Vol. 282, Issue 6, L1161-L1178, June 2002
INVITED REVIEW
Ionic mechanisms and Ca2+ regulation in airway
smooth muscle contraction: do the data contradict dogma?
Luke J.
Janssen
Asthma Research Group, Firestone Institute for Respiratory
Health, St. Joseph's Hospital; and Department of Medicine,
McMaster University, Hamilton, Ontario, Canada L8N 4A6
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ABSTRACT |
In general,
excitation-contraction coupling in muscle is dependent on
membrane depolarization and hyperpolarization to regulate the opening
of voltage-dependent Ca2+ channels and, thereby, influence
intracellular Ca2+ concentration
([Ca2+]i). Thus Ca2+ channel
blockers and K+ channel openers are important tools in the
arsenals against hypertension, stroke, and myocardial infarction, etc.
Airway smooth muscle (ASM) also exhibits robust Ca2+,
K+, and Cl
currents, and there are elaborate
signaling pathways that regulate them. It is easy, then, to presume
that these also play a central role in contraction/relaxation of ASM.
However, several lines of evidence speak to the contrary. Also, too
many researchers in the ASM field view the sarcoplasmic reticulum as
being centrally located and displacing its contents uniformly
throughout the cell, and they have focused almost exclusively on the
initial single [Ca2+] spike evoked by excitatory
agonists. Several recent studies have revealed complex spatial and
temporal heterogeneity in [Ca2+]i, the
significance of which is only just beginning to be appreciated. In this
review, we will compare what is known about ion channels in ASM
with what is believed to be their roles in ASM physiology. Also, we
will examine some novel ionic mechanisms in the context of
Ca2+ handling and excitation-contraction coupling in ASM.
excitation-contraction coupling; ion channels; membrane
potential
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INTRODUCTION |
AIRWAY HYPERREACTIVITY and variable
airflow obstruction are key features of asthma. Indeed, one might say
these are its most clinically relevant features. For this reason, it is
essential to have a good understanding of the mechanisms
underlying excitation-contraction (EC) coupling in airway smooth muscle
(ASM). A great deal of research is being focused on the
electrophysiology of ASM, given the importance of ion channels in
EC coupling in other muscle types. The goal of this review is to
stimulate a reevaluation of the existing literature on the
electrophysiology of ASM as it pertains to EC coupling, with a view to
redirect those research efforts.
Contraction in smooth muscle is a product of the interaction between
actin and myosin, as described by the classic sliding filament theory.
The degree of this interaction is determined by the net level of
phosphorylation of the 20-kDa myosin light chain, which, in turn,
is dependent on the relative activities of myosin light chain kinase
(MLCK) and myosin light chain phosphatase (MLCP). MLCK is activated by
Ca2+/calmodulin. Thus, in general, bronchoconstrictors act
by elevating intracellular Ca2+ concentration
([Ca2+]i) to increase MLCK activity and/or by
decreasing MLCP activity (which effectively increases the
Ca2+ sensitivity of the contractile apparatus).
Bronchodilators, on the other hand, generally produce the opposite
effects. A more complete description of the interactions between
actin, myosin, MLCK, and MLCP is beyond the scope of this review but
can be found elsewhere (87, 209-212).
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TRADITIONAL VIEWS |
EC coupling in nonairway muscles: adequate models for ASM?
In striated muscles as well as vascular and gastrointestinal smooth
muscle, EC coupling is largely dependent on membrane depolarization, although for very different reasons.
In cardiac muscle, Na+ channel opening depolarizes the
membrane, resulting in Ca2+ entry via voltage-dependent
("L-type") Ca2+ channels, producing a transient
elevation of [Ca2+]i immediately under the
plasmalemma (17). This initial rise in
[Ca2+]i does not trigger contraction
directly: instead, it activates ryanodine receptors on the sarcoplasmic
reticulum (SR), causing a massive discharge of Ca2+ from
the internal store, resulting in contraction (17).
Voltage-dependent Ca2+ influx also contributes to a number
of other cellular events, including refilling of the SR, activation of
plasmalemmal ion channels, and modulation of various enzyme activities, etc.
In vascular and gastrointestinal smooth muscle, on the other hand,
voltage-dependent Ca2+ influx through L-type channels is
sufficient for contraction (17). Moreover, many
electrophysiological studies of these tissues reveal a "window
current" spanning the physiologically relevant range of membrane
potentials, i.e., a range of potentials more positive than the
threshold for activation of the Ca2+ channels but over
which voltage-dependent inactivation is not complete, giving rise to a
persistent Ca2+ influx. Thus small hyperpolarizations lead
to decreased activation of the channels and a drop in
[Ca2+]i, while small depolarizations increase
Ca2+ channel activation and elevate
[Ca2+]i.
Despite the differences in the mechanisms underlying EC coupling in
these tissues, the central role played by dihydropyridine-sensitive Ca2+ channels in both cases provides the rationale for the
use of Ca2+ channel blockers and K+ channel
agonists in controlling cardiac and smooth muscle contractions in
hypertension, stroke, myocardial infarction, and gastrointestinal motility disorders, etc. More importantly, clinical studies attest to
the efficacy of these tools for these purposes (59, 66, 187).
Agonist-mediated EC coupling in ASM.
Excitation of ASM is similar in many respects to that of vascular or
gastrointestinal smooth muscles. First, it is accompanied by membrane
depolarization (60) mediated primarily by activation of
Cl and nonselective cation currents as well as
suppression of K+ currents (106, 117, 119, 120,
236). Patch-clamp studies have documented the large
voltage-dependent Ca2+ currents activated by membrane
depolarization (68, 140) (Fig. 1). Finally, these
Ca2+ currents are sufficient to produce contraction, as
manifest in the robust dihydropyridine-sensitive contractions evoked by
potassium chloride (108, 122) or K+ channel
blockers such as tetraethylammonium (TEA), 4-aminopyridine, or
charybdotoxin (45), although these contractions are
generally only a fraction of the size of those evoked by physiological
agonists such as carbachol, histamine, and endothelin, etc.
