MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Weibiao Cao,1 Uy Dong Sohn,2 Khalil N. Bitar,3 Jose Behar,1 Piero Biancani,1 and Karen M. Harnett1

1Department of Medicine, Rhode Island Hospital and Brown University Providence, Rhode Island 02902;2Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul, Korea 156-756; and3Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan 48109

Submitted 26 April 2002 ; accepted in final form 26 February 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.

second messenger system; signal transduction; mitogen-activated protein kinase; protein kinase C; lower esophageal sphincter


ESOPHAGEAL and lower esophageal sphincter (LES) circular muscles are functionally different and regulated by different signal transduction pathways. Esophageal muscle is relaxed in its basal state and contracts quickly and briefly in response to neural stimulation or swallowing. LES circular muscle exhibits relatively high resting tone, which is maintained in the absence of neural stimuli and relaxes in response to swallowing. We have previously shown that esophageal contraction in response to its endogenous neurotransmitter ACh and to other agonists is mediated by a protein kinase C (PKC)-dependent pathway (10), involving a calcium-insensitive PKC-{epsilon} (53). In the LES, contraction in response to a maximally effective concentration of ACh is calmodulin and myosin light-chain kinase (MLCK) dependent. LES spontaneous tone or contraction in response to a low concentration of ACh, however, is calmodulin independent and mediated through a calcium-sensitive PKC-{beta} (10, 32).

The precise mechanisms responsible for mediation of PKC-dependent contraction are not well established (5, 15, 38, 39, 58); however, MAPK has been described as a missing link in the signal transduction cascade between membrane-bound PKC and smooth muscle activation. MAPKs represent a point of convergence for cell surface signals regulating cell growth and division. MAPKs comprise a family of serine threonine kinases, which include extracellular signal-regulated kinases ERK1 and ERK2, the Jun NH2-terminal kinase/stressactivated protein kinase, and p38 MAPK (19). These MAPKs are activated by dual phosphorylation of Tyr185 and Thr187 residues, catalyzed by MEK, a mitogen-activated/extracellular-regulated protein kinase kinase that is specific for MAPK (6, 7). Several receptors that are coupled to heterotrimeric G proteins have been shown to activate MAPKs (20, 25, 42). These include bombesin (25), thromboxane A2/PGH2 (48), PGF2{alpha} (60), ANG II (24, 33), {alpha}1B-adrenergic (25), {alpha}2A-adrenergic (25, 57), M1 muscarinic (25), D2 dopamine (25), and A1 adenosine (25) receptors. The signal transduction pathways employed by these receptors are heterogeneous, and significant heterogeneity also exists between cell types (21).

ERK1 and ERK2 MAPKs are activated by diverse extracellular stimuli and by protooncogene products that induce proliferation or enhance differentiation (19). However, ERK MAPKs are constitutively expressed in differentiated smooth muscle (1, 2, 9), and phosphorylation of MAPK substrates in contractile cells may not necessarily be related to proliferation and progress through the cell cycle (27). For example, in ferret aorta, calcium-independent phenylephrine-induced contraction is mediated by phosphorylation of ERK1 MAPK and caldesmon (22).

The p38 MAPK is thought to be activated by inflammatory cytokines and environmental stress; it was identified as part of a protein kinase cascade activated by interleukin-1{beta} or physiological stress and ending in activation of MAPK-activated protein (MAPKAP) kinase 2 and phosphorylation of the 25-/27-kDa heat shock proteins HSP25/HSP27 (26, 50). MAPKAP kinase 2 phosphorylates HSP25/HSP27 in a cell-free preparation at the sites phosphorylated in intact cells in response to stress (56).

Phosphorylation of HSP27 has been described as an MAPK-mediated mechanism modulating contraction of intestinal (15) and vascular smooth muscle (45, 62). In rectosigmoid smooth muscle, p38 MAPK is activated during PKC-dependent contraction and cotranslocates with HSP27 (61).

In the current study, we examine the hypothesis that PKC-mediated contraction of LES and esophageal muscle depends on activation of MAPKs. We find that the PKC-dependent contractile pathway responsible for maintenance of LES resting tone and contraction in response to low concentrations of the endogenous neurotransmitter ACh depends on activation of MAPKs. Similarly, in esophageal smooth muscle, contraction in response to ACh depends on ERK MAPK phosphorylation and/or HSP27-linked p38 MAPK phosphorylation.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Adult male cats weighing between 3.5 and 5.5 kg were initially anesthetized with ketamine (Aveco, Fort Dodge, IA) and then were killed with an overdose of pentobarbital sodium (Schering, Kennilworth, NJ). The chest and abdomen were opened with a midline incision exposing the esophagus and stomach. The esophagus and LES were isolated and excised as previously described (11, 13).

Measurements of in vitro LES tone. LES strips (2 mm) were mounted in separate 1-ml muscle chambers and equilibrated for 2 h with continuous perfusion of oxygenated physiological salt solution (PSS), as previously described in detail (913, 32). During this time, the tension in LES strips increased, attaining a steady level at 2 h. The PSS contained the following (in mM): 116.6 NaCl, 21.9 NaHCO3, 1.2 NaH2PO4, 3.4 KCl, 2.5 CaC12, 5.4 glucose, and 1.2 MgCl2. The solution was equilibrated with a gas mixture containing 95% O2-5% CO2, at pH 7.4 and 37°C. After equilibration, LES strips were incubated for 30 min in solution containing vehicle (control) or the appropriate concentrations of PD-98059 and SB-203580. The vehicle for PD-98059 and SB-203580 was ethanol, which has no effect on strips at concentrations <0.1%. The highest concentration used in this study was 0.05%.

Smooth muscle tension was recorded on a chart recorder (Grass Instruments, Quincy, MA). Passive force was obtained at the end of the experiment by completely relaxing the strips with excess EDTA until no further decrease in resting force was observed. Basal LES tone is the difference between resting and passive force. Percent increase in basal tone was defined by the ratio between the increase in force after drug administration and basal LES tone. Percent basal LES tone was calculated by the ratio between the force after using the drugs and the basal LES tone.

Preparation of circular smooth muscle tissue. The esophagus and LES were excised, the circular muscle layer was cut into 0.5-mm-thick slices with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA), and tissue squares were made by cutting two times with a 2-mm blade block, the second cut at right angles to the first. This circular smooth muscle tissue was used for Western blot analysis of MAPK, measurement of MAPK activity and MAPK phosphorylation, and to obtain isolated smooth muscle cells.

