Spatial and temporal aspects of calcium sparks in porcine
tracheal smooth muscle cells
Christina M.
Pabelick1,
Y. S.
Prakash1,
Mathur S.
Kannan2, and
Gary C.
Sieck1,3
Departments of 1 Anesthesiology
and 3 Physiology and
Biophysics, Mayo Foundation, Rochester 55905; and
2 Departments of Veterinary
PathoBiology and Pediatrics, University of Minnesota, St. Paul,
Minnesota 55108
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ABSTRACT |
Spontaneous,
localized intracellular Ca2+
concentration
([Ca2+]i)
transients (Ca2+ sparks) in
skeletal, cardiac, and smooth muscle cells are thought to represent
Ca2+ release through
ryanodine-receptor (RyR) channels. In porcine tracheal smooth muscle
(TSM) cells, ACh induces propagating
[Ca2+]i
oscillations that also represent
Ca2+ release through RyR channels.
We used real-time confocal imaging to examine the spatial and temporal
relationships of Ca2+ sparks to
propagating
[Ca2+]i
oscillations in TSM cells. Ca2+
sparks within an intracellular region displayed different spatial Ca2+ distributions with every
occurrence. The amplitudes of Ca2+
sparks within a region were approximately integer multiples of the
smallest response. However, across different regions, the attributes of
Ca2+ sparks varied considerably.
Individual sparks were often grouped together and coupled across
adjacent regions. Fusion of individual sparks produced large local
elevations in
[Ca2+]i
that occasionally triggered a propagating
[Ca2+]i
wave. The incidence of sparks was increased by ryanodine and caffeine
but was unaffected by removal of extracellular
Ca2+. Exposure to ACh triggered
repetitive, propagating
[Ca2+]i
oscillations that always originated from foci with a high spark incidence. The
[Ca2+]i
oscillations disappeared with the removal of ACh, and
Ca2+ sparks reappeared. We
conclude that agonist-induced
[Ca2+]i
oscillations represent a spatial and temporal integration of local
Ca2+-release events through RyR
channels in TSM cells.
second messenger; sarcoplasmic reticulum; ryanodine
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INTRODUCTION |
PROPAGATING OSCILLATIONS of intracellular
Ca2+ concentration
([Ca2+]i)
have been reported in response to acetylcholine (ACh) in porcine (6,
12, 17, 18, 23) and guinea pig (24) tracheal smooth muscle
(TSM) cells. Such ACh-induced
[Ca2+]i
oscillations in porcine TSM cells arise from repetitive release of
sarcoplasmic reticulum (SR) Ca2+
through ryanodine-receptor (RyR) channels (6, 23). The propagation of
[Ca2+]i
oscillations suggests that agonist stimulation triggers localized Ca2+ release that further
stimulates release from adjacent regions, perhaps via a
Ca2+-induced
Ca2+ release (CICR) mechanism (2,
6, 23). Another important observation is that the propagation of
[Ca2+]i
oscillations is initiated within a limited region of the TSM cell and
propagation occurs in one direction (6, 23). The initiation of
[Ca2+]i
oscillations in a limited region of the cell may reflect localized differences in RyR-channel distribution (11) and/or sensitivity to CICR.
Spontaneous, localized Ca2+
transients (Ca2+ sparks) have been
observed in skeletal muscle fibers (8, 10, 25), cardiac myocytes (5,
14, 16), and vascular smooth muscle cells (15; also reviewed in Refs.
4, 21). Ca2+ sparks are thought to
represent unitary Ca2+ release
through RyR channels (5, 8, 25). Accordingly, the amplitude of
Ca2+ sparks likely reflects the
number of RyR channels that are more or less synchronous in their
Ca2+ release and the frequency
reflects channel kinetics. In a recent study, Sieck et al.
(23) demonstrated the existence of
Ca2+ sparks in porcine TSM cells,
which also most likely represents SR
Ca2+ release through RyR channels.
