1 Division of Urology and 2 Department of Pathobiology, University of Pennsylvania, and 3 Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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We have established a cell line from
hypertrophied rabbit urinary bladder smooth muscle (SM) that stably
expresses SM myosin (SMM). These cells, termed BSM, are spindle shaped
and form swirls, similar to the "hills and valleys" described for
cultured aortic SM cells. Western blotting revealed that BSM expresses
the amino-terminal SMM heavy chain isoform SM-B, the carboxy-terminal
SM1 and SM2 isoforms, and SM -actin. In addition, they express
cGMP-dependent protein kinase G, made by contractile SM cells in vitro
but not by noncontractile cells synthesizing extracellular matrix.
Immunofluorescence studies indicate a homogeneous population of cells
expressing
-actin and SMM, including the SM-B isoform, and
karyotyping demonstrates a stable 4N chromosomal pattern. These cells
also express calcium-dependent myosin light chain kinase and
phosphatase activity and contract in response to the muscarinic agonist
bethanechol. To our knowledge, BSM is the first visceral SM cell line
that expresses the SM-B isoform and might serve as a useful model to
study the transcriptional regulation of tissue-specific SMM isoforms in
differentiation and pathological SM.
contractility; SM-B; kinase; phosphatase; cGMP-dependent protein kinase
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INTRODUCTION |
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PHENOTYPIC MODULATION OF smooth muscle (SM) cells is associated with various pathological conditions including bladder dysfunction (27, 50), pulmonary hypertension (38), atherosclerosis, and restenosis after angioplasty (2). However, the exact mechanisms underlying the modulation of the SM phenotype in these tissues are not known, despite extensive studies on SM in vivo and in vitro (25, 28, 36, 48). One factor complicating elucidation of these mechanisms is the heterogeneity of SM cells with respect to myosin isoform expression and other biological properties (24, 49).
SM cell culture provides an excellent tool to study cellular function,
provided the cells grown in vitro are capable of expressing the in vivo
phenotype. However, cells enzymatically liberated from SM are also
heterogeneous and lose their differentiated properties when cultured
(6, 11, 29, 45). Most studies in which cultured bladder
myocytes have been used relied only on the expression of -SM actin
as a marker for SM (12, 39, 53). In a study by Kropp et
al. (32), SM myosin (SMM) expression and contractility were examined in cultured myocytes that had been passaged two to five
times, but they found only a small percentage of the isolated cells
staining for SMM, and the cells did not contract in response to
agonists that typically cause bladder SM tissue contraction.
Several attempts have been made to establish SM cell lines
derived primarily from vascular SM (31, 44). However,
Firulli et al. (23) showed that two of the major vascular
SM cell lines reported to date (PAC1 and A7r5), although retaining
expression of -actin, express virtually no SMM mRNA on passaging.
Moreover, karyotyping of both of these cell lines revealed alterations
in chromosome number and/or structure (23).
The two SMM heavy chain isoforms SM-A and SM-B are generated from a single gene through alternative splicing of the pre-mRNA at the 5' end, whereas splicing at the 3' end of this gene produces the SM1 and SM2 heavy chain isoforms (9, 10). The SM-B isoform contains a seven-amino acid insert in the amino-terminal region near the ATP-binding site. This isoform is found in visceral SM and small muscular arteries, but not in large arteries such as the aorta or pulmonary artery, which express ~100% of the SM-A isoform (20), and has a higher ATPase activity and increased in vitro motility of the myosin (20, 30) as well as increased shortening velocity of the muscle itself (20) compared with the SM-A isoform. A cell line generated from urinary bladder SM, having contained almost entirely SM-B isoform, is likely to provide a homogeneous population of cells with respect to this myosin isoform. Such a population of cells is valuable for studying the transcriptional regulation of the SM-B SMM heavy chain isoform.
SM cells also express an embryonic form of SMM heavy chain known as SMemb, which is encoded by a gene distinct from that encoding SMM (33). This SMemb myosin is expressed at high levels during embryonic development and then decreases dramatically after birth and is expressed at only trace levels in the adult SM (33). The SMemb isoform has been shown to be identical to the nonmuscle myosin B heavy chain isoform found in brain (47) but to be distinct from the nonmuscle myosin A isoform. Expression of SMemb is upregulated in fetal SM (33) and in adult SM in pathological conditions including atherosclerosis (3) and after vascular stent implantation (2).
