanical coupling) as well as by refilling of the internal
Ca2+ pool (15, 19, 25, 31). With
the K+ equilibrium potential at
80 to
90 mV, the opening of
K+ channels mediates membrane
hyperpolarization. K+ channels are
also important in setting the resting state of the cell and/or
inhibiting excitation (14, 26, 31). Because the
Cl
equilibrium potential
(ECl) in smooth
muscle is believed to range from
40 to
20 mV (2, 3),
opening of Cl
channels
leads to depolarization in resting cells (14, 18, 20) but
hyperpolarization in cells that have already been depolarized to
potentials more positive than
ECl.
In the absence of stimulation by neurotransmitters or other
pharmacological agents, canine ASM is mechanically and electrically quiescent (5). In addition to membrane depolarization and contraction, spasmogens such as cholinergic agonists (16), thromboxanes (17), leukotrienes (1), or K+ channel
blockers such as tetraethylammonium (TEA) and 4-aminopyridine (16)
evoke oscillations in membrane potential that are referred to as
"slow waves"; these generally have a frequency of
1 Hz and
amplitude of 10-25 mV. Similar oscillations occur spontaneously or
in response to arachidonic acid metabolites in human (6, 11, 12),
guinea pig (4, 27, 28, 33, 34), equine (8, 35), and bovine (34) ASM. In
all cases, the slow waves are insensitive to neuronal blockers such as
tetrodotoxin (6, 7, 27, 29). This suggests that a myogenic oscillatory
mechanism is resident in all ASM tissues and is invoked by blockade of
K+ channels or by excitatory
stimulation by acetylcholine and histamine (canine and bovine ASM), by
leukotrienes (human ASM), or by prostanoids (guinea pig and equine
ASM).
The depolarizing phase of slow waves is mediated by voltage-dependent
Ca2+ channels, since blocking
Ca2+ influx (by removal of
external Ca2+ or by addition of
blockers of voltage-dependent Ca2+
channels, such as Cd2+,
dihydropyridines, and verapamil) eliminates slow-wave activity and
leaves the smooth muscle cell in a relatively hyperpolarized state
(i.e., approximately equal to
45 mV; see Refs. 6, 7, 16, 27,
33). The Ca2+ channels are
generally believed to be L type in nature, since the slow waves are
sensitive to dihydropyridines (4, 7, 16, 33). The ionic conductance
changes underlying the repolarizing phase of the slow waves in ASM,
however, have not yet been resolved. In gastrointestinal smooth
muscle, repolarization is attributed to opening of
Ca2+-dependent K+ channels subsequent to influx
of Ca2+ through voltage-dependent
channels (9). In ASM, however, slow waves persist in the presence of 25 mM TEA or 5 mM 4-aminopyridine (16), arguing strongly against such a
role for K+ currents. Instead, the
repolarizing phase in ASM may be mediated by
Ca2+-dependent
Cl
currents, which are also
triggered by voltage-dependent
Ca2+ influx (22). Alternatively,
Na+-K+-ATPase,
which is also electrogenic, has been proposed to play a role, since
slow waves in guinea pig ASM are reduced upon cooling or exposure to
ouabain (28, 29, 33, 34).
In this study, we sought to characterize the roles played by
Ca2+-dependent
Cl
currents and
Na+-K+-ATPase
activity in slow waves in canine bronchial smooth muscle (BSM).
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METHODS |
Dissection. Adult mongrel dogs were
euthanized with pentobarbital sodium (100 mg/kg); whole pulmonary lobes
were excised and kept in oxygenated Krebs-Ringer solution (composition
given in Solutions and
chemicals) throughout the study.
Parenchymal tissue and vasculature overlying the bronchi were dissected
away (dissection carried out at 25°C), exposing the entire
bronchiolar tree. The outer diameters of the tissues used in these
studies ranged from 1 to 8 mm (3rd to 5th order). Ring segments
4-5 mm in length were excised from the lobe of lung and opened by
cutting perpendicularly to the axis of the smooth muscle bundles; care
was taken to not damage the epithelium.