Because propagating
[Ca2+]i
oscillations in TSM cells also represent SR
Ca2+ release through RyR channels,
it is likely that there is a spatial and temporal relationship between
the pattern of spontaneous Ca2+
sparks and agonist-induced
[Ca2+]i oscillations.
The temporal aspects of Ca2+
sparks have been characterized in several previous studies (8, 10,
14-16, 25) with line-scan confocal microscopy, with a temporal
resolution of ~2 ms. These studies have provided important
information on the amplitude and incidence of sparks. However, the
spatial resolution of
[Ca2+]i
measurements with line-scan confocal microscopy is relatively poor
(~1 µm width with a ×60, 1.4-numerical aperture oil-immersion objective). Furthermore, evaluation of the temporal aspects of sparks
and propagating
[Ca2+]i
oscillations necessitates measurements from different parts of the
cell. Therefore, line-scan confocal microscopy is inadequate to
evaluate the spatiotemporal relationships between spontaneous Ca2+ sparks and agonist-induced
[Ca2+]i
oscillations. In the present study, we used rapid real-time two-dimensional confocal imaging of
Ca2+ sparks and
[Ca2+]i
oscillations in TSM cells to determine the spatiotemporal relationships between these two phenomena.
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METHODS |
Cell preparation. Porcine tracheae
were obtained from a local abattoir, and TSM cells were isolated with
previously described techniques (7). The dissociated cells were plated
on collagen-coated glass coverslips and incubated for 1-2 h in a
5% CO2 incubation chamber at
37°C. Based on trypan blue exclusion, cell viability of >90% was
confirmed. A sample of cells dissociated from each animal was also
processed with an anti-smooth muscle myosin antibody (Sigma
Immunochemicals, St. Louis, MO) to estimate the relative proportion of
smooth muscle myocytes (immunoreactive) and fibroblasts, which was
found to be ~50:1.
Cells were incubated in 5 µM fluo 3-AM (Molecular Probes, Eugene, OR)
for 30-45 min at 37°C. The cells were then washed in Hank's
balanced salt solution (HBSS), and the coverslip was mounted on an open
slide chamber (RC-25F, Warner Instruments, Hamden, CT). The tissue
chamber was perfused at 2-3 ml/min at room temperature.
Real-time confocal imaging. Detailed
descriptions of the real-time two-dimensional confocal-imaging
technique have been recently published (18). Briefly, an Odyssey XL
real-time confocal system (Noran Instruments, Middleton, WI) equipped
with an Ar-Kr laser and mounted on a Nikon Diaphot microscope was used
to visualize fluo 3-loaded TSM cells. Image size was set to 640 × 480 pixels, and the pixel area for a Nikon ×40, 1.3-numerical
aperture oil-immersion objective lens was calibrated with a stage
micrometer (0.06 µm2/pixel). A
fixed combination of laser intensity and photomultiplier gain was set
to ensure that pixel intensities within regions of interest (ROIs)
ranged between 25 and 255 gray levels. To calibrate [Ca2+]i,
cells were exposed to 10 µM A-23187, a
Ca2+ ionophore, at varying levels
of extracellular Ca2+ ranging from
0 (HBSS with EGTA) to 10 µM, and fluorescence intensities were
measured. The relationship between fluorescence intensity and
[Ca2+]i
was found to be linear from 10 nM to 10 µM.
Images of fluo 3-loaded TSM cells were acquired at sampling frequencies
ranging between 15 and 480 frames/s to evaluate the extent of frequency
aliasing of the dynamic
[Ca2+]i
response. We found that, in TSM cells, acquisition rates of 120 frames/s for Ca2+ sparks and 30 frames/s for
[Ca2+]i
oscillations were sufficient to measure various parameters with
adequate resolution and without frequency aliasing.
