Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the role of mitogen-activated protein kinase (p44/42 MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60c-src inhibitor), and antibodies to pp60c-src and p120 ras GTPase-activating protein (p120 rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p120 rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p44/42 MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60c-src and p44/42 MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.
tonic smooth muscle; tyrosine kinase; tyrosine phosphorylation; p120 ras GTPase-activating protein; p190 rho GTPase-activating protein
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ANGIOTENSIN II (ANG II) is known to elicit widely diverse cellular responses such as growth, proliferation, and smooth muscle contraction. These responses are mediated largely via the AT1 receptor (46, 49, 51). Limited information is available on the actions and signal transduction mechanisms of ANG II in the gastrointestinal smooth muscle, specifically the tonic smooth muscles of sphincters. Lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles play an important role in the pathophysiology of a number of gastrointestinal motility disorders characterized either by the hypo- or hypertensive sphincteric smooth muscles (36, 44).
It is well known that a majority of the sphincteric smooth muscle tone may come from the myogenic properties of the smooth muscle (3, 8, 20). However, the basal tone of LES and IAS smooth muscles is prone to modulation by neurohumoral agents. Recent studies (41) conducted in our laboratory have shown that the rat is an appropriate animal model for investigating the actions of ANG II in the tonic smooth muscles of the IAS and LES. Our studies also show that the sphincteric smooth muscle contraction caused by ANG II is mediated primarily by its direct actions on the smooth muscle cells (SMC). ANG II-induced contraction of LES and IAS circular smooth muscles is inhibited specifically by the AT1 antagonist losartan. This observation combined with the presence of AT1 receptors in the LES and IAS smooth muscles suggests a specific role for AT1 receptors in the mediation of ANG II-induced contraction in these tissues (16).
The role of mitogen-activated protein kinase (p44/42 MAPKs) and extracellular signal-regulated kinases in the signal transduction of smooth muscle contraction by different agonists, including ANG II, is well established (1, 18, 38, 48). In vascular SMCs (VSMCs), activation of p44/42 MAPKs lies downstream of tyrosine kinase activation, activation of pp60c-src, tyrosine phosphorylation of p120 ras GTPase-activating protein (p120 rasGAP), and p190 rhoGAP (14, 25, 27, 43). Together, these findings suggest that ANG II-mediated tyrosine phosphorylation plays an important role in the signaling events of vascular smooth muscle. Although the role of tyrosine phosphorylation and p44/42 MAPKs in the different smooth muscles has been investigated recently (1, 21, 54), the relationship between upstream signaling events and activation of p44/42 MAPKs in ANG II-induced contraction in the tonic gastrointestinal smooth muscles has not been examined.
Caldesmon is a physiological substrate of p44/42 MAPKs and inhibits the ATPase activity of actomyosin complex in smooth muscles (31). Phosphorylated caldesmon positively regulates smooth muscle contraction by relieving the inhibition of actomyocin ATPase activity (18, 31) and, consequently, promoting smooth muscle contraction.
The purpose of this investigation is to identify critical events in the signal transduction mechanisms of ANG II-induced contraction of LES and IAS smooth muscles. Results of the present investigation show that the activation of pp60c-src and p44/42 MAPKs play an important role in ANG II-induced contraction of the tonic smooth muscles of the LES and IAS.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of smooth muscle strips. Smooth muscle strips were prepared as described previously (17). Adult Sprague Dawley rats (of either sex, ~250 g) were anesthetized with pentobarbital sodium (50 mg/kg ip). Laparotomy was performed, and the LES and IAS smooth muscle tissues were excised and transferred immediately to a beaker containing oxygenated (95% O2-5% CO2) Krebs solution (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose. The smooth muscle tissues were carefully freed of all extraneous structures, opened, and pinned flat with the mucosal side up on a dissecting tray containing oxygenated Krebs solution. The mucosal and submucosal layers along with the serosal connective tissue and visible blood vessels were removed by sharp dissection. The smooth muscle strips (1 × 10 mm) were secured at both ends with silk sutures and transferred to 2-ml muscle baths containing oxygenated Krebs solution at 37°C. One end of each smooth muscle strip was secured at the bottom of the muscle bath, and the other end was attached to a force transducer (model FT03; Grass Instruments, Quincy, MA) connected to a PowerLab recorder (CB Sciences, Milford, MA). Only those smooth muscle strips that developed a spontaneous steady tone and relaxed in response to electrical field stimulation were used for these experiments.