In addition to SMM expression, the expression of cGMP-dependent protein
kinase (PKG) type I has been suggested as another key marker of the SM
phenotype (14). SM cells isolated from rat aorta express
high levels of type I PKG. However, on passaging, PKG levels decrease
rapidly, such that after several passages PKG expression is
undetectable. This decreased expression of PKG correlates with a switch
to a more synthetic phenotype (18). Moreover, transfection
of these passaged cells with cDNA for PKG-1 returns the cells to a
contractile phenotype (14), supporting a role of PKG in
maintenance of the SM phenotype. Whereas PKG-1
is expressed
abundantly in many different cell types, its alternatively spliced
counterpart, PKG-1
, is expressed only in SM.
The contraction of SM is regulated by phosphorylation of the 20-kDa myosin regulatory light chain (LC20) (1). The primary enzyme responsible for phosphorylation of SMM is calcium-dependent myosin light chain kinase (MLCK), whereas dephosphorylation of the light chain is accomplished by myosin phosphatase. The phosphatase that is thought to be the major regulator of SM is PP1, which is a multimeric protein consisting of a 37-kDa catalytic subunit, a 21-kDa M21 subunit, and a 130/133-kDa (MYPT1) myosin-targeting subunit. Thus, for a SM cell to remain contractile, it must retain kinase and phosphatase activity. In vivo, bladder SM must be able to respond to neuronal signals. Stimulation of the muscarinic cholinergic receptor sites by acetylcholine plays a major role in bladder SM contraction, and thus the muscarinic agonist bethanechol has widely been used to assess bladder contractility (51).
Here we report a stable SM cell line (BSM), derived from the SM of
hypertrophied urinary bladder, that continues to express both -actin
and SMM even after >50 passages. These cells also retain expression of
PKG, another marker protein for the differentiated SM phenotype, and
have a stable chromosome pattern. In addition, BSM cells exhibit
calcium-dependent MLCK and phosphatase activity and retain the ability
to contract in response to bethanechol. This cell line should prove a
valuable tool to study transcriptional regulation of myosin isoforms
and the effects of factors or etiological agents that induce
pathological changes in SM. In addition, BSM might be a useful source
of SM cells for tissue engineering research utilizing rabbits as an
animal model and in perfecting the techniques for enhancement of
bladder or urethral function in diseases of the lower urinary tract.
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METHODS |
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Partial outlet obstruction. All studies involving animals were approved by the Children's Hospital of Philadelphia and the University of Pennsylvania's Animal Use Committee. Hypertrophied urinary bladder SM tissue was obtained from a 4-mo-old male New Zealand White rabbit with a 2-wk partial obstruction of the bladder outlet induced by surgical ligation of the urethra (34). The bladder from this rabbit, which had developed in vivo urinary bladder dysfunction, was surgically removed under sterile conditions and placed into sterile M199 medium containing 1% nutrient mix and 1% antibiotic-antimycotic (GIBCO BRL, Rockville, MD).
Cell dissociation and propagation. After removal of the mucosa and serosa, bladder body SM from a 2-wk partially obstructed rabbit bladder was minced into small pieces (1 × 1 mm) and incubated overnight in M199 medium (GIBCO BRL) with collagenase (1 mg/ml; GIBCO BRL) to dissociate bladder myocytes. The single cells were collected by centrifugation at 1,000 g at 4°C. The pellet was resuspended and washed in M199 and cultured in nutrient medium [M199 + 10% FCS (GIBCO BRL)] in 60-mm tissue culture dishes (Corning, Action, MA) at 37°C in a humidified atmosphere of 5% CO2-95% air. Primary cultures were grown for 2 days, and small colonies were picked with a Pasteur pipette and flushed to mechanically dissociate the cells. The dissociated cells were subcultured. This procedure was repeated several times until a stable morphology was obtained, as confirmed by light microscopy. Once cells reached sufficient densities, large-scale amplification was performed in T75 flasks (Corning) and cells were continuously passaged >50 times, after brief treatment with trypsin (0.05% trypsin-EDTA; GIBCO BRL). Cells from various passages were scraped off with a rubber policeman and analyzed for expression of SM-specific proteins by Western blotting and immunofluorescence microscopy. After passage 50, a growth curve was generated. Cells were seeded in six-well plates (Corning) at an initial density of 4 × 104 cells/well in growth medium as described above. Triplicate hemacytometer readings were recorded each day after seeding. NIH/3T3 cells were used as a control for the growth curve.
