1

Generation of a Cell Line with Smooth Muscle Phenotype

from Hypertrophied Urinary Bladder

Yongmu Zheng1, Wilfried T. Weber2, Shuqin Wang2, Alan J. Wein1,

Stephen A. Zderic3, Samuel Chacko1,2 and Michael E. DiSanto1*

1 Division of Urology and 2Department of Pathobiology

University of Pennsylvania

&

3Children’s Hospital of Philadelphia

Philadelphia, PA 19104

Running Title: Cell Line with Smooth Muscle Phenotype

*Corresponding Author Address

3010 Ravdin Courtyard

3400 Spruce Street

Philadelphia, PA 19104

U.S.A.

Phone (215) 662-6870 Fax (215) 349-5026

email: mdisanto@mail.med.upenn.edu

Copyright 2002 by the American Physiological Society.

AJP-Cell Articles in PresS. Published on March 27, 2002 as DOI 10.1152/ajpcell.00002.2002

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ABSTRACT

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. SDS-

PAGE and Western blotting revealed that BSM expresses the N-terminal SMM heavy chain

isoform SM-B, the C-terminal SM1 and SM2 isoforms, and SM α-actin. In addition, they

express cGMP-dependent protein kinase G, made by contractile SM cells in vitro, but not by

non-contractile cells synthesizing extracellular matrix. Immunofluorescence studies indicate a

homogeneous population of cells expressing α-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.

Key words: contractility, SM-B, kinase, phosphatase, PKG

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INTRODUCTION

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 upon culture

(6,11,29,45). Most studies in which cultured bladder myocytes have been used relied only upon

the expression of α-smooth muscle actin as a marker for smooth muscle (12,39,53) In a study by

Kropp et al., smooth muscle myosin expression and contractility were examined in cultured

myocytes that had been passaged 2-5 times (32), but they found only a small percentage of the

isolated cells staining for smooth muscle myosin and they did not contract in response to

agonists which typically cause bladder smooth muscle tissue contraction (12,39).

Several attempts have been made to establish SM cell lines derived primarily from

vascular SM (31,44). However, Firulli et al. (23) showed two of the major vascular SM cell

lines reported to date (PAC1 and A7r5), although retaining expression of α-actin, express

virtually no smooth muscle myosin (SMM) mRNA upon passaging. Moreover, karyotyping of

both of these cell lines revealed alterations in chromosome number and/or structure (23).

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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 while splicing at the 3’ end of this

gene produces the SM1 and SM2 heavy chain isoforms (9,10). The SM-B isoform contains a 7-

amino acid insert in the N-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 almost 100% of the SM-A isoform (20), and has a higher ATPase activity

and increased in vitro motility of the myosin (30) (20) as well as increased shortening velocity of

the muscle itself (20) compared to 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 distinct from

the nonmuscle myosin A (NMA) 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 another key marker of the SM phenotype (14). SM cells isolated from

rat aorta express high levels of type I PKG. However, upon passaging, PKG levels decrease

rapidly such that after several passages its expression is undetectable. This decreased expression

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of PKG correlates with a switch to a more synthetic phenotype (18). Moreover, transfection of

these passaged cells with cDNA for PKG-1α returns the cells to a contractile phenotype (14),

supporting a role of PKG in maintenance of the SM phenotype. Whereas PKG-1α is expressed

abundantly in many different cell types, its alternatively spliced counterpart, PKG-1β, 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) while dephosphorylation of the light chain is

accomplished by myosin phosphatase. The phosphatase which 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 smooth

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 α-actin and SMM even after more than 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

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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

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-month-old male New Zealand

White rabbit with a 2-week 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-week partially obstructed rabbit bladder was minced into small pieces (1 x 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 1000 X g at

4°C. The pellet was resuspended and washed in M199 and cultured in nutrient medium [M199 +

10% fetal calf serum (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 pipet 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 more than 50 times, after brief treatment with trypsin (0.05%

trypsin/EDTA) (GIBCO BRL). Cells from various passages were scraped off with a rubber

