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Am J Physiol Gastrointest Liver Physiol 290: G83-G95, 2006. First published September 22, 2005; doi:10.1152/ajpgi.00178.2005
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NEUROREGULATION AND MOTILITY

RhoA- and PKC-{alpha}-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells.

Suresh B. Patil and Khalil N. Bitar

Division of Pediatric, Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan

Submitted 19 April 2005 ; accepted in final form 15 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-{alpha}. Presently, we examined the cross talk between RhoA and PKC-{alpha} in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-{alpha}, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21 ± 3.52 and 42.19 ± 3.85%nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12 ± 46.60% and 290.59 ± 22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-{alpha} by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-{alpha} pathways, suggesting cross talk between RhoA and PKC-{alpha} resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.

myosin light chain; contraction; CPI-17; acetylcholine; Rho kinase; colon; phosphatase; protein kinase C; heat shock protein

DIRECT PHOSPHORYLATION of myosin light chain (MLC) by MLC kinase (MLCK) is the essential factor for Ca2+-mediated calmodulin-dependent smooth muscle contraction (1, 37). The ratio of myosin phosphatase (MLCP) to MLCK activity determines the level of MLC phosphorylation. MLC phosphorylation by MLCK promotes smooth muscle contraction, whereas MLC dephosphorylation by MLCP results in muscle relaxation (14, 51).

MLCP is composed of a 37-kDa catalytic subunit (PP1c) that is responsible for dephosphorylation of its highly specific substrate, MLC (15), and a 130-kDa regulatory myosin binding subunit (MYPT). Phosphorylation of MYPT by Rho kinase (ROCK) results in loss of ability of MLCP to dephosphorylate MLC (44). Recent studies in vitro have suggested that the small GTP-binding protein RhoA and its effector, ROCKII, play an important role in smooth muscle contraction (24, 45) and actin cytoskeleton organization (2, 13). MLCK-independent MLC phosphorylation and contraction is suggested to be mediated by RhoA (12, 38). In smooth muscle cells, RhoA/ROCKII functions by phosphorylating MYPT, thereby maintaining MLC phosphorylation.

Another mechanism for inhibition of MLCP involves a PKC-potentiated inhibitory protein of 17 kDa (CPI-17) (11). CPI-17 is a MLCP inhibitor protein whose inhibitory potency is increased by phosphorylation at Thr38 (34). CPI-17 can be phosphorylated by PKC and by ROCKII (28, 29, 35, 39). Therefore, MLCP could be regulated by both RhoA and PKC pathways.

It is not certain whether PKC and RhoA regulate MLCP independently. We have shown that in colonic smooth muscle, PKC-{alpha} and RhoA translocate and associate with each other in the particulate fraction in response to stimulation with acetylcholine (6). We have also shown that RhoA interacts directly with PKC-{alpha} in vitro (41). In human endothelial cells, activation of ROCK by RhoA requires PKC activation (17). These data indicate that there is strong interrelation between PKC-{alpha} and RhoA during agonist-induced smooth muscle contraction.

We have recently shown that preincubation of smooth muscle cells with Y-27632, a specific ROCKII inhibitor, partially inhibited contraction (~50%) concomitant with inhibition of association of ROCKII with heat shock protein (HSP)27 in the particulate fraction (41). These experiments suggest that the association and translocation of ROCKII with HSP27 is essential in maintaining sustained contraction (41). HSP27 is a member of the mammalian small HSP family. Evidence has shown that HSP27 is important in many cell functions, including smooth muscle contraction (8, 9). HSP27 colocalizes with actin filaments in cardiac (33), skeletal (4, 52), and smooth muscle (7, 21). Phosphorylation of HSP27 changes the actin cytoskeleton and actin-dependent events. Nonphosphorylated HSP27 reduced translocation of RhoA and PKC-{alpha} to the membrane during agonist-induced contraction of smooth muscle (40). It has also been suggested that translocation of ROCKII to the membrane is necessary for its activation. ROCKII has also been found to localize at the cell membrane in freshly isolated smooth muscle cells (31, 48).

MYPT has been shown to translocate to the membrane in PGF2{alpha}-induced contraction of ferret vascular smooth muscle cells, and the translocation remained sustained beyond 5 min (42). The aims of the studies were to investigate the agonist-induced possible association and translocation of MYPT with HSP27 and to examine the roles played by RhoA and PKC pathways in the regulation of MLCP activity. To examine the roles played by RhoA and PKC, we selectively inhibited ROCKII and PKCs by specific inhibitors in freshly isolated smooth muscle cells from the rabbit colon. To examine the interrelationship between RhoA and PKC-{alpha} in the regulation of MLCP activity, we used cultured smooth muscle cells transfected with negative dominant forms of either RhoA or PKC-{alpha}. The results suggest that 1) acetylcholine induced activation of both RhoA and/or PKC-{alpha} pathways, suggesting cross talk between RhoA and PKC-{alpha} resulting in phosphorylation of MYPT, inhibition of phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of MLCP activities and modulate sustained phosphorylation of MLC20.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals

Y-27632 was kindly supplied by Yoshitomi Pharmaceutical Industries (Osaka, Japan). Acetylcholine chloride was purchased from Sigma. Collagenase type II was from Worthington (Lakewood, NJ). Protein G-Sepharose was from Pharmacia Biotech (Uppsala, Sweden). Polyvinylidene difluoride (PVDF) membranes were from Bio-Rad. Enhanced chemiluminescence (ECL) detection reagents were from Amersham. Rabbit polyclonal PKC-{alpha} (1:100) was from Panvera/Invitrogen. Rabbit polyclonal phospho-PKC-{alpha} antibody was from Upstate. Mouse monoclonal anti-HSP27 (1:2,000) has been previously described (5, 20). Rabbit polyclonal CPI-17, phospho-specific CPI-17, and Thr696 phospho-specific MYPT antibodies were from Upstate. Peroxidase-conjugated anti-mouse secondary antibody or anti-rabbit secondary antibody was purchased from Bio-Rad. Human PKC-{alpha} negative (K368R) constructs were a gift from Dr. J. W. Soh (University of North Korea). Negative RhoA (R19N) constructs were a gift from Dr. Naranatt (University of Kansas Medical Center). Phosphatase activity assay kits (catalog no. E-6646) were from Invitrogen. The protein kinase activity assay kit (catalog no. EKS-420A) was purchased from Stressgen Bio Reagents Tech (San Diego, CA).

Isolation of Smooth Muscle Cells from Rabbit Colon

Smooth muscle cells from the rabbit rectosigmoid were isolated as described previously (20). Briefly, the circular smooth muscle layer from the distal colon from New Zealand rabbits was removed by sharp dissection. A 5-cm length of the rectosigmoid, orad to the junction, was dissected and digested with collagenase to yield isolated smooth muscle cells. The tissue was incubated for two successive 1-h periods at 31°C in 15 ml HEPES (pH 7.4) with (in mM) 115 NaCl, 5.7 KCl, 2.0 KH2PO4, 24.6 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 5.6 glucose containing 0.1% (wt/vol) collagenase (150 U/mg, Worthington CLS type II), 0.01 (wt/vol) soybean trypsin inhibitor, and 0.184 (wt/vol) DMEM. After the end of the second enzymatic incubation period, the medium was filtered through 500-µm Nitex. The partially digested tissue left on the filter was washed four times with 10 ml of collagenase-free buffer solution. Tissue was then transferred into 15 ml of fresh collagenase-free buffer solution, and cells were gently dispersed. After a hemocytometric cell count, the harvested cells were resuspended in collagenase-free HEPES buffer (pH 7.4). Each rectosigmoid yielded 10–20 x 106 cells.