Immunofluorescence microscopy.
Cells from various passages in M199 medium were grown on rat tail
collagen (Sigma, St. Louis, MO)-coated coverslips. All subsequent solutions were prepared in 1× PBS. Cell-containing coverslips were
fixed in a solution of 70% ethanol, 1% formaldehyde, and 5% glacial
acetic acid for 10 min. Cells were then washed three times with PBS,
incubated for 30 min in 1% BSA to block nonspecific binding, and
incubated for 1-2 h at room temperature with primary antibody
against the following SM-specific proteins: SMM (1:1,000), SM-B
(1:500), and -actin (1:1,000). After three washes in PBS, cells were
treated with a second antibody (FITC-conjugated goat anti-mouse IgG or
IgM for SM-B, both at 1:100) for 1 h, washed three times with PBS,
and mounted with a drop of Aqua-Mount (Lerner Labs, Stamford, CT). All
antibodies were purchased from Sigma, with the exception of the
SM-B-specific antibody that was generated in our laboratory against the
seven-amino acid insert of rabbit SM-B, as described and characterized
previously (21). Cells were viewed under a fluorescence
microscope (Leitz). Negative controls were treated in the same way,
except that the primary antibody was replaced with mouse preimmune
serum or the second antibody was omitted.
Protein extraction and Western blot analysis.
The cell pellet was homogenized in extraction buffer at 5 × 106 cells/ml buffer containing 50 mM Tris, pH 6.8, 20%
glycerol, and protease inhibitors [0.8 mM phenylmethylsulfonyl
fluoride (PMSF), 10 µM pepstatin, 1 µM antipain, and 0.1 mg/ml
trypsin inhibitor; all from Sigma]. After addition of SDS to a final
concentration of 1%, samples were boiled and spun at 15,000 g for 20 min. A small portion of the supernatant was used
for protein determination by the Bio-Rad DC (detergent compatible) kit.
Bromphenol blue and 25 mM dithiothreitol (DTT; which interferes with DC
assay) were added to the remainder of the sample for analysis by
SDS-PAGE. Bladder SM from normal adult 4-mo-old rabbits and NIH/3T3
cells were extracted in a similar manner as positive and negative
controls, respectively, for the expression of SMM isoforms. For all
samples, equal amounts (20 µg) of total extractable protein were
separated in mini 7.5% SDS-PAGE gels or to separate the SM1 and SM2
SMM isoforms, samples containing equal amounts of total protein (10 µg) were loaded onto highly porous 4.5% (1 mm thick, 16 cm long) polyacrylamide gels and separated by slow electrophoresis overnight at
4°C as previously described (21). Since SMemb has been
shown to comigrate with SM2, similar gels were transferred overnight to
Immobilon-P membranes (Millipore, Bedford, MA) as previously described
(21), blocked with 5% nonfat dry milk for 1 h, and incubated with a 1:20,000 dilution of SMM-specific primary antibody (clone hSM-V) from Sigma or a 1:10,000 dilution of antibody specific for SMemb [a gift from Dr. Robert Adelstein at the National Institutes of Health (NIH)]. After being washed three times in 1× PBS plus 0.1%
Tween 20, secondary horseradish peroxidase-linked antibody (1:10,000
dilution of sheep anti-mouse Ig for SMM antibody or 1:5,000 dilution of
donkey anti-rabbit Ig for SMemb) was added for 1 h. Antibody
reactivity was detected by enhanced chemiluminescence (Amersham
Pharmacia Biotech, Buckinghamshire, UK). In a similar manner, Western
blot analysis was also performed for SM-B (1:500) and SM -actin
(clone 1A4, 1:1,000; Sigma) and for MLCK (clone K36, 1:10,000; Sigma),
MYPT1 (clone ASC.M130, 1:10,000; Covance, Richmond, CA), and PKG
(KAP-PK005, 1:4,000; Stressgen Biotechnology, Victoria, BC, Canada),
except that IgM second antibody was used for SM-B and the blots were
developed with 3,3'-diaminobenzidine (DAB; Sigma).
