Superficial buffer barrier and preferentially directed release
of Ca2+ in canine airway smooth
muscle
Luke J.
Janssen,
Pierre A.
Betti,
Stuart J.
Netherton, and
Denise K.
Walters
Asthma Research Group and Smooth Muscle Research Group, Department
of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
We examined cytosolic concentration of
Ca2+
([Ca2+]i)
in canine airway smooth muscle using fura 2 fluorimetry (global changes in
[Ca2+]i),
membrane currents (subsarcolemmal
[Ca2+]i),
and contractions (deep cytosolic
[Ca2+]i).
Acetylcholine (10
4 M)
elicited fluorimetric, electrophysiological, and mechanical responses.
Caffeine (5 mM), ryanodine (0.1-30 µM), and
4-chloro-3-ethylphenol (0.1-0.3 mM), all of which trigger
Ca2+-induced
Ca2+ release, evoked
Ca2+ transients and membrane
currents but not contractions. The sarcoplasmic reticulum (SR)
Ca2+-pump inhibitor cyclopiazonic
acid (CPA; 10 µM) evoked Ca2+
transients and contractions but not membrane currents. Caffeine occluded the response to CPA, whereas CPA occluded the response to
acetylcholine. Finally, KCl contractions were augmented by CPA,
ryanodine, or saturation of the SR and reduced when SR filling state
was decreased before exposure to KCl. We conclude that
1) the SR forms a superficial buffer
barrier dividing the cytosol into functionally distinct compartments in
which
[Ca2+]i
is regulated independently; 2)
Ca2+-induced
Ca2+ release is preferentially
directed toward the sarcolemma; and 3) there is no evidence for
multiple, pharmacologically distinct Ca2+ pools.
sarcoplasmic reticulum; membrane currents; contraction
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INTRODUCTION |
CA2+ is sequestered
within the sarcoplasmic reticulum (SR) by sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) (1); this
uptake is selectively inhibited by agents such as thapsigargin or
cyclopiazonic acid (CPA) (17, 20). Agonists can release this stored
Ca2+ by stimulating phospholipase
C to generate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3],
which, in turn, activates
Ca2+-permeable channels on the SR
(1). Ca2+ can also be released
through channels on the SR membrane that are gated by an elevation in
the cytosolic concentration of
Ca2+
{[Ca2+]i;
Ca2+-induced
Ca2+ release (CICR)} (2).
Ryanodine, a plant-derived muscle-paralyzing alkaloid, binds to a
high-affinity site on this channel and induces channel opening; thus
these channels are also referred to as ryanodine receptors. At higher
concentrations, ryanodine also binds to low-affinity sites on the
channel and induces channel closure (2), thereby complicating the
interpretation of data obtained with this ligand. Recently,
4-chloro-3-ethylphenol (CEP) has been shown to mimic the ability of
ryanodine to open these channels but without the inhibitory effects on
channel function, although it may have nonspecific inhibitory effects
on the contractile apparatus (15). Caffeine at millimolar
concentrations increases the Ca2+
sensitivity of CICR channels such that basal levels of
[Ca2+]i
are sufficient to induce channel opening; however, like other methylxanthines, caffeine also inhibits phosphodiesterases (leading to
accumulation of cAMP) and blocks adenosine receptors. Thus there are a
wide variety of agents available to examine the uptake and release of
Ca2+ from the SR, although care
must be taken to consider their possible nonspecific actions.
According to the superficial buffer barrier (SBB) hypothesis, the
peripheral SR separates the cytosol into a subsarcolemmal compartment
and a deep cytosolic compartment and "buffers" elevations in
[Ca2+]i
in the latter space due to Ca2+
influx (21). It is also proposed that
Ca2+ from the SR is vectorially
"leaked" into the subsarcolemmal space and then extruded from the
cell by
Na+/Ca2+
exchange and/or the sarcolemmal pump. Although this hypothesis is
supported by data from studies of vascular smooth muscle (SM) (3, 21),
it has not been examined in airway SM.
Ca2+-sensitive dyes such as fura 2 provide a global estimate of
[Ca2+]i
throughout the entire cell. Patch-clamp recordings of
Ca2+-dependent membrane currents
and contractile responses, on the other hand, can serve as indirect
indexes of Ca2+ concentration
([Ca2+]) within the
subsarcolemmal space and the deeper cytosol, respectively (3, 9, 13,
14, 16); it should be noted, though, that these responses do not
precisely mirror changes in
[Ca2+]i
under all conditions so these data must be interpreted with caution.
For example, contractions can occur without any corresponding change in
[Ca2+] (19).
Similarly, although activation of
Ca2+-dependent
Cl
channels appears to be
solely Ca2+ dependent, this is
soon followed by phosphorylation and consequent inactivation of the
channels (23).
