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Experimental Biology and Medicine 228:972-981 (2003)
© 2003 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Involvement of Rho and Rho-Associated Kinase in Sphincteric Smooth Muscle Contraction by Angiotensin II

Satish Rattan1, Rajinder N. Puri and Ya-Ping Fan

Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60c-src, p190 RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of G{alpha}13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p190 RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for G{alpha}13 and pp60c-src in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.

Key Words: smooth muscle • angiotensin II • Rho-associated kinase • tyrosine phosphorylation • pp60c-src • G{alpha}13 • p190 RhoGAP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The tonic smooth muscles of internal anal sphincter (IAS) and lower esophageal sphincter (LES) and IAS serve important functions to prevent gastrointestinal reflux and anorectal incontinence, respectively (1–5). Although a majority of tone in these tissues is by virtue of the myogenic properties of the smooth muscles, the basal tone is prone to excitatory modulation to a number of neurohumoral substances (6–8). Our recent studies suggest that among different species, rat is an ideal model for the investigations of angiotensin (Ang) II effects in the IAS and LES (9). Our studies further suggest that Ang II-induced contraction of these smooth muscles is mediated via the activation of AT1 receptor and involves multiple pathways that include Ca2+ influx, Protein kinase C (PKC), 44/42mitogen-activated protein kinases (MAPKs), and Rho kinase (8–10). The exact sequence of intracellular events from AT1 receptor activation to the Rho kinase activation in the contraction of these smooth muscles is not known.

Rho belongs to ras superfamily of monomeric or small G-proteins (11–14) and is known to exert diverse cellular functions. Rho cycles between a biologically inactive GDP-bound state and an active GTP-bound state (11). The low intrinsic GTPase activity of Rho is accelerated by the GTPase-activating proteins (GAPs) (11, 13). The rate of GDP/GTP exchange is regulated by the guanine nucleotide exchange factors (GEFs) (11, 13). Two serine/threonine kinases, p160 ROCK and ROK{alpha}, containing similar kinase and coiled-coil structure domains, have been identified among different effectors of Rho (15). Rho- and Rho-associated kinase (ROK)-mediated signaling pathways play important roles in smooth muscle contraction (15–20). ROK inactivates myosin light chain (20 kDa; MLC20) phosphatase by catalyzing phosphorylation of the p130 myosin-binding subunit in a Ca2+-independent manner (21). ROK also phosphorylates MLC in a Ca2+-independent manner at Ser-19, the site of MLC20 phosphorylation (MLC-P) by the Ca2+-calmodulin-dependent MLC kinase (MLCK) (22). These two biochemical events may increase MLC-P at constant intracellular ([Ca2+]i), a phenomenon known as Ca2+ sensitization of MLC-P (22). G-protein-mediated Ca2+ sensitization of rabbit trachea smooth muscle has been shown to involve ROK (23). In addition to Rho, another monomeric G-protein ADP ribosylation factor (ARF) has also been shown to participate in signal transduction (19, 20, 24, 25).

The members of G{alpha}12 family (G{alpha}12/13) of GTPases appear not to affect any of the second messenger-generating functions of G{alpha}s and G{alpha}i (26). Lysophosphatidic acid-induced growth stimulation but not stress fiber formation is inhibited by pertussis toxin (27). This suggests that stress fiber formation (via Rho activation) is mediated by a G-protein(s) different from Gi such as G{alpha}12/G{alpha}13 (28). The role of G{alpha}12/G{alpha}13 in different cell systems has been reviewed elsewhere (29, 30). G{alpha}12/G{alpha}13 have been shown to interact directly with p115 RhoGEF (31, 32) and to activate Rho.