Cell isolation and permeabilization. Isolated smooth muscle cells were obtained by enzymatic digestion, as previously described (912). Briefly, esophageal and LES circular smooth muscle was digested in HEPES-buffered physiological solution containing 150 U/ml collagenase (CLS type II; Worthington Biochemicals, Freehold, NJ) for 2 h. The HEPES solution contained 114.7 mM NaCl, 5.7 mM KCl, 2.1 mM KH2PO4, 11 mM glucose, 24.5 mM HEPES, 1.9 mM CaCl2, 0/57 mM MgCl2, 0.3 mg/ml BME amino acid supplement (M. A. Bioproducts, Walkersville, MD), and 0.08 mg/ml soybean trypsin inhibitor (Worthington Biochemicals). The HEPES solution was oxygenated (100% O2) at 31°C, and the pH was adjusted to 7.4. At the end of the digestion period, the tissue was rinsed and then incubated in collagenase-free HEPES buffer. The cells dissociate freely in collagenase-free solution.

When permeable cells are required to allow the use of MAPK and HSP antibodies that do not diffuse across the intact plasma membrane, the partly digested muscle tissue is washed with a "cytosolic" enzyme-free PSS (cytosolic buffer) of the following composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO3, 5.0 MgSO4, 0.96 NaH2PO4, 1.0 EGTA, and 0.48 CaCl2. The cytosolic buffer contained 2% BSA and was equilibrated with 95% O2-5% CO2 to maintain pH of 7.2 at 31°C. Muscle cells dispersed spontaneously in this medium. A low concentration of calcium is present in the cytosolic buffer to avoid spontaneous contraction of the cells in the absence of agonists after the membrane becomes permeable. The cells are permeabilized by incubation for 3 min in cytosolic buffer containing saponin (75 µg/ml). After exposure to saponin, the cell suspension is spun at low gravity, and the resulting pellet is resuspended in saponin-free modified cytosolic buffer containing antimycin (10 µM), ATP (1.5 mM), and an ATP-regenerating system consisting of creatine phosphate (5 mM) and creatine phosphokinase (10 U/ml; see Ref. 14).

Agonist-induced contraction of isolated muscle cells. Once the cells had dissociated, 0.5-ml aliquots of the cell-containing fluid were added to tubes for exposure to agonists and measurement of contraction. The maximally effective concentration of ACh for contraction of intact or saponin-permeabilized single cells is 1010 to 109 M and lower than the dose required for a maximal response in undigested muscle tissue strips or squares (105 M; see Ref. 11) that were used for Western blots or for measurements of kinase activity. Intact esophageal circular smooth muscle cells were contracted with a maximally effective concentration of ACh alone or after 10 min of exposure to the indicated concentration of PD-98059 or SB-203580. The vehicle for PD-98059 and SB-203580 was ethanol, which has no effect on strips or cells at concentrations <0.1%. The highest concentration used in this study was 0.05%.

Permeabilized esophageal and LES cells were exposed to a maximally effective concentration of ACh or to the diacylglycerol analog 1,2-dioctanoylglycerol (DG, 106 M) for 30 s. When MAPK or HSP antibodies were used, permeabilized cells were incubated in the antiserum at the indicated concentration for 1 h before the addition of agonist (15). We have previously shown that specific steps in the signaling pathway can be selectively inhibited by antibodies against the appropriate proteins mediating the specific reaction (18, 52).

After exposure to agonist, the cells were fixed in acrolein at a final 1.0% concentration and kept refrigerated.

Cell measurements. A drop of the cell-containing medium was placed on a glass slide and covered by a coverslip. Thirty consecutive cells from each slide were observed through a phase-contrast microscope (Carl Zeiss) and a CCTV camera (model WV-CD51; Panasonic, Secaucus, NJ) connected to a Macintosh Computer (Apple, Cupertino, CA). The Image 1.59 software program (NIH, Bethesda, MD) was used to measure cell length and for data accumulation. The average length of 30 cells, measured in the absence of agonists, was taken as "control" length. The average cell length is the same between intact cells (66.9 ± 1.8 µm) and permeable cells (66.9 ± 3.8 µm). In addition, average cell length was measured after the addition of test agents. Shortening was defined as a percentage decrease in average length after agonists when compared with control length.

Western blot. Esophageal and LES circular muscle was homogenized in Triton X lysis buffer containing 50 mM Tris · HCl (pH 7.5), 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% (vol/vol) Triton X, 40 mM {beta}-glycerolphosphate, 40 mM p-nitrophenylphosphate, 200 µM sodium orthovanadate, 100 µM phenylmethylsulfonyl fluoride, 1 µg/ml lepeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin. The suspension was centrifuged at 15,000 g for 5 min, and the protein concentration in the supernatant was determined. The supernatant of each sample containing the same amount of protein (150 µg ERK1/ERK2, 120 µg for p38 MAPK) was mixed with 2% SDS loading buffer containing 62.5 mM Tris · HCl (pH 6.8), 5% 2-mercaptoethanol, 0.002% bromphenol blue, and 10% glycerol and boiled for 5 min. Prestained molecular weight marker was prepared in the same manner. After these supernatant samples were subjected to SDS-PAGE (200 volts, 45 min) using an acrylamide concentration of 10% in the separating gel for p38, ERK1, and ERK2 MAPK and 15% for HSP 27, the separated proteins were electrophoretically transferred to a nitrocellulose (NC) membrane (Bio-Rad, Melville, NY) at 30 volts overnight. Transfer of proteins to the NC membrane was confirmed with Ponseau S staining reagent (Sigma, St. Louis, MO). To block nonspecific binding, the NC membrane was incubated in 5% nonfat dry milk in PBS for 60 min followed by three rinses in milk-free buffer. Samples were incubated with anti-phosphorylated MAPK antibody (1:20,000; Promega, Madison, WI), anti-phosphorylated p38 MAPK antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-HSP27 antibody (1:100; Upstate Biotechnology, Waltham, MA) for 1 h with shaking followed by three washes with antibody-free buffer. This was followed by a 60-min incubation in horseradish peroxidase-conjugated goat anti-rabbit antibody (Amersham, Arlington Heights, IL). Detection was achieved with an enhanced chemiluminescence agent (Amersham). Molecular weight was estimated by comparison of sample bands with prestained molecular weight marker (Amersham). For the p38 and ERK1/ERK2 MAPK phosphorylation, after detecting the phosphorylated MAPKs, the membranes were incubated in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.6 mM Tris · HCl, pH 6.7) at 50°C for 30 min, washed three times (10 min each), and then reprobed by using anti-p38 MAPK antibody (1:500; Santa Cruz Biotechnology) and anti-ERK2 antibody (1:500; Santa Cruz Biotechnology), respectively.

MAPK immunoprecipitation. Esophageal circular muscle was homogenized and centrifuged as described above for Western blot analysis. MAPK was immunoprecipitated with ERK2 or p38 MAPK antibodies. Smooth muscle extract (500 µl of 1 mg protein) was added to 5 µl of anti-ERK2 or p38 MAPK antibody (Santa Cruz Biotechnology) and incubated for 1 h at 4°C. For each immunoprecipitation of MAPK, 4 mg protein A-Sepharose were swollen for 15 min in Triton X lysis buffer. The smooth muscle extract-MAPK antibody mixture was added to the protein A-Sepharose, and this mixture was rotated at 4°C overnight. The protein A beads were pelleted by centrifugation (2 min, 14,000 g, 4°C) and washed two times with 1 ml Triton X lysis buffer and two times with 1 ml of 50 mM HEPES buffer (pH 7.5) containing 10 mM magnesium acetate and 1 mM dithiothreitol. The immunoprecipitated ERK2 was used in determinations of MAPK activity. The immunoprecipitated p38 was used in Western blotting experiments.