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Fig. 1.
Acetylcholine-evoked depolarizations and Ca2+
currents. Current-voltage relationships in canine tracheal smooth
muscle cells from 2 different laboratories [from Kotlikoff
(140) ( ) and Muraki et al.
(164) ( )] were converted to
activation-voltage relationships by assuming that Ca2+
currents were maximally activated at +30 mV. Bars (left)
indicate membrane potentials recorded in canine trachealis during
stimulation with a range of concentrations of acetylcholine (Ach)
[from Farley and Miles (60)]. Even with maximally
effective concentrations of acetylcholine, membrane potential barely
enters the range at which substantial Ca2+ current is
activated. Similar results have been obtained for every mammalian
species studied (see Fig. 4). VR, resting membrane
potential; I/Imax, fraction of
maximal current.
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Given these similarities with other muscle types, it is understandable
that many treat Ca2+, K+, and Cl
channels as central players in EC coupling in ASM. However, a large
body of data speaks to the contrary.
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CHALLENGING THE DOGMA |
Do agonist-evoked contractions in ASM require voltage-dependent
Ca2+ influx?
Many groups have characterized the voltage-dependent Ca2+
currents in ASM (usually of the trachealis) and found these to be almost exclusively L-type in nature (77, 81, 84, 107, 140, 152,
164). It is particularly important to bear in mind a number of
biophysical properties of these currents. 1) All
electrophysiological studies find the threshold potential for these
currents to be in excess of 
40 mV, and almost all of them show little
or no Ca2+ current until membrane voltages rise above 20
mV (Fig. 1); peak activation occurs at +10 to +20 mV. 2)
These currents can develop substantial voltage- and
Ca2+-dependent inactivation, and they are also suppressed
by various second messenger signaling pathways (233, 242,
246). 3) L-type channels are selectively and potently
blocked by dihydropyridines (Fig. 2),
with an IC50 in the nanomolar range (158).
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Fig. 2.
Contractions in airway smooth muscle (ASM) are seemingly
unaffected by blockers of voltage-dependent Ca2+ channels
or of Cl channels. Despite the ability of niflumic acid
and nifedipine to abolish Cl and Ca2+
currents in ASM (A and B, respectively) [adapted
from Janssen and Sims (117) and Janssen
(107)], these agents have no substantial effect on
agonist-evoked contractions (C).
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Given these properties of the Ca2+ channels, several
observations call into question the view that voltage-dependent
Ca2+ channels play a central role in ASM contraction.
First, many have shown that agonist-evoked contraction of ASM is
seemingly unaffected under conditions in which voltage-dependent Ca2+ influx is prevented by Ca2+ channel
blockers (25, 26, 48, 61) (Fig. 2) by "clamping" the
membrane potential to very negative values far below the threshold for
Ca2+ channel activation (118) (Fig.
3) or even by removal of external Ca2+ (48, 61). Likewise, although
Cl channels are primarily responsible for the membrane
depolarization (106, 117, 119, 120, 147, 148, 237, 238),
agents such as niflumic acid, which are able to completely block the
Cl currents in ASM, have essentially no effect on resting
membrane potential (113) or agonist-evoked contractions
(Fig. 2). The only studies that do describe an inhibitory effect of
dihydropyridines on mechanical responses in ASM were carried out under
very nonphysiological conditions (complete depletion of the internal
Ca2+ pool) (8, 24-26, 114, 123, 197, 224)
or used supramaximally effective concentrations of dihydropyridines.
For example, in the case of nifedipine, most groups tend to use
106 M, and may even use 105 M
(231), even though the IC50 value reported for
this agent is in the nanomolar range (158); submicromolar
concentrations are sufficient to completely block the Ca2+
channels in ASM (77, 84, 243) and to suppress contractions in vascular or gastrointestinal smooth muscle (13, 38, 57, 70,
145). Thus nonspecific effects of the dihydropyridines (191) need to be kept in mind when interpreting such data.
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Fig. 3.
Substantial contraction can still be evoked at very negative
membrane potentials. Vertical axis indicates cell length in an ASM cell
held continuously under voltage clamp at 60 mV. Acetylcholine
(104 M) was applied at ~3-min intervals
( ). Numerous contractions (to almost 50% of resting
length) can still be evoked at this potential, which is far below
threshold for opening of voltage-dependent Ca2+ channels.
[From Janssen and Sims (118).]
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Second, the range of membrane potentials typically seen in ASM at rest
and during excitation ranges from 70 to 30 mV (1, 27, 40, 46,
60, 85, 95, 96, 99-102, 109, 111, 112, 125, 130, 132, 136, 155,
188, 215, 220), which is well below the range of potentials
required for Ca2+ channel activation (30 to +20 mV)
(68, 81, 84, 107, 140, 152, 164, 242, 243, 247) (Figs. 1
and 4). More to the point, the voltages
required to only marginally activate voltage-dependent Ca2+
channels (40 to 30 mV) are attained only with concentrations of
agonist that evoke nearly complete contraction, and Ca2+
currents are maximal at membrane potentials never seen during agonist
stimulation (+10 to +20 mV; see Figs. 1 and 4). Simultaneous electrophysiological and fura 2 fluorimetric recordings in equine ASM
have shown that the Ca2+ currents evoked by voltage step
commands to potentials in the physiologically relevant range produce
elevations in [Ca2+]i of <50 nM
(68), which pales in comparison with
bronchoconstrictor-evoked Ca2+ responses (both the peak and
plateau values typically measure several hundreds of nanomolars)
(249-251).