There is only limited information examining the role of G-protein-coupled receptor (GPCR)-mediated signaling events in the activation of Rho and ROK, and ARF in gastrointestinal smooth muscle contraction (19, 20, 33, 34). Ang II has been shown to be a potent contractile agonist in different sphincteric smooth muscles, especially in the IAS (7–9, 35) in different species examined, including humans (36). In all of the species examined, Ang II has been shown to cause an increase in the basal LES tone in the concentration range found in the blood (37). Additionally, Ang II-converting enzyme activity has been shown to be present in the iris sphincter (38). Ang II is known to produce its actions, including smooth muscle contraction of the LES and IAS, via multiple signal transduction pathways (8, 9, 39). The purpose of the present investigation is to examine the role of Rho kinase pathway and related sequence of events in Ang II-induced contraction of these tonic smooth muscles. Such information is important for increased understanding of the basic mechanisms for the contraction of these smooth muscles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Smooth Muscle Strips and Measurement of Isometric Tension.
Smooth muscle strips (1 x 10 mm) from LES and IAS of rats were prepared as described previously (40) in oxygenated (95% O2 and 5% CO2) Krebs’ solution (118.07 mM NaCl, 4.69 mM KCl, 2.52 mM CaCl2, 1.16 mM MgSO4, 1.01 mM NaH2PO4, 25 mM NaHCO3, and 11.10 mM glucose). The mucosal and submucosal layers were removed by sharp dissection. The LES and IAS circular smooth muscle strips thus prepared were used for the recording of isometric tension, using force transducer (model FT03; Grass Instruments, Quincy, MA) connected to a PowerLab recorder (CB Sciences, Milford, MA). The details of the procedure have been given before (40). The smooth muscle strips initially stretched with 10 mN (millinewton) force were allowed to equilibrate for 1 hr. During equilibration, the smooth muscle strips were replenished every 20 min with fresh solution. Only those smooth muscle strips that developed spontaneous steady tone and relaxed in response to electrical field stimulation were used in further experiments. The optimal length and the basal tone of the smooth muscle strips were determined as described previously. The increase in contraction in rat LES and IAS smooth muscle was calculated as percentage of maximal contraction induced by bethanechol (1 x 10-4 M) at the conclusion of the experiment, in relation to active tone, determined at the end of each experiment using 5 mM EGTA.

Use of animals was approved by the institution’s Animal Care and Use Committee.

Isolation and Permeabilization of Smooth Muscle Cells (SMC) from Rat LES and IAS.
SMC from the tonic smooth muscle strips were isolated by the method described previously (41). The sphincteric regions were identified and marked in situ using a water-perfused catheter assembly specially designed for rats and mice. The areas were marked as high-pressure zones that relaxed to appropriate stimuli. Rat LES and IAS smooth muscle strips were cut into small pieces (1- to 2-mm cubes) and were incubated in oxygenated Krebs’ solution containing collagenase (0.01% for LES and 0.013% for IAS) and soybean trypsin inhibitor (0.01%) at 37°C for two successive 1-hr periods. After each incubation, the mixture was filtered through a 500-µm Nitex mesh. The tissue trapped on the mesh was rinsed with 25 ml (5 x 5 ml) of collagenase-free Krebs’ solution. The tissue was finally incubated in collagenase-free Krebs’ solution at 37°C, and dispersion of the cells (0-1 hr) was monitored periodically by examining a 10-µl aliquot of the mixture under microscope. SMC were harvested by filtration through the Nitex mesh. The filtrate containing the cells was centrifuged at 350g for 10 min at room temperature. The cells in the pellet were resuspended in Krebs’ solution at a cell density of 3 x 104 cells/ml. To increase the cell yield, the LES and IAS tissue samples were pooled from three to four animals for different experiments. Each experimental protocol with an appropriate control was carried out under identical conditions.

Permeabilization of SMC was accomplished by the method previously used in our laboratory (40, 42). LES and IAS SMC were permeabilized by incubating them in cytosolic solution (20 mM NaCl, 100 mM KCl, 5 mM MgSO4, 0.96 mM NaH2PO4, 25 mM NaHCO3, 1 mM EGTA, 0.48 mM CaCl2, and 1% bovine serum albumin [BSA]) with saponin (75 µg/ml) for 3 min at room temperature. The cell suspension was centrifuged at 350g for 10 min. The pellet was suspended in a cytosolic solution supplemented with antimycin A (10 mM), ATP (1.5 mM), phosphocreatine (5 mM), and creatine phosphokinase (10 U/ml) and was centrifuged at 350g for 10 min. Cells were washed twice with the modified cytosolic solution to remove saponin and were resuspended in the fresh modified cytosolic solution.

Measurement of SMC Length by Scanning Micrometry.
Aliquots (30 µl) of the SMC were incubated with Ang II (5-min exposure) in the absence or presence of selective inhibitors (20 min of incubation before Ang II). Incubations were terminated by the addition of acrolein (0.1%). The mean length of 30 cells chosen randomly in each set was determined by micrometry using phase contrast microscopy. The images were stored digitally and the cell length was measured by the Image-Pro Plus V4.0 program (Media Cybernatics, Silver Spring, MD). Digital data was transferred directly to the Microsoft Excel computer program. The data were expressed as the percentage of shortening of the original SMC length of 30 randomly chosen cells in each set as mean ± SEM. Each n in such experiments represents the mean of 30 cells either from one animal or cells pooled from more than one animal. Different categories of experiments were repeated at least four times in cells from different groups of animals (n = 4).

