Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Endothelin-1 (ET-1) is a vasoconstrictor peptide originally derived from endothelial cells functioning as a local regulator of vascular tone and has been reported to possess a wide variety of other biological activities. Recent studies indicate the presence of endothelin-like immunoreactivity, endothelin-1 mRNA, and endothelin receptor in esophagus (Uchida et al., 1998a; Ohta et al., 2000) and colon (Inagaki et al., 1991).

ET-1 has a potent pharmacological effect on gastrointestinal smooth muscle, but its mechanisms remain uncharacterized. In gastrointestinal tract, ET-1 causes contraction of the esophagus (Uchida et al., 1998a), stomach (Allcock et al., 1995), and intestines (Kitsukawa et al., 1994). ET-1 plays a broader role in diverse physiological actions (Rubanyi and Polokoff, 1994), localizes in the enteric nervous system (Takahashi et al., 1990), and exerts important modulatory actions in gastrointestinal motility (Takahashi et al., 1990; Allcock et al., 1995). Most of the actions of ET-1 in gastrointestinal tract are contractile and occur via its direct action at the smooth muscle (Kitsukawa et al., 1994). It is also suggested that ET-1 levels are elevated in gastrointestinal disease (Rubanyi and Polokoff, 1994; Ohta et al., 2000).

ET-1 activates multiple signaling systems in vascular smooth muscle, involving such effects as phospholipase C (PLC), phospholipase D (PLD), phospholipase A2 (PLA₂), and protein kinase (PKC) (Simonson and Dunn, 1990). After binding to its G protein-coupled ET receptor, ET-1 stimulates PLC hydrolysis of phosphatidylinositol- 4,5-bisphosphate (PIP₂), generating the two second-messengers inositol triphosphate (IP₃) and DAG.

Increased phosphorylation of proteins on tyrosine residues following ET-1 stimulation has been observed in vascular smooth muscle cells, implicating protein-tyrosine kinases (PTKs) and/or phosphatases in the responses to this peptide (Koide et al., 1992). Initially, the role of tyrosine kinases in cell signaling was thought to be restricted to long-term effects such as growth and proliferation. However, it is now recognized that increased protein tyrosine phosphorylation occurs rapidly (within seconds) in response to both polypeptide growth factors and vasoconstrictor hormones (Abedi et al., 1995). Furthermore, the observation that epidermal growth factor and platelet-derived growth factor increase vascular tone, an effect blocked by genistein and typhostins, both inhibitors of tyrosine kinases, supports the possibility that tyrosine phosphorylation is involved in the contractile response (Hollenberg, 1994).

PKC is a family of homologous serine and threonine protein kinases. PKC is present in the cell cytoplasm and, upon agonist stimulation, rapidly translocates to the particulate or membrane fraction observed by Western immunoblot analysis and immunofluorescence studies (Sohn et al., 1997a,b). Agonist-stimulated PKC location occurs coincidentally with Ca²⁺ release from intracellular stores, but the specific role of increased cytosolic Ca²⁺ in PKC activation is not known. The most direct evidence for PKC-mediated contraction is provided by studies in which active PKC- was injected into saponin-permeabilized ferret aorta smooth muscle cells (Horowitz et al., 1996a). Active PKC- stimulates ferret aorta contraction, identical to phorbol ester-stimulated contraction, which is reversed by a PKC pseudosubstrate inhibitor. An earlier study revealed that Ca²⁺-independent contraction is preceded by translocation of PKC- from the cytosol to the plasma membrane (Khalil et al., 1992).

The p44/42 MAP kinase (MAPK) pathway consists of a protein kinase cascade linking growth and differentiation signals with transcription in the nucleus. A selective and potent inhibitor of the p44/42 MAPK cascade, PD98059, has been identified (Payne et al., 1991). As this compound does not inhibit the stress-activated protein kinase or p38 MAPK cascades, it will help to identify functional activities associated with MEK and p44/42 MAPK activation (Payne et al., 1991). Recently, it was reported that ET-1 activates MAPK (Liang et al., 2000), which is inhibited by PD98059.

