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CARDIOVASCULAR

Regulation of ₁-Adrenoceptor-Mediated Contractions of the Uterine Artery by Protein Kinase C: Role of the Thick- and Thin-Filament Regulatory Pathways

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

Received April 13, 2007; accepted June 8, 2007.

	Abstract

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Previously we demonstrated that activation of protein kinase C (PKC) enhanced ₁-adrenoceptor-induced contractions in nonpregnant uterine arteries (NPUA) by increasing the Ca²⁺ sensitivity but that it inhibited the contractions in pregnant uterine arteries (PUA) by decreasing intracellular Ca²⁺ mobilization. The present study tested the hypothesis that PKC activation differentially regulated the thick- and thin-filament regulatory pathways in ₁-adrenoceptor-induced contractions of NPUA and PUA in sheep. Simultaneous measurements of contractions and phosphorylation levels of 20-kDa regulatory myosin light chain (LC₂₀) in the same tissue revealed that the PKC activator phorbol-12,13-dibutyrate (PDBu) inhibited phenylephrine-induced phosphorylation of LC₂₀ and contractions in PUA. In NPUA, PDBu significantly potentiated phenylephrine-induced contractions without significantly changing phosphorylation levels of LC₂₀. Further studies in NPUA demonstrated that PDBu-mediated potentiation of phenylephrine-induced contractions was associated with a significant increase in phosphorylation levels of extracellular signal-regulated kinase (ERK_42/44) and caldesmon-Ser⁷⁸⁹, measured simultaneously with the tension in the same tissue. In addition, the ERK_42/44 inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] and the actin polymerization inhibitor cytochalasin B produced a concentration-dependent inhibition of PDBu-mediated potentiation of phenylephrine-induced contractions in NPUA. The results suggest that activation of PKC inhibits ₁-adrenoceptor-mediated contractions in PUA through down-regulation of the thick-filament pathway and decreased myosin light chain phosphorylation, but that it enhances the contractions in NPUA through its effect on the thin-filament regulatory pathway and activation of ERK/caldesmon and actin polymerization.

Recently, we have demonstrated that activation of protein kinase C (PKC) enhances ₁-adrenoceptor-induced contractions in nonpregnant uterine arteries by increasing Ca²⁺ sensitivity but that it inhibits contractions in pregnant uterine arteries by decreasing intracellular Ca²⁺ mobilization (Zhang et al., 2006). These findings present an intriguing dichotomy in mechanisms of PKC in the regulation of uterine artery contractility at different physiological states, i.e., pregnancy and nonpregnancy. It is not known to what extent that thick- and/or thin-filament regulatory pathways contribute to the dissociative mechanisms of PKC in regulation of ₁-adrenoceptor-induced contractions in nonpregnant and pregnant uterine arteries. The previous finding that PKC activator PDBu decreased phenylephrine-induced Ca²⁺ mobilization in pregnant uterine arteries (Zhang et al., 2006) suggests an inhibition of thick-filament pathway. This hypothesis needs to be tested by measuring phosphorylation levels of LC₂₀ in uterine arteries.

In addition to thick-filament regulation, previous studies have demonstrated importance of thin-filament regulatory pathway in PKC-mediated regulation of the Ca²⁺ sensitivity and contractions in uterine arteries (Xiao et al., 2004; Xiao and Zhang, 2005). Among other mechanisms, caldesmon functions as a thin-filament regulatory protein inhibiting smooth muscle contractions at given levels of [Ca²⁺]_i and LC₂₀ phosphorylation, and phosphorylation of caldesmon reverses its inhibitory effect (Katsuyama et al., 1992; Matsumura and Yamashiro, 1993; Morgan and Gangopadhyay, 2001; Wier and Morgan, 2003). Caldesmon is a thin-filament-associated protein that binds to both actin and myosin and inhibits actin-activated myosin ATPase activity. In intact vascular smooth muscle, extracellular signal-regulated kinase (ERK_42/44) has been demonstrated as a physiologically relevant caldesmon kinase that mediates caldesmon phosphorylation (Adam et al., 1989). It has been proposed that ERK_42/44-mediated phosphorylation of caldesmon reverses the inhibitory activity of caldesmon on actin-activated myosin ATPase, thereby increasing contractions at given levels of [Ca²⁺]_i and LC₂₀ phosphorylation (Horowitz et al., 1996; Morgan and Gangopadhyay, 2001). In uterine arteries, activation of PKC produces time-dependent increases in phosphorylation of ERK_42/44 and ERK_42/44-dependent phosphorylation of caldesmon at Ser⁷⁸⁹ (Xiao et al., 2004). Whether and to what extent these thin-filament mechanisms contribute to PKC-mediated enhancement of ₁-adrenoceptor-induced contractions in nonpregnant uterine arteries remains unclear.