MAPK activity. MAPK activity was measured by filter assay (49). The MAPK reaction was started by the addition of 40 µl reaction buffer to the protein A complex. The reaction buffer contained 50 mM HEPES buffer (pH 7.5), 10 mM magnesium acetate, 1 mM dithiothreitol, 20 µM ATP, 0.25 mg/ml myelin basic protein, and 10 µCi [{gamma}-32P]ATP/sample. After 30 min at 30°C, the reaction was stopped by the addition of 20 µl of 200 mM EDTA (pH 7.0). Samples were centrifuged (14,000 g, 2 min, 4°C), and 50 ml of the supernatant were spotted on p81 cation-exchange paper (Fisher Scientific, Pittsburgh, PA). The p81 filters were washed two times with 0.5% phosphoric acid and one time with acetone and dried, and the radioactivity was measured using a TriCarb 1900 CA liquid scintillation analyzer (Packard Instrument, Meriden, CT).

Protein determination. The amount of protein present in the supernatant of the homogenates of LES and esophageal tissues was determined by colorimetric assay (Bio-Rad) according to the method of Bradford (16). Briefly, the samples were diluted 1,000 times in distilled water and mixed with Bio-Rad Protein Assay dye reagent (1:4, vol/vol). Bio-Rad's protein assay is based on the color change of Coomassie brilliant blue G-250 dye in response to various concentrations of protein, which can be measured at 595 nm.

Statistical analysis. Data are expressed as means ± SE. Statistical differences between means were determined by Student's t-test. Differences between multiple groups were tested using ANOVA for repeated measures and checked for significance using Scheffé's F-test.

Drugs and chemicals. [{gamma}-32P]ATP was purchased from New England Nuclear (Boston, MA); PD-98059 and SB-203590 were from Calbiochem (San Diego, CA); soybean trypsin inhibitor and collagenase CLS type II were from Worthington Biochemicals; papain, saponin, BME amino acid supplement, EGTA, HEPES, creatine phosphate, creatine phosphokinase, ATP, antimycin A, protein A-Sepharose, and other reagents were purchased from Sigma.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAPK in esophageal and LES smooth muscle. The MAPKs ERK1 (p44) and ERK2 (p42) were identifiable by Western blot analysis in esophageal and LES circular smooth muscle (Fig. 1). To test whether ERK1/ERK2 participate in PKC-dependent contraction, the cells were permeabilized with saponin to allow diffusion of antibodies in the cytoplasm and then contracted with the PKC activator DG. ERK1 and ERK2 antibodies concentration dependently inhibited the DG-induced contraction of LES and esophageal muscle (P = 0.001, ANOVA; Fig. 1), supporting the view that MAPKs play a role in PKC-dependent contractile pathways. The ERK2 antibody was more effective and at a 10 µg/ml concentration reduced shortening in response to DG from 20.0 ± 0.2 to 7.1 ± 0.6% in LES and from 21.5 ± 0.6 to 9.0 ± 0.7% in esophageal cells.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1. MAPKs mediate PKC-dependent contraction of permeabilized esophageal (ESO) and lower esophageal sphincter (LES) circular muscle cells. Western blot analysis using ERK1 and ERK2 antibodies (Ab) is shown on top. Bottom: ERK1 and ERK2 antibodies dose dependently reduced ESO and LES contraction in response to a maximally effective concentration of the direct PKC agonist 1,2-dioctanoylglycerol (DG; P = 0.001, ANOVA). Values are means ± SE of 3–4 animals with 30 cells counted for each animal.

 

LES tone and response to ACh. We have previously shown that high and low ACh concentrations activate different signal transduction pathways in LES muscle. Low ACh activates a PKC-dependent contractile pathway, whereas high ACh concentrations are mediated through a calmodulin- and MLCK-dependent pathway (10, 52). To test whether inhibition of MAPKs is effective only against PKC-mediated contraction, LES cells were permeabilized with saponin, to allow diffusion of antibodies into the cytoplasm, and exposed to antibodies against ERK1 and ERK2. At low ACh concentration, when contraction is PKC dependent, the contraction was inhibited by the antibodies (Fig. 2). At high, (i.e., maximally effective) ACh concentration, when contraction is calmodulin- and MLCK-dependent and not dependent on PKC, the contraction was not affected, suggesting that ERK1 and ERK2 participate only in the PKC-dependent contractile pathway.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. MAPK mediates PKC-dependent contraction of saponin-permeabilized LES circular muscle cells. LES circular smooth muscle cells were permeabilized and then contracted with ACh (1013 to 109 M). Contraction was inhibited by antibodies raised against both ERK1 and ERK2 at low but not at high (i.e., maximally effective) ACh concentration (P < 0.02, ANOVA). We have previously shown that low ACh activates a PKC-dependent contractile pathway, whereas high ACh concentrations activate a calmodulin- and MLCK-dependent pathway (10, 51). These data suggest that ERK1 and ERK2 participate only in the PKC-dependent contractile pathway. Values are means ± SE of 3 animals with 30 cells counted for each animal.

 

If PKC-dependent contraction is mediated by MAPKs, LES circular muscle resting tone, which is also mediated by a PKC-dependent contractile pathway (32), should depend on MAPKs. To test whether MAPKs play a role in maintenance of LES tone, LES circular muscle strips were mounted in a muscle chamber and allowed to equilibrate and develop spontaneous tonic contraction. After a steady tone had developed, the strips were exposed to increasing concentrations of inhibitors of MAPKs. The ERK1 and ERK2 MAPKs are activated by a MAP kinase kinase (MEK), which phosphorylates ERK1 and ERK2 on two sites. The selective MEK inhibitor PD-98059 and the p38 MAPK inhibitor SB-203580 concentration dependently reduced (P = 0.001, ANOVA) LES tone (Fig. 3) when used alone. LES tone was reduced further when PD-98059 and SB-203580 were used in combination (P = 0.001, ANOVA). These data suggest the involvement of ERK1, ERK2, and p38 MAPKs in PKC-dependent LES tone and suggest that MAPKs may participate in PKC-dependent muscle contractions. To confirm that PKC-dependent contraction is mediated by ERK1/2 and p38 MAPK, LES cells were contracted with the diacylglycerol analog DG, which directly activates PKC. Similarly to spontaneous tone, DG-induced contraction of LES cells was reduced by PD-98059 and by SB-203580 (P = 0.001, ANOVA) and was further reduced when PD-98059 and SB-203580 were used in combination (P = 0.001, ANOVA; Fig. 4)



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. MAPKs mediate PKC-dependent LES basal tone of LES strips. The mitogen/extracellular signal-regulated kinase kinase (MEK) inhibitor PD-98059 or the p38 MAPK inhibitor SB-203580 produced a concentration-dependent reduction in LES tone (P = 0.001, ANOVA). LES tone was reduced further when PD-98059 and SB-203580 were used in combination (P = 0.001, ANOVA), suggesting that PKC-dependent tone is mediated by ERK1/2 and p38 MAPKs. Values are means ± SE of 3 animals.