Cells and Transfections

Smooth muscle cells isolated from the rabbit colon as described above were plated and cultured for two passages on 100-mm Corning dishes. Cells were transfected with vector (pcDNA-3.1) containing dominant negative RhoA (R19N; gift from Dr. Naranatt, University of Kansas Medical Center) or dominant negative PKC-{alpha} (K368R; generous gift from Dr. J. W. Soh, University of North Korea). Stable transfections were performed using lipofectamine as per the manufacturer’s instructions (Invitrogen/GIBCO Life Technologies). For each transfection, 1 µg DNA, 2 µl plus reagent, and 97 µl DMEM were used, followed by a 15-min incubation with 1 µl lipofectamine in 99 µl DMEM. Complexes were added to cells for 3 h, followed by the addition of 1 ml DMEM + 5% heat-inactivated FBS and penicillin-streptomycin. After 48 h, transfected cells were cultured in DMEM containing penicillin-streptomycin and gentamycin. After the cells attained confluence, they were subpassed and used for the experiments.

Preparation of Particulate Fractions

Freshly isolated smooth muscle cells were counted on a hemocytometer and diluted with HEPES buffer as needed. Cells were then treated with the agonists and/or antagonist for the indicated periods. After the treatment, cells were washed twice with buffer A [containing (in mM) 150 NaCl, 16 Na2HPO4, 4 NaH2PO4, and 1 sodium orthovanadate, pH 7.4] and sonicated in buffer B [containing (in mM) 1 Na3VO4, 1 NaF, 2 phenylmethylsulfonyl fluoride, 5 EDTA, 1 Na4MoO4, 1 dithiothreitol, 20 NaH2PO4, 20 Na2HPO4, and 20 Na4P2O7·10H2O with 50 µl/ml DNase-RNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain-HCl (pH 7.4), 0.08 mg/ml soybean trypsin inhibitor (STI), 60 µg/ml phospho-amidon, and 5 mg/ml Pefbloc]. Cell sonicates were centrifuged at 100,000 g for 60 min. The supernatant material from the high-speed centrifugation was collected as the soluble cytosolic fraction. The pellet material was resuspended by sonication twice for 30 s in buffer B plus 1% Triton X-100 and collected as the particulate fraction. The protein content was determined using Bio-Rad protein assay reagent.

Immunoprecipitation

Each sample (500 µg protein) obtained as described above was subjected to anti-MYPT antibody, anti PKC antibody, or anti-HSP27 antibody overnight at a ratio of 1:250. Samples were then mixed with protein G-Sepharose beads and rocked for 2 h. Beads were washed twice with Tris-buffered saline and boiled in 2x Laemmli sample buffer with 2-mercaptoethanol.

SDS-PAGE and Electrophoretic Transfer

For one-dimensional SDS-PAGE, samples were mixed in an equal volume of 2x sample buffer [50 mM Tris, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.1% (wt/vol) bromophenol blue, pH 6.8]. Proteins were separated by 12.5% or 15% SDS-PAGE and transferred onto nitrocellulose or PVDF membranes. Proteins were identified by chemiluminescence.

Western Immunoblot Analysis of Immunoprecipitates

Immunoprecipitates were size separated by SDS-PAGE and electrophoretically transferred to PVDF membranes. Immunoblotting was performed using antibodies specific to each of the proteins tested, namely, rabbit polyclonal anti-phospho-MYPT antibody (1:1,000), polyclonal anti-phospho-CPI-17 (1:1,000) antibody, monoclonal anti-HSP27 antibody (1:5,000), and polyclonal anti-phospho-PKC-{alpha} antibody (1:1,000) as primary antibody. The membrane was reacted with peroxidase-conjugated goat anti-mouse IgG antibody or goat anti-rabbit IgG as the case may be (1:2,500 dilution) for 1 h at 24°C. Enzymes on the membrane were detected with luminescent substrates. As a negative control, blots were incubated in the secondary antibody only.

PKC Assay

PKC assays were performed according to the manufacturer’s instructions (catalog no. EKS-420A, Stressgen Bioreagents). Briefly, cell lysates were immunoprecipitated with pan PKC antibody (catalog no. SC-10800, Santa Cruz Biotechnology; Santa Cruz, CA) and incubated in microtiter wells precoated with PKC substrate (CREB peptide), and the reactions were initiated by adding 2 µM ATP to each well. Samples were incubated for 90 min at 30°C. Reactions were stopped by emptying the contents of each well. A phospho-specific substrate rabbit polyclonal antibody was added to each well, and the mixture was further incubated at room temperature for 60 min. Microtiter wells were washed three times with 100 µl PBS (pH 7.4). Wells were then incubated with anti-rabbit horseradish peroxidase-conjugated IgG and incubated at room temperature for 45 min. At the end of the incubation period, wells were washed three times with PBS, tetramethylbenzidine substrate was added to each well, and samples were further incubated at room temperature for 60 min. Samples were carefully observed for color development, and reactions were stopped by adding Acid Stop solution. Absorbance at 450 nm was measured using a Bio-Rad microtiter plate reader. Activities of PKC were determined as the ratio between absolute absorbance at 450 nm and the quantity of the cell lysate used for the immunoprecipitation. Activities of the experimental samples were expressed as percent changes from control.

MLCP Assay

MLCP activity of in the cell lysates was determined using lysates immunoprecipitated with anti-MYPT antibody and using them for quantization of Pi released using 2-amino-6-mercapto-7-methypurine riboside (MESG). In the presence of Pi, the substrate MESG is converted enzymatically by purine nucleoside to ribose-1-phosphate and 2-amino-6-mercapto-7-methy purine, which results in a spectrophotometric shift from 330 to 360 nm. For data analysis, the values determined for the no-substrate control were subsubtracted from the substrate control reaction and expressed as nmoles of phosphate released. To confirm the amount of catalytic subunit present, immunoprecipitates of whole cell lysates were subjected to SDS-PAGE followed by Western blot analysis with anti-PP1c antibody at a dilution of 1:2,000 (Upstate; Fig. 1B).



