1 Department of Pharmacology, Meiji Pharmaceutical University, Tokyo 204-8588; and 2 Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan
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ABSTRACT |
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String-shaped reconstituted
smooth muscle (SM) fibers were prepared in rectangular wells by thermal
gelation of a mixed solution of collagen and cultured SM cells derived
from guinea pig stomach. The cells in the fiber exhibited an elongated
spindle shape and were aligned along the long axis. The fiber
contracted in response to KCl (140 mM), norepinephrine (NE;
107 M), epinephrine (10
7 M), phenylephrine
(10
6 M), serotonin (10
6 M), and histamine
(10
5 M), but not acetylcholine (10
5 M).
Phentolamine (10
7 M) produced a parallel rightward shift
of the NE dose-response curve. Moreover, NE-induced contraction
was partially inhibited by nifedipine and completely abolished by the
intracellular Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
acetoxymethyl ester, the myosin light chain kinase inhibitor ML-9, the
Rho kinase inhibitor Y-27632, and papaverine. A
[3H]quinuclidinyl benzilate binding study revealed that
the loss of response to acetylcholine was due to the loss of muscarinic receptor expression during culture. The expression of contractile proteins in the fibers was similar to that in cultured SM cells. These
results suggest that, although the fiber is not a model for fully
differentiated SM, contractile mechanisms are maintained.
cultured cells; isometric force; contractility
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INTRODUCTION |
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SMOOTH MUSCLE TISSUE is composed of several types of cells and of characteristic material in the extracellular space. Studies of the stimulus-contraction coupling of smooth muscle have largely been performed on intact tissues. Although considerable information about the function of these tissues has been developed, results obtained from intact tissue often result from the complex organization of smooth muscle cells into a multicellular unit rather than from the properties of the contractile cells themselves. On the other hand, the use of cultured smooth muscle cells has been limited by the inability to reliably induce contractile responses after subcloning of the cells. This contractile-response loss has been attributed to the tendency of smooth muscle cells to change their phenotypic properties when cultured in vitro (7, 29, 41). Several investigators, however, have shown that smooth muscle cells derived from a variety of tissues continue to express smooth muscle cell differentiation markers and retain contractile capabilities through multiple passages in vitro (4, 24, 26). In most of these cases, smooth muscle cell contraction was assessed on cells grown on deformable substrates rather than directly on nondeformable plastic dishes, thus permitting cell shortening. The objective of this study was to establish a reliable technique for the quantitative measurement of contraction of cultured smooth muscle cell populations.
Although little is known regarding the mechanisms and factors that regulate changes in the differentiated state of smooth muscle cells, one of the factors is mechanical stimulation (2, 16, 21, 36). Cultured smooth muscle cell-inoculated collagen gels spontaneously shrink with time because of cellular traction (3, 37, 38, 40, 42). When the spontaneous contraction was mechanically restricted, smooth muscle cells in gels exhibited elongated bipolar spindle shapes and were oriented parallel to the direction of stretch. We have developed a method of reconstituting hybrid smooth muscle fibers that retain the capabilities of responding to typical contractile agonists to produce isometric contraction. This technique allows functional study of a variety of smooth muscle cells undergoing biochemical and genetic manipulations. We present the results of our initial examination of contractile responses of this reconstituted smooth muscle tissue to typical contractile stimuli.
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METHODS |
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Cell isolation. Smooth muscle cells were isolated from the fundus of guinea pig stomach by the method described previously (28) under sterile conditions. The viability of isolated single cells, measured by trypan blue exclusion, was >90%. Approximately 50% of the intact smooth muscle cells retained the ability to shorten in response to ACh; these cells were spindle shaped, and their surfaces were smooth.
Cell culture.
