Agonist-induced isometric contraction of smooth muscle cell-populated collagen gel fiber

Kazuhiko Oishi1, Yuko Itoh1, Yasuhiro Isshiki1, Chikatoshi Kai1, Yasushi Takeda1, Kazuhiro Yamaura1, Hiromi Takano-Ohmuro2, and Masaatsu K. Uchida1

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


    ABSTRACT
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INTRODUCTION
METHODS
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DISCUSSION
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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; 10-7 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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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INTRODUCTION
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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 alpha -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 10-5 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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Fig. 1.   A: a schematic representation of a rectangular well. Each well (0.8 × 5.0 × 0.5 cm deep) has 2 poles glued 4 cm apart on the bottom. a, Top view; b, lateral view. B: photographs showing time-dependent contraction of smooth muscle cell-populated collagen gels. Collagen-gel suspension containing 3 × 106 cells/ml cells was poured into a rectangular well and placed in a CO2 incubator at 37°C in a humidified 5% CO2-95% air atmosphere. The collagen-gel suspension gelled within 30 min (a). One day after cells were cast, the diameter of the cross section was dramatically reduced and formed a string-shaped fiber (b). This shape was maintained for 3 (c) and 7 (d) days after casting.

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 alpha -actin is a monoclonal antibody raised against the synthetic NH2-terminal decapeptide of the smooth muscle alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 F2alpha , 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.


    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 × 10-5 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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Fig. 2.   Representative tracing of force development by smooth muscle cell-populated collagen gels and its characterizations. A: collagen-gel suspension containing 3 × 106 cells/ml cells was cast in DMEM with 10% fetal bovine serum (FBS), and the isometric force was continuously monitored using a force displacement transducer. B: medium was replaced with FBS-free DMEM 99 h after casting (a). After the tension reached a new steady-state value, 3 × 10-5 M ML-9 was added (b), and then 3 × 10-5 M genistein (c) and 2 × 10-6 M cytochalasin D (d) were likewise added. C: in another experiment, 3 × 10-5 M ML-9 was added 102 h after casting (b). After the tension reached a new steady-state value, 3 × 10-5 M genistein was added (c). Cytochalasin D (2 × 10-6 M) was likewise added (d). D: representative tracing of FBS-induced contraction of smooth muscle fiber and its inhibition by ML-9 and genistein. Seven-day-incubated fibers were cut into 2 pieces and mounted vertically in a 10-ml organ bath containing L-15 medium. The tension development was recorded isometrically with a force displacement transducer. Contractions were induced by adding FBS (a-c), and 3 × 10-5 M ML-9 (b) and 3 × 10-5 M genistein (c) were applied 10 min later. Data are representative traces of repeated experiments (n = 4).

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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Fig. 3.   Hematoxylin-eosin staining of longitudinal sections of smooth muscle cell-populated collagen gels. Collagen-gel suspension containing 3 × 106 cells/ml cells was cast and then fixed with fixative at various times (a, 0 days; b, 1 day; c, 3 days; d, 7 days). Longitudinal sections were cut with a cryostat and subjected to hematoxylin-eosin staining. e, Higher magnification of d. Bar indicates 100 µm and also represents the direction of isometric axis.

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 alpha -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 alpha -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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Fig. 4.   Immunoblot analysis of smooth muscle from the fundus of guinea pig stomach, cultured smooth muscle cells, and reconstituted smooth muscle fibers. Samples from isolated stomach smooth muscle tissue (a), cultured smooth muscle cells (b), and reconstituted smooth muscle fibers (c) were subjected to SDS-PAGE and immunoblotting. A: immunostaining with monoclonal antibodies against nonmuscle myosin heavy chains (lane 1), smooth muscle embryonic (SMemb) myosin heavy chains (lane 2), SM-2 (lane 3), and SM-1 (lane 4). B: immunostaining with monoclonal antibodies against smooth muscle alpha -actin. C: immunostaining with monoclonal antibodies against calponin. D: immunostaining with monoclonal antibodies against caldesmon (h, h-caldesmon; l, l-caldesmon). Note that the cells used to generate the reconstituted fibers (lanes c) were from the same passage (10) as those used in lanes b. The relevant proteins of the blots are displayed here; there were no nonspecific bands observed. Arrowheads in A, B, and C represent 200, 43, and 34 kDa, respectively.

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 (10-7 M) or Epi (10-7 M) addition. A similar magnitude of tonic contraction was also triggered by alpha -adrenoceptor-specific stimulant phenylephrine (10-6 M) (Fig. 5g). In contrast, beta -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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Fig. 5.   Representative traces showing contractile responses of smooth muscle fibers to various contractile stimulants. Tension development was recorded isometrically with a force displacement transducer. Contractions were induced by replacing the medium with a potassium-rich solution (A) or by adding agonists (B-H). NE, norepinephrine; Epi, epinephrine. The maximal tensions (mN) produced by KCl, serotonin, histamine, NE, Epi, and phenylephrine were 0.90, 1.49, 0.31, 0.88, 0.98, and 0.95, respectively.