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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 6-well plates (Corning) at an initial density of 4 X 104 cells/well in growth medium as

described above. Triplicate hemocytometer 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 1X phosphate buffered saline (PBS). Cell-containing coverslips were fixed in a

solution of 70% ethanol, 1% formaldehyde and 5% glacial acetic acid for 10 minutes. Cells were

then washed 3 times with PBS, incubated for 30 minutes in 1% BSA to block nonspecific

binding, and incubated for 1-2 hours at room temperature with primary antibody against the

following SM-specific proteins: SMM (1:1000), SM-B (1:500) and α-actin (1:1000). After 3

washes in PBS, cells were treated with second antibody (FITC-conjugated goat anti-mouse IgG

or IgM for SM-B both at 1:100) for 1 hour, washed 3 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 which was generated in our laboratory against the

7-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 pre-immune serum or the second

antibody was omitted.

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Protein Extraction and Western Blot Analysis: The cell pellet was homogenized in extraction

buffer at 5 X 106 cells per ml of buffer containing 50mM Tris, pH 6.8, 20% glycerol and

protease inhibitors (0.8 mM 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 X g for 20 min. A small portion of the supernatant was used for

protein determination by the BioRad DC (detergent compatible) kit. Bromophenol blue and 25

mM DTT (which interferes with DC assay) was added to the remainder of the sample for

analysis by SDS-PAGE. Bladder SM from normal adult 4-month-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 in order 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

hour and incubated with a 1:20,000 dilution of SMM-specific primary antibody (clone no. hSM-

V) from Sigma or a 1:10,000 dilution of antibody specific for SMemb (a gift from Dr. Robert

Adelstein at NIH). After washing 3 times in 1X 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 hr. Antibody

reactivity was detected by ECL (Amersham Pharmacia Biotech; Buckinghamshire, England). In

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a similar manner, Western Blot analysis was also performed for SM-B (1:500), SM α-actin

(clone no. 1A4, 1:1000; Sigma). MLCK (clone K36, 1:10,000; Sigma), MYPT1 (clone

ASC.M130, 1:10,000; Covance; Richmond, CA) and PKG (KAP-PK005, 1:4000; Stressgen

Biotechnology Corp.; 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 grams of cells using a mini electric

homogenizer in 2.0 ml extraction buffer [in mM: 60 KCl, 40 imidazole-HCl (pH 7.1), 2 EDTA, 2

dithiothreitol, 10 ATP, and protease inhibitor cocktail containing 0.2 phenylmethylsulfonyl

fluoride and in µM: 1 antipain, 1 pepstatin, 5 trypsin inhibitor] followed by centrifugation at

20,000 X g for 20 minutes. The cell extract present in the supernatant was collected and

dialyzed overnight in phosphorylation buffer [in mM: 10 imidizaole-HCl (pH 7.1), 5

dithiothreitol and protease inhibitor cocktail] as above. Next 20 µl of the cell extract was

assayed for kinase activity in a total volume of 150 µl containing 5 mM MgCl2, 0.1 mM CaCl2,

10 mM imidazole-HCl (pH 7.1), 2 mM ATP. Fifty micrograms of exogenous purified chicken

gizzard SMM prepared as described (17) was also added to each assay and the reaction started by

adding [γ-32P]ATP. The reaction was stopped at various times by adding 150 µl of the reaction

mix to 1 ml of 10% trichloroacetic acid (TCA)/2% sodium pyrophosphate, the mixture

centrifuged at 14,000 X 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

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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 mg of cell extract protein.

The same assay was conducted in the absence of Ca2+ with 2 mM EGTA to determine the Ca2+

dependence of the kinase activity.

Phosphatase Assay: The cell extracts prepared as described under the kinase assay were assayed

for phosphatase activity in [5 mM MgCl2, 0.1 mM CaCl2, 10 mM imidazole-HCl (pH 7.1)].