There may also be regional heterogeneity or specialization with respect
to the SR itself. For example, in some cell types, it seems that there
are multiple, pharmacologically distinguishable Ca2+ pools: subsets of SR are
sensitive to caffeine (i.e., express CICR sites), whereas the remainder
are sensitive to agonists [i.e., express
Ins(1,4,5)P3-gated
release sites] (1, 5). Furthermore, there is evidence that the
Ins(1,4,5)P3-sensitive
Ca2+ pools, but not the
caffeine-sensitive Ca2+ pools, are
sensitive to CPA (5). Alternatively,
Ca2+ release sites may be
concentrated on one side of the SR (e.g., that which faces the deep
cytosol or the subsarcolemmal space) and mediate a preferential or
vectorial release in a certain direction (21).
In this study, we sought to examine
Ca2+ handling in canine airway SM.
Using fura 2 fluorimetry, patch-clamp recordings, and contractions to
monitor changes in
[Ca2+]i,
we provide evidence that the SR does, in fact, divide the cytosol into
two functionally distinct compartments, that CICR is preferentially
directed toward the sarcolemma, and that there is no evidence for
pharmacologically distinct Ca2+
pools. Some of these data have been presented in abstract form (7).
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METHODS |
Preparation of tissues and cell
dissociation. Adult mongrel dogs were euthanized with
pentobarbital sodium (100 mg/kg). Tracheae were excised and kept in a
physiological solution. The trachealis was isolated by removing
connective tissue, vasculature, and epithelium, then cut into strips
parallel to the muscle fibers (
1 mm wide). For single-cell studies,
tracheal SM (TSM) strips (0.5-1.0 g wet weight) were transferred
to dissociation buffer (composition given in Solutions
and chemicals) containing collagenase
(type IV, 2.7 U/ml), elastase (type IV, 12.5 U/ml), and BSA (1 mg/ml),
then were either used immediately or stored at 4°C for use up to 48 h later; Janssen and Sims (8) 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 an
enzyme-containing solution were incubated at 37°C for 60-120 min, then gently triturated.
Fura 2 fluorimetry. Single cells were
studied with a Deltascan system (Photon Technology International,
South Brunswick, NJ). After settling onto a glass coverslip mounted
onto a Nikon inverted microscope, the cells were loaded with fura 2 (fura 2-AM; 2 µM for 30 min at 37°C), then superfused
continuously with Ringer buffer at 37°C (2-3 ml/min). The
cells were illuminated alternately (0.5 Hz) at the excitation
wavelengths, and the emitted fluorescences (measured at 510 nm) induced
by 340- (F340) and 380-nm
(F380) excitation were measured
with a photomultiplier tube assembly. The ratio of
F340 to
F380 was converted to
[Ca2+] with previously
published methods (6). The fluorescence ratio values under saturating
(maximum ratio) and Ca2+-free
(minimum ratio) conditions were obtained previously (13), and the
Ca2+-fura 2 dissociation constant
was assumed to be 224 nM (6). Background fluorescence, determined with
cells not loaded with fura 2 but otherwise handled in a similar
fashion, was subtracted from the raw data. Agonists were applied by
pressure ejection from a puffer pipette (Picospritzer, General Valve,
Fairfield, NJ).
Patch-clamp electrophysiology. Single
TSM cells were allowed to settle and adhere to the bottom of a
recording chamber (1-ml bath volume perfused at 2-3 ml/min) and
were studied within 6 h after dissociation. Membrane currents were
recorded with the nystatin perforated-patch method, which Janssen and
Sims have previously described in detail (8-10). The electrode
solution contained the following (in mM): 140 KCl, 0.4 CaCl2, 1 MgCl2, 1 EGTA, and 20 HEPES, pH
7.2, and nystatin (final concentration 200 µg/ml). Data were filtered
at 1 kHz and were stored on magnetic tape with a digital data recorder
(Medical Systems, Instrutech, Great Neck, NY), with simultaneous
sampling at 2 kHz with pCLAMP 6 software (Axon Instruments, Foster
City, CA). Corrections were not made for liquid junction potentials
(previously found to be only
2 mV) (8). Agonists were applied by
pressure ejection from a puffer pipette.
In some cases, cell shortening was recorded with a video camera mounted
on the microscope. Images were later played back and changes in cell
length were quantified with a line drawn through the central axis of
the cell (SigmaScan, Jandel, Corte Madera, CA) as Janssen and Sims have
previously described (9).
Microelectrode studies. Intact tissues
were carefully pinned out in a chamber having a bath volume of
10
ml; Krebs-Ringer buffer (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. Microelectrodes (tip
resistance of 30-80 M
when filled with 3 M KCl) were pulled
from borosilicate capillary tubes and used to impale single SM cells.
Membrane potential changes were observed on a dual-beam oscilloscope
(Tektronix D13, 5A22N differential amplifier, and 5B12 dual-time base)
and recorded on 0.25-inch magnetic tape with a Hewlett-Packard
instrumentation recorder. Portions of these data were played back,
digitized (Digidata 1200), and sampled with pCLAMP 6 software (Axon
Instruments), then fitted with pCLAMP 6 and/or exported to SigmaPlot
(Jandel) for graphic presentation.