ROIs with a fixed dimension of 5 × 5 pixels (1.5 µm2) were defined within the
boundaries of individual cells. The optical section thickness for
confocal measurements was set to 1 µm by controlling the slit size on
the Odyssey system. This corresponded to the optimal sectioning
capability of the ×40 lens as determined in a previous study
(19). The focus was adjusted such that measurements were obtained in a
plane through the maximum thickness of a cell as far as possible.
Overall,
[Ca2+]i
measurements were obtained from a volume of 1.5 µm3. Each ROI represented
0.05-0.10% of the volume of a TSM cell. To determine
intracellular heterogeneity in the dynamic
[Ca2+]i
regulation, up to 8 ROIs were defined. The distances between these ROIs
within a cell were measured with the length calibration for the
×40 lens. On-line
[Ca2+]i
measurements were made with the Odyssey system for acquisition rates of
30 frames/s, whereas
[Ca2+]i
measurements at higher acquisition rates were made post hoc from
acquired images with an image-processing software package [ANALYZE, Mayo Biomedical Imaging Resource (20)].
In experiments where the spatial distribution of
Ca2+ during sparks was determined,
a hardware zoom of ×3 or ×4 was used such that the scanning
dimensions were decreased by the same factor, but the image size was
maintained. Obviously, this was likely to result in greater dye
bleaching. Therefore, acquisitions were limited to ~1 min, at which
time the extent of dye bleaching was estimated to be <5%. Images
were acquired at 120 frames/s and processed to delineate
Ca2+ distribution. The centroid of
the distribution was then calculated as an index of the "origin"
of the spark within the confocal plane with ANALYZE.
Characterization of
[Ca2+]i
transients.
The amplitude of Ca2+ sparks and
oscillations was defined as the difference between the peak of the
transient and the basal level of
[Ca2+]i.
Rise time was normalized for amplitude, whereas fall time was
normalized for the difference between the peak of the response and the
basal
[Ca2+]i
level at the end of the transient. The incidence of
Ca2+ sparks was measured over
1-min intervals. The frequency of the [Ca2+]i
oscillations was measured as the inverse of the peak-to-peak interval
between oscillations.
Effect of
Ca2+ influx on
Ca2+ sparks.
To determine whether Ca2+ sparks
are dependent on Ca2+ influx, TSM
cells were exposed to nominally
Ca2+-free HBSS, and changes in the
incidence and amplitude of Ca2+
sparks were determined over a 15-min period.
Ca2+ at 2.5 mM was
then reintroduced into the extracellular medium, and the incidence and
amplitude of sparks were reevaluated. As mentioned in
Real-time confocal
imaging, a potential confounding factor in
these experiments was dye bleaching due to continued laser exposure.
Therefore, images were acquired at 1-min intervals for 15-30 s.
Effect of ryanodine and caffeine on
Ca2+ sparks.
To determine whether Ca2+ sparks
arise from SR Ca2+ release through
RyR channels, TSM cells were exposed to 0.1, 1, and 10 µM ryanodine,
and the changes in various parameters of
Ca2+ sparks were evaluated. In a
second set of experiments, TSM cells were exposed to 1, 10, and 50 µM
caffeine, and the changes in various parameters of
Ca2+ sparks were evaluated.
ACh-induced
[Ca2+]i
oscillations.
After evaluation of Ca2+ sparks,
TSM cells were exposed to 1 µM ACh to induce
[Ca2+]i
oscillations. A previous study (22) in porcine TSM has shown that the
ACh concentration at which the response is 50% of maximum for the
[Ca2+]i
response is ~1 µM. In a second set of experiments, after evaluation of Ca2+ sparks and
[Ca2+]i
oscillations, TSM cells were washed in HBSS for 15 min, and the
incidence and amplitude of Ca2+
sparks were reevaluated.
Statistical analysis. At least five
ROIs were outlined in each TSM cell for the evaluation of intracellular
heterogeneity in Ca2+ sparks and
[Ca2+]i
oscillations. Therefore, only one to two cells could be used from each
coverslip. Overall, a total of 72 cells was used in this study. The
number of cells for each protocol is indicated in
RESULTS. Significance for single
parameters was tested at a 0.05 level with unpaired
t-tests and correlations. The
normality of the distribution of spark amplitudes was evaluated with a
Kolmogorov-Smirnoff test. Values are expressed as means ± SE.