Purified gizzard myosin phosphorylated using [γ-32P]ATP was used as the substrate. The

reaction was started by adding 50 µg of exogenous myosin with covalently bound [γ-32P]

phosphate. The reaction was stopped by adding TCA to a final concentration of 10% and

sodium pyrophosphate to 2% final and the amount of phosphate released per min per mg protein

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, USA). 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, Japan)

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 Corporation) using Metafluor

acquisition and analysis software (Universal Imaging, USA). Stored images were analyzed off-

line using the Metafluor package. Contraction was induced by bath application of bethanechol

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(1-100 µM), and relaxation 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 hrs, 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 to normal rabbit chromosomes.

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RESULTS

Growth Characteristics: The BSM cells proliferated at a steady rate and exhibited a uniform

growth pattern in medium containing 10% fetal calf serum. The growth curve of BSM revealed

a doubling time of approximately 58 hours (Fig. 1), similar to that of NIH 3T3 cells cultured

under the same conditions (data not shown) and of other SM cell lines (23). Upon subculture, the

BSM cells grew logarithmically for approximately 3 days under our culture conditions and

reached a plateau at about 5 days. Similar results were also obtained at passage numbers 15 &

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).

Smooth Muscle Myosin & α-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 & 2) although amounts of SMM per mg of extracted protein was 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 versus nonmuscle

myosin as also indicated by the manufacturer.

The SM-A and SM-B SMM isoforms differ only by 7 amino acids and thus are not

separable by SDS-PAGE. Therefore, Western blot analysis was used to examine SM-B protein

expression in BSM. Antibody against the 7-amino acid insert, which is specific for the SM-B

isoform (21), reacted strongly with BSM (Fig. 3B, lanes 1-3) but again to a lesser extent than

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normal bladder (lane N). Western blot analysis also showed that BSM strongly expresses α-

actin (Fig. 3C, lanes 1-3) at levels comparable to those of normal bladder SM (lane N).

Unlike the normal adult bladder myosin (Fig. 4A, lane N) which separated into the two

expected bands for SM1 and SM2 upon slowly running highly porous 4.5% SDS-PAGE gels

similar to purified pig bladder myosin (lane P), BSM (lane 1) produced an additional third lower

band. Based upon the mobility of this band it appears to represent the nonmuscle myosin A

isoform (~ 196 kDa). However, Western blot analysis showed that antibody generated against

human nonmuscle myosin A failed to cross-react with the nonmuscle myosin A band as has been

reported for other species (7).

Western blot analysis of an identical highly porous gel showed that the two similar bands

obtained in both normal bladder and BSM reacted strongly with Sigma antibody that recognizes

all SMM isoforms while the third unique band found in BSM did not, confirming that this band

is not SMM heavy chain (Fig. 4B). Interestingly, the cell line (lane 1) expressed slightly more

SM2 relative to SM1 comparable to normal bladder (lane N). Since some of the middle SM2

band (in Fig. 4A, lane 1) could also represent SMemb as this myosin has been shown to

comigrate with SM2 in SDS-PAGE gels, we also analyzed blots of myosin electrophoresed in

highly porous gels using antibody specific for SMemb (a gift from Dr. Robert Adelstein at NIH).

Figure 4C shows that although normal rabbit bladder SM (lane N) expresses only trace amounts

of SMemb, significant expression of SMemb is found in BSM (lane 1), although the level of

expression in BSM is about half of hypertrophied bladder muscle (lane 2) while much less than

that in 27 day rabbit fetal aorta (lane 3).

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Relative Percentage of Smooth Muscle Myosin: A preliminary quantification of the percentage

of SMM heavy chain protein expressed by the BSM cell line per mg total myosin (SMM +

nonmuscle myosin) protein was performed using purified pig bladder SMM as a standard.

Purified myosin ranging from 0.1-3.0 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 no. hSM-V) that recognizes all SMM isoforms as described in the

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 about

30% of the SMM expressed by the normal bladder (lane N) per mg total extractable myosin.

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 α-actin (Fig. 6A), while a negative control in which the primary antibody was

replaced with mouse pre-immune 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. 6C & D). A similar staining pattern was observed using

primary antibody against the SMM heavy chain-specific isoform SM-B (Fig. 6E & F).

cGMP-Dependent Protein Kinase G (PKG) Expression: Western blot analysis (Fig. 7) revealed

that BSM (lane 1) expressed PKG like normal rabbit bladder (lane N). The nonmuscle NIH3T3

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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-1α and PKG-1β isoforms and that the

cell line clearly makes both isoforms.