Organ bath studies. TSM strips were
mounted vertically in 3-ml organ baths with silk (Ethicon 4-0) tied to
either end of the strip, one of which was fastened to a Grass FT.03
force transducer while the other was anchored. Isometric changes in
tension were digitized and recorded with an on-line program (DigiMed
System Integrator, MicroMed, Louisville, KY). Tissues were bathed in Krebs-Ringer buffer (see Solutions and
chemicals for composition) containing indomethacin (10 µM), 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 begun. At the conclusion of the experiments, tissue
dry weight was obtained and used to standardize the contractile responses.
Solutions and chemicals. The
dissociation buffer contained (in mM) 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 0.25 EDTA, 10 D-glucose, and 10 L-taurine, 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 with 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 with the exception of
fura 2-AM (Calbiochem, La Jolla, CA). All agents were prepared as
aqueous solutions except for CPA (DMSO), fura 2 (DMSO), and ryanodine
(95% ethanol).
Data analysis. Responses are reported
as means ± SE and were compared with two-tailed Student's
t-test (paired or unpaired as
appropriate), with P values < 0.05 being considered significant.
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RESULTS |
ACh-induced Ca2+ release.
We first investigated ACh-induced
Ca2+ release. In single cells
studied at 37°C, ACh
(10
4 M) induced a rapid
spikelike elevation in
[Ca2+]i
that reached a peak within 5-10 s after onset of the application, then decayed toward basal levels (Fig.
1A,
Table 1); the initial spikelike elevation and the subsequent "plateau" have previously been shown to represent release of internal
Ca2+ and influx of external
Ca2+, respectively (13, 18, 26).
In cells held under voltage clamp at
60 mV and studied with the
perforated-patch configuration so that intracellular signaling pathways
would remain intact, ACh
(10
4 M) evoked a large
transient inward current that peaked within 5 s after onset of the
application, then reversed completely to basal levels before the
application of ACh had ended (Fig. 1B, Table 1). This membrane current response has been shown previously (8)
to represent activation of
Ca2+-dependent
Cl
channels in response to
the release of internally sequestered Ca2+. Cells that responded to ACh
in this way also shortened to 28 ± 2% of their initial length
(data not shown, but see Ref. 9). Isometric contractile responses were
studied in intact tissues with the standard organ bath technique; under
these experimental conditions, ACh evoked powerful and sustained
contractions (Fig. 1C, Table 1).

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Fig. 1.
ACh releases Ca2+, activates
current, and causes contraction. A: in
a single cell loaded with fura 2, ratio of fluorescence during
excitation at 340 and 380 nm
(F340/F380)
was elevated {indicating a rise in cytosolic concentration of
Ca2+
([Ca2+]i)}
in presence of ACh (10 4 M)
and returned to prestimulation levels when ACh was removed.
B: in another single cell held under
voltage clamp at 60 mV, ACh
(10 4 M) evoked a transient
inward current that decayed to baseline even though ACh continued to be
applied. C: in an intact tissue, ACh
(10 4 M) elicited a
sustained contraction.
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These data indicate that ACh elevates
[Ca2+]i
in the deep cytosol (leading to contraction) as well as within the
subsarcolemmal region (causing activation of
Ca2+-dependent
Cl
channels).
Ca2+ release induced by
antagonism of SR Ca2+ pump.
CPA selectively blocks SERCA activity (17), which normally compensates
for a continuous leakage of
Ca2+ from the SR (1, 19). As a
result, Ca2+-pump inhibition leads
to a net release of internally sequestered Ca2+; these responses have been
described in detail elsewhere (9, 13, 18).
CPA (10 µM) induced an elevation in
[Ca2+]i
that was significantly smaller than the cholinergic response (Table 1);
this elevation peaked
1-2 min after the addition of CPA (Fig.
2A) and
subsided to baseline after 10-15 min. CPA also evoked a
contraction that was significantly smaller (Table 1) and developed more
slowly than the ACh-evoked response (Fig.
2C); after 10-15 min,
CPA-induced tone spontaneously decayed to baseline levels. However, CPA
did not significantly increase membrane current in any of 14 cells held
under voltage clamp at
60 mV (Fig.
2B, Table 1), although subsequent
exposure to ACh evoked no response (Fig.
2B; n = 6 cells), indicating that internally sequestered
Ca2+ had been released. Consistent
with this, CPA had no significant effect on membrane potential in
intact tissues studied with the intracellular microelectrode
electrophysiological technique (Fig. 2D); the mean membrane potential was
61 ± 2 mV before CPA and
64 ± 3 mV after exposure
to CPA (net change of 3 ± 4 mV; n = 8 cells).

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Fig. 2.
Cyclopiazonic acid (CPA) evokes a
Ca2+ transient and contraction but
not a membrane current. A: in a fura
2-loaded cell, CPA (10 5 M)
evoked an elevation in
[Ca2+]i
that reached a peak ~1 min after onset of application; this was
followed by a slow decay toward baseline level.
B: CPA did not evoke any membrane
current in another cell held under voltage clamp at 60 mV,
although response to ACh
(10 4 M; applied 15 min
later) was abolished. C: mechanical
response to CPA in an intact tissue.
D: intracellular microelectrode
recording showing lack of effect of CPA in an intact tissue.
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These observations suggest that, on application of CPA, there is an
elevation in
[Ca2+]i
within the deep cytosol but not in the region immediately beneath the sarcolemma.