For some studies, the smooth muscle tissues were treated with 1 × 10Isolation of SMC from rat LES and IAS. SMC from the tonic smooth muscle tissues were isolated essentially by the method described previously (6). Rat LES and IAS smooth muscle strips were cut into small pieces (1-2 mm cubes) and incubated at 37°C for two successive 1-h periods in oxygenated Krebs solution containing collagenase (0.01% for LES and 0.013% for IAS) and soybean trypsin inhibitor (0.01%). After each incubation, the mixture was filtered through a 500-µm Nitex mesh. Tissue trapped on the mesh was rinsed with 25 ml (5 × 5 ml) collagenase-free Krebs solution. Tissue was finally incubated in collagenase-free Krebs solution at 37°C, and dispersion of the cells (0-1 h) was monitored periodically by examining a 10-µl aliquot of the mixture under a microscope. SMC were harvested by filtration through Nitex mesh. Filtrate containing the cells was centrifuged at 350 g for 10 min at room temperature. Cells in the pellet were resuspended in Krebs solution at a cell density of 3 × 104 cells/ml.
Permeabilization of LES and IAS SMC. Permeabilization of LES and IAS SMC was accomplished by the method previously used in our laboratory (17). SMC from rat LES and IAS were permeabilized by incubating them in cytosolic solution (in mM): 20 NaCl, 100 KCl, 5 MgSO4, 0.96 NaH2PO4, 25 NaHCO3, 1 EGTA, 0.48 CaCl2, and 1% BSA with saponin (75 µg/ml) for 3 min at room temperature. Cell suspension was centrifuged at 350 g 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 centrifuged at 350 g for 10 min. Cells were washed twice with the modified cytosolic solution to remove saponin and resuspended in the fresh, modified cytosolic solution.
Measurement of cell length by scanning micrometry. Aliquots (30 µl) of SMC from the rat tissues were incubated with various agonists in the absence or presence of specific inhibitors. Incubations were terminated by the addition of 1% acrolein. The mean length of 30 cells chosen at random in each set was determined by micrometry using phase contrast microscopy. Images were stored digitally, and the cell lengths were measured by the Image-Pro Plus version 4.0 program (Media Cybernatics, Silver Spring, MD). Digital data were transferred directly to the Microsoft Excel computer program. Data are presented as percentage shortening of the SMCs after different treatments in each set as means ± SE compared with that of controls (untreated cells).
Gel electrophoresis and Western blot analysis.
Protein extracts from rat LES and IAS smooth muscles were prepared by
cutting the tissues into 1-2 mm cubic pieces and by incubating
them in a lysis buffer (1% SDS, 1 mM sodium orthovanadate, and 10 mM
Tris, pH 7.4) at 90°C for 3 min. Incubation mixtures were
homogenized, followed by centrifugation at 16,000 g for 15 min at 4°C. Protein in the supernatants was estimated by Lowry's method. Solutions of the protein extracts were prepared by mixing them
with an equal volume of a 2× sample buffer (125 mM Tris, pH 6.8, 10%
glycerol, 2% -mercaptoethanol, and 0.006% bromophenol blue). These
samples then were heated in a boiling water bath for 3 min. Protein
samples (40 µg protein/20 µl) were subjected to SDS-PAGE by the
Laemmli method (28). Unless otherwise noted, a
discontinuous gel system utilizing 4% stacking gel, pH 6.8, and 10%
running gel, pH 8.8 (or as stated), was used in all experiments involving gel electrophoresis.
Data analyses. Data were calculated as means ± SE by using the Sigma Plot computer program for personal computers. Differences between groups were examined by Student's t-test (P value) with P < 0.05 considered to be statistically significant.
Chemicals and drugs.
ANG II (human) and herbimycin A (Hb A) were obtained from Calbiochem
(San Diego, CA). Tyrosine kinase inhibitor genistein and the MAPK
kinase (MEK) inhibitor PD-98059 were obtained from Research
Biochemicals International (Natick, MA) and Biomol (Plymouth Meeting,
PA), respectively. Collagenase (140 U/mg), soybean trypsin inhibitor,
ATP, antimycin A, creatine, creatine phosphate, creatine phosphokinase,
bethanechol chloride (carbamyl--methylcholine 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).
Stock solutions of Hb A and PD-98059 were prepared in DMSO, and the
final concentration of DMSO in the incubation mixtures was 0.5%.
Antibodies. Anti-p120 rasGAP, anti-phosphotyrosine monoclonal antibodies, and anti-mouse antibody conjugated with HRP were obtained from Transduction Labs (Lexington, KY). Anti-pp60c-src polyclonal antibody was obtained from Chemicon International (Temecula, CA). Anti-phosphoserine, anti-caldesmon monoclonal antibodies, and anti-mouse antibody-HRP were obtained from Sigma. Anti-phospho-p44/42 MAPK monoclonal antibody was purchased from New England BioLabs (Boston, MA). Anti-rabbit antibody-HRP, enhanced chemiluminescense Western blotting reagents kit, and X-ray hyperfilm were purchased from Amersham Pharmacia Biotech.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of tyrosine kinase inhibitor genistein and MAPK
kinase inhibitor PD-98059 on ANG II-induced contraction of LES
and IAS SMC.