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  		Fig. 1. A: acetylcholine (ACh)-induced myosin phosphatase (MLCP) activity in rabbit colon smooth muscle cells. Cell lysates were immunoprecipitated (IP) with an antibody against the 130-kDa regulatory myosin binding subunit (MYPT) and assayed for Pi release from 2-amino-6-mercapto-7-methypurine riboside (MESG) substrate as detailed in MATERIALS AND METHODS. Stimulation of cells with the contractile agonist ACh (10–7 M) resulted in a significant and sustained decrease in MLCP activity (nM ATPase units/ml expressed as a percentage of controls) in smooth muscle cell lysates (66.21 ± 3.52% and 42.19 ± 3.85%, respectively, at 30 s and 4 min, n = 3, *P < 0.05). Preincubation of cells with the Rho kinase (ROCK)II inhibitor Y-27632 inhibited the ACh-induced (10–7 M) decrease in MLCP activity (89.52 ± 2.12% and 89.25 ± 6.27% at 30 s and 4 min, respectively, n = 3; in nM ATPase units/ml expressed as a percentage of controls). Preincubation of cells with the PKC inhibitor calphostin C inhibited the ACh-induced (10–7 M) decrease in MLCP activity (93.226 ± 4.23% and 109.79 ± 5.25%, respectively, at 30 s and 4 min, n = 3) compared with control, suggesting that there is a decrease in ACh-induced MLCP activity in rabbit colon smooth muscle cells. B: Western immunoblot (IB) showing equal amounts of PP1c immunoprecipitated with anti-MYPT antibody in the control state and in response to ACh.

 
Measurement of MLC20 Phosphorylation

MLC20 phosphorylation was measured as described previously (41). Briefly, freshly isolated cells were washed with PBS (pH 7.4) three times, and cells were suspended in isoelectric focusing (IEF) buffer containing 4% CHAPS, 7 M urea, 2 M thiourea, 10 mg/ml dithiothreitol, and 1% carrier ampholytes (pH 3–10) (Pharmalytes, Amersham-Pharmacia Biotech). The resulting samples were centrifuged at 4,000 rpm, and the insoluble material was discarded. The lysates were mixed with sample loading buffer (50% glycerol) and applied onto mini-IEF slab gels that contained a mixture of 1:4 ampholytes of pH 3–10 and pH 5–8. Samples were run at 10 V for 15 min, 200 V for 15 min, and overnight (~10 h) at 450 V. They were then electrophoretically transferred onto PVDF membranes. The membranes were Western blotted with a monoclonal anti-MLC antibody (1:2,000) followed by peroxidase-conjugated anti-mouse secondary antibody and detected with luminescent (ECL) substrates.

Data Analysis

Bands were quantified using a densitometer (model GS-700, Bio-Rad), and band volumes (absorbance units x mm2) were calculated. The volumes were then expressed as percent changes over the control. All observations were done with at least three separate experiments consisting of smooth muscle cells isolated from different animals. The difference between control and experimental values were compared using Student’s t-test and considered significant at P < 0.05. Blotting data are within the linear range of detection for each antibody used.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of Acetylcholine on Total MLCP Activity in Rabbit Colon Smooth Muscle Cells

Recently, we (41) have shown that in rabbit colon smooth muscle cells, RhoA/ROCKII association with HSP27 and translocation to the particulate fraction is crucial for maintenance of sustained contraction. There are reports suggesting that phosphatase inhibition could be a key event in signaling during smooth muscle contraction (47). We examined the changes in MLCP activities of smooth muscle cells in response to a contractile agonist. Cell lysates were immunoprecipitated with anti-MYPT antibody and analyzed for Pi released from the MESG substrate. Stimulation with the contractile agonist acetylcholine (10–7 M) resulted in a significant and sustained decrease in the MLCP activity, as indicated by Pi release, in smooth muscle cell lysates (66.21 ± 3.52 and 42.19 ± 3.85 %nM ATP/ml lysate immunoprecipitated, respectively, at 30 s and 4 min, n = 3, P < 0.05 compared with control; Fig. 1A). Preincubation of the cells with the ROCK inhibitor Y-27632 (10 µM) for 20 min inhibited acetylcholine-induced decreases (89.52 ± 2.12 and 89.25 ± 6.27 %nM ATP/ml lysate immunoprecipitated, respectively, at 30 s and 4 min, n = 3, P < 0.05). Preincubation of cells with calphostin C also inhibited acetylcholine-induced changes in MLCP activity (93.226 ± 4.23 and 109.79 ± 5.25 %nM ATP/ml lysate immunoprecipitated, respectively, at 30 s and 4 min; Fig. 1A). Western blot analysis of the samples immunoprecipitated indicated that both control and acetylcholine-treated samples contained the same amount of PP1c (Fig. 1B). The results indicate that acetylcholine induced a decrease in MLCP activity in rabbit colon smooth muscle cells treated with acetylcholine (10–7 M). ROCK inhibitor and PKC inhibitors inhibited acetylcholine-induced decreases in MLCP activity, suggesting that the inhibition of MLCP is both PKC and RhoA dependent.

Effect of Acetylcholine on PKC Activity in Rabbit Colon Smooth Muscle Cells

It was also suggested that the key events in cellular signal transduction are changes in kinase activities. We have shown that several kinases including ERK, MAPKAP and SRC are activated in smooth muscle cells (22). It is generally accepted that several signaling events are initiated by PKC. Therefore, lysates from freshly isolated rabbit colon smooth muscle cells treated with acetylcholine were used to quantify agonist-induced changes in PKC activity. Cell lysates were immunoprecipitated with anti-PKC antibody, which recognizes all isozymes of PKCs, and then analyzed in vitro for PKC activity using CREB peptide. Smooth muscle cells treated with acetylcholine for 30 s or 4 min showed an increase in PKC activity (298.12 ± 46.60 and 290.59 ± 22.07 %increase in PKC activity/mg protein immunoprecipitated, n = 3, P ≤ 0.05; Fig. 2). Preincubation of cells with a specific RhoA kinase inhibitor, Y-27632 (10 µM), partially inhibited acetylcholine-induced changes in PKC activity (142.02 ± 8.74 and 138.11 ± 8.22 %increase in PKC activity/mg protein immunoprecipitated compared with controls, n = 3). Preincubation of cells with the PKC inhibitor calphostin C (10 µm) totally inhibited acetylcholine-induced changes in PKC activity (105.734 ± 12.52 and 76.219 ± 17.06 %increase in PKC activity/mg protein immunoprecipitated at 30 s and 4 min, respectively). These results suggest that PKC activities are partially independent of RhoA pathways during acetylcholine-induced smooth muscle contraction.



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  		Fig. 2. ACh-induced PKC activity in rabbit colon smooth muscle cells. Cell lysates from freshly isolated rabbit colon smooth muscle cells were immunoprecipitated with anti-PKC antibody as described in MATERIALS AND METHODS. The immunoprecipitates were analyzed in vitro for PKC activities using CREB peptide substrate. Smooth muscle cells treated with ACh for 30 s or 4 min showed increased PKC activity (298.12 ± 46.60 and 290.59 ± 22.07 %increase in PKC activity/mg protein immunoprecipitated, n = 3,* P < 0.05). Preincubation of cells with the specific ROCK inhibitor Y-27632 (10 µM) partially inhibited ACh-induced changes in PKC activity (142.02 ± 8.74% and 138.11 ± 8.22% compared with controls, n = 3, P < 0.06). Preincubation of cells with the PKC inhibitor calphostin C (10 µM) totally inhibited ACh-induced changes in PKC activity (105.73 ± 12.52 and 76.21 ± 17.06 %/mg protein at 30 s and 4 min, respectively, n = 3, P < 0.05).