To test whether ET-1-induced contraction is mediated via a G protein-, phospholipase isozyme-, PKC-, PTK-, or MAPK-dependent pathway, we investigated the signals in mediating contraction induced by ET-1 in cat esophageal circular muscle cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

DEDA, R59949, PD98059, and SB202190 were purchased from Calbiochem (San Diego, CA); G protein antibodies (G_i1, G_i2, G_i3, G_q, G_s, and G_o), PKC isozyme antibodies (II, , and ) and PLC isozymes antibodies (1, 3, and 1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific p44/p42 monoclonal MAP kinase antibody was obtained from New England Biolabs (Beverly, MA); enhanced chemiluminescence agents were obtained from PerkinElmer Life Sciences (Boston, MA); nitrocellulose membrane was obtained from Bio-Rad (Hercules, CA); and SDS sample buffer was obtained from Owl Separation Systems (Woburn, MA). Selective myristoylated peptide inhibitors derived from pseudosubstrate sequences of PKC- (myr-PKC-) or PKC- (myr-PKC-) were gifts from Drs. D. A. Dartt and D. Zoukhri (Harvard Medical School, Boston, MA). HEPES, endothelin-1, collagenase type F, ammonium persulfate, ponceau S, bovine serum albumin, leupeptin, aprotinin, -mercaptoethanol, neomycin, pCMB, EGTA, EDTA, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Preparation of Dispersed Muscle Cells

Single muscle cells were isolated as previously described (Bitar and Makhlouf, 1982; Biancani et al., 1987). Muscle strips were incubated overnight in normal potassium-HEPES buffer containing 1 mg/ml papain, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin, and 0.5 mg/ml collagenase (type F, Sigma) and equilibrated with 95% O₂-5% CO₂ to maintain pH 7.0 at 31°C. The composition of the normal potassium-HEPES buffer was 1 mM CaCl₂, 250 µM EDTA, 10 mM glucose, 10 mM HEPES, 4 mM KCl, 131 mM NaCl, 1 mM MgCl₂, and 10 mM taurine. The next day we warmed up the tissue at room temperature for 30 min and incubated the tissue in a water bath at 31°C for 30 min. After incubation, the digested tissue was poured out over a 360-µm Nitex filter, rinsed in collagenase-free HEPES buffer to remove any trace of collagenase, and then incubated in this solution at 31°C, gassed with 95% O₂-5% CO₂. The cells were allowed to dissociate freely for 10 to 20 min. Suspensions of single muscle cells were harvested by filtration through a 500-µm Nitex mesh (Bitar and Makhlouf, 1982; Biancani et al., 1987). Before beginning the experiment, the cells were kept at 31°C for at least 10 min to relax the cells. Throughout the entire procedure, care was taken not to agitate the fluid to avoid cell contraction in response to mechanical stress.

Preparation of Permeabilized Smooth Muscle Cells

Cells were permeabilized, when required, to diffuse the agents such as G protein antibodies or PLC isozyme antibodies or PKC antibody, which do not diffuse across the intact cell membrane. After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed with an enzyme-free cytosolic buffer of the following composition: 20 mM NaCl, 100 mM KCl, 5.0 mM MgSO₄, 0.96 mM NaH₂PO₄, 1.0 mM EGTA, and 0.48 mM CaCl₂, and 2% bovine serum albumin. The cytosolic buffer was equilibrated with 95% O₂-5% CO₂ to maintain a pH of 7.2 at 31°C. Muscle cells dispersed spontaneously in this medium. The cytosolic buffer contained 0.48 mM CaCl₂ and 1 mM EGTA, yielding 0.18 µM free Ca²⁺ as calculated according to Fabiato and Fabiato (1979). After dispersion, the cells were permeabilized by incubation for 5 min in cytosolic buffer that contained saponin (75 µg/ml). After exposure to saponin, the cell suspension was spun at 350g, and the resulting pellet was washed with saponin-free modified cytosolic buffer that contained antimycin A (10 µM), ATP (1.5 mM), and an ATP-regenerating system that consisted of creatine phosphate (5 mM) and creatine phosphokinase (10 units/ml). After the cells were washed free of saponin, they were resuspended in modified cytosolic buffer.