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Fig. 4.
Membrane voltages and Ca2+ currents.
Estimates of membrane potentials at rest ( ) and during
stimulation with various excitatory agents (), obtained
using intracellular microelectrode techniques. Numerals in parentheses
indicate reference citation from which data were extracted. Cch,
carbachol; EFS, electric field stimulation; 4-AP, 4-aminopyridine; TEA,
tetraethylammonium; 5-HT, 5-hydroxytryptamine. Also shown are voltages
for threshold and peak ( and ,
respectively) activation of voltage-dependent Ca2+
currents, as determined using patch-clamp electrophysiological
techniques. The voltages at which those currents are half inactivated
are also indicated by the solid vertical bar. Shaded box indicates the
range of potentials at which membrane oscillations (slow waves) are
typically seen in ASM. The purpose of this figure is to highlight the
lack of overlap between the physiologically relevant range of
potentials and those potentials required for significant
voltage-dependent Ca2+ influx. TSM, tracheal smooth
muscle; BSM, bronchial smooth muscle; LTC, leukotriene
C4.
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Third, the Ca2+ currents and changes in
[Ca2+]i described above are overestimations
of those one should expect to see in vivo. Because they were evoked
under experimental conditions in which voltage-dependent inactivation
had not developed to any substantial degree, i.e., the depolarizing
steps used to evoke these currents are generally less than a few
hundred milliseconds in duration and are each separated by several
seconds to minimize inactivation. This inactivation is half-maximal
(meaning the currents are halved in size) whenever membrane potential
approaches 30 mV for more than a few seconds (77, 84, 107, 140,
164) and is nearly complete as membrane potential approaches 0 mV. Thus during slow wave activity, when the membrane potential is
slowly oscillating between 40 and 20 mV for many minutes or even
hours, substantial inactivation of these currents will have occurred.
One group has described a small persistent Ca2+ influx at
the lower limit of this voltage range (referred to as window current)
that can manifest as a detectable elevation of
[Ca2+]i (<100 nM) (67), but the
latter is again too small to account for the robust contractions evoked
by agonists.
Fourth, whenever the tissues are stimulated by bronchoconstrictors such
as cholinergic agonists, voltage-dependent Ca2+ currents
are further suppressed via phosphorylation of the channels by protein
kinase C (246) and/or through Ca2+-induced
inactivation of the Ca2+ channels (233). Thus
the small window current that might exist would be wiped out by many
bronchoconstrictors. Surprisingly, 
-agonists have been shown to
augment L-type Ca2+ currents in ASM (242) even
though they are powerful relaxants.
Finally, and perhaps most importantly, clinical studies have found
Ca2+ channel blockers to be ineffective as therapeutic
agents in asthma (14, 65, 76, 86, 162, 189, 202).
This begs the question: Why does ASM exhibit such large
Ca2+ and Cl currents? We will propose several
answers to this question in this review.
Do K+ channels play a major role in
agonist-evoked relaxations?
In general, K+ channels are divided into four major
classes: Ca2+ dependent (KCa), voltage
dependent (KV), ATP dependent (KATP), and
inward rectifier (KIR) (142, 169, 187), of
which there is substantial direct evidence in ASM for two of these
classes and very limited evidence for the other two classes.
McCann and Welsh (157) were the first to directly record
KCa in ASM, and there have since been innumerable studies
that add to this evidence. These channels are generally of the large
conductance subtype because they are highly sensitive to K+
channel blockers such as TEA, charybdotoxin, and iberiotoxin, with very
little effect of the small conductance blocker apamin (28, 141,
165, 207, 208). Also, direct patch-clamp recordings show these
channels to have unitary conductances of several hundred picoamperes
(207, 208, 216). One of these studies (216)
further found that these channels can undergo random conformational
changes that lead to subconductance states of 17, 33, 41, 52, 63, and 72% of the full conductance. These currents can appear to be very "noisy" with chaotic oscillations and spikes, in part due to marked spatial/temporal changes in [Ca2+]i and the
large unitary conductance of these channels.
KV currents in ASM have also been studied in detail at the
whole cell (69, 141) and single channel (28)
levels. Activation of these channels ensues after somewhat of a delay
(28) (thus they are also referred to as "delayed
rectifier" currents) and occurs much more "smoothly" than
KCa given their smaller unitary conductance (10-15 pS)
and insensitivity to [Ca2+]i relative to
KCa. The channels also exhibit voltage-dependent inactivation, which is roughly half-maximal at the resting membrane potential. KV channels are effectively blocked by
4-aminopyridine (1-5 mM) or dendrotoxin (1-100 nM) but
not by TEA, charybdotoxin, or glybenclamide unless unreasonably high
concentrations of these agents are used (169).
Although there are many studies providing indirect evidence for
KATP in ASM (in that relaxations are evoked by
KATP agonists such as cromakalim) and these mechanical
responses are antagonized by KATP blockers such as
glybenclamide (21, 22, 36, 42, 43, 97, 131, 165, 178),
direct electrophysiological evidence for these channels is essentially
nonexistent. Many groups that have characterized K+
currents in ASM in detail directly using patch-clamp techniques have
not reported a glybenclamide-sensitive component. Rather than an action
on some channel per se, some evidence suggests that KATP
agonists act instead by suppressing phosphodiesterase activity
(178, 203).
Dozens of studies of ASM cells from the larger airways have failed to
identify any inward rectifier K+ currents. However, one
recent study (208) of cells obtained from small human
bronchioles (outer diameter 0.3-1.0 mm) has done so. The
physiological relevance of this possible regional heterogeneity is unclear.