CICR. Caffeine (5 mM) elicited a
transient elevation in
[Ca2+]i
(Fig.
3A), the
magnitude of which was not significantly different from that of the
response to ACh (Table 1). In cells studied under voltage-clamp
conditions, caffeine also activated membrane currents, with similar
magnitudes and time courses as those for the response to ACh (Fig.
3B, Table 1). Intact tracheal tissues exhibited little or no contractile response on exposure to caffeine (5 mM; Fig. 3C, Table 1), although the
CPA-evoked contraction was essentially abolished in all tissues treated
with caffeine (Fig. 3C); the mean
CPA response was
0.2 ± 0.1 g/mg in caffeine-treated tissues
compared with 3.8 ± 2.0 g/mg in paired control tissues (n = 5). In contrast, pulmonary venous
tissues studied under identical experimental conditions exhibited
substantial contractions on exposure to caffeine
(n = 11; Fig.
3D), suggesting that the lack of
response in airway tissues is not due merely to an inability of
caffeine to diffuse quickly through an intact tissue.

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Fig. 3.
Caffeine elevates Ca2+
concentration ([Ca2+])
and activates membrane current but without any mechanical response.
A: caffeine (5 mM) evoked a
Ca2+ transient with similar
magnitude and time course as cholinergic response shown in Fig. 1.
B: under voltage-clamp conditions,
caffeine elicited a transient inward membrane current that reached a
peak with same time course as elevation in
[Ca2+]i
but then fell back to baseline even though caffeine continued to be
applied. C: caffeine failed to elicit
any contraction, yet it abolished contractile response to CPA (compare
with Fig. 2C).
D: under identical experimental
conditions, caffeine (5 mM) evoked contraction in a canine pulmonary
venous smooth muscle tissue.
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Ryanodine (3 × 10
5
M) evoked a Ca2+
transient in only three of seven cells, an example of which is given in
Fig. 4A;
the response in this cell had a time course similar to that evoked by
caffeine, with peak activation occurring within 5 s, followed by a
decay to a suprabasal plateau level. There was little or no change in [Ca2+]i
in the remaining four cells challenged with 3 × 10
5 M ryanodine nor in any
of six cells challenged with
10
6 M ryanodine (Fig.
4C). The effects of ryanodine
(10
5 to
10
4 M) on membrane currents
were tested in five cells and found to vary considerably between a
large inward current (>200 pA) shortly after onset of exposure
(n = 2 cells), a small inward current (35 pA) after a 30-s delay (n = 1 cell), or no inward current whatsoever
(n = 2 cells). Similarly, ryanodine
had mixed effects on mechanical tone. There was little or no change in
baseline tone in 16 of 20 tissues exposed to 3 × 10
5 M ryanodine (mean
change of 5.3 ± 4.3%; n = 7; Fig.
4D; see Fig. 7A for an example) nor in any of 22 tissues exposed to 10
6 M
ryanodine (mean change of
7 ± 4%;
n = 7; Fig.
4D). In the remaining four tissues
exposed to 3 × 10
5 M
ryanodine, however, the baseline was increased >50%, albeit after a
delay of up to 10 min (Fig. 4, B and
D).

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Fig. 4.
Responses to ryanodine. Occasionally, ryanodine evoked responses such
as a transient elevation in
F340/F380
in a fura 2-loaded cell (A) or a
transient contraction in a tissue strip
(B; however, note long latency). In
majority of cases, however, ryanodine evoked no response (e.g., see
Fig. 7A).
C: individual changes in
[Ca2+]i
in all cells tested (n = 5).
[ryanodine], Ryanodine concentration.
D: changes in baseline tone in
individual tissues (n = 7).
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Like caffeine, CEP (0.1 and 0.3 mM) produced a large elevation in
[Ca2+]i
(Fig. 5A,
Table 1); however, these responses were sustained in contrast to the
transient responses evoked by caffeine or ryanodine. CEP alone did not
trigger contractions (Fig. 5B, Table
1), even though the tissues were still able to respond to ACh (Fig.
5B), indicating that the contractile
apparatus was still functional.

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Fig. 5.
4-Chloro-3-ethylphenol (CEP) elevates
[Ca2+] but has no
direct effect on mechanical activity.
A: CEP (300 µM) triggered a
sustained elevation in
[Ca2+]i
in a cell loaded with fura 2. B: in an
intact tissue, however, CEP had no effect of its own on mechanical
activity; to verify that contractile apparatus was still functional,
however, we later applied ACh
(10 4 M), finding that it
could still evoke contraction (albeit smaller and more transient than
typical control response shown in Fig.
1C).
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These data suggest that agents that trigger CICR elevate
[Ca2+]i
in the subsarcolemmal space to a greater extent than that in the deep cytosol.
Barrier function of the SR. It has
been proposed that, in vascular SM, the superficial SR forms a barrier
to Ca2+ entry, allowing the SR to
modulate the elevation in
[Ca2+]i
that accompanies agonist stimulation (21). We investigated this
possibility by examining contractions evoked by KCl (which triggers
voltage-dependent Ca2+ influx)
under conditions in which SR buffering capacity was altered.
