Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3J7
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ABSTRACT |
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Chronic asthma is characterized by hypertrophy and hyperplasia of airway smooth muscle cells (SMC) that limit airflow by a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of protein changes was seen, no restoration in contractility occurred. Phenotypic modulation of postconfluent cultured airway SMC under prolonged serum deprivation (arrested cells) is reported here. Two phenotypically distinct groups of cells were identified in primary airway SMC cultures: 1) elongated spindle-shaped cells, which expressed large amounts of smooth muscle contractile and regulatory proteins, and 2) flat and stellate cells, which expressed very little. The first group showed a surprising shortening capacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain (MHC) isoform (SM-B) generally said to be associated with the higher shortening velocity disappeared from the cell, while the content of the key rate-limiting regulating enzyme, myosin light chain kinase (MLCK), increased 30-fold. We conclude that a functional, contractile phenotype of airway SMC can be obtained by prolonged serum deprivation. We speculate that the increased contractility could be the result of increased phosphorylation of the 20-kDa myosin light chain resulting from increased content of smooth muscle MLCK rather than any increase in endogenous MHC ATPase activity. This model may be useful for study of SMC differentiation and contraction.
smooth muscle myosin light chain kinase; smooth muscle myosin heavy chain isoform; airway; asthma
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INTRODUCTION |
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SMOOTH MUSCLE CELLS (SMC) exhibit a high degree of plasticity and do not undergo terminal differentiation (15, 24). The principal function of mature SMC is contraction, which depends on expression of a large number of different contractile and regulatory proteins present in precisely controlled concentrations (8). These cells undergo rapid phenotypic modulation in primary culture, manifested by a marked decrease in content of smooth muscle-specific contractile and regulatory proteins and by an increase of nonmuscle proteins (6, 20). Retention of differentiated contractile and regulatory proteins and functional surface receptors were reported in cultured SMC (18), and reexpression of smooth muscle marker proteins was identified in postconfluent cultures (6), but redevelopment of contractility of proliferative SMC has not been shown. Studies using magnetic twisting cytometry and atomic force microscopy suggested that cultured SMC retain the ability to stiffen in response to contractile agonists (9, 10). Ca2+ transients were also reported in cultured SMC in response to a variety of contractile agonists (13, 17). However, this indirect evidence does not prove the existence of contraction in cultured SMC. Increased stiffness of cultured cells as revealed by magnetic twisting cytometry and atomic force microscopy may well be the result of alteration of non-contraction-related cytoskeletal structures. Dissociation of the Ca2+ signal from contraction may occur in cultured SMC. Therefore, these reported properties of cultured SMC cannot be cited as clear evidence for the existence of contraction. Presently it is believed that cells in culture undergo dedifferentiation that only partially reverses at confluence. Functionally, they remain very poorly contractile. Bowers and Dahm (3) reported that the contractile phenotype of freshly isolated SMC could be maintained in a defined medium in which the proliferation of cells was minimal, but once the cells were cultured in proliferative media their contractility was lost rapidly. Gunther et al. (5) and Birukov et al. (2) reported a transient maintenance of smooth muscle contractility in proliferative cultures, but again contractility was lost with increased duration of cell growth. In contrast, with striated muscle cells, relatively little is currently known about molecular mechanisms that control smooth muscle differentiation, due in part to the extreme plasticity of this type of cell and to limitations with respect to the inducibility and/or retention of the differentiated phenotype in cultures.
Therefore, establishment of a contractile SMC culture would facilitate
studies of smooth muscle differentiation and control of SMC
contraction. This was the major objective of our studies. Phenotypic
modulation of primary tracheal SMC culture was examined during
prolonged serum deprivation. Contractility of cultured tracheal SMC was
estimated by direct measurement of zero-load shortening in lifted cells
in response to contractile agonists and electrical stimulation.
