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Am J Physiol Heart Circ Physiol 288: H2828-H2835, 2005. First published January 21, 2005; doi:10.1152/ajpheart.01189.2004
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{alpha}1-Adrenoceptor-mediated phosphorylation of MYPT-1 and CPI-17 in the uterine artery: role of ERK/PKC

DaLiao Xiao, Lawrence D. Longo, and Lubo Zhang

Center for Perinatal Biology, Department of Pharmacology and Physiology, Loma Linda University School of Medicine, Loma Linda, California

Submitted 29 November 2004 ; accepted in final form 17 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously demonstrated that ERK/PKC signaling pathways play a key role in regulation of Ca2+ sensitivity and contractility of the uterine artery. The present study tested the hypothesis that ERK and PKC differentially regulated myosin light chain phosphatase activity by phosphorylation of myosin phosphatase target protein-1 (MYPT-1) and CPI-17. Agonist-induced contractions and phosphorylation of MYPT-1/Thr696, MYPT-1/Thr850, and CPI-17/Thr38 were measured simultaneously in the same tissues of isolated near-term pregnant ovine uterine arteries. Phenylephrine produced time-dependent concurrent increases in the phosphorylation of ERK44/42 and MYPT-1/Thr850 that preceded contractions. In addition, phenylephrine induced phosphorylation of CPI-17/Thr38 that was concurrent with the contractions. In contrast, phenylephrine did not induce phosphorylation of MYPT-1/Thr696 in the uterine artery. PD-098059 inhibited phosphorylation of ERK44/42 and the initial peak phosphorylation of MYPT-1/Thr850 but did not affect CPI-17/Thr38 phosphorylation. Activation of PKC by phorbol 12,13-dibutyrate induced a time-dependent phosphorylation of CPI-17/Thr38 that preceded contractions of the uterine artery. In addition, phorbol 12,13-dibutyrate activated PKC-{alpha} and induced a coimmunoprecipitation of PKC-{alpha} with caldesmon. The results suggest that phosphorylation of MYPT-1/Thr850 and CPI-17/Thr38 play important roles in regulation of agonist-mediated Ca2+ sensitivity in the uterine artery, in part by ERK and PKC, respectively. In addition, phosphorylated CPI-17 may regulate Ca2+ sensitivity by interacting with caldesmon and reversing its inhibitory effect on myosin ATPase.

myosin light chain phosphatase; phenylephrine; calcium sensitivity; caldesmon

SMOOTH MUSCLE CONTRACTION is predominantly regulated by phosphorylation of myosin light chain (MLC), which is determined by the activities of MLC kinase (MLCK) and MLC phosphatase (MLCP) (15, 20). Increases in intracellular Ca2+ concentrations ([Ca2+]i) activate MLCK, resulting in phosphorylation of 20-kDa MLC (MLC20) and contractions. However, the force development of smooth muscle is not simply determined by [Ca2+]i (22, 35, 42, 59). Ca2+ sensitivity of myofilaments also plays an important role in the regulation of smooth muscle contraction. Regulation of Ca2+ sensitivity may include at least two components: thick filament regulation (i.e., Ca2+ sensitivity of MLC20 phosphorylation) and thin filament regulation (i.e., MLC20 phosphorylation-independent contractions). MLCP plays a key role in regulating Ca2+ sensitivity of MLC20 phosphorylation (13, 28, 29, 47). MLCP consists of three subunits: a 110- to 130-kDa regulatory subunit [myosin phosphatase target protein-1 (MYPT-1)], an ~37-kDa catalytic subunit (PP1c), and a 20-kDa subunit of unknown function. Inhibition of MLCP is regulated by at least three mechanisms: phosphorylation of MYPT-1, arachidonic acid-induced dissociation of the holoenzyme, and PKC-mediated phosphorylation of CPI-17 at Thr38, which becomes a potent inhibitor of PP1c. Two phosphorylation sites of MYPT-1 have been identified at Thr696 and Thr850.

In addition to the thick filament regulation, Ca2+ sensitivity is also regulated by the smooth muscle actin-binding protein caldesmon, which inhibits myosin ATPase activity (36). Studies using the antagonist peptide GS17C and the antisense have strongly suggested an important and physiologically relevant role for caldesmon in suppressing smooth muscle tone (7, 24, 37, 40). Removal of caldesmon-mediated inhibition can be achieved by phosphorylation of caldesmon and/or binding of other regulatory proteins, e.g., Ca2+/calmodulin, which reverse its inhibitory effect on myosin ATPase.