KCl elicited contractions in a dose-dependent fashion, with a maximal
response of 5.8 ± 0.8 g/mg dry weight (Fig.
6, A and C). After a 30-min exposure to CPA
(10 µM; which had no discernable effect of its own on tone in the
tissue represented in Fig. 6A), the
responses to 15 and 30 mM KCl were significantly enhanced, whereas
those to higher concentrations of KCl were not (Fig. 6, A-C).
In addition, the rate of rise of KCl-induced tone was significantly accelerated (Fig. 6, B and
D). Likewise, ryanodine
(3 × 10
5 M) also
induced a leftward shift in the KCl dose-response curve (Fig.
7,
A-C)
and accelerated the rate of rise of KCl-evoked contractions (Fig. 7,
B and
D) without directly inducing a
mechanical response of its own (Fig.
7A). The lower concentration of
ryanodine (10
6 M) had no
effect on mechanical activity and little or no affect on KCl responses
(n = 7 tissues; data not shown).

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Fig. 6.
CPA enhances KCl contractions. A:
representative tracing showing protocol used to examine effects of CPA
(+CPA; 30 µM) on contractions evoked by KCl added in cumulative
fashion (15-75 mM). B: expanded
traces of contractions (i and
ii) evoked by 15 mM KCl in
A, highlighting increased magnitude
and rate of rise in response after exposure to CPA.
C: mean KCl dose
([KCl])-response relationship from tissues exposed to CPA
or its vehicle (n = 5).
* P < 0.05. D: mean rate of rise of contractile
responses evoked by 15 mM KCl in tissues exposed to CPA or its vehicle
(n = 5) standardized as a percent
change from 1st or control response to KCl. *P < 0.05.
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Fig. 7.
Ryanodine enhances KCl contractions.
A: same protocol illustrated in Fig.
6A was used to examine effects of
ryanodine on KCl-evoked contractions.
B: expanded traces of contractions
(i and
ii) evoked by 15 mM KCl before and
after exposure to ryanodine. C: mean
KCl dose-response curves from tissues exposed to ryanodine or its
vehicle (n = 5).
* P < 0.05. D: mean rate of rise of contractions
evoked by 15 mM KCl in tissues exposed to ryanodine or its vehicle
(n = 5) standardized as a percent
change from 1st or control response to KCl. *P < 0.05.
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In the experiments summarized in Figs. 6 and 7, we often found that, in
a given tissue exposed to vehicle, the response to a second challenge
with KCl 30 min later was somewhat enhanced compared with the first or
control response, although this augmentation was much less than that in
tissues exposed to CPA or ryanodine (as indicated in Figs.
6D and
7D). It may be that during the first exposure to KCl (S1), the SR became more fully loaded and was therefore
less able to buffer the second response (S2). To test this directly, SR
unloading was facilitated by bathing some tissues in nominally
Ca2+-free medium during the 30-min
period separating S1 and S2 (external Ca2+ was reintroduced 1-2 min
before S2); under these conditions, S2 developed more slowly and was
reduced in height compared with S1 (Fig.
8). Overall, the rate of rise and magnitude
of S2 were decreased when S2 was preceded by 30 min in
Ca2+-free medium and increased
when external Ca2+ was present for
the full 30 min before S2 (Fig. 8, B
and C); one-tailed paired
t-test analysis showed these trends to
be significant.

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Fig. 8.
Ca2+ influx saturates sarcoplasmic
reticulum (SR) and compromises buffering capacity of SR.
A: contractile response to KCl (15 mM)
was assayed before (S1) and 30 min after (S2) bath in nominally
Ca2+-free medium; magnitude and
rate of rise in S2 were decreased compared with those of S1.
B and
C: absolute rate of rise and peak
magnitude, respectively, of paired responses to 15 mM KCl obtained
before (i.e., S1) and after (i.e., S2) 30 min in
Ca2+-free
(n = 7) or
Ca2+-containing
(n = 7) medium.
P values were obtained by paired
t-test analysis.
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 |
DISCUSSION |
Multiple Ca2+ pools?
Some tissues possess multiple, functionally distinct
Ca2+ pools expressing
Ins(1,4,5)P3-gated
and/or Ca2+-gated release sites
(1, 5); in some cases, the agonist-sensitive pool is CPA sensitive
(i.e., is refilled by SERCA), whereas the caffeine-sensitive pool is
not (5). We found that caffeine completely occluded the contractile
response to CPA (Fig. 3C), suggesting that the caffeine-sensitive and CPA-sensitive
Ca2+ pools overlap completely. ACh
and caffeine liberate the same intracellular pool of
Ca2+ in this tissue (8, 18), and
CPA can completely deplete the ACh-sensitive pool (9, 13, 18).
Likewise, ryanodine reduced the total cellular
Ca2+ content in canine TSM to the
same extent as carbachol, and carbachol had no additional effect on
tissue Ca2+ content after
pretreatment with ryanodine (4). CPA completely depletes the
caffeine-sensitive Ca2+ pool in
equine TSM (23). Thus airway SM cells do not appear to possess
multiple, heterogeneous Ca2+ pools
as may be the case in other preparations (1, 5). In porcine TSM, the
ACh-sensitive and caffeine-sensitive
Ca2+ pools appear to be linked
(allowing ACh response to be occluded by caffeine and vice versa) but
seem to be refilled by different mechanisms (14).