ANG II-induced contractions of the SMC were comparable with bethanechol
chloride, a muscarinic receptor agonist. ANG II-induced contraction of
SMC from LES and IAS was antagonized by preincubation of the SMC with
genistein (2) and PD-98059 (12)
(P < 0.05; n = 4 animals; Fig.
1). In control experiments, the basal
lengths of the LES and IAS SMC were 54.6 ± 3.4 and 39.8 ± 2.5 µm, respectively. Genistein and PD-98059 by themselves had no
significant effect on the basal lengths of the SMC. In the LES and IAS
SMC, ANG II caused a 15.0 ± 2.1 and 27.0 ± 2.7% shortening
that was subsequently antagonized to 5.0 ± 1.0 and 13.1 ± 1.8%, respectively, by genistein. Tyrphostin, another tyrosine kinase
inhibitor, had effects similar to those found with genistein (data not
shown). Results suggest that ANG II-induced contraction of LES and IAS
SMC involves a tyrosine kinase signal transduction pathway leading to
the activation of p44/42 MAPKs.
|
Relative distribution of phospho-p44/42 MAPKs in the
basal state and after ANG II treatment in LES and IAS smooth muscles:
effect of genistein and PD-98059.
We examined the effect of ANG II on the levels of phosphorylated
(phospho)-p44/42 MAPKs before and after the
specific inhibitors of tyrosine kinase and
p44/42 MAPKs. Western blots show the presence of
phospho-p44/42 MAPKs in the basal state and an
increase in their levels by ANG II treatment (Fig.
2). ANG II caused a two- to threefold
increase in the levels of phosphorylation of p44
MAPK in the LES and IAS, respectively. Corresponding increases in
phospho-p42 MAPK levels were approximately five-
and threefold, respectively. Data further showed that ANG II-induced
increases in phospho-p44/42 MAPKs were
significantly attenuated by genistein and PD-98059 (P < 0.05; n = 4 animals). Genistein or PD-98059 alone
had no effect on the basal levels of
phospho-p44/42 MAPKs in these tissues (data not
shown).
|
Effect of Hb A on ANG II-induced contraction of SMC from LES and
IAS.
Hb A is an irreversible and specific inhibitor of the src family of
tyrosine kinases (50) and by itself had no significant effect on the length of the SMC from LES and IAS. ANG II-induced contraction of SMC from LES and IAS, however, was significantly inhibited by Hb A (P < 0.05; n = 4;
Fig. 3). In these experiments, ANG II
caused 18.2 ± 2.1% shortening of the IAS SMC antagonized by Hb A
to 6.5 ± 1.1% in controls. Results suggest the involvement of
the src family of tyrosine kinase in the signal transduction pathway
for the contractile responses of LES and IAS smooth muscles to ANG II.
|
Effect of pp60c-src and p120 rasGAP
antibodies on ANG II-induced contraction of SMC from LES and IAS.
Antibodies to pp60c-src and
p120 rasGAP do not penetrate the
plasma membrane readily and require permeabilization of the SMC. We
first determined that the permeabilization procedure by itself had no significant effect on the SMC contraction caused by the agonists bethanechol chloride and ANG II [not significant (NS);
P > 0.05; n = 4 animals; Figs.
4, A and B]. ANG
II and bethanechol chloride caused 18 ± 1.0 and 25 ± 2.1%
shortening of the LES SMC in nonpermeabilized SMC, respectively.
Corresponding values for the IAS SMC were 15 ± 1.4, 19 ± 2, and 22 ± 2.2% shortening, respectively.
|
Relative distribution of phospho-p120 rasGAP in the LES
and IAS smooth muscles in the basal state and after ANG II.
The presence of p120 rasGAP in the
LES and IAS smooth muscles was determined first by Western blot studies
with an appropriate antibody. Western blot studies utilizing
anti-phosphotyrosine monoclonal antibody were carried out to determine
the presence of tyrosine phospho-p120
rasGAP in rat LES and IAS, in the basal state and after ANG
II treatment. ANG II (1 × 107 M) caused an increase
in phospho-p120 rasGAP in both the
LES and IAS tissues (Fig. 5). PD-98059, a specific inhibitor of p44/42 MAPKs, had no
significant effect, but the tyrosine kinase inhibitor genistein caused
an attenuation of ANG II-induced increase in the levels of
p120 rasGAP phosphorylation in these
smooth muscles.
|
Effect of genistein and PD-98059 on ANG II-induced phosphorylation
of caldesmon in LES and IAS smooth muscles.