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Fig. 6.   Dose-response relationships for NE (A) and serotonin (B) vs. force and their inhibition by antagonists. A: the first contraction was induced by adding NE cumulatively to achieve the final concentration desired (open circle ). The fiber was then washed for 60 min and treated with 10-7 M phentolamine for 10 min. The second contraction was induced by adding NE cumulatively to the final concentration desired in the presence of phentolamine (). The fiber was again washed for 60 min, and then the third contraction was likewise induced (triangle ). The maximal effect (pD2) for NE calculated by this dose-response curve was 7.66 ± 0.58. The pA2 value for phentolamine was 7.78 ± 0.43. B: The first contraction was induced by adding serotonin cumulatively to achieve the final concentration desired (open circle ). The fiber was then washed for 60 min and treated with 10-9 M methysergide for 10 min. The second contraction was induced by adding serotonin cumulatively to the final concentration desired in the presence of methysergide (). The maximal effect (pD2) for serotonin calculated by this dose-response curve was 7.27 ± 0.36. The pD'2 value for methysergide was 8.57 ± 0.31. Data points and error bars are means ± SE (n = 4). *P < 0.05, **P < 0.01, ***P < 0.005, compared with the first contraction (n = 4 in A and B).

To compare maximal force values with that produced by other cells, we prepared reconstituted fibers of a smooth muscle cell line (SM-3) derived from rabbit aorta and nonmuscle fibroblasts and measured maximal force values during isometric contractions. The SM-3 fibers contracted in response to 10-5 M prostaglandin F2alpha , which is reported to be a good stimulant (33). The fibroblast fibers also contracted in response to 5 U/ml thrombin, which is reported to be a contractile agonist (17). The maximal force values per fiber for the cultured smooth muscle cells (stimulated with 10-6 M serotonin), SM-3, and fibroblasts were 1.49 ± 0.09 mN (n = 4), 0.30 ± 0.06 mN (n = 3), and 0.32 ± 0.07 mN (n = 4), respectively. Notably, SM-3 and fibroblasts developed force much more slowly than reconstituted fibers (data not shown). The maximal force induced by carbachol (10-5 M) for the fundus strips in L-15 medium ranged between 15.4 and 40.8 mN, with a mean of 28.9 ± 3.84 mN, which was 76.3% of that in normal HEPES-buffered Tyrode solution.

To compare isometric force values in our system with those produced by smooth muscle tissues, we calculated the cellular cross-sectional areas in collagen fibers. Measurements were performed by computing the percentage of collagen fiber cross-sectional area occupied by cells in the sections. Cultured smooth muscle cells in 7-day-incubated collagen fiber exposed to 10-7 M NE produced an isometric force of 4.58 ± 0.06 mN/mm2 (n = 4), which was 16-fold lower and 2.6-fold higher than carbachol- and NE-triggered responses from isolated stomach smooth muscle tissue, respectively (Table 1).

                              
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Table 1.   Comparison of agonist-induced force normalized to cellular cross-sectional area in smooth muscle fiber and isolated smooth muscle tissue from the fundus of guinea pig stomach

Finally, the effect of phentolamine on NE-induced contraction was investigated. The nonselective alpha -adrenoceptor antagonist phentolamine, at a concentration that shows alpha -blocking activity (10-7 M), inhibited NE-induced contraction and produced a parallel rightward shift in the NE concentration-response curve (pA2 = 7.78 ± 0.43) (Fig. 6A). Contraction induced by serotonin was inhibited by the serotonergic antagonist methysergide (10-9 M) and reduced the maximal effect in the serotonin concentration-response curve (pD'2 = 8.57 ± 0.31) (Fig. 6B).

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 × 10-5 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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Fig. 7.   A: representative tracings showing inhibition of NE-induced contraction by ML-9. The first contraction was induced by adding 10-7 M NE (a). The fiber was then washed for 60 min and treated with 3 × 10-5 M ML-9 for 10 min. The second contraction was induced by adding 10-7 M NE (b). The fiber was again washed for 60 min, and then the third contraction was likewise induced (c). B: representative tracings showing inhibition of NE-induced contraction by Y-27632 (a), cytochalasin D (b), and papaverine (c). The contraction was induced by adding 10-7 M NE, and then 10-6 M Y-27632, 2 × 10-6 M cytochalasin D, or 10-5 M papaverine was applied 8-10 min later. Data are representative traces of repeated experiments (n = 4).

We next studied the Ca2+-dependence of NE-induced contraction. These experiments were performed in Ca2+-free HEPES-buffered Tyrode solution, and the results were expressed as a percentage of the control NE-induced contraction in the presence of 1.8 mM Ca2+ elicited at the beginning of the experiment. When the external solution was changed to Ca2+-free solution (with 1 mM BAPTA) and NE was added, NE-induced contraction was decreased to 57.0 ± 2.5% (n = 4) of the control, indicating that the contraction was partly dependent on extracellular Ca2+. When the fiber was pretreated with 20 µM BAPTA-AM for 15 min in Ca2+-free solution, NE-induced contraction was decreased further to 10.4 ± 3.9% (n = 4), indicating that the intracellular Ca2+ chelator BAPTA-AM is able to inhibit those parts of the contractions that are independent of extracellular Ca2+. Moreover, NE-induced contraction in the presence of Ca2+ was partially inhibited (49.3 ± 0.4% of control, n = 4) by the Ca2+ antagonist nifedipine (3 × 10-6 M), suggesting the involvement of voltage-dependent Ca2+ channels.

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 10-4 M ACh). The muscarinic antagonist atropine (10-7 M) inhibited ACh-induced contraction and produced a parallel rightward shift in the ACh concentration-response curve.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 (10-5 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), alpha 1-adrenergic receptors (35), and beta -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 alpha -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.


    ACKNOWLEDGEMENTS

We are especially grateful to Dr. F. Matsumura for providing anti-caldesmon antibody.


    FOOTNOTES

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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