Kinase assay.
Kinase activity was determined as previously described
(16). Briefly, kinase was extracted from the BSM cell line
by homogenizing 0.8 g of cells using a mini electric homogenizer
in 2.0 ml of extraction buffer [in mM: 60 KCl, 40 imidazole
hydrochloride (pH 7.1), 2 EDTA, 2 DTT, 10 ATP, and protease inhibitor
cocktail containing 0.2 PMSF; and in µM: 1 antipain, 1 pepstatin, and
5 trypsin inhibitor] followed by centrifugation at 20,000 g
for 20 min. The cell extract present in the supernatant was collected
and dialyzed overnight in phosphorylation buffer [in mM: 10 imidazole
hydrochloride (pH 7.1), 5 DTT, and protease inhibitor cocktail] as
above. Next, 20 µl of the cell extract were assayed for kinase
activity in a total volume of 150 µl containing (in mM) 5 MgCl2, 0.1 CaCl2, 10 imidazole hydrochloride
(pH 7.1), and 2 ATP. Fifty micrograms of exogenous purified chicken
gizzard SMM prepared as described (17) were also added to
each assay and the reaction started by adding
[-32P]ATP. The reaction was stopped at various times
by adding 150 µl of the reaction mix to 1 ml of 10% TCA-2% sodium
pyrophosphate, the mixture was centrifuged at 14,000 g for
20 min, and then the supernatant was dissolved directly in SDS-PAGE
sample buffer. The pH was adjusted to neutrality with Tris and the
samples separated in a 14% SDS-PAGE gel. The gel was then stained with
Coomassie blue and exposed to X-ray film. The radiolabeled band
representing the LC20 was then excised from each lane,
added to a scintillation vial, crushed with a spatula, mixed with
scintillation fluid, and counted in a scintillation counter. The
protein concentration of the extracts used for the kinase assay were
determined by the Bradford assay (15), and then the
specific activity of the kinase was calculated as the number of moles
phosphate bound to the LC20 per minute per milligram of
cell extract protein. The same assay was conducted in the absence of
calcium with 2 mM EGTA to determine the calcium dependence of the
kinase activity.
Phosphatase assay.
The cell extracts prepared as described under the kinase assay were
assayed for phosphatase activity in [in mM: 5 MgCl2, 0.1 CaCl2, 10 imidazole hydrochloride (pH 7.1)]. Purified
gizzard myosin phosphorylated using [-32P]ATP was used
as the substrate. The reaction was started by adding 50 µg of
exogenous myosin with covalently bound
[
-32P]phosphate. The reaction was stopped by adding
TCA to a final concentration of 10% and sodium pyrophosphate to 2%
final concentration, and the amount of phosphate released per
minute per milligram protein was determined by filter binding as
previously described (16).
Cell contraction studies. Muscle cells adhered to glass coverslips coated with rat tail collagen were placed into the temperature-controlled recording chamber on an inverted microscope (Nikon TE300). The bath solution consisted of (in mM) 155 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES and was adjusted to pH 7.4. Bright-field images were obtained with an intensified charge-coupled video camera (Hammamatsu model C2400-68) connected to the side port of the inverted microscope. Video images were averaged and digitized (0.5 Hz) with a video frame grabber (DVP32, Instrutech) using Metafluor acquisition and analysis software (Universal Imaging). Stored images were analyzed off-line using the Metafluor package. Contraction was induced by bath application of bethanechol (1-100 µM), and relaxation was subsequently induced by perfusion with agonist-free extracellular solution.
Chromosome preparation. At passage 50, dissociated cells from the BSM bladder SM cell line were exposed to colcemid for 2-3 h, and metaphase-arrested chromosome spreads were prepared by standard methodology and stained with Giemsa. A minimum of 20 randomly selected metaphase spreads were photographed, and chromosome number and structure were compared with normal rabbit chromosomes.
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RESULTS |
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Growth characteristics.
The BSM cells proliferated at a steady rate and exhibited a uniform
growth pattern in medium containing 10% FCS. The growth curve of BSM
revealed a doubling time of ~58 h (Fig.
