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ERK-mediated uterine artery contraction: role of thick and thin filament regulatory pathways

Center for Perinatal Biology, Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, California 92350

Submitted 16 October 2003 ; accepted in final form 16 December 2003

	ABSTRACT

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We have demonstrated that extracellular signal-regulated kinase (ERK) plays an important role in the regulation of uterine artery contraction. The present study tested the hypothesis that ERK regulates thick and thin filament regulatory pathways in the uterine artery. Isometric tension, intracellular free Ca²⁺ concentration ([Ca²⁺]_i), and 20-kDa myosin light chain (LC₂₀) phosphorylation were measured simultaneously in uterine arteries isolated from near-term (140 days gestation) pregnant sheep. Phenylephrine produced time-dependent increases in [Ca²⁺]_i and LC₂₀ phosphorylation that preceded the contraction, which were inhibited by the MEK (ERK) inhibitor PD-098059. In addition, PD-098059 decreased the intercept of the regression line of LC₂₀ phosphorylation vs. [Ca²⁺]_i but increased the rate of tension development vs. LC₂₀ phosphorylation. In contrast to phenylephrine, phorbol 12,13-bibutyrate (PDBu) produced contractions without changing [Ca²⁺]_i or LC₂₀ phosphorylation. PD-098059 potentiated PDBu-induced contractions without affecting [Ca²⁺]_i and LC₂₀ phosphorylation. PDBu produced time-dependent increases in phosphorylation of p42 and p44 ERK and ERK-dependent phosphorylation of caldesmon at Ser⁷⁸⁹ in the uterine artery. PD-098059 blocked PDBu-mediated phosphorylation of p42 and p44 ERK and caldesmon. The results indicate that ERK may regulate force by a dual regulation of thick and thin filaments in uterine artery smooth muscle. ERK potentiates the thick filament regulatory pathway by enhancing LC₂₀ phosphorylation via increases in [Ca²⁺]_i and Ca²⁺ sensitivity of LC₂₀ phosphorylation. In contrast, ERK attenuates the thin filament regulatory pathway and suppresses contractions independent of changes in LC₂₀ phosphorylation in the uterine artery.

isometric tension; intracellular free calcium concentration; myosin light chain phosphorylation; PD-098059; phenylephrine; phorbol 12,13-dibutyrate

Extracellular signal-regulated kinase (ERK) has been proposed to regulate smooth muscle contraction (2, 11, 16, 17, 26, 52, 55, 57). Different signaling transduction mechanisms have been reported in ERK-mediated regulation of smooth muscle contraction. Previous studies showed that the MEK (ERK) inhibitor PD-098059 did not affect agonist-induced [Ca²⁺]_i in isolated smooth muscle cells but inhibited agonist-induced smooth muscle contraction by decreasing Ca²⁺ sensitivity of contractile proteins (1, 2, 11, 17, 21, 38, 49). It has been proposed that ERK mediates smooth muscle contraction through the thin filament regulatory pathway by phosphorylation of caldesmon (CaD) (2, 21, 33). However, by measuring [Ca²⁺]_i and tension simultaneously in the intact tissue, our recent studies clearly demonstrated that PD-098059-mediated inhibition of phenylephrine-induced contraction was associated with a decrease in [Ca²⁺]_i and Ca²⁺ sensitivity in sheep uterine artery smooth muscle (55). This suggests that, in addition to Ca²⁺-independent pathways, as proposed previously, the ERK signaling pathway also involves Ca²⁺-dependent components of vascular contractions. Similarly, recent studies in porcine carotid artery suggest that ERK might regulate force in arterial smooth muscle by inhibiting LC₂₀ phosphorylation (8).