Contractility of smooth muscle is regulated by a variety of contractile
and regulatory proteins (8). Smooth muscle myosin light chain kinase
(MLCK) is believed to be the major protein that regulates smooth muscle
contraction. Recently, a new smooth muscle myosin heavy chain (MHC)
isoform (SM-B) that possesses an additional insert of seven amino acids in the NH2 terminus was identified
and reported to be important in determining smooth muscle mechanical
properties (7, 12). Other proteins such as smooth muscle -actin,
total smooth muscle MHC, tropomyosin, caldesmin, and calponin are also
important in determining SMC contractility. Restoration in expression
of these proteins may be a necessary preparation for recovery of
cultured SMC contractility. Therefore, temporal changes in expression
of these proteins were also examined during prolonged serum deprivation in airway SMC cultures. Our results demonstrated that prolonged serum
deprivation resulted in emergence of spindle-shaped phenotype in
cultured tracheal SMC. These cells expressed considerably increased amounts of smooth muscle contractile and regulatory proteins. They
remained elongated after being lifted from the plate and demonstrated
very similar morphology to that of freshly isolated contractile cells;
they shortened in response to contractile agonists and electrical
stimulation. Surprisingly, these cells showed a contractility that was
even greater than that of freshly isolated cells. These data indicate
that a functional contractile phenotype can be induced in tracheal SMC
cultures through prolonged serum deprivation.
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METHODS AND MATERIALS |
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SMC Culture
Tracheae were excised from anesthetized, 6- to 12-mo-old mongrel dogs and placed into ice-cold Ca2+-free Krebs solution. Trachealis muscle was dissected, cleaned of serosa, vasculature, and epithelia at room temperature, and washed four times in Hanks' balanced salt solution (HBSS) containing 100 mg/ml streptomycin and 100 U/ml penicillin under aseptic conditions. The muscle was then minced thoroughly with fine scissors and resuspended in digestion buffer [HBSS containing 600 U/ml collagenase (GIBCO BRL), 8 U/ml type IV elastase (Sigma), and 1 U/ml type XXVII Nagarse protease (Sigma)]. Cells were isolated by serial digestion (3 stages, 45 min each) with vigorous shaking at 37°C. The fractions were pooled, filtered through 70-µm nylon mesh, and then washed and diluted in culture medium (DMEM) containing 10% fetal bovine serum (FBS). After their number was estimated by counting with a model ZBI Coulter counter, the cells were plated into 100-mm plastic culture dishes at a density of 5,000 cells/cm2 and allowed to attach for 36 h. Cells were grown at 37°C in a humidified atmosphere consisting of 95% air-5% CO2. The medium was replaced with fresh medium containing 10% FBS and antibiotics every 72 h. For all the studies, only the primary cultures were used unless otherwise stated. Cultures normally reached confluence within 6-7 days. At confluence, cultures were switched to serum-free medium (F-12 Redu-serum) containing insulin, transferrin, and selenium and arrested for up to 15 days. Spindle-shaped contractile phenotype of cells emerged beginning from day 3 of deprivation and increased in number during continued deprivation. Cells were counted in five views (upper right, upper left, lower right, lower left, and center) directly from each culture dish under a phase-contrast microscope; five dishes were counted for each time point, and the final data were expressed as the arithmetic average of the counts obtained from five dishes.Preparation of SMC Protein Homogenates
Cultured SMC were collected by trypsinization beginning from the day of confluence and every three days during serum deprivation thereafter until day 15. The collected cells were washed twice by resuspension in ice-cold PBS following centrifugation. Crude protein homogenates were prepared in Tris lysis buffer containing 1.5% Nonidet P-40 (NP-40; pH 7.6) to which protease inhibitors (leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor) were added freshly. Cells were disrupted by pipetting several times. Samples were finally stored atWestern Blot Analysis
Proteins in cell homogenates were fractionated by SDS-PAGE on 8 × 10-cm minigels and then transferred to nitrocellulose, as described by Stephens et al. (22). Blots were blocked overnight at 4°C in 0.1% Tween 20-10 mM Tris-buffered saline (TTBS) containing 3% nonfat dried milk powder. Blots were then incubated in primary antibody [1:1,000 monoclonal mouse anti-smooth muscle MLCK (Sigma); 1:10,000 monoclonal mouse anti-smooth muscleFluorescent Immunocytochemistry