1), similar to that of NIH/3T3
cells cultured under the same conditions (data not shown) and of other
SM cell lines (23). When subcultured, the BSM cells grew
logarithmically for ~3 days under our culture conditions and reached
a plateau at ~5 days. Similar results were also obtained at passage
numbers 15 and 30. Phase-contrast microscopy revealed fibroblast-like
cells in sparse cultures (Fig.
2A); in confluent cultures,
these cells become spindle-shaped (Fig. 2B) and arranged in
a swirly pattern (Fig. 2C) similar to the "hills and
valleys" described for cultured aortic SM cells (43).
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SMM and -actin expression.
Western blot analysis using an antibody that specifically recognizes
all known isoforms of SMM revealed significant amounts of SMM in BSM
(Fig. 3A, lanes 1 and
2), although amounts of SMM per milligram of extracted
protein were less than that found in normal bladder SM (lane
N). No antibody reactivity was detected in the total protein
extract from NIH/3T3 cells, which produce predominantly nonmuscle
myosin (both A and B isoforms; lane T), confirming the
specificity of this antibody for smooth vs. nonmuscle myosin as also
indicated by the manufacturer.
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Relative percentage of SMM.
A preliminary quantification of the percentage of SMM heavy chain
protein expressed by the BSM cell line per milligram total myosin
(SMM + nonmuscle myosin) protein was performed using purified pig
bladder SMM as a standard. Purified myosin ranging from 0.1 to 3.0 µg
was electrophoresed on a 7.5% mini SDS-PAGE gel along with crude
extracts of both normal bladder and the BSM cell line containing equal
amounts of total myosin heavy chain as predetermined by Coomassie blue
staining. The gel was then transferred by Western blotting to membrane,
and the blot was reacted with the SM-specific Sigma myosin antibody
(clone hSM-V) that recognizes all SMM isoforms, as described in
METHODS (Fig. 5). The
purified pig myosin exhibited a linear increase in DAB staining
(lanes 1-6), and using this standard curve it was
concluded that the BSM cell line (lane C) contains ~30%
of the SMM expressed by the normal bladder (lane N) per
milligram total extractable myosin.
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Homogeneity of myosin and actin expression.
Immunofluorescence microscopy using the same antibodies that were used
in the Western analysis revealed strong staining of all BSM cells for
SM-specific -actin (Fig.
6A), whereas a negative control in which the primary antibody was replaced with mouse preimmune
serum showed only faint staining (Fig. 6B). A negative control in which the primary antibody was omitted also did not show
significant staining (data not shown). Staining of BSM for total SMM
was also intense and homogeneous throughout the cells except for the
nucleus (Fig. 6, C and D). A similar staining
pattern was observed using primary antibody against the SMM
heavy-chain-specific isoform SM-B (Fig. 6, E and
F).
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PKG expression.
Western blot analysis (Fig. 7) revealed
that BSM (lane 1) expressed PKG like normal rabbit bladder
(lane N). The nonmuscle NIH/3T3 cell line (lane
T) did not express any appreciable PKG protein. Additionally, it
can also be seen in Fig. 7 that our large gels were able to separate
the PKG-1 and PKG-1
isoforms and that the cell line clearly makes
both isoforms.
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Kinase and phosphatase levels.
Western blot analyses showed that the BSM cell line does express MLCK
(Fig. 8A) protein, but at a
much lower level than normal bladder per extractable milligram of
protein. Interestingly, BSM cells make protein corresponding to MYPT1
(Fig. 8B) at a higher level than normal bladder. Most
importantly, functional kinase activity was present in the BSM cell
extract as determined by the presence of covalently bound
[-32P]phosphate when [
-32P]ATP is
used for the kinase assay (Fig. 8C). In the presence of
calcium, BSM kinase activity was ~300 pmol phosphate incorporated into 20 kDa myosin light chain per minute per milligram protein extract, reaching a maximum level of phosphorylation within 1 min. The
majority of this activity was found to be calcium dependent, since the
same kinase assay conducted in the absence of calcium (with 2 mM EGTA)
incorporated only ~30 pmol phosphate per minute per milligram
extractable protein in the extract.
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Cell contraction.
