Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637; Department of Pediatrics, Mayo Clinic, Rochester, Minnesota 55905; and Department of Anesthesiology and Reanimation, Hacettepe University Medical School, Ankara, Turkey
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously demonstrated that after several days of
serum deprivation about one-sixth of confluent cultured canine tracheal myocytes acquire an elongated, structurally and functionally
contractile phenotype. These myocytes demonstrated significant
shortening on ACh exposure. To evaluate the mechanism by which these
myocytes acquire responsiveness to ACh, we assessed
receptor-Ca2+ coupling using fura 2-AM fluorescence imaging
and muscarinic receptor expression using Western analysis. Cells were
grown to confluence in 10% fetal bovine serum and then maintained
for 7-13 days in serum-free medium. A fraction of
serum-deprived cells exhibited reproducible intracellular
Ca2+ mobilization in response to ACh that was uniformly
absent from airway myocytes before serum deprivation. The
Ca2+ response to 104 M
ACh was ablated by inositol 1,4,5-trisphosphate (IP3)
receptor blockade using 10
6 M
xestospongin C but not by removal of extracellular Ca2+.
Also, 10
7 M atropine or
10
7 M
4-diphenylacetoxy-N-methylpiperidine completely blocked the response to ACh, but intracellular Ca2+ mobilization was
not ablated by 10
6 M pirenzepine
or 10
6 M methoctramine. In
contrast, 10
5 M bradykinin (BK) was
without effect in these ACh-responsive myocytes. Interestingly,
myocytes that did not respond to ACh demonstrated robust increases in
intracellular Ca2+ on exposure to
10
5 M BK that were blocked by
removal of extracellular Ca2+ and were only modestly
affected by IP3 receptor blockade. Serum deprivation
increased the abundance of M3 receptor protein and of
BK2 receptor protein by two- to threefold in whole cell
lysates within 2 days of serum deprivation, whereas M2
receptor protein fell by >75%. An increase in M3
receptor abundance and restoration of M3 receptor-mediated
Ca2+ mobilization occur concomitant with reacquisition of a
contractile phenotype during prolonged serum deprivation. These data
demonstrate plasticity in muscarinic surface receptor expression and
function in a subpopulation of airway myocytes that show mutually
exclusive physiological and pharmacological diversity with other cells
in the same culture.
canine tracheal smooth muscle; fura 2-acetoxymethyl ester; acetylcholine; bradykinin; airway remodeling
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ABOUT ONE-SIXTH OF CANINE TRACHEAL smooth muscle cells
in culture acquire a structurally and functionally contractile
phenotype after prolonged (>7-day) serum deprivation as recently
reported by us (2) and by Ma et al. (11). These
contractile myocytes are distinguished by their elongated, wormlike
morphology; their abundant cables of myofilaments replete with smooth
muscle -actin, smooth muscle myosin, and SM22; their expression of
immunoreactive surface muscarinic M3 receptors; and their
high-velocity shortening in response to electrical field stimulation or
acetylcholine (ACh) exposure. The remaining myocytes instead become
flattened and circular, express little contractile protein, and do not
contract in response to agonists (2, 11).
In this study, we sought to identify the mechanism through which serum-deprived cultured airway myocytes reacquire contractile responsiveness to ACh. Specifically, we tested the hypotheses that the reacquisition of ACh responsiveness is associated with the restoration of ACh coupling to intracellular Ca2+ mobilization and increased abundance of muscarinic M3 receptor protein. In our previous study, immunocytochemistry suggested that muscarinic M3 receptors are present in a perinuclear distribution within confluent canine tracheal myocytes before serum deprivation, even though they do not contract in response to ACh stimulation (2). Myocytes that became elongated during serum deprivation showed greater immunoreactivity for muscarinic M3 receptors, and these receptors appeared localized in punctate clusters at the cell surface. Thus it is uncertain whether the restoration of ACh-induced contraction in this subpopulation of serum-deprived cells is associated with greater abundance of M3 receptors, improved coupling of the receptor to Ca2+ mobilization, or both. To address these questions and the signaling pathways through which ACh elevates intracellular Ca2+ concentration ([Ca2+]i) in these cells, we evaluated responses to ACh or bradykinin (BK) in confluent canine tracheal smooth muscle cultures using the Ca2+-sensitive dye fura 2-acetoxymethyl ester (AM) before and after 7-13 days of serum deprivation and quantified muscarinic and BK receptor abundance using Western analysis.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Canine tracheal smooth muscle primary cultures. Tracheae were obtained from adult mongrel dogs, and primary airway smooth muscle cell cultures were established as described previously (2). Briefly, cleaned tracheal muscle was obtained by dissection and minced with scissors. Myocytes were enzymatically dispersed for 60 min at 37°C in buffered saline containing 600 U/ml collagenase, 10 U/ml elastase, and 2 U/ml Nagarse protease. Isolated cells were seeded on uncoated glass coverslips or in uncoated plastic culture plates at a density of 5-10 × 103 cells/cm2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids (NEAA), 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were grown at 37°C in a humidified incubator under 5% CO2. For passage of cultures at confluence, cells were lifted using 0.05% trypsin and 0.5 mM EDTA and reseeded into three new culture plates per confluent dish. Cells from passage 1 or 2 were used in the studies described.