 
Effect of Acetylcholine on Phosphorylation of PKC-{alpha} in Rabbit Colon Smooth Muscle Cells

Our data indicate that in smooth muscle cells of the colon, acetylcholine induces changes in activity of both kinase(s) and phosphatase(s). It is not certain whether changes in kinase and phosphatase activities involve activation and phosphorylation of PKC and RhoA signaling pathways individually. Therefore, experiments were conducted using specific inhibitors of PKC (calphostin C) and ROCKII (Y-27632).

Freshly isolated rabbit colon cells were stimulated with acetylcholine (10–7 M) for 30 s or 4 min in the presence or absence of Y-27632 or calphostin C. Cell lysates were Western blotted with anti-phospho-PKC-{alpha} antibody (Fig. 3). Acetylcholine (10–7 M) induced a significant and sustained increase in the phosphorylation of PKC-{alpha} in smooth muscle cells at 30 s and at 4 min (132.47 ± 12.62% and 138.18 ± 11.42%, P ≤ 0.05, n = 3, at 30 s and 4 min, respectively) compared with control. Preincubation of the cells with the ROCK inhibitor Y-27632 (10 µM) for 20 min inhibited the acetylcholine-induced increase (101.10 ± 2.5% and 103.11 ± 0.98% at 30 s and 4 min, respectively, n = 3) in the phosphorylation of PKC-{alpha}. Similarly, calphostin C inhibited acetylcholine-induced phosphorylation of PKC-{alpha} (83.82 ± 13.7% and 80.93 ± 13.06%, respectively, at 30 s and 4 min, n = 3). These data suggest that acetylcholine-induced phosphorylation of PKC-{alpha} may depend on RhoA activity as well. To examine whether the activities of RhoA and PKC-{alpha} would affect phosphorylation of MYPT independently, we examined phosphorylation of MYPT both in the presence of PKC and of RhoA/ROCKII inhibitors.



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  		Fig. 3. A: ACh-induced phosphorylation of PKC-{alpha} in rabbit colon smooth muscle cells. Particulate fractions from smooth muscle cells were immunoprecipitated with anti-PKC antibody and further subjected to SDS-PAGE followed by Western blot analysis with anti-phospho-PKC antibody. Stimulation with the contractile agonist ACh (10–7 M) resulted in a significant and sustained increase in phosphorylation of PKC-{alpha} in the particulate fraction of smooth muscle cells (132.47 ± 12.62% and 138.18 ± 11.42% at 30 s and 4 min, respectively, n = 3, *P < 0.05) compared with the control. Preincubation of the cells with the ROCK inhibitor Y-27632 (10 µM) for 20 min inhibited the ACh-induced increase in PKC-{alpha} phosphorylation (101.10 ± 2.5% and 103.11 ± 0.98% at 30 s and 4 min, respectively, n = 3, P < 0.05 compared with control). Preincubation of cells with calphostin C, a PKC inhibitor, significantly inhibited ACh-induced phosphorylation of PKC-{alpha} (83.82 ± 13.70% and 80.93 ± 13.06%, respectively, at 30 s and 4 min, n = 3), indicating a possible interdependence between RhoA and PKCs modulating phosphorylation of PKC-{alpha}. B: representative blot indicating equal loading and distribution of actin in the samples tested. Particulate fractions from smooth muscle cells were subjected to Western blot analysis with either anti-phospho-MYPT (Thr696) antibody, anti-phospho-PKC-{alpha} antibody, anti-heat shock protein (HSP)27 antibody, or anti-phospho-CPI-17 antibody. The blots were striped and reprobed with anti-actin antibody to demonstrate equal loading and distribution of actin in the samples tested.

 
Effect of Acetylcholine on Thr696 Phosphorylation of MYPT in Rabbit Colon Smooth Muscle Cells

There is evidence suggesting that activation of RhoA/ROCKII results in phosphorylation of MYPT at Thr696 (10, 23). We thus tested the effect of acetylcholine on MYPT phosphorylation. Acetylcholine (10–7 M) induced a significant and sustained increase in the phosphorylation of MYPT in freshly isolated rabbit colon smooth muscle cells (129.99 ± 12.74% and 162.41 ± 12.76%, respectively, at 30 s and 4 min, n = 3, P < 0.01) compared with control (Fig. 4). To test whether the observed increase in MYPT phosphorylation was RhoA/ROCKII mediated, cells were preincubated with the specific ROCK inhibitor Y-27632 (10 µM) for 20 min and then further stimulated with acetylcholine. Particulate fractions from cultured smooth muscle cells were subjected to SDS-PAGE followed by Western blot analysis with anti-phospho-MYPT (Thr696) antibody. Y-27632 inhibited acetylcholine-induced increases (105.29 ± 1.06% and 101.00 ± 5.90% at 30 s and 4 min, respectively, n = 3 compared with control) in the phosphorylation of MYPT. Preincubation of cells with the PKC inhibitor calphostin C also inhibited MYPT phosphorylation (98.36 ± 0.51% and 101.14 ± 2.78% at 30 s and 4 min, respectively). These results suggest that acetylcholine-induced phosphorylation of MYPT at Thr696 is regulated both by RhoA/ROCKII and PKC pathways.



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  		Fig. 4. ACh-induced phosphorylation of MYPT in rabbit colon smooth muscle cells. Particulate fractions from smooth muscle cells were subjected to SDS-PAGE followed by Western blot analysis with anti-phospho-MYPT (Thr696) antibody. Stimulation of rabbit colon smooth muscle cells with the contractile agonist ACh (10–7 M) resulted in a significant and sustained increase in the phosphorylation of MYPT (129.99 ± 12.74% and 162.41 ± 12.76% at 30 s and 4 min, respectively, n = 3, *P < 0.01) compared with control. Preincubation of the cells with the ROCK inhibitor Y-27632 (10 µM) for 20 min inhibited the ACh-induced increase (105.29 ± 1.06% and 101.00 ± 5.90% at 30 s and 4 min, respectively, n = 3) compared with control in the phosphorylation of MYPT. Preincubation of the cells with the PKC inhibitor calphostin C (10 µM) also inhibited the ACh-induced changes in MYPT phosphorylation (98.36 ± 0.51% and 101.14 ± 2.78% at 30 s and 4 min, respectively, n = 3). The results suggest that phosphorylation of MYPT is induced by both PKC and RhoA/ROCKII pathways.