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RESULTS |
Ca2+ sparks
in TSM cells.
Ca2+ sparks were observed in 35 TSM cells that were analyzed shortly (<2 h) after being plated on
coverslips and in 22 cells >15 h after being plated. These cells
represented 75-80% of the total number of cells studied.
The area of the cell occupied by a spark could not be easily defined in
terms of geometric patterns such as a circle or ellipse. The overall width of a spark ranged from 1.2 to 1.5 µm (Fig.
1). Based on a 1-µm optical section
thickness, individual sparks were thus localized to <1% of the cell
volume (0.15 ± 0.02%).

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Fig. 1.
Confocal image of a porcine tracheal smooth muscle (TSM) cell
(A) loaded with fluorescent
Ca2+ indicator fluo 3, displaying
a region of Ca2+ sparks (box).
Region in box has been magnified in B
to display spatial and temporal aspects of
Ca2+ sparks (red and yellow). Nos.
in boxes, time in ms. Note short duration of sparks and quiescent
periods between subsequent sparks. Bar, 0.5 µm.
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The time series of confocal images was imported into ANALYZE and
defined as a three-dimensional volume where the
X- and
Y-axes were taken to be in the plane
of the confocal image and time was the third axis as illustrated in
Fig. 2. The intensity of each voxel then
represented the
[Ca2+]i
level at one particular XY location at
a particular time. This "three-dimensional" representation
allowed visualization and analysis of
Ca2+ sparks along defined planes,
e.g., the X vs. time or
Y vs. time planes, with the pixel
intensity still representing the
[Ca2+]i
level. In our analysis, we visualized the spark data in the Y vs. time plane as illustrated in
Fig. 2, top. Any image in this view
represented the sparks at a particular
X. Therefore, by drawing a line across
such an image, the time profile of
Ca2+ sparks at a particular
XY could be obtained as illustrated in Fig. 2, bottom. Such a line profile of
pixel intensities was thus qualitatively similar to a line scan
reported in previous studies (8, 10, 14-16, 25). However, in
contrast to these previous studies, the advantage of the present
technique was that such line profiles could be simultaneously obtained
from different X and
Y regions within the sparking area by
selecting the image and location of the line profile.

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Fig. 2.
Image manipulation of Ca2+ spark
data. XY images obtained from confocal
microscope were stacked to yield a time series. Set of images was then
viewed in Y vs. time plane at any
given X. Line profiles of
Ca2+ sparks in this new
orientation provided data on time-dependent variation in spatial
aspects and intensity of spark. However, unlike line scans obtained in
other studies, line profiles could be simultaneously obtained from
several regions within spark. Note variation in amplitude and apparent
rate of occurrence in different line profiles (lines
1 and 2), suggesting
that area of a spark is not fixed.
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When intensities were measured with the line profile described above,
there was considerable variation in the apparent amplitude of the same
spark at different line locations (Fig. 2). In some cases, as the line
profile was moved, the apparent rate of occurrence was also changed,
suggesting that the area occupied by the spark (at least along the
Y direction shown in Fig. 2), varied
between subsequent events, even within a region of sparking.
In >80% of the cells examined, multiple foci for
Ca2+ sparks (2-5/cell) were
present (Fig. 3). The
incidence of individual Ca2+
sparks was coupled in 65% of adjacent ROIs that were separated by <5
µm (r2 = 0.93 ± 0.04; P < 0.05). However, the incidence of
sparks was not correlated for more distant ROIs.

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Fig. 3.
Ca2+ sparks in different regions
of interest (ROIs) of a single porcine TSM cell. Incidence of sparks in
adjacent regions was correlated but was not correlated across larger
distances. There was considerable interregional variation in spark
amplitude and occurrence.