To induce the contractile phenotype, cultured myocytes were grown to confluence and then serum-containing growth medium was replaced with serum-free Ham's F-12 medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 0.1 mM NEAA, 50 U/ml penicillin, and 50 µg/ml streptomycin. Fresh serum-free medium was provided every 48-72 h thereafter.
Measurement of intracellular free Ca2+ responses. To assess calcium responses, cells were incubated in buffered saline, pH 7.4 (in mM: 130 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 10 HEPES, and 10 dextrose), with 0.1% BSA containing 5 µM fura 2-AM for 30 min at room temperature. Cells were then washed twice with fresh buffer and incubated for another 30 min to allow for complete hydrolysis of the ester. The coverslip was then transferred to an inverted Nikon Diaphot microscope equipped with a Princeton Instruments RTE/CCD 12-bit digital camera (Trenton, NJ) controlled through a PC workstation. A Nikon ×20/0.75 objective lens was used, and image size was set to 720 × 540 pixels. At this setting, pixel intensities ranged between 25 and 2,500 gray levels. A fluorescent light excited the cells alternately at 340 and 380 nm with a Lambda 10 filter wheel (Sutter Instruments; Novato, CA). Emitted fluorescence (510 nm) was acquired for 300 ms at each excitation wavelength and used to calculate calcium concentrations (in nM) at each pixel from an in vitro calibration curve of known free Ca2+ (0-1.35 µM) and pentapotassium fura 2 (50 µM) (17). Calcium responses within individual cells were determined using Metamorph software (Jandel Scientific; Santa Barbara, CA) by circumscribing single myocytes and spatially averaging fura 2 fluorescence within the borders of each cell. Intracellular free Ca2+ was recorded for 20 s to establish a baseline and for at least 220 s to characterize peak and plateau responses after addition of either ACh or BK.
Coverslips were placed in a modified Sykes-Moore chamber (with a large glass coverslip forming the bottom of the open chamber) containing 400 µl of BSA-containing HEPES buffer (maximum volume 1 ml). For the addition of agonists, an equal volume (400 µl) of buffer containing twice the target concentration was pipetted into the chamber to ensure rapid mixing for the whole volume. For antagonists, cells were equilibrated with buffer containing the receptor blocker for at least 10 min before addition of agonist.
Intracellular free Ca2+ responses
to ACh and BK. Two coverslips of tracheal myocytes were prepared as
described in Measurement of intracellular free
Ca2+ responses. Cells
were exposed to either ACh (104 M final
concentration) or BK (10
5 M final
concentration) in random order, and responses of individual cells were
recorded. Myocytes were then washed with three volumes of buffer. After
5 min of equilibration, basal
[Ca2+]i again was recorded, the
other agonist was added to the chamber, and
[Ca2+]i response was measured. ACh
and BK responses were compared in up to 50 individual cells per coverslip.
Assessment of long axis-to-short axis ratio in ACh- and
BK-responsive tracheal myocytes. Cells were grown to confluence and serum-deprived as described in Measurement of free
Ca2+ responses. On day 12 of
serum deprivation, myocytes were loaded with fura 2-AM and peak
[Ca2+]i responses to ACh or BK
exposure were recorded as described. Three coverslips of cells were
exposed to 107 M BK and
10
6 M ACh sequentially, and
Ca2+ mobilization was recorded. Care was taken not to
disturb the visual field between agonist exposures as BK was washed out
from the chamber. Using Metamorph, we subtracted the baseline 380-nm image from the 380-nm exposure obtained at peak agonist response to
yield images that reflect peak calcium responses. Peak responses to BK
or to ACh stimulation were pseudocolored green and red, respectively,
to allow for identification of BK-responsive, ACh-responsive, or dually
responsive myocytes. Using Metamorph software, we measured the long
axis and the perpendicular short axis at the widest point (usually at
or near the midpoint of the long axis of each cell) of 15 individual
ACh-responsive and 15 individual BK-responsive myocytes on each of
these three coverslips (45 myocytes per agonist total) and calculated
the long axis-to-short axis ratio as an index of cell elongation. Areas
of overlap of red and green images appeared yellow when the two peak
response images from an individual coverslip were superimposed.