Isolated smooth muscle cells were grown in Dulbecco's modified
Eagle's medium (DMEM; high glucose) supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum (FBS)
on plastic culture dishes. Spindle-shaped smooth muscle cells attached
and spread within the first 2 days after seeding and began to
proliferate. When cultures reached confluence, cells were subcultured
with 0.05% trypsin for dissociation, and cultures through up to 30 passages were used in this study. The smooth muscle cells in culture
extended laterally and showed a highly flattened configuration. They
proliferated with a doubling time of ~2.3 days through at least 30 passages. On the basis of immunocytochemical analyses of cultured
smooth muscle cells, >95% of the cells express smooth muscle
-actin and smooth muscle embryonic (SMemb) myosin heavy chains,
which have been reported to be expressed in proliferating smooth muscle
cells (5, 18, 30). These results indicate that the
cells in culture were smooth muscle cells. Rat skin fibroblasts were
prepared from rat dermis. Small pieces of dermis (1-3
mm3) were explanted to 60-mm culture dishes and incubated
in DMEM. After 7-10 days in culture, outgrown fibroblast-like
cells were harvested with 0.05% trypsin and subcultured. Cultures
through up to 10 passages were used. A smooth muscle cell line (SM-3) was cultured in DMEM.
Transfection. Plasmid pEFm3 was constructed by subcloning a BamH I fragment encoding the human M3 muscarinic ACh receptor gene from pDSm3 (12) into pEF-BOS (23). The BamH I fragment was inserted into the Xba I site of pEF-BOS after the BamH I and Xba I fragments were converted to blunt ends. Plasmid pEFm3 was cotransfected into cultured smooth muscle cells with pEF-neo via the calcium phosphate method (8). Clones were selected in the presence of 600 µg/ml geneticin (G418) for 2 wk. Single colonies were isolated with the use of cloning rings and were propagated to generate continuous cell lines. Positive colonies were identified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB).
Binding experiments.
[3H]QNB binding was measured via the procedure described
by Inoue et al. (14). In brief, aliquots of cell
suspensions (3 × 103 cells/ml) were incubated with
various concentrations of [3H]QNB in the reaction medium,
which consisted of 137 mM NaCl, 1.8 mM CaCl2, and 4.2 mM
HEPES (pH 7.4 at 30°C) for 10 min at 30°C. The incubation mixture
was passed through Whatman GF/C glass fiber filters, which were then
washed twice with ice-cold reaction medium. Specific binding was
calculated as the difference between total binding and binding in the
presence of 105 M atropine. The radioactivity remaining
on the filter was counted in a liquid scintillation counter (Packard
TRI-CARB 4530).
Preparation of reconstituted smooth muscle fibers.
String-shaped reconstituted tissue fibers were prepared in rectangular
wells (0.8 × 5.0 × 0.5 cm deep) with two poles placed 4 cm
apart on the bottom of each well (Fig.
1A). Wells were constructed by
using an acrylic plastic mold (0.8 × 5.0 × 0.5 cm) with two holes (2-mm diameter). The mold, placed in a 100-mm polystyrene petri
dish (Iwaki Glass, Tokyo, Japan) was filled to a depth of 5 mm with
silicone (KE-106; Shinetsu Chemical, Tokyo, Japan) and allowed to
harden for 2 h at 100°C. String-shaped reconstituted tissue
fibers were formed by growing cells in collagen gels as described by
Kolodney and Wysolmerski (17) and as modified by Obara et
al. (27). Briefly, dispersed cells were suspended in an
ice-cold collagen solution containing 3 × 106
cells/ml cultured cells, 2.2 mg/ml collagen type I-A, and 0.24 mg/ml
collagen type IV in DMEM. An aliquot (2 ml) of the collagen-cell suspension was poured into a trough and placed in a CO2
incubator (humidified 5% CO2-95% air atmosphere) at
37°C. The collagen-gel suspension gelled within 30 min. After 2 h, an additional 15 ml of DMEM were added to each petri dish. The
preparations were incubated until the cells shrank the gel and formed a
string-shaped fiber. For the measurement of force development, one of
the poles was attached to a force displacement transducer (TB-612T;
Nihon Kohden, Tokyo, Japan), and the other was glued to the bottom of
the rectangular well. After the collagen-cell suspension was cast,
isometric force was continuously monitored. Measurements were performed
at 37°C in a humidified 5% CO2-95% air atmosphere.
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Tissue preparations. Guinea pigs (300-450 g) were stunned and exsanguinated, and the stomach was removed. The tissue was immersed in Leibovitz's L-15 medium, pH 7.4 at 37°C (20). Circular muscle layers of the fundus were carefully separated from the mucosa as described previously (31). The width and length of the preparations were ~2 and 15 mm, respectively.
Antibodies.
