Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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We
tested the hypothesis that prolonged serum deprivation would allow a
subset of cultured airway myocytes to reacquire the abundant
contractile protein content, marked shortening capacity, and elongated
morphology characteristic of contractile cells within intact tissue.
Passage 1 or
2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days.
During serum deprivation, two differentiation pathways emerged.
One-sixth of the cells developed an elongated morphology and aligned
into bundles. Elongated myocytes contained cables of contractile
myofilaments, dense bodies, gap junctions, and membrane caveoli,
ultrastructural features of contractile SM in tissue. These cells
immunostained intensely for SM -actin, SM myosin heavy chain (MHC),
and SM22 (an SM-specific actin-binding protein), and Western analysis
of culture lysates disclosed 1.8 (SM
-actin)-, 7.7 (SM MHC)-, and
5.8 (SM22)-fold protein increases during serum deprivation.
Immunoreactive M3 muscarinic
receptors were present in dense foci distributed throughout elongated,
SM MHC-positive myocytes. ACh
(10
3 M) induced a marked
shortening (59.7 ± 14.4% of original length) in 62% of elongated
myocytes made semiadherent by gentle proteolytic digestion, and
membrane bleb formation (a consequence of contraction) occurred in all
stimulated cells that remained adherent and so did not shorten.
Cultured airway myocytes that did not elongate during serum deprivation
instead became short and flattened, lost immunoreactivity for
contractile proteins, lacked the
M3 muscarinic-receptor expression
pattern seen in elongated cells, and exhibited no contractile response
to ACh. Thus we demonstrate that prolonged serum deprivation induces
distinct differentiation pathways in confluent cultured tracheal
myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.
phenotypic modulation; serum deprivation; contraction; ultrastructure; heterogeneity; muscarinic receptors; contractile proteins
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INTRODUCTION |
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IN NORMAL TISSUES, smooth muscle (SM) cells (SMCs) contract to regulate the luminal diameter of hollow organs. In disease states, SMCs also serve as effectors of fibrosis, muscle mass accumulation, and inflammation (13, 24). For example, myocytes of the "synthetic-proliferative" phenotype appear in atherosclerotic lesions and arise from phenotypic modulation of contractile cells and/or selection of preexisting subsets of myocytes that retain these potential functions (14). Synthetic-proliferative myocytes express little contractile apparatus and instead synthesize matrix proteins, growth factors, and cytokines.
Cell culture of SM has advanced the understanding of SM gene transcription (12, 22), pathways regulating proliferation (35), and cytokine and/or growth factor secretion (6). However, study of the contractile function in cultured SMCs has been hampered by their tendency to acquire the synthetic-proliferative phenotype. Contractile protein content increases at confluence, but even such confluent cells differ importantly from contractile myocytes in SM tissue (11). They do not shorten appreciably when stimulated by contractile agonists, and they do not exhibit intracellular calcium responses to muscarinic stimulation (15, 23, 25, 36). Furthermore, they assume a short, spindlelike morphology that is distinct from the elongated, wormlike shape of myocytes freshly isolated from tissue (11, 13). Notably, confluent cultured SMCs exhibit heterogeneity of morphology, contractile and cytoskeletal protein content (2, 17, 33), and mitogen sensitivity (10, 19). Thus, like in intact tissue, SM cultures include myocytes of differing potential for proliferative, secretory, or contractile function.
Based on previous reports (1, 5, 13, 33) that short-term serum deprivation promotes contractile protein gene expression and the heterogeneity of cultured SMCs cited above, we reasoned that prolonged serum withdrawal from confluent passaged airway myocytes might allow a select subset of cells to reacquire the abundant contractile protein content, marked shortening capacity, and elongated morphology characteristic of contractile phenotype cells within intact tissue. We tested this hypothesis using passaged, cultured canine tracheal myocytes and found that striking morphological elongation, contractile protein accumulation, acetylcholine (ACh)-induced intracellular calcium mobilization (21), and ACh-induced shortening occur in a subset of serum-deprived cells.