Reproducibility of calcium mobilization to ACh. Coverslips of
tracheal myocytes were prepared as described in Measurement of
intracellular free Ca2+ responses; ACh
(104 M final concentration) was added to
the chamber and responses of individual cells were
recorded. Myocytes that mobilized calcium in response to
ACh were noted, and the circumference of each responsive myocyte was
demarcated using Metamorph software. Cells were then washed with three
volumes of buffer. After 5 min of equilibration, basal
[Ca2+]i again was recorded, ACh
(10
4 M final concentration) again was
added to the chamber, and the [Ca2+]i response was measured.
First and second responses of individual ACh-responsive myocytes were compared.
Assessment of heterogeneity of
[Ca2+]i
responses to ACh among individual myocytes. Basal (preagonist),
peak, and plateau (postagonist) [Ca2+]i responses to dual ACh
exposures were recorded. Unlike the reproducibility study, two
different concentrations of ACh were employed during these
measurements. First, myocytes were exposed to ACh at a final concentration ranging from 1012 M to
10
4 M. The second
[Ca2+]i response was elicited by
exposure to 10
4 M ACh, to provide a near
maximal stimulus. Borders of cells that responded to
10
4 M ACh were circumscribed, and
calcium concentration responses to first and second stimulations were
determined. Comparison of the first to the second
[Ca2+]i responses among individual
myocytes revealed the fraction of potentially ACh-responsive myocytes
that mobilized intracellular Ca2+ on submaximal ACh stimulation.
Effect of muscarinic receptor antagonists on
[Ca2+]i
response to ACh. Basal and peak
[Ca2+]i responses were recorded in
individual myocytes stimulated with 104
M ACh. Myocytes were washed with two volumes of buffer and then bathed
in 400 µl of buffer containing either no antagonist,
10
6 M atropine (nonspecific muscarinic
receptor antagonist), 10
6 M pirenzepine
(M1 receptor specific), 10
6
M methoctramine (M2 receptor specific), or
10
6 M
4-diphenylacetoxy-N-methylpiperidine (4-DAMP; M3
receptor specific). After 10 min of equilibration, basal
and peak [Ca2+]i levels in response
to 10
4 M ACh were again recorded and
compared with preantagonist responses.
Calcium pool utilization during
Ca2+ mobilization in ACh- and
BK-responsive myocytes. Basal and peak
[Ca2+]i responses were recorded as
described during exposure to 5 mM caffeine. In additional experiments,
we assessed the influence of 106 M
xestospongin C [inositol 1,4,5-trisphosphate (IP3)
receptor antagonist] or 10
5 M
ryanodine (in greater concentration blocks ryanodine receptors) on ACh-
or BK-induced calcium mobilization in individual myocytes exposed to
10
6 M ACh or
10
7 M BK before and during incubation
with the above agents. Finally, the role of extracellular
Ca2+ was explored by recording
[Ca2+]i responses to ACh or BK in
the presence and then nominal absence of extracellular
Ca2+.
Western analysis of receptor protein expression after serum
deprivation. Temporal changes in receptor protein composition of
canine tracheal myocytes in culture were assessed by Western analysis.
After 0-7 days of serum deprivation, cultures were washed with PBS
and then total protein homogenates were prepared in extraction buffer
[0.3% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 7.6, 0.6 M
-mercaptoethanol, 20 µg/ml leupeptin, 250 µM
phenylmethylsulfonyl fluoride, and 50 mg/ml soybean trypsin
inhibitor]. Protein from whole cell lysates (15 µg/lane) was
size-fractionated by SDS-polyacrylamide gel electrophoresis and then
was electroblotted onto nitrocellulose membranes using semidry
transfer. Blots were blocked overnight at 4°C with 3% nonfat milk
in Tris-buffered saline, pH 7.4, containing 0.1% Tween 20 (TBST).
Blots were then incubated at room temperature for 2-4 h in primary
antibodies diluted 1:800 for M1, 1:1,000 for
M2, 1:1,000 M3, and 1:1,000 for BK2
receptor protein in TBST with 1% dry milk. Blots were incubated for 40 min in a secondary antibody, sheep anti-mouse IgG diluted 1:1,000 in
TBST. Streptavidin-horseradish peroxidase in TBST was used (1:5,000)
for the tertiary incubation. Immunoreactive bands were detected on
Hyperfilm-ECL using enhanced chemiluminescence reagents (Amersham Life
Sciences). To assess the change with time in the relative abundance of
individual receptor proteins from the resulting chemilumigrams, a
Hewlett-Packard scanner with Scanplot software was used (15).