The fact that the CEP-triggered
Ca2+ response is much larger and
more prolonged than that of ACh or caffeine does not contradict the
claim that all three agents are acting on the same
Ca2+ pool;
Ins(1,4,5)P3- and
caffeine-triggered Ca2+ release
are both subject to feedback regulatory mechanisms (e.g., suppression
when
[Ca2+]i
reaches micromolar levels) (1, 2), whereas CEP is reported to lack such
inhibitory effects on SR
Ca2+-channel function (15).
Multiple cytosolic regions? Although
we found no evidence for multiple functionally distinct
Ca2+ pools in canine airway SM
(see Multiple
Ca2+
pools?), this does not rule out the possibility of
heterogeneity within the cytosol. In fact, we found that ACh, CPA,
caffeine, ryanodine, and CEP all elevated
[Ca2+]i
(indicated directly with fura 2 fluorimetry) but that this elevation
did not seem to be uniform throughout the cell.
For example, CPA evoked contraction but did not activate
Ca2+-dependent
Cl
current in cells studied
under voltage-clamp conditions (Fig. 2B) nor alter membrane potential in
intact tissues (Fig. 2D); likewise,
in equine TSM, CPA did not increase the activity of Ca2+-dependent
K+ current (22), which may be
somewhat more sensitive to changes in
[Ca2+]i
than the Cl
current (12).
These observations suggest that on blockade of SERCA activity in airway
SM, there is a net increase in
[Ca2+]i
in the deep cytosolic space but not in the region around the ion
channels (Fig. 9). This does not
necessarily imply that the spontaneous leak of
Ca2+ from the SR is preferentially
directed toward the deep cytosol; instead, it may be that this leak
from the SR is uniform in all directions but that some
Ca2+ extrusion pathway prevents
subsarcolemmal
[Ca2+]i
from reaching levels that would increase membrane channel activity (Fig. 9). Ca2+ that was released
toward the deep cytosol, on the other hand, would mediate contraction
until it diffused to the periphery, whereupon it would be rapidly
ejected.

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Fig. 9.
Model summarizing data. SR (shaded ellipsoid) divides cytosol into 2 distinct spaces. At rest, there is a tonic leak of
Ca2+ from SR that is compensated
for by SR and plasmalemmal Ca2+
pumps (i). Inhibition of
sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) activity leads
to a net flux of Ca2+ toward deep
cytosol and contraction (ii).
Release of stored Ca2+ through
ryanodine-sensitive channels is preferentially directed toward
subsarcolemmal space (iii), whereas
that through inositol 1,4,5-trisphosphate
(IP3)-gated channels elevates
[Ca2+]i
throughout the cell (v). A fraction
of Ca2+ entering across sarcolemma
is shunted through SR by SERCA (which, in turn, passes it on to
plasmalemmal Ca2+ pump through
Ca2+-induced
Ca2+ release), thereby providing a
means of controlling change in
[Ca2+]i
(iv).
|
|
In marked contrast, those agents that cause CICR (i.e., caffeine,
ryanodine, and CEP) are relatively ineffective in triggering contraction in airway SM (although vascular SM exhibited substantial responses; Fig. 3D) but nonetheless
activate membrane current (Figs. 3-5). We do not feel that the
relative inability of these three agents to trigger contraction is due
to their nonspecific pharmacological actions because each has a unique
profile of nonspecific effects. For example, although caffeine might
cause a slowly developing accumulation of cAMP and thereby antagonize
mechanical activity, ryanodine and CEP do not alter phosphodiesterase
activity. Similarly, although caffeine and ryanodine may have
paradoxical effects on CICR due to adaptation and/or induction of a
subconductance state of the ryanodine receptor, CEP does not (15).
Finally, although high concentrations of CEP can directly antagonize
the contractile apparatus, this is not seen with lower concentrations
of CEP (15) nor with caffeine or ryanodine. Although it might be argued
that the inability of these agents to evoke contraction is due to their inability to increase Ca2+
sensitivity (as is the case for ACh), we would point out that CPA also
does not sensitize the contractile apparatus but did trigger
contractions with only a modest increase in
[Ca2+]i
(much less than that evoked by either ACh or caffeine). The property
that these diverse chemicals have in common, however, is the ability to
trigger CICR and elevate
[Ca2+]i
but to do so in a manner (or a cytosolic region) that does not result
in a significant mechanical response.
ACh, however, evokes membrane current as well as contraction (Fig. 1);
the same is true for histamine (10) and substance P (11). Thus
physiological agonists that trigger
Ins(1,4,5)P3-gated Ca2+ channels seem to release
Ca2+ toward both the sarcolemma
and the deeper cytosol (Fig. 9).
SBB function of the SR. This study
also provides strong evidence that the SR serves as an SBB in airway SM
(Fig. 9) as was first proposed for vascular SM (21). First, CPA induced
a marked and significant leftward shift in the KCl dose-response curve and accelerated the rate of rise in KCl-induced contractions (Fig. 6).