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

mg 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 [γ-32P] phosphate when [γ-

32P]ATP is used for the kinase assay (Fig. 8C). In the presence of calcium, BSM kinase activity

was ~ 300 picomoles phosphate incorporated into 20 kDa myosin light chain per min per mg

protein extract reaching a maximum level of phosphorylation within 1 minute. The majority of

this activity was found to be calcium dependent since the same kinase assay conducted in the

absence of Ca2+ (with 2 mM EGTA) incorporated only ~ 30 picomoles phosphate per min per mg

extractable protein in the extract.

The BSM cell line also expresses functional myosin phosphatase activity which

dephosphorylates covalently bound [γ-32P] phosphate from phosphorylated myosin used as a

substrate (Fig. 8D). Although [γ-32P] continued to be released from the myosin for at least 5

minutes, the highest activity was again obtained within the first minute in which ~ 2.3 nmoles of

[γ-32P] phosphate were released per minute per mg of extractable protein.

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

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washed with the bath solution described in the Methods. No detectable change in cell size or

shape was detected after 5 minutes in bath solution (Fig. 9A & D). Upon addition of 10 µM

bethanechol the cells began to contract, but maximum contraction was achieved with 100 µM

bethanechol by 1-2 minutes (Fig. 9B & E) demonstrating that these cells maintain the ability to

contract in response to agonist that produces contraction in the intact bladder SM strips.

Furthermore, the bethanechol-induced contraction was almost completely reversible within 2

minutes by simply washing the cells with bath solution (Fig. 9C & F).

Cytogenetic Analysis of the Cell Line: Chromosome analysis of BSM cells revealed a

tetraploidy (4N) with no readily discernable additional morphological differences compared to

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

In this study we report the derivation of a stable bladder SM cell line (BSM) that

maintains expression of SMM and α-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 more than 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 smooth muscle

myosin, MLCK and myosin phosphatase, and PKG and is contractile in response to physiologic

agonist. 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 α-actin as the major criterion to designate these cells as smooth

muscle cells (12,22,39,53). However, α-actin is not the actin isoform expressed in all SM cells,

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 γ-actin

(46). In our study, we focused on the expression of SMM as a marker of the differentiated state

of SM because: unlike α-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).

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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, 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 3X stronger compared to 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 almost

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

used as a measure of total SMM expression. Indeed, no antibody reactivity was observed with

NIH3T3 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. 6C & 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 about 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 (Figs. 3A & B and 4A & B). The fact that BSM

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retains expression of PKG and makes both PKG-1α and PKG-1β (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 A myosin is based on the fact that it migrates in

a position relative to SM1 and SM2 which would predict a size of about 196 kDa (the observed

size of nonmuscle myosin A in humans (40) and rats (29,45) and 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 nmoles/min/mg protein) (13) and normal rabbit

basilar artery (3.8 nmoles/min/mg protein) using [γ-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

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vivo. Further, we also show that the cells are also capable of relaxation since the cells returned to

almost their original shape upon 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 0.3-1.0%

of the SM cells in the normal rabbit aorta were either 3C or 4C (tetraploid). In human aorta,

Printseva et al. (41) found that about 10% of the SM cells in the media and intima of the aorta

were tetraploid; interestingly, the number of tetraploid cells increased to about 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

smooth muscle 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 α-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)

Acknowledgements: This work was supported by a National Institutes of Health O’Brien

Urology Research Center Grant, P50 DK52620.

22

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26

FIGURE LEGENDS

Fig. 1. Growth Curve for BSM. Twelve cultures (4 X 104 cells/60 mm dish) established from

passage 50 of the BSM cell line were fed with M199 medium containing 10% fetal calf serum.

Each day a culture was trypsinized and the dissociated cells were counted with a hemocytometer.

Results are the mean of three separate determinations. Doubling time was calculated as

approximately 58 hours and cells appeared to reach confluency at 4-5 days.