Recently, we demonstrated in the pregnant uterine artery that {alpha}1-adrenoceptor-mediated contractions are regulated through thick and thin filament pathways, with the thick filament regulatory pathway, i.e., MLC20 phosphorylation, predominating. However, PKC-mediated contractions were regulated predominantly through the thin filament pathway, i.e., independent of changes in MLC20 phosphorylation (56). We previously demonstrated that ERK plays an important role in regulation of {alpha}1-adrenoceptor-mediated contractions of the uterine artery (56, 57). Inhibition of ERK by PD-098059 significantly decreased the Ca2+ sensitivity of MLC20 phosphorylation in response to phenylephrine, i.e., less MLC20 phosphorylation at a given [Ca2+]i (56). However, ERK-mediated phosphorylation of the thin filament binding protein caldesmon at Ser789 may not lead to the reversal of caldesmon inhibitory effects on actin-activated myosin ATPase in pregnant ovine uterine arteries. In contrast, our previous studies suggested that, in the pregnant uterine artery, PKC-induced, thin filament-dependent contractions may be mediated by a direct phosphorylation of caldesmon or by phosphorylation of other regulatory protein(s) that may interact with caldesmon.

In the present study, we determined the temporal relations between phosphorylation of MYPT-1/Thr696, MYPT-1/Thr850, and CPI-17/Thr38 and contractions induced by phenylephrine and tested the hypothesis that ERK played a role in inhibition of MLCP in the uterine artery. Given that CPI-17 is a PKC substrate (8) and phosphorylated CPI-17 may serve as a regulatory binding protein, the present study also determined the temporal relation between PKC-mediated phosphorylation of CPI-17 and contractions. In addition, we determined the potential direct interaction between PKC and caldesmon by examining the coimmunoprecipitation of PKC-{alpha} isozyme with caldesmon in the uterine artery.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation. Near-term pregnant (~140 days gestation) sheep were anesthetized with thiamylal (10 mg/kg) administered via the external left jugular vein. The ewes were then intubated, and anesthesia was maintained on 1.5–2.0% halothane in O2 throughout the surgery. An incision was made in the abdomen, and the uterus was exposed. The uterine arteries were isolated and removed without being stretched and were placed into a modified Krebs solution (pH 7.4) of the following composition (in mM): 115.21 NaCl, 4.7 KCl, 1.80 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 22.14 NaHCO3, 0.03 EDTA, and 7.88 dextrose. The Krebs solution was oxygenated with 95% O2-5% CO2. After the tissues were removed, the animals were killed with euthanasia solution (T-61, Hoechst-Roussel, Somerville, NJ). All procedures and protocols used in the present study were approved by the Animal Research Committee of Loma Linda University and followed the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Contraction studies. Fourth branches of the main uterine arteries were separated from the surrounding tissue and cut into 2-mm rings. They were chosen because of their close characteristics to arterioles in contribution to vascular resistance. We used these arteries extensively in our previous studies (56, 57), and the present study would provide comparative results. We have shown that these small arteries contract to {alpha}1-adrenoceptor activation more effectively than the conduit vessel of the main uterine artery, in part because of a higher density of {alpha}1-adrenoceptors in the fourth-branch arteries (18). The arterial rings were attached to an isometric force transducer and bathed in the Krebs solution under 95% O2-5% CO2 at 37°C. Isometric tensions were measured as described previously (57). After 60 min of equilibration in the tissue bath, each ring was stretched to the optimal resting tension as determined by the tension developed in response to 120 mM KCl added at each stretch level. Tissues were then stimulated with phenylephrine and/or phorbol 12,13-dibutyrate (PDBu). Tension developed was recorded with an online computer. At the indicated times, arterial rings were snap frozen with liquid N2-cooled clamps and then immersed in a dry ice-acetone slurry containing 10% trichloroacetic acid and 10 mM DTT. The rings were stored at –80°C until analysis. In certain experiments, tissues were pretreated with PD-098059 (30 µM) or the vehicle (DMSO) for 30 min and then stimulated with phenylephrine.