Caldesmon, an actin-binding protein, negatively regulates actomyosin
ATPase activity and smooth muscle contraction. Caldesmon phosphorylation by p44/42 MAPKs at serine
residues relieves this inhibition. Western blots using an appropriate
anti-phosphoserine antibody showed the presence of both phosphorylated
87- and 150-kDa caldesmons in the LES and IAS smooth muscles in the
basal state and their significant increase after ANG II
(P < 0.05; Fig. 6).
PD-98059 and genistein caused significant attenuation in ANG
II-mediated increase in the levels of phosphorylated 87- and 150-kDa
caldesmons (P < 0.05; Fig. 6). Data suggest the role
of tyrosine phosphorylation signaling pathway in activating
p44/42 MAPKs, which led to increased caldesmon
phosphorylation. This may be partly responsible for the smooth muscle
contraction after ANG II treatment.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These studies show that a part of ANG II-induced contraction of
the SMC from the tonic smooth muscles of the LES and IAS is dependent
on pp60c-src activation and phosphorylation of
p120 rasGAP. Stimulation of
pp60c-src causes activation of ras
and then of p44/42 MAPKs. These events, in turn,
may be partly responsible for the smooth muscle contraction via
phosphorylation of caldesmon. These events are schematically displayed
in Fig. 7.
|
ANG II-induced activation of pp60c-src represents an important early upstream signaling event in the contraction of LES and IAS smooth muscles. This is evident from the findings that genistein, a tyrosine kinase inhibitor, and Hb A, an irreversible inhibitor of pp60c-src causes attenuation of ANG II-induced contraction of LES and IAS SMC. In addition, pp60c-src antibody causes significant attenuation of ANG II-induced contraction of the LES and IAS SMC. In addition, these treatments block intermediary events before smooth muscle contraction, such as rasGAP phosphorylation (monitored by p120 rasGAP phosphorylation at tyrosine residues), p44/42 MAPK activation (monitored by p44/42 MAPK phosphorylation), and phosphorylation of caldesmon at serine residues.
Earlier studies from our laboratory show that ANG II-induced contraction of the sphincteric smooth muscle is mediated primarily via the activation of AT1 receptor, because it was antagonized by AT1 antagonist losartan (16). Our data are consistent with those in different smooth muscle systems, which suggest that the activation of the AT1 receptors by ANG II followed by activation of pp60c-src represents a critical event in the ras-related signal transduction pathway (14, 25, 26, 33, 43, 45). Involvement of p44/42 MAPKs in ANG II-induced contraction of the SMC was identified recently with the use of knockout mice and retrovirally transduced VSMC (known to express pp60c-src) (24). We speculate that stimulation of pp60c-src tyrosine kinase may set off activation of the ras-raf-MEK-MAPK signaling cascade responsible for a part of ANG II-induced contraction of rat LES and IAS smooth muscles.
Activation of pp60c-src may play an important
role in signal transduction mechanisms associated with
Ca2+-dependent and Ca2+-independent contraction
of smooth muscles. In the Ca2+-dependent pathway, G
protein-coupled receptor (GPCR)-mediated activation of
Gq leads to an increase in intracellular
Ca2+ concentration, which activates nonreceptor tyrosine
kinases such as PYK2, a Ca2+-sensitive proline-rich
tyrosine kinase (42). PYK2 can associate with
pp60c-src through src homology domain
3. Activation of PYK2 by ANG II in rat VSMC has been shown to promote
increased PYK2-pp60c-src complex formation
(42). This process combined with a series of additional
steps leads to the activation of p44/42 MAPKs.
In a Ca2+-independent pathway, GPCR may couple to
pp60c-src through
-arrestin 1, an adapter
protein (35). This signaling mechanism was recently shown
to be involved in the activation of ras and
p44/42 MAPKs. In a Ca2+-independent
pathway, the requirement for Ca2+-dependent stimulation of
pp60c-src and of ras may be
circumvented by the protein kinase C (PKC)-mediated activation of
raf, which subsequently causes a direct activation of
p44/42 MAPKs (7).
Ca2+-independent contraction of differentiated smooth
muscles by phenylephrine was shown recently to involve activation of
p44/42 MAPKs (10). PKC-activator
phorbol 12-myristate 13-acetate can stimulate MAPKs without activating
ras in VSMC (39). The present studies were
carried out under normal Ca2+ conditions and do not address
the issue of the Ca2+-independent pathway in MAPK
activation. However, our earlier experiments have shown that ANG
II-mediated contraction of the tonic smooth muscles uses both
Ca2+-dependent and Ca2+-independent pathways.
Furthermore, such contraction, in part, also utilizes the PKC pathway
(16). In addition, whether ANG II-mediated influx of
Ca2+ and PKC activation contribute to PYK2 and
pp60c-src stimulation in the tonic smooth
muscles remains to be determined. It is possible that activation of
extracellular-regulated MAPKs by ras in signal transduction
mechanisms involving GPCRs may be tissue and stimulus specific.