 
Effect of Acetylcholine on Association and Translocation of Phospho-MYPT with HSP27

We have recently shown that acetylcholine induces translocation and association of ROCKII and HSP27 in the particulate fraction (41). To examine whether HSP27 and MYPT translocate and associate, particulate fractions from freshly isolated rabbit colon smooth muscle cells stimulated with acetylcholine (10–7 M) were immunoprecipitated with anti-phospho-MYPT (Thr696) antibody and further subjected to Western blot analysis using anti-HSP27 antibody. Acetylcholine induced a significant and sustained increase in the association of phospho-MYPT (Thr696) with HSP27 in the particulate fraction (179.62 ± 9.86% and 203.48 ± 120.85%, respectively, at 30 s and 4 min, n = 3, P < 0.01) compared with control (Fig. 5), indicating that HSP27 associated with phosphorylated MYPT and the ROCKII-mediated phosphorylation of MYPT was a membrane-bound event. Preincubation of the cells with the ROCK inhibitor Y-27632 (10 µM) for 20 min inhibited acetylcholine-induced increases in the association of phosphorylated MYPT with HSP27 (66.72 ± 4.47% and 43.21 ± 2.81%, respectively, at 30 s and 4 min) compared with control. Preincubation of cells with the PKC inhibitor calphostin C inhibited acetylcholine-induced increases in the association of phospho-MYPT with HSP27 (55.12 ± 1.52% and 61.09 ± 1.09% at 4 min, respectively, n = 4). These results suggest that the association of HSP27 with phosphorylated MYPT during acetylcholine-induced smooth muscle contraction is possibly a cytoskeleton-bound event that is regulated both by RhoA/ROCKII and PKC pathways.



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  		Fig. 5. ACh-induced association of phosphorylated MYPT with HSP27 in rabbit colon smooth muscle cells. Particulate fractions from smooth muscle cells were immunoprecipitated with anti-phospho-MYPT (Thr696) antibody and further subjected to SDS-PAGE followed by Western blot analysis with anti-HSP27 antibody. Stimulation with ACh (10–7 M) resulted in a significant and sustained increase in the association of phospho-MYPT with HSP27 in the particulate fraction (179.62 ± 9.86% and 203.48 ± 120.85% at 30 s and 4 min, respectively, n = 3, *P < 0.01) compared with control. Preincubation of the cells with the ROCK inhibitor Y-27632 (10 µM) for 20 min inhibited the ACh-induced increase (55.12 ± 1.52% and 61.09 ± 1.09% at 30 s and 4 min, respectively, n = 4) compared with control in the association of phospho-MYPT with HSP27 in the particulate fraction. Preincubation of cells with the PKC inhibitor calphostin C inhibited ACh-induced increases in the association of MYPT with HSP27 (66.72 ± 4.47% and 43.21 ± 2.81%, respectively, at 30 s and 4 min). These results suggest that association of phosphorylated MYPT (Thr696) with HSP27 is in the particulate fraction and is regulated both by PKC and RhoA/ROCKII pathways.

 
Effect of Acetylcholine on CPI-17 Phosphorylation in Rabbit Colon Smooth Muscle Cells

Reports suggest that MYPT phosphorylation is also regulated by CPI-17, a PKC-regulated molecule (32). To examine whether MYPT phosphorylation is affected by changes in CPI-17 phosphorylation, freshly isolated smooth muscle cells from the rabbit colon were stimulated with acetylcholine for 30 s and 4 min in the presence or absence of either calphostin C or Y-27632. Lysates were subjected to SDS-PAGE followed by Western blot analysis against anti-phospho-CPI-17 antibody. Acetylcholine induced an increase in CPI-17 phosphorylation (201.13 ± 31.86% and 166.89 ± 23.52% at 30 s and 4 min, respectively, n = 3, P < 0.01; Fig. 6). Preincubation of cells with Y-27632 greatly inhibited CPI-17 phosphorylation (113.89 ± 2.94% and 121.52 ± 4.14% at 30 s and 4 min, respectively, P < 0.03). Preincubation of cells with the PKC inhibitor calphostin C totally inhibited acetylcholine-induced increases in CPI-17 phosphorylation (90.52 ± 4.97% and 76.94 ± 9.26% at 30 s and 4 min, respectively, n = 3). These results suggest that both calphostin C and Y-27632 affect phosphorylation of CPI-17 during acetylcholine-induced smooth muscle contraction.



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  		Fig. 6. ACh-induced phosphorylation of CPI-17 in rabbit colon smooth muscle cells. Particulate fractions from smooth muscle cells were subjected to SDS-PAGE followed by Western blot analysis with anti-phospho-CPI-17 antibody. Stimulation of rabbit colon smooth muscle cells with the contractile agonist ACh (10–7 M) resulted in a significant and sustained increase in the phosphorylation of CPI-17 (201.13 ± 31.86% and 166.89 ± 23.52% at 30 s and 4 min, respectively, n = 3, *P < 0.01) compared with the control. Preincubation of the cells with the ROCK inhibitor Y-27632 (10 µM) for 20 min partially inhibited the ACh-induced increase (113.89 ± 2.95% and 121.52 ± 4.14% at 30 s and 4 min, respectively, n = 3) compared with control in the phosphorylation of CPI-17. Preincubation of cells with calphostin C, a PKC inhibitor, significantly inhibited ACh-induced phosphorylation of CPI-17 (90.52 ± 4.97% and 76.94 ± 9.26% at 30 s and 4 min, respectively, n = 3), suggesting that phosphorylation of CPI-17 is mainly regulated by the PKC pathway.

 
Effect of Expression of Negative Constructs of RhoA and PKC-{alpha} on MLCP Activity, PKC Activity, Phosphorylation of MYPT, CPI-17, and MLC20 in Cultured Smooth Muscle Cells

Effect of acetylcholine on MLCP activity in cultured smooth muscle cells transfected with negative dominant RhoA or dominant negative PKC-{alpha} constructs. Our data indicate that in smooth muscle cells of the colon, acetylcholine induces specific activation of both signal transduction pathways involving PKC and RhoA, resulting in phosphorylation, translocation of MYPT, and its association with HSP27 and also in specific phosphorylation of CPI-17, a substrate of PKC that also regulates MLCP by phosphorylating the PP1c {delta}-subunit at Ser38. However, it is not certain whether the specific isozyme PKC-{alpha} is involved in the regulation of CPI-17 and MLCP. Furthermore, Y-27632 inhibits ROCKII, which is downstream of RhoA. Whether inactivation of RhoA has any role in the regulation of MYPT is not certain. Therefore, smooth muscle cells transfected with mutant cDNA constructs that inhibit activation of RhoA (dominant negative RhoA n19) or PKC-{alpha} (dominant negative PKC K to R) were used to examine the pathways that lead to the regulation of MLCP.

Normal smooth muscle cells in culture and cells transfected with negative dominant RhoA were treated with acetylcholine for 30 s and 4 min. Lysates were immunoprecipitated with anti-MYPT antibody and analyzed for MLCP activity as described in MATERIALS AND METHODS. Similar to freshly isolated smooth muscle cells, acetylcholine induced a decrease in MLCP activity at 30 s and 4 min in normal cultured smooth muscle cells (68.06 ± 0.78% and 65.93 ± 1.4% at 30 s and 4 min, respectively, compared with untreated controls, n = 3, P < 0.05; Fig. 7). Acetylcholine-induced decreases were inhibited in cells transfected with negative dominant RhoA constructs (97.49 ± 0.77% and 95.85 ± 1.87%, respectively, at 30 s and 4 min compared with controls, n = 3). The inhibition of MLCP activity exerted by Y-27632 was of the same magnitude compared with the cultured cells transfected with negative dominant RhoA construct. This validates the use of negative dominant RhoA cells to further investigate MLCP regulatory pathways. Acetylcholine-induced decreases were also inhibited in cells transfected with dominant negative PKC-{alpha} constructs compared with untransfected controls (94.28 ± 1.43% and 97.03 ± 0.88%, respectively, at 30 s and 4 min, n = 3). These results suggest that both PKC-{alpha} and RhoA pathways are involved in the regulation of MLCP activity during acetylcholine-induced smooth muscle contraction.