Western immunoblotting analysis. Tissues were homogenized in a lysis buffer containing 150 mM NaCl, 50 mM Tris·HCl, 10 mM EDTA, 0.1% Tween 20, 0.1% {beta}-mercaptoethanol, 0.1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin, pH 7.4. Homogenates were centrifuged at 4°C for 5 min at 6,000 g, and the supernatants were collected. Protein was quantified in the supernatant. Samples with equal protein were loaded on 7.5% (MYPT-1/Thr696 and MYPT-1/Thr850) or 10% (CPI-17/Thr38 and phosphorylated ERK42/44) polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS) and separated by electrophoresis at 100 V for 2 h. Proteins were then transferred onto nitrocellulose membranes. Nonspecific binding sites in the membranes were blocked by overnight incubation at 4°C in a Tris-buffered saline solution containing 5% dry milk. The membranes were incubated with primary antibodies and then with secondary antibodies. Proteins were visualized with enhanced chemiluminescence reagents, and the blots were exposed to Hyperfilm. Results were quantified with the Kodak electrophoresis documentation and analysis system and Kodak 1D image analysis software.

Measurement of PKC-{alpha} translocation. Uterine arteries were mounted in tissue baths and equilibrated for ≥60 min in the Krebs solution under 95% O2-5% CO2 at 37°C. The rings were then exposed to PDBu (3 µM) or the vehicle (DMSO) for 30 min. The uterine arterial rings were snap frozen and homogenized in ice-cold homogenization buffer containing 20 mM Tris·HCl, 250 mM sucrose, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 10 mM {beta}-mercaptoethanol, and 1 mM benzamide. The homogenate was centrifuged at 100,000 g for 60 min at 4°C, and the supernatant was used as the cytosolic fraction. The pellet corresponding to the membrane particulate fraction was solubilized in a buffer containing Triton X-100 at a final concentration of 0.1% by stirring on ice for 45 min at 4°C and then centrifuged at 100,000 g for 60 min at 4°C to remove insoluble membrane particles. The supernatant was collected and was referred to as the membrane particulate fraction. Immunoreactive bands for PKC-{alpha} in cytosolic and particulate fractions were determined by Western blotting using PKC-{alpha} antibody (1:500; Santa Cruz Biotechnology) as described above.

Immunocytochemistry. Smooth muscle cells were freshly isolated from the uterine arteries using a collagenase-elastase digestion mixture with gentle shaking as described previously (44). After enzyme incubation, tissues were rinsed with Hanks' solution, and dissociated cells were placed over unsiliconized coverslips. The cells were settled by gravity and spontaneously adhered to the glass. Immunocytochemical staining of PKC-{alpha} in freshly isolated cells was determined as previously described (25). Briefly, after treatment, freshly isolated cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). The excess fixative was quenched with 0.1% mM glycine in 1% BSA-Hanks' solution. The cells were then permeabilized with 0.1% Triton X-100 in 1% BSA-Hanks' solution and washed in 1% BSA-Hanks' solution. Cells were blocked with 10% bovine serum in 1% BSA-Hanks' solution, reacted with the PKC-{alpha} antibody, washed in 1% BSA-Hanks' solution with 0.05% Triton X-100, and labeled with a secondary antibody conjugated with FITC. Cells were then washed in 1% BSA-Hanks' solution to remove excess label. The cell nuclei were stained with Hoechest 33258. Cells were then examined using a fluorescent microscope with the SPOT digital camera.

Coimmunoprecipitation. Coimmunoprecipitation of PKC-{alpha} and caldesmon was determined as described previously (34). Tissues were homogenized in a buffer containing 50 mM Tris, pH 7.4, 10% glycerol, 5 mM EGTA, 140 mM NaCl, 1% Triton X-100, the protease inhibitors leupeptin and pepstatin (5.5 µM each) and aprotinin (20 kallikrein-inactivating units), 1 mM Na3VO4, 10 mM NaF, 0.25% (wt/vol) sodium deoxycholate, 100 µM ZnCl2, 20 mM {beta}-glycerophosphate, and 2 µM PMSF. After centrifugation, the protein concentration was determined using the Bradford method (Bio-Rad). An equal amount of protein was incubated with the caldesmon antibody overnight at 4°C with gentle rotation. Then protein A/G PLUS-agarose (Santa Cruz Biotechnology) was added and incubated for an additional 2 h. The beads were washed extensively with the homogenization buffer, and the immune complex was eluted in SDS-PAGE sample buffer. Total immune complex samples and protein samples from total tissue homogenates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with the antibody against PKC-{alpha}. After they were washed and incubated with secondary antibody, immunoreactive proteins were visualized with the enhanced chemiluminescence detection system. The recovery of caldesmon was determined by immunoblot using the caldesmon antibody.