Anti-caldesmon monoclonal antibody, which recognizes both h- and
l-caldesmon, was suspended in hybridoma supernatant. This antibody was
kindly provided by Dr. F. Matsumura (44). Anti-smooth muscle -actin is a monoclonal antibody raised against the synthetic NH2-terminal decapeptide of the smooth muscle
-isoform
of actin (Progen Biotechnik, Heidelberg, Germany). Anti-calponin is an IgG1 monoclonal antibody (clone hCP) specific for smooth muscle calponin (Sigma, St. Louis, MO). Anti-SM-1 (clone 1C10), SM-2 (clone
2B8), and SMemb (3H2) are monoclonal antibodies that specifically recognize the isoforms of SM-1, SM-2, and SMemb smooth muscle myosin
heavy chains, respectively (Yamasa, Tokyo, Japan). Anti-nonmuscle SM is
a polyclonal antibody raised against human platelet myosin heavy chain
(Paesel+Lorei, Frankfurt, Germany). Alkaline phosphatase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibody developed in goat
were purchased from Promega (Madison, WI). Biotinylated anti-mouse IgG
secondary antibody developed in horse was from Vector Laboratories
(Burlingame, CA).
Immunocytochemistry of cultured smooth muscle cells.
Dissociated smooth muscle cells in culture were plated on sterile glass
coverslips in glass-bottomed culture dishes (Meridian Instruments) at a
density of 1.5 × 104 cells/dish. The cultures were
grown for 16 h in the medium. After being washed with
phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4 · 7H2O, and 1.4 mM
KH2PO4) for 3 min, cells were fixed in 4%
paraformaldehyde for 15 min and incubated in PBS containing 0.5%
Triton X-100 for 15 min at room temperature (RT). After fixation, cells
were stained with anti-smooth muscle -actin (1:10) or anti-SMemb
antibodies diluted (1:3,000) in PBS containing 2% horse serum for 60 min at RT. The cells were then incubated for 60 min at RT with
biotinylated horse anti-mouse IgG secondary antibody (Vector
Laboratories) diluted (1:500) in PBS, followed by a second 60-min
incubation in an avidin-biotin solution (Vectastain Elite ABC kit).
Both incubations were preceded by thorough rinses with PBS. The
reaction products were visualized with 3,3'-diaminobenzidine and
hydrogen peroxide (DAB kit). Cells were then washed three times with
PBS, and the slides were mounted in Permount. Cells were viewed and
photographed on an Olympus photomicroscope.
Immunoblotting.
Frozen smooth muscle fibers and cultured smooth muscle cells were
crushed in a frozen stainless mill at 170°C. The frozen powder was
extracted in 20 vols of ice-cold sample buffer (5% SDS, 5 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5% 2-mercaptoethanol, and 0.1 M
Tris · HCl, pH 6.8). Homogenates were then heated in a boiling
water bath for 3 min. Gel electrophoresis and immunoblotting were
performed as described previously (39). In brief, proteins from the resultant supernatant were loaded in lanes and separated by
SDS-polyacrylamide gel electrophoresis in a 6-18% polyacrylamide gel. The amount of samples loaded in each lane was normalized with the
same amount of 43-kDa actin band proteins. The actin content was
determined by scanning densitometry. The protein concentration was
measured with bicinchoninic acid (BCA Protein Assay kit; Pierce, Rockford, IL). The proteins were then electrotransferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane
was then cut into rectangular strips, one of which was treated with
amido black to stain the proteins. The other strips were soaked in a
5% nonfat milk solution to reduce nonspecific reactions and were then
reacted at RT for 1 h with one of the following antibodies:
anti-SM-1 (1:30,000), anti-SM-2 (1:30,000), anti-SMemb (1:30,000),
anti-nonmuscle SM (1:2,000), anti-smooth muscle
-actin (1:10),
anti-calponin (1:1,000), or anti-caldesmon (1:100). After being rinsed
four times with Tris-buffered saline containing 0.05% Tween 20 (T-TBS)
for 5 min, each strip was reacted at RT for 1 h with a solution of
alkaline phosphatase-labeled anti-mouse or rabbit secondary antibody
diluted by 1:3,000. After being rinsed again four times with T-TBS for
5 min, the strips were reacted with 5-bromo-4-chloro-3-indolyl
phosphate p-toluidine salt (BCIP)-nitroblue tetrazolium (NBT) to
produce a violet color. After an appropriate intensity of color had
appeared, the membrane strips were washed with distilled water to
terminate the reaction.