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The incidence of Ca2+ sparks
ranged from 3 to 28/min within an ROI (Table
1) but displayed a burst
pattern in 45% of the cells studied (Fig.
4). In 23% of cells displaying such burst
patterns, individual Ca2+ sparks
often summated into larger
[Ca2+]i
elevations (Fig. 5) that sometimes
initiated a single propagating wave throughout the cell. No consistent
periodicity in this summation pattern was observed.

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Fig. 4.
Fusion of Ca2+ sparks.
Ca2+ sparks often appeared in
bursts of 2-6 sparks followed by periods of quiescence. On some
occasions, sparks summated into a large
[Ca2+]i
response followed by a return to baseline and a continuation of spark
activity.
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Fig. 5.
Summation of Ca2+ sparks within an
ROI of a TSM cell. Individual sparks (red) occurring with <30-ms
delay resulted in a summation that yielded a greater elevation in
intracellular Ca2+ concentration
([Ca2+]i;
yellow). Nos. in boxes, time in ms. On some occasions, this summation
triggered a propagating
[Ca2+]i
wave (data not shown). Bar, 0.5 µm.
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The burst pattern of Ca2+ sparks
led us to evaluate the distribution of spark amplitudes within an ROI.
In 10 TSM cells, histogram analysis of the range of spark amplitudes
within an ROI revealed a multimodal distribution, typically with 3 peaks. These peaks were approximately two (110-120 nM)- and three
(190-200 nM)-fold multiples of the first peak (50-60 nM; Fig.
6). The distribution failed the
Kolmogorov-Smirnoff test (P = 0.038), indicating a nonnormal, multimodal distribution. The amplitude of the first peak
varied between ROIs and across cells (see below).

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Fig. 6.
Range of Ca2+ spark amplitudes.
Histogram analysis of range of spark amplitudes within an ROI
(summarized across 10 cells) revealed a multimodal distribution,
typically with 3 peaks corresponding to approximate multiples of 1st
peak.
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The analysis of spark amplitude suggested that sparks represented a
form of "quantal" Ca2+
release from RyR channels, with the first peak of the amplitude distribution representing the quantum. Accordingly, comparisons between
ROIs and cells were restricted to the first peak of the amplitude
distribution. This amplitude peak of individual
Ca2+ sparks ranged from 30 to 110 nM (Table 1). Within a cell, the amplitude of
Ca2+ sparks displayed
significantly greater variance across ROIs than for an individual ROI
(P < 0.05; Table
2). A similar heterogeneity in the
amplitude of Ca2+ sparks was
observed across TSM cells when data from different ROIs within a cell
were pooled and compared across cells
(P < 0.05; Table 2).
The rise time of Ca2+ sparks in
TSM cells ranged from 30 to 90 ms (Table 1). Within an ROI, the rise
time of individual Ca2+ sparks
(normalized for spark amplitude) was relatively constant (Table 2), but
again, across different regions of
Ca2+ sparking within a cell, there
was considerably greater variability (P < 0.05; Table 2). A similar
heterogeneity in the rise time of
Ca2+ sparks was observed across
TSM cells (P < 0.05; Table 2). The fall time of Ca2+ sparks ranged
from 60 to 350 ms (Table 1). The fall time of individual
Ca2+ sparks was relatively
constant (Table 2), but across different regions of
Ca2+ sparking within a cell, there
was greater variability in fall time
(P < 0.05; Table 2). Similarly, the
fall time of Ca2+ sparks was more
variable across TSM cells (P < 0.05;
Table 2).
Effect of
Ca2+ influx on
Ca2+ sparks.
In 25 TSM cells, the incidence of
Ca2+ sparks was initially
unaffected by inhibition of Ca2+
influx either by exposing cells to nominally free extracellular Ca2+ (Fig.
7) or by blocking
Ca2+ influx through
voltage-dependent Ca2+ channels
with 100 nM nifedipine. However, after ~5 min,
Ca2+ sparks disappeared in the
absence of Ca2+ influx, possibly
as a result of a depletion of SR
Ca2+ stores.