Materials. Phenylephrine, PDBu, and PD-098059 were obtained from Sigma (St. Louis, MO); phosphorylated MYPT-1/Thr696 and MYPT-1/Thr850 antibodies and caldesmon antibody from Upstate Biotechnology (Lake Placid, NY); phosphorylated ERK42/44 (Thr202/Tyr204) antibodies from Cell Signaling Technology (Beverly, MA); phosphorylated CPI-17/Thr38 antibody and PKC-{alpha} antibody from Santa Cruz Biotechnology (Santa Cruz, CA); and all electrophoretic and immunoblot reagents from Bio-Rad. General laboratory reagents were of analytic grade or better and were purchased from Sigma and Fisher Scientific. All drugs were prepared fresh each day and kept on ice throughout the experiment.

Data analysis. Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). Values are means ± SE. Differences were evaluated for statistical significance (P < 0.05) by two-way ANOVA and Student's t-test.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Phenylephrine-induced contractions and phosphorylation of ERK, MYPT-1, and CPI-17. Figure 1 shows temporal relations between phenylephrine-induced contractions and phosphorylation of ERK, MYPT-1/Thr850, and CPI-17/Thr38, measured simultaneously in the same tissues of pregnant uterine arteries. Phenylephrine produced time-dependent and concurrent increases in phosphorylation of ERK and MYPT-1/Thr850 that preceded the contractions. Phenylephrine-induced phosphorylation of CPI-17/Thr38 trailed phosphorylations of ERK and MYPT-1/Thr850 but was concurrent with the contractions.



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  		Fig. 1. Time course of phenylephrine-induced phosphorylation of ERK44, myosin phosphatase target protein-1 (MYPT-1)/Thr850, and CPI-17/Thr38 and contractions in uterine artery. Uterine arterial rings from pregnant sheep were stimulated with 3 µM phenylephrine. Contractile responses were terminated at times indicated, and phosphorylated levels of ERK44 (pERK44), MYPT-1/Thr850 (pMYPT-1/Thr850), and CPI-17/Thr38 (pCPI-17/Thr38) were determined in the same tissue by Western blotting. Values are means ± SE of tissues from 4 animals.

 
Effect of PD-098059 on phenylephrine-induced phosphorylation of ERK44/42. Our previous studies (56) demonstrated that PKC activation stimulates a time-dependent increase in ERK44/42 phosphorylation in the pregnant uterine artery, which is blocked by PD-098059, the MEK/ERK inhibitor. Phenylephrine-induced time-dependent increases in phosphorylation of ERK44 and ERK42 were also blocked by PD-098059 (Fig. 2).



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  		Fig. 2. Effect of PD-098059 on phenylephrine-induced phosphorylation of ERK44/42 in uterine artery. Uterine arterial rings from pregnant sheep were pretreated with 30 µM PD-098059 or vehicle (DMSO, control) for 30 min and then stimulated with 3 µM phenylephrine. Representative Western immunoblots show phosphorylated ERK44/42 induced by phenylephrine in the absence (top blot) and presence (bottom blot) of PD-098059 at 0–300 s. Values are means ± SE of tissues from 6 animals. Two-way ANOVA indicates a significant difference between control and PD-098059-treated samples (P < 0.05).

 
Effect of PD-098059 on phenylephrine-induced phosphorylation of MYPT-1/Thr850 and MYPT-1/Thr696. Previous studies suggested a differential regulation of phosphorylation of MYPT-1/Thr850 and MYPT-1/Thr696 in smooth muscle. Agonists stimulated phosphorylation of MYPT-1/Thr850, but not MYPT-1/Thr696 (47). Basal phosphorylation levels of MYPT-1/Thr696 were detected in the uterine artery (Fig. 3). Consistent with previous studies, phenylephrine did not increase MYPT-1/Thr696 phosphorylation in the uterine artery (Fig. 3). This was not altered by inhibition of ERK with PD-098059. In contrast, phenylephrine induced a time-dependent increase in phosphorylation of MYPT-1/Thr850, which was blocked in the presence of PD-098059 (Fig. 4).