The present study tests the hypothesis that different effects of PKC activation on ₁-adrenoceptor-induced contractions observed in nonpregnant and pregnant uterine arteries are due, in part, to its differential regulations on thick- and thin-filament regulatory pathways. To test this hypothesis, we first determine the relationship between tension and LC₂₀ phosphorylation by measuring tension development and phosphorylation levels of LC₂₀ simultaneously in the same tissue in the presence of PDBu and phenylephrine. We then determine the role of ERK_42/44 in the effect of PKC on phenylephrine-induced contractions by measuring phosphorylation levels of ERK_42/44 and caldesmon-Ser⁷⁸⁹ simultaneously with contractions induced by PDBu and phenylephrine. To determine the cause and effect relation between activation of ERK_42/44 and the effect of PKC on phenylephrine-induced contractions, we examine contractions in the presence of ERK_42/44 inhibitor PD098059. Given that actin polymerization plays an important role in thin-filament regulatory pathway, we also examine the effect of cytochalasin B, an inhibitor of actin polymerization, on PKC-mediated enhancement of ₁-adrenoceptor-induced contractions in nonpregnant uterine arteries.

	Materials and Methods

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Tissue Preparation. Nonpregnant and near-term pregnant (140-day gestation) ewes were anesthetized with 10 mg/kg thiamylal, administered via the external left jugular vein. The ewes were then intubated, and anesthesia was maintained with 1.5 to 2.0% halothane in O₂ throughout the surgery. An incision was made in the abdomen to expose the uterus. The uterine arteries were isolated and removed without stretching and were placed in a modified Krebs' solution, pH 7.4, of the following composition: 115.2 mM NaCl, 4.7 mM KCl, 1.80 mM CaCl₂, 1.16 mM MgSO₄, 1.18 mM KH₂PO₄, 22.14 mM NaHCO₃, 0.03 mM EDTA, and 7.88 mM dextrose. The Krebs' solution was oxygenated with a mixture of 95% O₂, 5%CO₂. The third (nonpregnant) and fourth (pregnant) branches of the main uterine arteries with similar external diameter were collected and used in the present studies, as described previously (Zhang et al., 2006). After the tissues were removed, animals were killed with T-61 euthanasia solution (Hoechst-Roussel, Somerville, NJ). All procedures and protocols used in the present study were approved by the Animal Research Committee of Loma Linda University (Loma Linda, CA) and followed the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Simultaneous Measurement of Tension and LC₂₀ Phosphorylation. LC₂₀ phosphorylation and contractile tension were measured simultaneously in the same tissue of nonpregnant and pregnant uterine arteries, as described previously (Xiao et al., 2004). In brief, pregnant and nonpregnant uterine arterial rings were equilibrated in the tissue bath and the optimal tension was obtained. The tissues were then treated with phenylephrine in the absence or presence of pretreatment with the PKC activator PDBu for 10 min. In our previous studies of simultaneous measurement of [Ca²⁺]_i and contractions (Zhang et al., 2006), we demonstrated that pretreatment of PDBu inhibited 3 µM phenylephrine-induced increase in [Ca²⁺]_i and contractions in pregnant uterine arteries. In nonpregnant uterine arteries, PDBu had no effect on phenylephrine-induced contractions at the concentration of 3 µM, but it potentiated it at lower concentrations of phenylephrine. To be consistent with the previous studies, the concentrations of phenylephrine used in the present study were 3 and 1 µM for pregnant and nonpregnant uterine arteries, respectively. Because the previous studies demonstrated that 0.1 and 1 µM PDBu produced the maximal effect in nonpregnant and pregnant uterine arteries, respectively (Zhang et al., 2006), these concentrations were used for PDBu in the present study. Tensions developed were continuously recorded with an online computer. To measure phosphorylation levels of LC₂₀ simultaneously in the same tissue, arterial rings were snap-frozen with liquid N₂-cooled clamps at the plateau of tension, and they were rapidly immersed in a dry ice-acetone slurry that contained a 10% trichloroacetic acid (TCA) and 10 mM DTT mixture. Tissues were then stored at –80°C until analysis of phosphorylated LC₂₀. Tissue LC₂₀ phosphorylation levels were measured as described previously (Xiao et al., 2004). Tissues were brought to room temperature in a dry ice-acetone-TCA-DTT mixture, and they were washed three times with ether to remove the TCA. Tissues were then extracted in 100 µl of sample buffer, pH 8.6, that contained 20 mM Tris base and 23 mM glycine, 8.0 M urea, 10 mM DTT, 10% glycerol, and 0.04% bromphenol blue. Samples (20 µl) of tissue extraction were electrophoresed at 12 mA for 2.5 h after a 30-min prerun in 1.0-mm minipolyacrylamide gels that contained 10% acrylamide, 0.27% bisacrylamide, 40% glycerol, and 20 mM Tris base, pH 8.8. Proteins were transferred to nitrocellulose membranes and subjected to immunoblot with a specific monoclonal anti-LC₂₀ antibody (1:500). Goat anti-mouse IgM conjugated with horseradish peroxidase was used as a secondary antibody (1:2000). Bands were detected using enhanced chemiluminescence, visualized on films, and analyzed using Kodak electrophoresis documentation and analysis system and Kodak 1D image analysis software (Eastman Kodak, Rochester, NY). Phosphorylated LC₂₀ were expressed as percentage of the intensity of the phosphorylated LC₂₀ band over the sum of the phosphorylated plus the unphosphorylated LC₂₀ bands.