Measurement of Contraction by Scanning Micrometry

Contraction of isolated muscle cells was measured by scanning micrometry (Sohn et al., 1995). An aliquot of cell suspension containing 10⁴ muscle cells/ml was added to HEPES medium containing the test agents. The reaction was terminated by addition of formalin (10% final concentration). The length of 40 to 50 muscle cells treated with a contractile agent was measured at random by scanning micrometry, phase contrast microscope (model ULWCD 0.30; Olympus, Tokyo, Japan), and digital closed-circuit video camera (CCD color camera; Toshiba, Tokyo, Japan) connected to a Macintosh computer (Apple, Cupertino, CA) with a software program, NIH Image 1.57 (National Institutes of Health, Bethesda, MD) compared with length of untreated cells. Contraction was expressed as the percentage decrease in mean cell length from control. The time course of contraction with agonists consists of peak contraction followed by a lower sustained plateau. Contraction in the present study refers to the initial peak contraction that occurred at 30 s with ET-1.

Phospho-MAP Kinase Western Blots. Previously frozen samples were homogenized in a buffer containing 20 mM Tris, 0.5 mM EDTA, 0.5 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 mM -mercaptoethanol (pH 7.5). Sample homogenates were then centrifuged for 10 min at 4°C, and the supernatants were collected. Aliquots were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. Membranes were blocked in PBS containing 5% dry milk for 2 h before an overnight incubation in a PBS solution containing 0.1% bovine serum albumin and a phosphospecific p44/p42 MAP kinase (Tyr-202/Tyr-204) antibody. Membranes were washed using PBS containing 0.05% Tween 20 and then incubated with horseradish peroxidase-conjugated secondary antibody (dilution 1:2000) for 1 h. Immunoreactive bands were visualized by enhanced chemiluminescence. Developed films from enhanced chemiluminescence were scanned and analyzed using NIH Image software; care was taken to avoid saturation of exposures for densitometry.

Protein Determination. The protein concentration in each reaction vial was measured spectrophotometrically using the Bio-Rad assay (Bio-Rad Chemical Division, Richmond, CA). The absorption was monitored at a wavelength of 595 nm.

Data Analysis. Data are expressed as the mean ± S.E.M. Statistical differences between means were determined by Student's t test. Differences between multiple groups were tested using analysis of variance for repeated measures and checked for significance using the Scheffe F test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Endothelin-1-Induced Contraction of Isolated Smooth Muscle Cells. It is known that ET-1 induces contraction in smooth muscle cells (Ibitayo et al., 1998; Wang and Bitar, 1998; Su et al., 1999). ET-1 induced contraction of smooth muscle cells that peaked at 30 s (26.2 ± 2.0% decrease in cell length from control) and was sustained for up to 30 min (Fig. 1), being decreased slowly. The response to ET-1 was concentration-dependent (Fig. 2). Freshly isolated smooth muscle cells were stimulated for 30 s with 10¹⁰ M~10⁶ M. The maximal response was seen at 10⁷ M. 

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Time course of contractile response of smooth muscle cells from cat esophagus. Freshly isolated cells were incubated in ET-1 (107 M) for the indicated times. Data are means ± S.E. of four experiments.

View larger version (8K): [in this window] [in a new window]   Fig. 2.   Dose-dependent contractile response of smooth muscle cells from cat esophagus. Freshly isolated smooth muscle cells were stimulated for 30s with the indicated concentration of ET-1.