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  		Fig. 3. Effect of PD-098059 on phenylephrine-induced phosphorylation of MYPT-1/Thr696 in uterine artery. Uterine arterial rings from pregnant sheep were pretreated with 30 µM PD-098059 or DMSO (control) for 30 min and then stimulated with 3 µM phenylephrine. Representative Western immunoblots show phosphorylated MYPT-1/Thr696 induced by phenylephrine in the absence (top blot) and presence (bottom blot) of PD-098059 at 0–300 s. Values are means ± SE of tissues from 3 animals.

 


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  		Fig. 4. Effect of PD-098059 on phenylephrine-induced phosphorylation of MYPT-1/Thr850 in uterine artery. Uterine arterial rings from pregnant sheep were pretreated with 30 µM PD-098059 or DMSO (control) for 30 min and then stimulated with 3 µM phenylephrine. Representative Western immunoblots show phosphorylated MYPT-1/Thr850 induced by phenylephrine in the absence (top blot) and presence (bottom blot) of PD-098059 at 0–300 s. Values are means ± SE of tissues from 6 animals. Two-way ANOVA indicates a significant difference between control and PD-098059-treated samples (P < 0.05).

 
Effect of PD-098059 on phenylephrine-induced phosphorylation of CPI-17/Thr38. Phenylephrine produced a time-dependent increase in phosphorylation of CPI-17 in the pregnant uterine artery (Fig. 5). PD-098059 showed a tendency to decrease phenylephrine-induced CPI-17 phosphorylation that did not reach a statistically significant level (Fig. 5).



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  		Fig. 5. Effect of PD-098059 on phenylephrine-induced phosphorylation of CPI-17/Thr38 in uterine artery. Uterine arterial rings from pregnant sheep were pretreated with 30 µM PD-098059 or DMSO (control) for 30 min and then stimulated with 3 µM phenylephrine. Representative Western immunoblots show phosphorylated CPI-17/Thr38 induced by phenylephrine in the absence (top blot) and presence (bottom blot) of PD-098059 at 0–300 s. Values are means ± SE of tissues from 4 to 5 animals. Two-way ANOVA indicates no significant difference between control and PD-098059-treated samples (P > 0.05).

 
PDBu-induced contraction and phosphorylation of CPI-17. Given that CPI-17 is a PKC substrate and phosphorylated CPI-17 plays an important role in the regulation of Ca2+ sensitivity of myofilaments, we determined the temporal relation between PDBu-induced phosphorylation of CPI-17/Thr38 and contractions measured simultaneously in the same tissues. PDBu produced a time-dependent increase in phosphorylation of CPI-17/Thr38 that preceded the contractions (Fig. 6).



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  		Fig. 6. Time course of phorbol 12,13-dibutyrate (PDBu)-induced phosphorylation of CPI-17/Thr38 and contractions in uterine artery. Uterine arterial rings from pregnant sheep were stimulated with 5 µM PDBu. Contractile responses were terminated at various times, and phosphorylated levels of CPI-17/Thr38 were determined in the same tissue by Western blotting. Values are means ± SE of tissues from 5 animals.

 
PDBu-induced translocation of PKC-{alpha} isozyme. We previously demonstrated that PDBu increases the particulate-to-cytosolic PKC activity ratio in the uterine artery, which is blocked by staurosporine (57). To determine the isozyme involved in PDBu-stimulated PKC activity, we determined the effect of PDBu on the distribution of PKC-{alpha} in cytosolic and particulate fractions. The tissues were pretreated with 0.1 µM staurosporine for 30 min before the addition of PDBu (3 µM for 30 min). PKC-{alpha} levels in the cytosolic and particulate fractions were determined by Western blotting using the PKC-{alpha} antibody. PDBu significantly increased the particulate-to-cytosolic PKC-{alpha} activity ratio in the uterine artery, which was blocked by staurosporine (Fig. 7). To confirm that the translocation of PKC-{alpha} was from cytosol to cell plasma membrane, we examined the immunohistochemical staining of PKC-{alpha} in freshly isolated smooth muscle from the uterine artery. In the resting cells, PKC-{alpha} was distributed in the cytosol (Fig. 8). PDBu induced a translocation of PKC-{alpha} from cytosol to plasma membrane that was accompanied by contractions of the cells.