Materials. Collagenase, purified from Clostridium histolyticum, was purchased from GIBCO BRL, Life Technologies (Grand Island, NY). Caffeine, elastase type IV, and Nagarse protease type XXVII were purchased from Sigma (St. Louis, MO). Insulin-transferrin-selenium culture medium supplement was obtained from Collaborative Biomedical Products (Bedford, MA). Nonessential amino acid nutrient supplement was purchased from GIBCO BRL, Life Technologies. Fura 2-AM used for measurement of intracellular Ca2+ concentration was purchased from Molecular Probes (Eugene, OR). Ryanodine and xestospongin C were purchased from Calbiochem-Novabiochem (La Jolla, CA). Primary antibodies for Western blotting included monoclonal anti-BK2 receptor (clone 20) from Transduction Laboratories (Lexington, KY) and polyclonal rabbit antibodies for muscarinic M1, M2, or M3 receptors from Research and Diagnostic Antibodies (Berkeley, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intracellular Ca2+ responses to
ACh and BK. Figure 1 shows
representative pseudocolor images derived from analysis of fura 2-AM fluorescence within cultured canine tracheal myocytes; these images were obtained at the time of peak intracellular Ca2+
mobilization responses to stimulation with ACh or BK. Confluent myocytes before serum deprivation (day 0) exhibited virtually no intracellular Ca2+ mobilization after stimulation with
ACh (104 M), but BK
(10
5 M) exposure induced a brisk and
substantial rise in [Ca2+]i.
|
In contrast to the day 0 cells, many but not all myocytes in
12-day serum-deprived cultures (Fig. 1) demonstrated substantial increases in [Ca2+]i after ACh
(106 M). In addition, many but not all
myocytes exhibited brisk increases in
[Ca2+]i after BK
(10
7 M) stimulation. These results were
typical of cultures deprived of serum for 7 or more days, and,
therefore, data from cultures studied at days 7-13 of
serum deprivation are considered together.
Mutually exclusive BK or ACh responsiveness in serum-deprived
myocytes. To determine whether the ACh-responsive and BK-responsive myocytes in serum-deprived cultures represent the same or different populations, we recorded peak intracellular Ca2+ responses
in additional 12-day serum-deprived cells on three coverslips
sequentially exposed to 107 M BK and
then to 10
6 M ACh. Figure
2 shows these peak calcium responses, with
responses to BK pseudocolored green and responses to ACh pseudocolored
red. Overlay of these two images from each coverslip demonstrates that two distinct subpopulations of cells are present and suggests that
these two subpopulations are mutually exclusive in their responsiveness
to ACh or BK (Fig. 2). The only overlap between BK and ACh responses
(seen as yellow in the overlay image of Fig. 2, middle row)
reflects divergent responses in physically overlapping myocytes. To
evaluate further whether BK or ACh responses occur in different
subpopulations, we quantified calcium responses to both agonists in
additional 13-day serum-deprived myocytes. As shown in Fig.
3, there is considerable heterogeneity of
BK and ACh responses among serum-deprived myocytes. Furthermore,
myocytes that exhibit substantial intracellular Ca2+
mobilization in response to ACh exposure have virtually no response to
BK (ACh responsive, Fig. 4), and myocytes
that exhibit great intracellular Ca2+ elevations during BK
exposure have virtually no response to ACh (BK responsive, Fig. 4).
Thus ACh-responsive and BK-responsive myocytes comprise different
subpopulations of serum-deprived cells. It is also interesting to note
that serum-deprived ACh-responsive cells maintained a significantly
elevated plateau [Ca2+]i after ACh
exposure (Figs. 3 and 4). Not until ACh was washed out of the cell
chamber did [Ca2+]i return to
baseline. This sustained response is in sharp contrast to
serum-deprived BK-responsive myocytes, which demonstrate a transient
peak response to BK without a sustained plateau (Figs. 3 and 4).
|
|
|
To evaluate whether the subpopulations identified above correspond to
the elongated (contractile) or nonelongated (noncontractile) phenotype
we previously described (2), we quantified the cell shape of ACh- or
BK-responsive myocytes shown in Fig. 2. As shown in Fig.
5, the long axis-to-short axis ratio of
ACh-responsive myocytes was 2.6-fold greater (P < 0.001, t-test) than that of BK-responsive cells. Thus ACh
responsiveness is a property of elongated serum-deprived myocytes, and
BK responsiveness occurs in more nonelongated myocytes.
|
Pharmacological characterization of muscarinic ACh receptors on
elongated serum-deprived myocytes. We tested the reproducibility of
[Ca2+]i responses of individual
ACh-responsive elongated myocytes to two sequential exposures to
104 M ACh. Intracellular
Ca2+ responses to the second ACh exposure were
systematically reduced compared with those observed after the initial
ACh exposure, averaging 71.7 ± 3.1% of the first response. The two
responses were closely correlated (r2 = 0.920; Fig. 6), and all cells that
responded to the first ACh activation exhibited a reduced but clear-cut
response to the second ACh exposure. This finding allowed us to
evaluate the influence of various muscarinic receptor antagonists on
ACh-induced Ca2+ mobilization by administering dual ACh
challenges in the absence and presence of muscarinic antagonist. As
shown in Table 1, ACh-responsive serum-deprived myocytes demonstrated robust increases in
[Ca2+]i that were completely
blocked in a second ACh exposure by 10
6
M atropine (nonspecific muscarinic receptor antagonist) or
10
6 M 4-DAMP (M3 specific)
but were unaffected by 10
6 M pirenzepine
(M1 specific antagonist). Methoctramine
(10
6 M), an M2 receptor
antagonist with partial anti-M3 receptor activity, caused
partial inhibition of [Ca2+]i
responses to ACh. Thus ACh acts through muscarinic M3
receptors to mobilize intracellular Ca2+ in serum-deprived
elongated tracheal myocytes.