Gel Electrophoresis and Western Blot Analysis.
While monitoring the isometric tension, the rat LES and IAS smooth muscles strips were quick-frozen either in the basal state, after Ang II (1 x 10-7 M) alone, or in the presence of selective inhibitors, at the time of maximal contraction (usually within 2 to 3 min of addition of Ang II). The inhibitors were added 10 to 20 min before Ang II challenge. The smooth muscle tissues were fixed at the time of sustained contraction with Ang II, before and after different inhibitors. Protein extracts from the smooth muscles were prepared by cutting the tissues into small pieces and incubating them with a homogenization buffer (1% SDS, 1 mM sodium orthovanadate, and 10 mM Tris, pH 7.4) at 90°C for 3 min. The incubation mixtures were homogenized followed by centrifugation at 16,000g for 15 min at 4°C. Protein contents in the supernatants were estimated by Lowry’s method. Solutions of the protein extracts were prepared by mixing them with an equal volume of a 2x sample buffer (125 mM Tris, pH 6.8, 10% glycerol, 2% ß-mercaptoethanol, and 0.006% bromphenol blue) and heating the samples in a boiling water bath for 3 min. Protein samples (40 µg of protein/20 µl) were subjected to SDS-PAGE by the method of Laemmli (43). Discontinuous gel system using 4% stacking gel, pH 6.8, and 10% running gel, pH 8.8, was used.

Proteins in the gels were electroblotted on to a nitrocellulose membrane at 100 V for 1 hr at 4°C. The membranes were transferred in a blocking buffer containing Tris-buffered saline (TBS; 20 mM Tris and 137 mM NaCl, pH 7.4), 1% BSA, and 0.1% Tween 20 and were left overnight at 4°C. The membranes were rinsed with a washing buffer containing TBS and 0.1% Tween 20 (TBS-Tween), treated with a primary antibody for 1 hr with gentle agitation, and washed three times with TBS-Tween. They were subsequently treated with a secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hr with gentle agitation and then washed three times with TBS-Tween.

The dilutions of primary and secondary antibodies were as follows: p190 RhoGAP (primary antibody, 1:1000) and an anti-mouse immunoglobulin (Ig) G-HRP (secondary antibody, 1:1000). Western blots of tyrosine-phosphorylated p190 RhoGAP were prepared by using mouse monoclonal anti-phosphotyrosine antibody (primary antibody, 1:1000) and an anti-mouse IgG-HRP (secondary antibody, 1:1000). MLC20 mouse monoclonal antibody (IgM class) to MLC20 (primary antibody, 1:500) and an anti-mouse IgM (µ-chain specific)-HRP (secondary antibody, 1:1000). Western blots of phospho-MLC20 were prepared by using mouse monoclonal antibody to antiphosphoserine (primary antibody, 1:8000) and an anti-mouse IgG-HRP (secondary antibody, 1:2000).

The membranes were blotted semi-dry by placing them in between two filter papers and they were developed with the enhanced chemiluminescence (ECL) Western blotting reagents according to the instructions provided by the supplier (Amersham Pharmacia Biotech, Piscataway, NJ). Protein bands were visualized by exposing the membranes to x-ray films that were scanned with a scanner (model SNAPSCAN 310; Agfa, Ridgefield Park, NJ) followed by densitometric analysis by using the Image-Pro Plus V4.0 software (Media Cybernetics).

Data Analysis.
Data were calculated as mean ± SEM using the Sigma Plot computer program for PCs. Differences between groups were examined by students t test (P value) with P < 0.05 considered significant. One-tail P values were computed by the same computer program.

Chemicals and Drugs.
Ang II (human), HA 1077 (Rho kinase inhibitor) (44) and G{alpha}13-CT peptide (a blocking peptide corresponding to the carboxyl terminus of G{alpha}13 protein) were obtained from Calbiochem (San Diego, CA). Tyrosine kinase inhibitor genistein and MEK inhibitor PD 98059 were obtained from Research Biochemicals International (Natick, MA) and Biomol (Plymouth Meeting, PA), respectively. Rho kinase inhibitor Y 27632 (18, 44) was a generous gift from Yoshitomi Pharmaceutical Industries (Osaka, Japan). Clostridium botulinum C3 3exozyme (known to specifically inactivate Rho by ADP-ribosylation; collagenase [CLS II] 140 U/mg) (45, 46), soybean trypsin inhibitor, ATP, antimycin A, creatine, creatine phosphate, creatine phosphokinase, bethanechol (carbamyl ß-methyl choline chloride), and saponin were purchased from Sigma (St. Louis, MO). All other chemicals used in this investigation were of reagent grade. All materials used in electrophoresis experiments, including the molecular mass markers (broad range), were obtained from Bio-Rad Laboratories (Hercules, CA).