Freshly isolated cells were plated in six-well dishes containing 22 × 22-mm rat tail collagen, and collagen type I-coated glass coverslips (Becton Dickinson). When the cells attained confluence, they were arrested for 10 days by withdrawing serum and the coverslips and attached cells were rinsed with PBS and fixed in 1% paraformaldehyde-PBS (pH 7.6) for 15 min at 4°C. They were subsequently permeabilized using 0.1% Triton X-100 in PBS for 15 min at 4°C and then rinsed with PBS and used immediately for immunostaining or stored in PBS containing 0.05% sodium azide for a maximum of 10-14 days. For immunostaining, the coverslips were incubated in blocking solution (PBS containing 5% normal goat serum and 0.1% Tween 20) for 2-4 h at 4°C in a humidified chamber. After rinsing with PBS containing 1% BSA and 0.1% Tween 20, the coverslips were incubated with primary antibodies diluted in PBS containing 1% BSA and 0.1% Tween 20 overnight in a cold room. The dilutions of antibodies were as follows: rabbit anti-nonmuscle MLCK, 1:200; mouse anti-smooth muscle MLCK, 1:25; mouse anti-smooth muscleRT-PCR
To study the expression of the message of a newly identified smooth muscle MHC isoform SM-B (which possesses a 7-amino acid insert in its NH2-terminal head) in cultured tracheal SMC at different time points of serum deprivation, RT-PCR was performed as described by Meer and Eddinger (14). Total RNA was extracted from cells with the use of TRIzol reagent (GIBCO BRL). RNA (1.0 µg) was then added to the RT reaction mixture to a final volume of 10 µl and incubated at 37°C for 2 h. PCR was performed using two oligonucleotide primers, corresponding to rabbit stomach smooth muscle myosin, flanking the 21-nt exon in the NH2-terminal head that encodes the difference sequence between inserted and noninserted smooth muscle MHC isoforms (1). Thirty cycles of amplification were performed. The PCR products generated with the use of these primers were 162 and 141 bp, corresponding to smooth muscle MHC mRNA with or without the 21-nt insert. These products were separated on 4% agarose gel and visualized with staining by ethidium bromide under ultraviolet light.Preparation of Contractile SMC
Fresh tracheal SMC. Fresh tracheal SMC preparation was carried out as described by Gregory and Sims (4). Tracheal smooth muscles were dissected out free of cartilage, epithelium, and vasculature and were cut into strips ~1-2 mm wide and 1 cm long. These were then incubated at 37°C in Hanks' solution with 10 mM taurine, 400 U/ml collagenase, 30 U/ml papain (Sigma), 1 mg/ml BSA, and 1 mM dithiothreitol for 45 min. After enzymatic digestion, the tissue was washed with Ca2+-free Krebs-Henseleit solution several times and the cells were finally dispersed by gentle trituration with a Pasteur pipette in Ca2+-free Krebs-Henseleit solution and stored on ice for study within 6 h. Most freshly isolated cells appear fully relaxed with smooth and phase-lucent sarcolemmal membranes as seen under an inverted microscope. Addition of relaxing agents such as norepinephrine, atropine, and isoproterenol did not cause further relaxation of cells. These cells showed reversible contractile responses to ACh, histamine, KCl, and single-pulse electrical stimulation.
Arrested cultured SMC. Arrested cultures were first washed three times with Ca2+- and Mg2+-free PBS prewarmed to 37°C. Cells were lifted by addition of PBS containing 0.05% trypsin and 0.53 mM EDTA and by occasional gentle shaking. Lifted cells were then stored at 4°C, without washing or any other disturbance, for further study. This was critical to keep serum deprivation-induced spindle-shaped cells in the relaxed state. Measurement of cell shortening was conducted within 1 h.
Measurement of Single Cell Shortening
Measurement of single cell shortening was conducted at room temperature. A drop of cell preparation was transferred to a custom-designed chamber containing 1 ml aerated Krebs-Henseleit solution and allow to settle for 5 min, after which the chamber was slowly perfused with fresh Krebs-Henseleit solution. Cell length was measured with an inverted microscope. Maximum shortening of cells was elicited by applying bipolar electric pulse stimulation (10 Hz, 40 V, 1 ms width), with one spot electrode placed ~10 µm away from the cell and the other at a random position in the chamber. Throughout the experiment, images of the cells were monitored and recorded via a charge-coupled device video camera mounted on the microscope. Cell shortening was then analyzed with a computer program (peak5) for maximal shortening capacity (Data Analysis
Data are expressed as means ± SE. Vo and ![]() |
RESULTS |
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Morphologically, two distinct groups of cells appeared in postconfluence primary SMC culture during long-term serum deprivation (Fig. 1): one group of cells appeared flat and bright under the inverted microscope; these comprised almost all the cells that were present before serum deprivation. The second group demonstrated the normal elongated spindle shape and were aligned side by side in bundles in most cases; they were dark but possessed a shining sarcolemma. This group began to appear after 2 days of serum deprivation and increased in number as deprivation was prolonged. After 15 days of deprivation, they comprised 28.5% ± 4.6 (SE) of all cells present but occupied almost 40% of total area of the culture dish. When cells were lifted using trypsin-EDTA, those in the first group "balled up" much as nonarrested cells do, whereas those in the second group retracted but remained elongated. Their average length was similar to that of the freshly isolated tracheal SMC (110 ± 8 µm).