 


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4. DG-induced contraction of LES cells (106 M) was signifi-cantly reduced by the MAP kinase kinase (MEK) inhibitor PD-98059 and the p38 MAPK inhibitor SB-203580 (P < 0.01, ANOVA). PD-98059 and SB-203580 in combination nearly abolished DG-induced contraction of LES cells (P = 0.001, ANOVA). Values are means ± SE of 3 animals with 30 cells counted for each animal.

 

Esophageal contraction. Because contraction of esophageal muscle in response to its physiological neurotransmitter ACh is also mediated through a PKC-dependent pathway, we examined the response of esophageal circular smooth muscle to ACh in the presence of PD-98059 and SB-203580 (Fig. 5).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. ACh-induced contraction (109 M) of ESO cells was concentration dependently reduced by the MEK inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580 (P < 0.01, ANOVA). PD-98059 and SB-203580 in combination nearly abolish ACh-induced contraction of ESO cells (P < 0.01, ANOVA). Values are means ± SE of 3 animals with 30 cells counted for each animal.

 

The selective MEK inhibitor PD-98059 concentration-dependently reduced contraction (P < 0.001, ANOVA) of intact esophageal smooth muscle cells in response to ACh. A maximally effective concentration of PD-98059 reduced ACh-induced contraction from 22.9 ± 1.4 to 9.7 ± 0.8%. Similarly, the selective p38 MAPK inhibitor SB-203580 concentration dependently reduced shortening of intact esophageal cells in response to a maximally effective concentration of ACh (109 M; Fig. 5; P < 0.001, ANOVA). A maximally effective concentration of SB-203580 reduced ACh-induced contraction from 23.9 ± 1.4 to 14.7 ± 0.4%.

SB-203580 and PD-98059, when used in combination, nearly abolished contraction of esophageal muscle (P < 0.01, ANOVA), supporting the view that esophageal contraction in response to ACh may be entirely mediated through activation of both ERKs and p38 MAPKs.

To confirm the contribution of ERK1, ERK2, and p38 MAPKs to ACh-induced contraction, esophageal smooth muscle cells were permeabilized and then contracted with a maximally effective concentration of ACh (109 M) in the presence of ERK1, ERK2, and p38 MAPK antibodies (Fig. 6). ACh-induced contraction was concentration dependently reduced (P = 0.001, ANOVA) by ERK1 and ERK2 antibodies. ERK2 antibodies caused a greater reduction in contraction than ERK1. ERK2 antibodies (10 µg/ml) reduced shortening in response to ACh from 23.3 ± 0.6 to 8.4 ± 0.1%. The reduction by the ERK2 antibody was similar to that produced by the MEK inhibitor PD-98059 at a maximally effective concentration (from 22.9 ± 1.4 to 9.7 ± 0.8%). Similarly, ACh-induced contraction was concentration dependently reduced (P = 0.001, ANOVA) by a p38 MAPK antibody (Fig. 6). The reduction produced by the p38 antibody at its maximally effective concentration (from 25.5 ± 0.3 to 13.4 ± 0.7) was similar to that produced by the p38 MAPK inhibitor SB-203580 (23.9 ± 1.4 to 14.7 ± 0.4%).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. ERK1/2 and p38 MAPKs mediate ACh-induced contraction of permeabilized ESO circular muscle cells. ACh-induced contraction (109 M) was concentration dependently reduced by ERK1, ERK2, and p38 antibodies (P = 0.001, ANOVA). Values are means ± SE of 3–4 animals with 30 cells counted for each animal.

 

Agonist-induced phosphorylation of HSP27 has been proposed as an MAPK-mediated mechanism of smooth muscle contraction (8, 15, 17, 43, 61). We therefore examined the role of HSP27 on esophageal contraction. HSP27 antibodies concentration dependently reduced contraction in response to a maximally effective concentration of ACh (109 M; P < 0.05, ANOVA; Fig. 7). HSP27 and p38 MAPK antibodies in combination caused no greater inhibition than either one alone (Fig. 7), suggesting that p38 MAPK and HSP27 act in the same signal transduction pathway.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7. Heat shock protein 27 (HSP27) contributes to ACh-induced contraction of permeabilized ESO circular muscle cells. ESO contraction in response to a maximally effective concentration of ACh (109 M) was significantly reduced by HSP27 antibodies (P = 0.001, ANOVA). HSP27 and p38 MAPK antibodies in combination caused no greater inhibition than either one alone, suggesting that p38 MAPK and HSP27 act in the same signal transduction pathway. Values are means ± SE of 3 animals with 30 cells counted for each animal.

 

MAPK activity and phosphorylation. To confirm that ACh-induced contraction of esophageal circular smooth muscle results from MAPK activation, we measured MAPK activity and phosphorylation. MAPK was purified by immunoprecipitation from esophageal circular muscle after incubation with 105 M ACh for 0, 30, 60, and 300 s. MAPK activity (ERK2) significantly increased from 0.12 ± 0.04 pmol · min1 · mg protein1 at the basal level to 0.31 ± 0.06 and 0.34 ± 0.06 pmol · min1 · mg protein1 (P < 0.05) at 30 and 60 s after ACh stimulation (Fig. 8).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 8. ACh stimulates MAPK activity in ESO circular smooth muscle. MAPK was purified by immunoprecipitation from ESO circular smooth muscle after incubation with 10 µM ACh for 0, 30, 60, or 300 s. MAPK activity significantly increased at 30 and 60 s after ACh stimulation (P < 0.05) and was down again by 5 min. Values are means ± SE of 3 animals.

 

Phosphorylation of ERK MAPK in response to ACh was measured by Western blot analysis using activated (phosphorylated) ERK2 antibodies (Fig. 9). Figure 9 shows that ERK2 phosphorylation of esophageal circular smooth muscle increased significantly after 1 min of stimulation with ACh (105 M; P < 0.01), confirming that ACh-induced contraction of esophageal circular smooth muscle is associated with MAPK activation. In addition, the PKC inhibitor chelerythrine (105 M), abolished the ACh-induced increase in ERK2 phosphorylation of esophageal circular smooth muscle, demonstrating that activation of ERK2 is PKC dependent.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 9. A: average ERK2 kinase activation (i.e., phosphorylation) in response to a maximally effective concentration of ACh (105 M for intact muscle) alone or in the presence of the PKC inhibitor chelerythrine (Chel, 105 M). Values are means ± SE of 3 animals. Chelerythrine caused a significant decrease compared with ACh alone. B: typical Western blot of phosphorylated ERK2 in ESO circular smooth muscle. The supernatant of tissue homogenate was probed with anti-ERK2 antibody (top) or anti-active [phosphorylated (p)] ERK2 antibody (bottom). Top shows total ERK2 and demonstrates equal protein loading between lanes.