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METHODS |
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Materials. Collagenase purified from
Clostridium histolyticum was purchased
from GIBCO BRL (Life Technologies, Grand Island, NY). 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 (NEAA) nutrient supplement was purchased from
GIBCO BRL (Life Technologies). Monoclonal mouse antibodies used for
Western blotting and immunocytochemical studies included anti-SM myosin
heavy chain (MHC; clone hSM-V), anti-SM -actin (clone 1A4), and
anti-
-actin (clone AC-74) from Sigma Immunochemicals. Polyclonal
guinea pig anti-SM22 was a gift from Dr. W. Gerthoffer (University of
Nevada, Reno). Polyclonal rabbit anti-M2 and
anti-M3
muscarinic-receptor antibodies were purchased from
Research and Diagnostic Antibodies (Richmond, CA). Fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG was obtained from Amersham Life Science (Arlington Heights, IL). FITC-conjugated sheep anti-guinea pig IgG and indocarbocyanine-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Canine tracheal SM cultures. Tracheae were obtained from adult mongrel dogs, and primary SMC cultures were established as previously described (11). 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 of collagenase, 10 U/ml of elastase, and 2 U/ml of Nagarse protease. Isolated cells were seeded on 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 NEAAs, 50 U/ml of penicillin, and 50 µg/ml of streptomycin. Cells were grown at 37°C in a humidified incubator under 5% CO2. Cultures were passaged at confluence by lifting the cells with 0.05% trypsin-0.5 mM EDTA and reseeding them into three new culture plates per confluent dish. Cells from passage 1 or 2 were used in the studies.
To induce phenotypic differentiation of tracheal myocytes, cultured cells were grown to confluence, then serum-containing growth medium was replaced with serum-free Ham's F-12 medium supplemented with insulin-transferrin-selenium medium (final concentrations: 5 µg/ml of insulin, 5 µg/ml of transferrin, and 5 ng/ml of selenium), 0.1 mM NEAAs, 50 U/ml of penicillin, and 50 µg/ml of streptomycin. Fresh serum-free medium was provided every 48-72 h thereafter. Cell morphology was assessed with a Nikon Diaphot II phase-contrast microscope equipped with a 35-mm camera.
Fluorescence immunocytochemistry.
Cells were grown as in Canine tracheal SM
cultures but on precleaned sterile glass
coverslips. All staining steps were carried out at 4°C unless
otherwise noted. Antibodies were diluted in PBS (pH 7.4) containing
0.1% Tween 20 (PBS-T) and 1% bovine serum albumin (BSA); for negative
controls, coverslips were incubated in this buffer containing no
primary antibody. Cells were fixed for 20 min in PBS containing 1%
paraformaldehyde, then were incubated in PBS containing 1% Triton
X-100 for 15 min. The coverslips were blocked for 1 h in PBS-T
containing 3% BSA and incubated overnight in primary antibody. Primary
antibodies included mouse monoclonal anti-SM MHC diluted 1:50; mouse
monoclonal anti-SM -actin diluted 1:500, guinea pig polyclonal
anti-SM22 diluted 1:100, or polyclonal rabbit antibodies specific for
either M2 or
M3 muscarinic-receptor subtypes
diluted 1:500. For SM MHC,
-actin, and SM22 detection, the cells
were washed in PBS-T, then incubated in FITC-conjugated anti-mouse IgG
or FITC-conjugated anti-guinea pig IgG for 2 h at room temperature
(RT). Cells that were double immunolabeled for SM MHC and
M2 or
M3 muscarinic receptors were
incubated in a secondary antibody cocktail containing both FITC-conjugated anti-mouse IgG and indocarbocyanine-conjugated anti-rabbit IgG. To stain the nuclei, the cells were washed, then incubated for 30-45 s at RT in PBS-T containing either 10 µg/ml of propidium iodide plus 50 µg/ml of RNase A or 10 µg/ml of Hoechst 33342. The coverslips were rinsed in water, then mounted on slides with
antifade adhesive (85% glycerol, 1 mM
p-phenylenediamine, and 100 mM Tris,
pH 7.4). The slides were stored at
20°C. Fluorescent immunostaining was assessed with a Nikon microscope equipped with epifluorescence optics and a Photometrics SenSys 12-bit digital video
camera. Digitized images were captured with Spectrum imaging software
(IP Laboratories, Vienna, VA).