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  		Fig. 7. ACh-induced MLCP activity in rabbit colon smooth muscle cells transfected with either negative RhoA or negative PKC-{alpha}. Cultured cells either transfected or untransfected with negative dominant RhoA (–ve RhoA) and negative dominant PKC-{alpha} (–ve PKC) constructs were treated with ACh (10–7 M) for 30 s and 4 min. Lysates were immunoprecipitated with anti-MYPT antibody and then analyzed for Pi release as described in MATERIALS AND METHODS. Released phosphate was converted to nM/ml and is represented here as %activity compared with control. ACh induced a decrease in the MLCP activity in normal cultured smooth muscle cells (68.06 ± 0.78% and 65.93 ± 1.4% at 30 s and 4 min, respectively, n = 3, *P < 0.05). In cells transfected with negative dominant RhoA, ACh did not induce any decreases in MLCP activity (97.49 ± 0.77% and 95.85 ± 1.87% at 30 s and 4 min, respectively, n = 3). In cells transfected with negative PKC-{alpha}, ACh-induced deceases in MLCP activity were inhibited (94.28 ± 1.43% and 97.03 ± 0.88% at 30 s and 4 min, respectively, n = 3). The results in cultured cells corroborate those obtained from freshly isolated cells (see Fig. 1). The results suggest that both RhoA and PKC-{alpha} mediate ACh-induced decreases in MLCP activity.

 
Effect of acetylcholine on PKC activity in cultured smooth muscle cells transfected with negative dominant RhoA or dominant negative PKC-{alpha} constructs. Cultured smooth muscle cells transfected with negative dominant RhoA were treated with acetylcholine for 30 s and 4 min. Cell lysates were assayed for PKC activities as described in MATERIALS AND METHODS. Stimulation of cells with acetylcholine induced an increase in PKC activity in normal cultured cells (148.93 ± 5.32 and 146.69 ± 5.17 %increase in PKC activity/mg protein immunoprecipitated at 30 s and 4 min, respectively; Fig. 8). Transfection with negative dominant RhoA did not affect acetylcholine-induced PKC activities (132.41 ± 1.81 and 131.41 ± 2.6 %/mg protein immunoprecipitated, respectively, at 30 s and 4 min, n = 3, P < 0.05). Acetylcholine-induced increases in PKC activity in cells transfected with negative dominant PKC-{alpha} were lower by ~30% at 30 s and 4 min (117.94 ± 2.78 and 120.19 ± 1.56 %/mg protein immunoprecipitated at 30 s and 4 min, respectively, n = 3, P < 0.05). These results suggest that PKC-{alpha} contributes ~30% to total PKC activity in smooth muscle cells. Results also suggest that inhibition of RhoA does not alter PKC activities in smooth muscle cells.



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  		Fig. 8. ACh-induced PKC activity in rabbit colon smooth muscle cells transfected with either negative RhoA or negative PKC-{alpha}. Cultured smooth muscle cells transfected with either negative dominant PKC-{alpha} or RhoA were treated with ACh for 30 s and for 4 min. Cell lysates were assayed for PKC activities as described in MATERIALS AND METHODS. Stimulation of cells with ACh induced an increase in PKC activity in normal cultured cells (148.93 ± 5.32 and 146.69 ± 5.17 %increase in PKC activity/mg protein, n = 3, *P < 0.05). In cells transfected with negative dominant RhoA, no changes in PKC activity were observed (132.41 ± 1.81 and 131.41 ± 2.6 %increase in PKC activity/mg protein, respectively, at 30 s and 4 min, n = 3, *P < 0.05), suggesting that PKC activities are independent of RhoA activity. In cells transfected with negative PKC-{alpha} constructs, ACh-induced PKC activity was only partially inhibited (117.94 ± 2.78 and 120.19 ± 1.56 %/mg protein immunoprecipitated at 30 s and 4 min, respectively, n = 3, P < 0.05). Results confirm that PKC-{alpha} contributes to only ~30% of the total PKC activity during ACh-induced smooth muscle contraction.

 
Effect of acetylcholine on phosphorylation of MYPT in cultured smooth muscle cells transfected with negative dominant RhoA or dominant negative PKC-{alpha} constructs. Particulate fractions from smooth muscle cells were subjected to SDS-PAGE followed by Western blot analysis with anti-phospho-MYPT (Thr696) antibody. Cultured rabbit colon smooth muscle cells stimulated with acetylcholine (10–7 M) showed a significant and sustained increase in the phosphorylation of MYPT (157.93 ± 19.73% and 153.85 ± 14.31% at 30 s and 4 min, respectively, compared with control, n = 5, P < 0.05; Fig. 9). In cells transfected with negative dominant RhoA, acetylcholine failed to induce MYPT phosphorylation (68.80 ± 9.65% and 67.02 ± 8.02%, respectively, at 30 s and 4 min, n = 3). In cells transfected with negative dominant PKC-{alpha}, acetylcholine also failed to induce MYPT phosphorylation (65.31 ± 22.39% and 78.52 ± 15.38%, respectively, at 30 s and 4 min compared with control, n = 3). These results suggest that both PKC-{alpha} and RhoA pathways are involved in phosphorylation of MYPT during acetylcholine-induced smooth muscle contraction and confirm the results obtained using the respective inhibitors (Y-27632 and calphostin C).



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  		Fig. 9. Acetylcholine-induced MYPT phosphorylation in rabbit colon smooth muscle cells transfected with negative RhoA or negative PKC-{alpha}. Particulate fractions from smooth muscle cells were subjected to SDS-PAGE followed by Western blot analysis with anti-phospho-MYPT (Thr696) antibody. Stimulation of rabbit cultured smooth muscle cells with ACh (10–7 M) resulted in a significant and sustained increase in the phosphorylation of MYPT (157.93 ± 19.73% and 153.85 ± 14.31% at 30 s and 4 min, respectively, compared with control, n = 5, P < 0.05). In cells transfected with negative dominant RhoA, ACh failed to induce MYPT phosphorylation (68.80 ± 9.65% and 67.02 ± 8.02%, respectively, at 30 s and 4 min, n = 3). In cells transfected with negative dominant PKC-{alpha}, ACh failed to induce MYPT phosphorylation (65.31 ± 22.39% and 78.52 ± 15.38%, respectively, at 30 s and 4 min compared with control, n = 3), suggesting that both RhoA and PKC{alpha} pathways converge to phosphorylate MYPT. Note the diminished levels of expression of MYPT in the cells transfected with negative RhoA or negative PKC-{alpha}.