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These elongated SMC showed normal reversible contraction on
stimulation with optimal doses of ACh
(105 M), histamine
(10
5 M), KCl (10 mM), and
low-level single-pulse electrical stimulation (10 V; Fig.
2). Maximal contraction could be induced
with repeated pulse electrical stimulation. Figure
3 shows typical curves of unloaded
shortening of freshly isolated and the newly induced contractile single
tracheal SMC under repeated pulse electrical stimulation. Surprisingly
these newly induced contractile cells evinced hypercontractility to
electrical stimulation (Fig. 4). Thus they
shortened maximally by 50% of their original length at room
temperature on repeated electrical stimulation, whereas freshly
isolated cells only shortened by 27%. These cells shortened faster,
with the Vo at
8% of cell length/s, almost double the value for freshly isolated
cells. In addition, arrested cells responded to electrical stimulation
at lower current intensity than freshly isolated cells, indicating a
higher sensitivity to electrical stimulation. The voltages inducing
half-maximal contraction for freshly isolated and arrested contractile
cells were 18 ± 3 and 11 ± 2 V, respectively. These were
significantly different (P < 0.05).
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In correspondence with the time-dependent increase in number of
elongated spindle-shaped cells, expression of contractile and
regulatory proteins increased, as revealed by Western blot analysis
(Fig. 5). After 15 days of arrest, total
smooth muscle MHC content increased 10.8 ± 1.1-fold, smooth muscle
-actin content increased 5.9 ± 1.0-fold, and smooth muscle MLCK
increased by 62.9 ± 13.5-fold compared with contents of cells in
nonarrested confluent cultures. The content of smooth muscle type MHC
reached the same level as in freshly isolated cells. Surprisingly, the contents of smooth muscle MLCK and
-actin were significantly (~30
and 2 times, respectively) higher than those of freshly isolated cells
(Fig. 6). The expression of SM-B (the
7-amino acid "inserted" isoform of smooth muscle
MHC) was not detected at all tested time points, both at protein and
message level (Fig. 7).
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To further confirm that the newly emerging spindle-shaped cells express
more smooth muscle contractile and regulatory proteins, an
immunocytochemical study was employed. Results of experiments employing
specific antibodies showed that spindle-shaped cells stained
intensively for smooth muscle type MHC, -actin, and smooth muscle
MLCK, whereas the flat cells stained quite faintly with smooth muscle
MLCK and were almost negative to smooth muscle MHC and
-actin
staining (Fig. 8). In addition, these cells
also stained strongly for nonmuscle MLCK and
-tubulin (Fig.
9). Centrally located cigar-shaped nuclei
were identified in spindle-shaped cells, but round nuclei were seen in
flat ones (Fig. 8).
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The above evidence demonstrated that serum deprivation resulted in phenotypic modulation of cultured airway SMC from synthetic to contractile, in spite of the fact that SM-B, the isoform reported to be responsible for conferring Vo, was considerably downregulated.