Ultrastructural analysis. For transmission electron microscopy, passaged cells were grown to confluence on Permanox culture plates (Nalge Nunc International, Naperville, IL) and were then serum deprived for 12 days. The cells were fixed onto the plates in PBS (pH 7.4) containing 4% paraformaldehyde plus 1.25% gluteraldehyde. The cells were postfixed with 1% osmium tetroxide, then embedded in LX-112 acrylic medium. Blocks were then prepared from areas where bundles of elongate cells were evident by light microscopy. Ultrathin en face longitudinal and transverse sections were prepared, then mounted onto Formvar-coated grids and further stained with 1% uranyl acetate and lead citrate. Cell ultrastructure was assessed with an electron microscope at an acceleration voltage of 60-80 kV.
Evaluation of cytoplasmic coupling through gap junctions. Myocytes deprived of serum for 7-10 days were bathed in HEPES-buffered Krebs solution containing 10 mM taurine and 0.1% BSA. To determine the degree of functional cell-to-cell cytoplasmic coupling, individual cells were microinjected at RT with buffer (40 mM NaCl and 50 mM HEPES, pH 7.4) containing 2% sulforhodamine 101 plus 1% FITC-conjugated 10-kDa dextran. Microinjections were performed during phase-contrast microscopy on a Leica DM-IRB/E epifluorescence microscope equipped with an Eppendorf transjector system; injection duration was 0.6 s, and injection pressure was 80 hPa. Five to seven minutes after injection, rhodamine and fluorescein fluorescences were recorded with a video camera (Optronics Engineering, Goleta, CA) and a Sony video printer.
Western analysis. Temporal changes in
the protein composition of cultured cells were assessed by Western
analysis as previously described (11). At 0-19 days after
initiation of serum deprivation, myocyte cultures were washed with PBS,
then total protein homogenates were prepared in extraction buffer that
consisted of 0.3% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 7.6),
0.6 M -mercaptoethanol, 20 µg/ml of leupeptin, 250 µM
phenylmethylsulfonyl fluoride, and 50 mg/ml of soybean trypsin
inhibitor. Protein lysates (7 µg/lane) were size fractionated by
SDS-polyacrylamide gel electrophoresis and then were electroblotted
onto nitrocellulose membranes with a semidry transfer. The blots were
blocked overnight at 4°C with 3% nonfat milk in Tris-buffered
saline (pH 7.4) containing 0.1% Tween 20 (TBS-T). Blots were then
incubated for 2-4 h at RT with the primary antibodies diluted in
TBS-T with 1% dry milk. The same antibodies used for
immunocytochemistry were used for immunoblot analysis of contractile
apparatus proteins. In addition, a mouse monoclonal anti-
-actin
primary antibody was employed. The blots were incubated for 40 min at
RT with either biotinylated sheep anti-mouse IgG or biotinylated sheep
anti-guinea pig IgG diluted (1:1,000) in TBS-T.
Streptavidin-horseradish peroxidase (1:5,000) in TBS-T was used in the
tertiary incubation. Immunoreactive bands were detected on
Hyperfilm-ECL using enhanced chemiluminescence reagents (Amersham Life
Sciences). To assess the relative abundance of individual proteins from
the resulting chemilumigrams, a Hewlett-Packard scanner with Scanplot
software system was used (34).
Contraction of serum-deprived tracheal
myocytes. Cultures were grown to confluence in plastic
dishes and maintained for 6-10 days in serum-free medium as in
Canine tracheal SM
cultures. All subsequent steps were
carried out at RT. The cells were washed twice with Hanks' balanced
salt solution containing 10 mM taurine, 1% BSA, and 1 mM
dithiothreitol, then incubated for 40-70 min in the presence of
400 units/ml of collagenase and 10 units/ml of papain, to loosen,
although not detach, myocytes from the substrata. The cells were washed
twice, then incubated in HEPES-buffered Krebs-Henseleit (HKH) solution
containing 10 mM taurine and 1% BSA. Semiadherent, elongated myocytes
were visualized by phase-contrast microscopy with a Leica DM-IRB/E
phase-contrast microscope with video camera and recorder. Individual
elongated myocytes were stimulated to contract as follows. A
micropipette filled with ACh
(103 M in HKH solution) was
positioned within 10 µm of the target cell with an Eppendorf
micromanipulator/transjector system. Ach was "puffed" along the
cell surface from the micropipette for up to 15 s, and cell shortening
was recorded on videotape. To confirm that contractile shortening
responses (see Contraction of serum-deprived tracheal
myocytes) were the direct result of ACh
stimulation, atropine (10
7
M) was added to some cultures before ACh exposure. In addition, the
response of cells puffed with HKH solution alone (i.e.,
without ACh) were recorded. Cell shortening was measured as the percent change from the original length with National Institutes of Health Image software.