Microelectrode studies. Tissues were
carefully pinned out, epithelial face upward, in a chamber having a
bath volume of 5 ml. Krebs-Ringer solution (composition given in
Solutions and chemicals) 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. Smooth muscle cells
were impaled from the epithelial surface of the tissue. Membrane
potential changes were observed on a dual-beam oscilloscope (Tektronix
D13; 5A22N differential amplifier; 5B12 dual-time base) and recorded on
0.25-in. magnetic tape with a Hewlett-Packard instrumentation recorder.
Although we have found that impalements can be maintained for several
hours provided no intervention is made that will induce a change in
tension (causing the microelectrode to be pulled out of the cell),
these generally last only 10-15 min in a typical experiment;
frequently, impalement can be reestablished within seconds, although it
is not clear whether this is the same cell or an adjacent one. However,
ASM is highly coupled by gap junctions (23); as such, membrane
potentials and electrical events in one cell are essentially identical
to those in the surrounding cells. In general, the effects of only one
drug or condition (i.e., niflumic acid, ouabain, high
K+) were examined in a given
tissue. Portions of these data were played back, digitized (Digidata
1200), and sampled using pCLAMP 6 software (Axon Instruments, La Jolla,
CA) and then fitted using pCLAMP 6 and/or exported to SigmaPlot
for graphical presentation.
Electrical field stimulation.
Electrical field stimulation (EFS) was achieved through two silver
plates on either side of the tissue (
1 cm apart). Electrical pulses
(pulse width of 0.5 ms) were provided by a Grass S88 stimulator; both
single pulses and pulse trains (frequencies ranging up to 20 pulses/s)
were used. The voltage used was that which gave maximal responses
(generally 50-100 V).
Solutions and chemicals. Tissues were
studied using Krebs-Ringer buffer (KRB) containing (in mM) 116 NaCl,
4.2 KCl, 2.5 CaCl2, 1.6 NaH2PO4,
1.2 MgSO4, 22 NaHCO3, 11 D-glucose,
and 0.01 indomethacin, which was bubbled continuously to maintain pH at
7.4. Isosmotic high-K+ KRB was prepared by
substituting NaCl with KCl. Unless indicated otherwise, KRB solutions
also contained TEA (25 mM). Chemicals were obtained from Sigma
Chemical. Initially, niflumic acid was made up as a concentrated
solution in 95% ethanol and was added to the bathing solution (final
bath concentration of ethanol was 0.1%); later, however, we dissolved
niflumic acid directly into KRB
(10
4 M, without any
ethanol) and superfused the tissues with this solution. Ouabain was
prepared as an aqueous solution.
Data analysis. The rates of change for
the various components (phases
i-v; see legend of Fig. 1) of the slow
waves were derived using Clampfit software (Axon Instruments). In Fig.
1, phase ii, phase
iii, and phase iv
could be well fit by linear functions, whereas a monoexponential
function was used to fit phase v. The time course of phase i, on the other
hand, was more complicated and varied; as a result, we were unable to
obtain a satisfactory fit using the algorithms supplied by Clampfit.
For each cell impaled, the values of phases
ii-v were obtained from three different portions of a trace, and the average for that cell was calculated. Responses are
reported as means ± SE; n refers
to the number of tissues tested. Statistical comparisons were made
using a paired Student's t-test, with
P values <0.05 being considered
significant.
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RESULTS |
Electrical slow waves are evoked by TEA in canine
BSM. In canine BSM at rest, the membrane potential was
62 ± 4 mV (n = 21), and
there was little or no spontaneous activity. Within 10 min after
introduction of 25 mM TEA, however, the membrane depolarized and
slow-wave activity was triggered. The slow waves were generally sinusoidal in appearance, with an exponential rise and fall
( phase i and
phase v, respectively, in Fig.
1). In many cases (8 of 13 tested),
superimposed upon the rising phase of the sinusoidal oscillations
(i.e., phase i) were action
potentials with a rapid spike-like depolarization
( phase ii in Fig.
1B,
right) followed by a rapid decay
( phase iii) to a much more
slowly decaying "plateau" or "shoulder" region
(phase iv). The mean threshold
potentials at which slow waves and action potentials were triggered
were
43 ± 4 and
35 ± 6 mV, respectively. In the
absence of any other experimental intervention, slow-wave activity
persisted for at least 1 h, with no notable change in frequency or
amplitude (Fig. 2).

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Fig. 1.