 

Phosphorylation of p38 MAPK in response to ACh was also measured by Western blot analysis using activated (i.e., phosphorylated) p38 MAPK antibodies (Fig. 10). Phosphorylation of p38 MAPK in esophageal circular smooth muscle significantly increased to 604 ± 169% of control after 15–60 s stimulation with ACh (105 M; P < 0.05) and decreased at 300 s, demonstrating that ACh-induced contraction of esophageal circular smooth is associated with activation of p38 MAPK.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 10. A: average time course of p38 kinase activation (i.e., phosphorylation) in response to a maximally effective concentration of ACh (105 M for intact muscle). The phosphorylation increase induced by ACh was significant at 15–60 s (P < 0.05) after exposure to ACh, decreasing at 300 s. Values are means ± SE of 3 animals. B: typical Western blot of phosphorylated p38 kinase in ESO circular smooth muscle (bottom). Top: reprobe of total ERK2 demonstrating equal protein loading between lanes.

 

To confirm that p38 MAPK and HSP27 act in the same signal transduction pathway, esophageal tissue squares were stimulated with ACh and then homogenized and immunoprecipitated with a phosphorylated p38 MAP antibody. The immunoprecipitate was then probed with an HSP27 antibody. The immunoprecipitate contained increased levels of HSP27 after stimulation with ACh compared with unstimulated muscle (Fig. 11). We interpret these findings to mean that ACh stimulation and consequent activation of p38 MAPK promote association of HSP27 with the phosphorylated (i.e., active) p38 MAPK. This interpretation is further supported by the finding that the time-dependent association of HSP27 and phosphorylated p38 MAPK (Fig. 11) closely mirrors the time course of p38 MAPK phosphorylation.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 11. ESO tissue squares were stimulated with ACh (105 M) and then homogenized and immunoprecipitated with a phosphorylated p38 MAP antibody. HSP27 levels were then detected in the immunoprecipitate. A: Western blot of the immunoprecipitate with an HSP27 antibody found greater amounts of HSP27 at 15, 30, and 60 s (P < 0.05) after stimulation with ACh than in unstimulated muscle. Values are means ± SE of 3 animals. B: typical Western blot. The data suggest that HSP27 is linked to phosphorylated p38 MAPK after ACh stimulation.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Contraction of esophageal circular muscle in response to its endogenous neurotransmitter ACh is mediated by PKC-{epsilon} (53). A PKC (PKC-{beta})-dependent contractile pathway is also present in LES circular muscle, where it mediates maintenance of LES resting tone and contraction in response to a low level of ACh (10, 32), whereas LES contraction in response to a maximally effective concentration of ACh is mediated by a calmodulin-dependent pathway (10, 51). In the current study, our data suggest that MAPK activation participates only in these PKC-dependent contractions of LES and esophageal circular smooth muscle and not in calmodulin-dependent LES contraction in response to maximally effective concentrations of ACh.

To demonstrate that MAPK participates in PKC-mediated contraction of esophageal and LES circular muscle, we tested for the presence of ERK1 and ERK2 by Western blot analysis. ERK1 and ERK2 are present in LES and esophageal circular smooth muscle, consistently with numerous studies that report expression of MAPKs in numerous cell types. In addition, when contraction was induced by the diacylglycerol analog DG, ERK1 and ERK2 antibodies concentration dependently reduced contraction in both esophageal circular muscle and LES. Similarly to diacylglycerol, DG is known to directly activate PKC, confirming that MAPK participates in PKC-mediated contraction of esophageal and LES circular muscle.

We next examined in detail the participation of MAPKs in contraction of LES and esophageal muscle.

MAPK and LES smooth muscle contraction. In LES circular muscle, we therefore tested the role of MAPKs in the response to high and low concentrations of ACh and in LES spontaneous tone. Our data show that inhibition of MAPKs by selective antibodies inhibits contraction induced by a low concentration ACh and by DG, both of which activate a PKC-dependent, calmodulin-independent contractile pathway. The antibodies had no effect on contraction induced by a high (i.e., maximally effective) concentration of ACh, which is mediated by calmodulin. Similarly, LES tone, which is PKC dependent, was concentration dependently reduced by the MEK inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580 when used alone. When used in combination, the two inhibitors further reduced tone. The same MAPK inhibitors, when used alone, reduced DG-induced contraction of intact LES cells and, in combination, almost abolished it.

PD-098059 has been reported to selectively inhibit the MAPK-activating enzyme MAPK/ERK (MEK) without inhibiting activity of MAPK itself. Inhibition of MEK by PD-098059 prevents activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells (4, 23). SB-203580 is a pyridinyl imidazole derivative that selectively inhibits the kinase activity of p38 (44). The effectiveness and selectivity of inhibitors and antibodies are supported by the similarity in the results.

In addition, selectivity of the antibodies is supported by the fact that ERK1/2 antibodies inhibited contraction of permeable LES cells by a low concentration of ACh but not contraction induced by a high concentration of ACh. These findings are consistent with numerous studies (18) showing that antibodies against particular proteins in the contractile pathway are as effective as selective inhibitors of the same protein. This finding may be because of the relatively high molecular weight of immunoglobulins. It is entirely possible that, once antibodies bind to a protein, they may inhibit its effect because of their size, regardless of their particular binding site on the protein.

The data suggest that in LES circular muscle MAPKs (ERKs and p38) may participate in PKC-dependent contractions, such as LES tone or contraction in response to the PKC activator DG, or in response to low concentrations of ACh. Contraction in response to a maximally effective concentration of ACh, which is calmodulin dependent, was not affected by MAPK antibodies, confirming MAPK involvement only in PKC-mediated contraction.

MAPK and esophageal smooth muscle contraction. Similarly to spontaneous LES tone, contraction of esophageal muscle in response to its endogenous neurotransmitter ACh is PKC dependent (10, 32, 53) and therefore may depend on activation of MAPKs. As expected, either inhibition of MEK, the upstream activator of MAPK, by PD-980959 or inhibition of p38 kinase by SB-203580 reduced ACh-induced contraction of esophageal smooth muscle when used alone. In combination, SB-203580- and PD-980959-induced inhibition was additive, resulting in almost complete abolition of ACh-induced contraction. These results with MAPK inhibitors were confirmed in saponin-permeabilized cells with ERK1, ERK2, and p38 kinase antibodies. Similarly to the inhibitors, ERK or p38 kinase antibodies dose dependently reduced ACh-induced contraction of esophageal cells. The effectiveness and selectivity of inhibitors and antibodies is supported by the similarity in the results.