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RESULTS |
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Morphological changes of serum-deprived confluent cultured canine tracheal myocytes. At confluence, canine tracheal myocyte cultures exhibited a typical hill-and-valley appearance (5, 20) characteristic of confluent SMCs growing in serum (Fig. 1A). Individual cells assumed a flattened, spindle, or oblong shape, and elongated or worm-shaped cells were very rarely seen. With serum deprivation, though, individual unflattened elongated cells became evident, usually by day 4 of serum withdrawal, and the hill-and-valley appearance of the cultures was lost. The fraction of individual elongate cells was estimated by counting cells in five ×100 phase-contrast fields from each of four different cultures deprived of serum for 7 days. Elongated morphology was seen in 17.3 ± 8.6% (SD) of the cells. Furthermore, elongated cells tended to appear in parallel and end to end, forming a multilayered pattern of elongated cell bundles up to eight cells wide that coursed randomly through the culture plate (Fig. 1, B and C). The myocytes that did not become elongate with serum deprivation became very flattened and rather circular and were oriented randomly into a continuous monolayer.
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Immunocytochemical analysis of contractile apparatus
protein expression. Immunostaining patterns were
markedly different at confluence and after 10 days of serum deprivation
(Fig. 2). At confluence
("day 0" of serum deprivation),
virtually all canine tracheal myocytes exhibited weak immunoreactivity
for SM -actin, SM MHC, and SM22 (an SM-specific actin-binding
protein). Occasional spindle-shaped cells exhibited stronger
immunostaining for
-actin or SM MHC (Fig. 2,
A and
D). Cells that stained strongly for
-actin were counted in five ×100 fields on each of three
coverslips from three different day 0 cultures (45 fields total); they comprised only 0.9 ± 0.5% of the
total cell population. In marked contrast, serum-deprived myocytes
exhibited a much more heterogeneous muscle protein expression.
Elongated cells demonstrated strikingly positive immunoreactivity for
all three muscle proteins studied, whereas the flattened, circular
cells showed virtually no immunoreactivity for these proteins (Fig. 2).
-Actin-positive, elongated myocytes were counted in five ×100
fields from each of three coverslips from three different cultures;
after 7 and 14 days of serum deprivation, they comprised 15.2 ± 2.3 and 18.8 ± 5.9%, respectively, of all cells. These data are
similar in value to those obtained by counting elongated cells under
phase contrast after 7 days of serum deprivation. Thus the fraction of
elongated,
-actin-positive cells appears stable by 7 days of serum
deprivation. In elongated cells, SM
-actin (Fig.
2C), SM MHC (Fig.
2F), and SM22 (Fig.
2I) appeared primarily along stress
fibers or in thick cables oriented parallel to the long axis of the
cells. In addition, occasional myocytes of semielongated morphology and
moderate muscle protein abundance, similar to the more immunoreactive
cells seen in day 0 cultures, were
observed in serum-deprived cultures. Negative control slides incubated
only in secondary antibody showed no appreciable staining (data not
shown).