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  		Fig. 7. PDBu-induced PKC-{alpha} translocation in uterine artery. Uterine arterial rings from pregnant sheep were pretreated in the absence or presence of 0.1 µM staurosporine (Stau) for 30 min and then stimulated with 3 µM PDBu for 30 min. Representative Western immunoblots show PKC-{alpha} determined by Western blotting in cytosolic and membrane particulate fractions. Values are means ± SE of tissues from 5 animals. *P < 0.05 vs. control (C).

 


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  		Fig. 8. PDBu-induced redistribution of PKC-{alpha} in uterine artery smooth muscle cells. Freshly isolated smooth muscle cells from pregnant uterine arteries were treated with PDBu (5 µM) or vehicle control for 20 min. After fixation, cells were labeled with anti-PKC-{alpha} antibody. Representative fluoromicroscopy images show cells stained for PKC-{alpha} (green) and nuclei (blue). At rest, uterine artery smooth muscle cells showed typical elongated spindle shape and a relatively homogeneous cytosolic staining for PKC-{alpha}. PDBu stimulated translocation of PKC-{alpha} from cytosol to cell plasma membrane, which was accompanied with a shortening of smooth muscle cells. Result was confirmed by 3 experiments.

 
Coimmunoprecipitation of PKC-{alpha} and caldesmon. Our recent studies suggested that PKC may directly phosphorylate caldesmon in the uterine artery (56). The direct interaction of PKC-{alpha} and caldesmon was determined by coimmunoprecipitation of PKC-{alpha} and caldesmon. A significant increase in PKC-{alpha} was detected in the caldesmon immunoprecipitates in PDBu-stimulated tissues compared with controls, suggesting an increase in the direct interaction between the activated PKC-{alpha} and caldesmon in the uterine artery (Fig. 9). The recovery of caldesmon was not significantly different between control and PDBu-treated tissues (Fig. 9).



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  		Fig. 9. Coimmunoprecipitation of PKC-{alpha} and caldesmon in uterine artery. Uterine arterial rings from pregnant sheep were treated with 5 µM PDBu or vehicle (control) for 5 min. Immunoprecipitation (IP) of caldesmon was performed using caldesmon antibody. Immune complex was separated by SDS-PAGE, transferred to nitrocellulose membrane, and blotted with PKC-{alpha} and caldesmon antibodies. Representative Western immunoblots (IB) show PKC-{alpha} in caldesmon immunoprecipitates and recovery of caldesmon in control and PDBu-treated samples. Values are means ± SE of tissues from 3 animals. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of the present study are as follows: 1) Phenylephrine induces the phosphorylation of ERK and MYPT-1/Thr850 that precedes the contractions. 2) Phenylephrine-induced phosphorylation of MYPT-1/Thr850 is mediated in part by activation of ERK. 3) Phenylephrine produces concurrent phosphorylation of CPI-17/Thr38 and contractions, and the phosphorylation of CPI-17/Thr38 is not mediated by ERK activation. 4) Phenylephrine does not increase phosphorylation of MYPT-1/Thr696. 5) PDBu activates PKC-{alpha} isozyme and induces a time-dependent increase in CPI-17/Thr38 phosphorylation that precedes the contractions. 6) Activated PKC-{alpha} coimmunoprecipitates with the actin-binding protein caldesmon in the uterine artery.

It has been demonstrated in many studies that different agents that produce smooth muscle contraction activate ERK at the same time (2, 5, 11, 12, 54). Recently, we showed that the MEK/ERK inhibitor PD-098059 selectively inhibits phenylephrine-induced contractions in the pregnant, but not nonpregnant, uterine arteries, suggesting that pregnancy enhances the role of ERK in {alpha}1-adrenoceptor-mediated contractions in the uterine artery (57). In the present study, we found that phenylephrine produced a time-dependent increase in phosphorylation of ERK44 and ERK42 that was inhibited by PD-098059. This finding supports a role for ERK in the regulation of {alpha}1-adrenoceptor-mediated contractions in the uterine artery. Different signaling transduction mechanisms have been reported in the ERK-mediated regulation of smooth muscle contraction. Previous studies have shown that PD-098059 does not affect agonist-induced [Ca2+]i in isolated smooth muscle cells but does inhibit agonist-induced smooth muscle contraction by decreasing the Ca2+ sensitivity of contractile proteins (1, 2, 5, 12, 17). Our previous studies also demonstrated that inhibition of ERK by PD-098059 significantly decreases the Ca2+ sensitivity of MLC20 phosphorylation in response to phenylephrine, i.e., less MLC20 phosphorylation at a given [Ca2+]i in the uterine artery (56). It has been proposed that ERK mediates smooth muscle contraction through the thin filament regulatory pathway by phosphorylation of caldesmon (2, 17, 36). However, our recent studies demonstrated that, in the pregnant uterine artery, phosphorylation of ERK-specific site Ser789 of caldesmon may not lead to reversal of the caldesmon inhibitory effect on myosin ATPase. Instead, phosphorylation of caldesmon/Ser789 may stabilize its inhibitory effect on actin-activated myosin ATPase (56).