Histological observation of the smooth muscle fiber. The gel matrix including cultured smooth muscle cells was fixed with freshly prepared 4% paraformaldehyde in PBS for at least 3 h at 4°C, followed by cryoprotection in 30% sucrose in PBS at 4°C for 24-48 h. The gels were immersed in Tissue-Tek O.C.T. (optimum cutting temperature) compound (Miles, Elkhart, IN) and frozen in liquid nitrogen. Longitudinal sections (10 µm) were cut using a cryostat. The sections were then subjected to hematoxylin-eosin staining.
Determination of cellular cross-sectional area.
The gels and smooth muscle tissue from the fundus of guinea pig stomach
were fixed in the same fixative and cryoprotected. The gels or tissues
were cut into cross sections, followed by immunostaining with
anti-smooth muscle -actin as described earlier in
Immunocytochemistry of cultured smooth muscle cells.
The fractional area occupied by the cells in each section was computed
by using NIH Image software. Notably, >95% of cells in the fibers
were smooth muscle
-actin positive. Cellular cross-sectional areas of the reconstituted fiber and guinea pig fundus were 15.0% and 89.9%, respectively. The force of each collagen gel or smooth muscle
tissue was normalized to its cellular cross-sectional area determined
in this manner.
Measurement of isometric force. Smooth muscle fibers prepared as described earlier in Preparation of reconstituted smooth muscle fibers were cut into two pieces (each 20 mm long) and mounted vertically in a 10-ml organ bath containing Leibovitz's L-15 medium, pH 7.4 at 37°C. The fibers were equilibrated in the same medium for 1 h at a resting tension of 2 mN. Tissue preparations were also mounted vertically in a 10-ml organ bath containing L-15 medium or normal HEPES-buffered Tyrode solution and were equilibrated in the same medium for at least 1 h at a resting tension of 5 mN. The tension development was recorded isometrically with a force displacement transducer (TB-612T; Nihon Kohden). Contractile studies were performed by adding various chemical agents to the final concentration desired or by replacing the medium with potassium-rich solution (140 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, and 4.2 mM HEPES, pH 7.4 at 37°C). In some experiments, to observe Ca2+ dependence of contraction, we used normal and Ca2+-free HEPES-buffered Tyrode solutions as bathing solutions. The normal solution had the following composition: 137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, and 4.2 mM HEPES, pH 7.4 at 37°C. The Ca2+-free solution had the same composition as normal solution except that CaCl2 was omitted. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (20 µM) and BAPTA (1 mM) were added to the Ca2+-free solution for the chelation of cytosolic and extracellular Ca2+, respectively.
Drugs used.
Collagen type I-A and IV were purchased from Nitta Gelatin (Osaka,
Japan); ACh (Ovisot) was from Daiichi Pharmaceutical (Tokyo, Japan);
norepinephrine (NE), epinephrine (Epi), phenylephrine, isoproterenol,
serotonin, histamine, BAPTA, BAPTA-AM, nifedipine, and papaverine
hydrochloride were from Wako Pure Chemicals (Osaka, Japan);
phentolamine, carbachol, ML-9, cytochalasin D, genistein, thrombin,
prostaglandin F2, and the DAB kit were from Sigma; and
BCIP and NBT solutions were from Promega. Leibovitz's L-15, DMEM, and
geneticin (G418) were from GIBCO-BRL (Gaithersburg, MD); the
bromodeoxyuridine (BrdU)-labeling kit was from
Boehringer-Mannheim (Mannheim, Germany); and methysergide maleate was
from Funakoshi (Tokyo, Japan). Y-27632 was generously donated by
Yoshitomi Pharmaceutical (Osaka, Japan), pDSm3 was donated by Dr. J. Silvio Gutkind (National Institutes of Health/National Institute of
Dental Research, Bethesda, MD), and pEF-BOS was donated by Dr. Sigekazu
Nagata (Osaka Bioscience Institute, Osaka, Japan).
[3H]QNB was from NEN (Boston, MA). All other chemicals
were of reagent grade.