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Ultrastructural features of serum-deprived cultured canine tracheal myocytes. Transmission electron micrographs (×7,000-40,000 initial magnification) revealed that elongated myocytes exhibited ultrastructural features typical of mature, contractile SMCs in tissue (4, 11, 20). As shown in Fig. 3A, elongated cells contained large cytoplasmic cables of contractile myofilaments, with characteristic electron-dense structures scattered through the cables. These dense structures likely represent dense bodies as found in SM tissue. Between the contractile filament cables, the cytoplasm contained numerous mitochondria. Typical of SMCs of the contractile phenotype (32), many caveoli were evident along the cell surface membrane (Fig. 3C); they often organized into linear arrays of 6-12. Intercellular contacts between elongated cells were predominantly zonae adherens, and a few typical gap junctions were observed (Fig. 3, D and E). In addition, transverse sections revealed that bundles of elongate cells were composed of up to six cell layers. In marked contrast, contractile filament cables, dense bodies, and caveoli were absent from nonelongated serum-deprived myocytes, which instead contained numerous mitochondria and cytoplasmic vacuoles and grew as a monolayer (Fig. 3B).
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Evaluation of cell-cell cytoplasmic coupling. To test whether the gap junctions found in elongated, serum-deprived myocytes by electron microscopy resulted in cell-cell cytoplasmic coupling, we microinjected individual cells with both a small-molecular-mass (606.7 Da), rapidly diffusible marker (sulforhodamine 101) and a large (10-kDa) nondiffusible marker (FITC-conjugated dextran). As shown in Fig. 4, microinjection of a single elongated cell labeled only that cell with nondiffusible fluorescent dextran, but sulforhodamine quickly diffused into three adjacent cells; when present, coupling with two to five cells was typically observed. However, this finding was inconstant in that cytoplasmic coupling with adjacent cells was present in only 6 of the 10 elongated cells so studied. Nonelongated cells also demonstrated sporadic cell-cell cytoplasmic coupling.
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Western analysis of muscle protein
expression. Western analysis of culture lysates
revealed substantial accumulation of SM MHC and SM22 during serum
deprivation that reached a plateau after 12 days (Fig.
5). When normalized to the quantity present
at confluence (day 0), SM MHC and
SM22 exhibited the greatest relative increases; the abundance of SM MHC
and SM22 was increased significantly in lysates collected after 8 days
of serum deprivation (P < 0.05 by
ANOVA and Fisher's least significant difference test). The relatively
smaller increase in SM -actin (<2.3-fold;
P < 0.05 by ANOVA and Fisher's
least significant difference test) likely reflects its greater initial
concentration in confluent cells before serum deprivation (Fig. 5; Ref.
11). Given that the elongated cells contain most, if not all, of these
muscle proteins in serum-deprived cultures (Fig. 2), the relative
increases in mixed culture lysates shown in Fig. 5 represent an
underestimation of the changes in protein content within elongated
myocytes. In contrast to these increasing muscle-specific protein
contents,
-actin abundance in culture lysates remained constant
throughout serum deprivation (P > 0.75 by ANOVA; Fig. 5).
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Contraction of serum-deprived tracheal myocytes. Detachment of elongated cells from the substrata and striking contraction were observed in 62.3% of all elongated cells treated with ACh (n = 77 cells from 3 different cultures; Fig. 6A). These cells shortened by an average of 59.7 ± 14.4% from the original adherent myocyte length, and most cells shortened by 60-80% (Fig. 6B). The duration of shortening was variable and appeared to be dependent on the degree of myocyte attachment to the substratum and contiguous cells. No reelongation of shortened myocytes was seen up to 10 min after maximal contraction was reached. In all serum-deprived elongated myocytes that did not shorten, membrane blebs developed immediately after the first puffs of ACh were received (Fig. 6C). The formation of ACh-induced membrane blebs and myocyte shortening was completely inhibited in cells preincubated with atropine, indicating that ACh-induced contractions resulted directly from muscarinic receptor-mediated signaling. In addition, none of the 10 elongated myocytes that were puffed with HKH solution alone showed either of the above indications of contraction. Thus the ACh-induced contractions observed cannot be attributed to any mechanical effect of the puff. Nonelongated myocytes exhibited no morphological response to ACh exposure.