Recently, we demonstrated that ERK plays an important role in the regulation of uterine artery contraction and that the effect of ERK on the uterine artery is altered during pregnancy (55). In the present study, we tested the hypothesis that ERK regulates uterine artery contraction through thick and thin filament regulatory pathways. We examined the effects of the MEK (ERK) inhibitor PD-098059 on phenylephrine- and phorbol 12,13-dibutyrate (PDBu)-induced increases in [Ca²⁺]_i, LC₂₀ phosphorylation, and contractile tension in uterine arteries obtained from pregnant sheep. We also examined phosphorylation of ERK and ERK-dependent phosphorylation of the thin filament regulatory protein CaD. The present study provides evidence that ERK potentiates the thick filament regulatory pathway by enhancing LC₂₀ phosphorylation but attenuates the thin filament regulatory pathway by suppressing contractions independent of changes in LC₂₀ phosphorylation in the uterine artery.

	METHODS

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Tissue preparation. 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 in the abdomen was made, and the uterus was exposed. The uterine arteries were isolated, removed without stretching, and placed into 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 National Institutes of Health guidelines for the care and use of laboratory animals.

Contraction studies. The fourth branches of the main uterine arteries were separated from the surrounding tissue and cut into 2-mm ring segments. The small branches of the main uterine artery were chosen, because they are much closer in characteristics to arterioles and play a substantial role in vascular resistance. Isometric tension was measured in Krebs solution in a tissue bath at 37°C, as described previously (55). After 60 min of equilibration, 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 pretreated with 30 µM PD-098059 or vehicle (DMSO) for 30 min and then stimulated with phenylephrine or PDBu. Tension was recorded with an online computer. To measure LC₂₀ phosphorylation simultaneously in the same tissues, arterial rings were snap frozen with liquid N₂-cooled clamps at the indicated times and immersed in a dry ice-acetone slurry containing 10% trichloroacetic acid (TCA) and 10 mM DTT. The rings were stored at –80°C until they were used.

Measurement of LC₂₀ phosphorylation. Tissues were brought to room temperature in the dry ice-acetone-TCA-DTT mixture and then washed three times with ether to remove the TCA. Tissues were then extracted in 100 µl of sample buffer containing 20 mM Tris base and 23 mM glycine (pH 8.6), 8.0 M urea, 10 mM DTT, 10% glycerol, and 0.04% bromphenol blue, as previously described (8, 28). Samples (20 µl) were electrophoresed at 12 mA for 2.5 h after a 30-min prerun in 1.0 mm mini-polyacrylamide gels containing 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 LC₂₀ antibody (1:500; Sigma, St. Louis, MO). Goat anti-mouse IgG conjugated with horseradish peroxidase was used as a secondary antibody (1:2,000; Calbiochem). Bands were detected with enhanced chemiluminescence, visualized on films, and analyzed with the Kodak electrophoresis documentation and analysis system and Kodak 1D image analysis software. Moles of phosphate per mole of light chain were calculated by dividing the density of the phosphorylated band by the sum of the densities of the phosphorylated plus the unphosphorylated bands.

Simultaneous measurement of [Ca²⁺]_i and tension. Simultaneous recordings of contraction and [Ca²⁺]_i in the same tissue were conducted as described previously (56). Briefly, each arterial ring was attached to an isometric force transducer in a 5-ml tissue bath mounted on an intracellular Ca²⁺ analyzer (model CAF-110, Jasco, Tokyo, Japan). The tissue was equilibrated in Krebs buffer under a resting tension of 0.5 g for 40 min. The ring was then loaded with 5 µM fura 2-AM for 3 h in the presence of 0.02% Cremophor EL at room temperature (25°C). After loading, the tissue was washed with Krebs solution at 37°C for 30 min to allow for hydrolysis of fura 2 ester groups by endogenous esterase. Contractile tension and fura 2 fluorescence were measured simultaneously at 37°C in the same tissue. The tissue was illuminated alternately (125 Hz) at excitation wavelengths of 340 and 380 nm, respectively, by means of two monochromators in the light path of a 75-W xenon lamp. Fluorescence emission from the tissue was measured at 510 nm by photomultiplier tube. The fluorescence intensity at each excitation wavelength and the ratio of these two fluorescence values were recorded with a time constant of 250 ms and stored with the force signal on a computer.