Slow waves in canine bronchial smooth muscle (BSM) depolarized using
tetraethylammonium (TEA; 25 mM). A:
slow waves were sinusoidal in appearance
(left); in some cells, action
potentials were superimposed on the rising phase of the sinusoid
(right).
B: individual electrical events
indicated by * in A are displayed on a
faster time scale; the rising and falling phases of the sinusoidal
waves ( phases i and
v, respectively) were best fit by
monoexponential functions, whereas the rising and falling phases of the
action potentials ( phases ii,
iii, and
iv) could be well fit by linear
functions. t1/2,
Time required for the slow wave per se, excluding the action potential,
to decay to one-half of its peak value.
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Fig. 2.
Slow-wave time course is consistent over time.
A: typical tracings obtained shortly
after onset of TEA-induced slow waves
(left) and 120 min later
(right); there were no other
pharmacological interventions during this period.
B: although there was a slight
decrease in slow-wave frequency over this period, the time course of
the slow waves did not seem to change, as indicated by superimposition
of the individual electrical events marked by * in
A. t,
Time.
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Niflumic acid abolishes TEA-evoked slow
waves. The depolarizing phase of the slow waves
( phase i) is mediated by
dihydropyridine-sensitive voltage-dependent
Ca2+ channels (16). The
mechanism(s) underlying the repolarizing phases
( phases iv and
v), however, are unclear but may
involve Ca2+-dependent
Cl
currents. We tested this
hypothesis by examining what effect niflumic acid, a blocker of the
Ca2+-dependent
Cl
channels (22), might
have on slow waves induced by TEA (25 mM). We used 100 µM niflumic
acid because this was found to be sufficient to maximally block
Ca2+-dependent
Cl
current in this tissue
(22).
Under control conditions, the decay of the shoulder region
( phase iii) was roughly
linear, whereas the subsequent portion of the slow wave
( phase iv) decayed
exponentially (Figs. 1-3 and Table
1). The time required for the membrane
potential to drop midway between the "crest" or shoulder to the
"trough"
(t1/2) was 559 ± 37 ms, and the frequency of the slow waves was 0.54 ± 0.04 Hz. Within minutes after introduction of niflumic acid, however,
t1/2 was markedly
prolonged and slow frequency markedly reduced. These effects seemed to
be secondary to a prolongation of the shoulder region of the slow waves
(Fig. 3 and Table 1, phase iv)
without any change in the other components of the slow waves. With more
prolonged exposure to niflumic acid, slow waves appeared sporadically
and eventually ceased altogether (Fig. 3). In some cases, these could
be triggered again (albeit only temporarily) by EFS. Eventually,
however, slow waves were abolished entirely. At this point, mean
membrane potential was
46 ± 5 mV (not significantly different from the mean control value of
43 ± 4 mV, given in Electrical slow waves are evoked by TEA in canine
BSM, at which slow waves are triggered).

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Fig. 3.
Slow waves are prolonged and eventually abolished by niflumic acid.
A: representative traces showing
slow-wave activity observed during perfusion with TEA-Krebs-Ringer
buffer (KRB; left) and how this
activity is suppressed (middle) and
eventually abolished (right) upon
perfusion with TEA-KRB containing niflumic acid (100 µM).
B: individual electrical events
indicated by * in A are superimposed
to highlight the prolongation of the electrical event; this is
primarily due to a prolongation of the plateau phase.
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Lack of effect of ouabain on TEA-evoked slow
waves. The repolarization phase of the slow waves may
also be mediated by
Na+-K+-ATPase
activity; to test this hypothesis, we examined the effect of
10
5 M ouabain, which is
sufficient to maximally inhibit the
Na+-K+
pump (34). After 30 min of exposure to ouabain, membrane potential was
further decreased by
10 mV above the level existing in the presence
of TEA. In addition, the mean frequency of the slow waves was increased
and t1/2 somewhat
decreased (Fig.
4A and
Table 1), although these changes did not reach statistical significance (Table 1). More importantly, however, the rates of recovery
( phases iv and
v) were not slowed; in fact,
phase v was significantly faster in
the presence of ouabain (Fig. 4 and Table 1).

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Fig. 4.
A: representative traces showing
TEA-induced slow-wave activity before
(left) and during
(right) exposure to ouabain (10 µM). B: individual electrical events
indicated by * in A were digitally
made equivalent in size, while maintaining constant aspect ratio, and
then superimposed to facilitate comparison; ouabain did not slow down
the repolarization phase.