Involvement of MAPKs in PKC-mediated ACh-induced contraction of esophageal muscle is confirmed by MAPK activity measurements that show increased MAPK activity in response to ACh and abolition of ERK2 phosphorylation by the PKC inhibitor chelerythrine. These results are consistent with other studies reporting MAPK activation by a variety of contractile agonists in vascular, airway, and intestinal smooth muscles (27, 28, 34). For example, in tracheal smooth muscle cells, ACh stimulates ERK1 and ERK2 phosphorylation, which is significantly reduced by PD-98059 (31).

In the current study, our data indicate that PKC-dependent contraction is associated with activation of MAPK. A connection between MAPK activation and PKC-mediated smooth muscle contraction has been proposed (36, 37) for ferret aorta, where MAPK is activated in response to phenylephrine (46). In this preparation, PKC-{epsilon} and ERK1 MAPK translocate from the cytosol to the sarcolemma in response to adrenergic stimuli; PKC-{epsilon} remains associated with the sarcolemma, whereas MAPK redistributes to the cytosol coincident with contraction (38, 39). There is some confusion, however, about the role of MAPK activation in smooth muscle contraction. In swine carotid artery, agonists such as histamine and direct PKC activation by phorbol 12,13-dibutyrate (PDBu) induced MAPK activation and phosphorylation, but inhibition of the MAPK cascade by PD-098059 did not affect histamine or PDBu-induced contraction (30). In tracheal smooth muscle, M2 receptor activation of ERK MAPKs and phosphorylation had little or no effect on isometric force (31). It has been proposed that these conflicting reports may be because of tissue-specific differences related to the calcium dependence of the particular contraction. Calcium-independent contractions of smooth muscle may be more dependent on MAPK-mediated pathways than calcium-dependent contraction. For example, the swine carotid artery does not contract in response to agonist activation in the absence of extracellular calcium (30). However, calcium-independent contractions induced by phenylephrine in the ferret aorta (22), histamine in bovine tracheal smooth muscle (41), and PGF2{alpha} in the iris sphincter smooth muscle (63) have all been significantly reduced by PD-098059.

No such confusion exists for contraction of LES and esophageal smooth muscle. In these tissues, there is a clear distinction between calcium-calmodulin-MLCK-dependent contraction and PKC-dependent contraction, which is not dependent on activation of the calcium-sensitive MLCK. The contractile paradigm for the calcium-calmodulin-MLCK-dependent contraction has been understood for some time. Understanding of the mechanisms responsible for the PKC-dependent contractile pathways is still evolving, and considerable confusion is derived from attempts to explain PKC-dependent contractile pathways as variants of the MLCK-dependent pathway, involving increased sensitization to calcium, or other mechanisms (29, 40, 54, 55). In the esophagus and LES, the two pathways are clearly distinct and independent of each other. Even in the LES, where both pathways exists at low calcium levels, which are insufficient to fully activate calmodulin, contraction is PKC dependent, whereas when calcium levels are sufficient to fully activate calmodulin, the activated calmodulin inhibits PKC and activates MLCK. In both tissues, ERK1, ERK2, and p38 MAPKs participate only in the PKC-dependent contractile pathway and not in the calmodulin- and MLCK-dependent pathway. In addition, the finding that combined inhibition of the ERK1, ERK2, and p38 MAPKs results in almost complete inhibition of LES tone and DG contraction, and of esophageal contraction, supports the view that PKC-dependent contraction of these muscles is almost entirely mediated through activation of these MAPKs.

In addition to a role of the ERK MAPKs, our data demonstrate a role of the p38 MAPK in PKC-mediated contraction. The contractile pathway mediated by p38 MAPK may be linked to HSP27 because HSP27 and p38 MAPK antibodies in combination caused no greater inhibition of contraction than either one alone. In a different smooth muscle preparation, it has been proposed that p38 MAPK may be activated during PKC-dependent contraction and cotranslocated with HSP27 (61). HSP27 is an actin filament-binding protein (47) that may contribute to PKC-dependent contraction in intestinal smooth muscle (61). Phosphorylation of HSP27 increases after stimulation with contractile agonists such as carbachol (43), thrombin (17), C2-ceramide and endothelin-1 (17), and cyclosporin A (8). Smooth muscle contraction is reduced by inhibition of HSP27 phosphorylation (8, 15) or by changes in the intracellular distribution of HSP27 (59).

To confirm the link between HSP27 and p38 MAPK, esophageal muscle was stimulated with ACh, then homogenized and immunoprecipitated with p38 antibody. Muscle homogenates, immunoprecipitated with a phosphorylated p38 MAPK antibody, contained increased levels of HSP27 after stimulation with ACh compared with unstimulated muscle, suggesting that ACh stimulation and consequent activation of p38 MAPK promote association of HSP27 with the phosphorylated (i.e., active) p38 MAPK. This interpretation is further supported by the finding that the time-dependent association of HSP27 and phosphorylated p38 MAPK (Fig. 11) closely mirrors the time course of p38 MAPK phosphorylation (Fig. 10). These results are consistent with reports of ANG II-induced contraction of rat vascular smooth muscle, where p38 MAPK-dependent phosphorylation of HSP27 contributes to contraction (45).

Our data suggest that contraction of esophageal circular smooth muscle is mediated by ERKs and p38 MAPKs, consistent with the finding that contraction is almost abolished by a combination of ERK and p38 MAPK inhibitors.

In addition, the actin-binding proteins calponin and caldesmon may be involved in PKC- and MAPK-dependent contraction (51). Calponin and caldesmon bind to actin and inhibit the Mg2+-ATPase of phosphorylated smooth muscle myosin, thereby preventing cross-bridge cycling and smooth muscle contraction. Actin binding and ATPase inhibition are abolished by phosphorylation of these thin-filament proteins by PKC and calcium- and calmodulin-dependent protein kinase II and restored by dephosphorylation (5). Caldesmon may mediate MAPK-dependent contraction because it is phosphorylated during contraction (3), the phosphorylation sites on caldesmon have been identified as MAPK sites (2), and a caldesmon inhibitory peptide contracts permeable smooth muscle cells (35, 51). Calponin may function as an adapter protein connecting the PKC cascade to the ERK cascade because it coimmunoprecipitates with ERK1 and PKC in ferret aorta homogenates, colocalizes in cells with ERK1 and PKC, and binds activated PKC in a gel overlay assay (46). We have reported that, in permeable cells of the esophagus, calponin and caldesmon play a role in MLCK-independent, PKC-dependent contraction. Both calponin and caldesmon inhibit DG-induced (i.e., PKC-dependent) contraction of esophageal muscle but do not affect LES contraction in response to a maximally effective concentration of ACh, which is calmodulin dependent (51).