Western immunoblotting analysis. Arterial rings were equilibrated in the tissue bath, and the optimal tensions were obtained as described above. The tissues were then incubated for 30 min with 30 µM PD-098059 or vehicle alone in the organ bath (37°C). After incubation, they were stimulated with 5 µM PDBu. The reaction was stopped at various times by snap freezing the tissues in liquid N₂, and the tissues were stored at –80°C until they were used. Protein levels of phosphorylated p42 and p44 ERK and CaD at Ser⁷⁸⁹ were determined by Western blot analysis, as previously described (55). Briefly, samples with equal protein were loaded on 10% (p42 and p44 ERK) and 7.5% (CaD) SDS-polyacrylamide gels, separated by electrophoresis, and transferred to nitrocellulose membranes. The membranes were incubated with the antibodies against phosphorylated p42 and p44 ERK (Thr²⁰²/Tyr²⁰⁴) and phosphorylated CaD (Ser⁷⁸⁹) and then with secondary antibodies of horseradish peroxidase-conjugated goat anti-rabbit. 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. The data were normalized by actin.

Materials. Phenylephrine, PDBu, PD-098059, and monoclonal antimyosin (20-kDa light chains) were obtained from Sigma; phosphorylated p42 and p44 ERK (Tyr²⁰²/Tyr²⁰⁴) antibodies from Cell Signaling Technology (Beverly, MA); phosphorylated CaD (Ser⁷⁸⁹) antibody from Santa Cruz Biotechnology (Santa Cruz, CA); all electrophoretic and immunoblot reagents from Bio-Rad; and fura 2-AM from Molecular Probes (Eugene, OR). Other reagents were of analytic grade or better and were purchased from Sigma or Fisher Scientific. All chemicals were prepared fresh each day and kept on ice throughout the experiment.

Data analysis. Data were analyzed by computer-assisted linear or nonlinear regression to fit the data using GraphPad Prism (GraphPad Software, San Diego, CA). Values are means ± SE. Differences were evaluated for statistical significance (P < 0.05) by one-way ANOVA and Student's t-test.

	RESULTS

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Effect of PD-098059 on phenylephrine-induced increases in [Ca²⁺]_i, LC₂₀ phosphorylation, and tension. Phenylephrine produced time-dependent increases in [Ca²⁺]_i, LC₂₀ phosphorylation, and tension development in the uterine artery (Fig. 1). The tension development was preceded by [Ca²⁺]_i and LC₂₀ phosphorylation. Although the agonist-induced tension was sustained for up to 5 min, LC₂₀ phosphorylation and [Ca²⁺]_i progressively declined to a steady state of 50% and 65%, respectively, of their peak values. PD-098059 significantly inhibited the phenylephrine-induced increase in [Ca²⁺]_i in the uterine artery and decreased the peak level to 56% of control (Fig. 1). Likewise, PD-098059 significantly decreased the rate of phenylephrine-mediated LC₂₀ phosphorylation and the maximum phosphorylation in the uterine artery. The peak phosphorylation level was significantly decreased to 35.6 ± 8.7% of control, and the time to the peak was delayed by 20 s compared with control. Although PD-098059 did not change the time to the peak of phenylephrine-induced force development, it slowed the initial rate of tension development and significantly decreased the contractile tension compared with control.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Effect of PD-098059 on phenylephrine-induced intracellular Ca2+ concentration ([Ca2+]i; A), phosphorylation of 20-kDa myosin light chain (LC20; B), and contraction (C) in the uterine artery. Arterial rings were pretreated with 30 µM PD-098059 or the vehicle DMSO (control) for 30 min and then stimulated with 3 µM phenylephrine. [Ca2+]i, LC20 phosphorylation, and contraction were recorded simultaneously in the uterine artery. Representative Western immunoblot shows LC20 and phosphorylated LC20 (LC20-P) induced by phenylephrine at 0–300 s. Values are means ± SE from 4–9 animals. *All PD-098059 values at 5 s are significantly decreased compared with control (P < 0.05).