Cells from the BSM cell line were grown on collagen-coated coverslips
for 2 days and then placed on the stage of an inverted microscope
equipped for video photography and washed with the bath solution
described in METHODS. No detectable change in cell size or
shape was detected after 5 min in bath solution (Fig. 9, A and D). When
10 µM bethanechol was added to them, the cells began to contract, but
maximum contraction was achieved with 100 µM bethanechol by 1-2
min (Fig. 9, B and E), demonstrating that these
cells maintain the ability to contract in response to agonist that
produces contraction in intact bladder SM strips. Furthermore, the
bethanechol-induced contraction was almost completely reversible within
2 min by simply washing the cells with bath solution (Fig. 9,
C and F).
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Cytogenetic analysis of the cell line.
Chromosome analysis of BSM cells revealed a tetraploidy (4N) with no
readily discernable additional morphological differences compared with
normal rabbit chromosomes (Fig. 10).
Thus cells contained 88 chromosomes, twice the number of chromosomes
reported for normal rabbit cells (42).
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DISCUSSION |
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In this study we report the derivation of a stable bladder SM cell
line (BSM) that maintains expression of SMM and -actin along with
the ability to contract in response to agonist stimulation. As starting
material, we used SM from adult male rabbit urinary bladder wall, which
was induced to undergo hypertrophy by partial ligation of the urethra
(34). Cells of BSM are capable of large-scale amplification, have now been passaged >50 times, maintain a uniform high expression of SM-specific marker proteins (including SMM), and
exhibit a seemingly stable tetraploid (4N) chromosomal pattern.
To our knowledge, this is the first report of a SM cell line that expresses SMM, MLCK and myosin phosphatase, and PKG and is contractile in response to physiological agonists. A unique feature of this cell line is its expression of myosin isoform SM-B, which has a high ATPase activity in the presence of actin. The vascular SM cell lines presently available do not express SM-B, since they have all been generated from large arteries that do not contain SM-B (20).
The majority of studies conducted on cultured vascular as well as
visceral-type SM cells have relied on expression of -actin as the
major criterion to designate these cells as SM cells (12, 22, 39,
53). However,
-actin is not the actin isoform expressed in
all SM cells, it has been identified in many other cell types including
fibroblasts (19), and its expression is actually decreased during development of visceral SM, which expresses predominantly
-actin (46). In our study, we focused on the expression
of SMM as a marker of the differentiated state of SM because, unlike
-actin, SMM is expressed in all SM cells (52) and its
expression is increased in visceral SM during development
(7). Moreover, SMM heavy chain expression appears to be
exclusively limited to SM cells (35).
Our BSM cell line clearly retains strong expression of SMM heavy chain at the protein level based on Western blot and immunofluorescence analyses. For the Western blot analysis of SMM heavy chain, we used a monoclonal antibody that reacts with all four isoforms of myosin (SM1A, SM1B, SM2A, and SM2B) and does not cross-react with nonmuscle myosin heavy chain. This conclusion is based on the fact that both SM1 and SM2 of the bladder, which is predominantly SM-B and thus the isoforms are SM1B and SM2B, react with the Sigma antibody. The reaction for SM2 is about three times stronger compared with the SM1 isoform, producing a ratio similar to what is found for Coomassie blue staining, suggesting that the antibody reacts equally with SM1 and SM2. In addition, we (unpublished data) and others (25) have found that the antibody also reacts equally well with SM1 and SM2 from aorta, and, since the aorta is ~100% SM-A, the isoforms are SM1A and SM2A.
Thus it appears that the Sigma antibody does react with all four known
isoforms and can be used as a measure of total SMM expression. Indeed,
no antibody reactivity was observed with NIH/3T3 cells in Western blot
analysis by us (Fig. 3A) and others (25). Furthermore, immunofluorescence microscopy using this antibody showed
that all the cells in the cultures established from BSM show positive
staining (Fig. 6, C and D), indicating the
homogeneity of the cell population. Thus, although we cannot be certain
that our cell line was derived from a single clone, the phenotype with respect to SMM appears stable. Together, our results demonstrate SMM
expression by the BSM cell line at the protein and cellular levels.