Activation of the PKC contractile pathway may result in phosphorylation of MEK, which in turn may phosphorylate MAPK. MAPK may then phosphorylate either calponin or caldesmon or some intermediate protein, resulting in caldesmon/calponin phosphorylation (2). Caldesmon and calponin, when phosphorylated, change conformation and no longer inhibit actomyosin interaction, allowing contraction to occur. It is possible that two distinct parallel pathways may contribute to PKC-dependent contraction, one involving p38 kinase and HSP25/27 and the other one involving ERKs and calponin/caldesmon.


    ACKNOWLEDGMENTS
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-28614.

These data were presented, in part, at the American Gastroenterological Association meetings May 17–21, New Orleans, LA and May 20–23, 2001, Atlanta, GA.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. Harnett, G. I. Motility Research Laboratory, SWP5 Rhode Island Hospital and Brown Univ., 593 Eddy St., Providence RI 02903 (E-mail: Karen_Harnett{at}Brown.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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adam L, Franklin M, Raff G, and Hathaway D. Activation of mitogen-activated protein kinase in porcine carotid arteries. Circ Res 76: 183–190, 1995.[Abstract/Free Full Text]
  2. Adam L and Hathaway D. Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-Caldesmon. FEBS Lett 322: 56–60, 1993.[ISI][Medline]
  3. Adam LP, Haeberle JR, and Hathaway DR. Phosphorylation of caldesmon in arterial smooth muscle. J Biol Chem 264: 7698–7703, 1989.[Abstract/Free Full Text]
  4. Alessi DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogenactivated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489–27494, 1995.[Abstract/Free Full Text]
  5. Allen BG and Walsh MP. The biochemical basis of the regulation of smooth muscular contraction. Trends Biochem Sci 19: 362–368, 1994.[ISI][Medline]
  6. Anderson N, Maller J, Tonks N, and Sturgill T. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343: 651–653, 1990.[ISI][Medline]
  7. Babu YS, Bugg CE, and Cook WJ. X-ray diffraction studies of calmodulin. Methods Enzymol 139: 632–642, 1987.[ISI][Medline]
  8. Beall A, Epstein A, Woodrum D, and Brophy CM. Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins. Biochim Biophys Acta 1449: 41–49, 1999.[ISI][Medline]
  9. Biancani P, Billett G, Hillemeier C, Nissenshon M, Rhim BY, Sweczack S, and Behar J. Acute experimental esophagitis impairs signal transduction in cat LES circular muscle. Gastroenterology 103: 1199–1206, 1992.[ISI][Medline]
  10. Biancani P, Harnett KM, Sohn UD, Rhim BY, Behar J, Hillemeier C, and Bitar KN. Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh. Am J Physiol Gastrointest Liver Physiol 266: G767–G774, 1994.[Abstract/Free Full Text]
  11. Biancani P, Hillemeier C, Bitar KN, and Makhlouf GM. Contraction mediated by Ca2+ influx in the esophagus and by Ca2+ release in the LES. Am J Physiol Gastrointest Liver Physiol 253: G760–G766, 1987.[Abstract/Free Full Text]
  12. Biancani P, Walsh JH, and Behar J. Vasoactive intestinal polypeptide: a neurotransmitter for lower esophageal sphincter relaxation. J Clin Invest 73: 963–967, 1984.[ISI][Medline]
  13. Biancani P, Zabinski M, Kerstein M, and Behar J. Lower esophageal sphincter mechanics: anatomic and physiologic relationships of the esophagogastric junction of the cat. Gastroenterology 82: 468–475, 1982.[ISI][Medline]
  14. Bitar KN, Bradford P, Putney JW, and Makhlouf GM. Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-triphosphate in isolated gastric smooth muscle cells. J Biol Chem 261: 16591–16596, 1986.[Abstract/Free Full Text]
  15. Bitar KN, Kaminski MS, Hailat N, Cease KB, and Strahler JR. HSP27 is a mediator of sustained smooth muscle contraction in response to bombesin. Biochem Biophys Res Commun 181: 1192–1200, 1991.[ISI][Medline]
  16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[ISI][Medline]
  17. Brophy CM, Woodrum D, Dickinson M, and Beall A. Thrombin activates MAPKAP2 kinase in vascular smooth muscle. J Vasc Surg 27: 963–969, 1998.[ISI][Medline]
  18. Cao W, Chen Q, Sohn U, Kim N, Kirber M, Harnett K, Behar J, and Biancani P. Ca2+-induced contraction of cat esophagealcircular smooth muscle cells. Am J Physiol Cell Physiol 280: C980–C992, 2001.[Abstract/Free Full Text]
  19. Cobb MH and Goldsmith EJ. How MAP kinases are regulated. J Biol Chem 270: 14843–14846, 1995.[Free Full Text]
  20. Crespo P, Xu N, Simonds WF, and Gutkind JF. Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature 369: 418–420, 1994.[ISI][Medline]
  21. Della Rocca GJ, van Biesen T, Daaka Y, Luttrell DK, Luttrell LM, and Lefkowitz RJ. Ras-dependent mitogen-activated protein kinase activation by G-protein-coupled receptors. J Biol Chem 272: 19125–19132, 1997.[Abstract/Free Full Text]
  22. Dessy C, Kim I, Sougnez CL, Laporte R, and Morgan KG. A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha-adrenoceptor stimulation. Am J Physiol Cell Physiol 275: C1081–C1086, 1998.[Abstract/Free Full Text]
  23. 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]
  24. Duff JE, Berk BC, and Corson MA. Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun 188: 257–264, 1992.[ISI][Medline]
  25. Faure M, Voyno-Yasenetskaya TA, and Bourne HR. cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem 269: 7851–7854, 1994.[Abstract/Free Full Text]
  26. Freshney NW, Rawlison L, Guesdon F, Jones E, Cowley S, Hsuan J, and Saklatvala J. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78: 1039–1049, 1994.[ISI][Medline]
  27. Gerthoffer WT, Yamboliev IA, Pohl J, Haynes R, Dang S, and McHugh J. Activation of MAP kinases in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 272: L244–L252, 1997.[Abstract/Free Full Text]
  28. 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]
  29. Gong MC, Kinter MT, Somlyo AV, and Somlyo AP. Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle. J Physiol 486: 113–122, 1995.[Abstract]
  30. Gorenne I, Su X, and Moreland RS. Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery. Am J Physiol Heart Circ Physiol 275: H131–H138, 1998.[Abstract/Free Full Text]
  31. Hedges JC, Oxhorn BC, Carty M, Adam LP, Yamboliev IA, and Gerthoffer WT. Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle. Am J Physiol Cell Physiol 278: C718–C726, 2000.[Abstract/Free Full Text]