Effect of PD-098059 on LC₂₀ phosphorylation-[Ca²⁺]_i relation. Contractions of smooth muscle can be regulated by altering the dependence of LC₂₀ phosphorylation on [Ca²⁺]_i or the dependence of force on LC₂₀ phosphorylation. To determine the potential role of ERK in the regulation of Ca²⁺, sensitivity of LC₂₀ phosphorylation at fixed [Ca²⁺]_i, we examined the effect of PD-098059 on the relation of phenylephrine-stimulated increases in [Ca²⁺]_i and LC₂₀ phosphorylation in the uterine artery. PD-098059 did not affect the slope [i.e., LC₂₀ phosphorylation ÷ ratio of fluorescence at 340 nm to fluorescence at 380 nm; from 13.5 ± 2.8 (n = 9, control) to 11.6 ± 1.2 (n = 5, treatment group), P > 0.05; Fig. 2] but significantly decreased the intercept (from 0.164 ± 0.054 to 0.0097 ± 0.0019, P < 0.05). This downward shift in the curve has the effect of showing a decreased LC₂₀ phosphorylation at any given [Ca²⁺]_i.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Effect of PD-098059 on phenylephrine-mediated LC20 phosphorylation-[Ca2+]i relation in the uterine artery. Arterial rings were pretreated with 30 µM PD-098059 or DMSO for 30 min and then stimulated with 3 µM phenylephrine. Increases of LC20 phosphorylation stimulated by phenylephrine were plotted to show responses as a function of [Ca2+]i [fura 2 signal, i.e., ratio of fluorescence at 340 nm to fluorescence at 380 nm (Rf340/380)] at each corresponding time point (from 3 to 30 s). Values are means ± SE from 4–9 animals.

Effect of PD-098059 on force-LC₂₀ phosphorylation relation. Although the majority of stimuli result in a unique relation between LC₂₀ phosphorylation and force in smooth muscle, collateral regulation via thin filaments in modulating contraction can increase or decrease the force-to-LC₂₀ phosphorylation ratio. We further examined the role of ERK in the regulation of contractions independent of changes in LC₂₀ phosphorylation by evaluating the effect of PD-098059 on the relation between phenylephrine-stimulated increases in LC₂₀ phosphorylation and force measured simultaneously in the same tissue. Phenylephrine-induced tensions were plotted against their corresponding phosphorylated LC₂₀ levels of the initial rising phases in control and PD-098059-treated tissues, respectively (Fig. 3A). PD-098059 caused a leftward shift in the force-LC₂₀ phosphorylation relation (Fig. 3A), suggesting that tension developed at a given level of LC₂₀ phosphorylation in PD-098059-treated tissues was increased compared with that in control tissues. Because the relation between phenylephrine-stimulated LC₂₀ phosphorylation and force development was nonlinear in control and PD-098059-treated tissues, analysis was performed to determine the levels of LC₂₀ phosphorylation of control and PD-098059-treated tissues at corresponding developed tensions (from 0.4 to 2.8 g). There was a linear relation between the levels of LC₂₀ phosphorylation of control and PD-098059-treated tissues at corresponding tensions (Fig. 3B), with the slope (0.456 ± 0.008) significantly lower than the line of identity, confirming that the amount of force produced per phosphorylated LC₂₀ was enhanced by PD-098059.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Effect of PD-098059 on phenylephrine-induced force-LC20 phosphorylation relation in the uterine artery. Arterial rings were pretreated with 30 µM PD-098059 or DMSO (control) for 30 min and then stimulated with 3 µM phenylephrine. A: increases of tension stimulated by phenylephrine plotted to show responses as a function of phosphorylated LC20 measured simultaneously in the same tissue at each corresponding time point (0–10 s for control and 0–30 s for PD-098059-treated rings; see Fig. 1). B: relation between levels of LC20 phosphorylation from control and PD-098059-treated tissues at corresponding developed tensions, calculated from curves in A. Dashed line, line of identity. Values are means ± SE from 4–9 animals.