Preliminary quantification using purified pig bladder SMM as a positive
standard revealed that the BSM cell line expressed ~30% of the SMM
expressed by normal bladder SM as a percentage of equal amounts of
total myosin (Fig. 5). This level seems to agree with the lower
expression levels of SMM in our gels and Western blots as well (Fig. 3,
A and B, and Fig. 4, A and
B). The fact that BSM retains expression of PKG and makes
both PKG-1 and PKG-1
(expressed only in SM) at the protein level
further supports maintenance of the SM phenotype.
The presence of SMemb and nonmuscle myosin A is not surprising, since cells undergoing increased growth are known to express elevated levels of SMemb (4). Hypertrophied SM from partially obstructed rabbit bladder also exhibits increased expression of nonmuscle myosin A (unpublished data). However, we cannot say with absolute certainty that this band is nonmuscle myosin A, since it does not react with the nonmuscle myosin A antibody we used. Our belief that this band is nonmuscle myosin A is based on the fact that it migrates in a position relative to SM1 and SM2, which would predict a size of ~196 kDa [the observed size of nonmuscle myosin A in humans (40) and rats (29, 45)], and on the fact that others have reported that the antibody to human nonmuscle myosin A does not recognize the sheep nonmuscle myosin A (7).
We showed that the cell line expresses protein for both MLCK and the
MYPT1 subunit of myosin phosphatase while the cell extracts revealed
activities for both these enzymes. The maximum phosphatase activity
obtained is at a level similar to phosphatase activity values reported
for normal sheep pulmonary artery (2.0 nmol · min1 · mg protein
1)
(13) and normal rabbit basilar artery (3.8 nmol · min
1 · mg protein
1)
using [
-32P]phosphorylase-a as substrate
(26). Although the MLCK activity is lower than other
reported values (13), in our assay the kinase was capable
of maximally (mol/mol) phosphorylating the 20-kDa myosin light chain.
It is possible that the addition of exogenous calmodulin would have
increased the activity further.
We also demonstrate that these cells are indeed contractile by showing that the cells shorten in response to the muscarinic agonist bethanechol. Contraction to bethanechol suggests that the BSM cells do have muscarinic receptors, similar to that present in bladder myocytes in vivo. Further, we also show that the cells are also capable of relaxation, since the cells returned to almost their original shape on removal of the bethanechol from the cells by washing.
The BSM cell line is tetraploid. Although tetraploidy is quite often associated with cancer-related cell lines, there are noncancerous cell lines, including the A7r5 vascular smooth muscle cell line (23), that exhibit tetraploidy as well. Anthony et al. (5) found between 0.3 and 1.0% of the SM cells in the normal rabbit aorta were either 3C or 4C (tetraploid). In human aorta, Printseva and Tjurmin (41) found that ~10% of the SM cells in the media and intima of the aorta were tetraploid; interestingly, the number of tetraploid cells increased to ~20% in response to hypertension. A similar increase in polyploidy in response to hypertension was also found in rats, and this same study found that increased polyploidy was accompanied by an increase in SM cell hypertrophy (37). Therefore, it is not surprising that this cell line, derived from hypertrophied bladder smooth muscle, is tetraploid.
In summary, we have described the isolation of the first SM cell line
(BSM) that stably expresses the SMM SM-B isoform and contracts in
response to bethanechol. Furthermore, BSM expresses -actin, MLCK,
MYPT1, and PKG. This cell line will provide a valuable tool for
studying transcriptional regulation of myosin isoforms and for
perfecting tissue engineering in rabbits for urinary bladder augmentation and replacement with tissue-engineered bladders
(8).
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ACKNOWLEDGEMENTS |
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We thank Dr. Bruce Freedman and Dr. Qinghua Liu for help in performing the cell contraction studies.
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FOOTNOTES |
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This work was supported by a National Institute of Diabetes and Digestive and Kidney Diseases O'Brien Urology Research Center Grant P50 DK-52620, DK-55529, and DK-55042.
Address for reprint requests and other correspondence: M. E. DiSanto, 3010 Ravdin Courtyard, 3400 Spruce St., Philadelphia, PA 19104 (E-mail: mdisanto{at}mail.med.upenn.edu).
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. Section 1734 solely to indicate this fact.
First published March 27, 2002;10.1152/ajpcell.00002.2002
Received 4 January 2002; accepted in final form 21 March 2002.
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