The Effect of Pertussis Toxin (PTX) on Contraction Induced by ET-1. It has been shown that ET-1 has its own receptor coupled with PTX-sensitive G protein (Kasuya et al., 1992; Kimura et al., 1999). The cells were preincubated for 60 min with PTX (400 ng/ml). PTX abolished contraction induced by ET-1 (10⁷ M), implying that contraction in cat esophagus smooth muscle cells activated by ET-1 was coupled to a pertussis toxin-sensitive G protein (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of PTX on contraction induced by ET-1 (107 M). The cells were preincubated for 60 min with PTX (400 ng/ml) (Student's t test; , P < 0.001 versus control). Data are means ± S.E. of four experiments.

Characterization of G Protein Subtype-Coupled Receptor of ET-1. Previously, we have shown that G_i1-2, G₀, G_i3, G (40 kDa), G_s (46 kDa), and G_q (42 kDa) proteins exist in cat esophageal cells (Sohn et al., 1995; Yang et al., 2000). To identify the specific G protein involved in cat esophagus contraction, muscle cells were permeabilized with saponin preincubated in cytosolic medium containing G protein antibody (1:200) to allow diffusion of the antibodies into the cytosolic region of the cell membrane (Sohn et al., 1997a). These antibodies block receptor-induced activation of G protein by binding to the terminal peptide region of G protein that interacts with the receptor. After permeabilization, the G_i3 and G antibody inhibited contraction, but G_i1-2, G_o, G_q, and G_s did not (Fig. 4).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Inhibition of ET-1-induced contraction in permeabilized esophageal circular muscle cells by antibodies to G protein isoforms. Results are expressed as percentage decrease in cell length, compared with control. Data are means ± S.E. of four experiments (Student's t test; , P < 0.01 versus control).

PLC-3 Mediates ET-1-Induced Contraction

ET-1-induced contraction of esophageal smooth muscle cells was not affected by PLA₂ inhibitor DEDA (10⁵ M) (Yang et al., 2000) and PLD inhibitor CMB (10⁵ M) (Sohn et al., 1993; Yang et al., 2000), but was significantly abolished by PLC inhibitor neomycin (10⁵ M) (Sohn et al., 1993; Yang et al., 2000) that abolished phosphoinositide hydrolysis in cells (Fig. 5). These results suggested that contraction of esophageal smooth muscle cells might be partially mediated by phosphatidylinositol-specific PLC.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Different phospholipases mediate ET-1-induced contraction of smooth muscle cells of feline esophagus. The cells were contracted with ET-1 (107 M) after a 10-min preincubation with the PLC inhibitor, neomycin (105 M) or the PLD inhibitor, CMB (105 M) and after a 1-min preincubation with the PLA2 inhibitor, DEDA (105 M). Preincubation for 10 min with neomycin caused 46.2% inhibition. Data are mean ± S.E. of five experiments (Student's t test; , P < 0.05 versus control).

We previously showed that Western blot analysis of homogenates obtained from dispersed smooth muscle cells using polyclonal antibodies to PLC isozymes demonstrated the presence of immunoreactive protein bands corresponding to 150 kDa (PLC-1 and PLC-3 antibody) and 145 kDa (PLC-1 antibody) (Yang et al., 2000).

Incubation of permeabilized circular muscle cells for 1 h with PLC-3-specific antibody (1:200) (Yang et al., 2000) inhibited ET-1-induced (10⁷ M) contraction (P < 0.05). No other PLC-specific antibodies had any significant effect on the contraction (Fig. 6).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Inhibition of ET-1-induced contraction in permeabilized esophageal circular muscle cells by antibodies to PLC isoforms. Results are expressed as percentage decrease in cell length, compared with control. Data are means ± S.E. of three experiments (Student's t test; , P < 0.05 versus control).