Effect of PD-098059 on PDBu-stimulated [Ca²⁺]_i, LC₂₀ phosphorylation, and tension. Compared with phenylephrine, PDBu stimulated a much slower development of tension, which reached the maximum at 20 min. In contrast to phenylephrine, PDBu did not significantly increase [Ca²⁺]_i (data not shown) or LC₂₀ phosphorylation in the uterine artery (Fig. 4). Simultaneous measurement of [Ca²⁺]_i and contractions showed that PD-098059 increased PDBu-induced contractions without changing [Ca²⁺]_i (data not shown). The effect of PD-098059 on PDBu-induced contraction and LC₂₀ phosphorylation was examined in the tissues in which contractile tension and LC₂₀ phosphorylation were measured simultaneously. PD-098059 augmented the PDBu-mediated contractions over the time period examined (Fig. 4) but did not change LC₂₀ phosphorylation.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Effect of PD-098059 on phorbol 12,13-dibutyrate (PDBu)-stimulated tension and LC20 phosphorylation in the uterine artery. Arterial rings were pretreated with 30 µM PD-098059 or DMSO for 30 min and then stimulated with 5 µM PDBu. PDBu-induced contraction and LC20 phosphorylation (LC20-P) were measured simultaneously in the same tissues. Representative Western immunoblot shows LC20 and no phosphorylated LC20 induced by PDBu at 0–30 min. Phenylephrine (PE)-induced phosphorylated LC20 serves as a positive control. Values are means ± SE from 7 animals. *P < 0.05 vs. control tensions.

Effect of PD-098059 on PDBu-stimulated phosphorylation of p42 and p44 ERK and CaD. PDBu produced time-dependent increases in phosphorylation of p42 and p44 ERK (Fig. 5) and ERK-dependent phosphorylation of CaD at Ser⁷⁸⁹ in the uterine artery (Fig. 6). PD-098059 blocked PDBu-mediated phosphorylation of p42 and p44 ERK and CaD (Figs. 5 and 6).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effect of PD-098059 on PDBu-stimulated phosphorylation of p42 and p44 ERK in the uterine artery. Arterial rings were pretreated with 30 µM PD-098059 or DMSO for 30 min and then stimulated with 5 µM PDBu. PDBu-induced increase in phosphorylated p42 (A) and p44 (B) ERK (p-ERK42 and p-ERK44) was determined by Western immunoblot analysis. Values are means ± SE from 6 animals.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Effect of PD-098059 on PDBu-stimulated phosphorylation of caldesmon (Ser789) in the uterine artery. Arterial rings were pretreated with 30 µM PD-098059 or DMSO for 30 min and then stimulated with 5 µM PDBu. PDBu-induced increase in phosphorylated caldesmon at Ser789 (p-CaD789) was determined by Western immunoblot analysis. Values are means ± SE from 4–6 animals.

	DISCUSSION

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ERK and thick filament regulation. Smooth muscle contraction is regulated by thick and thin filament regulatory pathways. Thick filament regulation is mediated by Ca²⁺-dependent mechanisms that lead to activation of MLCK and LC₂₀ phosphorylation and by Ca²⁺-independent mechanisms that involve inactivation of MLCP and a decrease in LC₂₀ dephosphorylation. In the present study, we examined the temporal relations among [Ca²⁺]_i, LC₂₀ phosphorylation, and isometric force in the pregnant uterine artery and, consistent with previous findings, demonstrated that phenylephrine produced a time-dependent increase in [Ca²⁺]_i and LC₂₀ phosphorylation that preceded the contraction. We found that the decrease in phenylephrine-induced force development caused by PD-098059 was preceded by a reduction in LC₂₀ phosphorylation, suggesting a role for ERK in thick filament regulation. Similar results were obtained in porcine carotid artery, in which PD-098059-mediated inhibition of endothelin-1-induced contraction was associated with a reduction in LC₂₀ phosphorylation (8).