Antibodies.
Anti-p190 RhoGAP (antibody to full length), and antiphosphotyrosine monoclonal antibodies, and anti-mouse antibody conjugated with HRP were obtained from Transduction Laboratories (Lexington, KY). Anti-pp60c-src polyclonal antibody was obtained from Chemicon International (Temecula, CA). Antiphosphoserine, antimyosin light chain (20 kDa; IgM class; MLC20) monoclonal antibodies and anti-mouse (IgM, µ-chain)-HRP, and anti-sheep antibody-HRP were obtained from Sigma. G{alpha}13-CT antibody (antibody to the carboxyl terminus of the G{alpha}13 subunit) was obtained from Calbiochem (St. Louis, CA). ARF antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to p115 RhoGEF was kindly provided by Dr. G. Bollag (ONYX, Richmond, CA). Anti-rabbit antibody-HRP, ECL western blotting reagent kit, and x-ray Hyper film were purchased from Amersham Pharmacia Biotech. Different agents and antibodies were used in the concentrations known to selectively block their respective actions (18, 44–49).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ROK Inhibitors on Ang II-Induced Contraction of SMC from LES and IAS.
Rho kinase inhibitors HA 1077 (1 x 10-6 M) and Y 27632 (1 x 10-6 M) alone caused no significant shortening of the SMC from LES and IAS (P > 0.05; n = 4; Fig. 1Go). Ang II (1 x 10-7 M) produced 18.5% ± 1.2% shortening of SMC from LES and 16.4% ± 1.5% those of the IAS. HA 1077 and Y 27632 inhibited Ang II-induced contraction of LES SMC to 2.6% ± 0.3% and 3.8% ± 0.4%, respectively, and 4.5% ± 0.4% and 3.2% ± 0.4%, respectively in the IAS (*P > 0.05; n = 4). These inhibitors in these concentrations caused no significant effect on PKC activity in the LES and IAS SMC (data not shown).



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  		Figure 1. Effect of ROK inhibitors HA 1077 and Y 27632 on Ang II-induced contraction of SMC from LES and IAS. Pretreatment with HA 1077 and Y 27632 cause a significant attenuation (*P < 0.05; n = 4) of Ang II-induced shortening of the SMC. On the other hand, the inhibitors by themselves had no significant effect of the basal cell lengths by themselves.

 
Effect of C. botulinum C3 Exozyme on Ang II-Induced Contraction of SMC from LES and IAS.
SMC were permeabilized to facilitate the transport of large molecular mass molecules such as C3 exozyme, pp60c-src, and p190 RhoGAP antibodies across the plasma membrane. Permeabilization of LES and IAS SMC had no significant effect on the SMC contraction induced by Ang II, bethanechol, and KCl (data not shown). The C3 exozyme (50 µg/ml) by itself caused no significant effect on the length of the permeabilized SMC from either the LES or IAS (P > 0.05; Fig. 2Go). Ang II-induced shortening of the permeabilized SMC from both LES (19.4% ± 1.5%) and IAS (18.6% ± 1.7%) was significantly (*P < 0.05; n = 4) inhibited by the C3 exozyme in SMC from these tissues (4.8% ± 0.5% and 5.7% ± 0.6%, respectively). Above data suggest a role for RhoA/ROK in Ang II-induced contraction of SMC from LES and IAS.



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  		Figure 2. Effect of Rho inhibitor Clostridium botulinum C3 exozyme on Ang II-induced contraction of SMC from LES and IAS. Permeabilized SMC from LES and IAS were incubated with Ang II in the presence or absence of the C3 exozyme (50 µg/ml, 0.5 hr). Note that the C3 exozyme causes significant inhibition (*P < 0.05; n = 4) of Ang II-induced contraction of the SMC. However, by itself it has no significant effect on the basal cell lengths.