Data analysis. All data are presented as means ± SE. Standard analysis of variance and unpaired t-tests were used to assess differences.
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RESULTS |
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A collagen gel containing 3 × 106 cells/ml cultured smooth muscle cells was prepared. The collagen-cell suspension gelled within 30 min after the inoculated collagen-cell suspension was placed in a CO2 incubator. Within 1 day after they were cast, smooth muscle cells began to contract the gel from an initial thickness of 5.0 mm to ~1.2 mm in diameter and maintained this string shape for >7 days (Fig. 1B).
Tension development.
Figure 2A shows a
representative tracing of the time course of tension developed by
smooth muscle cells. Typically, a steady increase in force occurred
16 h after casting and then plateaued at a steady-state value of
~6.2 mN within 120 h after casting. Cells maintained this
steady-state tension for >2 wk in culture. The BrdU-labeling study
revealed that, in 3-day-incubated fibers, only 2.1 ± 0.5%
(n = 3) of cells were BrdU-positive, indicating that
little cell replication occurred within the collagen matrix. The
removal of FBS from the medium gradually decreased the steady-state tension; a tension of 6.3 mN was decreased to 2.8 mN (Fig.
2B). Subsequent addition of the myosin light chain kinase
(MLCK) inhibitor ML-9 (3 × 105 M) and the tyrosine
kinase inhibitor genistein (3 × 10
5 M) did not
influence the FBS-independent tension. In another set of experiments,
the steady-state tension was partially inhibited by 3 × 10
5 M ML-9. Subsequent addition of genistein further
decreased this value: a tension of 5.9 mN was decreased to 5.2 mN by
ML-9 and decreased further to 3.7 mN by genistein (Fig. 2C).
To determine whether the effect of FBS is due to activation of a
contractile system, the effect of FBS on contraction of the fiber was
studied. Seven-day-incubated smooth muscle fibers were mounted in an
organ bath containing L-15 medium, and isometric contraction was
measured with a force displacement transducer. As shown in Fig.
2D, 10% FBS induced sustained contraction. The contraction
induced by 10% FBS was partially inhibited by subsequent addition of
3 × 10
5 M ML-9 and almost completely by 3 × 10
5 M genistein.
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Alignment of cells.
To evaluate the organization of cells populating the collagen fiber,
the smooth muscle cells were stained with hematoxylin-eosin. Smooth
muscle cells were randomly dispersed when the collagen-cell suspension
was cast (Fig. 3a). Three days
after they were cast, the smooth muscle cells in the fiber exhibited
elongated bipolar spindle shapes and were oriented parallel to the
direction of the isometric axis (Fig. 3, b-d). As shown
in Fig. 3e, the surface of a smooth muscle cell in the fiber
is separated from that of neighboring cells, and it is unlikely that
there was any cell-to-cell contact. The cells maintained this
longitudinally oriented shape for at least 2 wk.
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Expression of smooth muscle phenotype marker proteins.
Immunoblot analysis revealed that smooth muscle from the fundus of
guinea pig stomach expressed differentiated smooth muscle cell marker
proteins including SM-1 and SM-2 myosin heavy chains, smooth muscle
-actin, calponin, and h-caldesmon, but not the developing (immature)
smooth muscle cell marker proteins SMemb and nonmuscle myosin heavy
chains or l-caldesmon (Fig. 4). Smooth muscle cells passaged 10 times did not react with anti-SM-2 antibody, although the cells continued to express smooth muscle
-actin and
calponin (Fig. 4). The expression of SM-1 myosin heavy chains and
h-caldesmon decreased, and SMemb expression increased. The cells also
began to express nonmuscle myosin heavy chains and l-caldesmon, which
are marker proteins for developing smooth muscle cells. There was no
significant difference in the expression of these marker proteins in
cell cultures after 10 or 30 passages (data not shown). These results
suggest that these smooth muscle cells continue to express some
differentiated smooth muscle cell marker proteins after multiple
passages in cell culture. The expression of these parameters in
reconstituted fibers closely resembles that of cultured smooth muscle
cells except for the decrease in h-caldesmon and calponin in the fibers
(Fig. 4). These results suggest that mechanical stress loading on
cultured smooth muscle cells does not result in an increase in the
expression of smooth muscle cell differentiation markers toward the
contractile state but, rather, results in a decrease in some markers of
differentiated smooth muscle cells.