Simultaneous Measurement of Tension and Phosphorylation of ERK_42/44 and Caldesmon-Ser⁷⁸⁹. Phosphorylation levels of caldesmon-Ser⁷⁸⁹ and ERK_42/44 and contractions were measured simultaneously in the same tissue of nonpregnant uterine arteries. Uterine arterial rings were equilibrated in the tissue bath and the optimal tensions were obtained. Tissues were then subjected to stimulation with 1 µM phenylephrine in the absence or presence of pretreatment with 0.1 µM PDBu. Tensions developed were continuously recorded with an online computer. To measure phosphorylation levels of caldesmon-Ser⁷⁸⁹ and ERK_42/44 simultaneously in the same tissue, arterial rings were snap-frozen at the plateau of tension, as described above. Tissues were then homogenized in an icecold lysis buffer, pH 7.5, that contained 20 mM Tris-HCl, 250 mM sucrose, 5 mM EDTA, 5 mM EGTA, 0.2% Triton X-100, 10 mM -mercaptoethanol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 50 µM leupeptin, 1 mM dithiothreitol, and 2 µg/ml aprotinin. Homogenates were centrifuged at 6000g for 5 min at 4°C, and the supernatants were collected. Proteins were determined with a protein assay kit (Bio-Rad, Hercules, CA). Samples with 5 µgof protein were subjected to electrophoresis on 7.5% [phosphorylated (p)-CaD-Ser⁷⁸⁹] or 10% p-ERK_42/44 sodium dodecyl sulfate-polyacrylamide gel, and then they were transferred electrophoretically to nitrocellulose membranes. The membranes were incubated at room temperature for 1 h in Tris-buffered saline solution that contained 5% dried milk and 0.5% Tween 20, followed by incubation with primary anti-p-CaD-Ser⁷⁸⁹ or anti-p-ERK_42/44 antibodies overnight at 4°C and secondary antibody of anti-rabbit IgG for 1 h at room temperature. Bands were detected using enhanced chemiluminescence, visualized on Hyperfilm (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and analyzed with the Kodak 1D image analysis software.