 
Effect of Antibodies to pp60c-src and p190 RhoGAP on Ang II-Induced Contraction of SMC from LES and IAS.
Western blots obtained by using a polyclonal antibody to pp60c-src and monoclonal antibody to p190 RhoGAP showed the presence of these proteins in LES and IAS smooth muscles in their basal states (data not shown). Antibodies to pp60c-src and p190 RhoGAP (introduced into the cells by permeabilization) alone had no significant effect on the length of SMC from LES and IAS. In such experiments, Ang II-induced shortening of SMC from LES (21.3% ± 1.2%) and IAS (18.6% ± 1.4%; Fig. 3Go) was significantly inhibited by the antibodies to pp60c-src and p190 RhoGAP (4.6% ± 0.4% and 6.4% ± 0.8%, respectively; *P < 0.05; n = 4). For these studies, control experiments with Ang II were performed in the presence of nonimmune IgG (5 µg/ml). Herbimycin A (Hb A; c-src inhibitor; 1 x 10-6 M) produced inhibition of Ang II-induced contraction of the SMC similar to that by pp60c-src antibody. In the IAS, Ang II-induced shortening of SMC of 18.1% ± 2.1% was attenuated by Hb A to 6.5% ± 1.1% (*P < 0.05; n = 4).



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  		Figure 3. Effect of antibodies to pp60c-src and p190 RhoGAP on Ang II-induced contraction of SMC from LES and IAS. Control experiments with Ang II alone were carried out in the presence of nonimmune IgG (5 µg/ml). Permeabilized cells from LES and IAS were incubated with an antibody to p190 RhoGAP (5 µg/ml) for 1 hr at room temperature followed by Ang II for 5 min. Note that pp60c-src and p190 RhoGAP antibodies (5 µg/ml) cause significant attenuation in Ang II-induced contraction of SMC (*P < 0.05; n = 4).

 
Effect of PD 98059 and Genistein on Ang II-Induced Tyrosine Phosphorylation of p190 RhoGAP.
Western blots prepared by using monoclonal antibody to phosphotyrosine show the presence of basal levels of tyrosine phosphorylated p190 RhoGAP in unstimulated rat LES and IAS (Fig. 4AGo). In addition, Ang II treatment caused an increase in the levels of tyrosine phosphorylation of p190 RhoGAP in the LES and IAS. Densitometric analyses of the western blots are shown in Figure 4BGo. PD 98059 (1 x 10-5 M) being a specific inhibitor of MEK (47, 50), had no significant effect on Ang II-mediated increase in the levels of tyrosine-phosphorylated p190 RhoGAP in the smooth muscles. Conversely, genistein, a general inhibitor of tyrosine kinases, caused a significant inhibition of the increase in the tyrosine phosphorylation of p190 RhoGAP in LES and IAS (**P < 0.05; n = 4; Fig. 4Go).



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  		Figure 4. Effect of PD 98059 and genistein on tyrosine phosphorylation of p190 RhoGAP in LES and IAS smooth muscles contracted by Ang II. (A) The western blot. (B) The relative distribution of phosphorylated p190 RhoGAP by densitometric analyses. For these experiments, the tissue protein extracts were first immunoprecipitated with p190RhoGAP antibody followed by immunoblotting with antiphosphotyrosine as described previously (49). Afterward, the membranes were stripped and reprobed with p190 RhoGAP antibody to determine the uniformity of loading of the specific protein. The controls correspond to basal levels of tyrosine phosphorylated p190 RhoGAP in these tissues (taken as 1). Note a significant increase in the levels of p190 RhoGAP tyrosine phosphorylation by Ang II (*P < 0.05; n = 4), that was not significantly modified by PD 98059, but was by genistein (**P < 0.05; n = 4).

 
Effect of Antibodies to G{alpha}13-CT, G{alpha}13-CT Peptide, and ARF antibody on Ang II-Induced Contraction of SMC from LES and IAS.
Western blots using an anti-G{alpha}13-CT antibody showed the presence of G{alpha}13 in rat LES and IAS smooth muscles (data not shown). Neither the anti-G{alpha}13-CT antibody nor the G{alpha}13-CT peptide had any significant effect on the percentage of shortening of the SMC from the LES and IAS and IAS\ in the basal state. In these experiments, Ang II in control experiments (in the presence of nonimmune IgG) caused contraction of permeabilized cells from LES and IAS to 17.4% ± 1.2% and 16.8% ± 1.7%, respectively. G{alpha}13-CT antibody and G{alpha}13-CT peptide significantly inhibited Ang II-induced contraction of permeabilized cells from LES (5.1% ± 0.6% and 4.5% ± 0.5%, respectively) and IAS (5.8% ± 0.8% and 6.1 ± 0.7%, respectively; *P < 0.05; n = 4; Fig. 5Go). ARF antibody caused a partial inhibition of Ang II-induced contraction of the SMC (Fig. 5Go). On the other hand, The combination of ARF and G{alpha}13-CT antibodies lead to further attenuation of the contraction.