General dogma has it that relaxants act by opening K+
channels and hyperpolarizing the membrane. However, again, several
lines of evidence speak to the contrary.
For example, bronchodilators such as -agonists and nitric oxide can
still evoke substantial or even complete relaxation in the presence of
K+ channel blockers (6, 7, 45, 102, 116, 127, 128,
165, 219) (Fig. 5). Although a
rightward shift in the concentration-response relationship for the
bronchodilator is sometimes seen (127, 128, 163), this
should be interpreted carefully. Such a parallel shift is a hallmark of
competitive inhibition, yet the K+ channel blocker and
bronchodilator agonist are not competing at a common receptor, and one
should not expect that stimulating the receptor more aggressively (by
using higher concentrations of agonist) would displace the blocker from
the channel and thus unmask the relaxation. Instead, it may be that the
K+ channel blockers are depolarizing nerve endings in the
tissues, causing them to release excitatory agonists (97,
132) that then antagonize the bronchodilator response. Such
functional antagonism can be overcome by using higher concentrations of
bronchodilator agonist (64, 110, 185). Tetrodotoxin is not
a guarantee against this because it only prevents depolarization caused
by Na+ channel activation but not that caused by
suppression of outward K+ currents in the nerve endings.
Instead, agents such as 
-conotoxin should be used to prevent the
subsequent Ca2+ influx and neurotransmitter release.
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Fig. 5.
Substantial relaxation can still be evoked during blockade of
K+ channels. Relaxations were evoked by the NO donor
soluble N-ethylmaleimide-sensitive factor attachment protein
(SNAP) in ASM tissues pretreated with the K+ channel
blockers charybdotoxin (ChTx; 0.1 µM), TEA (30 mM), or 4-AP (1 mM).
These data suggest that K+ channels are not absolutely
necessary for NO-evoked relaxation. [From Janssen et al.
(116).]
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Also, bronchodilators can often cause relaxations when
voltage-dependent Ca2+ influx has already been abolished
beforehand using Ca2+ channel blockers (22, 102, 130,
218). Others have found that relaxations are not evoked by
artificially imposed hyperpolarizing current (39, 48);
again, bronchoconstrictors can still evoke substantial contraction
during voltage clamp at resting membrane potentials (117,
118) (Fig. 3).
Clearly, then, membrane hyperpolarization alone is neither necessary
nor sufficient for relaxation in ASM. Consistent with this,
K+ channel openers have been found to be ineffective as a
therapy for asthma (41, 62, 135, 204). Thus a better
understanding of agonist-evoked relaxation demands a new emphasis on
mechanisms other than K+ channel activation. Many (perhaps
all) bronchodilators that activate K+ channels are also
known to exert other effects on ASM, including decreased
Ca2+ sensitivity of the contractile apparatus (126,
174, 209, 210, 212), inhibition of
D-myo-inositol 1,4,5-trisphosphate
(IP3) binding to its receptor on the SR (196),
suppression of IP3-induced Ca2+ release
(133, 241), and enhancement of Ca2+
uptake/extrusion (133).
EC coupling in ASM owes much more to voltage-independent
mechanisms.
In contrast to the questionable significance of membrane
voltage-regulated Ca2+ influx, a number of
voltage-independent mechanisms are primarily responsible for
contraction in ASM.
The most widely recognized involves the release of Ca2+
sequestered within the SR. Cholinergic agonists, histamine, endothelin, leukotrienes, and thromboxane A2 activate phospholipase C,
which, in turn, generates the second messengers diacylglycerol and
IP3 (32-34). The latter of these two
messengers activates Ca2+-permeable ion channels on the
membrane of the SR, releasing its store of Ca2+ and,
thereby, triggering contraction (212). The SR also
expresses another group of Ca2+-permeable ion channels that
are activated by Ca2+ itself, caffeine, ryanodine, or by
cyclic ADP ribose (71, 201, 209, 212). The physiological
role of these "ryanodine receptors" is still debated (see
Bronchodilators: is K+
channel activation a causal event or an epiphenomenon?).
Refilling of the SR involves primarily the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). However, the existence of at least one
other novel refilling pathway has been suggested (25, 26, 118, 186) as summarized in Voltage-dependent
Ca2+ channels and SR
refilling.
More recently, a great deal of attention has been focused on
agonist-induced changes in the sensitivity of the contractile apparatus
to [Ca2+]. One such mechanism that is gaining a great
deal of momentum both in the vascular and ASM fields involves
activation of the monomeric G protein Rho, which in turn translocates
to the membrane and activates Rho kinase (31, 98, 124,
253). The latter phosphorylates and thereby inactivates MLCP,
leading to a net accumulation of phosphorylated myosin light chains,
and thus contraction (211). Others are investigating
mechanisms such as extracellular regulated kinase
(ERK)-mediated phosphorylation of caldesmon and calponin
(72, 75), integrin-mediated tyrosine phosphorylation of
focal adhesion kinase, paxillin, and talin (73, 159, 160, 177,
221, 223, 240), and protein kinase C activation (2, 29,
93). Endothelin-stimulated activation of ERK was recently shown
to be dependent on Ca2+ influx (232),
indicating some "cross talk" between Ca2+-dependent and
Ca2+-independent pathways.
The superficial buffer barrier and spatial/temporal heterogeneity
of [Ca2+] in ASM.
Several recent findings have revolutionized the way Ca2+
handling in ASM should be considered.
First, until recently, the internal Ca2+ pool has usually
been modeled as being more or less centrally located and releasing its
store of Ca2+ roughly uniformly throughout the entire
cytosol. However, we had long been puzzled by the observation that both
acetylcholine and caffeine evoke substantial elevations of
[Ca2+]i as well as membrane currents, but
only the former would reliably evoke contraction (74, 108, 117,
119). That is, caffeine only evokes a contraction when it causes
a massive discharge of Ca2+ (e.g., when applied
instantaneously in high millimolar concentrations), but not when this
discharge occurs gradually (e.g., using lower concentrations and/or
introducing caffeine more slowly via the bath perfusion).