Fig. 2. Phase Contrast Microscopy of BSM Cells at Passage 50. Panel A: Cells in sparse

culture (2 days post subculture) have a fibroblast-like shape. Panels B: Cells in confluent

cultures (more than 4 days post subculture) have a more spindle-shaped appearance. Panel C:

Some of the cells in confluent cultures are arranged in a swirly pattern similar to the “hills and

valleys” described for cultured aortic cells. Magnification 100X.

Fig. 3. Western Blot Analysis of Contractile Protein Expression in BSM. Equal amounts of

total protein (20 µg) extracted from the various samples were loaded onto a 7.5% SDS-

polyacrylamide gels, separated by electrophoresis and transferred to Immobilon-P membrane

overnight at 4°C. Blots were probed using antibodies specific for contractile proteins and

reactivity quantitated using DAB as described in the Methods. Panel A: Extracts from normal

bladder SM (lane N), passage 15 (lane 1) and passage 50 (lane 2) BSM as well as NIH 3T3 cells

(lane T) were probed with primary antibody from Sigma which recognizes all forms of SMM.

Lane M was loaded with prestained myosin marker. Panels B & C: Extracts from the BSM cell

line at passage 15 (lane 1), 30 (lane 2) and 50 (lane 3) and normal bladder SM (lane N) were

27

probed with primary antibody specific for the 7-amino acid insert found only in the SM-B SMM

isoform and SM α-actin, respectively.

Fig. 4. Relative Expression of Smooth Muscle Myosin Heavy Chain Isoforms in BSM.

Protein extracts containing equal amounts (10 µg) of total extractable protein and purified pig

bladder myosin (1 µg) were separated on highly porous 4.5% SDS-PAGE gels as described in

Methods. Panel A: Normal bladder SM (lane N) produced two bands of the predicted size for

SM1 and SM2 similar to purified pig bladder myosin (lane P) while in addition to these two

bands, BSM at passage 50 (lane 1) produced an additional third band which migrated slightly

faster than SM2 and appears to be nonmuscle myosin A. Panel B: Western blot analysis of the

4.5% gel described in Panel A using SMM-specific antibody from Sigma. Panel C: Equal

amounts of total extractable protein (10 µg) from normal bladder SM (lane N), BSM at passage

50 (lane 1), 2 week obstructed/hypertrophied bladder (lane 2) and fetal 27 day embryonic aorta

(lane 3) were separated on highly porous SDS-PAGE gels and reacted with antibody specific for

SMemb/nonmuscle myosin B.

Fig. 5. Relative Expression of Smooth Muscle Myosin by BSM. Purified pig bladder SMM

ranging from 0.1-3.0 µg was separated on a 7.5% mini SDS-PAGE gel along with crude extract

supernatants of both normal bladder and the BSM cell line (passage 50) containing equal

amounts of total myosin heavy chain, as predetermined by Coomassie blue staining and

densitometric analysis. The gels were transferred by Western blotting to membrane and the blot

reacted with the SM-specific Sigma myosin antibody (clone no. hSM-V). Purified pig bladder

SMM in the amounts of 0.1 µg (lane 1), 0.2 µg (lane 2), 0.5 µg (lane 3), 1.0 µg (lane 4), 2.0 µg

28

(lane 5), and 3.0 µg (lane 6) and extracts from the BSM cell line (lane C) and normal bladder SM

(lane N) were loaded. Lane M was loaded with prestained myosin marker.

Fig. 6. Immunofluorescence Analysis of BSM. Immunofluorescent staining of the cell line at

passage 50 was performed as described in Methods. All staining was performed with FITC-

conjugated Ig secondary antibody. Panel A: Staining with α-actin primary antibody (Mag

400X); Panel B: Negative staining in which the primary antibody was replaced with mouse pre-

immune serum (Mag 400X); Panel C: Staining with Sigma antibody recognizing all isoforms of

the SMM heavy chain (Mag 400X); Panel D: same as C but Mag is 100 X; Panel E: Staining

with antibody specific for the 7-amino acid insert in SM-B (Mag 400X); Panel F: Same as E but

Mag is 100X.