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Fig. 7.
Role of extracellular and intracellular
Ca2+ on
Ca2+ sparks.
Ca2+ sparks persisted for several
minutes after removal of extracellular
Ca2+, suggesting that sparks arise
from intracellular stores (A).
However, with continued exposure to
Ca2+-free extracellular medium,
sparks eventually disappeared after 10-15 min. During ongoing
spark activity in presence of extracellular
Ca2+, exposure to low
concentrations of ryanodine resulted in increased spark activity
(B). High ryanodine concentrations
inhibited spark activity (data not shown).
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Effect of ryanodine and caffeine on
Ca2+ sparks.
In response to 0.1 µM ryanodine, the incidence of
Ca2+ sparks in 20 TSM cells
increased (443 ± 88% of control value;
P < 0.05; Fig.
8) and so did the amplitude (529 ± 67%
of control value; P < 0.05). The
fusion of individual Ca2+ sparks
precluded a more rigorous analysis. In contrast, exposure to 1 µM
ryanodine did not change either the frequency or amplitude (94 ± 26 and 101 ± 66%, respectively, of control values). However, exposure
to 10 µM ryanodine induced an
[Ca2+]i
transient and inhibited spark activity. Exposure to 1, 10, and 50 µM
caffeine all increased the incidence of
Ca2+ sparks in 15 TSM cells (135 ± 5, 288 ± 23, and 370 ± 26%, respectively, of control
values; P < 0.05). The amplitude of
the sparks was significantly changed with 50 µM caffeine (385% of
control value; P < 0.05).

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Fig. 8.
Effect of ryanodine on Ca2+
sparks. In response to 0.1 µM ryanodine, incidence of
Ca2+ sparks as well as amplitude
increased compared with control values. In contrast, exposure to 1 µM
ryanodine did not change either frequency or amplitude (compared with
control value). * Significant difference from control value,
P < 0.05.
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ACh-induced
[Ca2+]i
oscillations.
The spatial and temporal patterns of ACh-induced
[Ca2+]i
oscillations were studied in 35 TSM cells where multiple foci of
Ca2+ sparks were observed. Within
each oscillation, the first detectable change in
[Ca2+]i
always occurred at a site where spontaneous
Ca2+ sparks had been previously
recorded. In >80% of these cells, the intracellular site that
previously displayed the highest incidence of
Ca2+ sparks was also the origin of
the first significant change in [Ca2+]i
above basal levels on ACh exposure (Fig.
9). After the rise in
[Ca2+]i
at this initiation site, the oscillation spread toward other parts of
the cell. In some cases, two
[Ca2+]i
waves were initiated, typically from the long ends of the cell, and
propagated independently toward the center of the cell. In these
instances, the origins of the two
[Ca2+]i
waves were also sites where a high incidence of
Ca2+ sparks had been previously
observed.

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Fig. 9.
Relationship between Ca2+ sparks
and ACh-induced
[Ca2+]i
oscillations. In a cell with ongoing
Ca2+ sparks, exposure to ACh
resulted in initiation of
[Ca2+]i
oscillations. Site of initiation of
[Ca2+]i
oscillations was intracellular focus with highest spark incidence.
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In 10 of the 35 TSM cells, ACh was washed out for 15 min with HBSS.
After the wash, no
[Ca2+]i
oscillations were observed. However, in all these cells,
Ca2+ sparks reappeared in all the
sites where they had been observed before ACh exposure.
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DISCUSSION |
Real-time confocal imaging was used to examine the spatial and temporal
relationships of Ca2+ sparks to
propagating
[Ca2+]i
oscillations in porcine TSM cells.