Electronmicroscopy of vascular smooth muscle cells shows the SR to form
sheets around the internal periphery of the cell (170),
thereby dividing the cytosol into two spaces (Fig.
6), the peripheral space immediately
underneath the plasmalemma where ion channels are found (many of them
being regulated by Ca2+), and the deep cytosolic space
where the contractile apparatus is found. Another recent study suggests
that the same anatomical arrangement can be found in ASM
(49). In this way, the cell can dissociate the influence
of [Ca2+] on mechanical and electrical activities. This
"superficial buffer barrier" accounts for the paradoxical effects
of caffeine (108, 124): ryanodine receptors may direct
Ca2+ release preferentially into the peripheral space (and,
thereby, activate ion channels), and the contractile apparatus only
becomes activated when this release is so massive that it "spills
over" into the deep space. It can also explain how subnanomolar
concentrations of acetylcholine can evoke substantial ionic current but
without any change in tension (124). That is, the ionic
currents indicate that Ca2+ is in fact being released
(because they are totally dependent on that process) (108, 117,
118, 121, 143, 238) although this release cannot be discerned
using fluorimetric techniques that measure the average change in
[Ca2+]i throughout the entire cell
(124, 198, 200, 249). However, the anticipated mechanical
response is quelled by the barrier function of the SR. Thus it is
essential to use more refined Ca2+ imaging techniques, ones
that can resolve subcellular regions, for further studies of
Ca2+ handling in ASM.
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Fig. 6.
Superficial buffer barrier model. The sarcoplasmic
reticulum (SR) divides the cytosol into 2 spatially and functionally
distinct compartments: the subplasmalemmal space and the deep cytosolic
space. Bottom left: excitatory agonists release
Ca2+ into the deep cytosol to trigger contraction, as well
as into the subplasmalemmal space to activate
Ca2+-dependent Cl channels. Bottom
right: relaxants, on the other hand, promote Ca2+
uptake and Ca2+ extrusion but also trigger Ca2+
release via ryanodine receptors. The latter effect allows for unloading
of the SR, increasing its buffering capacity, without triggering
contraction. It may also lead to activation of
Ca2+-dependent K+ channels and membrane
hyperpolarization.
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Another change in our understanding of Ca2+ handling was
stimulated by advances made in other cell types using highly
sophisticated Ca2+ imaging equipment. In the ASM field,
many focus their attention on the magnitude of the solitary, brief,
spikelike Ca2+ transient evoked by a high concentration of
agonist and relate that to much more persistent cellular events, such
as contraction. In other words, this Ca2+ transient is
being interpreted as a persistent elevation of
[Ca2+]i throughout the entire cell. Instead,
the sustained changes in [Ca2+]i evoked by
lower concentrations of agonist might be more physiologically relevant.
It is now known that agonists can evoke recurring changes in
[Ca2+]i ("oscillations"), which propagate
throughout the cell ("waves") (225), and are further
refined or shaped as they progress through the cytosol
(139). These oscillations convey information within their
amplitudes (peak height as well as their mean or temporally averaged
amplitude) as well as in their frequency, and this information may be
decoded by Ca2+/calmodulin-dependent kinase (19, 20,
30, 35, 50, 52, 55, 184), MLCK (50), the SR
Ca2+ pump (50), calpain (229),
adenylyl cyclase (44), or mitochondria (79).
For example, gene expression of several proinflammatory cytokines is
differentially regulated by Ca2+ oscillations in a
frequency- and amplitude-dependent fashion (54, 146).
Ca2+ oscillations have been reported in ASM from the human
(56), rat (193, 194, 227), guinea pig
(117, 200), pig (171, 180-183), and dog
(117), but their underlying mechanism and their
physiological relevance are still poorly understood.
Finally, a great deal of work has been done in the cardiac and vascular
smooth muscle fields looking at Ca2+ sparks: small and
transient elevations of [Ca2+]i produced by
localized bursts of Ca2+ from a handful of ryanodine
receptors (23, 82, 103, 104, 168). These are proposed to
be the triggers for spontaneous transient ion currents, which, in turn,
are believed to modulate mechanical activity (168, 254).
They also represent the fundamental event underlying Ca2+
oscillations and relaxations in vascular smooth muscle
(168). There have been limited studies of sparks in ASM
(172, 173, 199, 254), but their physiological role(s) is
unclear. Although they may lead to activation of K+ and
Cl channels (254), electromechanical
coupling is of limited importance in ASM. Instead, their primary
function may be to discharge the SR contents, without evoking
contraction, toward the plasmalemmal Ca2+ pump to increase
the buffering capacity of the SR.
Thus Ca2+ signals are organized into complex temporal and
spatial patterns that are lost using techniques and models that focus solely on the spikelike elevation averaged across the entire ASM cell.
Many previous reports of Ca2+ responses in ASM were
severely limited, because whole cell photometry was used: this approach
averages the changes in [Ca2+]i across the
entire cell. None have yet compared bronchoconstrictor-induced changes
in the deep cytosol vs. the subplasmalemmal space. Also, the sampling
rate of most of these studies (on the order of 1 Hz) was much too slow
to adequately resolve events with time courses on the millisecond
scale, such as Ca2+ oscillations and Ca2+
sparks (199). Finally, the few studies able to resolve
subcellular regions generally used only a maximally effective
concentration of excitatory agonist: we have previously obtained
evidence (using patch-clamp recordings) that concentrations of
acetylcholine that were subthreshold for mechanical or fluorimetric
responses nonetheless evoked substantial membrane currents
(124), suggesting important differences between global and
subplasmalemmal measurements of [Ca2+]. The concentration
of agonist used is also important with respect to the likelihood of
observing Ca2+ oscillations, since models of these
phenomena indicate a critical dependence on variables such as
[IP3] and basal [Ca2+] (199,
225).
| |
NOVEL IONIC MECHANISMS |
Voltage-dependent Ca2+ channels and
SR refilling.