The Roles of Protein Kinase C and Tyrosine Kinase in ET-1-Induced Contraction. Cells were preincubated with either the tyrosine kinase inhibitor genistein (10⁵ M) for 20 min or the protein kinase C inhibitor H-7 for 15 min (10⁵ M) (Sohn et al., 1995; Uchida et al., 1998b) or chelerythrine (10⁵ M) (Yang et al., 2000) or DAG kinase inhibitor R59949 (10⁵ M)(Sohn et al., 2000) for 1 min, respectively, before the addition of ET-1. ET-1-induced contraction was inhibited by preincubation with genistein as follows: percentage decrease in cell length was 27.7 ± 2.1 versus 9.8 ± 2.3% and 10.3 ± 2.3 versus 16.4 ± 2.0% in the cells preincubated with H-7 and chelerythrine, respectively (Fig. 7).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Contractile response of smooth muscle cells from cat esophagus to ET-1 (107 M) in the presence of protein kinase C inhibitors (H-7, 105 M; chelerythrine, 105 M), protein tyrosine kinase inhibitor (genistein, 105 M), and diacylglycerol kinase inhibitor (R59949, 105 M) was examined. Preincubation of cells for 1 min with chelerythrine or for 15 min with H-7 caused 40.8 and 62.8% inhibition, respectively. Preincubation of cells for 20 min with genistein caused 64.6% inhibition. Data are means ± S.E. of five experiments (Student's t test; , P < 0.01 versus control).

Inhibition of ET-1-Induced Contraction of Permeabilized Esophagus Cells by PKC Antibodies. We previously have shown that PKC isozymes, detected by Western blot analysis, including the PKC-II, -, and - isozymes, are present in the circular smooth muscle of the esophagus (Sohn et al., 1997b). To test the theory that PKC-mediated contraction may be isozyme-specific, we examined the effect of PKC isozyme antibodies on contraction induced by ET-1. Figure 8 shows that ET-1-induced contraction of permeabilized esophagus cells was significantly inhibited, from 20.4 ± 0.4 to 6.0 ± 0.3, by antibodies raised against PKC- (1:200) (Cao et al., 2001) and not by antibodies raised against the PKC-II or - isozyme.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   PKC- mediates ET-1-induced contraction of permeabilized circular muscle cells of esophagus. ET-1-induced contraction of esophagus cells was significantly inhibited by antibodies raised against PKC- and not by antibodies raised against the PKC- or -II isozymes (, P < 0.001 versus control).

Different N-Myristoylated Pseudosubstrate Peptides Inhibit ET-1-Induced Contraction of Esophagus. To characterize further the specific PKC isozymes that mediate contraction of the smooth muscle cell type, we used as an inhibitor N-myristoylated peptides derived from the pseudosubstrate sequences of PKC-, -, , and - (myr- PKC-, myr-PKC-, myr-PKC-, and myr-PKC-) and examined their effect on ET-1-induced contraction of intact esophagus smooth muscle cells. ET-1-induced contraction was inhibited by the myristoylated peptide corresponding to the pseudosubstrate sequence of PKC- and was not inhibited by myr-PKC-, myr-PKC-, myr-PKC-, and PKI (Fig. 9). These data support the hypothesis that PKC- mediates contraction of esophagus.

View larger version (38K): [in this window] [in a new window]   Fig. 9.   The effect of myristoylated pseudosubstrate peptides on ET-1-induced contraction of intact smooth muscle cells from the circular layer of the esophagus. ET-1-induced contraction of esophagus was significantly inhibited by myr-PKC- and not inhibited by myr-PKC-, myr-PKC-, or myr-PKC- (, P < 0.01 versus control). The PKA inhibitor, PKI, had no effects on esophagus. These data suggest that PKC- may mediate contraction of esophagus. Values are means ± S.E.M. of four animals with 35 cells counted at random for each data point.

View larger version (9K): [in this window] [in a new window]   Fig. 10.   Agonist-induced contraction was dose dependently inhibited by myr-PKC- in the esophagus. Values are means ± S.E.M. of four animals with 35 cells counted at random for each data point.