Ca2+ sparks displayed relatively
constant rise times and amplitudes within a focus of a TSM cell, but
across different foci, these attributes displayed considerable
variability. Individual sparks were often grouped together and coupled
across adjacent regions. Fusion of individual sparks produced large
local elevations in [Ca2+]i
that triggered a propagating
[Ca2+]i
wave in an unstimulated cell. The incidence of sparks was increased by
both ryanodine and caffeine but was largely unaffected by removal of
extracellular Ca2+. These data
suggest that Ca2+ sparks in TSM
cells represent SR Ca2+ release
through RyR channels. Exposure to ACh triggered repetitive, propagating
[Ca2+]i
oscillations that originated from foci with a high incidence of
Ca2+ sparks. The
[Ca2+]i
oscillations disappeared with removal of ACh, but
Ca2+ sparks reappeared. These
results indicate that agonist-induced [Ca2+]i
oscillations represent a spatiotemporal integration of local Ca2+-release events through RyR
channels in TSM cells.
In the present study, we used rapid confocal imaging of
Ca2+ sparks in TSM cells. Although
the temporal resolution of this imaging technique was comparable to
that used with line scans in previous studies (8, 10, 14-16, 25),
a distinct advantage was that in addition to the temporal aspect,
two-dimensional information on
Ca2+ distribution within the area
of the spark could also be obtained. With this feature, we found that
the area occupied by a spark varied from event to event within a
sparking region. This observation would be entirely missed by a line
scan where the shifting of the spark area would only appear as a change
in the apparent amplitude of the spark. These findings are also of
significance with regard to the proposed mechanisms underlying
Ca2+ sparks. Previous observations
in skeletal muscle fibers, cardiac myocytes, and vascular smooth muscle
cells (1, 5, 8, 15, 25) led to suggestions that
Ca2+ sparks represent elemental or
unitary Ca2+ release through RyR
channels (5, 13, 15, 25). The fact that both ryanodine and caffeine
modulated the incidence and amplitude of
Ca2+ transients in TSM cells also
indicates the involvement of RyR channels. The concept of
Ca2+ sparks representing elemental
Ca2+ release was also generally
supported by the relatively constant amplitude and rise time of
individual Ca2+ sparks in porcine
TSM cells. More importantly, the modes of the spark amplitude
distribution were found to be multiples of a basic amplitude,
resembling quantal neurotransmitter release at neuromuscular junctions.
Accordingly, these data suggest that individual sparks represent
all-or-none SR Ca2+ release that
can occasionally fuse into larger events.
If Ca2+ sparks are elemental units
of
[Ca2+]i
regulation, it may be expected that the spatiotemporal patterns of
Ca2+ sparks in different tissues
reflect the kinetics of Ca2+
regulatory mechanisms such as release, reuptake, and passive diffusion.
In a previous study on vascular smooth muscle cells, Nelson et al. (15)
reported Ca2+ sparks that
displayed rise and fall times of the same order of magnitude as those
observed in the present study on TSM cells. Cannell et al. (3) reported
that Ca2+ sparks in cardiac
myocytes lasted for 100-200 ms. In a separate study, Prakash et
al. (19) recently observed Ca2+
sparks in rat cardiac myocytes, where the amplitude of sparks was
comparable to that observed in TSM cells, but the rise and fall times
were considerably shorter, with 10- to 35-ms rise times and 60- to
100-ms fall times. These data suggest that the differences in temporal
aspects of Ca2+ sparks between
tissues most likely reflect the kinetics of the release and reuptake
mechanisms. In this regard, the shifting in the area occupied by the
spark may reflect heterogeneities in the kinetics of RyR channels
within a group of channels that contribute to a spark.