Several studies show voltage-dependent Ca2+ channels in ASM
to be important for refilling and maintenance of the SR (25, 26, 118, 149, 186). For example, we used agonist-evoked
Cl currents to assess the filling state of the SR, and we
found that we could completely deplete the SR using cyclopiazonic acid (SERCA inhibitor) and then refill the SR in a dihydropyridine-sensitive fashion using a series of depolarizing pulses (Fig.
7) (118). More surprisingly,
though, this refilling occurred in the maintained presence of
cyclopiazonic acid (which was completely sufficient to functionally
deplete the SR), suggesting that this refilling pathway did not involve
SERCA. In other words, Ca2+ was crossing the plasmalemma
and entering the SR without being pumped by the
Ca2+-ATPase.
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Fig. 7.
Ca2+ currents may contribute to refilling of
the SR. Acetylcholine-evoked Cl currents
() were used to assess the filling state of the SR in
canine tracheal smooth muscle. Exposure to cyclopiazonic acid (CPA)
completely depleted the SR (ii). In the maintained presence
of cyclopiazonic acid, a series of depolarizing pulses led to partial
refilling of the SR (iii). This refilling was enhanced by
the Ca2+ channel agonist BayK8644 and inhibited by
nifedipine (iv). [From Janssen and Sims
(118).]
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|
These paradoxical findings can be explained using a recently proposed
model that describes a physical interaction between the SR and
plasmalemmal store-operated Ca2+ channels (18, 138,
176, 190, 191, 252). Briefly, agonist-induced depletion of the
internal store triggers activation of tyrosine kinase(s), Ras, and
reorganization of the cytoskeleton in such a way that it directly
couples IP3 receptors on the SR with Ca2+
channels on the plasmalemma. Similarly, a functional interaction between voltage-dependent Ca2+ channels and the nucleus has
recently been described. Dolmetsch et al. (53) found that
gene transcription could be stimulated by Ca2+ influx
through L-type Ca2+ channels but not through N- or P/Q-type
Ca2+ channels (which are also present but seem to
contribute to other physiological responses in these cells). Their data
show that calmodulin, which is tethered at the mouth of the L-type
Ca2+ channels and is activated by Ca2+ influx
through them, signals to the nucleus via a mitogen-activated protein
kinase pathway (53).
Several observations made in ASM are consistent with a physical
coupling between plasmalemmal Ca2+ channels and SR
membranes. First, excitatory stimulation of ASM is accompanied by
activation of tyrosine kinases (206, 228) and Ras/Rho
(58, 80, 83, 226) as well as cytoskeletal rearrangement (78, 83, 226). Second, inhibition of tyrosine kinases
compromises SR refilling (151). Third, ASM depleted of
focal adhesion kinase (which regulates cytoskeleton stability) shows
marked suppression of acetylcholine-evoked Ca2+ transients
and contractions as well as changes in voltage-dependent Ca2+ channel function without any disruptive changes in the
contractile apparatus per se (assessed by addition of Ca2+
to permeabilized strips) (222). Fourth, our observation
that the SR can be refilled by voltage-dependent Ca2+
influx in the maintained presence of cyclopiazonic acid is difficult to
explain otherwise, because there is no other Ca2+ pump on
the SR other than SERCA.
At first glance, this novel model of SR refilling also suffers from the
criticism raised earlier in this review that membrane potential rarely
reaches the threshold for opening of the voltage-dependent Ca2+ channels. However, if there is a very close apposition
or even physical coupling between the plasmalemmal and SR membranes, it is unclear what transmembrane potentials the plasmalemmal
Ca2+ channels would experience. That is, the close
proximity and physical interaction of a large polypeptide (the
IP3 receptor) and the SR membrane with the inner face of
the Ca2+ channel, with the resultant changes in membrane
surface charge and/or induction of conformational changes in the
Ca2+ channel, could easily alter their voltage
characteristics, allowing them to open at physiologically relevant
potentials (167). Also, access of Ca2+ and of
protein kinase C to the inner face of the Ca2+ channels
might be hindered during this interaction, thereby preventing Ca2+ channel inactivation.
Bronchodilators: is K+ channel
activation a causal event or an epiphenomenon?
The traditional view has been that bronchodilators act by decreasing
[Ca2+]i throughout the cell. Surprisingly,
however, we and others have described elevations in
[Ca2+]i in response to relaxant agents
(63, 115, 116, 245). Yamaguchi et al. (245)
resolved the effects of isoproterenol on [Ca2+] in
greater detail and showed that it increases [Ca2+] in the
peripheral regions of the ASM cells and decreases it in their more
central regions. One interpretation of these findings is that
bronchodilators act by triggering Ca2+ release from the SR,
completely contrary to current dogma, but not in the same fashion as
bronchoconstrictors. That is, rather than elevating
[Ca2+]i globally throughout the cell (via
IP3-induced Ca2+ release), the data suggest
that ryanodine receptors are involved and direct Ca2+ into
the subplasmalemmal space where it is extruded from the cell by the
plasmalemmal Ca2+-ATPase. In the process,
Ca2+-dependent K+ channels may or may not be
activated. Although extensive evidence has been given for such a
mechanism in vascular smooth muscle (103, 105, 137, 168),
this model has not been examined in ASM. However, there has been one
report of K+ currents activated by Ca2+ sparks
in ASM (although relaxants were not used in this study) (254), and the isoproterenol-induced elevation of
[Ca2+]i was shown to involve ryanodine
receptors (245). Isoproterenol (171), cAMP
(171), and cGMP (37) suppress the frequency
of cholinergic Ca2+ oscillations in ASM.