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DISCUSSION |
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The concept of plasticity of SMC is widely accepted and considered to be a necessary part of the SMC differentiation program that evolved because it conferred a survival advantage to the organism (19). It is well established that the SMC can change its phenotype from contractile to synthetic, and vice versa, in response to environmental influences (15). The mature contractile SMC was reported to undergo a rapid modulation of its phenotype to the immature synthetic type during culture in the presence of serum (6, 20). This was demonstrated by a considerable decrease in smooth muscle-specific contractile and regulatory proteins and an increase in nonmuscle type proteins. Direct evidence for reversal of modulation of cultured SMC, i.e., redevelopment of contractile properties, has never been demonstrated, although reaccumulation of smooth muscle MHC was found in postconfluent cultures (this is the biochemical concomitant of contractile phenotype) (6). Recovery of contractile responsiveness was reported in cultured SMC (13, 17). However, increased intracellular Ca2+ transient and/or myosin light chain (MLC) phosphorylation in response to contractile agonists were used as indexes of contractility. These are indirect methods for inferring contractility and have limited usefulness. Our results demonstrate that mature contractile SMC can be induced in postconfluent primary cultures under long-term serum deprivation. After "lifting," these cells retained their normal spindle shape and shortened isotonically in response to contractile agonists such as ACh, histamine, KCl, and electrical stimulation, which also indicates that appropriate receptors were present. The data demonstrate that cultured airway SMC (synthetic phenotype) retain the capability to reverse their phenotype and undergo differentiation to contractile phenotype as a result of serum deprivation. Our studies provide novel direct evidence that under appropriate conditions cultured noncontractile SMC developed into a contractile phenotype.
Molecular mechanisms that control the differentiation program of SMC
have not yet been identified. The SMC is believed to be remarkably
plastic in that it can undergo rapid and reversible changes of its
phenotype in response to a variety of different stimuli. Consistent
with this property, differentiation of SMC appears to be highly
dependent on environmental influences (16). Our results demonstrated
that the differentiated phenotype of cultured airway SMC could be
induced and maintained as the result of serum deprivation. This
implicates the importance of growth arrest in determining SMC
differentiation and may provide a model to study differentiation of
SMC. The redevelopment of normal contractility of cultured SMC was
accompanied by reappearance of smooth muscle contractile, structural,
and regulatory proteins, such as smooth muscle MHC, -actin, and
smooth muscle MLCK, and morphological reversion to normal spindle
shape. Our data also show that the contents of nonmuscle MLCK and
-tubulin remain elevated in these contractile cells. This provides
an additional difference between arrested contractile and freshly
isolated cells. This suggests that downregulation of nonmuscle type
proteins may not be required in induction of contractile type of cells
from cultured noncontractile SMC under serum deprivation. Communication
among cells may also be important in differentiation under serum
deprivation, because induced contractile cells were found in most cases
formed into bundlelike clusters aligned in parallel. Instead of
invoking communication between cells, the grouping of the contractile
cells in bundles can also be attributed to their forming a colony that
originated from a common precursor. We currently cannot
distinguish these two possibilities.
Induction of contractile phenotype in cultured tracheal SMC was found
to be accompanied by considerable increased expression of smooth muscle
-actin, smooth muscle MHC, and smooth muscle MLCK during prolonged
serum deprivation. Surprisingly, we did not find corresponding changes
in expression of the smooth muscle MHC isoform (SM-B) that possesses a
seven-amino acid insert in its NH2
terminus. Actually, we did not detect any expression at all of SM-B at
either protein or message levels in cultured tracheal SMC before or
during serum deprivation, but it was prominently detected in freshly
isolated tracheal SMC. Expression of SM-B has been reported to be
important in determining smooth muscle mechanical properties (25). It
confers a cycling velocity on the muscle that is three times faster
than those of the other isoforms (12). Our finding seems contradictory
to this notion. The newly induced spindle-shaped cultured tracheal SMC
showed an elevated contractility as a result of arrest but did not
express SM-B, whereas freshly isolated cells did express SM-B isoform but showed lower contractility, indicating that proteins other than
SM-B are responsible for the supercontractility of newly induced
contractile cells. Dissociation of the content of SM-B expression from
contractile properties of smooth muscle was also found by other
investigators. Haase and Morano (7) reported a decrease of SM-B
expression while the
Vo of smooth
muscle increased in pregnant rat myometrium fibers. Siegman et al. (21)
recently found that no correlation between the amount of SM-B and
shortening velocity existed in mouse megacolon. The latter pointed out
that to compare the velocities regulated by the different MHC isoforms full phosphorylation of the 20-kDa MLC
(MLC20) must be ensured. When
this was carried out using adenosine
5'-O-(3-thiotriphosphate), no
difference in velocity due to the different isozymes was seen. The
correlation reported by others between
Vo and MHC
isozyme is therefore not the effect of the isozyme itself.