 
Effect of acetylcholine on phosphorylation of CPI-17 cultured smooth muscle cells transfected with dominant negative RhoA or dominant negative PKC-{alpha}. Particulate fractions from cultured smooth muscle cells were subjected to SDS-PAGE followed by Western blot analysis with anti-CPI-17 antibody. Stimulation of rabbit cultured smooth muscle cells with acetylcholine (10–7 M) resulted in a significant and sustained increase in the phosphorylation of CPI-17 (158.54 ± 20.16% and 150.07 ± 16.72% at 30 s and 4 min, respectively, n = 6, P < 0.05; Fig. 10). In cells transfected with negative dominant RhoA, acetylcholine failed to induce CPI-17 phosphorylation (76.12 ± 21.66% and 64.15 ± 21.47%, respectively, at 30 s and 4 min compared with control, n = 3). In cells transfected with negative dominant PKC-{alpha}, acetylcholine also failed to induce increases in CPI-17 phosphorylation (51.51 ± 23.35% and 73.24 ± 39.46% at 30 s and 4 min, respectively, n = 3; Fig. 10). These results suggest that both PKC-{alpha} and RhoA pathways are involved in the phosphorylation of CPI-17 during acetylcholine-induced smooth muscle contraction.



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  		Fig. 10. ACh-induced CPI-17 phosphorylation in rabbit colon smooth muscle cells transfected with negative PKC-{alpha}. Particulate fractions from smooth muscle cells were subjected to SDS-PAGE followed by Western blot analysis with anti-phospho-CPI-17 antibody. Stimulation of rabbit cultured smooth muscle cells with ACh (10–7 M) resulted in a significant and sustained increase in the phosphorylation of CPI-17 (158.54 ± 20.16% and 150.07 ± 16.72% at 30 s and 4 min, respectively, n = 6, P < 0.05). In cells transfected with negative dominant RhoA, ACh failed to induce CPI-17 phosphorylation (76.12 ± 21.66% and 64.15 ± 21.47%, respectively, at 30 s and 4 min compared with control, n = 3). In cells transfected with negative dominant PKC-{alpha}, ACh failed to induce CPI-17 phosphorylation (51.51 ± 23.35% and 73.24 ± 39.46% at 30 s and 4 min, respectively, n = 3) compared with nontransfected cells treated with ACh, suggesting that both RhoA and PKC-{alpha} pathways converge in phosphorylation of CPI-17. Note the diminished levels of expression of CPI-17 in the cells transfected with negative RhoA or negative PKC-{alpha}.

 
Effect of acetylcholine on MLC phosphorylation in cultured smooth muscle cells transfected with negative RhoA constructs or dominant negative PKC-{alpha}. Cultured smooth muscle cells transfected with negative RhoA constructs were stimulated with acetylcholine for 30 s or 4 min. Cells were then washed with PBS and lysed in 8 M urea buffer. Samples were then separated by IEF (pH 3–9) and Western blotted with anti-MLC antibody. Acetylcholine induced an increase in MLC phosphorylation at 30 s and 4 min in normal cultured cells (125.89 ± 8.22% and 150.21 ± 10.48% at 30 s and 4 min, respectively, n = 4, P < 0.05). Cultured smooth muscle cells transfected with negative RhoA constructs showed a decline in acetylcholine-induced MLC phosphorylation at 4 min (125.77 ± 5.93% and 117.48 ± 3.34% at 30 s and 4 min, respectively, P < 0.05 compared with normal cells treated with acetylcholine for 4 min), suggesting that RhoA activation is responsible for the maintenance of sustained smooth muscle contraction. It has been shown that inhibition of PKCs by calphostin C in smooth muscle cells inhibits MLC phosphorylation (43). To test the role of specific isoform of PKC-{alpha}, cultured smooth muscle cells transfected with negative constructs of PKC-{alpha} were stimulated with acetylcholine for 30 s and 4 min. Cells were then lysed in 8 M urea buffer and subjected to IEF followed by Western blot analysis using anti-MLC antibody (Fig. 11). Cells transfected with negative constructs to PKC-{alpha} showed a decline in MLC phosphorylation at 4 min (119.48 ± 4.63% and 114.23 ± 1.30%, P < 0.05 compared with normal control cells treated with acetylcholine at 4 min, n = 4; Fig. 11), indicating a role for PKC-{alpha} in the maintenance of sustained smooth muscle contraction.



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  		Fig. 11. ACh-induced myosin light chain (MLC) phosphorylation in rabbit colon smooth muscle cells transfected with negative RhoA or negative PKC-{alpha}. Cultured smooth muscle cells either transfected with negative PKC-{alpha} or negative RhoA were stimulated with ACh (10–7 M) for 30 s or 4 min. Cells were washed with PBS and lysed in 8 M urea buffer. Samples were then separated by IEF (pH 3–9) and Western blotted with anti-MLC antibody (1:3,000). The band intensities are represented graphically as percentages over the control. ACh induced an increase in MLC phosphorylation at 30 s and 4 min in normal cultured cells (125.89 ± 8.2% and 150.21 ± 10.48% at 30 s and 4 min, respectively, n = 4, P < 0.05). Cultured smooth muscle cells transfected with negative RhoA or negative PKC-{alpha} did not show a decline in MLC phosphorylation at 30 s (125.77 ± 5.9% and 119.48 ± 4.6% at 30 s for smooth muscle cells transfected with either negative RhoA or negative PKC-{alpha}, respectively; n = 4). However, at 4 min, MLC phosphorylation was significantly inhibited in cells transfected with negative RhoA or negative PKC-{alpha} constructs (117.48 ± 3.34% and 114.23 ± 1.30% at 4 min for smooth muscle cells transfected with either negative RhoA or negative PKC-{alpha}, respectively, n = 4, P < 0.05), indicating the requirement of both RhoA and PKC pathways in the regulation of MLC phosphorylation.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main regulatory mechanism of smooth muscle contraction involves calmodulin-dependent phosphorylation of myosin by MLCK. Other mechanisms that are independent of calmodulin/MLCK may help to sustain MLC phosphorylation and may be operating (38). In smooth muscle, MLCP activity could be regulated by both RhoA and PKC pathways.

The small GTP-binding protein RhoA is an important signaling protein that mediates various actin-dependent cytoskeletal functions including smooth muscle contraction (19, 46, 49). Studies with purified ROCKII have demonstrated that isolated MLC forms a substrate for ROCKII in vitro (36) and that activated ROCKII induced contraction by direct phosphorylation of myosin at Ser19, the MLCK site (30). ROCKII can phosphorylate both smooth muscle and nonmuscle MLC in solution (25), suggesting that direct phosphorylation of MLC by ROCKII is not physiologically significant. We have reported that Y-27632 inhibited agonist-induced increases in MLC phosphorylation in rabbit colon smooth muscle cells (41). The precise mechanism by which RhoA/ROCKII pathways induce MLC20 phosphorylation and contraction remains to be determined. A model has been proposed in which RhoA/ROCKII regulate MLC20 phosphorylation primarily by inhibition of MLCP (3, 26, 45). MLCP is a heterotrimeric enzyme composed of a 37-kDa catalytic subunit (PP1c, a member of the type 1 protein Ser/Thr phosphatase family, specifically the {delta}-isoform), a 110- to 130-kDa myosin-targeting subunit called MYPT1 (which anchors the phosphatase to myosin filaments), and a 20-kDa subunit whose function remains unclear (15). Immunocytochemical evidence suggests that dissociation of the subunits of MLCP may occur after agonist stimulation of intact smooth muscle cells (42). Dissociation of the subunits would decrease the phosphatase activity toward phosphorylated myosin (48). MYPT can be phosphorylated at two different sites: Thr696 and Thr850. Phosphorylation of MYPT is important in regulating the activity of MLCP. It has been demonstrated in vitro that the phosphorylation of Thr852 by ROCK leads to dissociation of MLCP from myosin (50), whereas phosphorylation of MYPT at Thr696 results in inactivation of MLCP.