It appears, then, that bronchodilators simultaneously trigger uptake of
Ca2+ from the deep cytosol into the SR as well as release
of SR Ca2+ into the subplasmalemmal space, followed by
extrusion of Ca2+ from that peripheral space into the
extracellular space (Fig. 6). Our proposal that relaxants stimulate
Ca2+ release might be counterintuitive, but it should be
expected. The cells must be able to discharge internally sequestered
Ca2+, but without triggering contraction, to
increase/maintain the Ca2+-buffering capacity of the SR.
Data presented in an earlier study (108) suggest that
ryanodine receptors are involved and that this Ca2+ release
is preferentially directed into the subplasmalemmal space. The
important point to be made in all of this is that K+
channels appear to be more like bystanders than key players in the
process of relaxation.
What role do plasmalemmal Cl
channels play in ASM physiology?
There is substantial electrophysiological evidence for a population of
Cl channels activated during excitatory stimulation
(81, 106, 117, 119, 120, 147, 148, 237, 238). These
channels exhibit a small unitary conductance (far below 20 pS)
(121), and their activation is Ca2+ dependent
(via Gq/G11-stimulated release of
internal Ca2+) (238) but voltage independent
(121). Moreover, these inactivate in a voltage-sensitive
fashion (121) via phosphorylation by
Ca2+/calmodulin-dependent kinase II (237)
(which is in turn triggered by the elevation of
[Ca2+]i that activated the channels in the
first place).
Given that excitatory stimulation is generally associated with such
large Cl currents that can depolarize the membrane and
are tightly regulated by second messenger signaling events (121,
237, 241, 254), much as is the case in vascular smooth muscle,
it is easy to conclude that the Cl currents play a key
role in contraction of ASM by depolarizing the membrane and thus
triggering voltage-dependent Ca2+ influx. However, the
truth of the matter is that agonist-evoked contractions are not
affected by Cl channel blockers (Fig. 2). Also, given
that the equilibrium potential for Cl is approximately
40 to 30 mV (4, 5), activation of Cl
channels will indeed depolarize the membrane but will essentially clamp
it at potentials barely sufficient for activation of voltage-dependent Ca2+ channels. Again, it bears repeating that substantial
contractions can still be evoked during voltage clamp at very negative
potentials (118) (Fig. 3), indicating that depolarization
is not necessary for contraction anyway. Why, then, are
Cl currents so prominent in ASM?
Recently, another type of Cl channel has been isolated
from ASM with properties diametrically opposite to those described above (195). That is, they have a large unitary
conductance (several hundred picosiemens) and their activation is
voltage dependent but Ca2+ independent. All of these
properties are similar to those of the Cl channels
present on the SR of skeletal and cardiac muscle and facilitate
Ca2+ flux by neutralizing charge buildup on the SR
membranes (3).
This finding prompts us to propose an entirely novel and testable
hypothesis: that agonists activate Cl currents in the
plasmalemma of the ASM cell to facilitate Ca2+
release/uptake. That is, Ca2+ efflux from the SR leads to a
net negative charge on the inner face of the SR membrane that hinders
Ca2+ release (Fig.
8A) unless alleviated by
compensatory fluxes of Cl out of the SR (Fig.
8B) (134, 179). However, the accumulation of
Cl outside the SR opposes further Cl efflux
from the SR (and thus Ca2+ release; Fig. 8B). A
sudden opening of Cl channels on the plasmalemma, with
subsequent loss of Cl from the subplasmalemmal space,
would instantaneously alter the equilibrium potential for
Cl across the SR membrane, thereby boosting efflux of
Cl (and Ca2+) from the SR (Fig.
8C). Consistent with this, we have noted anecdotally that
agonist-evoked membrane currents and contractions were lost in cells
studied using a low internal [Cl] solution (20 mM)
(117).
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Fig. 8.
Hypothesized role for membrane Cl channels.
A: release of Ca2+ from the SR results in
accumulation of negative charge () on the membrane of the SR, which
in turn hinders further Ca2+ release. B: efflux
of Cl from the SR compensates for the charge buildup and
allows greater Ca2+ release. However, accumulation of
Cl in the cytosol hinders both processes. C:
opening of Cl channels on the plasmalemma allows a
massive Cl efflux from the cell, instantaneously
increasing the driving force on Cl to exit the SR,
thereby allowing complete dissipation of the charge buildup and thus
facilitating further Ca2+ release.
|
|
Voltage-independent Ca2+ influx
pathways?
Agonists activate a membrane conductance that is nonselective for
various monovalent cations (117, 235, 239). It is unclear whether this conductance is Ca2+ permeable and, thereby,
serves as a source of Ca2+ for contraction as is the case
in vascular and gastrointestinal smooth muscle (51, 90,
94). Elevation of [Ca2+]i is necessary
but not sufficient for activation of these nonselective cation
channels; instead, they are activated by Gi/Go
(activated by M2 muscarinic or H1 histaminergic
receptors). While the Cl current that is concurrently
activated by these agonists decays within seconds, the nonselective
cation channels remain open as long as the agonists are present,
resulting in a persistent noninactivating inward current.