Another novel finding of this investigation is that ANG II-induced contraction of the LES and IAS smooth muscles is accompanied by an increase in the levels of tyrosine phosphorylation of p120 rasGAP. Genistein, but not PD-98059, attenuated these increases in p120 rasGAP phosphorylations. These findings highlight the fact that genistein and PD-98059 work only at specific steps of the ras-related pathway. Data show also that p44/42 MAPK activation is downstream from p120 rasGAP. In addition, p120 rasGAP antibody causes specific inhibition of ANG II-induced contraction of LES and IAS SMC.
Tyrosine phosphorylation of p120 rasGAP may be an important step between pp60c-src activation and ras activation (11, 15, 37, 47). Change of rasGDP (inactive form) to rasGTP (active form) is critical for ras activation in the ras-related pathway. Guanine exchange factors (GEFs) and GAPs are also known to play important roles in this regulation. GEFs serve as positive regulators for the conversion of rasGDP to rasGTP, whereas activation of p120 ras GTPase serves as a negative regulator by an increase in the rate of hydrolysis of rasGTP to rasGDP. Tyrosine phosphorylation of p120 rasGAP may lead to sustained activation of ras and then to MAPKs via multiple mechanisms, such as increased complex formation with other phosphoproteins, such as p62 and p190, and rasGAP downregulation (11, 15, 37, 47).
It is known that in stimulated cells, the low intrinsic level of GTPase activity of ras is enhanced by p120 rasGAP, which may act as a vectorial manager (4). Both p120 rasGAP and p190 rhoGAP are substrates for phosphorylation by pp60c-src (30, 43). Our data are consistent with previous data demonstrating that pp60c-src antibody causes an attenuation of ANG II-mediated tyrosine phosphorylation of p120 rasGAP and contraction in rat VSMC (43). In addition, cells transformed with cytoplasmic (avian v-src) and receptor-like tyrosine kinases show the presence of an immune complex containing tyrosine-phospho-p120 rasGAP and p190 rhoGAP (15). Our data support the hypothesis that p120 rasGAP is a regulator of rasGTPase activity and that ras transmits signals from pp60c-src tyrosine kinases to serine/threonine kinases (p44/42 MAPKs) (27, 32, 43).
ANG II-mediated activation of p44/42 MAPKs plays an important role in ANG II-induced contraction of tonic smooth muscles of LES and IAS. The suggestion is based on the following observations. First, ANG II causes increases in the basal levels of phospho-p44/42 MAPKs in these smooth muscle tissues. Second, tyrosine kinase inhibitor genistein (2) and a specific inhibitor of MEK 1/2, PD-98059 (12), attenuate ANG II-induced increases in phospho-p44/42 MAPKs. Third, an increase in phospho-p44/42 MAPKs levels is associated with ANG II-induced contraction of the LES and IAS SMC. The exact role of pp60c-src and p44/42 MAPKs in ANG II-induced contraction of adjoining phasic smooth muscles of the esophagus and rectum are difficult to ascertain, because ANG II does not produce a reproducible contraction of these tissues in most of the species examined (40). Our data with PD-98059 are similar to those obtained in other smooth muscles and SMC (10, 48). Investigations of the cause and effect relationship between smooth muscle contraction and activation of p44/42 MAPKs have been carried out only recently (10, 52).
Although phosphorylation of myosin light chain (20 kDa) (MLC20-P) is the major determinant of smooth muscle contraction after ANG II treatment in different smooth muscles, phosphorylation of other contractile proteins such as caldesmon may also play an important role under certain conditions (5, 9, 22, 23, 31, 34). Caldesmon is an effector of p44/42 MAPKs and inhibits the ATPase activity of the actomyosin complex in smooth muscles (31). Phosphorylation of caldesmon may promote smooth muscle contraction by relieving inhibition of the ATPase activity (10, 18, 31). Our data support this hypothesis because ANG II causes significant increase in caldesmon phosphorylation. In addition, genistein and PD-98059 cause significant attenuation of LES and IAS SMC contraction as well as that of caldesmon phosphorylation. Two types of caldesmon, low-molecular-mass caldesmon (l-caldesmon; 87 kDa) (29), and high-molecular-mass caldesmon (h-caldesmon; 140-150 kDa) (53), have been shown to be present in the smooth muscles. Our data suggest that activation of p44/42 MAPKs causes an increase in phosphorylation of l- as well as h-caldesmon in ANG II-induced contraction of LES and IAS smooth muscles.