Contraction Studies. Nonpregnant uterine arteries were dissected and cut into 2-mm ring segments. Isometric tension was measured in the Krebs' solution in a tissue bath at 37°C, as described previously (Zhang et al., 2006). In brief, tissues were equilibrated for 60 min, and then they were gradually stretched to the optimal resting tension as determined by the tension developed in response to 120 mM KCl added at each stretch level. Contractile tensions were recorded with an online computer. Tissues were then stimulated with cumulative additions of phenylephrine in approximate one-half log increments to produce a concentration-response curve. After washing away phenylephrine, tissues were relaxed to the baseline, and they were recovered at the resting tension for 30 min. As described previously (Zhang et al., 2006), the second concentration-response curves of phenylephrine-induced contractions were then repeated in the absence or presence of 0.1 µM PDBu for 10 min with or without the ERK inhibitor PD098059 at 10, 30, or 60 µM for 20 min or the actin polymerization inhibitor cytochalasin B at 5, 10, and 30 µM for 20 min. There was no significant time-related shift of phenylephrine-response curves. Contractions were expressed as percentage of the KCl response. Previous studies demonstrated that KCl-induced contractions were the same in uterine arteries from pregnant and nonpregnant animals (Xiao and Zhang, 2002, 2004).

Materials. Phenylephrine, PDBu, PD098059, cytochalasin B, and monoclonal anti-LC₂₀ antibody were obtained from Sigma-Aldrich (St. Louis, MO). Anti-p-CaD-Ser⁷⁸⁹ antibody and the secondary antibodies (goat anti-mouse IgM and anti-rabbit IgG conjugated with horseradish peroxides) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p-ERK_42/44 antibody was from Cell Signaling Technology Inc. (Danvers, MA). All other electrophoretic and immunoblot reagents were from Bio-Rad (Hercules, CA). General laboratory reagents were of analytical grade or better, and they were purchased from Sigma-Aldrich and Fisher Scientific (Pittsburgh, PA). All drug solutions were prepared freshly each day, and they were kept on ice throughout the experiment.

Data Analysis. Data were expressed as means ± S.E.M. Differences were evaluated for statistical significance (P < 0.05) using one-way ANOVA followed by Newman-Keuls post hoc testing. For the analysis of concentration-response curves, data were analyzed by computer-assisted nonlinear regression to fit the data using Graph-Pad Prism (GraphPad Software Inc., San Diego, CA). Values of pD₂ (–log EC₅₀) and the maximal response obtained were used in the statistical analysis.

	Results

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Effect of PDBu on Phenylephrine-Induced LC₂₀ Phosphorylation and Contractions. Figure 1 shows the effects of PDBu on phenylephrine-induced LC₂₀ phosphorylation and contractions, measured simultaneously in the same tissue, in nonpregnant and pregnant uterine arteries. In both nonpregnant and pregnant uterine arteries, phenylephrine induced contractions with corresponding increases in LC₂₀ phosphorylation in the same tissue. In contrast, PDBu increased contractions in the absence of any significant changes of phosphorylation levels of LC₂₀. These findings were consistent with the previous results that showed LC₂₀ phosphorylation-dependent, and -independent contractions caused by phenylephrine and PDBu, respectively, in the uterine arteries (Xiao and Zhang, 2005). In pregnant uterine arteries, PDBu significantly decreased phenylephrine-induced contractions from 70.9 ± 6.6 to 45.5 ± 6.5% K⁺ maximum (P < 0.05), with a corresponding reduction of phenylephrine-induced LC₂₀ phosphorylation levels from 30.7 ± 1.8 to 13.5 ± 1.7% (P < 0.05) (Fig. 1). In contrast, in nonpregnant uterine arteries, PDBu significantly enhanced phenylephrine-induced contractions from 10.5 ± 2.4 to 54.0 ± 15.8% K⁺ maximum (P < 0.05), consistent with the previous finding (Zhang et al., 2006). However, phenylephrine-induced phosphorylation levels of LC₂₀, measured simultaneously in the same tissue, were not significantly affected with PDBu (Fig. 1).