Previous studies in isolated smooth muscle cells suggested that ERK does not regulate [Ca²⁺]_i (38, 49). However, our recent studies in intact arterial rings, in which [Ca²⁺]_i and contractile tension were measured simultaneously in the same tissue, demonstrated clearly that PD-098059-mediated inhibition of phenylephrine-induced contraction was associated with a decrease in [Ca²⁺]_i and Ca²⁺ sensitivity in the uterine artery (55). This suggests that, in addition to the Ca²⁺-independent pathway as previously proposed (1, 2, 11, 17, 21, 33), the ERK signaling pathway also involves the Ca²⁺-dependent components of vascular contractions. Consistent with our previous findings, the present study demonstrated that PD-098059 inhibited the phenylephrine-induced increase in [Ca²⁺]_i. The mechanisms involved in ERK-mediated regulation of [Ca²⁺]_i are not clear. Given the finding that PD-098059 did not affect force development elicited by KCl depolarization (8, 52, 55), it is likely that ERK may regulate the release of Ca²⁺ from intracellular pools but not Ca²⁺ entry via L-type Ca²⁺ channels. Because LC₂₀ phosphorylation is primarily regulated by [Ca²⁺]_i, our results suggest that ERK may regulate LC₂₀ phosphorylation, in part, through modulating [Ca²⁺]_i.

In addition, we found that PD-098059 significantly decreased Ca²⁺ sensitivity of LC₂₀ phosphorylation in response to phenylephrine, i.e., less LC₂₀ phosphorylation at the given [Ca²⁺]_i in the presence of PD-098059. Because alterations in the activities of MLCK or MLCP at fixed [Ca²⁺]_i will affect the Ca²⁺ sensitivity of LC₂₀ phosphorylation, the results suggest that, in addition to the regulation of MLCK activity through changes in [Ca²⁺]_i, ERK may also regulate MLCK or/and MLCP activities independent of changes in [Ca²⁺]_i. The finding that PD-098059 decreased the intercept without affecting the slope of the LC₂₀ phosphorylation-[Ca²⁺]_i relation suggests that ERK may have a positive tonic effect on Ca²⁺ sensitivity of LC₂₀ phosphorylation but may not affect agonist-mediated Ca²⁺ sensitivity. MLCK and MLCP can be involved in the regulation of Ca²⁺ sensitivity of LC₂₀ phosphorylation, and MLCP may be primarily involved in agonist-induced Ca²⁺ sensitization (39). Although the present study cannot rule out effects on MLCP, we speculate that the tonic effect of ERK on Ca²⁺ sensitivity observed in the present study may be mediated by an increase in MLCK activity. It has been demonstrated that purified, constitutively active ERK1/2 can phosphorylate chicken gizzard MLCK and increase its phosphotransferase activity in vitro (29). In addition, transient transfection of a mutationally activated MEK1 increased MLCK and LC₂₀ phosphorylation in COS-7 cells, which were reversed by pretreatment with PD-098059 (29).

ERK and thin filament regulation. The present findings that [Ca²⁺]_i and the degree of LC₂₀ phosphorylation decline from their peak values to lower steady-state levels during phenylephrine-induced sustained uterine artery contraction are in agreement with previous results (8, 12, 34, 54). Previously, the dissociation of force and LC₂₀ phosphorylation was postulated to be a consequence of the "latch" state (25, 35). However, recent studies suggested an additional thin filament regulation in smooth muscle contraction (21, 33). The present study demonstrated that PD-098059 increased force development at given levels of LC₂₀ phosphorylation mediated by phenylephrine. This suggests that, in the uterine artery, ₁-adrenoceptor-mediated contractions are regulated, in addition to the thick filament pathway, by thin filament pathways. More importantly, the present results suggest that ERK inhibits this ₁-adrenoceptor-mediated thin filament regulation of contractions. Nevertheless, PD-098059 inhibited the phenylephrine-induced contraction, suggesting that the thick filament regulatory pathway, i.e., LC₂₀ phosphorylation, predominates in ₁-adrenoceptor-mediated uterine artery contractions.