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Lack of effect of high
K+ on TEA-evoked
slow waves.
Although the persistence of slow waves in the presence of TEA argues
against a causal role for large-conductance
Ca2+-dependent
K+ channels in mediating
repolarization, it is possible that other types of
K+ channels might be responsible,
including delayed rectifier K+
channels (10, 31) and small-conductance
K+ channels; in this light, it is
interesting that niflumic acid has been reported to increase the
activity of small-conductance K+
channels (32), which might account for its inhibitory effect on slow
waves. We therefore raised external
K+ concentration
([K+]o)
isosmotically to 36 mM to elevate the
K+ equilibrium potential to
approximately
30 mV, thereby decreasing the outward driving
force on K+ but not converting it
to an inward driving force when the membrane potential was
30 mV
(i.e., the peak of the oscillations). Upon introduction of
high-K+ KRB, the troughs of the
oscillations became progressively less negative (i.e., membrane
depolarized), resulting in a significant decrease in slow-wave
amplitude (Fig. 5 and Table 1); there was also an increase in slow-wave frequency (Fig. 5 and Table 1). Despite
these changes, the rate of repolarization of the slow waves was not
significantly decreased (Table 1). In one tissue during prolonged
exposure to high-K+ medium, the
membrane continued to depolarize until slow waves were barely
discernable, began to become desynchronized, and were eventually lost;
even seconds before they disappeared, however, the rate of
repolarization was not slowed.

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Fig. 5.
A: slow waves in a tissue perfused
with standard TEA-KRB (left) and
after perfusion with high-K+ KRB
containing TEA; elevating external
K+ concentration
([K+]o)
to 36 mM increased slow-wave frequency and decreased their magnitudes
(by elevating the troughs to less negative potentials).
B: individual slow waves indicated by
* in A are superimposed here to show
that elevation of
[K+]o
had no effect on the rate of repolarization of the slow waves (note:
the slow wave obtained in the presence of high
K+ was digitally increased in
size, while maintaining constant aspect ratio).
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Niflumic acid abolishes slow waves evoked by
EFS. Usually, canine BSM exhibits a single spike-like
excitatory junction potential in response to EFS (Fig.
6B);
occasionally, however, the excitatory junction potential is followed by
a series of slow waves (Fig. 6A). In
this experiment, it was not our goal to ascertain the conditions that
dictated whether or not slow waves would be evoked by EFS; instead, we
investigated whether these slow waves were mediated by the same
mechanisms as those seen during K+
channel blockade (see Niflumic acid abolishes TEA-evoked slow waves). In four cells that did in fact exhibit
EFS-evoked slow waves, niflumic acid abolished the latter but not the
excitatory junction potential evoked by EFS (Fig. 6).

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Fig. 6.
A: in this cell perfused with normal
KRB (i.e., not containing TEA), electrical field stimulation (5 pulses
at 20 pulses/s; indicated by ) evoked an excitatory junction
potential followed by a series of recurring oscillations or slow waves
with varying amplitude. B: after
exposure to niflumic acid (100 µM), excitatory junction potential
could still be evoked, but the oscillations were completely
abolished.
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DISCUSSION |
Slow waves have been recorded in the ASM of many species, including the
dog (16), human (6, 11, 12), guinea pig (4, 27, 28, 33, 34), horse (8,
35), and cow (34). The depolarizing phase of slow waves involves
opening of voltage-dependent Ca2+
channels. The ionic conductance changes underlying the repolarizing phase of the slow waves, however, have been unclear but may involve Ca2+-dependent
Cl
currents,
TEA-insensitive K+ channels, or
electrogenic
Na+-K+-ATPase.
These mechanisms are easily distinguished using a variety of
pharmacological approaches.
Contribution of
K+ channels and
Na+-K+-ATPase
to TEA-induced slow waves.