View larger version (52K): [in this window] [in a new window]   Fig. 1. Effect of PDBu on phenylephrine-induced LC20 phosphorylation and contractions in nonpregnant (NPUA) and pregnant (PUA) uterine arteries. PDBu-(0.1 µM for NPUA and 1 µM for PUA) and/or phenylephrine (PE; 1 µM for NPUA and 3 µM for PUA)-induced LC20 phosphorylation and contractions were measured simultaneously in the same tissues, as described under Materials and Methods. Top and middle, p-LC20 and unphosphorylated (unp)-LC20 were detected by Western immunoblotting, and levels of p-LC20 are expressed as percentage of p-LC20/(p-LC20 + unp-LC20). Bottom, simultaneously measured contractions are expressed as percentage of 120 mM KCl-induced contraction (% K+). Data are means ± S.E.M. of tissues from four animals. *, P < 0.05, versus control. , P < 0.05, versus phenylephrine treatment alone.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Effect of PDBu on phenylephrine-induced phosphorylation of ERK42/44 and contractions in nonpregnant uterine arteries. PDBu-(0.1 µM) and/or 1 µM PE-induced phosphorylation of ERK42/44 and contractions were measured simultaneously in the same tissue, as described under Materials and Methods. Top and middle, phosphorylated ERK42 and ERK44 (p-ERK42 and p-ERK44) were detected by Western immunoblotting. The levels of p-ERK42 and p-ERK44 induced by PDBu and/or phenylephrine are expressed as -fold of control. Bottom, simultaneously measured contractions are expressed as percentage of 120 mM KCl-induced contraction (% K+). Data are means ± S.E.M. of tissues from four animals. *, P < 0.05, versus control. , P < 0.05, versus PDBu plus phenylephrine treatment.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Effect of PDBu on phenylephrine-induced phosphorylation of caldesmon-Ser789 in nonpregnant uterine arteries. PDBu-(0.1 µM) and/or 1 µM PE-induced phosphorylation levels of caldesmon-Ser789 were measured in the same tissues described in Fig. 2. p-CaD-Ser789 was detected by Western immunoblotting, and it is expressed as -fold of control. Data are means ± S.E.M. of tissues from four animals. *, P < 0.05, versus control. , P < 0.05, versus PDBu plus phenylephrine treatment.

Effect of Inhibition of ERK_42/44 and Actin Polymerization on PDBu-Potentiated Phenylephrine-Induced Contractions. The finding that PDBu significantly increased phenylephrine-induced phosphorylation of ERK_42/44 that correlated with the increased tension development (Fig. 3) suggests a role for ERK_42/44 as a thin-filament mechanism in PDBu-mediated potentiation of phenylephrine-induced contractions. To determine the cause and effect relationship, we examined the effect of ERK_42/44 inhibition on PDBu-mediated potentiation of phenylephrine-induced contractions in nonpregnant uterine arteries. Figure 4 shows that phenylephrine produced concentration-dependent contractions of nonpregnant uterine arteries. In the presence of 0.1 µM PDBu, the concentration-response curve of phenylephrine-induced contractions was markedly shifted to the left with a significant increase in the pD₂ value from 5.2 ± 0.1 to 7.7 ± 0.3 (P < 0.05), representing a more than 300-fold increase in the potency of phenylephrine-induced contractions. Pretreatment of tissues with the ERK inhibitor PD098059 (10, 30, and 60 µM) produced a concentration-dependent inhibition of PDBu-mediated potentiation of phenylephrine-induced contractions (Fig. 4). Given that actin polymerization may play an important role in PKC/ERK_42/44-mediated thin-filament regulation, we also examined the effect of cytochalasin B, an inhibitor of actin polymerization, on PDBu-mediated enhancement of phenylephrine-induced contractions in nonpregnant uterine arteries. As shown in Fig. 5, pretreatment of tissues with cytochalasin B (5, 10, and 30 µM) produced a concentration-dependent inhibition of PDBu-mediated potentiation of phenylephrine-induced contractions.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effect of ERK inhibition on PDBu-potentiated phenylephrine-induced contractions in nonpregnant uterine arteries. A, effect of 0.1 µM PDBu and/or ERK inhibitor PD098059 at 10, 30, and 60 µM on concentration-response curves of phenylephrine-induced contractions. B, data analysis of the pD2 values and the maximal responses (Tmax) of phenylephrine-induced contractions. Phenylephrine-induced contractions are expressed as percentage of 120 mM KCl-induced contraction (% K+). Data are means ± S.E.M. Control, n = 6; PDBu, n = 10; PD098059 at 10, 30, and 60 µM, all n = 5. *, P < 0.05, versus control. , P < 0.05, versus PDBu alone.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Effect of inhibition of actin polymerization on PDBu-potentiated phenylephrine-induced contractions in nonpregnant uterine arteries. A, effect of 0.1 µM PDBu and/or actin polymerization inhibitor cytochalasin B at 5, 10, and 30 µM on concentration-response curves of phenylephrine-induced contractions. B, data analysis of the pD2 values, and the maximal responses (Tmax) of phenylephrine-induced contractions. Phenylephrine-induced contractions are expressed as percentage of 120 mM KCl-induced contraction (% K+). Data are means ± S.E.M. Control, n = 6; PDBu, n = 10; cytochalasin B at 5, 10, and 30 µM, all n = 5. *, P < 0.05 versus control. , P < 0.05, versus PDBu alone.