The idea that ERK may inhibit thin filament regulatory pathways is further supported by the results of PKC-mediated contractions in the uterine artery. PKC activation of smooth muscle contraction has been well demonstrated from studies showing that phorbol esters, known to activate PKC, induce slow sustained contractions in many types of vascular smooth muscle (7, 10, 23, 24, 40, 44). In our previous studies in the uterine artery, we showed that PDBu increased PKC activity and induced contractions (55). Phosphorylation of LC₂₀ has been reported in phorbol ester-induced contractions (22, 41–43). However, the significance of phorbol ester-induced LC₂₀ phosphorylation in the contraction is controversial, and there is an inconsistency in the dependence of LC₂₀ phosphorylation on [Ca²⁺]_i. For instance, it has been reported that PDBu induces LC₂₀ phosphorylation in Ca²⁺-depleted rat aortas (22, 43), but 12-deoxyphorbol-13-isobutyrate does not induce LC₂₀ phosphorylation in ferret aortas, even in the presence of extracellular Ca²⁺ (24). In addition, previous studies showed dissociation between LC₂₀ phosphorylation and tension development in response to phorbol esters (14, 31, 47). The present study demonstrated that PDBu induced contractions independent of changes in [Ca²⁺]_i or LC₂₀ phosphorylation levels. This suggests that, in the uterine artery, PKC-induced contraction is mediated predominantly through thin filament regulatory pathways. Phorbol ester-induced contractions have been shown to involve regulatory components, including CaD and calponin, associated with thin filaments (33, 45, 46, 53). Phosphorylation of CaD by PKC reverses its inhibitory effect on myosin ATPase (48). The present finding that PD-098059 enhanced PKC-mediated contraction without changing [Ca²⁺]_i or LC₂₀ phosphorylation levels reinforces the conclusion that ERK inhibits the thin filament regulatory pathway in theuterine artery.

In intact vascular smooth muscle, ERK has been demonstrated as a physiologically relevant CaD kinase mediating CaD phosphorylation (2–5). CaD functions as a thin filament regulatory protein and exerts an inhibitory effect on vascular smooth muscle contractions (13, 27, 36). It has been proposed that ERK-mediated phosphorylation of CaD reverses the inhibitory activity of CaD on actin-activated myosin ATPase, hence activating the thin filament pathway (15, 17, 21, 33). Nonetheless, the importance of CaD phosphorylation at ERK-specific sites (particularly at Ser⁷⁸⁹) in smooth muscle contraction remains controversial. Krymsky et al. (30) showed that ERK phosphorylation of gizzard smooth muscle CaD did not reverse its inhibitory effects on myosin ATPase. In addition, Nixon et al. (37) demonstrated that CaD phosphorylation by recombinant, activated ERK2 had no effect on the Ca²⁺ sensitivity of Triton-permeabilized vascular smooth muscle preparations and concluded that the phosphorylation of CaD by ERK is temporally associated with, but not involved in, force generation in smooth muscle. In porcine carotid artery stimulated with endothelin-1, D'Angelo and Adam (8) examined the interrelation among ERK activity, phosphorylation of CaD and LC₂₀, and isometric force. They demonstrated that the inhibitory effect of PD-098059 on force could not be correlated with a corresponding effect on ERK-mediated CaD phosphorylation, because force in arterial strips stimulated with endothelin-1 in the absence or presence of PD-098059 tended to approximate each other over time, despite significant differences in the level of CaD phosphorylation.