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Agonist-stimulated contraction.
After 7 days of incubation in a CO2 incubator at 37°C,
isometric contractions of the fibers were studied. Although the
following experiments were performed using 7-day-incubated fibers,
3-day-incubated fibers possessed identical attributes. Figure
5A shows representative contractile responses to KCl-induced depolarization characterized by a
sustained increase in tension. Figure 5, e and f,
shows representative contractile responses of the fiber to adrenoceptor
stimulants characterized by sustained tension after NE
(107 M) or Epi (10
7 M) addition. A similar
magnitude of tonic contraction was also triggered by
-adrenoceptor-specific stimulant phenylephrine (10
6 M)
(Fig. 5g). In contrast,
-adrenoceptor stimulant
isoproterenol (10
5 M) caused a slight decrease in tension
(Fig. 5h). Serotonin (10
6 M) also induced
tonic contractions of similar magnitude (Fig. 5b). Histamine
(10
5 M) caused a biphasic effect: a phasic contraction
followed by a gradual decrease in tension (Fig. 5c). The
muscarinic agonist ACh (10
5 M), a typical contractile
agonist of guinea pig stomach smooth muscle tissues, caused little
contractile response (Fig. 5d). Cumulative dose-response
curves demonstrated dose-response relationships for NE and serotonin
vs. force (Fig. 6). Contracted fibers
readily relaxed after being washed for 30 min, and contraction could be repeatedly evoked by the same concentration of agonists.
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Contractile properties.
To clarify whether the characteristics of contraction of this fiber
reflect typical contractilities of natural smooth muscle tissues, we
first studied the effects of an MLCK inhibitor, a Rho kinase inhibitor,
cytochalasin D, and papaverine on NE-induced contraction. When the
fibers were pretreated with the MLCK inhibitor ML-9 (3 × 105 M), the NE-induced contraction was almost completely
inhibited (24.3% of control), suggesting the involvement of myosin
phosphorylation catalyzed by MLCK (Fig.
7A). NE-induced contraction
was completely abolished by the addition of the Rho kinase inhibitor
Y-27632 (10
6 M) (43) and cytochalasin D
(2 × 10
6 M), suggesting the involvement of Rho
kinase and actin filaments (Fig. 7, Ba and Bb).
NE-induced contraction was also completely abolished by the addition of
10
5 M papaverine, indicating that the cells in the fibers
exhibit relaxation response to papaverine (Fig. 7Bc).
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Lack of contractile response to ACh. To clarify whether the loss of response to muscarinic agonists is due to a loss of muscarinic receptors, we first measured the specific binding of a muscarinic receptor ligand, [3H]QNB. In freshly isolated smooth muscle cells from the fundus of guinea pig stomach, a Scatchard plot of the specific binding of [3H]QNB yielded a straight line, and the maximal number of binding sites (Bmax) and the dissociation constant (Kd) were 1.13 ± 0.09 pmol/mg protein and 0.24 ± 0.05 nM, respectively (n = 4). In contrast, in binding to cultured smooth muscle cells, Bmax and Kd were 0.08 ± 0.01 pmol/mg protein and 1.19 ± 0.03 nM, respectively (n = 4). The Bmax value was ~14-fold lower than that estimated in freshly isolated cells. These results indicate that the number of maximal binding sites decreased during cell culture.
To examine whether contractile responses to ACh are rescued by overexpression of muscarinic receptors, we transfected the smooth muscle cells with cDNA encoding human M3 muscarinic receptors and measured the isometric force of the fiber in response to ACh. In the [3H]QNB binding to M3 receptor-transfected cells, Bmax and Kd were 1.36 ± 0.03 pmol/mg protein and 0.41 ± 0.01 nM, respectively (n = 4). The value of Bmax was ~17-fold higher than that in control cultured cells and was almost the same as that in freshly isolated smooth muscle cells. The M3 receptor-expressed fiber developed a predominant contractile force in response to ACh (2.53 ± 0.08 mN at 10 ![]() |
DISCUSSION |
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Our results show that these string-shaped reconstituted fibers possess contractile ability and respond to typical contractile agonists. The present method permits for the first time the quantitative measurement of contractile force generated by cultured smooth muscle cells of a defined type in response to typical contractile agonists.