Role of MAP Kinases on ET-1-Induced Smooth Muscle Cell Contraction and Immunoblotting of MAP Kinase using Phosphospecific p44/p42 Monoclonal MAP Kinase Antibody. It has been known that MAP kinase (MAPK) mediates ET-1-induced cell responses (Sbrissa et al., 1997; Kimura et al., 1999). We investigated the role of MAPK activation in cat esophageal smooth muscle contraction. To test which MAPK is involved in ET-1-induced contraction, we used specific MAPK inhibitors. Preincubation of PD98059, a p44/p42 MAPK inhibitor, for 30 min blocked the contraction induced by ET-1 in a concentration-dependent manner. The maximal inhibition was observed in 10⁵ M. Preincubation for 30 min SB202190, a p38 MAPK inhibitor, also inhibited ET-1-induced contraction (Fig. 11). To determine whether the contractile effects of ET-1 are related to the activation of MAPK in isolated smooth muscle cells of cat esophagus, cells were stimulated with ET-1 and immunodetection of MAPK was performed. As shown in Fig. 12, ET-1 (10⁷ M) induced an increase in the intensity of the detection bands identified by immunological methods as phosphospecific MAPK monoclonal p44/p42 peptides. Preincubation of PD98059 for 30 min induced a decrease in the intensity of the detection bands as compared with ET-1-stimulated cells.

View larger version (12K): [in this window] [in a new window]   Fig. 11.   Effect of MEK inhibitor PD98059 and p38 MAPK inhibitor SB202190 on ET-1-induced cat esophageal smooth muscle cell contraction. Preincubation in the indicated concentration of PD98059 and SB202190 for 30 min decreased the contraction induced by ET-1 (107 M). The peak response was obtained with 105 M. Data are means ± S.E. of four experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 12.   Immunoblotting of MAPK using p44/42 phosphospecific monoclonal antibody. Samples were prepared from isolated cat esophageal smooth muscle and stimulated with ET-1 (107 M). A, at 30 s, ET-1 (107 M) increased in the intensity of the detection bands, indicating an increase in MAPK activity. When cells were pretreated with 105 M PD98059 for 30 min, the intensity of the detection bands was decreased as compared with ET-1. B, we measured the density of detection bands using the NIH Image program. Data are means ± S.E. of three experiments (Student's t test; , P < 0.05 versus control; #, P < 0.05 versus ET-1).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The endothelins are a family of 21-residue peptides consisting of three structurally related isoforms called endothelin (ET)-1, ET-2, and ET-3 (Huggins et al., 1993). Many of the details of the biosynthetic pathways involved in the production of mature ET isoforms remain to be elucidated. These mature, active forms are produced from the corresponding approximately 200-residue prepropolypeptides that are encoded by three separate genes. These prepropeptides are first processed by a furin-like processing protease into biologically inactive intermediates called big ET-1, -2, and -3 (Laporte et al., 1993). Big endothelins are then proteolytically activated via cleavage at the common Trp21 residue by a highly specific endopeptidase(s) called endothelin-converting enzyme (Opgenorth et al., 1992). The production of endothelins is tightly regulated at the level of mRNA transcription. Endothelins acts on two pharmacologically and molecularly distinct subtypes of the heptahelical superfamily of receptors called ETA and ETB receptors (Arai et al., 1990). Both receptor subtypes are expressed in a wide variety of cell types, with distinct but partially overlapping tissue distributions.

It is suggested that ET-1, possibly produced by mucosal epithelial cells, is present in the rat gastrointestinal tract and causes contraction of gastrointestinal smooth muscles (Takahashi et al., 1990). ET is thought to have important physiological and pathophysiological roles in both absorptive and secretory functions of the gastrointestinal mucosa. Rabbit gastric epithelial cells in culture produce ET-1, which can act in a paracrine manner on blood vessels and other tissues in the mucosa (Ota et al., 1991).

