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 2 Department of Pathobiology, University of Pennsylvania, and 3 Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 alpha -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 alpha -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-1alpha returns the cells to a contractile phenotype (14), supporting a role of PKG in maintenance of the SM phenotype. Whereas PKG-1alpha is expressed abundantly in many different cell types, its alternatively spliced counterpart, PKG-1beta , 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 alpha -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.


    METHODS
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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 alpha -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 alpha -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 [gamma -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 [gamma -32P]ATP was used as the substrate. The reaction was started by adding 50 µg of exogenous myosin with covalently bound [gamma -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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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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Fig. 1.   Growth curve for bladder smooth muscle (BSM) cells. Twelve cultures (4 × 104 cells/60-mm dish) established from passage 50 of the BSM cell line were fed with M199 medium containing 10% FCS. Each day a culture was trypsinized and the dissociated cells were counted with a hemacytometer. Results are the mean of 3 separate determinations. Doubling time was calculated as ~58 h and cells appeared to reach confluency at 4-5 days.



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Fig. 2.   Phase-contrast microscopy of BSM cells at passage 50. A: cells in sparse culture (2 days postsubculture) have a fibroblast-like shape. B: cells in confluent cultures (>4 days postsubculture) have a more spindle-shaped appearance. 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 ×100.

SMM and alpha -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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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 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 3,3'-diaminobenzidine as described in METHODS. 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 that recognizes all forms of smooth muscle myosin (SMM). Lane M was loaded with prestained myosin marker. B and C: extracts from the BSM cell line at passages 15 (lane 1), 30 (lane 2), and 50 (lane 3) and normal bladder SM (lane N) were probed with primary antibody specific for the 7-amino acid insert found only in the SM-B SMM isoform and SM alpha -actin, respectively.

The SM-A and SM-B SMM isoforms differ only by seven 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 seven-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 normal bladder (lane N). Western blot analysis also showed that BSM strongly expresses alpha -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 on 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 on 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).


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Fig. 4.   Relative expression of SMM 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. 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 that migrated slightly faster than SM2 and appears to be nonmuscle myosin A. B: Western blot analysis of the 4.5% gel described in A using SMM-specific antibody (clone hSM-V) from Sigma. C: equal amounts of total extractable protein (10 µg) from normal bladder SM (lane N), BSM at passage 50 (lane 1), 2-wk 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.

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, whereas 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). Because 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 one-half of hypertrophied bladder muscle (lane 2) while much less than that in 27-day rabbit fetal aorta (lane 3).

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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Fig. 5.   Relative expression of SMM 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 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 (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.

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 alpha -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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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. A: staining with alpha -actin primary antibody (magnification ×400). B: negative staining in which the primary antibody was replaced with mouse preimmune serum (magnification ×400). C: staining with Sigma antibody recognizing all isoforms of the SMM heavy chain (magnification ×400). D: same as C but magnification is ×100. E: staining with antibody specific for the 7-amino acid insert in SM-B (magnification ×400). F: same as E but magnification is ×100.

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-1alpha and PKG-1beta isoforms and that the cell line clearly makes both isoforms.


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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 NIH/3T3 cells (lane T) were separated on large 7.5% SDS-PAGE gels, transferred to Immobilon-P membrane, and reacted with antibody specific for PKG as described in METHODS. Note that this large gel was able to resolve the PKG-1alpha and PKG-1beta 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 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 [gamma -32P]phosphate when [gamma -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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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 membrane, and reacted with antibody specific for myosin light chain kinase (MLCK; A) or myosin phosphatase (B). The MLCK activity (C) and myosin phosphatase activity (D) in the crude cell extract prepared from the SM cell line at passage 50 were determined. Column-purified gizzard myosin in the nonphosphorylated and phosphorylated forms were used as substrates for the kinase and phosphatase, respectively. Kinase assays were performed using [gamma -32P]ATP, and the 32P covalently bound to the 20-kDa myosin regulatory light chain (LC20) was determined as described in METHODS. The moles of Pi (32P) incorporated into LC20 per minute per milligram of extractable protein were plotted (C). Phosphorylated myosin used as a substrate for the phosphatase assay was prepared using [gamma -32P]ATP. The gamma -32P liberated from the phosphorylated myosin was determined using a scintillation counter, and the moles of 32P liberated per minute per milligram of protein was determined and plotted (D).

The BSM cell line also expresses functional myosin phosphatase activity, which removes covalently bound [gamma -32P]phosphate from phosphorylated myosin used as a substrate (Fig. 8D). Although gamma -32P continued to be released from the myosin for at least 5 min, the highest activity was again obtained within the first minute in which ~2.3 nmol [gamma -32P]phosphate were released per minute per milligram 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 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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Fig. 9.   Bethanechol-induced contraction of cell line. The BSM cells post-passage 50 were cultured on collagen-coated coverslips for 2 days in either sparse culture (A-C) or monolayer (D-F). The coverslips were then placed on an inverted microscope equipped for video photography. The cells were washed with bath solution for 5 min and then photographed (A and D). The cells after 1-2 min exposure to 100 µM bethanechol are shown in B and E and then after washing for 2 min with bath solution (C and 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.

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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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 revealed a tetraploid (4N) cell line, with no obvious additional morphological differences compared with normal rabbit chromosomes.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we report the derivation of a stable bladder SM cell line (BSM) that maintains expression of SMM and alpha -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 alpha -actin as the major criterion to designate these cells as SM cells (12, 22, 39, 53). However, alpha -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 gamma -actin (46). In our study, we focused on the expression of SMM as a marker of the differentiated state of SM because, unlike alpha -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-1alpha and PKG-1beta (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 · min-1 · mg protein-1) (13) and normal rabbit basilar artery (3.8 nmol · min-1 · mg protein-1) using [gamma -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 alpha -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

We thank Dr. Bruce Freedman and Dr. Qinghua Liu for help in performing the cell contraction studies.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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