|
|
Heterogeneous and graded activation of contractile phenotype
myocytes by ACh. To evaluate whether individual elongated myocytes in serum-deprived cultures exhibit a graded intracellular
Ca2+ response to increasing ACh concentrations, we exposed
cells sequentially first to ACh at 1012
M, 10
10 M,
10
8 M,
10
6 M, or
10
4 M and second to
10
4 M ACh. For each cell that exhibited
a clear-cut rise in [Ca2+]i during
the second ACh stimulation, we determined the rise over baseline
[Ca2+]i induced by the first ACh
exposure. Because there is no widely accepted standard for the
magnitude of [Ca2+]i increase that
constitutes a "response," we calculated the fraction of
ACh-responsive myocytes whose peak
[Ca2+]i increment over baseline
during the first ACh exposure exceeded 100 nM, 200 nM, and 300 nM.
Figure 7 demonstrates that 1)
maximal stimulation with 10
4 M ACh
results in a wide range of peak
[Ca2+]i elevations in elongated,
serum-deprived myocytes; 2) submaximal stimulation with lesser
ACh concentrations results in graded peak [Ca2+]i elevations in these cells
and there is substantial heterogeneity of response among individual
cells; and 3) the fraction of ACh-responsive myocytes that
exhibit [Ca2+]i responses
increases with ACh concentration whether the threshold for response was
set at 100, 200, or 300 nM.
|
Mechanisms of intracellular Ca2+
coupling. Neither ACh-responsive elongated nor BK-responsive
nonelongated 13-day serum-deprived myocytes mobilized Ca2+
in response to 5 mM caffeine. Also, both subpopulations failed to
respond to ryanodine alone, and in the presence of 50 µM ryanodine, second responses to 107 M BK or
10
6 M ACh were unaffected in
BK-responsive and ACh-responsive myocytes, respectively (Fig.
8). However, 10 µM xestospongin C, a
blocker of IP3 receptors on the sarcoplasmic reticulum
(SR), almost completely inhibited Ca2+ mobilization in
response to a second ACh exposure in ACh-responsive myocytes (Fig. 8).
Xestospongin C also partially reduced Ca2+ response to a
second BK exposure in BK-responsive cells to 50% of that observed
during the first exposure to agonist (Fig. 8). The simultaneous
presence of ryanodine had no additive effect on the reduction of
Ca2+ mobilization caused by xestospongin C in either
ACh-responsive or BK-responsive myocytes.
|
Removal of extracellular Ca2+ had a profound effect on the second BK response in BK-responsive cells. In the absence of extracellular Ca2+, intracellular Ca2+ mobilization by BK was completely blocked in serum-deprived cells (Fig. 8). However, removal of extracellular Ca2+ did not affect the second peak Ca2+ mobilization caused by ACh exposure in serum-deprived ACh-responsive myocytes. Thus initial intracellular Ca2+ coupling mechanisms that determine peak calcium responses differ importantly in the two subpopulations of serum-deprived myocytes. Elongated myocytes initially mobilize calcium from the SR on ACh stimulation through downstream activation of IP3 receptors, whereas nonelongated myocytes respond to BK both through initial influx of extracellular Ca2+ and an intracellular IP3 receptor-dependent mechanism.
Western analysis of receptor expression during contractile
phenotype modulation. Western analysis of whole culture lysates using anti-muscarinic M3 receptor antibody revealed a
single immunoreactive band of ~70 kDa, which was detected in
confluent serum-fed (day 0) myocyte cultures but progressively
increased in abundance after 2 or 7 days of serum deprivation (Fig.
9). In contrast, M2 receptor abundance (detected as a ~70- kDa immunoreactive band with
anti-muscarinic M2 receptor antibody) decreased
significantly with prolonged serum deprivation (Fig. 9). Muscarinic
M1 receptors were not detectable at any time by Western
analysis (data not shown). Immunoblots using BK2
receptor-specific antibody revealed a protein doublet at ~42 kDa
(Fig. 9). In whole culture lysates, BK2 receptor
abundance increased significantly within 2 days of serum
deprivation but tended to decrease somewhat thereafter.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cultured airway smooth muscle typically loses many structural and functional characteristics of smooth muscle cells within intact tissue (1, 6, 7, 14, 16). When maintained under growth-promoting conditions, airway myocytes assume a nonelongated spindle shape, contain sparse contractile apparatus proteins, and exhibit virtually no ability to contract. However, we (2) and Ma et al. (11) recently reported that prolonged serum deprivation of confluent cultured airway myocytes promotes a monolayer culture and restores contractile structure and function to a subset of cells. Myocytes that acquire the contractile phenotype accumulate abundant contractile apparatus proteins, exhibit an elongated wormlike morphology, and shorten substantially on electrical field stimulation (11) or ACh exposure (2). The current study was performed to identify mechanisms through which ACh-induced contraction is restored in these contractile cultured cells.