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  		Figure 5. Effect of G{alpha}13-CT antibody and G{alpha}13-CT peptide (5 µg/ml) on Ang II-induced contraction of SMC from LES and IAS. Preliminary western blot studies confirmed the presence of G{alpha}13 in LES and IAS tissues. Permeabilized SMC from LES and IAS were incubated with G{alpha}13-CT antibody, G{alpha}13-CT peptide, or ARF antibody (5 µg/ml) for 1 hr at room temperature followed by Ang II. G{alpha}13-CT, G{alpha}13-CT peptide, and ARF antibodies significantly inhibit Ang II-induced contraction of SMC from LES and IAS (*P < 0.05; n = 4). The combination of ARF and G{alpha}13-CT antibodies causes further attenuation (**P < 0.05; n = 4).

 
Effect of Y 27632 and Genistein on MLC20 Phosphorylation (MLC20-P) in LES and IAS Smooth Muscles Contracted by Ang II.
Western blots obtained by using antiphosphoserine monoclonal antibody showed that Ang II-induced contraction of LES and IAS was associated with an increase in the levels of serine phosphorylation of MLC20 (Fig. 6Go). Preincubation of LES and IAS smooth muscles with Y 27632 or genistein resulted in significant decrease in the levels of phosphorylation of MLC20-P (**P < 0.05; n = 4). The results show that Ang II-induced contraction of smooth muscles involves a tyrosine phosphorylation signaling pathway(s), which activates ROK, a serine/threonine kinase.



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  		Figure 6. Effect of Y 27632 and genistein on Ang II-mediated increase in MLC20 phosphorylation in LES and IAS contracted by Ang II. (A) The western blot. (B) The relative distribution of phosphorylated MLC20 by densitometric analyses. For these experiments, the tissue protein extracts were first immunoprecipitated with MLC20 antibody followed by immunoblotting with antiphosphoserine as described previously (49). Afterward, the membranes were stripped and reprobed with MLC20 antibody to determine the uniformity of protein loading. The controls correspond to basal levels of tyrosine phosphorylated MLC20 in these tissues (taken as 1). Ang II caused a significant increase in MLC20-P (*P < 0.05; n = 4), which were attenuated significantly by Y 27632 and genistein (**P < 0.05; n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies suggest the role of monomeric G-proteins Rho and ARF as one of the pathways in Ang II-induced contraction of LES and IAS smooth muscles. Ang II-induced contraction of the SMC is inhibited by the ROK inhibitors, Y 27632 and HA 1077, at concentrations consistent with their specific inhibitory effects on ROK. Such concentrations tested in different systems may not cause significant inhibition of PKC, cAMP-dependent protein kinase, and MLCK (18, 19, 44). In addition, the C3 exozyme, a specific inhibitor of Rho (45, 46), attenuates Ang II-induced contraction of LES and IAS SMC. ROK is one of the effectors of Rho and is activated by its direct interaction with the small G-protein (15).

The participation of Rho signaling pathway after Ang II treatment in the LES and IAS smooth muscles is further supported by Ang II-induced increase in the levels of tyrosine phosphorylation of p190 RhoGAP. In addition, antibodies to pp60c-src and p190 RhoGAP and Hb A attenuate Ang II-induced contraction of the LES and IAS SMC. These results are in agreement with the earlier studies in different systems to suggest that p120 rasGAP and p190 RhoGAP are substrates for pp60c-src (48, 51). The data also suggest that Ang II-mediated stimulation of pp60c-src may be an upstream step in GPCR-mediated signaling mechanisms in Ang II effects. Activation of pp60c-src has been shown to be associated with the formation of a complex between p120 rasGAP and p190 RhoGAP (52). This coordinates ras- and Rho-mediated downstream signaling (53–55) in stimulated cells.