In the present study, we demonstrated that PDBu produced parallel time courses in increasing phosphorylation of p42 and p44 ERK and CaD at Ser⁷⁸⁹ and that the phosphorylation was blocked by PD-098059, suggesting ERK-mediated phosphorylation of CaD at Ser⁷⁸⁹ in the uterine artery. These are in agreement with previous findings (9). The novel finding of the present study is that PD-098059 had opposite effects on PDBu-induced CaD phosphorylation at Ser⁷⁸⁹ and contractions. It blocked phosphorylation of CaD at Ser⁷⁸⁹ but potentiated the contractions. This suggests that ERK-dependent phosphorylation of CaD at Ser⁷⁸⁹ may not be involved in the PDBu-induced contraction but, rather, may inhibit it in the uterine artery. Although we were unable to determine phosphorylation of CaD at Ser⁷⁵⁹ because of the lack of appropriate antibody, a previous study demonstrated that the major site of ERK-dependent phosphorylation in CaD was Ser⁷⁸⁹. Although all the phosphate incorporated into CaD by the ERK is in Ser⁷⁸⁹ or Ser⁷⁵⁹, not all the phosphate in CaD is accounted for by these two sites (3, 9). In addition to ERK-dependent phosphorylation sites, phorbol esters and activation of PKC may increase phosphorylation of CaD at other sites, directly or indirectly, through unknown mechanisms (4, 9, 48, 50, 51). Although CaD phosphorylation in vitro by PKC was at sites that were different from those involved in PDBu-induced phosphorylation of CaD in intact canine aortas (3), endogenous CaD kinase activity in sheep aorta smooth muscle was purified and identified as a proteolytic fragment of PKC (51). PKC phosphorylated sheep aorta CaD in native thin filaments and in the isolated state at Ser¹²⁷, Ser⁵⁸⁷, Ser⁶⁰⁰, Ser⁶⁵⁷, Ser⁶⁸⁶, and Ser⁷²⁶. PKC-mediated phosphorylation of intact CaD and of its COOH-terminal fragment containing 658–756 significantly decreased their ability to inhibit acto-heavy meromyosin ATPase (51). Taken together, we propose that, in sheep uterine artery, PKC induces phosphorylation of CaD at Ser⁷⁸⁹ through activation of ERK, as well as at other site(s) through unknown mechanisms (Fig. 7). It is the phosphorylation of CaD at site(s) other than Ser⁷⁸⁹ that may be important in reversing the inhibitory effect of CaD on myosin ATPase and leading to contractions. Phosphorylation of ERK-dependent Ser⁷⁸⁹ may not lead to the reversal of CaD inhibitory effects on myosin ATPase. Instead, phosphorylation of Ser⁷⁸⁹ may inhibit PKC-mediated phosphorylation of the other site(s).

View larger version (12K): [in this window] [in a new window]   Fig. 7. Proposed model: ERK-dependent phosphorylation of caldesmon at Ser789 inhibits PKC-mediated contractions. We propose that PKC induces phosphorylation of caldesmon at Ser789 through activation of ERK, as well as at other site(s) through unknown mechanisms in sheep uterine artery. 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 leading to contractions. Phosphorylation of ERK-dependent Ser789 may not lead to reversal of caldesmon inhibitory effects on myosin ATPase. Instead, phosphorylation of Ser789 may inhibit PKC-mediated phosphorylation of the other site(s).

In summary, we have shown in the pregnant uterine artery that ₁-adrenoceptor-mediated contraction is regulated through thick and thin filament pathways, with the thick filament regulatory pathway, i.e., LC₂₀ phosphorylation, predominating. However, PKC-mediated contraction is regulated predominantly through thin filament pathways, i.e., independent of changes in LC₂₀ phosphorylation. ERK activation differentially regulates thick and thin filament pathways in the uterine artery. ERK potentiates the thick filament regulatory pathway by elevating LC₂₀ phosphorylation via increases in [Ca²⁺]_i and Ca²⁺ sensitivity of LC₂₀ phosphorylation. In contrast, the present study indicates that ERK attenuates thin filament regulatory pathways and suppresses contractions independent of changes in LC₂₀ phosphorylation in the uterine artery. Although it is not clear whether the ERK-mediated inhibition of the thin filament regulatory pathway is unique to the pregnant uterine artery or is a more generalized mechanism, this novel finding suggests an intriguing hypothesis that, among ERK's effects, CaD phosphorylation at ERK-specific sites may stabilize its inhibitory effect on actin-activated myosin ATPase.

	ACKNOWLEDGMENTS

 
The authors thank Dr. K. G. Morgan for insightful discussion.

GRANTS

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.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Zhang, Center for Perinatal Biology, Dept. of Physiology and Pharmacology, 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.

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