The present studies focus primarily on the role of the ras-related MAPK pathway in ANG II-induced contraction of LES and IAS smooth muscle. However, multiple intracellular pathways such as phospholipase C, phospholipase D, protein kinase C, Ca2+-calmodulin-myosin light-chain kinase, MAPKs other than p44/42, rho kinase, and changes in ion channel activation may also contribute to the smooth muscle contractions by ANG II. The contribution of these pathways and their interactions with ras-related activation of MAPK after ANG II-induced contraction of the LES and IAS smooth muscles is not known. It also remains to be determined whether the proposed pathway for the partial contraction of ANG II is applicable to other smooth muscle contractile agents such as muscarinic agonists. In different smooth muscles, there appears to be an overlap of signal transduction pathways involved in contraction by muscarinic receptor activation (e.g., by bethanechol chloride and carbachol) and ANG II (3, 13, 19, 49).
In summary, data suggest an important role of pp60c-src tyrosine kinase in the activation of ras and p42/44 MAPKs in ANG II-induced contraction of LES and IAS smooth muscles. In addition, LES and IAS smooth muscles are found to have significant levels of phosphorylated p44/42 MAPKs, p120 rasGAP, and caldesmon in the basal state. Our findings support the notion that the ras/pp60c-src/MAPK pathway is involved in the agonist-stimulated as well as in the basal tone of the smooth muscles. Identification of intracellular mechanisms in the contraction of LES and IAS smooth muscles in the basal state and after contractile neurohumoral agonists will provide important information on the regulation and modulation of these tonic smooth muscles. This information may be vital in the understanding of the pathophysiology and therapeutic rationale for gastroesophageal and anorectal motility disorders.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. John J. Gartland for editing the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and by an institutional grant from Thomas Jefferson University.
Address for reprint requests and other correspondence: S. Rattan, Dept. of Medicine, Division of Gastroenterology, Thomas Jefferson University, 1025 Walnut St., Rm. 901 College; Philadelphia, PA 19107 (E-mail: Satish.Rattan{at}mail.tju.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00025.2002
Received 18 January 2002; accepted in final form 25 March 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adam, LP,
Franklin MT,
Raff GJ,
and
Hathaway DR.
Activation of mitogen-activated protein kinase in porcine carotid arteries.
Circ Res
76:
183-190,
1995
2.
Akiyama, T,
Ishida J,
Nakagawa S,
Ogawara H,
Watanabe SI,
Itoh N,
Shibuya M,
and
Fukami Y.
Genistein, a specfic inhibitor of tyrosine-specific protein kinases.
J Biol Chem
262:
5592-5595,
1987
3.
Biancani, P,
Sohn UD,
Rich HG,
Harnett KM,
and
Behar J.
Signal transduction pathways in esophageal and lower esophageal sphincter circular muscle.
Am J Med
103, Suppl:
23S-28S,
1997[ISI][Medline].
4.
Boguski, MS,
and
McCormick F.
Proteins regulating ras and its relatives.
Nature
366:
643-654,
1993[ISI][Medline].
5.
Burton, DJ,
and
Marston SB.
Control of shortening speed in single guinea-pig Taenia coli smooth muscle cells by Ca2+, phosphorylation and caldesmon.
Pflügers Arch
437:
267-275,
1999[ISI][Medline].
6.
Chakder, S,
Bandyopadhyay A,
and
Rattan S.
Neuronal NOS gene expression in gastrointestinal myenteric neurons and smooth muscle cells.
Am J Physiol Cell Physiol
273:
C1868-C1875,
1997
7.
Cobb, MH,
and
Goldsmith EJ.
How MAP kinases are regulated.
J Biol Chem
270:
14843-14846,
1995
8.
Culver, PJ,
and
Rattan S.
Genesis of anal canal pressures in the opossum.
Am J Physiol Gastrointest Liver Physiol
251:
G765-G771,
1986
9.
D'Angelo, G,
Graceffa P,
Wang CLA,
Wrangle J,
and
Adam LP.
Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth musclequantitation using novel anti-phosphopeptide antibodies.
J Biol Chem
274:
30115-30121,
1999
10.
Dessy, C,
Kim E,
Sougnez CL,
Laporte R,
and
Morgan KG.
A role for MAP kinase in differentiated smooth muscle contraction evoked by -adrenoceptor stimulation.
Am J Physiol Cell Physiol
275:
C1081-C1086,
1998
11.
Di Salvo, J,
Nelson SR,
and
Kaplan N.
Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium.
Proc Soc Exp Biol Med
214:
285-301,
1997[Abstract].
12.
Dudley, DT,
Pang L,
Decker SJ,
Bridges AJ,
and
Saltiel AR.
A synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc Natl Acad Sci USA
92:
7686-7689,
1995[Abstract].
13.
Eglen, RM,
Hedge SS,
and
Watson N.
Muscarinic receptor subtypes and smooth muscle function.
Pharmacol Rev
48:
531-565,
1996[ISI][Medline].
14.