We found that ACh stimulates intracellular Ca2+ mobilization within elongated, serum-deprived, contractile airway myocytes but does not do so in serum-fed confluent myocytes or in serum-deprived cells that fail to elongate (Figs. 1-5). Conversely, BK elicited intracellular Ca2+ responses in serum-fed confluent myocytes and in nonelongated serum-deprived cells but not in elongated serum-deprived myocytes. Thus responsiveness to ACh and responsiveness to BK were mutually exclusive in our culture system (Figs. 2 and 4). Pharmacological studies demonstrate clearly that, as in intact trachealis tissue (14), muscarinic M3 receptors mediate ACh-induced calcium responses in elongated, contractile myocytes (Table 1). Furthermore, increasing ACh concentration, which increases force generation by intact tissue in a graded fashion (13), also increases overall calcium mobilization in cultured contractile airway myocytes both by increasing the number of cells exhibiting any response and by increasing the magnitude of response within individual myocytes (Figs. 6 and 7).
Interestingly, elongated, ACh-responsive, serum-deprived myocytes and nonelongated, BK-responsive cells mobilize calcium in response to agonist by separate mechanisms. The ACh-responsive phenotype mobilizes calcium from a pool that is almost totally inhibited by xestospongin C pretreatment; peak fluorescence from these elongated myocytes during ACh exposure was not affected by removal of extracellular Ca2+ (Fig. 8). Thus calcium from the SR is mobilized in the presence of ACh, and its release is mediated through IP3 receptors on the SR. These results are in striking contrast with serum-deprived, nonelongated, BK-responsive cells. Xestospongin C only partially inhibited calcium mobilization in BK-responsive cells, whereas removal of extracellular Ca2+ totally inhibited fluorescence in response to agonist (Fig. 8). Thus extracellular Ca2+ may be the major source of the fluorescence response in the BK-responsive subpopulation, and calcium-stimulated calcium release may operate in these cells. Furthermore, the lack of response to caffeine or ryanodine in either subpopulation suggests that canine tracheal myocytes in culture may not express ryanodine receptors on the SR or that this mechanism of calcium release is not predominant in canine tracheal myocytes in culture (Fig. 8).
These findings have several implications. In elongated, contractile myocytes, muscarinic M3 receptors are found in clusters at the cell surface (2). In contrast, M3 receptors localize to the perinuclear region in serum-fed confluent or serum-deprived nonelongated airway myocytes (2), neither of which mobilize calcium on ACh exposure (Fig. 1). Thus translocation of muscarinic M3 receptors to the cell membrane appears to be one mechanism through which M3 receptor-calcium coupling is restored in elongated myocytes during serum deprivation. It may also be that an increase in total M3 receptor number during serum deprivation contributes to the restoration of ACh responsiveness (Fig. 9), although the relative importance of each mechanism remains uncertain.
Second, airway smooth muscle cells exhibit plasticity of cell surface receptor expression and function. In intact canine trachealis tissue, ACh but not BK elicits contraction (R. Mitchell, unpublished observations). Culture and passage of canine tracheal myocytes provoke the disappearance of functionally coupled M3 receptors and the emergence of BK-induced calcium mobilization. Yet, during prolonged serum deprivation, myocytes that reacquire the elongated contractile phenotype also regain the spectrum of agonist responsiveness present within intact tissue. The present study did not address the mechanisms by which M3 or BK receptor abundance is controlled in individual myocytes. However, the observed increase in total M3 receptor protein (and accompanying decrease in M2 receptor protein) suggests that either increased de novo synthesis or diminished degradation of muscarinic M3 receptors occurs during serum deprivation (Fig. 9). Because the muscarinic M3 receptor gene promoter has not been cloned, the nuclear mechanisms controlling M3 receptor gene transcription remain unknown.
Third, elongated, contractile cultured airway myocytes exhibit a wide range of responses to ACh as reflected in their heterogeneous intracellular Ca2+ responses to high concentrations of ACh and their heterogeneous responses to threshold concentrations (Fig. 7). Perhaps this variation among individual elongated myocytes reflects greater or lesser progression toward a "fully" contractile phenotype, but it also resembles the dispersion of other characteristics recently identified among individual myocytes acutely isolated from intact airway muscle (3-5). If this variation among cultured contractile airway myocytes reflects smooth muscle behavior in vivo, then the graded contractile responses to increasing ACh concentration characteristic of intact airway muscle strips (10, 12, 13) may stem both from increasing activation of individual myocytes and from recruitment of increasing numbers of myocytes participating in contraction. To our knowledge, this possibility has not yet been tested directly in intact airway smooth muscle or acutely isolated myocytes.