Several investigations of contraction in cultured smooth muscle cells have been performed on cells grown on deformable substrates (1, 13, 26). The magnitude of cellular force in these experiments was inferred from the degree of wrinkling of the substrates. In our system, force can be measured directly. The technique offers at least several principal advantages for studies underlying smooth muscle contraction. Investigators can measure the contraction of a single cell type isolated from various smooth muscle tissues. Furthermore, cell culture allows biochemical and genetic manipulations to be performed in an attempt to dissect the molecular pathways involved in the control of mechanical functions. The major disadvantage of this system is that cells are modified compared with their in vivo counterparts. Nevertheless, this method provides a very powerful means for functional and developmental study of smooth muscle.
Smooth muscle cells readily transform their differentiated phenotype
when grown in vitro. The so-called phenotypic modulation process was
first described by Chamley-Campbell et al. (7), who found
that vascular smooth muscle cells exhibit gradual loss of
morphologically and immunocytologically identifiable myofilaments as
well as contractility when grown in culture. It is, however, now clear
that there is not a complete and irreversible loss of smooth
muscle-specific contractile proteins in cultured smooth muscle cells.
Several investigators have shown that smooth muscle cells derived from
a variety of blood vessels and species can be grown under conditions in
which they continue to express smooth muscle contractile proteins
(24, 30, 32) and contractile responsiveness (4, 24,
26). Consistent with this, we found that these cells express
some marker proteins typically considered indicative of a smooth muscle
contractile phenotype, including SM-1 myosin heavy chains, smooth
muscle -actin, calponin, and h-caldesmon (Fig. 4).
Mechanical stress loading on smooth muscle cells did not result in an increase in the expression of smooth muscle cell differentiation marker proteins but, rather, resulted in a decrease in the expression of h-caldesmon and calponin. Kuro-o et al. (18, 19) demonstrated that vascular smooth muscle cells from adult rabbits express both SM-1 and SM-2 myosin heavy chains. In contrast, fetal smooth muscle cells expressed the SMemb and SM-1 myosin heavy chains but not SM-2. Our results as well as these suggest that, although mechanical stress loading in collagen gels can convert smooth muscle cells into a morphologically differentiated phenotype, another factor is needed to maintain the immunocytochemically differentiated phenotype.
The steady-state tension generated in formation of the fiber was developed in DMEM with 10% FBS. It has been reported that serum could induce myosin light chain phosphorylation and develop contraction via the tyrosine kinase pathway (15). We therefore examined the possible involvement of serum-induced contraction in the steady-state tension. Removal of FBS from the medium gradually decreased the tension to 44.5%, and the serum-dependent component of tension was partially inhibited by an MLCK inhibitor, ML-9. Subsequent addition of a tyrosine kinase inhibitor, genistein, further decreased tension. These results suggest that the serum-induced contractile force is partially involved in the steady-state tension. This is confirmed by the observations that FBS induced a tonic contraction and that this contraction was inhibited partially by ML-9 and almost completely by genistein.
Isometric force measurements revealed contraction of reconstituted
fibers in response to typical contractile agonists including -adrenoceptor agonists and serotonin. The smooth muscle fiber developed 5.0-fold higher force than SM-3 cell fiber and 4.7-fold higher force than fibroblast fiber, indicating that the cultured smooth
muscle cells we prepared maintained their ability to produce relatively
high forces compared with smooth muscle cell lines and nonmuscle
fibroblasts. The maximal force value for the fiber was, however,
9.9-fold less than that of stomach smooth muscle tissues (Table 1).
Immunoblot analysis showed that the distribution of contractile
proteins in the tissue substantially differed from that of the fiber
(Fig. 4). Therefore, one of the explanations of the much smaller force
produced by the fiber is downregulation of some contractile proteins
and receptors for contractile agonists. This is supported by the
finding that overexpression of muscarinic M3 receptor
resulted in a predominant contractile force in response to ACh. Another
explanation may be the change in the level of myosin light chain
phosphorylation. Alternatively, the fibers may not be at optimal length
for maximal tension development. Further studies, however, are required
to explain the small contractile force.