In the present study, we have demonstrated that phosphorylation of MYPT-1/Thr850 precedes phenylephrine-mediated contractions. The finding that MYPT-1/Thr850 consisted of two bands in the present study is in agreement with previous findings. It has been shown that MYPT-1 in smooth muscle consists of two isoforms and bands, but often the separation is not distinct (43, 55). The present finding that the time course of MYPT-1/Thr850 phosphorylation resembled that of phenylephrine-induced MLC20 phosphorylation (56) and preceded contractions suggests that phenylephrine-mediated Ca2+ sensitization is partly regulated through phosphorylation of MYPT-1/Thr850, resulting in an inhibition of MLCP activity in the uterine artery. Although MYPT-1/Thr850 was originally shown not to be an inhibitory site in vitro (9), a recent study demonstrated that its phosphorylation state might affect localization of MLCP and its catalytic subunit on myosin (52). The present finding that PD-098059 blocked ERK and MYPT-1/Thr850 phosphorylation induced by phenylephrine suggests that ERK-mediated regulation of Ca2+ sensitivity of MLC20 phosphorylation is mediated in part through MYPT-1/Thr850 phosphorylation in the uterine artery. It has been shown that Rho kinase (ROK) is activated in response to G protein activation and is responsible for the phosphorylation of MYPT-1/Thr850 and inhibition of MLCP (9, 26, 27). ROK is activated by the Ras-related monomeric GTPase Rho, which increases force at constant [Ca2+]i (14). However, the precise mechanism of ROK activation that leads to phosphorylation of MYPT-1 in smooth muscle is not known. There are concerns as to the access of ROK to its substrate MYPT-1, because Rho and ROK have to be recruited from a cytosolic pool to the cell membrane for activation (10, 50). The present finding suggests that ERK may be a link between activated ROK and phosphorylation of MYPT-1 in the uterine artery. Consistent with the present finding, previous studies demonstrated that RhoA was involved in the angiotensin II-induced ERK activation and contractions in intact rat mesenteric resistance arteries (33). In addition, Krepinsky et al. (31) also reported that the early activation of RhoA was essential for stretch-induced ERK activation in mesangial cells.

At least two phosphorylation sites of MYPT-1 have been identified at Thr696 and Thr850 (47). The present finding that phenylephrine did not increase phosphorylation of MYPT-1/Thr696 in the uterine artery is consistent with previous findings that MYPT-1/Thr850 is phosphorylated by ROK through agonist activation and that MYPT-1/Thr696 is constitutively phosphorylated by kinases other than ROK and does not respond to agonists (27, 38, 47). These findings suggest an exciting hypothesis that the different phosphorylation sites may regulate MLCP activities differently at basal and agonist-stimulated states. Basal levels of phosphorylated MYPT-1/Thr696 have been demonstrated in the uterine artery in the present study, yet the mechanisms involved in the regulation of MYPT-1/Thr696 in the uterine artery are not clear.