Ang II-mediated increase in the levels of tyrosine phosphorylation of p190 RhoGAP regulates the GTPase activity of Rho and may serve to coordinate downstream signaling by Rho. Findings by Nobes et al. (56) show that tyrphostin (an inhibitor of tyrosine phosphorylation) inhibits lysophosphatidic acid-mediated stress fiber formation without affecting stress fiber formation by Rho. Taken together, the results suggest that Rho serves to transmit signals from tyrosine kinases (e.g., pp60c-src) to serine/threonine kinases (e.g., ROK).

The exact mechanism by which p190 RhoGAP antibody inhibits Ang II-induced contraction of LES and IAS SMC is not known. However, it is possible that the antibody used here, by an unidentified mechanism, affects the tyrosine phosphorylation of p190 RhoGAP, which attenuates the GTPase activity of Rho (57). This may result in the activation of Rho, followed by Rho kinase, the inhibition of MLC-phosphatase, an increase in MLC20-phosphorylation, and finally, the contraction of smooth muscle. The findings are similar to {alpha}6ß1 integrin-induced invasiveness and tyrosine phosphorylation of p190 RhoGAP in adherent LOX melanoma cells that were attenuated by p190 RhoGAP antibody (49).

Present studies also show an involvement of G{alpha}13 in Ang II-induced contraction of LES and IAS SMC. A G{alpha}13-CT antibody as well as the carboxyl terminus peptide (G{alpha}13-CT peptide; used to produce the G{alpha}13-CT antibody) inhibit Ang II-induced contraction of LES and IAS SMC. Additionally, we have observed significant levels of G{alpha}13 and G{alpha}12 in the rat tonic smooth muscles in the basal state (data not shown). The G{alpha}13-CT antibody and G{alpha}13-CT peptide have been shown previously to inhibit Ang II-mediated increase in [Ca2+]i in rat portal vein myocytes (58). More recently, it has been shown that Rho guanine nucleotide exchange factors p115 RhoGEF (31, 32, 59) and PDZ-RhoGEF (60) form complexes with G{alpha}13, which in turn activate Rho.

In different smooth muscles examined, Ang II-induced contraction via AT1 receptors involves multiple signal transduction systems such as release of intracellular Ca2+ ([Ca2+]i), Ca2+ influx, Ca2+-calmodulin/MLCK, phospholipases, PKC, MAPKs, Janus kinases, tyrosine kinases, and small GTP-binding proteins (39, 61–63). AT1 receptor activation leads to coupling with GTP-binding proteins such as Gq and G12/13. Activation of Gq stimulates PLC-ß to generate inositol-1,4,5-trisphospate (IP3) and diacylglycerol (DAG). IP3 causes the release of intracellular Ca2+ from IP3-sensitive Ca2+ stores. This is followed by the influx of Ca2+ by the opening of voltage-sensitive Ca2+ channels, formation of Ca2+-calmodulin (Ca2+/CaM) complex, leading to the activation of Ca2+/CaM-MLCK, causing smooth muscle contraction in the initial stages. DAG causes smooth muscle contraction by PKC activation. The initial surge of DAG is derived from phosphatidylinositol-4,5-bisphosphate, and sustained increased levels of DAG are provided by the activation of phospholipase D (PLD). The second phase of smooth muscle contraction with Ang II is primarily contributed by Ca2+ influx and activation of PLD. PLD causes the production of phosphatidic acid and DAG. DAG contributes to the prolonged activation of PKC and smooth muscle contraction (61). In addition to IP3 formation and intracellular release of Ca2+, tyrosine kinase-dependent increases in [Ca2+]i have also been reported. PKC-independent contraction of Ang II-induced contraction of the SMC (that may involve protein kinase D or PKD) (64) has also been demonstrated (39).

Intracellular mechanisms that couple AT1 receptors to PLD have been identified such as G-protein ß{gamma} subunits and associated G{alpha}12 subunits. These G proteins activate PLD via src-dependent Rho pathways in vascular SMC (24, 65). The downstream signaling in this pathway may be PKC dependent or independent and may involve mobilization of [Ca2+]i and Ca2+ influx. In addition, Rho A may regulate Ca2+ sensitivity via PKC/MAP kinase pathway or via PKC-mediated effect on MLC phosphatase (22, 66–70).