  32. Hillemeier AC, Bitar KB, Sohn UD, and Biancani P. Protein kinase C mediates spontaneous tone in the cat lower esophageal sphincter. J Pharmacol Exp Ther 277: 144–149, 1996.[Abstract]
  33. 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]
  34. Katoch SS and Moreland RS. Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery. Am J Physiol Heart Circ Physiol 269: H222–H229, 1995.[Abstract/Free Full Text]
  35. Katsuyama H, Wang C-LA, and Morgan KG. Regulation of vascular smooth muscle tone by caldesmon. J Biol Chem 267: 14555–14558, 1992.[Abstract/Free Full Text]
  36. Khalil RA, Lajoie C, Resnick M, and Morgan KG. Ca2+-independent isoforms of protein kinase C differentially translocate in smooth muscle. Am J Physiol Cell Physiol 263: C714–C719, 1992.[Abstract/Free Full Text]
  37. Khalil RA, Menice CB, Wang C-LA, and Morgan KG. Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells. Circ Res 76: 1101–1108, 1995.[Abstract/Free Full Text]
  38. Khalil RA and Morgan KG. Protein kinase C: a second E-C coupling pathway in vascular smooth muscle? News Physiol Sci 7: 10–15, 1992.[Abstract/Free Full Text]
  39. Khalil RA and Morgan KG. PKC-mediated redistribution of mitogen-activated protein kinase during smooth muscle cell activation. Am J Physiol Cell Physiol 265: C406–C411, 1993.[Abstract/Free Full Text]
  40. Kitazawa T, Masuo M, and Somlyo AP. G-protein-mediated inhibition of myosin light chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci USA 88: 9307–9310, 1991.[Abstract]
  41. Koch A, Nasuhara Y, Barnes PJ, Lindsay MA, and Giembycz MA. Extracellular signal-regulated kinase 1/2 control Ca(2+)-independent force development in histamine-stimulated bovine tracheal smooth muscle. Br J Pharmacol 131: 981–989, 2000.[Abstract/Free Full Text]
  42. Koch WJ, Hawes BE, Allen LF, and Lefkowitz RJ. Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras. Proc Natl Acad Sci USA 91: 12706–12710, 1994.[Abstract/Free Full Text]
  43. Larsen J, Yamboliev I, Weber L, and Gerthoffer W. Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle. Am J Physiol Lung Cell Mol Physiol 273: L930–L940, 1997.[Abstract/Free Full Text]
  44. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, and Landvatter SW. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739–746, 1994.[ISI][Medline]
  45. Meloche S, Landry J, Huot J, Houle F, Marceau F, and Giasson E. p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle. Am J Physiol Heart Circ Physiol 279: H741–H751, 2000.[Abstract/Free Full Text]
  46. Mennice B, Hulvershorn J, Adam LP, Wang C-LA, and Morgan KG. Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem 272: 25157–25161, 1997.[Abstract/Free Full Text]
  47. Miron T, Vancompernolle K, Vandekerckhove J, Wilchek M, and Geiger B. A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein. J Cell Biol 114: 255–261, 1991.[Abstract]
  48. Morinelli TA, Zhang LM, Newman WI, and Meier KE. Thromboxane A2/prostaglandin H2-stimulated mitogenesis of coronary artery smooth muscle cells involves activation of mitogen-activated protein kinase and S6 kinase. J Biol Chem 269: 5693–5698, 1994.[Abstract/Free Full Text]
  49. Reuter CW, Catling AD, and Weber MJ. Immune complex kinase assays for mitogen-activated protein kinase and MEK. Methods Enzymol 255: 245–256, 1995.[ISI][Medline]
  50. Rouse J, Cohen P, Trigon S, Morange M, Alonzo-Llamanzares A, Zamanillo D, Hunt T, and Nebreda A. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78: 1027–1037, 1994.[ISI][Medline]
  51. Sohn U, Cao W, Tang D, Stull J, Haeberle J, Wang C-LA, Harnett KM, Behar J, and Biancani P. Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle. Am J Physiol Gastrointest Liver Physiol 281: G467–G478, 2001.[Abstract/Free Full Text]
  52. Sohn UD, Harnett KM, De Petris G, Behar J, and Biancani P. Distinct muscarinic receptors, G-proteins, and phospholipases in esophageal and lower esophageal sphincter circular muscle. J Pharmacol Exp Ther 267: 1205–1214, 1993.[Abstract]
  53. Sohn UD, Zoukhri D, Dartt D, Sergheraert C, Harnett KM, Behar J, and Biancani P. Different PKC isozymes mediate lower esophageal sphincter (LES) tone and phasic contraction of esophageal (ESO) circular smooth muscle in the cat. Mol Pharmacol 51: 462–470, 1997.[Abstract/Free Full Text]
  54. Somlyo A. Modulation of the Ca2+ switch: by G proteins, kinases, and phosphatases. News Physiol Sci 8: 2–6, 1993.[ISI]
  55. Somlyo AP, Wu X, Walker L, and Somlyo AV. Pharmacomechanical coupling: the role of calcium, G-proteins, kinases, and phosphatases. Rev Physiol Biochem Pharmacol 134: 203–236, 1998.
  56. Stokoe D, Engel K, Campbell DG, Cohen P, and Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313: 307–313, 1992.[ISI][Medline]
  57. Van Biesen T, Hawes BE, Luttrel DK, Krueger KM, Touhara K, Porfiri E, Sakaue M, Luttrel LM, and Lefkowitz RJ. Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway. Nature 376: 781–784, 1995.[ISI][Medline]
  58. Walsh MP. Regulation of vascular smooth muscle tone. Can J Physiol Pharmacol 72: 919–936, 1994.[ISI][Medline]
  59. Wang B and Sims SM. CCK regulates nonselective cation channels in guinea pig gastric smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 274: G709–G717, 1998.[Abstract/Free Full Text]
  60. Watanabe T, Waga I, Honda Z, Kurokawa K, and Shimizu T. Prostaglandin F2{alpha} stimulates formation of p21-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via G-protein-coupled pathway. J Biol Chem 170: 8984–8990, 1995.
  61. Yamada H, Strahler J, Welsh MJ, and Bitar KN. Activation of MAP kinase and translocation with HSP27 in bombesininduced contraction of rectosigmoid smooth muscle. Am J Physiol Gastrointest Liver Physiol 269: G683–G691, 1995.[Abstract/Free Full Text]
  62. Yamboliev I, Hedges J, Mutnick J, Adam L, and Gerthoffer W. Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway. Am J Physiol Heart Circ Physiol 278: H1899–H1907, 2000.[Abstract/Free Full Text]
  63. Yousufzai SY, Gao G, and Abdel-Latif AA. Mitogen-activated protein kinase inhibitors suppress prostaglandin F(2alpha)-induced myosin-light chain phosphorylation and contraction in iris sphincter smooth muscle. Eur J Pharmacol 407: 17–26, 2000.[ISI][Medline]