	Discussion

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We have previously demonstrated that activation of PKC enhances ₁-adrenoceptor-induced contractions in nonpregnant uterine arteries by increasing the Ca²⁺ sensitivity but that it inhibits the contractions in pregnant uterine arteries by decreasing intracellular Ca²⁺ mobilization (Zhang et al., 2006). The finding that 0.1 and 1 µM PDBu induced comparable contractions in nonpregnant and pregnant uterine arteries, respectively, but produced opposite effects on ₁-adrenoceptor-induced contractions in the two vessels (Zhang et al., 2006), suggests that PDBu-mediated contractions per se may not be related to its specific downstream effects on ₁-adrenoceptor-induced contractions. Although PDBu was used as an experimental tool to increase PKC activity in the present study, physiological activation of PKC in vascular smooth muscle can be seen in response to pressure or stretch, which plays an important role in the regulation of myogenic tone (Davis and Hill, 1999). The present follow-up study attempted to elucidate the role of the thick- and/or thin-filament regulatory pathways that contribute to the dichotomy of PKC mechanisms in the regulation of ₁-adrenoceptor-induced contractions in nonpregnant and pregnant uterine arteries. In the uterine artery, ₁-adrenoceptor-mediated contractions are regulated through both thick-filament regulatory pathway (i.e., LC₂₀ phosphorylation-dependent) and thin-filament regulatory pathway (i.e., LC₂₀ phosphorylation-independent) (Xiao et al., 2004; Xiao and Zhang, 2005). In agreement with the previous findings, the present study showed that phenylephrine induced contractions with corresponding increases in phosphorylation of LC₂₀ in both pregnant and nonpregnant uterine arteries. In contrast, PDBu produced contractions in the absence of significant changes in LC₂₀ phosphorylation in the uterine arteries. This is consistent with the previous studies that demonstrated dissociation between LC₂₀ phosphorylation and tension development in response to phorbol esters in vascular smooth muscle, including the uterine arteries (Fujiwara et al., 1988; Sutton and Haeberle, 1990; Laporte et al., 1994; Xiao and Zhang, 2005). Most importantly, the present study showed that PDBu-mediated inhibition of phenylephrine-induced contractions in pregnant uterine arteries was associated with a significant decrease in phosphorylation of LC₂₀, measured simultaneously in the same tissue but that PDBu potentiated phenylephrine-induced contractions in nonpregnant uterine arteries in the absence of any significant changes in LC₂₀ phosphorylation. Given the previous finding that PDBu inhibited ₁-adrenoceptor-mediated increases in [Ca²⁺]_i in pregnant uterine arteries (Zhang et al., 2006), the present study suggests that the inhibitory effect of PKC activation on ₁-adrenoceptor-induced contractions in pregnant vessels was mediated by suppressing the Ca²⁺-dependent thick-filament pathway. Although multiple mechanisms have been suggested in PKC-mediated inhibition of ₁-adrenoceptor-induced intracellular Ca²⁺ mobilization resulting in decreased myosin light chain kinase activity (Zhang et al., 2006), the potential direct effect of PKC on myosin light chain kinase-specific activity remains to be further investigated.