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Expression of muscarinic-receptor subtypes. M2 muscarinic receptors were ubiquitously expressed on myocytes in confluent cultures before serum deprivation and on myocytes of both elongated SM MHC-positive and flattened SM MHC-negative phenotypes in serum-deprived cultures (Fig. 7). In marked contrast, M3 muscarinic-receptor expression changed markedly after serum deprivation. M3 muscarinic receptors were prominently expressed in dense foci distributed throughout elongated SM MHC-positive myocytes in serum-deprived cultures (Fig. 7); such foci were distinctly absent from flattened SM MHC-negative cells in serum-deprived cultures and from myocytes from confluent, serum-fed cultures (Fig. 7). Interestingly, minor M3 muscarinic-receptor immunoreactivity was uniformly present in a polar, perinuclear region in all myocytes under each condition.
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DISCUSSION |
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In this study, we demonstrate the induction of an unequivocally functional contractile phenotype in a distinct, stable subpopulation of cultured, passaged tracheal SMCs. These myocytes express salient features of contractile cells normally present in intact airway tissue including 1) elongated cellular morphology; 2) a high intracellular abundance of contractile apparatus-associated proteins; 3) characteristic ultrastructural features including myofilaments, caveoli, and cytoplasmic dense bodies; 4) pharmacomechanically coupled M3 muscarinic surface receptors; and 5) contraction and marked cell shortening on ACh exposure. Prior studies (11, 13, 28-31) have shown that growth to confluence, serum withdrawal, manipulation of underlying matrix proteins, or cyclic deformational strain can partially skew cultured airway SMCs toward the contractile phenotype. However, no prior study has demonstrated the thorough reconstitution of contractile myocyte morphology, ultrastructure, and function reported here. Furthermore, our observation that only a small subset of cultured airway SMCs follows this differentiation pathway on serum deprivation is novel and discloses remarkable heterogeneity as a characteristic feature of these cells in culture. Ma et al. (18) have just reported similar findings in experiments that parallel the studies reported here.
The morphological, ultrastructural, and biochemical changes that occur
when contractile phenotype SMCs modulate toward the synthetic-proliferative phenotype have been well documented (5, 8, 11).
In contrast, the factors that orchestrate maturation and coordinated
cytostructural remodeling of noncontractile myocytes into functionally
contractile SMCs are relatively unexplored. Smith and colleagues
(28-30) demonstrated that cyclic stretch of cultured airway SMCs
in serum-containing medium partially restored contractile features.
Cyclic stretch induced myocyte elongation and alignment perpendicular
to the direction of strain, increased numbers of actin stress fibers
and focal adhesions, and accumulation of contractile
apparatus-associated proteins. Boerth et al. (3) reported that stable
transfection of rat aortic SMCs with a gene encoding catalytically
active cGMP-dependent protein kinase induced myocyte elongation and
partially restored contractile protein content to the levels
in intact tissues. Schuger et al. (26), using attached organotypic
cultures from embryonic mouse lungs, observed that elongated,
contractile protein-rich SMCs differentiate from mesenchymal cells, and
some align in concentric layers around epithelial cysts. Similar
changes in SM were observed in peribronchial mesenchymal cells during
bronchial SMC differentiation in cultured embryonic mouse lung explants
(27). Epithelial-mesenchymal contact via basement membranes rich in
laminin-1 was an important determinant of this rearrangement. Our
findings confirm the association between acquisition of the contractile
phenotype and cytostructural remodeling. However, our results also show
that a subset of airway SMCs can acquire a functionally contractile
state and can realign into organized cell clusters, even in the absence
of external physical stimuli or the paracrine influence of other cell
types. Thus it appears that airway SMCs can independently orchestrate
myocyte differentiation, phenotypic expression, and arrangement of
cells into organized units.
Our findings confirm and extend accumulating evidence that airway SMCs are heterogeneous both in vivo (7, 9) and in culture (2, 10, 17). Acutely dissociated tracheal or bronchial SMCs include myocytes with a wide range of cell size, contractile protein content, nuclear ploidy, and proliferative potential (9). Heterogeneity among cultured airway myocytes is not well understood, but these cells are known to vary in their sensitivity to mitogens and to the growth-inhibitory influence of heparin (9, 10). Here, we show that only a distinct subset of tracheal myocytes acquires a functionally contractile phenotype during prolonged serum withdrawal. Both the number of contractile phenotype myocytes and their contractile protein content increase no further after 7 days of serum deprivation, reflecting stability of the two subpopulations identified. Thus cultured airway SMCs are heterogeneous not only with respect to their proliferative potential but also in their capacity to coordinate the expression of that complement of genes that renders them functionally contractile. It is important to note that those myocytes that do not elongate and instead become flattened during serum deprivation still remain viable. They remain attached to culture substrata, they contain intracellular organelles of normal appearance, and they synthesize DNA in response to serum restimulation (8). Thus we believe they represent cells modulated toward the synthetic-proliferative phenotype.