Smooth muscle MLCK is an important candidate that could be responsible
for the correlation between
Vo and MHC
isozyme activity. Considerable increase in smooth muscle MLCK content
was found in newly induced contractile cells. After 15 days of serum
deprivation, smooth muscle MLCK content in cultured cells increased
62.9-fold compared with cells in nonarrested confluent culture. Our
previous studies showed that the content of smooth muscle MLCK in
confluent tracheal SMC culture decreased by 50% compared with that of
freshly isolated cells (6). Arrested cells expressed smooth muscle MLCK
at a content of 30 times that of freshly isolated cells; this may
contribute to the increased contractility of arrested cells. Smooth
muscle MLCK is known to be a primary regulator of smooth muscle
contraction through
Ca2+/calmodulin-dependent
phosphorylation of regulatory MLC
(MLC20). Increased smooth muscle
MLCK content and activity would lead to increase of
MLC20 phosphorylation with
concomitant increase in velocity during smooth muscle activation. The
importance of smooth muscle MLCK in regulating smooth muscle
contractility was supported by our previous studies on ragweed
pollen-sensitized canine airway smooth muscle, in which an increased
smooth muscle contractility was found to be closely correlated with the
increased content and activity of smooth muscle MLCK (11). Stephens and
Jiang (23) recently reported on the basis of a motility assay that in
vitro motility of the smooth muscle myosin head increased with increase
of smooth muscle MLCK concentration, which further demonstrated the
significance of smooth muscle MLCK in regulating smooth muscle contractility. Increased expression of smooth muscle -actin may also
contribute to the increased contractility, by providing more attachment
sites for myosin heads.
In conclusion, our data demonstrate that a functional, fully contractile phenotype is induced in cultured tracheal SMC as a result of prolonged serum deprivation. These newly induced contractile SMC possess even greater contractility than freshly isolated tracheal SMC. This model could provide a tool for studies to determine the relationship between smooth muscle differentiation and contraction. The development of hyperreactivity in cultured SMC, stemming from growth arrest as a result of serum deprivation, represents a novel finding. The responsible mechanisms need to be investigated. The existence of cellular heterogeneity also requires that these mechanisms be delineated for the different subpopulations of cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert Low for his generous gift of SM-B antibody and Dr. R. S. Adelstein for primers of RT-PCR. We are grateful to Dr. J. Dodd and members of her lab for expert assistance with RT-PCR.
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FOOTNOTES |
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This work was supported by operating grants from the Medical Research Council of Canada and Inspriaplex Canada.
Y. Wang is a research fellow from Norman Bethune University of Medical Sciences, China.
Address for reprint requests: X. Ma, Rm. 425, BMSB, 730 William Ave., Winnipeg, MB, Canada R3E 3J7.
Received 2 September 1997; accepted in final form 18 December 1997.
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REFERENCES |
---|
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---|
1.
Babij, P.
Tissue specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain.
Nucleic Acids Res.
21:
1467-1471,
1993[Abstract].
2.
Birukov, K. G.,
M. G. Frid,
J. D. Rogers,
V. P. Shirinsky,
V. E. Koteliansky,
J. H. Campbell,
and
G. R. Campbell.
Synthesis and expression of smooth muscle phenotype markers in primary culture of rabbit aortic smooth muscle cells: influence of seeding density and media and relation to cell contractility.
Exp. Cell Res.
204:
46-53,
1993[Medline].
3.
Bowers, C. W.,
and
L. M. Dahm.
Maintenance of contractility in dissociated smooth muscle: low-density cultures in a defined medium.
Am. J. Physiol.
264 (Cell Physiol. 33):
C229-C236,
1993
4.
Gregory, R. W.,
and
S. M. Sims.
Muscarinic stimulation of trachea smooth muscle cells activates large-conductance Ca2+-dependent K+ channel.
Am. J. Physiol.
265 (Cell Physiol. 34):
C658-C665,
1993
5.
Gunther, S.,
R. W. Alexander,
W. J. Atkinson,
and
M. A. Gimbrone, Jr.
Functional angiotensin II receptors in cultured vascular smooth muscle cells.
J. Cell Biol.
92:
289-298,
1982[Abstract].
6.
Halayko, A. J.,
H. Salari,
X. Ma,
and
N. L. Stephens.
Markers of airway smooth muscle cell phenotype.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L1040-L1051,
1996
7.
Haase, H.,
and
I. Morano.
Alternative splicing of smooth muscle myosin heavy chain and its functional consequences.
J. Cell. Biochem.
60:
521-528,
1996[Medline].
8.