| |
IONIC MECHANISMS IN ASM PATHOPHYSIOLOGY |
Inflammation plays a central role in asthma and airway
hyperreactivity, and it is becoming increasingly clear that cytokines and inflammatory mediators exert a variety of effects on various aspects of ASM function. Several researchers have sought to examine whether ionic mechanisms are altered in asthma or in animal models of
airway hyperresponsiveness. For example, it might be possible that
membrane potentials are higher or Ca2+ currents greater in
tissues/cells from asthmatics or hyperresponsive animals, as can happen
for vascular smooth muscle cells and hypertension (150,
217). However, immunological stimulation of excised guinea pig
tracheal tissues causes first a small and transient membrane depolarization, followed by a marked and prolonged membrane
hyperpolarization (213, 215). In excised tissues exposed
to allergen in vivo, membrane potentials were slightly more
hyperpolarized (<5 mV) when the guinea pigs had been acutely
sensitized to allergen but markedly depolarized (by >10 mV) when the
animals had been chronically exposed to allergen (156,
214). While canine ASM is normally very polarized (resting
potentials of approximately 60 mV) and does not show spontaneous
phasic electrical activity, ASM from dogs with "aspirin-induced
asthma" exhibited marked membrane depolarization and slow wave
activity (101). This might be related to the suppression of delayed rectifier K+ current (more specifically,
enhanced inactivation of the channels), which is reported to occur in
allergen-sensitized canine bronchial smooth muscle (234).
Despite all these observed changes in electrophysiological activity in
vitro, their significance to airway physiology/pathophysiology is
unclear given that electromechanical coupling is relatively unimportant
in ASM and that Ca2+ channel blockers and K+
channel openers are generally ineffective in the treatment of asthma in
the clinical setting (14, 41, 62, 65, 76, 86, 135, 162, 189, 202,
204).
On the other hand, asthma and airway hyperresponsiveness might be
associated with changes in Ca2+ handling. Perhaps basal
levels of [Ca2+]i are higher or
Ca2+ release is greater. Several proinflammatory cytokines
such as interleukin-1, tumor necrosis factor-
, interferon-
,
platelet-derived growth factor, and eosinophil major basic protein
markedly augment excitatory agonist-evoked Ca2+ transients
(9, 10, 12, 244, 248), phosphoinositide turnover (10, 248), and contractions (11, 154, 175,
244). In some cases, these effects involve mitogen-activated
protein kinase cascades (ras, raf, MEK, Rho, Rho kinase) and induction
of gene expression, protein synthesis, and proliferation (10, 88, 89, 226).
Oxidizing pollutants such as ozone and acrolein induce airway
hyperreactivity. Although these act in part through inflammatory cells,
they can also alter EC coupling and other cellular events in isolated
ASM cells or tissues (15, 16, 91, 92, 153, 192). This
direct action on the ASM per se seems to involve changes in inositol
phosphate metabolism and Ca2+ handling, with induction or
augmentation of Ca2+ oscillations (91, 92, 192,
194).
| |
FUTURE DIRECTIONS |
On the basis of the arguments laid out in this review, we propose
the following recommendations in future studies of ASM physiology.
Ionic mechanisms.
It seems that too many groups are studying ionic mechanisms in ASM in
the same fashion as they would a vascular or gastrointestinal smooth
muscle preparation. There has been far too much emphasis on
voltage-dependent mechanisms, to an extent that is not warranted by
clinical studies of Ca2+ channel blockers or K+
channel agonists. As outlined above, ASM is distinct from skeletal, cardiac, and vascular smooth muscle in many respects pertaining to EC
coupling, and a great deal remains to be learned about its unique
physiology and pathophysiology. There needs to be greater consideration
of other roles for the ion channels that do not depend on, or relate
to, electromechanical coupling.
Ca2+ handling.
The hypothesis that Ca2+ may play an important causal role
in airway hyperreactivity and asthma was proposed more than two decades ago (161, 230) but still has not been explored in
sufficient detail. We believe this has resulted in an overly simplistic
model of Ca2+ handling in ASM and an imbalanced
understanding of electromechanical coupling mechanisms, as outlined
above. A breakdown in the superficial buffer barrier and/or changes in
Ca2+ sparks/Ca2+ oscillations may be more
relevant to airway hyperreactivity than increases in basal
[Ca2+]i or peak magnitudes of agonist-evoked
Ca2+ transients.
EC coupling.
A great deal more attention needs to be focused on recently discovered
EC coupling mechanisms in ASM, particularly Rho/Rho-activated kinase-mediated regulation of MLCP as well as pathways directed at thin
filaments. Moreover, most studies use only a maximally effective
concentration of agonist in their studies. The full range of agonist
concentrations should be examined, from subthreshold to maximally
effective concentrations, since there is now evidence that different EC
coupling mechanisms may contribute to differing degrees depending on
the level of excitation (124, 194). Also, experimental
strategies and tools need to be wielded with a greater degree of
sophistication. For example, we should be increasingly wary of the use
of micromolar concentrations of dihydropyridine blockers, or of
contractions as an index of [Ca2+]i, or of
focusing almost exclusively on the temporally and spatially averaged
spikelike elevations of [Ca2+] evoked by a maximally
effective concentration of agonist. Finally, the vast majority of
studies of airway function are done using tracheal tissues/cells. A
greater use of smaller airways is advocated because evidence is
accumulating for marked regional differences across the airway tree
(47, 124, 208).
In summary, a more imaginative approach to the study of EC coupling in
ASM, one that does not lean so heavily on electromechanical mechanisms,
could lead to major advances in our understanding of the unique
physiology (and pathophysiology) of ASM.
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
L. J. Janssen, L-314, St. Joseph's Hospital, 40 Charlton
Ave. E., Hamilton, ON, Canada L8N 4A6 (E-mail:
janssenl{at}mcmaster.ca).
10.1152/ajplung.00452.2001
| |
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TOP
ABSTRACT
INTRODUCTION