Multiple foci for Ca2+ sparks were
frequently observed in individual TSM cells. Adjacent regions of
Ca2+ sparking were often coupled,
whereas more distant regions were not. This observation suggests that
localized SR Ca2+ release may
induce Ca2+ release from
surrounding regions, perhaps via CICR. Indeed, we frequently observed
groups of three to four individual
Ca2+ sparks separated by periods
of quiescence. These events may represent localized facilitation of
sparking from different groups of RyR channels. The spatial limitation
of foci may be due to SR Ca2+
reuptake acting as a barrier to the initiation of a propagating [Ca2+]i
oscillation from the region of sparking (5). In other cases, we
observed larger
[Ca2+]i
responses, with individual sparks superimposed on both the rising and
falling phases of the larger response. Similar events have been
observed previously in cardiac myocytes (5). These events may represent
facilitation of Ca2+ release from
a larger SR store, most likely via CICR.
In the present study, we observed that, in many TSM cells, regions of
increased incidence of Ca2+ sparks
corresponded with the site of initiation of propagating ACh-induced
[Ca2+]i
oscillations. These regions also displayed spontaneous summation of
individual sparks, leading to larger
[Ca2+]i
transients. In most cases, the amplitudes of the spontaneous, summated
responses were comparable, if not identical, to those of ACh-induced
responses. Therefore, our results suggest that Ca2+ sparks in TSM cells may arise
from "trigger" sites that reflect areas of high RyR-channel
density, as suggested by Lesh et al. (11), in vascular smooth muscle
and/or sensitivity and act as "primers" for agonist stimulation.
However, other potential mechanisms cannot be ruled out. For example,
in previous studies (6, 23), we used a
-escin-skinned
TSM cell preparation to demonstrate that ACh-induced
[Ca2+]i
oscillations require inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3]-induced SR Ca2+ release at least for
initiation, although the steady-state phase is not affected by
inhibitors such as heparin. Accordingly, the site of initiation may
also reflect higher density and/or sensitivity of muscarinic receptors
and
Ins(1,4,5)P3-receptor
channels in the SR. Furthermore, the distribution of different
receptors and channels is likely to play a role in the interaction
between
Ins(1,4,5)P3-induced SR Ca2+ release and oscillations
through RyR channels. These issues need to be examined in future studies.
A study (15) in vascular smooth muscle has suggested that
Ca2+ sparks may be important in
the control of resting membrane potential via
Ca2+-activated
K+ channels. In this scenario,
increased spark incidence would actually lead to a relaxation of smooth
muscle as a result of membrane hyperpolarization. Accordingly, it may
be expected that regions with a higher incidence of
Ca2+ sparks would play a greater
role in hyperpolarization of the membrane, making the cell less
responsive to agonist stimulation. Whether sparks locally regulate
membrane potential in TSM cells, as has been shown in vascular smooth
muscle, is not known. In the present study, we did not attempt to
determine the location of Ca2+
sparks relative to the plasma membrane because the optical section thickness of 1 µm and the XY
resolution of 0.25 µm were suboptimal. Nonetheless, a previous study
(9) in airway smooth muscle has indicated that
Ca2+-activated
K+ channels do not significantly
contribute to membrane potential. In this regard, the functional
significance of Ca2+ sparks may
vary between airway and vascular smooth muscle.
In conclusion, the results of the present study support a hypothesis
that Ca2+ sparks represent
Ca2+-release events from finite SR
Ca2+ pools via RyR channels.
Ca2+ sparks may arise from regions
with RyR channels of high sensitivity or high density, which also serve
as initiation sites for agonist-induced [Ca2+]i oscillations.
 |
ACKNOWLEDGEMENTS |
We thank Thomas Keller for technical assistance in cell preparation
and Vishal Verma for data analysis.
 |
FOOTNOTES |
This research was supported by a fellowship from Abbott Laboratories
(to C. M. Pabelick); by National Heart, Lung, and Blood Institute Grant
HL-057498 (to M. S. Kannan); by National Institute of General Medical
Sciences Grants GM-56686 (to G. C. Sieck) and GM-57816 (to Y. S. Prakash); and by the Mayo Foundation.
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: G. C. Sieck,
Anesthesia Research, Mayo Clinic, Rochester, MN 55905 (E-mail:
sieck.gary{at}mayo.edu).
Received 31 December 1998; accepted in final form 24 June 1999.
 |
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