G_i3 and PLC-3 Mediate ET-1-Induced Contraction. G proteins transduce ligands binding to a cell surface receptor into intracellular signals. ET receptors are coupled to PTX-sensitive G protein (Emala et al., 1999; Husain and Abdel-Latif, 1999; Kimura et al., 1999). It has been reported that PTX, at 100 ng/ml, completely blocked the ET-1-induced MAPK activation in rat puerperal uterine contraction (Kimura et al., 1999), and pretreatment of PTX caused a significant reduction of ET-1-induced contraction in porcine coronary artery smooth muscle (Kasuya et al., 1992), Ras-associated MAPK activity in rat ventricular myocytes (Chiloeches et al., 1999), and p38 MAPK activity in cat iris sphincter smooth muscle cells (Husain and Abdel-Latif, 1999). We found that PTX, which inactivates G_i/G_o proteins, blocked the ET-1-induced contraction, suggesting that ET receptor couples with the G_i/G_o families of PTX-sensitive G proteins, which are consistent with those observed in rat mesangial cells (Simonson and Dunn, 1990), rat ventricular myocytes (Kelly et al., 1990), and rat uterine myometrial cells (Kimura et al., 1999).

11

i3

ET-1-Induced Contraction Was Mediated by a Protein Tyrosine Kinase- or Protein Kinase C-Dependent Pathway in Cat Esophagus. Many vasoconstrictor agonists increase protein tyrosine phosphorylation and extracellular signal-regulated kinase activity in smooth muscle preparations (Khalil et al., 1995; Ohanian et al., 1997). Furthermore, tyrosine kinase inhibitors block agonist-induced contraction (Horowitz et al., 1996b; Watts et al., 1996; Ohanian et al., 1997) as does extracellular signal-regulated kinase inhibition (Sbrissa et al., 1997), suggesting that this pathway is important for smooth muscle contraction. Evidence from studies with growth factor and ₁-adrenoceptor agonists suggests that activation of tyrosine kinases may be involved in contraction (Hollenberg, 1994), although the mechanisms that regulate tyrosine kinase activity, and the point at which this pathway may be involved in a contractile response, remain unclear. Catalan et al. (1999) suggested that the effect of ET-1 on tyrosine phosphorylation was dose- and time-dependent and caused a rapid tyrosine phosphorylation of three groups of proteins in the molecular mass range 70 to 100 kDa, 100 to 150 kDa, and 150 to 200 kDa. In this study, we investigated the regulation of tyrosine phosphorylation following ET-1 stimulation and the role of this pathway in the contractile response. Genistein is known as protein tyrosine kinase inhibitor, and its level is used from 1 to 30 µM for inhibition (Liu and Sturek, 1996; Su et al., 1999). Genistein reduced contraction in response to ET-1 in cat esophagus cells, suggesting that tyrosine kinases are involved in an ET-1-induced contraction pathway.

ET-1-Induced Contraction Is Mediated via MAP Kinase-Dependent Pathway. MAP kinase (MAPK) has been implicated in a signal-transduction cascade that regulates cell proliferation and differentiation in various cell types (Cobb et al., 1991). In the present study, the occurrence of MAPK was confirmed by immunological studies. Using anti-phosphospecific p44/p42 MAPK antibody, we were able to clearly demonstrate two forms of proteins with relative molecular masses of 42 and 44 kDa, which were in the same range of MAPK found in most tissues (Bitar and Yamada, 1995; Yamada et al., 1995). The activation of MAPK by ET-1 was rapid, within 30 s. Kimura et al. (1999) showed that ET-1-induced MAPK activation is neither extracellular Ca²⁺- nor intracellular Ca²⁺-dependent.

View larger version (18K): [in this window] [in a new window]   Fig. 13.   Cell contraction pathway by ET-1 in cat esophagus. ET-1-induced contraction is mediated through PKC- and PTK pathways. ET receptor is coupled with Gi3 and G protein and activates PLC-3. ET-1-induced contraction is mediated via p44/p42- and p38-MAPK pathways.