The maximal forces induced by carbachol (105 M) for the
fundus strips ranged between 15.4 and 40.8 mN, with a mean of 28.9 ± 3.84 mN. This yielded a maximal force value of 71.3 ± 9.48 mN/mm2 cellular cross-sectional area, which was
lower than that for the longitudinal smooth muscle of rat stomach
fundus (10) and taenia cecum (11). We used
Leibovitz's L-15 medium as the bathing solution. When normal
HEPES-buffered Tyrode solution was substituted for L-15 medium,
carbachol induced a force of 37.9 ± 5.11 mN, which was 1.3-fold
higher than that force in L-15 medium. Therefore, the small contractile
force per cellular cross-sectional area for the fundus was partly due
to the different bathing solutions used. The cellular cross-sectional
area of the fundus strip was 89.9%, which was 1.4-fold higher than the
estimated value reported for the taenia caecum of the guinea pig
(11), 2.0- to 2.9-fold higher than that for dog carotid
artery (9), and 4.5-fold higher than that for rat
mesenteric resistance artery (25). Another cause might be
the different methods of estimating the cellular cross-sectional area.
The present finding that the contraction evoked by NE is inhibited by ML-9, Y-27632, and cytochalasin D indicates the importance of myosin phosphorylation catalyzed by MLCK, Rho kinase, and actin filaments in the contraction. Moreover, the contraction was found to be dependent on both extracellular and intracellular Ca2+, was partially sensitive to nifedipine, and was completely abolished by papaverine. These findings strongly suggest that, although the reconstituted fiber is not a model for fully differentiated smooth muscle with respect to the expression of smooth muscle cell markers, contractile mechanisms are, at least partly, maintained in the reconstituted fibers.
It has been shown that Y-27632 inhibits Ca2+ sensitization of smooth muscle contraction (43) and also inhibits Rho A-induced stress fiber formation in fibroblasts (22). We found that Y-27632 not only inhibited the NE-induced contraction but also depressed the force below the baseline level. Similar force depression was also observed by the addition of cytochalasin D, indicating that the effect of Y-27632 may be partly due to modulation of actin filaments.
However, there was virtually no contractile response to ACh. In
contrast, isolated smooth muscle tissue from guinea pig stomach responded significantly to ACh. The [3H]QNB binding study
revealed that the number of muscarinic receptors was decreased in
smooth muscle cells in culture. Moreover, the collagen fibers
consisting of M3 receptor-transfected cells developed an
enhanced contractile force in response to ACh. These results suggest
that the loss of response to muscarinic agonists is due to a loss of
expression of muscarinic receptors and that the smooth muscle cell
receptor phenotype is altered during culture. These findings may also
explain why the responses to NE are much higher in the fibers than they
are for stomach tissue. There is evidence of developmental regulation
of some contractile agonist receptors in vascular smooth muscle cells
including muscarinic M1, M2, and M3
receptors (6), 1-adrenergic receptors
(35), and
-adrenergic receptors (34). No
studies, however, have been reported indicating when and how these
contractile agonist receptors are expressed during embryonic
development of smooth muscle cells.
In conclusion, cultured smooth muscle cell-populated collagen gel
fibers were prepared. Contractions were dependent on the agonist used.
Increasing the concentration of agonists increased contractile force in
a typical dose-dependent manner. Contraction could be repeatedly evoked
after extensive washing. The competitive -adrenoceptor
antagonist phentolamine produced a parallel rightward shift of the NE
concentration-response curve. NE-induced contraction was partially
inhibited by nifedipine and completely abolished by the intracellular
Ca2+ chelator BAPTA-AM, MLCK inhibition, Rho kinase
inhibitor, cytochalasin D, and papaverine. These results suggest that
this method permits the quantitative measurement of isometric
contraction produced by a cultured smooth muscle cell population.
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ACKNOWLEDGEMENTS |
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We are especially grateful to Dr. F. Matsumura for providing anti-caldesmon antibody.
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FOOTNOTES |
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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
Address for reprint requests and other correspondence: K. Oishi, Dept. of Pharmacology, Meiji Pharmaceutical Univ., 2-522-1, Noshio, Kiyose, Tokyo 204-8588, Japan (E-mail: oishikz{at}my-pharm.ac.jp).
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.
Received 3 May 1999; accepted in final form 15 June 2000.
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