We were able to rule out any role for
K+ channels or
Na+-K+-ATPase
in mediating the repolarization phase of the slow waves, since phase iv was not significantly slowed
by elevating the
[K+]o
to 36 mM or by ouabain (10
5
M should maximally block
Na+-K+-ATPase
activity). Elevation of
[K+]o
should markedly decrease the outward driving force on
K+ at a membrane potential of
30 mV (i.e., during the peaks of the slow waves) and thus
minimize the ability of K+
channels to mediate hyperpolarization. In fact, to our surprise, we
found that the
t1/2 of the slow
waves was significantly decreased, and slow-wave frequency increased,
by high-K+ medium and that ouabain
also seemed to accelerate slow-wave activity. This acceleration may be
secondary to the depolarization that both interventions mediate;
depolarization would influence the kinetics of the voltage-dependent
Ca2+ channel activation and
inactivation.
Contribution of
Ca2+-dependent
Cl
currents to TEA-induced slow
waves.
The Cl
channel blocker
niflumic acid, on the other hand, markedly and significantly slowed
phase iv and prolonged
t1/2, indicating that this component of the slow waves is mediated by
Cl
channels. Although this
concentration of niflumic acid (100 µM) is sufficient to immediately
block Cl
channels when
applied by pressure ejection in the vicinity of an isolated canine
tracheal smooth muscle cell (22), its effect was delayed in this study,
presumably because of the time required for the drug to travel from the
physiological saline solution reservoir and to diffuse into the tissue.
Inevitably, however, slow waves were abolished after introduction of
niflumic acid, consistent with the hypothesized role of
Cl
channels in mediating
slow waves.
The final phase of hyperpolarization ( phase
v), however, was unaffected, suggesting that the
final component of the repolarization phase is mediated by some other
mechanism (possibly deactivation of the
Ca2+ channels). Although niflumic
acid may also enhance the activity of small-conductance
K+ channels (32), we have already
ruled out a role for K+ channels
in general (see Lack of effect of high K+ on
TEA-evoked slow waves). In addition, there have been
numerous studies of the ion currents in canine ASM (26, 31), and none describe a small-conductance K+
current in this tissue. Previously, we have shown that niflumic acid
does not affect the voltage-dependent
Ca2+ current in this tissue (15,
22). Consistent with the conclusion that
Ca2+-dependent
Cl
currents play a central
role in slow waves in canine ASM,
Ba2+ is much less effective than
Ca2+ in activating
Ca2+-dependent
Cl
currents, and we have
previously shown that slow waves are greatly reduced or even abolished
in the presence of Ba2+ (16).
Cl
current would not be
expected to contribute greatly to the depolarizing phase
( phase i) of these slow waves,
since it must first be triggered by
Ca2+ influx (i.e.,
phase ii), and then, once triggered,
it requires ~200 ms to be maximally activated (22), at which time the
action potential has peaked and membrane potential is falling
( phase iii).
Slow waves in the absence of TEA. Slow
waves can also be observed under conditions other than specific
blockade of K+ currents. For
example, they are occasionally evoked during neurogenic release of
cholinergic agonists (Fig. 6) and are commonly seen in canine airway
tissues during stimulation with exogenously added cholinergic agonist
(16), with inflammatory mediators such as leukotrienes (1) or
thromboxane A2 (17) or with
aspirin (13); in addition, they are abolished by the cholinergic
antagonist atropine (16). In this study, we found these to also be
mediated by Ca2+-dependent
Cl
currents, since they are
blocked by niflumic acid (Fig. 6B). In single freshly dissociated canine ASM held under voltage clamp at
60 mV, recurring oscillations of membrane current are sometimes observed spontaneously (21) or after stimulation with acetylcholine (14, 18) or histamine (20). These membrane current oscillations were
shown to be mediated by
Ca2+-dependent
Cl
current and are believed
to indicate oscillations of cytosolic Ca2+ concentration
([Ca2+]i),
possibly due to Ca2+-induced
feedback (both positive and negative) on phospholipase C and/or
the Ca2+ release sites on the
sarcoplasmic reticulum. Thus the first component of each slow wave
( phase i) that triggers
voltage-dependent Ca2+ influx
( phases ii and
iii) and the subsequent activation
of Ca2+-dependent
Cl
current
( phase iv) may be an elevation
of
[Ca2+]i.
Contribution of voltage-dependent
Ca2+ currents to
slow waves.