Previously, Yang and co-workers studied canine tracheal myocytes in culture that had been growth arrested by reduction of serum in the maintenance medium (18, 19, 21). They found that growth arrest increased the number of ligand-binding surface muscarinic receptors (18), that both M2 and M3 receptors were present (20), and that pharmacological activation of these receptors increased IP3 production (19-21). Interestingly, calcium was mobilized in these cultures both by carbachol and by BK in Yang's studies (20-25). Importantly, all cells in culture were treated as a single population rather than as discrete subpopulations of contractile vs. noncontractile (i.e., elongated vs. nonelongated) myocytes. Our studies confirm and extend Yang's work by demonstrating that serum deprivation induces heterogeneity of myocytes in culture and that only the contractile, elongated phenotype demonstrates M3 receptor-mediated calcium mobilization in response to cholinergic agonist. Thus restoration of ACh-mediated calcium signaling is not a general consequence of serum deprivation. Our data also show that the functional coupling of muscarinic M3 receptors to calcium mobilization is mediated through IP3 receptors on the SR in serum-deprived, cultured airway myocytes.
There are limitations to the interpretation of our data. M3 receptor protein was detected before serum deprivation but was apparently internalized (2) and not functionally coupled to Ca2+ mobilization. Prior studies of cultured confluent human airway myocytes suggest that muscarinic M2 receptors are associated with the sarcolemma because isoproterenol-induced cAMP formation is inhibited by carbachol and muscarinic M2 receptor antagonists block this inhibition (16). We also found that methoctramine, an M2 receptor antagonist, reduced calcium mobilization in response to ACh in elongated serum-deprived myocytes (Table 1); this partial antagonism in part may be due to low-affinity binding of methoctramine to M3 receptors (20). Furthermore, we could not quantify the distribution of M3 receptor protein among elongated vs. nonelongated serum-deprived myocytes. Thus it is conceivable that the increase in overall M3 receptor protein observed during serum deprivation occurred in part within the nonelongated, noncontractile subpopulation. Similarly, we suspect, but cannot prove, that the increase in BK2 receptor protein evident from analysis of whole culture lysates was localized primarily to the noncontractile myocytes. Also, we could not detect any significant response in either elongated or nonelongated myocytes to caffeine and ryanodine. However, Janssen et al. (8, 9) have noted that freshly dissociated canine tracheal smooth muscle cells rarely contract to these agents, and in only 3 of 7 cells tested (8) was there some transient calcium mobilization in response to concentrations of ryanodine similar to those used in our studies. This finding limits precise interpretation of the lack of a response to caffeine and ryanodine in our serum-deprived myocytes, which thus may indicate a possible continued perturbation in excitation-contraction coupling in the elongated, contractile myocytes. Finally, we cannot be sure that every phenotypically contractile, elongated serum-deprived myocyte does respond to ACh and does not respond to BK. However, we selected visual fields in which both elongated and flattened nonelongated myocytes were present so that we could directly compare [Ca2+]i responses to both ACh and BK. Quantification of cell shape (Fig. 5) confirms that ACh-responsive and BK-responsive subpopulations correspond to the contractile and noncontractile phenotypes we characterized previously (2), and the differences in [Ca2+]i responses and the mechanisms of calcium mobilization to ACh and BK between elongated tracheal myocytes and nonelongated cells in the same image field were striking.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grants HL-56399 and HL-64095, Inspiraplex, Merck-Frosst Canada, Sprague Memorial Institute, and Blowitz-Ridgeway Foundation.
![]() |
FOOTNOTES |
---|
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: J. Solway, Section of Pulmonary and Critical Care, Dept. of Medicine, Univ. of Chicago, 5841 S. Maryland Ave., MC 6026, Chicago IL 60637 (E-mail: jsolway{at}medicine.bsd.uchicago.edu).
Received 5 April 1999; accepted in final form 7 December 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Chamley-Campbell, JH,
Campbell GR,
and
Ross R.
Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens.
J Cell Biol
89:
379-383,
1981[Abstract].
2.
Halayko, AJ,
Camoretti-Mercado B,
Forsythe SM,
Vieira JE,
Mitchell RW,
Wylam ME,
Hershenson MB,
and
Solway J.
Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes.
Am J Physiol Lung Cell Mol Physiol
276:
L197-L206,
1999
3.
Halayko, AJ,
Rector E,
and
Stephens NL.
Characterization of molecular determinants of smooth muscle cell heterogeneity.
Can J Physiol Pharmacol
75:
917-929,
1997[ISI][Medline].
4.
Halayko, AJ,
Rector E,
and
Stephens NL.
Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition.