We do not feel that CPA did this by elevating
[Ca2+]i
and thereby displacing the muscle into a steeper region of the
[Ca2+]-tension
relationship because we tested the KCl responses 30 min after the
addition of CPA (Fig. 6), long after the transient elevation in
[Ca2+]i
caused by CPA would have subsided (12).
Similarly, saturating the SR by preexposing the tissues to a high
concentration of KCl 30 min before examining the responses to a second
challenge with KCl mimicked the effects of CPA (i.e., enhanced rate of
rise and magnitude of second response; Fig. 8); the opposite changes
were seen when SR unloading was facilitated by bathing the tissue in
Ca2+-free medium during the
intervening 30-min period (Fig. 8). We interpret these findings to
indicate that voltage-dependent
Ca2+ influx during the first
exposure to KCl increased uptake and saturated the SR, thereby
compromising the ability of the SR to buffer the response to the second
KCl exposure. Furthermore, the data indicate that the SR can discharge
some of its Ca2+ load while at
rest (see Vectorial
Ca2+ release and SR
unloading) to be able to serve as a
Ca2+ buffer.
Vectorial Ca2+ release and
SR unloading.
Generally, there is an ongoing spontaneous release of
Ca2+ from the SR that may account,
in part, for the spontaneous transient outward currents often recorded
from SM preparations (8). SERCA compensates for this spontaneous leak;
as such, agents such as CPA unmask this spontaneous release (Fig.
2A). The nature of this leak pathway
is unclear but may involve the stochastic flickering of the
Ca2+-permeable channels on the SR.
A mechanism has been proposed whereby the SR can unload sequestered
Ca2+ by preferentially releasing
it into the subsarcolemmal space, followed by ejection of that
Ca2+ out of the cell via
Na+/Ca2+
exchange and/or the sarcolemmal
Ca2+-ATPase (21). The data
obtained in this study indicate that this vectorial release involves
CICR because caffeine and CEP have a much greater effect on
subsarcolemmal
[Ca2+]i
than that in the deep cytosol (Figs. 3-5). Previously, Janssen and
Sims (8, 9) have shown that caffeine can trigger a transient contraction when puffed directly onto a single cell but not when introduced more slowly (e.g., by addition to the medium bathing the
isolated cell). These observations are also consistent with the model
proposed in Fig. 9. For example, when caffeine triggers an
instantaneous and massive dumping of the SR into the subsarcolemmal space, there may be some "spillover" of the released
Ca2+ into the deeper cytosol,
perhaps via the same pathway taken by external
Ca2+ during KCl-induced
contraction. When caffeine-triggered
Ca2+ release is more gradual,
however, Ca2+ homeostatic pathways
might be better able to buffer the change in
[Ca2+]i
and thereby circumvent contraction.
Likewise, a high concentration of ryanodine, which is sufficient to
suppress CICR, augmented KCl-evoked contractions (Fig. 7) in the same
fashion as did preventing Ca2+
uptake with CPA (Fig. 6) or saturating the SR by preexposure to high
KCl (Fig. 8). Others (4) have shown that contractile responses to
serotonin in TSM are similarly augmented by ryanodine. We interpret all
of these findings to indicate that the high concentration of ryanodine
prevents SR unloading, ultimately leading to saturation of the SR and
abrogation of its ability to buffer subsequent elevations in
[Ca2+]i.
In other words, it may be that CICR mediates the continuous and
spontaneous release of Ca2+ that
is unmasked by CPA and that this release is directed into the
subsarcolemmal space for subsequent extrusion.
Although
Ins(1,4,5)P3-induced
release seems to be directed toward both the plasmalemma and the deep
cytosol, it is conceivable that these also participate in
preferentially directed transport of
Ca2+ to the sarcolemma. First,
there is likely a gradient of
[Ins(1,4,5)P3] in the cytosol, high at the sarcolemma and low in the deep cytosol, because
Ins(1,4,5)P3 is
generated at the membrane by phospholipase C and metabolized by
cytosolic enzymes such as
Ins(1,4,5)P3
kinase and
Ins(1,4,5)P3
phosphatase as it diffuses to the deeper cytosolic spaces (21). Thus
generation of
Ins(1,4,5)P3 will
likely have a greater and more prolonged effect on those portions of
the SR close to and facing the sarcolemma. Second, phospholipase
C activity and
Ins(1,4,5)P3-gated
Ca2+-channel opening are both
enhanced by Ca2+ (1, 25); as a
result, the higher
[Ca2+]i
in the subsarcolemmal space (due to CICR) would also lead to higher
activity of
Ins(1,4,5)P3-induced
Ca2+ release sites on those
portions of the SR facing that space.
Ca2+ that is directed toward the
sarcolemma in these ways must ultimately be ejected; in vascular SM,
this seems to involve both
Na+/Ca2+
exchange and sarcolemmal
Ca2+-ATPase activities (21).
However, Janssen et al. (13) have previously shown that
Na+/Ca2+
exchange seems to play little or no role in the regulation of [Ca2+]i
in canine airway SM.