Eguchi, S,
Matsumoto T,
Motley ED,
Utsunomiya H,
and
Inagami T.
Indentification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells.
J Biol Chem
272:
14169-14175,
1996.
15.
Ellis, C,
Moran M,
McCormick F,
and
Pawson T.
Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases.
Nature
343:
377-381,
1990[ISI][Medline].
16.
Fan, YP,
Puri RN,
and
Rattan S.
Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action.
Am J Physiol Gastrointest Liver Physiol
282:
G461-G469,
2002
17.
Fan, YP,
Chakder S,
and
Rattan S.
Mechanism of action of cholera toxin on the opossum internal anal sphincter smooth muscle.
Am J Physiol Gastrointest Liver Physiol
277:
G152-G160,
1999
18.
Gerthoffer, WT,
Yamboliev IA,
Shearer M,
Pohl J,
Haynes R,
Dang S,
Sato K,
and
Sellers JR.
Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle.
J Physiol
495:
597-609,
1996[Abstract].
19.
Harnett, KM,
Cao WB,
Kim N,
Sohn UD,
Rich H,
Behar J,
and
Biancani P.
Signal transduction in esophageal and LES circular muscle contraction.
Yale J Biol Med
72:
153-168,
1999[ISI][Medline].
20.
Hillemeier, C,
Bitar KN,
Marshall JM,
and
Biancani P.
Intracellular pathways for contraction in gastroesophageal smooth muscle cells.
Am J Physiol Gastrointest Liver Physiol
260:
G770-G775,
1991
21.
Hirano, I,
Kakkar R,
Saha JK,
Szymanski PT,
and
Goyal RK.
Tyrosine phosphorylation in contraction of opossum esophageal longitudinal muscle in response to SNP.
Am J Physiol Gastrointest Liver Physiol
273:
G247-G252,
1997
22.
Horiuchi, KY,
Wang Z,
and
Chacko S.
Inhibition of smooth muscle actomyosin ATPase by caldesmon is associated with caldesmon-induced conformational changes in tropomyosin bound to actin.
Biochemistry
34:
16815-16820,
1995[ISI][Medline].
23.
Ibitayo, AI,
Sladick J,
Tuteja S,
Louis-Jacques O,
Yamada H,
Welsh M,
and
Bitar KN.
HSP27 in signal transduction and association with contractile proteins in smooth muscle cells.
Am J Physiol Gastrointest Liver Physiol
277:
G445-G454,
1999
24.
Ishida, M,
Ishida T,
Thomas SM,
and
Berk BC.
Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dedpendent on c-Src in vascular smooth muscle cells.
Circ Res
82:
7-12,
1998
25.
Ishida, M,
Marrero MB,
Schieffer B,
Ishida T,
Bernstein KE,
and
Berk BC.
Angiotensin II activates pp60c-src in vascular smooth muscle cells.
Circ Res
77:
1053-1059,
1995
26.
Ishida, Y,
Kawahara Y,
Tsuda T,
Koide M,
and
Yokoyama M.
Involvement of MAP kinase activators in angiotensin II-induced activation of MAP kinases in cultured vascular smooth muscle cells.
FEBS Lett
310:
41-45,
1992[ISI][Medline].
27.
Kudoh, S,
Komuro I,
Hiroi Y,
Zou Y,
Harada K,
Sugaya T,
Takekoshi N,
Murakami K,
Kadowaki T,
and
Yazaki Y.
Mechanical stretch induces hypertrophic responses in cardiac myocytes of angiotensin II type 1a receptor knockout mice.
J Biol Chem
273:
24037-24043,
1998
28.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
29.
Mak, AS,
Watson MH,
Litwin CME,
and
Wang JH.
Phosphorylation of caldesmon by cdc2 kinase.
J Biol Chem
266:
6678-6681,
1991
30.
Marrero, MB,
Schieffer B,
Paxton WG,
Schieffer E,
and
Bernstein KE.
Electroporation of pp60 c-src antibodies inhibits the angiotensin II activation of phospholipase C-1 in rat aortic smooth muscle cells.
J Biol Chem
270:
15734-15738,
1995
31.
Matsumura, F,
and
Yamashiro S.
Caldesmon.
Curr Opin Cell Biol
5:
70-76,
1993[Medline].
32.
McCormick, F.
Activaors and effectors of ras p21 proteins.
Curr Opin Genet Dev
4:
71-76,
1994[Medline].
33.
Melloy, CJ,
Taylor DS,
and
Weber H.
Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells.
J Biol Chem
268:
7338-7345,
1993
34.
Menon, C,
and
Chacko S.
Expression of smooth muscle caldesmon in developing chicken gizzard.
Tissue Cell
30:
118-126,
1998[ISI][Medline].
35.
Miller, WE,
Maudsley S,
Ahn S,
Khan KD,
Luttrell L,
and
Lefkowitz RJ.
-Arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC.
J Biol Chem
275:
11312-11319,
2000
36.
Mittal, RK,
and
Balaban DH.
The esophagogastric junction.
N Engl J Med
336:
924-932,
1997
37.
Moran, MF,
Polakis P,
McCormick F,
Pawson T,
and
Ellis C.
Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activationg protein.
Mol Cell Biol
11:
1804-1812,
1991[ISI][Medline].
38.
Nohara, A,
Ohmichi M,
Koike K,
Masumoto N,
Kobayashi M,
Akahane M,
Ikegami H,
Hirota K,
Miyake A,
and
Murata Y.
The role of mitogen-activated protein kinase in oxytocin-induced contraction of uterine smooth muscle in pregnant rat.
Biochem Biophys Res Commun
229:
938-944,
1996[ISI][Medline].
39.
Okuda, M,
Kawahara Y,
and
Yokoyama M.
Angiotensin II type 1 receptor-mediated activation of ras in cultured rat vascular smooth muscle cells.
Am J Physiol Heart Circ Physiol
271:
H595-H601,
1996
40.
Rattan, S,
Fan YP,
and
Puri RN.
Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles.
Life Sci
70:
2147-2164,
2002[ISI][Medline].
41.
Rattan, S,
and
Puri R.
Mechanism of action of angiotensin II (Ang II) in the internal anal sphincter (IAS) smooth muscle (Abstract).
Gastroenterology
120:
A396,
2001.
42.
Sabri, A,
Govindarajan G,
Griffin TM,
Byron KL,
Samarel AM,
and
Lucchesi PA.
Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle.
Circ Res
83:
841-851,
1998
43.
Schieffer, B,
Paxton WG,
Chai Q,
Marrero MB,
and
Bernstein KE.
Angitotensin II controls p21ras activity via pp60c-src.
J Biol Chem
271:
10329-10333,
1996
44.
Schiller, LR.
Fecal incontinence.
In: Sleisenger & Fordrtran's Gastrointestinal and Liver Disease, edited by Feldman M.. Philadelphia, PA: Saunders, 2000, p. 160-173.
45.
Takahashi, T,
Kawahara Y,
Okuda M,
Ueno H,
Takeshita A,
and
Yokoyama M.
Angiotensin II stimulates mitogen-activated protein kinases and protein synthesis by a ras-independent pathway in vascular smooth muscle cells.
J Biol Chem
272:
16018-16022,
1997
46.
Timmermans, PBMWM,
Wong PC,
Chiu AT,
Herblin WF,
Benfield P,
Carini DJ,
Lee RJ,
Wexler RR,
Saye JAM,
and
Smith RD.
Angiotensin II receptors and angiotensin II receptor antagonists.
Pharmacol Rev
45:
205-251,
1993[ISI][Medline].
47.
Tocque, B,
Delumeau I,
Parker F,
Maurier F,
Multon MC,
and
Schweighoffer F.
ras-GTPase activating protein (GAP): a putative effector for ras.
Cell Signal
9:
153-158,
1997[ISI][Medline].
48.
Touyz, RM,
Mabrouk ME,
He G,
Wu XH,
and
Schiffrin EL.
Mitogen-activated protein/extracellular signal-regulated kinase inhibition attenuates angiotensin II-mediated signalling and contraction in spontaneously hypertensive rat vascular smooth muscle cells.
Circ Res
84:
505-515,
1999
49.
Touyz, RM,
and
Schiffrin EL.
Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells.
Pharmacol Rev
52:
639-672,
2000
50.
Uehara, Y,
Fukazawa H,
Murakami Y,
and
Mizuno S.
Irreversible inhibition of V-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds.
Biochem Biophys Res Commun
163:
803-809,
1989[ISI][Medline].
51.
Unger, T,
Chung O,
Csikos T,
Culman J,
Gallinat S,
Gohlke P,
Hohle S,
Meffert S,
Stoll M,
Stroth U,
and
Zhu YZ.
Angiotensin receptors.
J Hypertens
14:
S95-S103,
1996[ISI].
52.
Watts, SW.
Serotonin ativates the mitogen-activated protein kinase pathway in vascular smooth muscle: use of the mitogen activating protein kinase kinase inhibitor PD098059.
J Pharm Pharmacol
279:
1541-1550,
1996.
53.
Weber, LP,
Van Lierop JE,
and
Walsh MP.
Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments.
J Physiol
516:
805-824,
1999
54.
Yamada, H,
Strahler J,
Welsh MJ,
and
Bitar KN.
Activation of MAP kinase and translocation with HSP27 in bombesin-induced contraction of rectosigmoid smooth muscle.
Am J Physiol Gastrointest Liver Physiol
269:
G683-G691,
1995