Am J Physiol Lung Cell Mol Physiol
274:
L17-L25,
1998
5.
Halayko, AJ,
Salari H,
Ma XF,
and
Stephens NL.
Markers of airway smooth muscle cell phenotype.
Am J Physiol Lung Cell Mol Physiol
270:
L1040-L1051,
1996
6.
Hall, IP,
and
Kotlikoff M.
Use of cultured airway myocytes for study of airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
268:
L1-L11,
1995
7.
Hirst, SJ.
Airway smooth muscle cell culture: application to studies of airway wall remodeling and phenotype plasticity in asthma.
Eur Respir J
9:
808-820,
1996
8.
Janssen, LJ,
Betti PA,
Netherton SJ,
and
Walters DK.
Superficial buffer barrier and preferentially directed release of Ca2+ in canine airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
276:
L744-L753,
1999
9.
Janssen, LJ,
and
Sims SM.
Ca2+-dependent Cl current in canine tracheal smooth muscle cells.
Am J Physiol Cell Physiol
269:
C163-C169,
1995
10.
Koenig, SM,
Mitchell RW,
Kelly E,
White SR,
Leff AR,
and
Popovich KJ.
-Adrenergic relaxation of dog trachealis: contractile agonist-specific interaction.
J Appl Physiol
67:
181-185,
1989
11.
Ma, X,
Wang Y,
and
Stephens NL.
Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells.
Am J Physiol Cell Physiol
274:
C1206-C1214,
1998
12.
Madison, JM,
Jones CA,
Tom-Moy M,
and
Brown JK.
Affinities of pirenzepine for muscarinic cholinergic receptors in membranes isolated from bovine tracheal mucosa and smooth muscle.
Am Rev Respir Dis
135:
719-724,
1987[ISI][Medline].
13.
Mitchell, RW,
Koenig SM,
Popovich KJ,
Kelly E,
Tallet J,
and
Leff AR.
Pertussis toxin augments beta-adrenergic relaxation of muscarinic contraction in canine trachealis.
Am Rev Respir Dis
147:
327-331,
1993[ISI][Medline].
14.
Tom-Moy, M,
Madison JM,
Jones CA,
de Lanerolle P,
and
Brown JK.
Morphologic characterization of cultured smooth muscle cells isolated from the tracheas of adult dogs.
Anat Rec
218:
313-328,
1987[ISI][Medline].
15.
Vincent, SG,
Cunningham PR,
Stephens NL,
Halayko AJ,
and
Fisher JT.
Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard scanjet scanner and Scanplot software.
Electrophoresis
18:
67-71,
1997[ISI][Medline].
16.
Widdop, S,
Daykin K,
and
Hall IP.
Expression of muscarinic M2 receptors in cultured human airway smooth muscle cells.
Am J Respir Cell Mol Biol
9:
541-546,
1993[ISI][Medline].
17.
Wylam, ME,
Gungor N,
Mitchell RW,
and
Umans JG.
Eosinophils, major basic protein, and polycationic peptides augment bovine airway myocyte Ca2+ mobilization.
Am J Physiol Lung Cell Mol Physiol
274:
L997-L1005,
1998
18.
Yang, CM.
Muscarinic receptor expression in the primary culture of tracheal smooth muscle cells.
J Recept Res
10:
235-247,
1990[ISI][Medline].
19.
Yang, CM,
and
Chou S-P.
Primary culture of canine tracheal smooth muscle cells in serum-free medium: effects of insulin-like growth factor I and insulin.
J Recept Res
13:
943-960,
1993[ISI][Medline].
20.
Yang, CM,
Chou SP,
and
Sung TC.
Muscarinic receptor subtypes coupled to generation of different second messengers in isolated tracheal smooth muscle cells.
Br J Pharmacol
104:
613-618,
1991[Abstract].
21.
Yang, CM,
Chou SP,
Sung TC,
and
Chien HJ.
Regulation of functional muscarinic receptor expression in tracheal smooth muscle cells.
Am J Physiol Cell Physiol
261:
C1123-C1129,
1991
22.
Yang, CM,
Hsia H-C,
Chou S-P,
Ong R,
Hsieh J-T,
and
Luo S-F.
Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells.
Br J Pharmacol
111:
21-28,
1994[Abstract].
23.
Yang, CM,
Hsia HC,
Hsieh JT,
Ong R,
and
Luo SF.
Bradykinin-stimulated calcium mobilization in cultured canine tracheal smooth muscle cells.
Cell Calcium
16:
59-70,
1994[ISI][Medline].
24.
Yang, CM,
Hsu MC,
Tsao HL,
Chiu CT,
Ong R,
Hsieh JT,
and
Fan LW.
Effects of cAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.
Cell Calcium
19:
243-254,
1996[ISI][Medline].
25.
Yang, CM,
Yo YL,
and
Wang YY.
Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.
Br J Pharmacol
110:
983-988,
1993[Abstract].