Horowitz, A.,
C. B. Menice,
R. Laporte,
and
K. G. Morgan.
Mechanisms of smooth muscle contraction.
Physiol. Rev.
76:
967-1003,
1996
9.
Hubmayr, R.,
S. Shore,
J. Fredberg,
E. Planus,
R. Panettieri,
and
N. Wang.
Cytoskeletal mechanics of human airway smooth muscle cells (Abstract).
Am. Rev. Respir. Dis.
151:
A125,
1995.
10.
Jain, M.,
D. Berger,
B. Camoretti-Mercado,
S. Shore,
K. Robison,
P. Schumacker,
L. Alger,
Q. Niu,
and
J. Solway.
Detection of individual tracheal myocyte contraction using atomic force microscopy (Abstract).
Am. Rev. Respir. Dis.
153:
A168,
1996.
11.
Jiang, H.,
K. Rao,
A. J. Halayko,
X. Liu,
and
N. L. Stephens.
Ragweed sensitization-induced increase of myosin light chain kinase content in canine airway smooth muscle.
Am. J. Respir. Cell Mol. Biol.
7:
567-573,
1992[Medline].
12.
Kelley, C. A.,
J. R. Takahashi,
J. H. Yu,
and
R. S. Adelstein.
An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature.
J. Biol. Chem.
268:
12848-12854,
1993
13.
Li, X.,
P. Tsai,
E. D. Wieder,
A. Kribben,
V. Van Putten,
R. W. Schrier,
and
R. A. Nemenoff.
Vascular smooth muscle cells grown on Matrigel: a model of the contractile phenotype with decreased activity of mitogen-activated protein kinase.
J. Biol. Chem.
269:
19653-19658,
1994
14.
Meer, D. P.,
and
T. J. Eddinger.
Heterogeneity of smooth muscle myosin heavy chain expression at the single cell level.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1819-C1824,
1996
15.
Owens, G. K.
Regulation of differentiation of vascular smooth muscle cells.
Physiol. Rev.
75:
487-517,
1995
16.
Owens, G. K., M. S. Vernon, and C. S. Madsen. Molecular regulation of smooth muscle
differentiation. J. Hypertens. 4, Suppl. 5: S55-S64, 1996.
17.
Panettieri, R. A.,
R. K. Murray,
L. R. DePalo,
P. A. Yadvish,
and
M. L. Kotlikoff.
A human airway smooth muscle cell line that retains physiological responsiveness.
Am. J. Physiol.
256 (Cell Physiol. 25):
C329-C335,
1989
18.
Rothman, A.,
T. J. Kulik,
M. B. Taubman,
B. C. Berk,
C. W. Smith,
and
B. Nadal Ginard.
Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures.
Circulation
86:
1977-1986,
1992[Abstract].
19.
Schwartz, S. M.,
G. R. Campbell,
and
J. H. Campbell.
Replication of smooth muscle cells in vascular disease.
Circ. Res.
58:
427-444,
1986[Abstract].
20.
Shanahan, C. M.,
P. L. Weissderg,
and
J. C. Metcalfe.
Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells.
Circ. Res.
73:
193-204,
1993[Abstract].
21.
Siegman, M. J.,
T. M. Butler,
S. U. Mooers,
L. Trinkle-Mulcahy,
S. Narayan,
L. Adam,
S. Chacko,
H. Haase,
and
I. Morano.
Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1571-G1580,
1997
22.
Stephens, N. L.,
A. J. Halayko,
and
B. Swynghedauw.
Myosin heavy chain isoform distribution in normal and hypertrophied rat aortic smooth muscle.
Can. J. Physiol. Pharmacol.
69:
8-14,
1991[Medline].
23.
Stephens, N. L., and H. Jiang. Velocity of
translation of single actin filaments (AF) by myosin heads from
antigen-sensitized airway smooth muscle. Mol. Cell.
Biochem. In press.
24.
Thyberg, J.
Differentiated properties and proliferation of arterial smooth muscle cells in culture.
Int. Rev. Cytol.
169:
183-265,
1996[Medline].
25.
White, S.,
A. F. Martin,
and
M. Periasamy.
Identification of a novel smooth muscle myosin heavy chain cDNA: isoform diversity in the S1 head region.
Am. J. Physiol.
264 (Cell Physiol. 33):
C1252-C1258,
1993