Our results also disclose phenotype-specific cellular localization of M3 muscarinic receptors. M3 muscarinic receptors appeared in prominent foci along the surface of contractile phenotype, SM MHC-positive myocytes in serum-deprived cultures. In contrast, flattened, SM MHC-negative serum-deprived myocytes, as well as confluent, serum-fed myocytes, demonstrated only polar, perinuclear staining. This striking disparity in M3 muscarinic-receptor cellular localization, as well as differences in contractile apparatus protein content, may contribute to the divergence of contractile responses to ACh observed in elongated versus flattened serum-deprived myocytes. Serum-fed SMCs do not generate inositol trisphosphate on ACh stimulation (36), and our flattened, noncontractile serum-deprived myocytes do not exhibit ACh-induced increases in intracellular free ionized calcium (21). Thus, even though perinuclear M3 muscarinic receptors are present in these myocytes, it seems that they do not generate intracellular signals associated with contraction. Apparently, their transport to the cell surface and coupling to intracellular second messengers only occurs in myocytes undergoing modulation to the fully contractile, elongated phenotype. The consistent intracellular distribution of M2 muscarinic receptors in myocytes under all conditions studied contrasts markedly with the phenotype-specific localization of M3 muscarinic receptors and underscores the likely importance of M3 muscarinic-receptor cellular redistribution during contractile phenotype modulation.
In our experiments, elongated SMCs were stimulated to contract while still semiadherent to the culture substratum. This approach allows for a simple and obvious determination of the presence or absence of ACh-induced contraction, but it precludes a precise determination of the unloaded velocity of shortening because an external load is imposed by contiguous cells and matrix interactions. Even so, the extent of shortening observed in most of the contractile cells that did pull free of substratum adhesions was considerably greater than that reported for acutely dissociated airway myocytes (16). Furthermore, we did not observe spontaneous reelongation after contraction as occurs in acutely dispersed cells (16). Together, these findings suggest that the internal elastic load against shortening within our cultured contractile cells may be smaller than that of myocytes acutely dispersed from tissue. Alternatively, differential effects of dissociation enzymes on cultured myocytes and those dissociated from tissue might instead account for the observed differences in shortening and responsiveness. Either of these possibilities could explain the increased velocity of shortening in similarly cultured tracheal myocytes recently described by Ma et al. (18).
Our results raise additional broad questions for which further study seems warranted. These include 1) what are the transcriptional, translational, and posttranslational regulatory mechanisms that control the marked accumulation of contractile apparatus proteins in serum-deprived tracheal myocytes; 2) what regulates intracellular localization of muscarinic receptors in airway myocytes; 3) what underlying factors (internal or external) lead to the distinct differentiation pathways followed by elongated or nonelongated myocytes during serum deprivation, and are these pathways reversible; 4) how is cellular elongation accomplished in the absence of externally applied forces, and what is the relationship of this process to normal smooth muscle development during embryogenesis; and 5) why do individual elongated cells appear to align into bundles? The answers to these questions will no doubt identify additional important aspects of airway SM biology.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. Gerthoffer (University of Nevada, Reno) for the generous gift of the anti-SM22 antibody.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grants HL-56399 and HL-54685; Inspiraplex; Merck-Frosst Canada; Sprague Memorial Institute; the American Lung Association of Metropolitan Chicago; and the American Heart Association of Chicago.
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: J. Solway, Section of Pulmonary and Critical Care, Dept. of Medicine, Univ. of Chicago, 5841 S. Maryland Ave., MC 6026, Chicago, IL 60637.
Received 29 May 1998; accepted in final form 13 October 1998.
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