The finding of the dissociation between LC₂₀ phosphorylation and PDBu-mediated increase in phenylephrine-induced contractions in nonpregnant arteries indicates a thin-filament mechanism in the PKC-mediated regulation of ₁-adrenoceptor-induced contractions in nonpregnant uterine arteries. The finding that PKC has differential regulatory effects on thick- and thin-filament pathways in ₁-adrenoceptor-induced contractions in nonpregnant and pregnant uterine arteries is intriguing, and it provides insight into understanding adaptation of uterine vascular contractile mechanisms in pregnancy. Consistent with the present study, previous studies suggested a transition from thin-filament to thick-filament regulatory mechanisms in the uterine artery during pregnancy and that the Ca²⁺-dependent thick-filament pathway, i.e., changes in LC₂₀ phosphorylation, predominated in ₁-adrenoceptor-mediated contractions in pregnant uterine arteries (Annibale et al., 1989, 1990; Xiao et al., 2004; Xiao and Zhang, 2005). This is supported by the findings of increased ₁-adrenoceptor densities and synthesis of inositol-1,4,5-trisphosphate in pregnant, compared with nonpregnant, uterine arteries (Xiao et al., 2003).

The present finding that PDBu significantly increased phenylephrine-induced phosphorylation of ERK_42/44 that correlated with the increased tension development, measured simultaneously in the same tissue, suggests a role for ERK_42/44 activation as a thin-filament mechanism in PDBu-mediated potentiation of phenylephrine-induced contractions in nonpregnant uterine arteries. Previous studies have demonstrated that serotonin, endothelin-1, and angiotensin II potentiate ₁-adrenoceptor-induced contractions of vascular smooth muscle and that activation of PKC plays a key role in these nonadrenoceptor-mediated potentiations (Henrion et al., 1992; Watts, 2000; Matsumura et al., 2001). In addition, the involvement of ERK_42/44 has been suggested (Watts, 2000). Activation of ERK_42/44 is dependent on a dual phosphorylation on Tyr¹⁸⁵ and Thr¹⁸⁷ by mitogen-activated/extracellular-regulated kinase kinase (Anderson et al., 1990). ERK_42/44 has been proposed to regulate smooth muscle contractions (Adam et al., 1995; Katoch and Moreland, 1995; Watts, 1996; Dessy et al., 1998; Xiao and Zhang, 2002; Zhao et al., 2003). In intact vascular smooth muscle, ERK has been demonstrated as a physiologically relevant caldesmon kinase that mediates caldesmon phosphorylation (Adam et al., 1989). Caldesmon functions as a thin-filament regulatory protein and exerts an inhibitory effect on vascular smooth muscle contractions (Ngai and Walsh, 1984; Earley et al., 1998; Morgan and Gangopadhyay, 2001). It has been proposed that ERK-mediated phosphorylation of caldesmon reverses the inhibitory activity of caldesmon on actin-activated myosin ATPase, thereby activating the thin-filament pathway (Horowitz et al., 1996; Morgan and Gangopadhyay, 2001). The finding that low concentrations of PDBu (0.1 µM) and phenylephrine (1 µM) alone produced small but not significant increases in ERK_42/44 phosphorylation, but increased caldesmon phosphorylation at the ERK_42/44-specific site Ser⁷⁸⁹, is intriguing, and it suggests a typical phenomenon that small signal is amplified through the intracellular signaling cascades. Most importantly, the present study demonstrated that PDBu was able to enhance phenylephrine-induced phosphorylation of both ERK_44/42 and caldesmon-Ser⁷⁸⁹ and contractions in the same tissue. The cause and effect relation between activation of ERK_42/44 and the enhancement of contractions in PKC-mediated effect was demonstrated with the present study showing that inhibition of ERK_42/44 with PD098059 abolished PDBu-mediated increases in phenylephrine-induced contractions. Our previous studies demonstrated that 30 µM PD098059 inhibited PDBu-stimulated phosphorylation of ERK_44/42 and caldesmon-Ser⁷⁸⁹ in ovine uterine arteries (Xiao et al., 2004). It should be noted that although only phosphorylation of caldesmon-Ser⁷⁸⁹ was determined in the present study because of the availability of the antibody, caldesmon can be phosphorylated at several other sites. It has been shown that PKC phosphorylates sheep aorta caldesmon both in native thin filaments and in the isolated state at multiple sites of Ser¹²⁷, Ser⁵⁸⁷, Ser⁶⁰⁰, Ser⁶⁵⁷, Ser⁶⁸⁶, and Ser⁷²⁶, and PKC-mediated phosphorylation of both intact caldesmon and of its C-terminal fragment of 658 to 756 significantly decreases its ability to inhibit acto-heavy meromyosin ATPase (Vorotnikov et al., 1994). Whether and to what extent these phosphorylation sites contribute to PKC-mediated potentiation of ₁-adrenoceptor-induced contractions in the uterine arteries remains an intriguing question for further studies.