Present studies are in general agreement with those of Murthy et al. (19) and Cao et al. (20). These investigators showed that both G{alpha}13/Rho and ARF are involved in parallel in signal transduction, in CCK-induced contraction in the rabbit intestinal SMC, and in PGF2{alpha}-induced contraction in the cat esophageal SMC, respectively. Our data supports this thesis because antibodies to both G{alpha}13 and ARF caused attenuation of Ang II-mediated contraction of SMC. The combination of these antibodies causes further attenuation. Earlier studies (19, 20) also showed that actions of Rho and ARF are mediated via PLD/DAG/PKC-{alpha} pathways. Our preliminary findings that G{alpha}13 antibody causes inhibition of Rho kinase activity and that PLD inhibitor propranolol (in appropriate concentrations) causes inhibition of Ang II-induced contraction of the SMC provide further support to the hypothesis.

Different tyrosine kinases associated with GPCRs have been suggested to be involved in the upstream events following AT1 receptor activation in different systems (16, 39, 71). It has been suggested that in addition to GEFs, activation of Rho kinase may be dependent on the activation of tyrosine kinase (56). Conversely, activation of Rho kinase may also affect indirectly tyrosine kinase downstream of Rho (72). Present data shows inhibition of Ang II-induced contraction of the SMC with the tyrosine kinase inhibitor. In addition, Rho kinase inhibitors Y 27632 and HA 1077, C. botulinum C3 exozyme, pp60c-src, Hb A, and RhoGAP antibodies decrease phosphorylation of RhoGAP and MLC20. These findings suggest that tyrosine kinase activation may be partly upstream. However, the exact nature of this tyrosine kinase is not known. The studies support the possibility of multiple intracellular pathways in the contraction of these smooth muscles because of a considerable crossover and interaction between different pathways. It is possible that Ca2+-dependent pathways involving PLC and MLCK as well as the G{alpha}13/RhoA/ROK and ARF pathways and changes in cytoskeletal organization are required for efficient and sustained contraction of these smooth muscles after Ang II (19, 34, 73). The relative contribution of different pathways in the signal transduction of Ang II-induced contraction of LES and IAS smooth muscles and possible differences in the adjoining phasic smooth muscles remains to be determined.

The main points of this study are outlined in Figure 7Go. Ang II-induced contraction of the LES and IAS smooth muscles involves activation of Rho kinase downstream of pp60c-src and G{alpha}13. Activation of G{alpha}13 may also lead to the activation of Rho through p115 RhoGEF. Stimulation of p190 RhoGAP augments the GTPase activity of Rho and coordinates Rho-mediated downstream signaling. Ang II-mediated activation of ROK with the intermediary pathways such as PLD and PKC contribute to an increase in the levels of MLC20 phosphorylation, and in turn, to the smooth muscle contraction. Such information may be relevant for the increased understanding of intracellular pathways in the hypertensive sphincters responsible for certain gastrointestinal motility disorders.



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  		Figure 7. Hypothetical diagram to show Rho kinase signal transduction pathway involving Ang II-induced contraction of the sphincteric smooth muscle. Using AT1 antagonist losartan, our earlier studies (8, 9) have shown that Ang II causes contraction of the SMC via activation of GPCR AT1 receptor. AT1 receptor activation may lead to the activation of a tyrosine kinase and complex formation with pp60c-src. pp60c-src-induced tyrosine phosphorylation of p190 RhoGAP and activation of Rho-guanine exchange factor (RhoGEF) lead to the formation of active form of Rho-GTP. In addition, G{alpha}13-mediated activation of Rho kinase may also be facilitated via p115 RhoGEF. p190 RhoGAP, and p115 RhoGEF complex formation, leading to Rho-GTP transformation leads to activation of RhoA/ROK. The latter may inhibit MLC phosphatase, leading to increased phosphorylation of MLC20. The net effect of these events is an increase in the basal tone of the smooth muscle and contraction of the SMC. Different pharmacological tools used and parameters followed to test the events are listed in the figure. We have shown previously that multiple pathways including PKC, MLCK, and MAPK are involved in the smooth muscle contraction by Ang II. The role of Gq/PLCb/IP3-DAG/Ca2+-MLCK-PKC in relation to Ang II-induced contraction of the tonic smooth muscles is also of particular interest. For simplicity, these pathways have not been depicted in this diagram. The relative contribution and nature of exact interaction and interplay between different pathways in relation to RhoA/ROK activation in Ang II-induced contraction of IAS and LES smooth muscles remain to be determined.

 


   Footnotes
 
This work was supported by the National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and by an institutional grant from Thomas Jefferson University.

1 To whom requests for reprints should be addressed at Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, 1025 Walnut Street, Room 901 College, Philadelphia, PA 19107. E-mail: Satish.Rattan{at}mail.tju.edu Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication August 5, 2002. Accepted for publication April 11, 2003.




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