The finding that phenylephrine-induced phosphorylation of CPI-17 was concurrent with the contractions suggests that phosphorylated CPI-17 may also be important in phenylephrine-mediated Ca2+ sensitization of myofilaments. Previous studies suggested that phosphorylated CPI-17/Thr38 was a potent inhibitor of the catalytic subunit (PP1c) of MLCP (47). However, the finding that phenylephrine-induced CPI-17 phosphorylation trailed phenylephrine-induced MLC20 phosphorylation (56) suggests that it may not be important in the regulation of Ca2+ sensitivity of MLC20 phosphorylation in the uterine artery. This is further supported by the results of PKC-mediated responses in the uterine artery. In the present study, we found that activation of PKC by PDBu induced phosphorylation of CPI-17 that preceded the contractions. CPI-17 was initially recognized as a PKC substrate and phosphorylated by PKC (8). However, interestingly, our previous studies demonstrated that activation of PKC produces contractions without changes in MLC20 phosphorylation levels in the uterine artery (56). These findings suggest that PKC-mediated phosphorylation of CPI-17 may not contribute to regulation of MLCP activity and MLC20 phosphorylation in the uterine artery. Woodsome et al. (55) reported that PDBu significantly increased CPI-17/Thr38 phosphorylation and MLC20 phosphorylation in the femoral artery but increased CPI-17/Thr38 phosphorylation without increasing MLC20 phosphorylation in vas deferens smooth muscle. These studies suggest that the role of phosphorylated CPI-17 in the regulation of MLCP activity and Ca2+ sensitivity of MLC20 phosphorylation is tissue specific. It is not clear whether or how phosphorylated CPI-17 regulates Ca2+ sensitivity in the uterine artery. In addition to the thick filament regulation through MLCP, Ca2+ sensitivity is also regulated by the smooth muscle actin-binding protein caldesmon, which inhibits myosin ATPase activity (36). Removal of caldesmon-mediated inhibition can be achieved by phosphorylation of caldesmon and/or binding of other regulatory proteins, e.g., Ca2+/calmodulin, which reverses its inhibitory effect on myosin ATPase. Given that phosphorylated CPI-17 is a regulatory binding protein and binds to target proteins, e.g., the catalytic subunit of MLCP, it is speculated that it may regulate Ca2+ sensitivity in the uterine artery by binding to caldesmon and reversing its inhibitory effect on myosin ATPase.

In the present study, we demonstrated that PDBu activated PKC-{alpha} isozyme in the uterine artery by stimulating its translocation from the cytosol to cell plasma membranes. This is consistent with our previous finding that PDBu increased the particulate-to-cytosolic PKC activity ratio in the uterine artery (57). PKC-{alpha} has been implicated in smooth muscle contractions and is involved in the regulation of myogenic tone of vascular smooth muscle (6, 58). Previous studies demonstrated that activation of PKC may increase phosphorylation of caldesmon at sites other than ERK-dependent phosphorylation sites (48, 51, 53). In sheep aorta, PKC phosphorylated caldesmon in native thin filaments and in the isolated state at multiple sites, i.e., Ser127, Ser587, Ser600, Ser657, Ser686, and Ser726, decreased caldesmon's inhibitory effect on myosin ATPase (53). Our recent studies in pregnant ovine uterine arteries suggested that PKC induced phosphorylation of caldesmon at Ser789 indirectly through activation of ERK, as well as at other site(s) through unknown mechanisms (56). It is the phosphorylation of caldesmon at site(s) other than Ser789 that may be important in reversing the inhibitory effect of caldesmon on myosin ATPase and may lead to contractions. In the present study, we demonstrated coimmunoprecipitation of activated PKC-{alpha} with caldesmon, indicating a direct interaction between activated PKC-{alpha} and caldesmon in the uterine artery. This provides support for PKC-mediated direct phosphorylation of caldesmon in the uterine artery.

In summary, we have demonstrated in pregnant ovine uterine arteries that {alpha}1-adrenoceptor-mediated Ca2+ sensitization of MLC20 phosphorylation is regulated in part through the ERK-dependent phosphorylation of MYPT-1/Thr850. {alpha}1-Adrenoceptor- and PKC-mediated phosphorylation of CPI-17/Thr38 may not be involved in the regulation of MLCP activity and MLC20 phosphorylation. The potential role of phosphorylated CPI-17/Thr38 in the regulation of Ca2+ sensitivity through thin filament regulation in the uterine artery presents an intriguing avenue for future investigation. The present study has also demonstrated a role for PKC-{alpha} isozyme in the PKC-mediated responses and suggested a role for PKC-{alpha} in direct phosphorylation of caldesmon in the uterine artery. Future studies are needed to determine {alpha}1-adrenoceptor-mediated contractions and the thick and thin filament regulatory pathways in the uterine arteries at different stages of gestation and relate them to the known changes in arterial tone during pregnancy.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants HL-57787, HL-67745, and HD-31226 and by the Loma Linda University School of Medicine. D. Xiao is a recipient of American Heart Association Predoctoral Fellowship Award 0215040Y.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Zhang, Center for Perinatal Biology, Dept. of Pharmacology & Physiology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (E-mail: lzhang{at}som.llu.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
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 RESULTS
 DISCUSSION
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