Fig. 7. Expression of cGMP-Dependent Protein Kinase (PKG) by BSM. Protein extracts

containing equal amounts (20 µg) of total extractable protein from normal rabbit bladder SM

(lane N), BSM at passage 50 (lane 1) and NIH3T3 cells (lane T) were separated on large 7.5%

SDS-PAGE gels, transferred to Immobilon-P and reacted with antibody specific for PKG as

described in Methods. Note that this large gel was able to resolve the PKG-1α and PKG-1β

isoforms.

Fig. 8. Kinase and Phosphatase Expression and Activity. Equal amounts of total extractable

protein (20 µg) from normal rabbit bladder and post passage 50 BSM cells were separated on

7.5% SDS-PAGE gels, blotted to Immobilon-P and reacted with antibody specific for MLCK

(Panel A) or myosin phosphatase (Panel B). The MLCK activity (Panel C) and myosin

29

phosphatase activity (Panel D) in the crude cell extract prepared from the SM cell line at passage

50 were determined. Column purified gizzard myosin in the unphosphorylated and

phosphorylated forms were used as substrates for the kinase and phosphatase, respectively.

Kinase assays was performed using [γ-32P]ATP and the 32P covalently bound to the LC20 was

determined as described under the Methods. The moles of Pi (32P) incorporated into LC20 per

min per mg cell extract was plotted (Panel C). Phosphorylated myosin used as a substrate for the

phosphatase assay was prepared using [γ-32P]ATP. The γ-32P liberated from the phosphorylated

myosin was determined using a scintillation counter and the moles of 32P liberated/min/mg

extract was determined and plotted (Panel D).

Fig. 9. Bethanechol-induced Contraction of Cell Line. The BSM cells post 50 passage were

cultured on collagen-coated coverslips for 2 days in either sparse culture (Panels A-C) or

monolayer (Panels D-F). The coverslips were then placed on an inverted microscope equipped

for video photography. The cells were washed with bath solution for 5 minutes and then

photographed (Panel A & D). The cells after 1-2 minute exposure to 100 µM bethanechol are

shown in Panels B & E and then after washing for 2 minutes with bath solution (Panels C & F).

Although the two different types of arrows are used to follow an individual cell through the

treatment cycle, notice that the majority of the cells shortened to some degree. The dark

circular spots on the coverslips served as markers to keep track of individual cells.

Fig. 10. Cytogenetic Analysis of the Cell Line. Representative metaphase-arrested

chromosome spread prepared from passage 50 cells of the BSM cell line. Chromosome analysis

30

revealed a tetraploid (4N) cell line, with no obvious additional morphological differences

compared to normal rabbit chromosomes.

Days in Culture0 2 4 6 8 10 12

Cel

l Num

ber

2 X 104

4 X 104

6 X 104

8 X 104

1 X 105

Doubling Time = ~ 58.5 hours

Figure 1

A B C

Figure 2

Figure 3

1 2 3 N

42 kDa

200 kDa 200 kDa

M N 1 2 T 1 2 3 N

A B

C

Figure 4

200 kDa

N 1 2 3

204 kDa200 kDa

N 1 P N 1

SM1SM2?

SM1SM2

SMemb

A B

C

Figure 5

200 kDa

M N 1 2 3 4 5 6 C

Figure 6

A C E

B D FB

78 kDa76 kDa PKG-1α

PKG-1β

Figure 7

N 1 T

N

M

MLCK

Time (minutes)0 1 2 3 4 5

Pi R

elea

sed

(n

M/m

g p

rote

in)

0

1

2

3

4

Time (minutes)0 1 2 3 4 5

Pi I

nco

rpo

rate

d (

pM

/mg

pro

tein

)

0

100

200

300

400

+ Calcium- Calcium

PhosphataseA B

C D

Figure 8

N 1

160 kDa 130 kDa

N 1

BA C

FED

0 Min 3 Min Post Bethanechol

3 Min Post Wash

Figure 9

Figure 10