The Ca2+ channels that contribute
to slow-wave activity are generally believed to be L type in nature, in
part because slow waves are dihydropyridine sensitive, even though the
threshold potentials for these channels and for slow waves are
substantially different (approximately equal to
30 and
45
mV, respectively, in canine ASM). In addition, L-type currents are
suppressed during agonist stimulation, whereas slow waves are not;
consistent with this, oscillations in canine ASM depolarized using TEA
have the appearance of action potentials, whereas those in canine ASM
depolarized using cholinergic agonists are generally smaller and
sinusoidal in appearance (this study and Ref. 16). T-type
Ca2+ currents, however, may
contribute to slow-wave activity, since 1) they are also dihydropyridine
sensitive but unaffected by cholinergic agonists;
2) slow waves sweep the membrane
continuously between
45 and
30 mV, which overlaps the
threshold and peak potentials for T-type "window current"; and
3) recovery from inactivation of
T-type currents occurs within 1 s, consistent with a role in oscillations that have a frequency of
1 Hz (recovery of L-type currents can take up to 30 s; see Refs. 15 and 30).
Sequence of molecular events underlying slow waves in
canine ASM. We would therefore propose the following
mechanism to account for the data presented in this and our previous
studies (15, 16, 22) regarding the ionic mechanisms underlying
slow-wave activity in canine ASM. An as yet poorly understood event
serves as a pacemaker and triggers membrane depolarization
( phase i), which in turn
activates T-type and L-type Ca2+
currents and a consequent action potential
( phase ii). The resultant Ca2+ influx leads to activation of
the Ca2+-dependent
Cl
current (22), which
opposes the depolarizing influence of the Ca2+ currents and draws the
membrane potential toward
ECl
( phases iii and
iv). As the membrane continues to
hyperpolarize beyond approximately
30 to
40 mV, the
L-type Ca2+ currents deactivate
( phase iv), leading to the
fast repolarizing phase of the slow waves ( phase
v). The T-type currents, which inactivate rapidly and
nearly completely at
40 mV (15), would have already decreased to
negligible levels at the peak of the slow wave. Deactivation of
Ca2+ current is unaffected by
Cl
channel blockers but is
slowed by Ba2+, accounting for the
insensitivity of the fast repolarizing phase to niflumic acid (this
study) and the changes in slow-wave activity induced by
Ba2+ (16). Finally, the subsequent
deactivation of the Ca2+ channels
leads to deactivation of the
Cl
current, and the
membrane is again primed for the next slow wave. Recovery of the T-type
currents from inactivation occurs within 1 s (15, 30), consistent with
their role in slow waves, which typically exhibit a frequency of 1 Hz.
This sequence of molecular events would account for the slow waves seen
in isolated airway tissues (this study and Refs. 6, 7, 16, 27, 33) or
in vivo electrophysiological recordings of ASM (24). In dissociated ASM
cells studied under current-clamp conditions, however, irregular
fluctuations in membrane potential are observed (18). It seems, then,
that the regular, synchronized appearance of the slow waves is a
product of the syncytial nature of this tissue and not of the
individual cells themselves. That is, the high degree of coupling by
gap junctions leads to electrotonic spread of the electrical changes,
allowing synchronization of each event (since they are triggered by
voltage-dependent Ca2+ influx) and
"smoothing" of the subsequent membrane potential changes.
Conclusion and physiological
significance. We conclude that the repolarization phase
of slow waves in canine BSM is mediated by
Ca2+-dependent
Cl
currents. Slow waves are
observed after stimulation with physiological agonists as well as under
pathophysiological conditions such as aspirin-induced or
allergen-induced airway hyperresponsiveness (1, 6, 11-13, 18, 20).
Slow waves are associated with voltage-dependent
Ca2+ influx and may maintain an
excited state in the smooth muscle after stimulation with agonists or
during inflammation; they may also play a role in the pathophysiology
underlying airway hyperreactivity. Thus
Cl
channel blockers, which
abolish slow waves and reduce excitatory electrical events, may prove
to be useful in the reversal of bronchoconstriction and treatment of
airway disease.
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ACKNOWLEDGEMENTS |
These studies were supported by grants from the Medical Research
Council of Canada and the Ontario Thoracic Society and by a Career
Award to L. J. Janssen from the Pharmaceutical Manufacturers Association of Canada and the Medical Research Council of Canada.
 |
FOOTNOTES |
Address for reprint requests: L. J. Janssen, Dept. of Medicine,
McMaster Univ., Hamilton, Ontario, Canada L8N 3Z5.
Received 29 December 1997; accepted in final form 19 May 1998.
 |
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