In addition to its role in inhibiting actin-activated myosin ATPase, caldesmon has been shown to be important in maintaining actin filament stability and in inhibiting Arp2/3-dependent actin polymerization (Galazkiewicz et al., 1989; Matsumura and Yamashiro, 1993; Yamakita et al., 2003; Hai and Gu, 2006). The inhibitory effect of caldesmon on actin polymerization can be reversed by phosphorylation with either ERK_44/42 or cdc2 kinase (Yamakita et al., 2003; Hai and Gu, 2006). In addition, ERK_44/42 can phosphorylate an actin-binding protein, cortactin, resulting in activation of the Arp2/3 complex and actin polymerization (Martinez-Quiles et al., 2004). It has been demonstrated that the polymerization of actin filaments from monomeric globular actin (G-actin) to filamentous actin (F-actin), occurring independently of changes in intracellular Ca²⁺ concentrations and LC₂₀ phosphorylation, is an important cellular mechanism of thinfilament regulation in smooth muscle contraction (Jones et al., 1999; Mehta and Gunst, 1999; Gunst and Fredberg, 2003; Ozaki et al., 2004), including contractions induced by PDBu and ₁-adrenoceptor agonists (Nunes, 2002; Tang and Tan, 2003; Zhao et al., 2004; Chen et al., 2006). The present finding that inhibition of actin polymerization with cytochalasin B produced a concentration-dependent inhibition of PDBu-mediated potentiation of phenylephrine-induced contractions in nonpregnant uterine arteries suggests a thin-filament mechanism of actin polymerization in PKC-mediated regulation of ₁-adrenoceptor-induced contractions. It has been demonstrated that the inhibition of contractions by actin polymerization inhibitors does not result from the disruption of thick-filament regulatory pathway (Obara and Yabu, 1994; Mehta and Gunst, 1999).

In summary, the present study demonstrated that PKC activation by PDBu differentially regulated ₁-adrenoceptor-mediated thick- and thin-filament pathways and that it resulted in the opposite effects on ₁-adrenoceptor-mediated contractions in pregnant and nonpregnant uterine arteries. The transition from a thin- to thick-filament mechanism in PKC-mediated regulation of ₁-adrenoceptor-induced contractions in nonpregnant and pregnant uterine arteries provides insight into the adaptation of uterine artery contractile mechanisms in pregnancy. Further studies are needed to investigate the mechanisms of steroid hormones in PKC-regulated ₁-adrenoceptor signaling pathways in the uterine arteries and their adaptation to pregnancy.

	Footnotes

 
This work was supported in part by the National Institutes of Health Grants HL57787 and HD31226 and by the Loma Linda University School of Medicine.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.124313.

ABBREVIATIONS: LC₂₀, 20-kDa regulatory myosin light chain; PKC, protein kinase C; PDBu, phorbol-12,13-dibutyrate; ERK, extracellular signal-regulated kinase; CaD, caldesmon; TCA, trichloroacetic acid; DTT, dithiothreitol; p-, phosphorylated; ANOVA, analysis of variance; NPUA, nonpregnant uterine arteries; PUA, pregnant uterine arteries; PE, phenylephrine; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one.

Address correspondence to: Dr. Lubo Zhang, Department of Pharmacology and Physiology, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, CA 92350. E-mail: lzhang{at}llu.edu

	References

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