Physiological significance. As
proposed by van Breemen et al. (21), the SBB can serve several
functions. First, it is a physiologically regulated barrier to
diffusion of Ca2+ through the
subsarcolemmal space to allow for fine control of the changes in
[Ca2+] and activation
level of the contractile apparatus. In cardiac muscle, a similar
arrangement, the transverse tubules in close apposition to the
ryanodine receptor-studded SR, allows for a marked amplification of the
elevation in
[Ca2+]i
caused by voltage-dependent Ca2+
influx (i.e., CICR) (1). However, Janssen and Sims (12) have previously
shown that this does not occur to a great extent in canine TSM because
Ca2+-dependent
Cl
currents triggered by
voltage-dependent Ca2+ influx were
not altered by depletion of the SR with CPA.
Ultimately, the SBB can allow differential regulation of
[Ca2+]i
in the subsarcolemmal and deep cytosolic spaces; in this way, enzymes
confined to the sarcolemma (for example, cyclooxygenases, phospholipases, protein kinases, and nitric oxide synthases) and ion
channels can be activated without necessarily evoking a change in
tension. In fact, others (24) have shown that
-agonists elevate
[Ca2+]i
in the periphery of bovine TSM cells while simultaneously decreasing [Ca2+]i
in the deeper cytosolic regions and mediate relaxation. This relaxant
response may involve the mechanism proposed by Nelson et al. (16), in
which localized elevations in subsarcolemmal [Ca2+]i,
referred to as Ca2+ sparks,
activate Ca2+-dependent
K+ channels, leading to membrane
hyperpolarization, deactivation of
Ca2+ channels, and relaxation. In
other words, relaxants may act by causing a localized increase in
[Ca2+], which, in
turn, triggers a more globalized decrease in
[Ca2+]i.
Spasmogens, on the other hand, release internally sequestered Ca2+ and activate
Ca2+-dependent
Cl
and nonselective cation
channels, which, in turn, depolarize the membrane and thereby open
voltage-dependent Ca2+ channels,
leading to contraction (8, 9, 11, 13, 18). Clearly then,
agonist-mediated responses involve a complicated interaction between
the SR and the sarcolemma. The mechanism(s) by which an elevation in
[Ca2+] in the
subsarcolemmal space leads to activation of
Cl
channels in the presence
of a spasmogen (8, 9, 11) but to activation of
K+ channels in the presence of a
relaxant (24) needs to be examined.
Experimental implications. These
findings have important ramifications for studies of agonist-induced
responses. First, physiologically important information is lost when
global changes in
[Ca2+]i
are monitored by photometry of whole cells or intact tissues in which
[Ca2+]i
in the subsarcolemmal and deep cytosolic spaces becomes averaged. In
addition, care must be taken when comparing data obtained with an
indicator dye that tends to partition in membranes (e.g., aequorin) with data obtained with dyes that partition more uniformly throughout the cell. These data also underscore the need for caution when using
contractions and membrane currents as indexes of global [Ca2+]i.
The differential regulation of
[Ca2+]i
in the subsarcolemmal and deep cytosolic spaces may also account, in
part, for the frequently reported discrepancies between myosin light
chain phosphorylation and changes in
[Ca2+]i
in SM (19). Finally, this confirmation of the SBB hypothesis in airway
SM is of utmost importance with respect to the physiological and
pathophysiological changes that take place at or near the membrane,
including those that involve second messenger signaling pathways that
are Ca2+ dependent, such as
phospholipases A2 and C, protein
kinase C, cyclooxygenases, nitric oxide synthases, and caveolae.
Summary and conclusion. We found that
agents that induce CICR directly (e.g., caffeine, ryanodine, and CEP)
increase subsarcolemmal [Ca2+]i
and membrane current activity but are much less effective in elevating
[Ca2+]i
in the deeper cytosol and tone, suggesting that CICR is preferentially directed toward the sarcolemma (Fig. 9). CPA, on the other hand, has
the opposite effects: transient elevation of
[Ca2+]i
in the deep cytosol, contraction, and augmentation of KCl-evoked responses but not of membrane currents (Fig. 9). Cholinergic
stimulation elevates
[Ca2+]i
in both cytoplasmic regions and thereby triggers membrane currents as
well as contraction (Fig. 9). Thus the SR in canine TSM forms an SBB
and allows for a complex regulation of
[Ca2+] (Fig. 9).
 |
ACKNOWLEDGEMENTS |
We acknowledge the technical assistance of M. Ostrowski.
 |
FOOTNOTES |
These studies were supported by a grant from the Medical Research
Council of Canada as well as Career Awards from the Pharmaceutical Manufacturer's Association of Canada (Health Research Foundation) and
the Medical Research Council of Canada (to L. J. Janssen).
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. Janssen,
Dept. of Medicine, HSC-3U1, McMaster Univ., 1200 Main St. West,
Hamilton, Ontario, Canada L8N 3Z5 (E-mail:
janssenl{at}fhs.csu.mcmaster.ca).
Received 8 October 1998; accepted in final form 29 January 1999.
 |
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