We have recently shown that Y-27632 inhibits acetylcholine-induced smooth muscle contraction and phosphorylation of MLC. The ROCKII inhibitor Y-27632 inhibited phosphorylation of MYPT and MLCP activity in smooth muscle cells. MYPT phosphorylation was also inhibited in cells transfected with negative dominant RhoA. These results suggest that MLCP activities are directly regulated by the RhoA pathway through phosphorylation of MYPT.

MLCP activity was also shown to be modulated by CPI-17. CPI-17 is a substrate for PKC (10). MYPT phosphorylation is inhibited by calphostin C. Inhibition of PKC-{alpha} inhibits CPI-17 but also inhibits RhoA activation, which results in inhibition of phosphorylation of MYPT at Thr696, thus resulting in activation of MLCP (i.e., an increase in MLCP activity) (Fig. 5).

Phosphorylation of CPI-17 is reduced by treatment with 10 µM Y-27632 in smooth muscle cells from the femoral artery (27). Our present results indicate that Y-27632 inhibited acetylcholine-induced phosphorylation of CPI-17 (Fig. 6). Furthermore, cells expressing negative RhoA also exhibited inhibition of CPI-17 phosphorylation in response to acetylcholine. PKC phosphorylation of CPI-17 in vitro is inhibited by the ROCKII inhibitor Y-27632 (27). These indicate that both PKC and RhoA pathways may mediate the phosphorylation of CPI-17, thereby regulating MLCP activity. This evidence also confirms a possible interrelation between RhoA and PKC-{alpha}.

Data from other investigators indicate that Y-27632 inhibited activation of PKC in human umbilical venous endothelial cells (16). This effect may not be due to direct inhibition of PKC by Y-27632. Y-27632 has been shown to inhibit PKC, albeit at higher doses (Ki for PKC is 26 µM compared with ROCK, which is 0.14 µM). Rho inhibitors block PKC translocation and activation in endothelial and epithelial cells, suggesting a RhoA requirement for PKC activation/translocation (16). The present results indicate that Y-27632 inhibited acetylcholine-induced phosphorylation of CPI-17. Experiments involving cells expressing negative RhoA corroborate these results. The PKC inhibitor calphostin C inhibited both CPI-17 and MYPT phosphorylation, indicating that PKC activation of RhoA may be required during agonist-induced contraction. These indicate that PKC activities may be RhoA interrelated.

To confirm the specific roles of each RhoA and PKC-{alpha} and their interrelation in regulation of MLCP, inactive (negative dominant) mutations of RhoA (R/N19) or PKC-{alpha} (K/R368) were transfected to smooth muscle cells in culture. The results indicate that the cells transfected with negative dominant PKC-{alpha} exhibited partially inhibited acetylcholine-induced PKC activity (Fig. 9) and CPI-17 phosphorylation.

In cells transfected with negative dominant RhoA, acetylcholine-induced decreases in total phosphatase activity were inhibited. Acetylcholine-induced phosphorylation of MYPT and its translocation was also reduced. Similarly, acetylcholine-induced increases in PKC activities and phosphorylation of CPI-17 were inhibited. These results suggest a possibility of independent regulation of MYPT by RhoA and PKC.

Our present results indicate that both in freshly isolated and cultured smooth muscle cells, MYPT translocated to the particulate fraction in response to acetylcholine. Our results also indicate that phosphorylated MYPT associated and translocated with HSP27.

Translocation of MLCP to the membrane has been shown in ferret vascular smooth muscle cells treated with PGF2{alpha}, and the contraction remained sustained (42). Shin et al. (42) showed that Y-27632 inhibited the translocation of both ROCKII and MYPT1 to the membrane. It is possible that ROCKII translocation with RhoA to the contractile apparatus after activation at the membrane is essential for inactivation of MYPT through ROCKII (18, 48). We have recently shown that RhoA and ROCKII associate and translocate to the particulate fraction upon stimulation with contractile agonists in rabbit colon smooth muscles. The present results suggest that phosphorylated MYPT was present in the particulate fraction and associated with HSP27 (Fig. 6). Furthermore, MYPT phosphorylation and translocation were inhibited by both Y-27632 and calphostin C. These suggest a parallel regulation of MYPT by RhoA and PKC.

We have previously shown that RhoA interacts directly with HSP27 in vitro (40). We have also recently shown that smooth muscle cells transfected with a nonphosphorylated form of HSP27 failed to exhibit sustained contraction and that the translocation of RhoA was diminished in these cells (40). In the present experiments, MYPT failed to translocate and associate with HSP27 in the presence of Y-27632. These results imply that HSP27-mediated translocation and formation of a complex with Rho/ROCKII and MYPT are crucial for the maintenance of sustained contraction.

We have also shown that HSP27 plays a significant role in mediating the translocation and association of RhoA with ROCKII. It has previously been reported (6) that HSP27 modulates the association of translocated PKC-{alpha} and RhoA in rabbit colon smooth muscle cells. In addition, our results suggest that formation of a complex of MYPT-Rho/ROCKII and HSP27 in the particulate fraction is a key event for interaction of the key regulatory proteins such as PKC and of RhoA. Thus HSP27 may function as a facilitator in thin filament regulation of smooth muscle contraction. Our previous results indicated that HSP27 facilitated association of RhoA and ROCKII, and the present results suggest that HSP27 and phosphorylated MYPT interact in the particulate fraction. Taken together, it is apparent that HSP27 contributes significantly for facilitating several signaling and contractile proteins and thus help maintain smooth muscle contraction. There are several indications in other cell systems that suggest that RhoA- and PKC-mediated pathways interact. This was suggested to occur through the binding of RhoA to PKC-{alpha} (41). The current report indicates that inhibition of either of the pathways does not completely inhibit MLCP activity or phosphorylation of the regulatory proteins PKC or CPI-17. This is suggestive of the fact that both RhoA and PKC are activated independently of each other upon stimulation with contractile agonists and each of the pathways subsequently converges at MYPT regulation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5 RO1 DK-042876.


   ACKNOWLEDGMENTS
 
Meticulous secretarial assistance, technical editing, and figure preparation of Shilow Blea is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Bitar, Div. of Pediatric Gastroenterology, Univ. of Michigan Medical School, 1150 W. Medical Center Dr., MSRB 1, Rm. A520, Ann Arbor, MI 48109-0656 (e-mail: bitar{at}umich.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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